Food Research International 38 (2005) 10591065 www.elsevier.

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Eect of sucrose on the anthocyanin and antioxidant capacity of mulberry extract during high temperature heating
P.J. Tsai
a b

a,*

, L. Delva a, T.Y. Yu a, Y.T. Huang a, L. Dufosse

Department of Food Science, National Pingtung University of Science and Technology, 1 Hsueh Fu Road, Nei Pu Hsiang, 91207 Pingtung, Taiwan, ROC le Technologique de Cre de Bretagne Occidentale, IUP Innovation en Industries Alimentaires, Po ach Gwen, Laboratoire ANTiOX, Universite F-29018 Quimper Cedex, France Received 2 June 2004; accepted 20 March 2005

Abstract This study aimed to elucidate how sucrose aects the anthocyanin and antioxidant 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 anthocyanin 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 browning depends 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. 2005 Elsevier Ltd. All rights reserved.
Keywords: Sucrose; Anthocyanin; Antioxidant capacity; Mulberry extract

1. Introduction Anthocyanins 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 anthocyanin pigment in strawberries (Ohta, Watanabe, & Osaiima,
*

Corresponding author. Tel.: +886 8 7740408; fax: +886 8 7740378. E-mail address: pijen@mail.npust.edu.tw (P.J. Tsai).

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 alkaline

0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.03.017

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media (Ajandouz & Puigserver, 1999; Farine, Villard, Moulin, Marchis Mouren, & Puigserver, 1997). Davies and Labuza (2005) reported that caramelization can take place at a temperature above 80 C in 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 during heating may aect both the color and antioxidant capacity. Mulberry fruit is rich in anthocyanins (Yang & Tsai, 1994). The eects of temperature and pH on the kinetics of the antiradical capacity of its extract have been reported (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 during heating using acidic medium. In this study, anthocyanin extracted from mulberries was heated in dierent pH (24) and sucrose concentrations (20%, 40%, 60%) at 90 C 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 investigate the changes of antioxidant capacity. Finally, SAS was applied to calculate the correlation among the mentioned factors.

The degradation index of anthocyanin (DI value) was calculated as A420 nm/A520 nm(Mazza, Fukumoto, Delaquis, Girard, & Ewert, 1999). Hue: hue angleab = arctan(b/a). 2.3. Copigmented, monomeric, and polymeric anthocyanins Copigmented, monomeric and polymeric anthocyanins were determined using a modication of the method from Mazza et al. (1999). 2.4. Furfural detection The 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 detector was set at 280 nm (Li, Sawamura, & Kusunose, 1988). 2.5. Anthocyanin remaining percentage The 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 capacity TEAC was measured by the method of Miller, Diplock, and Rice-Evans (1995). 2.7. FRAP assay FRAP 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 analysis

2. Materials and methods 2.1. Sample preparation Three 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 without sucrose (0%, 20%, 40% and 60%) and then heated at 90 C for 2, 4, 9,17, 26, 45 and 68 h. 2.2. Color measurement

Statistical 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 discussion 3.1. Eect of sucrose on the color during 90 C heating A 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). 3.1.1. L, a, b value During 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 samples

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showed 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. A520 Usually, 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 anthocyanin is almost completely degraded at this level. However, A520 increased obviously 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 samples 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.

3.1.3. DI value DI is the index of anthocyanin degradation. Increase of DI after the pigment degradation usually includes the decrease of red color (A520) and the increase of browning (A420). In this study, DI increased during heating in samples without sucrose. But for the ones with sucrose, DI reached a maximum value at 17 h (Fig. 1b). Since anthocyanin is the main source of red color of Mulberry, it is obvious that the observed decrease from 0 to 17 h of heating is the result of the anthocyanin degradation. The same tendency is observed at pH 3 or pH 4 and sucrose 20% or 60% samples. However, decreasing of DI after 17 h indicates that severe browning occurred at this period should be accompanied by a lower ratio of A420 and A520. 3.1.4. Distribution of monomeric, copigmented and polymeric anthocyanins The changes in the distribution of mulberry anthocyanin during heating were investigated. Fig. 2 shows that at pH 3 during heating from 0 h to 26 h polymeric anthocyanin increased signicantly from 10% to 62%, while monomeric anthocyanin decreased from 85% to 25%. Copigmented anthocyanin changed from 5% to 13%. However, after 26 h stability was observed. We think that an increase of A520 in calculating ASO2 and Aext for polymeryric anthocyanin might be involved in the stability of the data after 26 h of heating. Similar trends were found in all the samples where sucrose was added. Apparently, most of the anthocyanins extracted from mulberry exist as monomers, and the browning products derived from sucrose did not aect the distribution of anthocyanin by the methods used in this study.

2.5

Absorbance at 520 nm

pH2, 40%

pH3, 40% pH4, 40%

1.5 1

pH2, 0% pH3, 0% pH4, 0%

0.5

90 80

Percentage

20

30

40

50

60

70

80

70 60 50 40 30 20 10 0 0 10 20 30 40 50 60
Copigment (no Suc.) Monomer (no Suc.) Polymer (no Suc.) Copigment (40%suc) Monomer (40%suc) Polymer (40%suc)

a
6

Heating time (hr)

Degradation Index of the anthocyanin

5
pH2, 40%

4 3 2 1 0 0 20 30 40 50 60 70 80

pH3, 40% pH4, 40% pH2, 0% pH3, 0% pH4, 0%

Heating time (hr)

Heating time (hr)

Fig. 1. Evolution of the red color at pH (2, 3, 4) as expressed by the (a) A520 and (b) degradation index in the model containing 40% sucrose + anthocyanin and 0% sucrose + anthocyanin during 68 h heating.

Fig. 2. The changes of anthocyanin patterns of mulberry anthocyanin model system (pH 3) during heating. Copigment (no suc): % copigmented anthocyanin in model system without sucrose; monomer (no suc): % monomeric anthocyanin in model system without sucrose; polymer (no suc): % polymeric anthocyanin in model system without sucrose. Copigment (40%suc): % copigmented anthocyanin in model system with 40% sucrose; monomer (40%suc): % monomeric anthocyanin in model system with 40% sucrose; polymer (40%suc): % polymeric anthocyanin in model system with 40% sucrose.

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3.1.5. Causative pigment of browning There are quite a few ways to explain the browning eect of sucrose. In fact, the pH and temperature play an important role in the complexity of non-enzymatic browning reactions. Under our experimental pH conditions (pH 24) and temperature (90 C), caramelization product formation seems to be more probable. Therefore, wavelength scanning of the brown products and the intermediates of caramelization (A230, A285 and furfural) was investigated. 3.1.5.1. Wavelength scanning. The behavior of browning pigment possessing high A520 was studied by performing the scanning of the samples over a wide range of wavelength. As shown in Fig. 3a, the characteristic peak at 520 nm disappeared after heating. Instead, A420 greatly increased along with the increasing of A520. The changes depend on the sucrose concentration and pH. For example, at pH 2, and 17 h, samples with 40% of sucrose exhibited higher A420 and A520 compared to those with 20% and 60%. From this nding, it might
20%S uc,17hr 40%S uc,17hr 60%S uc,17hr 20%S uc+ACN,17hr 40%S uc+ACN,17hr 60%S uc+ACN,17hr 20%S uc+ACN,0hr 40%S uc+ACN,0hr 60%S uc+ACN,0hr

be assumed that sucrose at 40% exhibits more browning than at 20% or 60% and this could be explained by water availability which related to sucrose clusters formation at high sugar concentration (Leung, Magnuson, & Bruinsma, 1979) and the higher relaxation rate at 60% than at 40% (Tsai et al., 2004). For the pH eect, when the sucrose concentration was xed at 40%, pH 2 exhibited a higher A420 and A520 (Fig. 3b). This agreed with the fact that reaction rate or acid hydrolysis of sucrose increased with H+ during thermal process (Pinheiro Torres & Oliveira, 1999). 3.1.5.2. Caramelization intermediates. A230, A285 and furfural were investigated to monitor the formation of the caramelization browning. Fig. 4 shows that, in all models, furfural content increased with increasing heating time and reached a maximum at 26 h. A sucrose + anthocyanin model exhibited the highest furfural content and the lowest content was observed in the pure anthocyanin system. It is obvious that the furfural mainly comes from the sucrose degradation (Manley-Harris & Richards, 1995). The possible reaction between furfural and anthocyanin may facilitate the discoloration of anthocyanin, which is then masked by the browning compounds formed later in heating. Fig. 5 shows the pH dependence of caramelization intermediates as expressed by A230 and A285 in this study. A285 exhibited the same trend as furfural. The increase in the A520 during browning could also result from the formation of pyranoanthocyanin as the new pigment formed in red wine (Mateus, Silva, Rivas-Gonzalo, Santos-Buelga, & De Freitas, 2003). However, the scanning did not reveal any peak formation around 500600 nm, and the formation of pyroanthocyanins was not taken into consideration in this study.

2.5

Absorbance

1.5

0.5

0 64

40

44

48

52

56

60

a
2.5 2

Wavelength (nm)

68

Absorbance

1.5 1 0.5 0

Furfural (ppm)

40 %Suc,17hr, pH2 40 %Suc,17hr, pH3 40 %Suc,17hr, pH4 40 %Suc +A CN,17hr, pH2 40 %Suc +A CN,17hr, pH3 40 %Suc +A CN,17hr, pH4

Furfural(ppm)

30 25 20 15 10 5 0 0hr 4hr 9hr 17hr 26hr 45hr 68hr

3000 2500 2000 1500 1000 500 0 0hr

Time(hr) ACN

ACN ACN+SUC SUC

00

0 64

44

48

52

56

60

Wavelength (nm)

Fig. 3. The wavelength scanning of the system (a) with dierent sucrose concentration (20%, 40% and 60%) at pH 2 in sucrose + anthocyanin models at 0 and 17 h and (b) with dierent pH (pH 2, 3 and 4) with 40% sucrose or sucrose + anthocyanin models at 17 h. 40%suc: sucrose at the concentration of 40% in the pure sucrose model; 40%suc + ACN: sucrose at the concentration of 40% in the sucroseanthocyanin model.

68

4hr

9hr

17hr

26hr

45hr

68hr

Time (hr)

Fig. 4. The changes of furfural in three systems (mulberry anthocyanin system with or without sucrose, and the sucrose only) at pH 2 during being heated at 90 C. ACN: pure anthocyanin model; ACN + SUC: anthocyanin model associate with sucrose; SUC: pure sucrose model.

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Fig. 5. The changes of A230 and A285 on mulberry anthocyanin with or without sucrose during heating at 90 C at pH 2 for (a) and (b), pH 3 for (c) and (d) and pH 4 for (e) and (f). 20%suc, 40%suc, 60%suc: the same as in Fig. 3; 20%suc + ACN, 40%suc + ANC, 60%suc + ANC: the same as in Fig. 3.

3.2. The eect of sucrose on antioxidant capacity In order to elucidate the contribution of sucrose to the antioxidant capacity of the model systems two kinds of antioxidant assay (TEAC and FRAP) were carried out. Results showed that, FRAP reducing power increased in the pure sucrose + anthocyanin samples after they were heated (Fig. 6). Similar changes were found for the sucrose only system under dierent sucrose concentrations or pH values. On the other hand, the obtained trolox value (around 2.73.1 mM) was quite stable during heating in all samples (data not shown). It indicated that, in this study, sucrose browning products may increase the reducing power but had no eect on the ABT radical scavenging. Further analysis proved that furfural at dierent concentration (50

3000 ppm) did not show the reducing power (data not shown). This might lead to the assumption that the increase of the antioxidant activity during heating comes from other complexes derived from caramelization, like 5-HMF (Granados et al., 1996) or disaccharide (Ratsimba, Fernandez, Defaye, Nigay, & Voilley, 1999) instead of furfural. 3.3. Statistical analysis Statistical analysis was conducted to elucidate the relationship among all the parameters mentioned above. Table 1 showed that, in the sucrose + anthocyanin model, FRAP is signicantly correlated with furfural and A420 (with correlation coecient 0.92 and 0.86, respectively). While the correlation between DI and

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crease, and the transformation of the browning intermediates is too complicated to use A285/A420 as an index. Given this, further investigation is needed to identify the compounds responsible for the vast increase of the reducing power.

4. Conclusion Sucrose exhibited a hyperchromic eect on anthocyanin before heating, and the browning derived products may enhance anthocyanin discoloration during the early stage of heating. After that, DI value reached a maximum at 17 h, then dropped down due to a huge A520 from the brown products, which also aects the calculation of the distribution of anthocyanin. The browning promoted the changes of antioxidant properties. The high correlation between FRAP with the caramelization intermediate and A420, while low correlation with DI indicated that browning and FRAP increase can be preponderantly due to the caramelization of sucrose and not to the anthocyanin degradation during heating.

Fig. 6. The changes of reducing power in anthocyanin + sucrose system as expressed by FRAP assay at dierent pH values and dierent sucrose concentration during heating. pH 2, 20%, 40%, 60%: anthocyanin + sucrose system with 20%, 40% and 60% sucrose at pH 2; pH 3, 20%, 40%, 60%: anthocyanin + sucrose system with 20%, 40% and 60% sucrose at pH 3; pH 4, 20%, 40%, 60%: anthocyanin + sucrose system with 20%, 40% and 60% sucrose at pH 4.

FRAP was lower (0.48). In the pure sucrose model, FRAP was also found highly correlated with A285 and A420 (with correlation coecient 0.88 and 0.94, respectively), but not with their ratio (A285/A420, with correlation coecient 0.44; Table 2). This nding suggests that heating derivatives from sucrose are more important than anthocyanin degradation in FRAP in-

5. Note added in proof Addendum: After the submission of this paper, an article appeared in which Benjakul et al. (Food Chemistry 90:231239, 2005) showed the ability of sugar in increasing the antioxidant capacity in sh mince, supporting our nding of this study.

Table 1 Correlation coecients of the parameters in the pure sucrose + ACN model A520 A520 A420 A230 A285 DI FRAP Furfural
*,**,***

A420 0.42* 1.00

A230 0.35 0.23 1.00

A285 0.35** 0.77*** 0.33* 1.00

DI 0.48*** 0.42** 0.04 0.30* 1.00

FRAP 0.21 0.86*** 0.19 0.89*** 0.48*** 1.00

Furfural 0.11 0.83 0.90* 0.91* 0.52 0.92* 100

1.00

: Signicance at 5%, 1% and 0.1% levels, respectively.

Table 2 Correlation coecients of the parameters in the pure sucrose model A230 A230 A285 A420 A230/A420 A285/A420 FRAP Furfural
*,**,***

A285 0.42* 1.00

A420 0.21 0.82*** 1.00

A230/A420 0.54*** 0.46*** 0.56*** 1.00

A285/A420 0.36** 0.50*** 0.26 0.35** 1.00

FRAP 0.24 0.88*** 0.94*** 0.55*** 0.44* 1.00

Furfural 0.95* 0.95* 0.90* 0.49 0.52 0.92* 1.00

1.00

: Signicance at 5%, 1% and 0.1% levels, respectively. DI: degradation index of anthocyanin; FRAP: antioxidant activity of ferric reducing ability of plasma assay.

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