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Food Research International 38 (2005) 1059–1065

Effect of sucrose on the anthocyanin and antioxidant capacityof mulberry extract during high temperature heating

P.J. Tsai a,*, L. Delva a, T.Y. Yu a, Y.T. Huang a, L. Dufosse b

a Department of Food Science, National Pingtung University of Science and Technology, 1 Hsueh Fu Road, Nei Pu Hsiang, 91207 Pingtung,

Taiwan, ROCb Laboratoire ANTiOX, Universite de Bretagne Occidentale, IUP Innovation en Industries Alimentaires, Pole Technologique de Creac�h Gwen,

F-29018 Quimper Cedex, France

Received 2 June 2004; accepted 20 March 2005

Abstract

This study aimed to elucidate how sucrose affects the anthocyanin and antioxidant capacity at low pH under high temperature.The interactive role of different sucrose concentrations (20%, 40%, 60%) and pH values (2, 3, 4) on a mulberry anthocyanin modelsystem at different heating times was investigated. A520 (red color) decreased from 0 to 4 h and increased thereafter, degradationindex of anthocyanin (DI) increased in the pure anthocyanin system during 68 h of heating. The samples with sucrose showed a DIpeak 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 duringheating, 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 significant browning and theincrease 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 caramelizationintermediate 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 antioxi-dant, but they are quite unstable during processingand storage. Temperature, pH, oxygen, and water activ-ity are considered to be important factors influencing itsstability. During heating, degradation and polymeriza-tion usually lead to its discoloration (Markakis, 1982;Tsai & Huang, 2004). Sugar protection via hyperchro-mic effect was reported to stabilize the anthocyanin pig-ment in strawberries (Ohta, Watanabe, & Osaiima,

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

* 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 ro-selles (Tsai, Hsieh, & Huang, 2004) due to reduced wateractivity or availability. In contrast, thermal degradationproducts of sucrose like furfural, caramel or maillardreaction products (MRP) are also well known to bebrowning agents (Granados, Mir, Serrana, & Martinez,1996) and furfural has been found to be involved inanthocyanin deterioration (Debick-pospisil, Lovric, Tri-najstic, & Sabljic, 1983). Temperature and duration ofheating, pH and concentration of reactant are parame-ters that should be taken into consideration (Davies &Labuza, 2005) for the browning reaction. In general,acidic media favor sucrose hydrolysis and carameliza-tion while the maillard reaction is favored by alkaline

1060 P.J. Tsai et al. / Food Research International 38 (2005) 1059–1065

media (Ajandouz & Puigserver, 1999; Farine, Villard,Moulin, Marchis Mouren, & Puigserver, 1997). Daviesand Labuza (2005) reported that caramelization cantake place at a temperature above 80 �C in both a liquidand a dry mixture. It has been proven that some degra-dation products of anthocyanin have antioxidant capac-ity (Tsai & Huang, 2004; Wang, Cao, & Prior, 1997).MRPs were also proven to be powerful as antiradicalagents (Manzocco, Calligaris, Mastrocola, Nicoli, &Lerici, 2001). However, information related to the anti-oxidant capacity of caramels is very scarce. Therefore,the degradation of sucrose and anthocyanin duringheating may affect both the color and antioxidantcapacity.

Mulberry fruit is rich in anthocyanins (Yang & Tsai,1994). The effects of temperature and pH on the kineticsof the antiradical capacity of its extract have been re-ported (Suh, Kim, Lee, Lee, & Choi, 2004). However,to the best of our knowledge, no report exists on the ef-fect of sucrose on the antioxidant capacity of anthocya-nin during heating using acidic medium.

In this study, anthocyanin extracted from mulberrieswas heated in different pH (2–4) and sucrose concentra-tions (20%, 40%, 60%) at 90 �C for different time peri-ods. L, a, b, anthocyanin degradation index (DI),A520 and three fractions of anthocyanins (monomeric,copigmented and polymeric) were measured to monitorthe changes of color and pigment. Furfural content,absorbance at 420 nm, as well as some caramelizationintermediates were used as indicators of the sucrosetransfer. FRAP and TEAC (trolox equivalent antioxi-dant capacity) were used to investigate the changes ofantioxidant capacity. Finally, SAS was applied to calcu-late the correlation among the mentioned factors.

2. Materials and methods

2.1. Sample preparation

Three model systems, excluded with light and oxygen,were used, mulberry anthocyanin, sucrose + anthocya-nin and sucrose alone. The mulberry anthocyanin modelsystem was modified from the method described in Tsaiand Huang (2004). The three model systems were pre-pared at different pH (pH 2, 3, and 4) with or withoutsucrose (0%, 20%, 40% and 60%) and then heated at90 �C for 2, 4, 9,17, 26, 45 and 68 h.

2.2. Color measurement

A Laiko Colourimeter (Laiko, CDM08, Japan) wasused to obtain L, a, b and color difference of the sam-ples. The higher L, a, b means higher lightness, red col-or, and yellow color, respectively (Rommel, Heatherbell,& Wrolstad, 1990).

The degradation index of anthocyanin (DI value) wascalculated as A420 nm/A520 nm(Mazza, Fukumoto, Dela-quis, Girard, & Ewert, 1999). Hue: hue angleab = arc-tan(b/a).

2.3. Copigmented, monomeric, and polymericanthocyanins

Copigmented, monomeric and polymeric anthocya-nins were determined using a modification of the meth-od 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 stan-dard. The mobile phase includes 0.3% tetrahydrofuran(THF) with the flow rate 1.0 ml/min and the detectorwas set at 280 nm (Li, Sawamura, & Kusunose, 1988).

2.5. Anthocyanin remaining percentage

The extract was applied to Hitachi #3056 C18 HPLCcolumn and the anthocyanin eluted with a gradient mix-ture of 5% acetic acid and acetonitrile (Tsai & Ou, 1996).The anthocyanin remaining percentage was calculatedwith the peak area of samples at 0 h as 100%.

2.6. Trolox equivalent antioxidant capacity

TEAC was measured by the method of Miller, Dip-lock, and Rice-Evans (1995).

2.7. FRAP assay

FRAP assay is a method of measuring the ability ofreductants (antioxidants) to reduce Fe+3 to Fe+2. Thiswas described by Benzie and Strain (1996).

2.8. Statistical analysis

Statistical analyses of the data were done using SASsoftware (SAS Institute Inc., Cary, NC., USA). Generallinear model procedures were used to determine treat-ment effects. Correlations among the variables were alsocalculated.

3. Results and discussion

3.1. Effect of sucrose on the color during 90 �C heating

3.1.1. L, a, b value

During heating, the mulberry extract changed colorfrom red to brown. The a value decreased, L, b andhue value increased. In the beginning, pH 2 samples

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showed the highest a value and lowest hue, but after68 h heating, all samples showed similar L, a, b andhue value. For example, in sample at pH 2 with 40% su-crose, a, b and hue changed from 40.64, 22.73 and 29.22to 5.24, 39.66 and 82.47, respectively (data not shown).

3.1.2. A520

Usually, A520 is used to express the red color ofanthocyanin. As shown in Fig. 1a, samples at lowerpH and with higher sucrose concentration exhibitedhigher A520 as it has been established by severalresearchers (Bridle & Timberlake, 1997; Tsai et al.,2004). During heating, A520 decreased and the samplesheated for 4 h showed no red color (only about 2.01%anthocyanin remained). This finding suggests that theanthocyanin is almost completely degraded at this level.However, A520 increased obviously after that time. Forexample, at pH 2, in the model containing 40% sucrose,A520 increased sharply from 0.219 at 9 h to 1.051 at26 h and reached 2.114 when the samples were furtherheated for 68 h. Furthermore, pH 2 exhibited a higherA520 than others during browning. One probable expla-nation is that at pH 2, the degree of sucrose hydrolysiswas higher than at pH 3 and pH 4 (Buera, Chirife, &Karel, 1995). In this regards, the unexpected increaseobserved in the absorbance at 520 nm after 9 h mightbe due to the development of color by other reactions in-stead of anthocyanin itself.

0

0.5

1

1.5

2

2.5

Heating time (hr)

Heating time (hr)

Ab

sorb

ance

at

520

nm

pH2, 40%

pH3, 40%

pH4, 40%

pH2, 0%

pH3, 0%

pH4, 0%

0

1

2

3

4

5

6

0 20 30 40 50 60 70 80

0 20 30 40 50 60 70 80

Deg

rad

atio

n In

dex

of

th

e an

tho

cyan

in pH2, 40%

pH3, 40%

pH4, 40%

pH2, 0%

pH3, 0%

pH4, 0%

a

b

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 hheating.

3.1.3. DI value

DI is the index of anthocyanin degradation. Increaseof DI after the pigment degradation usually includes thedecrease of red color (A520) and the increase of brown-ing (A420). In this study, DI increased during heating insamples without sucrose. But for the ones with sucrose,DI reached a maximum value at 17 h (Fig. 1b). Sinceanthocyanin is the main source of red color of Mul-berry, it is obvious that the observed decrease from 0to 17 h of heating is the result of the anthocyanin degra-dation. The same tendency is observed at pH 3 or pH 4and sucrose 20% or 60% samples. However, decreasingof DI after 17 h indicates that severe browning occurredat this period should be accompanied by a lower ratio ofA420 and A520.

3.1.4. Distribution of monomeric, copigmented and

polymeric anthocyanins

The changes in the distribution of mulberry anthocy-anin during heating were investigated. Fig. 2 shows thatat pH 3 during heating from 0 h to 26 h polymericanthocyanin increased significantly from 10% to 62%,while monomeric anthocyanin decreased from 85% to25%. Copigmented anthocyanin changed from 5% to13%. However, after 26 h stability was observed. Wethink that an increase of A520 in calculating ASO2 andAext for polymeryric anthocyanin might be involved inthe stability of the data after 26 h of heating. Similartrends were found in all the samples where sucrosewas added. Apparently, most of the anthocyanins ex-tracted from mulberry exist as monomers, and thebrowning products derived from sucrose did not affectthe distribution of anthocyanin by the methods used inthis study.

0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50 60

Heating time (hr)

Per

cen

tag

e

Copigment (no Suc.)

Monomer (no Suc.)

Polymer (no Suc.)

Copigment (40%suc)

Monomer (40%suc)

Polymer (40%suc)

Fig. 2. The changes of anthocyanin patterns of mulberry anthocyaninmodel 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 withoutsucrose. Copigment (40%suc): % copigmented anthocyanin in modelsystem with 40% sucrose; monomer (40%suc): % monomeric antho-cyanin 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 browningeffect of sucrose. In fact, the pH and temperature playan important role in the complexity of non-enzymaticbrowning reactions. Under our experimental pH condi-tions (pH 2–4) and temperature (90 �C), caramelizationproduct formation seems to be more probable. There-fore, wavelength scanning of the brown products andthe intermediates of caramelization (A230, A285 andfurfural) was investigated.

3.1.5.1. Wavelength scanning. The behavior of browningpigment possessing high A520 was studied by perform-ing the scanning of the samples over a wide range ofwavelength.

As shown in Fig. 3a, the characteristic peak at520 nm disappeared after heating. Instead, A420 greatlyincreased along with the increasing of A520. Thechanges depend on the sucrose concentration and pH.For example, at pH 2, and 17 h, samples with 40% of su-crose exhibited higher A420 and A520 compared tothose with 20% and 60%. From this finding, it might

0

0.5

1

1.5

2

2.5

Ab

sorb

ance

Ab

sorb

ance

20%Suc,17hr40%Suc,17hr60%Suc,17hr20%Suc+ACN,17hr40%Suc+ACN,17hr60%Suc+ACN,17hr20%Suc+ACN,0hr40%Suc+ACN,0hr60%Suc+ACN,0hr

0

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1.5

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400440

480 520

560600

640680

400440

480 520

560600

640680

Wavelength (nm)

Wavelength (nm)

40 %Suc,17hr, pH240 %Suc,17hr, pH340 %Suc,17hr, pH440 %Suc +ACN,17hr, pH240 %Suc +ACN,17hr, pH340 %Suc +ACN,17hr, pH4

a

b

Fig. 3. The wavelength scanning of the system (a) with differentsucrose concentration (20%, 40% and 60%) at pH 2 insucrose + anthocyanin models at 0 and 17 h and (b) with differentpH (pH 2, 3 and 4) with 40% sucrose or sucrose + anthocyanin modelsat 17 h. 40%suc: sucrose at the concentration of 40% in the puresucrose model; 40%suc + ACN: sucrose at the concentration of 40% inthe sucrose–anthocyanin model.

be assumed that sucrose at 40% exhibits more browningthan at 20% or 60% and this could be explained by wateravailability which related to sucrose clusters formationat high sugar concentration (Leung, Magnuson, & Bru-insma, 1979) and the higher relaxation rate at 60% thanat 40% (Tsai et al., 2004). For the pH effect, when thesucrose concentration was fixed at 40%, pH 2 exhibiteda higher A420 and A520 (Fig. 3b). This agreed with thefact that reaction rate or acid hydrolysis of sucrose in-creased with H+ during thermal process (Pinheiro Tor-res & Oliveira, 1999).

3.1.5.2. Caramelization intermediates. A230, A285 andfurfural were investigated to monitor the formation ofthe caramelization browning. Fig. 4 shows that, in allmodels, furfural content increased with increasing heat-ing time and reached a maximum at 26 h. Asucrose + anthocyanin model exhibited the highest fur-fural content and the lowest content was observed inthe pure anthocyanin system. It is obvious that the fur-fural mainly comes from the sucrose degradation (Man-ley-Harris & Richards, 1995). The possible reactionbetween furfural and anthocyanin may facilitate the dis-coloration of anthocyanin, which is then masked by thebrowning compounds formed later in heating. Fig. 5shows the pH dependence of caramelization intermedi-ates as expressed by A230 and A285 in this study.A285 exhibited the same trend as furfural.

The increase in the A520 during browning could alsoresult from the formation of pyranoanthocyanin as thenew pigment formed in red wine (Mateus, Silva,Rivas-Gonzalo, Santos-Buelga, & De Freitas, 2003).However, the scanning did not reveal any peak forma-tion around 500–600 nm, and the formation of pyroan-thocyanins was not taken into consideration in thisstudy.

0

500

1000

1500

2000

2500

3000

0hr 4hr 9hr 17hr 26hr 45hr 68hr

0hr 4hr 9hr 17hr 26hr 45hr 68hr

Time (hr)

Fu

rfu

ral (

pp

m)

ACN

ACN+SUC

SUC

05

1015202530

Time(hr)

Fu

rfu

ral(p

pm

)

ACN

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

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 inFig. 3.

P.J. Tsai et al. / Food Research International 38 (2005) 1059–1065 1063

3.2. The effect of sucrose on antioxidant capacity

In order to elucidate the contribution of sucrose tothe antioxidant capacity of the model systems two kindsof antioxidant assay (TEAC and FRAP) were carriedout. Results showed that, FRAP reducing powerincreased in the pure sucrose + anthocyanin samplesafter they were heated (Fig. 6). Similar changes werefound for the sucrose only system under different su-crose concentrations or pH values. On the other hand,the obtained trolox value (around 2.7–3.1 mM) wasquite stable during heating in all samples (data notshown). It indicated that, in this study, sucrose brown-ing products may increase the reducing power but hadno effect on the ABT radical scavenging. Further analy-sis proved that furfural at different concentration (50–

3000 ppm) did not show the reducing power (data notshown). This might lead to the assumption that the in-crease of the antioxidant activity during heating comesfrom other complexes derived from caramelization, like5-HMF (Granados et al., 1996) or disaccharide (Rats-imba, Fernandez, Defaye, Nigay, & Voilley, 1999) in-stead of furfural.

3.3. Statistical analysis

Statistical analysis was conducted to elucidate therelationship among all the parameters mentioned above.Table 1 showed that, in the sucrose + anthocyaninmodel, FRAP is significantly correlated with furfuraland A420 (with correlation coefficient 0.92 and 0.86,respectively). While the correlation between DI and

Fig. 6. The changes of reducing power in anthocyanin + sucrosesystem as expressed by FRAP assay at different pH values and differentsucrose concentration during heating. pH 2, 20%, 40%, 60%: antho-cyanin + sucrose system with 20%, 40% and 60% sucrose at pH 2; pH3, 20%, 40%, 60%: anthocyanin + sucrose system with 20%, 40% and60% sucrose at pH 3; pH 4, 20%, 40%, 60%: anthocyanin + sucrosesystem with 20%, 40% and 60% sucrose at pH 4.

1064 P.J. Tsai et al. / Food Research International 38 (2005) 1059–1065

FRAP was lower (0.48). In the pure sucrose model,FRAP was also found highly correlated with A285and A420 (with correlation coefficient 0.88 and 0.94,respectively), but not with their ratio (A285/A420, withcorrelation coefficient 0.44; Table 2). This finding sug-gests that heating derivatives from sucrose are moreimportant than anthocyanin degradation in FRAP in-

Table 1Correlation coefficients of the parameters in the pure sucrose + ACN model

A520 A420 A230 A2

A520 1.00 0.42* �0.35 0A420 1.00 �0.23 0A230 1.00 �0A285 1DIFRAPFurfural

*,**,***: Significance at 5%, 1% and 0.1% levels, respectively.

Table 2Correlation coefficients of the parameters in the pure sucrose model

A230 A285 A420 A

A230 1.00 �0.42* �0.21A285 1.00 0.82*** �A420 1.00 �A230/A420A285/A420FRAPFurfural

*,**,***: Significance at 5%, 1% and 0.1% levels, respectively. DI: degradationability of plasma assay.

crease, and the transformation of the browning interme-diates is too complicated to use A285/A420 as an index.Given this, further investigation is needed to identify thecompounds responsible for the vast increase of thereducing power.

4. Conclusion

Sucrose exhibited a hyperchromic effect on anthocya-nin before heating, and the browning derived productsmay enhance anthocyanin discoloration during the earlystage of heating. After that, DI value reached a maxi-mum at 17 h, then dropped down due to a huge A520from the brown products, which also affects the calcula-tion of the distribution of anthocyanin. The browningpromoted the changes of antioxidant properties. Thehigh correlation between FRAP with the caramelizationintermediate and A420, while low correlation with DIindicated that browning and FRAP increase can be pre-ponderantly due to the caramelization of sucrose andnot to the anthocyanin degradation during heating.

5. Note added in proof

Addendum: After the submission of this paper, anarticle appeared in which Benjakul et al. (Food Chemis-try 90:231–239, 2005) showed the ability of sugar inincreasing the antioxidant capacity in fish mince, sup-porting our finding of this study.

85 DI FRAP Furfural

.35** �0.48*** 0.21 �0.11

.77*** 0.42** 0.86*** 0.83

.33* 0.04 �0.19 0.90*

.00 0.30* 0.89*** 0.91*1.00 0.48*** 0.52

1.00 0.92*100

230/A420 A285/A420 FRAP Furfural

0.54*** �0.36** �0.24 0.95*0.46*** 0.50*** 0.88*** 0.95*0.56*** 0.26 0.94*** 0.90*1.00 �0.35** �0.55*** 0.49

1.00 0.44* 0.521.00 0.92*

1.00

index of anthocyanin; FRAP: antioxidant activity of ferric reducing

P.J. Tsai et al. / Food Research International 38 (2005) 1059–1065 1065

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