Food Bioprocess Technol
DOI 10.1007/s11947-014-1296-8
ORIGINAL PAPER
Effect of Storage on Anthocyanin Degradation in Black Mulberry
Juice and Concentrates
Togzhan Boranbayeva & Feryal Karadeniz & Emel Yılmaz
Received: 21 February 2013 / Accepted: 5 March 2014
# Springer Science+Business Media New York 2014
Abstract Anthocyanins present in fruits and vegetables are
receiving increased attention because of their potential anti-
oxidant activity but are very susceptible to degradation during
processing and storage. Effect of storage on kinetics of antho-
cyanin degradation and hydroxymethylfurfural (HMF) forma-
tion in black mulberry juice and concentrate was determined
during 8 months of storage at temperatures of 5°, 20°, 30°, and
40 °C. The monomeric anthocyanin degradation was in ac-
cordance with the first-order reaction kinetics and the activa-
tion energies of anthocyanin degradation in black mulberry
juice and concentrate were found as 56.48 and 49.75 kJ mol−1,
respectively. HMF formation in black mulberry juice and
concentrate increased linearly with storage time and tempera-
ture and followed zero-order reactions. The activation ener-
gies of HMF formation in black mulberry juice and concen-
trate were found as 75.70 and 104.11 kJ mol−1, respectively.
The losses of antioxidant activity for black mulberry juice and
concentrate during storage at different temperatures were in
the ranges of 4.87–16.01 and 4.47–33.57 %, respectively.
Antioxidant activity in black mulberry juice and concentrate
was correlated with total monomeric anthocyanins.
Keywords Mulberry juice and concentrate . Kinetics .
Antioxidant activity . Anthocyanin . HMF . Storage
T. Boranbayeva
Department of Food Engineering, M. Auezov South Kazakhstan
State University, Shymkent, Kazakhstan
F. Karadeniz (*)
Faculty of Engineering, Department of Food Engineering, Ankara
University, Ankara 06110, Turkey
e-mail: karadeniz@ankara.edu.tr
E. Yılmaz
Faculty of Engineering and Architecture, Department of Food
Engineering, Trakya University, Edirne 22180, Turkey
Introduction
Mulberry belongs to the genus Morus of the family Moraceae.
There are 24 species of Morus and one subspecies, with at
least 100 known varieties (Pawlowska et al. 2008). Mulberry
(Morus species) is grown wild or cultivated in many countries
for its foliage, which is a primary source of food for silkworms
(Bombyx mori L.). In general, there are three types of mulber-
ry, including white (Morus alba), black (Morus nigra), and
red (Morus rubra) (Aramwit et al. 2010). The Anatolia region
of Turkey has growing conditions suitable for cultivating high
quality mulberry fruits. The production of mulberry in Turkey
in 2011 was 78.702 tonnes (FAO 2011) and its cultivation in
Turkey has been known for more than 400 years. The red-
colored fruits are eaten fresh and are also used in marmalades,
juices, liquors, natural dyes, and in the cosmetics industry
(Ercisli and Orhan 2007).
Epidemiological and clinical investigations have associ-
ated diets rich in fruits and vegetables with reduced risk of
heart disease, neurological and chronic diseases, and vari-
ous forms of cancer (Hertog et al. 1993). These physiolog-
ical functions of fruits and vegetables may be partly attrib-
uted to their high phenolic content (Cieslik et al. 2006) such
as anthocyanins. Nowadays, interest in anthocyanins has
increased because they are potential natural alternatives to
artificial colorants in the food and pharmaceutical indus-
tries and their antioxidant activity plays an important role in
the prevention of diseases (Alasalvar et al. 2005; Patras
et al. 2010). Anthocyanins are highly unstable and easily
susceptible to degradation. Anthocyanin stability is strong-
ly affected by pH, temperature, anthocyanin concentration,
oxygen, light, enzymes, and other accompanying sub-
stances such as ascorbic acid, sugars, sulfites, copigments,
and metallic ions. The amount of anthocyanins found in
foods decreases during processing and storage as tempera-
ture rises (Maccarone et al. 1985).
 Food Bioprocess Technol

Another important chemical change occurred in foods dur-

ing processing and storage is nonenzymatic browning reac-
tions, which involve caramelization, ascorbic acid degradation,
and the Maillard reaction (Cornwell and Wrolstad 1981).
Brown pigment formation is desired during food processing
as in the manufacture of coffee, tea, and beer and in the toasting
and baking of bread. This reaction improves the desirable
sensory characteristics of these foods, e.g., color, aroma, and
flavor. Conversely, it is undesirable in concentrated foods, as
maximum reaction rate occurs at water activities of 0.6–0.7
(Eskin 1990). Maillard reactions can also cause losses in nutri-
tional value of foods, formation of undesirable compounds, e.g.
furfural, hydroxy methyl furfural and brown pigment (Martins
et al. 2001). The Maillard reaction, takes place between α-
amino groups and reducing sugars, is the most important reason
for browning in fruit juice during storage (Toribio and Lozano
1984). Maillard reaction is influenced by many factors, includ-
ing reactant concentration, temperature, time, initial pH, and
water activity (Benjakul et al. 2005).
The stability of anthocyanin content during processing and
storage of foods is one of the most important quality attributes
of food products. Several studies have reported the effect of
processing and storage on the stability of fruit juice with high
anthocyanin content (Garzon and Wrolstad 2002; Wang and
Xu 2007; Jiménez et al. 2010). However, the literature
contains no references on the degradation of anthocyanin
in mulberry juice and concentrate during storage. In
addition to this, for the quality of stored fruit juices,
hydroxymethylfurfural (HMF) is an important criterion.
Therefore, this study was conducted to determine the kinetics
of anthocyanin degradation and HMF formation in black
mulberry juice and concentrate during storage at 5°, 20°,
30°, and 40 °C, as knowledge of the kinetic parameters, such
as reaction order, rate constant, and activation energy is
essential for predicting and controlling food quality. Further-
more, antioxidant activity was also analyzed to determine the
correlations among antioxidant activity, anthocyanins and
HMF.

Fig. 1 Black mulberry juice concentrate production flow chart

Materials and Methods

Materials pH, Titratable Acidity, and Water-Soluble Solids Content

Black mulberry juice (100 %, 15.2°Bx) and concentrate
(65.7°Bx) were supplied by one of the fruit juice and concen-
trate producers in Turkey (Göknur Foodstuffs Import Export
Trading and Production Company) just after production date.
Fruit juice samples have been stored in 200-mL glass bottles,
wheres concentrate samples have been stored in 100 g jars at
5°, 20°, 30°, and 40 °C for 8 months, and analyses were
carried out each month as two replicates. Black mulberry juice
concentrate production flow chart is given Fig. 1.
pH was measured at 20 °C with a pH meter (Consort P407,
Schott Gerate, Belgium) (International Federation of Fruit
Juice Producers (IFU) 1989). After determination of pH, the
samples were titrated with 0.1 N NaOH up to pH 8.1 and
results were expressed as gram citric acid per 100 mL juice
and gram citric acid per 100 g concentrate (IFU 1996). Water-
soluble solid contents of black mulberry juice and concen-
trates were determined with an Abbe refractometer (NOW;
Nippon Optical Work Co., Ltd, Tokyo, Japan) at 20 °C, and
Food Bioprocess Technol
results were expressed as °Bx (IFU 2000). Before analyzing
the pH value, total soluble solids (°Bx) and titratable acidity
(g citric acid per 100 g sample) were determined. These values
were found as 3.91, 15.2, and 0.26 in fruit juices and 3.29,
65.7, and 2.69 in concentrate, respectively. pH, titratable
acidity, and soluble solids were determined during 8 months
of storage, and no significant changes were observed
(p<0.01).
Total Monomeric Anthocyanins Analysis
The total monomeric anthocyanins were determined using the
pH differential method described by Giusti and Wrolstad
(2005). The dilution factor for the samples was determined
by diluting with potassium chloride buffer, pH 1.0, until the
absorbance of the sample at the λvis-max (520 nm) is within
the linear range of the spectrophotometer. The absorbance of
samples at 520 nm for anthocyanin content and 700 nm for
haze was measured on a UV–VIS spectrophotometer (Model
UV2 Unicam, England) against a blank cell filled with dis-
tilled water. The monomeric anthocyanin concentrations were
calculated as mg cyanidin-3-glucoside (cy-3-glu) equivalents.
A ¼ ðAvismax −A700 ÞpH 1:0 −ðAvismax −A700 ÞpH 4:5
The monomeric anthocyanin pigment concentration in the
original sample was calculated using the following formula:
Monomeric anthocyanin pigment ðmg=LÞ
¼ ðA  MW  DF  1; 000Þ=ε x 1Þ
where MW is the molecular weight (449.2 for cyanidin-3-
glucoside), DF is the dilution factor, and ε is the molar
absorptivity (26,900 for cyanidin-3-glucoside).
Determination of HMF
HMF was determined quantitatively, following the procedure
by IFU (1984) based on the colorimetric reaction between
barbituric acid and HMF, forming a red-colored complex. The
intensity of red color is dependent upon the concentration of
HMF, which was measured at 550 nm using a spectrophotom-
eter. A calibration curve of HMF was used to quantify HMF
contents of samples.
Determination of Antioxidant Activity
The antioxidant activity of black mulberry juice and concen-
trates was determined by Trolox Equivalent Antioxidant Ca-
pacity method described by Re et al. (1999). A 7-mM solution
of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) in double-distilled water was prepared and ABTS
radical cation (ABTS+) was formed after addition of potassium
persulphate (Merck) to the mixture in a final concentration
of 2.45 mM. The mixture was allowed to stand in the dark at
room temperature for 12–16 h before use. The solution was
diluted with ethanol to an absorbance reading of 0.7 (±0.02) at
734 nm. The ethanolic ABTS+ (1 mL) solution is added to
three different concentrations of antioxidant compound (5, 10,
and 15 μL), and absorbance readings at 734 nm were taken at
30 °C, exactly 1 min after initial mixing and up to 6 min. The
percentage inhibition of absorbance is calculated and plotted
as a function of concentration of Trolox for the standard
reference data. The results were expressed in equivalents of
micromole Trolox (TE) per 100 mL of juices and per 100 g of
concentrates.
Calculation of Kinetic Parameters
The loss of monomeric anthocyanin content in black mulberry
juice and concentrate followed a first-order kinetic model. The
degradation of total monomeric anthocyanins was calculated
by using the standard equation for a first-order reaction given
below:
lnC ¼ lnC o –kt
where C is the concentration at time t; Co the concentration at
time zero; k the first-order rate constant (month−1); and t the
storage time (month).
The accumulation of HMF in black mulberry juice and
concentrate was calculated by using the standard equation
for a zero-order reaction given below:
C ¼ C o –kt
where C is the concentration at time t; Co the concentration at
time zero; k the zero-order rate constant (mg HMF month−1);
and t the storage time (month).
Temperature dependence of both reactions was determined
by Arrhenius equation given below:
k ¼ k o xe–Ea =RT
where Ea is the activation energy (kJ mol−1); k the rate con-
stant; ko the frequency factor; R the universal gas constant
(8.314×10−3 kJ mol−1 K−1); and T the absolute temperature
(°K).
−T 1
Q10 ¼ ðk 2 =k 1 Þ10=T 2
where k2 is the rate constant of the HMF formation and
monomeric anthocyanin degradation at T2 temperature; k1 is

the rate constant of the HMF formation and/or monomeric
anthocyanin degradation at T1 temperature.
The half-life value of monomeric anthocyanin degradation
was also calculated using the equation given below:
t 1=2 ¼ ln 2=k
where k is the rate constant (per month).
Statistical Analysis
A statistical program (SPSS 9.05 for Windows and MINITAB
14.0) was used for data processing. All experimental data
were evaluated using one way analysis of variance (ANOVA
technique). Results were expressed as mean values±standard
error of mean (SD).
Results and Discussion
Total Monomeric Anthocyanin Content
Total monomeric anthocyanin content of black mulberry juice
and concentrate decreased with temperature and storage time.
Anthocyanin degradation in black mulberry juice and concen-
trate during storage is shown in Figs. 2 and 3, respectively.
The initial total monomeric anthocyanin contents of black
mulberry juices were 23.38, 23.13, 23.29, and 23.13 mg
cy-3-glu L−1 at 5°, 20°, 30°, and 40 °C, respectively, whereas
these values for concentrates were found as 234.84, 235.86,
233.96, and 235 mg cy-3-glu kg−1, respectively. Özgen et al.
(2009) found total monomeric anthocyanins of black mulber-
ry fruits as 253–830 mg cy-3-glu kg−1 f.w. Gerasopoulos and
Fig. 2 Monomeric anthocyanin degradation of black mulberry juice
during storage at different temperatures
Food Bioprocess Technol
Fig. 3 Monomeric anthocyanin degradation of black mulberry concen-
trate during storage at different temperatures
Stravroulakis (1997) reported values of 100 mg kg−1 f.w. in
M. nigra and 10 mg kg−1 f.w. in M. alba cultivated in Greece.
In this research, the anthocyanin content of black mulberry
juices was lower than the values reported on the previous
studies. The reason for that might be due to the preparation
of juices from the concentrates by reconstitution. As anthocy-
anins are decomposed easily because of heat treatment in the
concentrate production (Patras et al. 2010), it is not surprising
to get lower results for juices. As known, single strength fruit
juices always have better quality attributes than that of
reconstituted fruit juices.
The losses of total monomeric anthocyanin at 5°, 20°,
30,° and 40 °C for black mulberry juice and concentrate
during storage were in the ranges of 1.96–74.55 and
1.26–98.12 %, respectively (Table 1). Linear regression
confirmed that the degradation of anthocyanin in black
mulberry juice and concentrate followed a first-order
reaction. As the determination coefficient of the equa-
tions obtained from the results for 5 °C is too low, the
results were not used for calculation of kinetic parame-
ters. The heat dependence of the rate constants of antho-
cyanin degradation was represented by Arrhenius equa-
tion on the basis of linear regression analysis of natural
logarithms of rate constant against reciprocal absolute
temperature 1/T in Kelvin (Fig. 4). Activation energies
for anthocyanin degradation in juice samples and con-
centrate in the range of 20–40 °C were calculated as
56.48 and 49.75 kJ mol−1, respectively (Table 2). Previ-
ous studies showed that thermal degradation of anthocy-
anins in different fruits and fruit juices followed first-
order reaction kinetics (Ochoa et al. 1999; Garzon and
Wrolstad 2002; Wang and Xu 2007).
The Q10 values of black mulberry juice determined at the
temperature ranges of 20–30 and 30–40 °C were 2.02 and
2.17, respectively, whereas these values for concentrates were
Food Bioprocess Technol

Table 1 The variation of total monomeric anthocyanin content of black mulberry juice and concentrate during storage at different temperatures

Black mulberry Storage (month) Temperature (°C)

5 20 30 40

Juice (mg cy-3-glu/L) 0 23.38±0.17 A a 23.13±0.09 A A 23.29±0.26 A a 23.13±0.25 A a

1 22.88±0.17 A a 22.92±0.04 A A 22.17±0.29 B b 20.25±0.29 C b
2 23.26±0.13 A a 23.21±0.17 A A 18.08±0.13 B c 16.87±0.09 C c
3 23.09±0.13 A a 22.71±0.25 A A 16.53±0.25 B d 15.32±0.29 C d
4 23.59±0.04 A a 21.38±0.26 B B 16.33±0.13 C de 12.65±0.13 D e
5 23.01±0.13 A a 20.37±0.25 B B 15.74±0.04 C e 8.64±0.04 D f
6 23.00±0.21 A a 19.96±0.00 B B 13.74±0.30 C f 8.19±0.09 D f
7 23.55±0.00 A a 18.25±0.13 B B 13.28±0.17 C f 7.39±0.04 D g
8 22.92±0.04 A a 16.70±0.00 B B 11.86±0.00 C g 5.89±0.05 D h
Concentrate (mg cy-3-glu/kg) 0 234.84±1.82 A ab 235.86±4.10 A a 233.96±1.66 A a 235.00±0.81 A a
1 235.89±0.41 A ab 195.59±1.65 B b 165.51±1.24 C b 122.38±0.41 D b
2 233.43±0.41 A b 175.45±4.52 B c 118.64±0.83 C c 82.53±1.22 D c
3 237.54±0.41 A ab 152.03±3.29 B d 99.56±2.49 C d 41.06±2.85 D d
4 249.70±13.9 A a 153.66±0.36 B d 48.56±0.91 C fg 18.30±1.22 D f
5 235.29±0.51 A ab 123.54±2.13 B e 59.26±0.11 C e 26.88±0.89 D e
6 232.39±1.75 A b 109.61±3.58 B f 50.20±0.91 C f 17.11±0.87 D f
7 232.16±2.03 A b 102.22±1.37 B f 42.80±0.08 C g 7.33±0.02 D g
8 231.88±0.97 A b 76.70±1.38 B g 25.20±1.32 C h 4.42±0.55 D h

Significant differences between sample means were assessed by Duncan multiple test. Means within a line followed by different upper case letters are
significantly different (p<0.01). Means within a column followed by different lower case letters are significantly different (p<0.01)

found as 2.01 and 1.84, respectively. This means that a 10 °C
increase in the temperature results in an increase approximate-
ly two times in the rate of anthocyanin degradation in juice
and concentrate samples.
The t1/2 values for anthocyanin degradation at 20°, 30°,
and 40 °C were calculated as 17.4, 8.6, and 4 months
respectively for juice samples and 5.5, 2.7 and 1.5 months
respectively for concentrate samples. According to t1/2
values, anthocyanins in concentrates are decomposed more
quickly. Wang and Xu (2007) reported that anthocyanins in
the 65.0°Bx blackberry juice concentrate degraded more
rapidly than that of 8.90°Bx blackberry juice, with the
activation energies of 65.06 and 75.5 kJ mol−1, respectively.
The degradation of anthocyanins in raspberry pulp
followed by a first-order reaction and the value of activa-
tion energy was reported as 12.45 kcal mol−1 (Ochoa et al.
1999). Garzon and Wrolstad (2002) showed that the t1/2
values for anthocyanin degradation in strawberry juice and
concentrate at 25 °C were 8 and 4 days, respectively.

Hydroxymethylfurfural Formation

HMF content of both black mulberry juice and concentrate

Fig. 4 Arrhenius plots for anthocyanin degradation and HMF formation
of black mulberry juice and concentrate
increased significantly (p<0.01) with temperature and storage
time (Table 3). HMF contents of black mulberry juice
increased from initial values of 45.7, 45.85, 45.7, and 45.99
to 46.96, 57.2, 76.34, and 124.41 mg L−1 at the end of storage
time at 5, 20, 30, and 40 °C, respectively. There was a
significant increase (p<0.01) of HMF in black mulberry juice
at the end of 8 months storage at all temperatures except at
5 °C. At the beginning of storage, HMF contents of black
mulberry concentrate were 93.22, 93.28, 94.69, and
93.21 mg kg−1 at 5, 20, 30, and 40 °C, respectively. After
 Food Bioprocess Technol

Table 2 Kinetic parameters of

anthocyanin degradation and Parameters Black mulberry Temperature Rate constant Activation energy
HMF formation in black mulberry (°C) (k) (kJ mol−1)
juice and concentrate during
storage Anthocyanin (first-order reaction) Juice 20 0.0173 (0.8815) 56.48
30 0.0350 (0.9594)
40 0.0761 (0.9814)
Concentrate 20 0.0549 (0.9726) 49.75
30 0.1101 (0.9356)
40 0.2021(0.9581)
HMF (zero-order reaction) Juice 20 1.3852 (0.8601) 75.70
30 3.7042 (0.9351)
40 10.080 (0.9851)
Concentrate 20 9.6904 (0.8184) 104.11
30 38.212 (0.9485)
The values within the parentheses 40 148.42 (0.9898)
are determination coefficients

8 months storage, HMF contents of these samples were found
as 93.28, 177.44, 369.65, and 1,215.70 mg kg−1, respectively.
HMF formation in the black mulberry juice and con-
centrate fitted well to the zero-order reaction kinetics.
Zero- (Burdurlu et al. 2006), first- (Sancho et al. 1992),
and second-order (Shallenberger and Mattick 1983)
kinetic models were also reported for HMF accumulation
in foods.
The heat dependence of the rate constants of HMF forma-
tion was represented by the Arrhenius equation (Fig. 4).
Activation energies for HMF formation in black mulberry
juice and concentrates in the range of 20–40 °C were calcu-
lated as 75.70 and 104.11 kJ mol−1, respectively (Table 2).
Various researchers had been studied on kinetics of HMF
formation in foods and big variations were observed in acti-
vation energies. For example, activation energy for HMF in
carob pekmez was reported as 114.87 kJ mol−1 in the
temperarture range of 5–45 °C (Özhan et al. 2010), whereas
it was determined as 28–39.6 kcal mol−1 in apple juice model
solution (Resnik and Chirife 1979).

Table 3 The variation of HMF content of black mulberry juice and concentrate during storage at different temperatures

Black mulberry Storage (month) Temperature (°C)

5 20 30 40

Juice (mg HMF/L) 0 45.70±0.00 A a 45.85±0.15 A g 45.70±0.60 A h 46.00±0.30 A ı

1 46.59±0.30 C a 47.40±0.37 C ef 48.82±0.60 B g 51.26±0.52 A h
2 45.85±0.15 C a 46.59±0.30 C fg 49.56±0.15 B g 61.20±0.52 A g
3 45.77±0.22 C a 46.29±0.15 C fg 53.34±0.08 B f 71.36±0.00 A f
4 46.37±0.23 D a 48.15±0.23 C e 54.97±0.37 B e 80.94±0.67 A e
5 46.29±0.15 D a 50.15±0.30 C d 59.94±0.15 B d 87.83±0.00 A d
6 46.22±0.08 D a 52.16±0.08 C c 61.72±0.30 B c 98.29±0.52 A c
7 46.29±0.15 D a 54.97±0.07 C b 71.74±0.23 B b 118.10±0.30 A b
8 46.96±0.08 D a 57.20±0.38 C a 76.34±0.23 B a 124.41±0.08 A a
Concentrate (mg HMF/kg) 0 93.22±1.48 A a 93.29±1.48 A d 94.69±2.96 A i 93.21±4.44 A i
1 92.48±0.74 C a 94.03±0.74 C d 116.88±1.48 B h 247.08±4.44 A h
2 93.00±0.76 C a 95.47±0.46 C d 176.11±2.64 B g 360.29±7.31 A g
3 93.68±1.50 C a 95.82±0.17 C d 206.72±0.12 B f 508.13±4.96 A f
4 94.03±0.84 C a 99.57±0.26 C d 287.64±3.96 B e 751.30±2.06 A e
5 93.52±1.66 D a 111.98±1.65 C c 326.72±0.39 B d 873.50±1.09 A d
6 93.52±1.66 D a 131.87±0.95 C b 348.75±1.20 B c 989.94±4.37 A c
7 94.03±0.84 D a 159.31±0.71 C a 359.40±2.19 B b 1,177.2±1.55 A b
8 93.29±1.57 D a 167.44±3.01 C a 369.65±2.23 B a 1,215.7±4.41 A a

Means within a line followed by different upper case letters are significantly different (p<0.01). Means within a column followed by different lower case
letters are significantly different (p<0.01)
Food Bioprocess Technol

Table 4 The variation of antioxidant activity of black mulberry juice and concentrate during storage at different temperatures

Black mulberry Storage (month) Temperature (°C)

5 20 30 40

Juice (μmol TE/100 mL) 0 336.24±0.00 A a 337.12±0.00 A a 337.27±0.00 A a 336.64±0.00 A a

1 336.25±0.09 A a 334.70±0.94 A ab 335.04±1.72 A a 326.81±0.75 B b
2 336.56±0.11 A a 335.93±0.32 A ab 328.94±0.70 B b 321.78±0.66 C c
3 337.24±0.67 A a 332.39±1.62 B abc 325.68±1.03 C bc 313.77±0.42 D d
4 336.00±0.68 A a 331.37±0.56 B bc 323.89±1.19 C c 307.15±2.10 D e
5 334.75±0.96 A a 329.07±1.69 B c 318.99±0.39 C d 300.66±1.39 D f
6 332.69±1.20 A a 327.61±1.14 B c 314.05±0.75 C e 293.92±1.36 D g
7 333.32±1.91 A a 320.26±3.69 B d 301.89±0.69 C f 288.63±0.86 D h
8 319.88±0.76 A b 312.37±0.84 B e 298.37±0.93 C f 282.73±1.36 D ı
Concentrate (μmol TE/100 g) 0 3,099.4±0.00 A a 3,094.1±0.00 A a 3,086.9±0.00 A a 3,101.6±0.00 A a
1 3,083.4±1.59 A a 3,062.8±3.87 A a 3,033.4±2.26 A a 2,960.9±9.49 B a
2 3,076.4±16.1 A a 3,048.1±9.27 A a 2,966.3±16.5 B b 2,843.4±10.1 C b
3 3,053.9±15.2 A ab 2,983.9±32.7 B b 2,883.1±0.02 C c 2,690.3±8.94 D c
4 3,050.5±21.9 A ab 2,901.7±2.14 B c 2,753.2±1.96 C d 2,567.2±5.11 D d
5 3,036.2±10.4 A ab 2,842.3±2.40 B c 2,666.0±26.3 C e 2,428.5±31.0 D e
6 2,993.7±1.23 A bc 2,751.6±42.0 B d 2,578.3±8.89 C f 2,304.3±10.4 D f
7 2,949.5±5.71 A c 2,639.8±12.9 B e 2,434.8±1.24 C g 2,172.7±6.90 D g
8 2,960.8±9.72 A c 2,580.7±29.9 B e 2,313.5±13.3 C h 2,060.4±1.47 D h

Means within a line followed by different upper case letters are significantly different (p<0.01). Means within a column followed by different lower case
letters are significantly different (p<0.01)

The Q10 values of black mulberry juice for HMF deter-
mined at the temperature ranges of 20°–30 and 30°–40 °C
were 2.74 and 2.65, whereas these values for concentrate
were 4.03 and 3.93, respectively. It is pointed out that the
rate of HMF formation in concentrate samples increased
4.03 times when the temperature increased from 20 to
30 °C.
Antioxidant Activity
storage. Klimczak et al. (2007) found that antioxidant
activity measured by ferric reducing ability of plasma
(FRAP) assay of orange juice stored at 18, 28, and
38 °C decreased 23, 34, and 57 %, respectively at the
end of storage.
Correlations Among Antioxidant Activity, Monomeric
Anthocyanin Content, and HMF

The significant decrease (p<0.01) in the antioxidant activ-
ity of black mulberry juice and concentrate during storage
at different temperatures and times is shown in Table 4.
The initial value of the antioxidant activity in black mul-
berry juice was 336.24, 337.12, 337.27, and 336.64 μmol
TE 100 mL−1 at temperatures of 5°, 20°, 30° and 40 °C
respectively, and in concentrates it was found as 3,099.4,
3,094.1, 3,086.9, and 3,101.6 μmol TE 100 g−1, respec-
tively. The loss of antioxidant activity of black mulberry
juice and concentrate during storage at 5, 20, 30, and
40 °C were in the ranges of 4.87–16.01 and 4.47–
33.57 %, respectively. The loss of antioxidant activity
was also reported by some investigations (Klimczak
et al. 2007; Koca and Karadeniz 2008). Koca and
Karadeniz (2008) showed that the antioxidant activity of
carrot was reduced by 31 % during 6 months of cold
Antioxidant activity in black mulberry juice and concentrate
was correlated with total monomeric anthocyanins (r=0.956,
r=0.886, respectively, p<0.01) during storage. The correla-
tion coefficient, among total monomeric anthocyanin, TEAC
and FRAP were reported to be significant (p<0.05) for
M. nigra accessions (Özgen et al. 2009). Significant negative
correlations were found between HMF and total monomeric
anthocyanin in black mulberry juice and concentrate, respec-
tively (r=−0.928, −0.759, p<0.01) which indicates decompo-
sition of anthocyanins coincide with the chemical browning
reactions. In addition, significant negative correlations were
also found between antioxidant activity and HMF (r=−0.835
for juice, r=−0.860 for concentrate, p<0.01). This negative
correlation may be attributed to the loss of anthocyanins
which are the main compounds responsible for antioxidant
activity in fruits.

Conclusions
Kinetics of anthocyanin degradation and HMF formation in
black mulberry juice and concentrates were investigated dur-
ing storage. The present data shows that the degradation of
black mulberry anthocyanins follows first-order reaction
kinetics. The activation energies of anthocyanin degradation
in black mulberry juice and concentrate were found as 56.48
and 49.75 kJ mol−1, respectively. As increased temperature
and storage time has an important affect on the degradation of
black mulberry anthocyanin content, mulberry concentrates
and fruit juices should be kept lower than 5 °C. HMF accu-
mulation fitted to a zero-order kinetic model and activation
energies in black mulberry juice and concentrate were found
as 75.70 and 104.11 kJ mol−1, respectively. It was observed
that the increase of HMF in black mulberry concentrate at
30 °C was approximately 4.03 times higher than that of at
20 °C. Antioxidant activities of black mulberry juice and
concentrate were found positively correlated with total mono-
meric anthocyanins.
Acknowledgments The authors wish to thank Göknur Foodstuffs
Import Export Trading and Production Company (Niğde, Turkey) for
preparation of research samples.
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