JFS: Food Chemistry and Toxicology

Involvement of Blueberry Peroxidase in the

Mechanisms of Anthocyanin
Degradation in Blueberry Juice
F. KADER, M. IRMOULI, J.P. NICOLAS , AND M. M ETCHE
Food Chemistry and Toxicology

ABSTRACT: Addition of hydrogen peroxide (H2O2) to blueberry juice changed the red coloration toward brown.
Addition of ascorbic acid prevented the formation of brown polymers. An extract of peroxidase (POD) prepared from
blueberry fruits was able to oxidize CG into the corresponding o-quinone but only in the presence of H2O2. The
chlorogenoquinone plays a dominant role in anthocyanin degradation. We demonstrated that peroxidase extract in
the absence of CG showed a weak degradation activity toward blueberry anthocyanins, cyanidin 3-glucoside, and
pelargonidin 3-glucoside. Nevertheless, addition of CG increased anthocyanin degradation, leading to formation
of brown polymers. Therefore, blueberry POD could participate in the development of browning during blue-
berry-juice storage.
Key Words: blueberry juice, Vaccinium corymbosum, peroxidase, chlorogenic acid, anthocyanins

Introduction coside is 5.6 times greater than that of pelargonidin 3-glu-

P EROXIDASE (EC 1.11.1.7; DONOR: HYDROGEN-PEROXIDE OXI-

doreductase; POD) belongs to the group of oxidoreduc-
tases. POD is an iron-containing enzyme that is widely dis-
tributed in the plant kingdom (Vamos-Vigyazo 1981). Little
information is available on blueberry POD. Frenkel (1972)
studied the evolution of POD activity in highbush blueberries
during ripening, and the author found that this activity did
not correspond with indoleacetic-acid oxidase activity.
Miesle and others (1991) reported that total POD activity in-
creased during blueberry development. Most of the enzyme
was ionically bound to cell walls throughout development,
with the number of isoenzymes increasing with maturity.
The occurrence of enzymatic browning during the prepara-
tion of fresh blueberry puree and juice has been observed by
Kader and others (1997b).
Both polyphenol oxidase (PPO) and POD have been im-
plicated in enzymatic browning of plant tissues (Vamos-Vi-
gyazo 1981). PPO appeared to be the main enzyme involved
in these reactions, but the role of POD in enzymatic brown-
ing is still not well established. In the presence of H 2O 2,
POD is able to oxidize o-diphenolic compounds to their
corresponding o-quinones, which then are polymerized to
brown polymers. However, the low cellular concentration
in H 2O2 is the factor that limits POD activity (Nicolas and
others 1994). In addition, the main problem with POD-ac-
tivity studies is to detect a source of H2O2 required for the
enzymatic reaction.
The mechanism of anthocyanin degradation by POD re-
mains unknown. Few studies have been devoted to this
subject since POD activity does not contribute significantly
to anthocyanin degradation in fruits. Grommeck and
Markakis (1964) have studied the degradation of cyanidin 3-
gentiobioside, cyanidin 3-rhamnoglucoside, and pelargoni-
din 3-glucoside by horseradish POD in the presence of
H2O2. They reported that anthocyanin degradation is maxi-
mum for a range of pH values from 4.5 to 5.5 and for a
range of H 2O 2 concentrations from 10 -4 to 10 -3M. Under
these conditions, the degradation of cyanidin 3-rhamnoglu-
910 JOURNAL OF FOOD SCIENCE—Vol. 67, No. 3, 2002
coside. More recently, Calderon and others (1992) studied
the degradation of anthocyanins and anthocyanidins by
Gamay grapevine POD in the presence of H 2O2. Their re-
sults showed that anthocyanidins are good substrates for
grapevine POD. However, anthocyanins such as Cyanin and
Gamay anthocyanins are not oxidized because the hydroxyl
function in the position 3 is not free as in the aglycone
forms. According to these authors, the carbinol-pseudo-
base form is the true substrate for this enzyme. The subcel-
lular localization of Gamay POD and ␤-glycosidases sup-
port the possible involvement of these enzymes in
anthocyanin turnover and degradation in the plant. The im-
plication of Gamay POD in the catabolism of anthocyanins
requires the previous action of ␤-glycosidases to remove
the sugar moieties from the glycosides in order to liberate
the aglycone forms that are oxidized by POD in the pres-
ence of H2O2 (Zapata and others 1995; Calderon and others
1992). It is well known that POD is capable of oxidizing phe-
nolic compounds (Zapata and others 1992), anthocyanins
(Grommeck and Markakis 1964), anthocyanidins (Calderon
and others 1992), and flavonols (Richard and Nicolas 1989).
Lopez-Serrano and Ros Barcelo (1996) suggested that
strawberry POD isoenzyme may play a role in anthocyanin
turnover and degradation. Hence, the participation of POD
in enzymatic browning cannot be denied.
More recently, Skrede and others (2000) studied the
changes in anthocyanins and polyphenolics during juice pro-
cessing of highbush blueberries. The authors showed that
blueberry PPO is involved in the process of anthocyanin deg-
radation. The same results were obtained by Kader and oth-
ers (1997b) in crushed fresh blueberries.
The purpose of this work was to study the implication of
blueberry POD in the mechanism of anthocyanin degrada-
tion in blueberry juices obtained from frozen fruits. The ef-
fects of H2O2 addition on the color and browning of blue-
berry juices were examined. The use of model systems
should be useful in understanding the mechanism of antho-
cyanin degradation.
© 2002 Institute of Food Technologists

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 Peroxidasic Degradation of Blueberry Anthocyanins . . .

Materials and Methods
Fruit sampling and storage
Approximately 5 kg of ripe blueberries of the Coville vari-
ety were harvested during July 1996 in a commercial planta-
tion in Northeastern France (Vosges). Sample ripeness was
judged on the basis of skin color of representative berries.
The berries were cleaned and then packaged in polyethylene
containers (4 ⬚C). The fruits were fast frozen and then stored
in the dark at –25 ⬚C under nitrogen.
Reagents
Cyanidin-3-glucoside (Cy 3-glc, Kuromanin, HPLC grade)
from Millipore. The retentate (25 mL) was stored at 4 ⬚C un-
der nitrogen and was used as a source of POD.
POD activity was assayed with o-dianisidine as a substrate
according to a spectrophotometric procedure at 460 nm.
The assays were performed using 0.880 mL of McIlvaine
buffer pH 3.5, 0.010 mL of 0.5% o-dianisidine (w/v in metha-
nol), 0.010 mL of the enzyme extract. The reaction was initi-
ated by adding 0.100 mL of 50 mM H2O2. The increase in ab-
sorbance at 460 nm was measured on a Shimadzu UV-260
spectrophotometer at 25 ⬚C. One unit of enzymatic activity
was defined as an increase of 0.1 unit of absorbancy per min
per mg of protein at 460 nm (initial rate). The activity report-
ed is the mean of 4 initial rate values.

Food Chemistry and Toxicology

and pelargonidin 3-glucoside (Pg 3-glc, Callistephin, HPLC Protein concentration was determined according to the
grade) were purchased from Extrasynthèse (Genay, France). method using bicinchoninic acid (BCA) as a specific chro-
Chlorogenic acid (CG) and the bicinchoninic acid (BCA) pro- mogenic reagent (Smith and others 1985). Bovine serum al-
tein reagent were obtained from Sigma Chemical Co. (St. bumin was used as a standard.
Quentin Fallavier, France). All other chemicals were of ana-
lytical grade from Merck (Darmstadt, Germany). Cy 3-glc (1 Oxidation of CG by blueberry POD
mM), Pg 3-glc (1 mM), and CG (1 mM) were dissolved indi- The reaction mixture contained 1.0 mL of McIlvaine buff-
vidually in McIlvaine buffer pH 3.5. This buffer was prepared er pH 3.5, 0.200 mL of 1 mM CG in the same buffer, and
from 0.1 M citric acid adjusted to the correct pH by adding 0.100 mL of the diluted enzymatic extract (20 mU). The reac-
0.2 M dibasic potassium phosphate. tion was initiated by adding 0.100 mL of 50 mM H2O2. Oxida-
tion of CG by blueberry POD was monitored at 400 nm at
Preparation of blueberry juice 25 ⬚C. For each assay, 2 analyses were conducted on dupli-
One kg of berries was thawed at 4 ⬚C overnight. The fruits cate reactions. Each data point is therefore the mean of 4
were homogenized in a blender (Braun Mx 32) at high speed measurements.
for 2 min at 4 ⬚C. The mixture was centrifuged for 5 min at
17,500 ⫻ g at 4 ⬚C. The pellet was discarded, and the juice su- Extraction of blueberry anthocyanins
pernatant was filtered through a porous glass filter (100 to The skins were removed from 75 g of frozen berries and
160 ␮m) under suction and then stored at 4 ⬚C under nitro- extracted by maceration with 100 mL of methanol-HCl (99:1,
gen. The juice obtained per kg of berries amounted to 585 v/v) for 2 h at room temperature in the dark. This procedure
mL. was performed twice. The fractions were pooled and then
suction-filtered through Whatman paper No. 1. The extract
Effect of H2O2 alone on the color stability of fresh was evaporated under vacuum at 25 ⬚C. The residue was dis-
blueberry juice solved in 20 mL of 0.01 M HCl. Chlorogenic acid was elimi-
The reaction mixture contained 1.050 mL of McIlvaine nated by 4 successive extractions with ethyl acetate (1:1, v/v).
buffer pH 3.5, 0.250 mL of blueberry juice, and 0.100 mL of The aqueous phase (anthocyanin extract) was concentrated
50 mM H2O2. under vacuum at 25 ⬚C. This extract (10 mL) was used as an
anthocyanin source. Anthocyanin concentration was deter-
Effect of H2O2 in combination with ascorbic acid on mined using the pH differential method (pH 4.5 and 1.0)
the color stability of fresh blueberry juice (Wrolstad 1976). The results were expressed in equivalents
The reaction mixture contained 0.950 mL of McIlvaine malvidin 3-glucoside (0.15 g/L, 0.3 mM), which is 1 of the
buffer pH 3.5, 0.250 mL of blueberry juice, 0.100 mL of 50 most abundant pigments in blueberry fruits (Kader and oth-
mM ascorbic acid, and 0.100 mL of 50 mM H2O2. ers 1996).
After equilibrium, the reactions were started by adding
H2O2. Anthocyanin degradation was monitored using a Shi- Effect of blueberry POD alone on anthocyanin
madzu UV-260 spectrophotometer. The change in absorbance degradation
was recorded at a specific wavelength (520 nm) and every The reaction mixture contained 1.0 mL of McIlvaine buff-
minute across a spectrum between 350 and 600 nm. For each er pH 3.5, 0.200 mL of 1 mM Pg 3-glc or 1 mM Cy 3-glc or 0.3
assay, 2 analyses were conducted on duplicate reactions. Each mM blueberry anthocyanin, 0.100 mL of the diluted enzy-
data point is therefore the mean of 4 measurements. matic extract (20 mU), and 0.100 mL of 50 mM H2O2.

Blueberry POD extraction Effect of blueberry POD in combination with CG on

POD was extracted according to the procedure described
by Miesle and others (1991). All the procedures were carried
out at 4 ⬚C. Frozen blueberries (250 g) were homogenized in
a Braun Mx 32 homogenizer with 500 mL of 50 mM sodium
phosphate buffer (pH 6.0), containing 1 mg sodium bisulfite/
mL to retard browning and 0.2 M calcium chloride. The re-
sulting mixture was centrifuged at 5,000 ⫻ g for 20 min at
4 ⬚C. The supernatant was filtered through a porous glass fil-
ter (100 to 160 ␮m) under suction. The resulting solution was
ultrafiltered through a membrane of cellulose triacetate
(cut-off point 30 kDa, Sartorius) using a Minitan “S” system
anthocyanin degradation
The reaction mixture contained 0.8 mL of McIlvaine buff-
er pH 3.5, 0.200 mL of 1 mM Pg 3-glc or 1 mM Cy 3-glc or 0.3
mM blueberry anthocyanin extract, 0.200 mL of 1 mM CG,
0.1 mL of the diluted enzymatic extract (20 mU), and 0.100
mL of 50 mM H2O2.
After 5 min, the reaction was started by adding H2O2. The
degradation of blueberry anthocyanins, Cy 3-glc, and Pg 3-
glc was monitored at a specific wavelength, 520, 510, and 495
nm, respectively, and at 25 ⬚C. For each assay, 2 analyses
were conducted on duplicate reactions. Each data point is

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 Peroxidasic Degradation of Blueberry Anthocyanins . . .

therefore the mean of 4 measurements. The initial rate of fect against brown color development, however under these
degradation was calculated from the decreases in absor- conditions, we observed a slight decrease in absorbance at
bance at the maximum wavelength (520, 510, and 495 nm) 520 nm (Figure 1). The loss of color could be attributed ei-
per min and was expressed as a positive value. The rate of ther to the effect of H2O2 on blueberry anthocyanins (Ia-
degradation expressed in % was calculated as follows: (A20 cobucci and Sweeny 1983) or to the decolorizing effect of
min/A0 min) ⫻ 100. ascorbic acid and/or dehydroascorbic acid on anthocyanins
(Meschter 1953; Iacobucci and Sweeny 1983), or even to an
Results and Discussion endogenous enzymatic-decolorizing system that uses H2O2
as cosubstrate. It is important to note that ascorbic acid is
Effect of H2O2 addition on the color stability of not oxidized by blueberry POD in the presence of H2O2
blueberry juice (Tsumura and others 1993). Because of the chemical com-
The addition of H2O2 to a diluted blueberry juice resultedplexity of the blueberry juice, the use of model solutions
in a decrease in absorbance at 520 nm, which means that an- should be useful in understanding the mechanisms of antho-
Food Chemistry and Toxicology

thocyanins are degraded (Figure 1). After 20 min, the color cyanin degradation.
of the reaction mixture turned brown. The corresponding In the absence of ascorbic acid, we observed a slight de-
UV-vis spectrum showed an increase in the band between crease in absorbance at 520 nm (Figure 1). This could be at-
380 to 420 nm (Figure 2). The decrease in absorbance at 520 tributed to the formation of brown polymers, which also ab-
nm corresponding to the red color was followed by a con- sorb at this wavelength. Thus, the decrease in absorbance at
comitant increase in the brown color, suggesting that antho- 520 nm due to the degradation of anthocyanins is counter-
cyanins are degraded by a mechanism of oxidation. The ad- balanced by the absorbance of the newly formed brown
dition of H2O2 (cosubstrate essential for the POD activity) polymers. These results provide evidence that blueberry
must be the cause of all of these modifications. The spectro-POD could be involved in the process of anthocyanin degra-
photometric analyses of H2O2-free control solutions con- dation and in the subsequent formation of brown polymers.
taining diluted blueberry juice and McIlvaine buffer showed This could be very important because we observed the de-
that no anthocyanin degradation occurred since the initial velopment of a brown color during storage (at 4 ⬚C) of blue-
value of absorbance at 520 nm remained stable. berry juices prepared from frozen fruits. These observations,
We have shown earlier that CG is the main hydroxycin- however, set the problem of the source of H2O2 required for
namic acid in highbush blueberry fruit (Kader and others the peroxidation processes.
1996). We can suppose that blueberry POD in the presence of
added H2O2 is able to oxidize CG into the corresponding o- Oxidation of CG by blueberry POD in the presence of
quinone (chlorogenoquinone), which could react with an- H2O2
thocyanins, leading to the formation of brown polymers. In Since CG is found in a relatively high concentration in
order to check this hypothesis, we supplemented the reac- blueberry fruits, we investigated the oxidation of CG by blue-
tion mixture with ascorbic acid. Under these conditions, the
reaction mixture did not develop a brown color. This result
suggests that o-quinones generated during the oxidation of
o-diphenolic substrates by POD plus H2O2 play an important
role in the mechanism of anthocyanin degradation (brown-
ing). The results show that ascorbic acid has a protective ef-

Figure 1—Variation in the absorbance at 520 nm of diluted

blueberry juices (pH 3.5) treated with hydrogen peroxide
(䊐) or with hydrogen peroxide in combination with ascor-
bic acid (䉬). Hydrogen peroxide and ascorbic acid were
added to a final concentration of 3.6 mM. Each data point
is the mean of 4 determinations.
Figure 2—Absorbance spectra of diluted blueberry juices
(pH 3.5) treated with hydrogen peroxide to a final concen-
tration of 3.6 mM. Spectra were taken at 1-min intervals.

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 Peroxidasic Degradation of Blueberry Anthocyanins . . .

Table 1—Kinetic data concerning the degradation of blueberry anthocyanins, Cy 3-glc, and Pg 3-glc by blueberry POD
in the presence (⫹) and absence (–) of CG (mean SD, n = 4) 6
Blueberry anthocyanins Cy 3-glc Pg 3-glc
–CG CG 1 –CG 1CG –CG 1CG
Initial rate ofdegradationa 0.015 ⫾ 0.001 0.024 ⫾ 0.001 21.7 ⫾ 1.7 0.225 ⫾ 0.01 19.3 ⫾ 1.15 0.064 ⫾ 0.003
% of degradationb 15.5 ⫾ 1.1 29 ⫾ 1.7 0.049 ⫾ 0.002 77.5 ⫾ 3.8 0.027 ⫾ 0.002 53.1 ⫾ 2.5
Aspect of the medium partial brown partial brown partial brown
after 1 h of reaction decoloration decoloration decoloration
a Initial rate of degradation calculated at the maximum wavelength in the visible (absorbance decrease per min)
20 min /A 0 min ) ⫻ 100
b % of degradation calculated after 20 min of reaction (A

berry POD in model solutions. Figure 3 shows the effect of (⫾ CG), no degradation of anthocyanins was observed in the

Food Chemistry and Toxicology

blueberry POD on CG at pH 3.5 in the presence of H2O2. CG H2O2-free control solutions, meaning that blueberry POD is
in the presence of H2O2 is oxidized by blueberry POD into involved in the process of anthocyanin degradation.
the corresponding o-quinone, which displays maximum ab- The degradation of blueberry anthocyanins at pH 3.5 by
sorption at 400 nm (the color of the reaction mixture turns blueberry POD plus H2O2 was first examined in the absence
yellow). The absorbance increased sharply, reached a maxi- of CG and monitored spectrophotometrically at 520 nm (Fig-
mum after 6 min of reaction, and then decreased due to the ure 4). The results show that blueberry POD presents a low
polymerization of the quinone. It should be noted that no activity of degradation toward blueberry anthocyanins. After
reaction occurred when the crude enzyme was heated at 20 min of reaction, most of the red color remained in the re-
100 ⬚C for 10 min. The addition of ascorbic acid to the reac-
action mixture. After 20 min of reaction, the rate of anthocy-
tion mixture after 3 min of reaction resulted in an instanta-
anin degradation was low (15.5%), and the initial rate of deg-
neous bleaching. In the presence of excess reducing agent, radation was 0.015 unit (Table 1). No degradation occurred
chlorogenoquinone formed from CG is reduced to the origi- when the enzymatic extract was inactivated by heating for 10
nal o-diphenol (Rouet-Mayer and others 1990). min at 100 ⬚C. This result means that (a) the degradation of
In conclusion, the oxidation of CG by the enzymatic ex- blueberry anthocyanins has an enzymatic origin, and (b)
tract was strictly dependent on H2O2 and was therefore con- H2O2 has no effect on blueberry anthocyanins under these
sidered to be due to POD. Controls were carried out in air- conditions. Indeed, Hrazdina (1970) and Hrazdina and Fran-
saturated solutions but in the absence of H2O2. Under these zese (1974) have shown that malvin at pH 3.0 is oxidized by
conditions, CG was not oxidized, which means that the enzy- H2O2 leading to the colorless malvone. In the absence of CG,
matic extract did not contain PPO activity. This shows that the reaction mixture showed a partial decolorization, and no
PPO is not extracted by this procedure as mentioned by Kad- brown product was detected. The loss of red color can be at-
er and others (1997a). tributed to a peroxidase-decolorizing system that uses H2O2
as cosubstrate. The results clearly show that blueberry POD
Degradation of blueberry anthocyanins by POD in the is able to act directly on blueberry anthocyanins. However,
presence and absence of CG the rate of anthocyanin degradation remained low, meaning
It is important to note that whatever the conditions used that anthocyanins are poor substrates of blueberry POD.
The addition of CG increased the initial value of absor-

Figure 3—Oxidation of chlorogenic acid by blueberry POD
plus hydrogen peroxide in McIlvaine buffer (pH 3.5). Chlo-
rogenic acid oxidation was initiated by hydrogen peroxide
addition and followed at 400 nm. Each data point is the
mean of 4 determinations.
Figure 4—Variation in the absorbance of blueberry antho-
cyanins at 520 nm during their degradation by blueberry
POD plus hydrogen peroxide at pH 3.5 in the presence (䊐)
or in the absence (䉬) of chlorogenic acid. Each data point
is the mean of 4 determinations.

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 Peroxidasic Degradation of Blueberry Anthocyanins . . .

bance at 520 nm in comparison to the CG-free solutions In the absence of CG, Cy 3-glc and Pg 3-glc have the same
(Figure 4). This can be attributed to a copigmentation effect.behavior, the rates of degradation after 20 min of reaction
Anthocyanins have been shown to form weak complexes being 21.7% and 19.3%, respectively. Nevertheless, the com-
with a number of compounds such as chlorogenic acid. parison of the initial rates of degradation revealed that Cy 3-
Complex formation or copigmentation results in a batho- glc is degraded 1.8 times faster than Pg 3-glc (Table 1). From
chromic shift of the maximum of absorption and an increase this we can assume that Cy 3-glc is a better substrate for
in the absorbance in the visible range (Mazza and Brouillard blueberry POD than Pg 3-glc. Other assays were carried out
1987). The presence of CG in the reaction mixture increased to confirm the participation of blueberry POD in the process
the rate of anthocyanin degradation (Figure 4). The rate of of anthocyanin degradation. In the POD-free control solu-
anthocyanin degradation after 20 min of reaction was 29% tions, no degradation of anthocyanins occurred, confirming
that they were not oxidized by H2O2. In addition, no degra-
instead of 15.5% in the absence of CG. The initial rate of deg-
radation was also higher (0.024 unit instead of 0.015 in the dation occurred when the enzymatic extract was inactivated
absence of CG) (Table 1). The reaction mixture after 20 min by heating at 100 ⬚C for 10 min, which confirms that the ob-
Food Chemistry and Toxicology

of reaction presented a slight brown color, which tended to served degradation has an enzymatic origin. Our results
intensify after 1 h. These results suggest that anthocyanins demonstrate that blueberry POD may contribute to the deg-
are oxidized by the chlorogenoquinone formed from the ox- radation of anthocyanins when H2O2 is added to the reaction
idation of CG by POD in the presence of H2O2. Thus, o- mixture. In the absence of CG, the loss of color is associated
quinone seems to play a dominant role in the process of an- with a decolorizing system since no brown color was ob-
thocyanin degradation and stimulates this degradation. The served in the reaction mixture. Most of the red color re-
presence of CG in the reaction mixture resulted in (a) an in- mained in the reaction mixture. On the other hand, these re-
crease of the pigment degradation and (b) the formation of sults clearly explained the loss of color observed in blueberry
brown polymers, which has also been observed when H2O2 juice treated with ascorbic acid plus H2O2. This effect can be
was added to a diluted blueberry juice. attributed to an endogenous peroxidase-decolorizing sys-
In order to understand the mechanism of anthocyanin tem. Thus, blueberry POD can oxidize anthocyanins into col-
degradation by POD, we have used simplified model systems orless products without the mediating effect of o-diphenolic
containing pure anthocyanins. This procedure simplifies the substrates such as CG. However, in the absence of CG, the
composition of the reaction mixture such as blueberry an- rate of anthocyanin degradation remained very low.
thocyanin extracts, which are very complex. Whatever the pigment studied, the presence of CG in the
reaction mixture drastically increased the rate of anthocya-
Degradation of Cy 3-glc and Pg 3-glc by blueberry nin degradation (Table 1). In the case of Pg 3-glc, the initial
POD in the presence and absence of CG rate of degradation is 2.4 times greater than in absence of CG
Two anthocyanins differing by their B-ring substitution (0.064 unit instead of 0.027 in the absence of CG), and after
pattern, namely Cy 3-glc (3',4'-dihydroxylated) and Pg 3-glc 20 min, the rate of degradation reached 53.1% (instead of
(4'-monohydroxylated), were chosen and can be considered 19.3% in the absence of CG). Under the same conditions, the
as an o-diphenolic and non-o-diphenolic anthocyanin, re- degradation of Cy 3-glc is higher than that of Pg 3-glc. The
spectively. The behavior of these 2 anthocyanins must be rate of degradation after 20 min of reaction reached 77.5%.
correlated to their B-ring substitution pattern. The degrada- The initial rate of Cy 3-glc degradation is greater when CG is
tion of these anthocyanins was carried out at pH 3.5 in the added to the reaction mixture (0.225 unit instead of 0.049 in
presence and absence of CG by blueberry POD plus H2O2 the absence of CG), the latter being multiplied by 4.6. In the
(Figures 5A and 5B). presence of CG, we observed the formation of a brown color

Figure 5—Degradation of cyanidin 3-glucoside (A) and pelargonidin 3-glucoside (B) by blueberry POD plus hydrogen
peroxide at pH 3.5 in the presence (䊐) or in the absence (䉬) of chlorogenic acid. Each data point is the mean of 4
determinations.

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 Peroxidasic Degradation of Blueberry Anthocyanins . . .
(browning reaction), which is more intense with Cy 3-glc
than with Pg 3-glc. The chlorogenoquinone formed through
the enzymatic oxidation of CG is the first product formed
and reacts with anthocyanins according to a mechanism that
depends on the structure of the pigment. The addition of
ascorbic acid to the reaction mixture containing POD + CG +
anthocyanins had a protective effect on anthocyanin degra-
dation. This effect can be explained by the fact that the chlo-
rogenoquinone is readily reduced back to CG. Thus, the
chlorogenoquinone does not react with the pigments as long
as ascorbic acid is present in the reaction mixture.
The degradation of Cy 3-glc was much faster than that of
Pg 3-glc, suggesting that Cy 3-glc reacts more readily with the
enzymatically generated chlorogenoquinone. This is likely to
be due to its o-diphenolic moiety. The presence of an o-
diphenolic function on the nucleus B could mean that Cy 3-
glc degradation proceeds by a mechanism of coupled oxida-
tion. Since Pg 3-glc has no o-diphenolic function, no coupled
oxidation mechanisms occurred. However, in the presence
of CG, the rate of Pg 3-glc degradation increased, which
means that Pg 3-glc may react with chlorogenoquinone and/
or secondary products of degradation (formed from the
chlorogenoquinone) to form condensation products. This
reaction took place much more slowly than that of coupled
oxidations, which explains the kinetics data. However, the re-
actions actually involved remain to be elucidated by charac-
terizing the reaction products.
Conclusions
T HE STUDY SHOWS THAT BLUEBERRY POD CAN BE INVOLVED
in the process of anthocyanin degradation in blueberry
juice. In the absence of CG, the loss of color is associated
with a peroxidase-decolorizing system. However, the pres-
ence of CG increased the rate of anthocyanin degradation,
and the reaction mixture turned brown (browning reaction).
The chlorogenoquinone formed from CG seems to play a
dominant role in the process of anthocyanin degradation. Cy
3-glc (o-diphenolic anthocyanin) is degraded by a coupled
oxidation mechanism, whereas Pg 3-glc (non-o-diphenolic
anthocyanin) is degraded by a process of nonoxidative con-
densation. In order to justify the process of anthocyanin
degradation by blueberry POD, it appears necessary to eluci-
date the mechanisms of H2O2 formation in blueberry juice. Authors Kader, Irmouli, and Metche are with Laboratoire de Biochimie
This study is currently being carried out. Appliquée, Ecole Nationale Supérieure d’Agronomie et des Industries
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MS 20000835 Submitted 8/15/00, Accepted 5/17/2001, Received 5/16/01
Alimentaires, 2 Avenue de la Forêt de Haye BP 172-54505 Vandoeuvre-lès-
Nancy Cedex, France. Authors Nicolas and Metche are with Laboratoire de
Biochimie Nutritionnelle, Unité INSERM 308, Faculté de Médecine de Nancy,
9 Avenue de la Forêt de Haye BP 184-54500 Vandoeuvre-lès-Nancy Cedex,
France. Direct correspondence to M. Metche (E-mail: Jean-
Pierre.Nicolas@medecine.uhp-nancy.fr).
Vol. 67, No. 3, 2002—JOURNAL OF FOOD SCIENCE 915
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