Effect of enzyme-aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant juices

Journal of the Science of Food and Agriculture
J Sci Food Agric 85:2548– 2556 (2005)
DOI: 10.1002/jsfa.2284
Effect of enzyme-aided pressing on
anthocyanin yield and pro?les in bilberry
and blackcurrant juices
Johanna Buchert,1? Jani M Koponen,2 Marjaana Suutarinen,1 Annikka Mustranta,1
Martina Lille,1 Riitta T ¨orr ¨onen2 and Kaisa Poutanen1,2
1VTT Biotechnology, PO Box 1500, 02044 VTT, Finland
2Food and Health Research Centre, Department of Clinical Nutrition, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
Abstract: Bilberries (Vaccinium myrtillus) and blackcurrants (Ribes nigrum) were treated with extensive
dosages of commercial cell wall degrading enzyme preparations, i.e. Econase CE, Pectinex Ultra SP-L,
Pectinex Smash, Pectinex BE 3-L and Biopectinase CCM. The enzymes were dosed based on the
polygalacturonase activity. The juice yield was improved in both berries as a result of the enzymatic
treatment. The improvement was more pronounced with blackcurrants owing to their thicker cell walls.
The impact of the enzymatic treatment on anthocyanins present in the juices was investigated using
HPLC-DAD. The enzyme preparations affected the contents and composition of anthocyanins in the
juices. Pectinex Ultra SP-L, Pectinex Smash, Pectinex BE 3-L and Biopectinase CCM increased the
total content of anthocyanins by 13– 41% in the bilberry juices and by 18– 29% in the blackcurrant juices.
Econase CE, however, produced a dramatic decrease in the total anthocyanin content in the bilberry
juice due to its enzyme pro?le, whereas no such effect was observed with the blackcurrant juice. All
the enzyme mixtures tested produced a total or extensive loss of anthocyanidin galactosides in bilberry
juice. Commercial enzyme preparations used in the production of berry juices can improve extraction of
anthocyanins into the juice. However, they may effectively hydrolyse certain glycosides and thus affect the
pro?le of extracted anthocyanins.
? 2005 Society of Chemical Industry
Keywords: bilberry; blackcurrant; enzyme; juice; anthocyanins; phenolics
INTRODUCTION
anticarcinogenic potential.10 In mice, prevention of
Bilberries (wild European blueberries) (Vaccinium
obesity and amelioration of hyperglycaemia was
myrtillus L.) and blackcurrants (Ribes nigrum L. cv.
observed after feeding with an anthocyanin-rich diet.11
¨
Ojebyn) are used for juice manufacture, especially in
Anthocyanins are absorbed to some extent both
Scandinavia. Bilberries and blackcurrants are rich in
by humans and animals, and are found as intact
anthocyanins, which are a large and important group
glycosides in the blood and urine.12 – 17 The chemical
of ?avonoid compounds. Anthocyanins are mostly
structure, ie the nature of the sugar conjugate and the
responsible for the red and blue colours of these
phenolic aglycon, is known to affect bioavailability and
berries. Bilberry and blackcurrant anthocyanins are
to have an impact on the anthocyanin absorption and
glycosides containing glucose, galactose, arabinose or
excretion.15 – 17
rutinose as the sugar moiety linked to the aglycon
During juice manufacture the berries are crushed
(anthocyanidin). Blackcurrants contain mainly cyani-
and pressed, whereafter the juice is separated for
din 3-O-glucoside, cyanidin 3-O-rutinoside, delphini-
further use. Pectinolytic enzymes are currently used in
din 3-O-glucoside and delphinidin 3-O-rutinoside.1,2
industrial berry processing to facilitate juice extraction.
In bilberries anthocyanins are composed of 3-O-
With these enzymes, the cell wall network is disrupted
galactosides, 3-O-glucosides and 3-O-arabinosides
and consequently the juice yield is enhanced.18,19
of delphinidin, cyanidin, petunidin, peonidin and
Furthermore, enzymatic treatment is known to
malvidin.3,4
enhance the extractability of phenolic components
Anthocyanidins and their glycosidic forms are
from the cell wall matrix.20 Enzyme manufacturers
strong antioxidants in vitro mainly owing to their
provide various types of enzyme mixtures for berry
phenolic hydroxyl groups.5 – 9 They may also have
processing. Most of these enzyme preparations contain
? Correspondence to: Johanna Buchert, VTT Biotechnology, PO Box 1500, 02044 VTT, Finland
E-mail: Johanna.Buchert@vtt.?
Contract/grant sponsor: Commission of the European Communities; contract/grant number: QLK1-CT-2002-02364
(Received 28 September 2004; revised version received 4 March 2005; accepted 5 April 2005)
Published online 19 August 2005
? 2005 Society of Chemical Industry. J Sci Food Agric 0022–5142/2005/$30.00
2548

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Enzyme-aided pressing of bilberries and blackcurrants
endo-acting pectinases, cellulases and hemicellulases
birchwood glucuronoxylan27 and ?-glucan28 as sub-
in various ratios. In addition to these enzymes,
strate, respectively. Endopolygalacturonase, pectin
exo-acting enzymes are also present in the enzyme
lyase and ?-glucosidase activities were measured
mixtures, potentially affecting the chemistry of the
using polygalacturonic acid,29 citrus pectin30 and
phenolic glycosides extracted.21 Wrolstad et al22 and
4-nitrophenyl-?-D-glucopyranoside31
as
substrate,
Wightman and Wrolstad23 have screened various
respectively. Pectin methylesterase was assayed by
commercial pectinase preparations for their effect on
titration of the liberated carboxyl groups of citrus
cranberry juice anthocyanin hydrolysis. Arabinoside
pectin using an automatic titrator.32 ?-Galactosidase
pigments were not degraded whereas galactosides were
activity
was
measured
using
o-nitrophenyl-?-D-
degraded by 2 – 100% depending on the enzyme used.
galactopyranoside as substrate.31
?-Arabinosidase
The impact of ?-glucosidase activity on boysenberry
activity was analysed according to Poutanen et al33
and raspberry anthocyanins has also been veri?ed.23,24
by using p-nitrophenyl-?-L-arabinofuranoside as sub-
In this work, the effect of extensive dosages
strate.
of various commercial cell wall degrading enzyme
preparations on the pressability of juice from bilberries
Juice pressing
and blackcurrants was investigated with special
Frozen berries (60 g) were separated from stalks and
emphasis on improving the yield and maximizing the
leaves, etc. Bilberries and blackcurrants were thawed
extraction of anthocyanins into the juice.
for 20 and 35 min, respectively. After thawing, the
berries were mashed for 8 s (16 s for blackcurrants)
in a multifunctional chopper (Multitrio, Moulinex,
MATERIALS AND METHODS
Ireland). Fifty grams of the mashed berries were
Raw materials
weighed into a 0.5 L plastic bag and kept in
Bilberries (Vaccinium myrtillus L.) were obtained
a 45 ?C water bath until the temperature of the
from Kiantama Ltd, Suomussalmi, northern Finland.
mash reached 45 ?C. Enzymes were dosed based
Blackcurrants (Ribes nigrum L. cv.
¨
Ojebyn) were
on endopolygalacturonase activity corresponding to
obtained from Peltohermanni Ltd, Ilomantsi, eastern
1000 nkat g?1 in the treatment. Each enzyme was
Finland. Both berries were of 2002 harvest. Prior to
diluted with distilled water to the constant volume of
the experiments, they were stored frozen at ?23 ?C for
10 mL before adding to the mashed berries, with the
3 months.
exception of the Econase CE dosage of 1000 nkatg?1.
Owing to the low endopolygalacturonase activity of
Enzymes and enzyme activities
the Econase CE preparation, a high enzyme volume
Various enzyme preparations obtained from com-
had to be added to the berry mash (39 mL per 50 g
mercial suppliers were used (Table 1). The enzymes
berries). Separate reference treatment was therefore
used were Econase CE (supplied by AB Enzymes,
used in this treatment (39 mL of water instead of
Rajam¨aki, Finland), Pectinex Smash (supplied by
enzyme). The incubation was continued at 45 ?C
Novozymes, Dittinger, Switzerland), Pectinex BE-
for 2 h. The treatments were carried out at the
3L (supplied by Novozymes, Dittinger, Switzer-
intrinsic pH of the berries (about pH 3) and the
land), Pectinex Ultra SP-L (supplied by Novozymes,
pH was checked from the mash and the juice after
Bagsvaerd, Denmark) and Biopectinase CCM (sup-
the treatment. Reference treatments were carried out
plied by Quest International Ireland Ltd, Carriga-
correspondingly but omitting the enzyme. Reference
line, Ireland). The activity pro?les of the enzyme
treatments were also carried out using denatured
preparations were determined at pH 3.5 using stan-
enzymes. The enzymes were denatured by boiling
dard assays. The overall cellulolytic activity was
in a water bath for 5 min.
measured with ?lter paper as substrate. The activ-
The juice was extracted by a juice pressing device
ity was expressed as ?lter paper units (FPU).25
attached to a TA-HDi texture analyser (Stable Micro
Endoglucanase, mannanase, xylanase and ?-glucanase
Systems, Godalming, UK). The pressing device was
activities of the enzyme preparations were analysed
speci?cally developed for this work and built by
using hydroxyethylcellulose,25 locust bean gum,26
Protoshop Oy (Espoo, Finland) (Fig 1(a)). The inner
Table 1. Activity pro?les of enzymes measured at pH 3.5
CE (FPU
EG
XYL
MAN
PG
?-GLU
?-GAL
?-ARA
PME
Enzyme
mL?1)
(nkat mL?1) (nkat mL?1) (nkat mL?1) (nkat mL?1) (nkat mL?1) (nkat mL?1) (nkat mL?1) (nkat mL?1)
Econase CE
47
16 780
30 040
2 110
1 280
63
64
640
0
Pectinex Smash
0
1 990
590
30 915
34 885
42
1 910
774
7 807
Pectinex BE-3L
0
986
21 630
1 287
4 900
338
2 804
2 988
2 090
Biopectinase CCM
0
1 467
1 762
3 139
22 540
136
691
1 688
5136
Pectinex Ultra SP-L
0
1 653
900
16 160
29 300
8
1 464
715
2 537
CE = overall cellulolytic activity; EG = endoglucanase; XYL = xylanase; MAN = mannanase; PG = endopolygalacturonase; ?-GLU = ?-glucosidase;
?-GAL = ?-galactosidase; ?-ARA = ?-arabinosidase; PME = pectin methylesterase.
J Sci Food Agric 85:2548– 2556 (2005)
2549

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J Buchert et al.
(a)
Analysis of total phenolics and sugars
Total phenolics were determined from the raw
materials and juices using the Folin – Ciocalteau
method according to Singleton and Rossi.34 The
phenolic compounds were extracted from the berries
with methanol. The sugars present in the juice were
analysed by the HPLC assay procedure using a
modi?ed gradient.35 Initial eluent conditions consisted
of 85% water and 15% 100 mmol L?1 NaOH. The
NaOH concentration was decreased with a concave
curve from 15% to 0% between 0 and 4 minutes.
From 4 to 35 minutes the gradient was held at 100%
water. The column was washed with 300 mmol L?1
sodium acetate in 100 mmol L?1 NaOH for 3 min and
after that with 300 mmol L?1 NaOH for 4 min. After
these steps the gradient was returned to 85% of water
and 15% of 100 mmol L?1 NaOH and held at these
initial conditions for 15 min.
HPLC analysis of anthocyanins
The frozen juices were thawed and a 4.7 mL
sample was weighed for the analysis. After adding
concentrated HCl (50 µL), the sample volume was
adjusted to 5 mL with methanol. Frozen berry samples
were homogenised with a household blender, and
the homogenised sample (5 g) was extracted with 3 ×
(b)
15 mL of acidi?ed methanol (MeOH:HCl, 99:1, v/v).
After vigorous mixing for 1 min, the suspension was
centrifuged at 3400 × g for 10 min. The supernatants
from three extractions were combined and made to
50 mL. The pH of the ?nal samples was below 1.
Prior to HPLC analysis, the samples were ?ltered
through a syringe ?lter (0.45 µm TITAN, Gloucester,
UK). A 10 µL injection of the ?ltrates was separated
on a LiChroCart Purospher Star RP-18e column
(250 mm × 4.6 mm, i.d. 5 µm; 4 mm × 4 mm guard
column; Merck, Darmstadt, Germany) using a HP
Figure 1. (a) Juice pressing device developed for the texture
1100 series HPLC (Waldbronn Analytical Division,
analyser; (b) net discs for bilberries (left) and blackcurrants (right).
Waldbronn, Germany) equipped with a quaternary
pump, an autosampler and a diode-array detector
diameter of the pressing cell was 70 mm and the
(DAD) linked to an HP-ChemStation data handling
height was 105 mm. It had a ?xed metallic base
system. The analysis of anthocyanins was performed
that was perforated, the holes having a diameter of
using 10% formic acid in water as solvent A and
3 mm. On the ?xed base a thin net disc (edge 0.5 cm)
acetonitrile:methanol (85:15 v/v, HPLC grade) as
(Spinea Ltd, Helsinki, Finland) with the desired mesh
solvent B. The ?ow rate of the mobile phase
size could be placed. For the bilberries a net with
was 1.0 mL min?1 for 0 –10 min and 0.8 mL min?1
0.5 mm × 0.5 mm holes (thread thickness 0.29 mm)
for 10 – 70 min. The gradient used was as follows:
was used. For the blackcurrants a net with somewhat
0 – 2 min, 6% B; 2 – 4 min, 6 to 7% B; 4 – 11 min, 7%
larger holes was needed (0.75 mm × 0.75 mm, thread
B; 11 – 20 min, 7 to 9% B; 20 – 30 min, 9 to 10% B;
thickness 0.3 mm) (Fig 1(b)). The berry mash was
30 – 50 min, 10 to 12% B; 50 – 55 min, 12 to 16% B;
poured into the pressing cell and compressed with
55 – 64 min 16% B; 64 – 67 min, 16 to 90% B, followed
a speed of 0.5 mm s?1 until a force of 245 kg was
by an isocratic gradient for 3 min and then returning
reached. This force, which equates to a pressure
to the initial conditions for 5 min before the next
of 0.62 MPa (6.2 bar), was maintained for 5 s and
injection. Anthocyanins were detected at 520 nm.
thereafter the piston was moved up to its starting
Identi?cation of anthocyanins was based on ref-
position. After pressing, the weights of the resulting
erence compounds (UV-visible spectra and reten-
juice and press cake were determined and the
tion times), the literature1,3,4 and our previous
corresponding yields were calculated. The treatments
studies.36 – 38 Quanti?cation of anthocyanins was car-
were carried out in duplicate. The samples were frozen
ried out using representative anthocyanidin 3-O-
and stored at ?25 ?C before the analyses.
glucosides (Polyphenols AS, Sandnes, Norway) as
2550
J Sci Food Agric 85:2548– 2556 (2005)

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Enzyme-aided pressing of bilberries and blackcurrants
external standards, and the contents were expressed in
dosage (1000 nkat g?1) based on endopolygalactur-
mg kg?1 of fresh weight, for the weight of the aglycon.
onase activity. The dosage was selected to be able
to compare different enzyme preparations and to
see a clear impact of the role of minor activities in
RESULTS AND DISCUSSION
the preparations. The reference treatments were car-
Activity pro?les of the enzyme preparations
ried out at similar conditions but by adding water
Enzyme activity pro?les of the enzyme preparations
instead of enzyme preparation. After the treatments
measured at pH 3.5 and the calculated enzyme
the berries were pressed and the juice yields were mea-
activities in the enzyme mixtures with constant
sured (Tables 3 and 4). With the pressing device used,
endopolygalacturonase activity of 1000 nkat g?1 used
the bilberry juice yields obtained after the enzyme
for the treatments are seen in Tables 1 and 2, respec-
treatments were 116 – 118% of the corresponding ref-
tively. The enzyme preparations were clearly different
erences (Table 3). As mentioned, the enzyme dosages
with respect to the activity pro?les. The highest
used were, however, very high and maximal bilberry
?-glucosidase activity as related to endopolygalac-
juice yield was obtained already at much lower enzyme
turonase activity was in the Pectinex BE-3L and
dosages (below 50 nkat g?1; results not shown). The
Econase CE preparations, whereas Pectinex Ultra SP-
pH of the bilberry mash after enzyme addition was
L contained practically no ?-glucosidase (Table 2).
found to be 2.6 – 2.7, with the exception of Econase
The ?-glucosidase activities of Pectinex Smash and
Biopectinase CCM were also relatively low com-
CE treatment which (owing to the high volume of the
pared with those of Econase CE and Pectinex BE-L.
enzyme preparation used) raised the pH to 3.3.
All enzyme mixtures contained signi?cant amounts
Blackcurrant cell walls are known to be thicker
of ?-galactosidase, with the highest relative activ-
and to contain more pectins than those of bilberry.39
ity in Pectinex BE-3L. Clear differences in the ?-
Thus the effect of the enzymatic treatment on the
arabinosidase activities were found, and Econase CE
pressability was more pronounced compared with
and Pectinex BE-3L contained higher relative activities
bilberry pressing (Table 4). The highest juice yields
than the other enzymes used. Of the enzyme prepara-
compared with reference treatment (133 – 135%) were
tions tested, Pectinex BE-3L was relatively the richest
obtained by Pectinex BE-3L and Biopectinase CCM,
with respect to ?-glucosidase, ?-galactosidase and ?-
whereas the effect obtained by Econase CE was
arabinosidase activities. Econase CE was devoid of
clearly lower (114%). The lower effect of the Econase
any pectin methylesterase activity, which was present
CE preparation is partially expected to be due to
at practically similar levels in the other preparations
lack of pectin methylesterase activity in the mixture
used (Table 1).
(Tables 1 and 2), as polygalacturonase is unable to
attack cell wall pectins ef?ciently without concomitant
Effect of enzyme treatment on juice yield
demethylation of the pectins. Signi?cant variation in
Both berries, ie bilberry and blackcurrant, were
juice yields have usually been obtained depending on
treated for 2 h at 45 ?C with a relative high enzyme
pressing device. The juice yields reported by Landbo
Table 2. Calculated enzyme activities with PG activity of 1000 nkat g?1 berry sample
Enzyme
PG (nkat g?1)
?-GLU (nkat g?1)
?-GAL (nkat g?1)
?-ARA (nkat g?1)
Wt%
Econase CE
1000
49
50
500
78
Pectinex Smash
1000
1.2
56
22
3.4
Pectinex BE-3L
1000
69
572
610
2.9
Biopectinase CCM
1000
6
31
75
20
Pectinex Ultra SP-L
1000
<0.5
50
24
4.4
PG = endopolygalacturonase; ?-GLU = ?-glucosidase; ?-GAL = ?-galactosidase; ?-ARA = ?-arabinosidase.
Table 3. Enzyme-aided pressing of bilberry
pH after
pH of
Juice
Juice yield
Enzyme
enzyme addition
juice
Juice (g)
yielda (%)
(% of reference)
Reference (10 mL H2O)
2.74
2.97
40.1
66.8
100
Pectinex Ultra SP-L
2.59
2.84
47.6
79.3
119
Pectinex Smash
2.61
2.85
47.2
78.7
118
Pectinex BE-3L
2.60
2.84
47.9
79.8
119
Biopectinase CCM
2.66
2.94
47.6
79.3
119
Reference (39 mL H2O)b
2.82
3.07
67.7
76.1
100
Econase CE
3.33
3.48
78.6
88.3
116
a Juice yield = [(mass of juice)/(mass of mash into the pressing system)] × 100.
b Reference to Econase CE treatment.
J Sci Food Agric 85:2548– 2556 (2005)
2551

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J Buchert et al.
Table 4. Enzyme-aided pressing of blackcurrant
pH after
pH of
Juice
Juice yield
Enzyme
enzyme addition
juice
Juice (g)
yielda (%)
(% of reference)
Reference (10 mL H2O)
2.57
2.84
31.0
51.6
100
Pectinex Ultra SP-L
2.49
2.76
39.3
65.5
127
Pectinex Smash
2.48
2.75
40.0
66.7
129
Pectinex BE-3L
2.45
2.72
42.0
70.0
135
Biopectinase CCM
2.48
2.76
41.2
68.7
133
Reference (39 mL H2O)b
2.61
2.85
60.7
68.2
100
Econase CE
2.89
3.14
68.9
77.4
114
a Juice yield = [(mass of juice)/(mass of mash into the pressing system)] × 100.
b Reference to Econase CE treatment.
Table 5. Phenolic and carbohydrate composition of bilberry juices
Total phenolicsa
Fru
Glc
Gal
Man
Ara
Xyl
GalA
Total sugars
Enzyme
(g L?1)
Increase (%)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
Reference (10 mL H2O)
3.33
—
31.0
23.0
0
0
0
0
0
54.0
Pectinex Ultra SP-L
4.22 (3.05)
27
31.0
23.0
1.1
0
0.4
0
3.9
59.4
Pectinex Smash
4.21 (2.42)
26
30.0
22.0
0.9
0
0.4
0
3.5
56.8
Pectinex BE-3L
4.50 (2.89)
35
30.0
23.0
1.3
0
0.6
0
3.8
58.7
Biopectinase CCM
4.38 (3.13)
32
32.0
25.0
1.4
0
0.7
0
3.8
62.9
Reference (39 mL H2O)b
2.33
—
22.0
15.5
0
0
0
0
0
37.5
Econase CE
2.67 (1.87)
15
20.0
16.5
1.5
2.9
0.4
0
0
41.3
Fru = fructose; Glc = glucose; Gal = galactose; Man = mannose; Ara = arabinose; Xyl = xylose; GalA = galacturonic acid.
a The value obtained with denatured enzymes is in parentheses.
b Reference to Econase CE treatment.
and Meyer40 ranged from 66.4% to 78.9% by wet
(a)
weight of mash in experimental blackcurrant berry
600
juice production with various pectolytic enzymes.
500
400
Effect of enzyme treatment on bilberry juice
2. 3.
5.
14.
mAU 300
1.
4.
7.
quality
200
8.
The bilberry juices produced were analysed with
6.
11.
15.
100
12.
10.
respect to the phenolics and monosaccharides present
9.
13.
0
(Table 5). The total phenolic content of bilberry juice
10
20
30
40
50
60
as measured by the Folin – Ciocalteau method was
min
increased by 35%, from 3.3 to 4.5 g L?1 with the
(b)
u.i.
u.i.
most effective enzyme used, Pectinex BE-3L. Pectinex
600
Ultra SP-L, Pectinex Smash and Biopectinase CCM
u.i.
500
improved the extractability of phenolics by 26 – 32%.
u.i.
The added enzyme amount was not found to affect the
400
2.
14.
5.
Folin measurement. The galacturonic acid content of
mAU 300
bilberry juice was 3.5 – 3.9 g L?1 after all other enzyme
8.
4.
200
15.
u.i.
treatments but Econase CE, indicating that Econase
7.
11.
100
CE does not contain exo-pectinase activity.
13.
0
The content of individual anthocyanins of the
10
20
30
40
50
60
initial berries and the juices were analysed by HPLC
min
(Fig 2, Table 6). The most abundant anthocyanidin
glycosides in whole bilberry were delphinidin 3-O-
Figure 2. HPLC chromatograms (520 nm) of anthocyanins in bilberry
juices: (A) reference juice (no enzyme treatment); (B) enzyme-treated
arabinoside and delphinidin 3-O-galactoside (each
(Pectinex BE-3L) juice. Peak identi?cation: 1. delphinidin 3-O-galacto-
14% of total anthocyanins), followed by cyanidin 3-O-
side, 2. delphinidin 3-O-glucoside, 3. cyanidin 3-O-galactoside,
galactoside (12%) (Table 6). The total anthocyanin
4. delphinidin 3-O-arabinoside, 5. cyanidin 3-O-glucoside,
yields in bilberry juices were enhanced in all
6. petunidin 3-O-galactoside, 7. cyanidin 3-O-arabinoside,
other treatments except with Econase CE (Fig 3).
8. petunidin 3-O-gluco-side, 9. peonidin 3-O-galactoside,
10. petunidin 3-O-arabinoside, 11. peonidin 3-O-glucoside,
The most ef?cient enzyme preparation to increase
12. malvidin 3-O-galactoside, 13. peonidin 3-O-arabinoside,
the anthocyanin extraction was Pectinex BE-3L,
14. malvidin 3-O-glucoside, 15. malvidin 3-O-arabinoside,
increasing the yield by 41% (when identi?ed and
u.i. unidenti?ed.
2552
J Sci Food Agric 85:2548– 2556 (2005)

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Enzyme-aided pressing of bilberries and blackcurrants
e
e
e
e
e
)
r
a
2
1
3500
?
A
562
389
4
Unknown
1823
525
520
392
639
3000
b
Arabinosides
2500
Glucosides
4
2000
Galactosides
3
895
683
1500
Total
Glc
2316
1220
1181
1260
1160
1000
anthocyanins
500
0
0
0
0
al
35
Concentration (mg kg
0
G
645
457
2074
Ref.
Econase CE
Pectinex Smash
Pectinex BE-3L
anins
c
c
Pectinex Ultra SP-L
Biopectinase CCM
Ref. (for Econase)
cy
57
98
705
796
628
42
1401
Figure 3. Distribution of anthocyanidin glycosides in bilberry juices.
Unidenti?ed
ntho
a
a
unidenti?ed compounds were calculated). With the
69
94
95
84
48
30
Ar
143
105
others the increase was 13 – 16%. Econase CE, on
the other hand, decreased the total amount of
lc
G
13
detectable anthocyanins from about 1570 mg kg?1
460
191
251
242
268
234
148
Malvidin
to below 180 mg kg?1, ie by 89%. In all enzyme
glycosides
0
0
0
0
treatments, with the exception of Econase CE, a
Gal
35
82
167
110
signi?cant amount of unknown anthocyanin type
of components was formed (Figs 2 and 3, Table 6).
r
a
0
These components were not detected in the berries,
A
32
19
20
20
19
21
13
and their chemical structure will be characterized
0
in our further studies. We tentatively assume that
Glc
88
70
203
114
116
117
108
these new components represent aglycons liberated
Peonidin
glycosides
by enzymatic hydrolysis of anthocyanidin glycosides
al
0
0
0
0
0
as reported by Wightman and Wrolstad.23,41 In
G
60
29
22
addition to the formation of unknown components, the
treatments.
a
d
d
d
d
d
enzymatic treatments also caused clear changes in the
D
Ar
206
62
ND
ND
ND
ND
42
N
anthocyanin pro?les of the juices (Fig 3). As a result of
all enzymatic treatments, practically no anthocyanidin
nzymatic
lc
e
galactosides were detected (Table 6), which is in line
G
389
140
195
187
204
186
105
the
with the high galactosidase activities found in the
Petunidin
glycosides
nthocyanins.
a
preparations (Table 2). Hydrolysis of anthocyanidin
0
0
0
0
after
Gal
82
57
223
galactosides has also been reported for enzymatically
r
y
found
processed cranberries.22,41 The hydrolysis could also
ilber
ycon.
as
be visualised in the increased galactose content of the
b
r
a
r
epresenting
f
A
585
191
175
176
110
227
136
gl
o
a
juice after especially Econase CE, Biopectinase CCM
same
the
and Pectinex BE-3L treatment (Table 5).
j
uices
of
t
he
Glc
Pectinex BE-3L and Econase CE contained the
574
237
306
302
307
295
183
ht
g
not
and
Cyanidin
highest relative
glycosides
compounds
?-arabinosidase activity (Table 2),
wei
of
peaks.
which can be visualised in ef?cient arabinoside
r
i
es
al
0
0
0
0
G
766
194
140
t
he
um
ber
s
hydrolysis with hydrolysis degrees of 31% and 90%,
le
for
respectively (Table 6). The amount of petunidin
1
the
ho
a
?
redominantly
erlapping
w
as
p
v
3-O-arabinoside could not be analysed owing to
Ar
857
221
236
229
158
307
150
kg
o
i
n
to
mg
are
overlapping with an unknown peak formed during
in
peaks.
ue
the treatment. Glucosides were only hydrolysed by
sides
lc
3700508
arabinoside.
ples
d
G
1
691
239
354
334
364
337
177
=
calculated
am
Econase CE (Table 6, Fig 3), although Pectinex
lyco
s
g
r
a
Delphinidin
glycosides
A
amples
as
BE-3L contained even higher glucosidase activity
;
s
w
0
0
0
0
0
e
xcluded
d
overlapping
e
compared with Econase CE (Table 2). Thus the
Gal
859
230
156
yanidin
to
een
glucosidase(s) present in Econase CE and Pectinex
c
ucosi
reference
b
uplicate
treatment.
f
gl
d
BE-3L clearly have different modes of action.
f
t
he
wing
o
has
CE
antho
O)
O)
=
o
nthocyanins
i
n
f
2
2
l
c
a
o
H
H
f
G
o
a
Effect of enzyme treatment on blackcurrant juice
berry)
eans
mL
SP-L
mL
e;
d
m
peaks
conase
ash
CCM
quality
-3L
s
E
a
etermined
rabinoside
hole
(
10
l
tra
(
39
E
d
a
t
o
U
Sm
BE
C
ctosi
content
Blackcurrant juice (reference juice without enzymes)
Contents
(w
a
.
e
not
6
s
contained a lower concentration of total phenolics
a
gal
t
otal
unknown
n
=
=
o
than the corresponding bilberry juice (Tables 5 and 7).
r
eatment
c
Expressed
The
The
ND
Petunidin
Reference
Table
T
Bilberry
Reference
Pectinex
Pectinex
Pectinex
Biopectinase
Reference
E
Gal
a
b
c
d
e
f
The enzymatic improvement of phenolic extraction
J Sci Food Agric 85:2548– 2556 (2005)
2553

---

J Buchert et al.
Table 7. Phenolic and carbohydrate composition of blackcurrant juices
Phenolicsa
Increase
Fru
Glc
Gal
Man
Ara
Xyl
GalA
Total sugars
Enzyme
(g L?1)
(%)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
(g L?1)
Reference (10 mL H2O)
2.88
—
36.0
32.0
0
0
0
0
0.8
68.8
Pectinex Ultra SP-L
4.07 (3.21)
41
42.0
35.0
1.1
0
0.9
0
11.1
90.1
Pectinex Smash
3.77 (3.09)
31
43.0
34.0
0.9
0
0.9
0
10.9
89.7
Pectinex BE-3L
4.20
46
40.0
33.0
1.3
0
1.8
0
10.3
86.4
Biopectinase CCM
4.60 (2.68)
60
41.0
34.0
1.4
0
1.7
0
10.5
88.6
Reference (39 mL H2O)b
2.04
—
26.0
20.0
0
0
0
0
0.5
46.5
Econase CE
2.34 (2.21)
15
26.0
23.0
1.5
2.9
0.9
0.5
2.3
57.1
Fru = fructose; Glc = glucose; Gal = galactose; Man = mannose; Ara = arabinose; Xyl = xylase; GalA = galacturonic acid.
a The value obtained with denatured enzymes is in parentheses.
b Reference to Econase CE treatment.
(a) 700
Table 8. Contentsa of anthocyanidin glycosides in whole berries and
juices of blackcurrant
600
2.
4.
500
Delphinidin
Cyanidin
Total antho-
400
glycosides glycosides
cyanins
mAU 300
Treatment
Glc
Rut
Glc
Rut
Glc
Rut
1.
200
3.
Blackcurrant (whole
347
810
241
949
589
1759
100
berry)
0
Reference (10 mL H2O)
198
587
133
635
331
1222
10
20
30
40
50
60
Pectinex Ultra SP-L
392
651
350
606
743
1258
min
Pectinex Smash
337
650
285
604
622
1254
Pectinex BE-3L
321
650
229
634
550
1284
(b) 700
2.
Biopectinase CCM
304
707
204
756
508
1463
4.
600
Reference (39 mL H2O)b
143
421
95
439
238
860
Econase CE
115
520
66
541
181
1061
500
400
Glc = glucoside; Rut = rutinoside.
1.
a
mAU
Expressed as means of duplicate samples in mg kg?1 for the weight
300
3.
of the aglycon.
200
b Reference to Econase CE treatment.
100
0
)1? 2500
10
20
30
40
50
60
Rutinosides
min
2000
Glucosides
1500
Figure 4. HPLC chromatograms (520 nm) of anthocyanins in
1000
blackcurrant juices: (A) reference juice (no enzyme treatment);
500
(B) enzyme-treated (Pectinex BE-3L) juice. Peak identi?cation:
0
1. delphinidin 3-O-glucoside, 2. delphinidin 3-O-rutinoside,
Concentration (mg kg
3. cyanidin 3-O-glucoside, 4. cyanidin 3-O-rutinoside.
Ref.
Econase CE
Pectinex Smash
Pectinex BE-3L
was, however, higher than with bilberry treatments.
Pectinex Ultra SP-L
Biopectinase CCM
Ref. (for Econase)
Biopectinase CCM increased the phenol content by
60%, followed by Pectinex BE-3L (46%). Econase
Figure 5. Distribution of anthocyanidin glycosides in blackcurrant
juices.
CE treatment resulted in only a 15% increase. After
the enzymatic treatments with Pectinex Ultra SP-
L, Pectinex Smash, Pectinex BE-3L or Biopectinase
into the juice (Table 8). The anthocyanin yields were
CCM the blackcurrant juices contained signi?cant
increased from 1550 mg kg?1 to 2000 mg kg?1 and
amounts of galacturonic acid, indicating extensive
1970 mg kg?1, ie by 27 – 29%, by Pectinex Ultra SP-
hydrolysis of cell wall pectins. This could be expected
L and Biopectinase CCM, respectively (Fig 5). The
owing to the high concentration of pectins in these
relative ratio of delphinidin glycosides versus cyanidin
berries.40 Econase CE treatment also resulted in slight
glycosides remained about the same in the juices as
liberation of galacturonic acid to the juice.
compared with the whole berry. Of the glycosides,
The individual anthocyanins were analysed by
the rutinosides of delphinidin and cyanidin were more
HPLC (Fig 4) from the berries and juices, and the
abundant than the corresponding glucosides in all
results are presented in Table 8. Enzyme-aided press-
cases (Table 8, Fig 5). With all other enzymes with the
ing resulted in improved liberation of anthocyanins
exception of Econase CE, the anthocyanidin glucoside
2554
J Sci Food Agric 85:2548– 2556 (2005)

---

Enzyme-aided pressing of bilberries and blackcurrants
extraction was increased more pronouncedly than the
2 Nielsen IL, Haren GR, Magnussen EL, Dragsted LO and
rutinoside extraction. Landbo and Meyer40 found that
Rasmussen SE, Quanti?cation of anthocyanins in commercial
black currant juices by simple high-performance liquid
the relative quantitative distribution of the four major
chromatography. Investigation of their pH stability and
blackcurrant anthocyanins did not change with enzy-
antioxidative potency. J Agric Food Chem 51:5861–5866
matic maceration of the berries. In the present study,
(2003).
however, the relative contents of delphinidin and
3 Baj A, Bombardelli E, Gabetta B and Martinelli EM, Qual-
cyanidin glucosides were increased (total glucosides
itative and quantitative evaluation of Vaccinium myrtillus
21% in reference juice versus 26 – 37% in pectinase-
anthocyanins by high-resolution gas chromatography and
high-performance liquid chromatography. J Chromatogr
treated juices) and those of rutinosides decreased (total
279:365–372 (1983).
rutinosides 79% in reference juice versus 63 – 74% in
4 Kalt W, McDonald JE, Ricker RD and Lu X, Anthocyanin
pectinase-treated juices). No hydrolysis of extracted
content and pro?le within and among blueberry species. Can
rutinosides was observed by either of the ?ve enzyme
J Plant Sci 79:617–623 (1999).
mixtures used, indicating that rutinosidase activity or
5 Tsuda T, Watanabe M, Ohshima K, Norinobu S, Choi S-
W, Kawakishi S and Osawa T, Antioxidative activity of
rhamnosidase activities were not present in the prepa-
the anthocyanin pigments cyanidin 3-O-?-D-glucoside and
rations used. In the Econase CE treatment a partial
cyanidin. J Agric Food Chem 42:2407–2410 (1994).
hydrolysis of the extracted delphinidin and cyanidin
6 Rice-Evans CA, Miller NJ and Paganga G, Structure-
glucosides was observed, whereas the other enzymes
antioxidant activity relationships of ?avonoids and phenolic
did not have any activity against glucosides. Similarly
acids. Free Rad Biol Med 20:933–956 (1996).
7 Wang H, Cao G and Prior RL, Oxygen radical absorbing
as in the case of bilberries, the glucosidase present in
capacity of anthocyanins. J Agric Food Chem 45:304–309
Pectinex BE-3L was unable to hydrolyse the gluco-
(1977).
sides present in blackcurrant (Table 8). The enzyme
8 K¨ahk ¨onen MP
and
Heinonen M,
Antioxidant
activity
of
treatments clearly had less signi?cant effects on the
anthocyanins and their aglycons. J Agric Food Chem
anthocyanin pro?les in blackcurrant juices than in
51:628–633 (2003).
bilberry juices (Figs 2 – 5).
9 Matsumoto H, Nakamura Y, Hirayama M, Yoshiki Y and
Okubo K, Antioxidant activity of black currant aglycons
and their glycosides measured by chemiluminescence in a
neutral pH region and in human plasma. J Agric Food Chem
CONCLUSION
50:5034–5037 (2002).
The results indicate that the enzyme product used in
10 Hou D-X, Potential mechanisms of cancer chemoprevention by
pressing strongly affects both juice and anthocyanin
anthocyanins. Curr Mol Med 3:149–159 (2003).
yields. Careful understanding of enzyme activities is
11 Tsuda T, Horio F, Uchida K, Aoki H and Osawa T, Dietary
cyanidin 3-O-?-D-glucoside-rich purple corn color prevents
required as maximisation of anthocyanin extraction
obesity and ameliorates hyperglycemia in mice. J Nutr
and juice yield may not be obtained with the same
133:2125–2130 (2003).
enzyme mixture. The anthocyanin yield is affected by
12 Matsumoto H, Inaba H, Kishi M, Tominaga S, Hirayama M
potential improvement in the extractability, but exo-
and Tsuda T, Orally administered delphinidin 3-rutinoside
glycosidases may also cause hydrolysis of the liberated
and cyanidin 3-rutinoside are directly absorbed in rats and
anthocyanins.
humans and appear in the blood as the intact forms. J Agric
Food Chem 49:1546–1551 (2001).
13 Matsumoto H, Hanamura S, Kawakami T, Sato Y and
Hirayama M, Preparative-scale isolation of four anthocyanin
ACKNOWLEDGEMENTS
components of black currant (Ribes nigrum L.) fruits. J Agric
We thank Anne Ala-Kahrakuusi, Riitta Alander and
Food Chem 49:1541–1545 (2001).
Annika Majanen for their valuable work in carrying out
14 Mazza G, Kay CD, Cottrell T and Holub BJ, Absorption of
juice manufacture and chemical analyses. Dr Tuulikki
anthocyanins from blueberries and serum antioxidant status
in human subjects. J Agric Food Chem 50:7731–7737 (2002).
Sepp¨anen-Laakso is thanked for the analysis of the
15 McGhie TK, Ainge GD, Barnett LE, Cooney JM and Jensen
total phenolics.
DJ, Anthocyanin glycosides from berry fruit are absorbed and
excreted unmetabolized by both humans and rats. J Agric
Food Chem 51:4539–4548 (2003).
NOTE
16 Nielsen IL, Dragsted LO, Ravn-Haren G, Freese R and Ras-
This study was carried out with ?nancial support
mussen SE, Absorption and excretion of black currant antho-
from the Commission of the European Communities,
cyanins in humans and Watanabe heritable hyperlipidemic
rabbits. J Agric Food Chem 51:2813–2820 (2003).
speci?c RTD programme ‘Quality of Life and
17 Talav´era S, Felgines C, Texier O, Besson C, Lamaison J-L and
Management of Living Resources’, QLK1-CT-2002-
R´em´esy C, Anthocyanins are ef?ciently absorbed from the
02364, ‘Novel enzyme-aided extraction technologies
stomach in anesthetized rats. J Nutr 133:4178–4182 (2003).
for maximised yield and functionality of bioactive
18 Pilnik W and Voragen AG, The signi?cance of endogenous and
components in consumer products and ingredients
exogenous pectic enzymes in fruit and vegetable processing,
in Food Enzymology, vol. 1, ed by Fox PF. London and New
from by-products’. It does not necessarily re?ect the
York, Elsevier Applied Science, pp. 303–336 (1991).
Commission’s views and in no way anticipates the
19 Voragen AG, Pilnik W, Thibault J-F, Axelos MA and Renard
Commission’s future policy in this area.
CM, Pectins, in Food Polysaccharides and Their Applications,
ed by Stehen AM. New York — Basel — Hong Kong, Marcel
Dekker Inc., pp. 287–339 (1995).
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