Innovative Food Science and Emerging Technologies 5 (2004) 503 – 513

www.elsevier.com/locate/ifset

Effects of different enzymatic maceration treatments on enhancement of

anthocyanins and other phenolics in black currant juice
Anne-Katrine Landbo, Anne S. Meyer*
Food Biotechnology and Engineering Group, BioCentrum-DTU, Building 221, Technical University of Denmark, 2800 Lyngby, Denmark

Received 9 January 2004; received in revised form 13 August 2004; accepted 17 August 2004

Abstract

Pre-press maceration treatments with 10 different pectinolytic enzyme preparations were investigated in experimental black currant juice
production using response surface design templates. Enzyme dosage, maceration time, reaction temperature, and degree of berry crushing were
varied, and the juice yields, anthocyanins, total phenols, and turbidity in the resulting juices were compared for a total of 250 different enzymatic
treatments. The yields of anthocyanins and total phenols in the juices ranged from 900 to 2200 and 3050 to 5100 mg/kg wet weight black currant
mash, respectively. Juice yields ranged from 66.4% to 78.9% by wet weight of mash. Turbidity levels ranged from 25 to 916 formazan
nephelometric units (FNU). The reaction parameters induced larger variations in the responses than the different enzyme preparations, but the
cloned Aspergillus niger/Aspergillus aculeatus preparation Pectinex BER consistently tended to be among those giving the best responses
regarding anthocyanin yields, phenols, and low juice turbidity. The optimal maceration was achieved using an enzyme dosage of 0.18% by wet
weight of berries with a reaction at 60 8C for 30 min on the most finely crushed berry mash. This treatment gave similar profiles of anthocyanins
in the juices with all the 10 enzyme preparations. The same 10 juices all exhibited antioxidant activity against human low-density lipoprotein
oxidation in vitro, but the antioxidant potency varied depending on the enzyme preparation used in the pre-press maceration.
D 2004 Elsevier Ltd. All rights reserved.

Keywords: Enzymes; Black currant; Maceration; Anthocyanins; Juice; HPLC

Industrial relevance: It has been found previously by the authors that enzyme catalyzed degradation of cell wall polysaccharides in the skin fraction of black
currants released various polyphenols located within or tightly associated to the cell wall materials. Consequently the effectiveness of 10 commercial enzyme
preparations was evaluated. The potential of targeted processing for high antioxidant activity has been shown. The results can lead to the tailoring of specific
enzyme mixtures for optimum antioxidant juice products.

1. Introduction and the total content of anthocyanins is at least 2000 mg/kg on

In Northern Europe, black currants (Ribes nigrum) are used
in a variety of foods and beverages, but are primarily
processed for jam and berry juice products. In recent years,
the harvest of black currants in Europe has been stable at
~500,000–600,000 tonnes/year (Hummer & Barney, 2002).
Black currants are rich in phenolic compounds, notably
anthocyanins and hydroxycinnamic acids (Koeppen &
Herrmann, 1977). The dark red coloration of the black currant
berries and of the products derived from them is thus a result of
the very high level of anthocyanins present. The water-soluble
anthocyanins in black currants are mainly found in the skin
* Corresponding author.
E-mail address: am@biocentrum.dtu.dk (A.S. Meyer).
1466-8564/$ - see front matter D 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2004.08.003
a fresh mass basis of skins (Koeppen & Herrmann, 1977).
Several epidemiological data have shown a consistent
protective effect of fruit, wine, vegetable, and tea con-
sumption on cardiovascular diseases and coronary heart
disease mortality (Criqui & Ringel, 1994; Joshipura et al.,
2001; Ness & Powles, 1997). Although some uncertainty
still exists regarding the exact relationship between the
specific components present in these plant foods and
beverages and their effects and mechanisms in relation to
cardiovascular disease risk, the current literature suggests
that the cardioprotective action of fruit consumption is at
least partly attributable to the antioxidant activity of the
present ascorbic acid, flavonoids, and other phytochemicals
(Hertog et al., 1995; Knekt, Järvinen, Reunanen, & Maatela,
1996; Ness & Powles, 1997).
504 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
The flavonoids and other phenolic compounds present in
red wines, dark red berries, and in their various derived food
products and beverages have been demonstrated individu-
ally—as well as in mixtures—to exert potent antioxidant
activities against oxidation of human low-density lipopro-
teins (LDL) in test tube assays (Teissedre, Frankel, Water-
house, Peleg, & German, 1996; Meyer, Donovan, Pearson,
Waterhouse, & Frankel, 1998). Hence, one of the mecha-
nisms that have been advanced to explain the cardiopro-
tective effects of fruit and wine consumption is that the
phenolic compounds may slow the oxidative modification
of LDL to retard atherogenic plaque formation and in turn
prevent heart disease (The International Task for Prevention
of Coronary Heart Disease, 1998).
Black currants contain particularly high levels of pectic
polysaccharides and are relatively acid (pH 2.6–2.8)
(Grassin & Fauquembergue, 1996). Mechanical crushing
of the berries results in a highly viscous fruit puree from
which it is difficult to extract juice directly by pressing. For
this reason, addition of pectinolytic enzyme preparations to
the fruit pulp prior to pressing is a prerequisite for obtaining
a satisfactory juice yield and an efficient use of the press in
industrial production of black currant juice and concen-
trates. Pectic enzymes used in the fruit juice industry, as well
as increasingly in the manufacture of wines, mostly
originate from fungal sources, notably from Aspergillus
niger spp. (Grassin & Fauquembergue, 1996). These
enzyme preparations are generally multicomponent, i.e.
they harbor various pectinolytic as well as other plant cell
wall degrading enzyme activities.
During the pressing of liquefied black currant mash, the
juice is separated from the skins and seeds. Even though the
resulting juice contains relatively high levels of phenolics
and has an intense dark-purple colour from anthocyanins,
the press residue left behind is still rich in anthocyanins and
other phenolics (Landbo & Meyer, 2001).
Markham, Ryan, Gould, and Rickards (2000) presented
evidence for the occurrence of flavonol-glucosides in cell
walls of flower petals. The presence of cell wall localized
flavonoids and other phenolic compounds has also been
reported in Norway spruce needles (Hutzler et al., 1998).
However, the possible existence and eventual localization of
polyphenolic flavonoids, including anthocyanins, within the
compact skin cell walls in various fruits and berries have
received very limited attention.
We previously demonstrated that enzyme catalyzed
degradation of the cell wall polysaccharides in the skin
fraction of black currant juice press residues could enhance
the extraction of antioxidant phenols (Landbo & Meyer,
2001). The data obtained indicate that various phenolics,
including some flavonoids, are located within—or at least
tightly associated to—this cell wall material. We therefore
hypothesized that it might be possible to exploit the ability
of plant cell wall degrading enzymes to release phytochem-
icals from the fruit skins also in the active processing of
black currant juice. This enzyme assisted release might be
done by introducing a more forced enzymatic hydrolysis in
the pre-press fruit maceration step where pectinase enzymes
are already used for optimizing the juice yields.
The aim of this study was therefore to investigate the
possibilities for achieving enhanced extraction of anthocya-
nins and other co-existing phenols from black currants into
the juice by improved pre-press enzymatic treatment of
black currant mash. Effects on juice yield, anthocyanins,
total phenols, and juice turbidity of the enzyme reaction
parameters; time of hydrolysis, degree of berry crushing,
enzyme dose, and maceration temperature were compared in
a central composite experimental design template with 25
different experimental combinations for 10 commercial
multicomponent enzyme preparations intended for wine
and juice processing. Subsequently, 10 juice samples were
selected and tested for their ability to inhibit oxidation of
human LDL in vitro.
2. Materials and methods
2.1. Materials
Gallic acid and human LDL were purchased from Sigma-
Aldrich (St. Louis, MO). Methanol, Folin-Ciocalteu phenol
reagent, sodium carbonate, copper sulphate, and buffer salts
were obtained from Merck (Darmstadt, Germany). Antho-
cyanin standard compounds were purchased from poly-
phenols (Sandnes, Norway). The 10 pectinolytic enzyme
preparations were obtained from different enzyme manu-
facturing companies. Enzyme preparation names and
summaries of the available information regarding the
enzyme preparations’ activities are given in Table 1. The
enzyme preparations were selected for this study because
they are all intended for use in the juice and wine industry.
Frozen black currant berries, Ribes nigrum cv. Ben
Lemond, were obtained from a Danish commercial juice
processing factory, Vallb Saft (Kbge, Denmark), and stored
at 20 8C until use. Prior to juice processing, the berries
were gently defrosted and crushed in two different ways to
obtain pulps that had been crushed to different extents. The
finest crushing was obtained by using a meat grinder
(Jupiter, type: 863, Germany) and the more coarsely crushed
mash was obtained with a co-mill (Quadro Comil, JP
Process AB, Sweden). In the maceration experiments the
differently crushed samples are referred to as pulp 1 (P1)
(finest crushing) and pulp 2 (P2) (coarser crushing),
respectively. After crushing, ~220-g aliquots of the sample
material were vacuum packed, heated at 75 8C for 90 s,
cooled rapidly, and frozen at 20 8C until further use.
2.2. Temperature stability of anthocyanins
For this preliminary study, another batch of pasteurized
(85 8C for 40 s) black currant juice was used: maceration
conditions 50 8C, 2 h, Grindamylk Pectinase (Danisco
 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513 505

Table 1
Enzymes employed in the experiments
Enzyme name Activitya Main activity pH optimum Temp. optimum Production strain
b
Macer8k [FJ] 1500 PGU/g Pectin lyase Not given 40–50 8C Aspergillus strain
Pectinex Superpressc 1000 FDU 55 8C/ml Pectin transeliminase, Not given 55 8C A. niger
polygalacturonase,
pectin esterase, hemicellulases
Pectinex BEc 1000 FDU 55 8C/ml Pectin esterase, pectin lyase Not given ~50 8C A. niger/A. aculeatus
and polygalacturonase
Pectinex Ultra SP-Lc 26 000 PG/ml Pectolytic og hemicellulotic 3.5 35 8C A. aculeatus
Rapidase BE Superd 180 000 AVJP/g Pectinases and hemicellulases Active from 2 to 6 45–55 8C A. niger
Rohapect B5Le 160 PA Pectinases 3–4 45 8C A. niger
Rapidase EX Colord Not given Pectinases Not given 40–55 8C A. niger
Klerzyme Colord Not given Pectinases and proteases Active from 2 to 5 Active from A. niger
10 to 60 8C
Rapidase Vino Superd Not given Not given Not given 50–60 8C A. niger
Vinozyme Gc 4000 FDU 20 8C/g Pectin lyase, polygalacturonase, Not given Not given A. niger
hemicellulase and cellulase
The table also shows information concerning supplier, activity, main activity, pH optimum range, temperature optimum range and production strain.
a
Activity given by the suppliers’ data sheets.
b
Supplied by Biocatalysts Pontypridd, UK.
c
Supplied by Novozymes, Bagsv&rd, DK.
d
Supplied by DSM Gist-Brocades Delft, NL.
e
Supplied by Rfhm Enzyme, Darmstadt, Germany.

Brabrand, DK) at 0.05% E/S, where the E/S% addition level
refers to dosage in ml enzyme preparation/100 g wet mash.
Aliquots of 2-ml black currant juices were held at four
different temperatures (7, 30, 50, and 70 8C) for five
different holding times (10, 60, 90, 180, and 300 min). After
different time/heat treatments, the total levels of anthocya-
nins in the black currant juice samples were quantified by
the pH differential method as described below.
2.3. Maceration
Pulp samples were gently defrosted in a thermostatic bath
at ~20 8C. Portions of 40 g of pulp were weighed out in
pyrex glass bottles and the relevant enzyme preparation was
added at the % E/S ratio required according to the
experimental design template. Samples were mixed thor-
oughly and placed under agitation in a thermostatically
controlled water bath in accordance with reaction time and
temperature given in the experimental design. Immediately
after each treatment the juice was extracted from the
macerated black currant mash by pressing using nylon filter
bags and a stainless steel hydraulic press at 100 bar (HST
Tinkturen Press, Germany), where the pressure was held for
30 s. The juice yield was determined by weighing. After
extraction, the juice samples were immediately heated for
30 s at 90 8C in a water bath. The resulting juice was
analysed for anthocyanins, total phenols, and juice turbidity
as described below.
2.4. Determination of total phenols, anthocyanins, and juice
turbidity
Total phenols in the juices were determined by the
Folin-Ciocalteu procedure with total phenols expressed as
mg/l gallic acid equivalents (GAE) (Singleton & Rossi,
1965). Total anthocyanins were determined by the pH
differential method and anthocyanin concentrations in
black currant juice were calculated as cyanidin-3-rutino-
side equivalents (Wrolstad, 1976). Turbidity in formazin
nephelometric units (FNU) was measured by nephelom-
etry at 908 light scattering, 860 nm, with a Nephla reader
calibrated against hexamethylene tetramine formazine.
Prior to the FNU measurements all samples were diluted
with the same dilution factor to obtain a 8Brix value of
~3 8Brix.
2.5. HPLC
Anthocyanins and vitamin C were profiled by means of
the HPLC procedure described by Lamuela-Raventòs and
Waterhouse (1994). Based on spectral identification, the
areas of each of the four main anthocyanin peaks at 520 nm
were quantified by calibration with the authentic com-
pounds: delphinidin-3-glucoside, delphinidin-3-rutinoside,
cyanidin-3-glucoside, and cyanidin-3-rutinoside. The vita-
min C was measured at 280 nm and quantified by
calibration with l(+)-ascorbic acid.
2.6. Inhibition of human LDL oxidation
The antioxidant activity of black currant juices to
inhibit copper-catalyzed oxidation of human LDL (37 8C,
5 AM CuSO4) was assayed by monitoring formation of
conjugated diene hydroperoxides (234 nm) over 5 h
(Esterbauer, Striegl, Puhl, & Rotheneder, 1989). Immedi-
ately prior to assay, the juices were diluted with doubly
distilled water to allow testing at similar addition levels
(10 Al) at different equimolar concentrations of 5, 7.5 and
506 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
Table 2
Factor settings used in the experimental plan for assessing the juice yield,
clarity, anthocyanin and total phenol content when applying different
multicomponent enzyme preparations in the maceration step on black
currant pulp
Factor Minima Centerpoint Maxima
a holding juice samples at different temperatures for various
Enzyme dose (%E/S) 0.004 0.02 0.1 0.18
Temperature (8C) 29.2 32 46 60
Time (min) 12 30 120 210
Mash particle sizeb P0.9c P1 P1.5d P2
a
ml enzyme solution/100 g substrate.
b
Black currant berries were crushed with two different mills (P1 the
finest crushed, P2 the more coarsely crushed).
c
P0.9 consisted of 10% P1-pulp milled twice and 90% original
P1-pulp.
d
P1.5 was a mixture of the two pulps (P1 and P2) 50:50.
e
P2.1 consisted of 10% berries mashed by hand and 90% P2-pulp.
10 AM GAE in the LDL reaction mixture. After triplicate
analyses, the antioxidant activities of the juices were
evaluated on the basis of the net lag time prolongation, i.e.
the lag time subtracted the lag time of the control sample,
where the control sample contained all the ingredients
except the juice.
2.7. Experimental statistical design
The experimental plan was a randomised, quadratic
central composite circumscribed (CCC) response surface
design with the factors enzyme dose (% E/S), maceration
time (min), reaction temperature (8C), and degree of berry
crushing (P1 and P2) as experimental variables (Table 2).
The factor ranges in the experimental plan were attempted
chosen so that some of the combinations reflected an
industrial juice-processing situation (low enzyme dose,
long maceration time and medium to high temperature).
The temperature range was also decided on the basis of the
results obtained in the temperature stability evaluation of
the blackcurrant anthocyanins. Each enzyme preparation
was tested according to the same experimental plan that
comprised 25 different process combinations including 2
star points for each variable and 3 replicated centerpoints.
Mashing experiments without enzyme addition were made
separately.
2.8. Statistics
The computer program Modde (Umetri, Ume3, Sweden)
was used to aid the statistical design of the response surface
experiments and to fit and analyse the data by multiple
linear regression. Significance of the results was established
at PV0.05. Differences in the responses in the maceration
design templates and in the LDL antioxidant activities were
determined by one-way analysis of variance, where the 95%
confidence intervals were calculated from pooled standard
deviations (Minitab Statistical Software, Addison-Wesley,
Reading, MA).
3. Results and discussion
3.1. Thermal stability of anthocyanins
The thermal stability of anthocyanins was evaluated by
0.196
62.8 length of time. The total levels of anthocyanins in black
228 currant juice were almost the same in black currant juice
P2.1e samples held from 10 to 180 min at 7, 30 and 50 8C (Fig. 1).
However, samples held beyond 180 min at 50 8C tended to
have lower levels of anthocyanins than samples held at
lower temperatures for the same time (Fig. 1). In black
currant juice samples held at 70 8C, a linear decrease in
anthocyanin levels was observed in response to the holding
time. The linear relation was: total anthocyanins (mg/l)=
2.6 time (min)+2700, r= 0.98, pb0.01.
3.2. Enzymatic maceration results
Significant variations in juice yields, juice turbidity
levels, anthocyanin, and total phenol contents were
observed in response to the different enzymatic maceration
treatments, as well as in the separate non-enzyme treated
macerations (Table 3). The obtained extraction yields of
anthocyanins in the 250 different samples ranged from ~900
to 2200 mg/kg wet weight black currant mash equivalent to
a span of concentrations of anthocyanins in the juices of
1340–3220 mg/l juice (Table 3). Total phenols yields ranged
from 3050 to 5100 mg GAE/kg wet weight equivalent to
levels in the juices of ~4550–7400 mg GAE/l (Table 3). The
juice yields obtained in the 250 different macerations ranged
from 66.4% to 78.9% based on the weight of macerated
berries and also the turbidity levels of the juices varied
widely from 25 to 916 FNU (Table 3). The maceration
treatment carried out without enzyme addition, but with the
optimal combination of reaction parameters for the enzy-
matic maceration (see below), resulted in low juice yield
and in juice with relatively low anthocyanins and total
phenols levels (Table 3).
Fig. 1. Anthocyanin stability in black currant juice samples held at four
different temperatures (7, 30, 50, and 70 8C) and five different holding
times (10, 60, 90, 180, and 300 min).
 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513 507

Table 3
Response ranges for anthocyanin content, total phenol content, juice yield and clarity obtained from the 10 experimental plans: for each enzyme, the given
ranges describe the results of 25 different maceration treatments
Enzyme Anthocyanin contents Total phenol contents Juice yield Clarity
Yield (mg/kg)a Yield (mg/l)b Yield (mg/kg)a Yield (mg/l)b % w/wc FNUd
Macer8k [FJ] 1510–2160 2140–3220 3520–5080 5730–7390 66.9–77.0 49–305
Pectinex Superpress 1430–2140 2340–3210 3350–4790 5480–6900 66.5–77.4 34–764
Pectinex BE 1500–2190 2260–3160 3180–4460 4880–6380 69.2–78.9 33–734
Pectinex Ultra SP-L 1480–2170 2290–3180 3250–4690 5020–6840 69.6–78.2 47–466
Rapidase BE Super 1430–1980 2160–2800 3560–4620 5250–6500 69.0–77.5 25–356
Rohapect B5L 1660–2130 2440–3090 3500–4470 5370–6490 69.1–76.4 69–499
Rapidase EX Color 1370–2060 2150–3130 3260–4730 5350–6940 66.4–75.3 34–572
Klerzyme Color 1430–1980 2300–2870 3450–4680 5450–6950 67.8–77.2 59–916
Rapidase Vino Super 1500–2030 2170–2890 3450–5040 5690–7130 66.2–77.7 30–254
Vinozyme G 900–2110 1340–3060 3050–4600 4540–6550 69.2–77.5 36–895
No enzymee 1300F70 2120F116 3900F90 6100F110 68.9F1.8 ndf
a
mg/kg wet pulp.
b
mg/l juice. The minimum and maximum values shown have been rounded off to the nearest 10. For the anthocyanins, the average coefficient of variation
on the means (average CV) was 2.8%. For the total phenols, the average CV was 2.6%.
c
Percentage juice yields in weight/weight wet pulp.
d
Formazan nephelometric units.
e
Maceration without enzyme addition using the optimal combination of maceration parameters, 30 min., 60 8C, finest degree of chrushing (triplicate
determination).
f
The turbidity was beyond the Nephla reader’s range at 8Brix 3.

3.3. Total phenols and anthocyanins with increased maceration temperature in general increased
the total phenols yields, and hence the total phenols levels in
Multiple linear regression analyses of the data showed the black currant juice, within the factor levels tested (Table
that increased enzyme dosage and maceration time together 4). The anthocyanins yields also tended to increase with

Table 4
Enzymes for which the reaction parameters (enzyme dose, maceration time, temperature and substrate particle size) resulted in significantly ( pb0.05) increased
responses when evaluated by multiple linear regression analysis (MLR)
High factor level of Responses increased
Anthocyanins Total phenols Juice yield Clarity (FNU)a
(mg/kg mash) (GAE mg/kg mash) (% weight/weight wet mash)
Enzyme doseb Rapidase BE Super, All enzymes All enzymes All enzymes
Rohapect B5L,
Klerzyme Color,
Rapidase Vino Super
Timec Klerzyme Color, All enzymes but All enzymes but All enzymes
Vinozyme G Rohapect B5L Rohapect B5L,
Vinozyme G
Temperatured All enzymes but All enzymes Macer 8 FJ, –
Pectinex Ultra SP-L, Pectinex Superpress,
Rapidase BE Super, Klerzyme Color,
Rohapect B5L Rapidase Vino Super,
Vinozyme G

Substrate particle size

Coarsely crushed mash P2e – – Rapidase BE Super, Rapidase BE Super,
Vinozyme G Rohapect B5L,
Rapidase Vino Super
Finely crushed mash P1f All enzymes but Pectinex Superpress, – –
Klerzyme Color Pectinex Ultra SP-L,
Rapidase BE Super,
Rohapect B5L, Vinozyme G
a
Formazan nephelometric units.
b
0.18%.
c
210 min.
d
60 8C.
e
P2: coarser crushing.
f
P1: finest crushing.
508 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
increased enzyme dosage and increased maceration temper-
ature, but the effects of these parameters as well as the
influence of the maceration time varied depending on the
enzyme preparation used for the maceration (Table 4).
Increased maceration time thus only increased the antho-
cyanin content after treatment with two of the enzymes,
Klerzyme Color and Vinozyme G (Table 4), while shorter
maceration time penetrated as significantly positive for
anthocyanin levels with Macer 8 FJ and Rapidase BE Super
enzyme treatments. The positive effect of long maceration
time with the Klerzyme Color and Vinozyme G treatments,
respectively, may indicate that these enzyme preparations
contained activities that were able to catalyze digestion of
polysaccharides in the skin cell wall material that led to an
increased release of anthocyanins into the juice. In contrast,
the positive effects of the shorter treatment time with the
Macer 8 FJ and Rapidase BE Super macerations indicate
that these preparations harbored enzyme activities that
catalyzed the degradation of anthocyanins with longer
treatment time. Previous experiments with extraction of
phenols from black currant pomace (Landbo & Meyer,
2001) showed that treatment with the Macer 8 FJ
preparation contributed to an increase in the concentration
of total phenols but a decrease in the concentration of
anthocyanins in the resulting extracts. The negative influ-
ence of the Macer 8 FJ preparation on anthocyanins was
interpreted as being most likely due to enzyme-catalyzed
degradation of the anthocyanin glucosides caused by either
polyphenol oxidase activity or h-glucosidase side activities
in the multicomponent Macer 8 FJ enzyme preparation.
Presence of h-glucosidase side activities would cause
deglycosylation of the anthocyanin glucosides, which in
turn results in unstable anthocyanin aglycons and a
subsequent decrease in anthocyanin levels. The reason
why the apparent negative effects of the Macer 8 FJ and
the Rapidase BE Super enzyme preparations on anthocya-
nins did not penetrate directly as statistically significant
could be that the acid black currant juice dampened the
activity of the anthocyanin-degrading side activities as
compared to the higher pH employed in the experiments
with black currant pomace. With respect to the positive
effects on total phenols yields of enzyme dosage and
prolonged treatment time (Table 4), the data indicate, that
increased enzymatic polysaccharide digestion did help in
releasing possible cell wall sited phenolics. The data agree
with recent results obtained in wine making experiments,
where addition of pectinolytic enzyme preparations at the
vinification step also resulted in increased levels of phenolic
substances—notably low molecular weight phenols and
flavan-3-ol derivatives—in the resulting wines (Revilla &
González-SanJosé, 2003).
3.4. Juice yield and juice turbidity
Multivariate statistical analyses of the data revealed that
increased enzyme dosage and prolonged maceration time
always enhanced the overall juice yield and improved the
juice clarity irrespective of the enzyme preparation
employed. The only exceptions were the treatments with
Rohapect B5L and Vinozyme G, respectively, where
extended maceration time had no effect in relation to juice
yield (Table 4). With the high maceration temperatures, five
of the enzyme preparations, namely Macer 8 FJ, Pectinex
Superpress, Klerzyme Color, Rapidase Vino Super, and
Vinozyme G, gave significantly increased juice yields
(Table 4). This latter result is in complete agreement with
the available information on the temperature optima of the
main pectinase activities in these preparations (Table 1). The
results thus confirmed that the main pectinolytic activities of
these preparations were particularly active at the higher
maceration temperatures employed. With none of the
enzyme preparations, a lowered maceration temperature
penetrated as having significance on juice yield. Consider-
ing that all the enzyme preparations were mainly pectino-
lytic and had broad temperature optima in the range of
temperatures employed (Table 1) the data obtained are
consistent with the premise that an enhanced juice yield is a
result of the enzyme catalyzed degradation of the pectin in
the plant cell wall matrix and in the middle lamella that acts
as putty between the cells, but also binds water. All the
enzyme preparations employed were thus very effective in
catalyzing the desired degradation of the pectin to release
the juice from the black currant mash. On this basis, it is
therefore not surprising that within the reaction conditions
employed here, the effectiveness of these enzymes just
improved with higher enzyme dosage and longer reaction
time.
Furthermore, the enzyme activities present were appa-
rently also able to catalyze degradation of the turbidity-
causing polysaccharides provided that the reaction time was
long and the enzyme dosage high enough. However, higher
reaction temperature did not enhance the degradation of the
substances responsible for turbidity with any of the enzyme
preparations (Table 4). Rather, the treatments with the
enzymes Pectinex Ultra SP-L, Rapidase BE Super, Roha-
pect B5L, and Rapidase EX Color generally gave increased
clarity (i.e. less turbidity) at the low maceration temper-
atures (data not shown). In agreement with the available
knowledge on cloudiness and turbidity in fruit juices, we
assume that the turbid substances in the freshly pressed
black currant juice samples mainly consisted of suspended
proteinacious pectin particles and that the pectin was of
lower molecular weight than the pectin bound in the native
cell walls and in the middle lamellae (Grassin & Fauquem-
bergue, 1996; Hilz, Schols, & Voragen, 2003). Fractions of
other fruit cell wall material might also have contributed to
part of the turbidity, however. The turbidity causing material
was thus apparently released from the plant cell walls and
the middle lamellae during the enzymatic maceration.
However, the pectinases responsible for the concerted,
partial degradation of pectin in the native cell walls and in
the middle lamallae may not exhibit the same high activity
 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
on pectin polysaccharide chains of lower molecular weights
and/or with different branching patterns (Grassin & Fau-
quembergue, 1996). Further degradation of the released
polysaccharide material might therefore have been much
slower than the degradation causing the release of the
turbidity causing polysaccharides, and this could explain
why elevation of the reaction temperature did not improve
the efficiency enough to obtain a statistically significant
improvement. Alternatively, the activities responsible for the
degradation of the low molecular weight pectin fragments
were not as thermostable as the main pectinase activities.
Furthermore, from the wide ranges in turbidities obtained, it
seemed likely that the different, undeclared side activities
present in the enzyme preparations induced release of
differently composed plant cell wall polysaccharides. To
obtain a positive effect of temperature increase on clarity,
the side activities able to catalyze the degradation of these
polysaccharides would thus be required to have activity
optima in the high temperature ranges employed in the
experimental design.
The finding that some of the enzyme preparations
exhibited improved clarifying effects at the lower macer-
ation temperatures may be due to some of the pectinolytic
activities and/or the undeclared side activities in these
enzyme preparations responding differently to the exper-
imental parameters than the main pectin degrading activities
acting on the polysaccharides in the native cell wall
material.
3.5. Influence of reaction parameters and degree of berry
crushing
The milling degree of the black currant berries prior to
enzymatic maceration did not result in a universal effect on
the juice clarity or juice yield (Table 4). The coarser
crushing (P2) had an increasing effect on these two
responses for two and three of the enzyme preparations
respectively (Table 4), while the finest crushed mash gave
increased phenol concentration for half of the enzyme
preparations, namely for Pectinex Superpress, Pectinex
Ultra SP-L, Rapidase BE Super, Rohapect B5L, and
Vinozyme G. However, the concentration of anthocyanins
was significantly influenced by the particle size of the
crushed berries. Hence, the degree of berry crushing
penetrated as a main factor for the anthocyanin concen-
tration, with the finest crushed mash as the best. Only
Klerzyme Color treatments did not result in increased
anthocyanin levels with the finest crushed berry mash
(Table 4).
The data agree well with our previous results on enzyme
assisted release of phenolics directly from black currant
pomace: When the particle size of black currant pomace was
reduced from 500 to 1000 to b125 Am the phenol yields
after enzymatic maceration increased up to five times
(Landbo & Meyer, 2001). These findings imply that with
a finer crushing degree a larger portion of the phenols—that
509
presumably are entrapped in the plant cell wall and lignin
network—get accessible for extraction into the juice.
3.6. Determination of the optimal enzymatic maceration for
high anthocyanin and phenols yields
With respect to the yields of anthocyanins, a one-way
analysis of variances (ANOVA) on the data obtained with
each enzyme preparation showed that the 10 different
enzymes gave relatively similar results with similar reaction
conditions. The analysis thus revealed that within the
parameter setting employed here, a treatment comprising
the finest crushing (P1), 30 min of the enzymatic reaction
with 0.18% (E/S) at 60 8C was the most optimal for
obtaining high anthocyanin yields. This treatment gave
anthocyanin yields in the highest 95% confidence interval
group for eight enzyme preparations; the lowest yield was
obtained with Pectinex Superpress that gave anthocyanins
yields below the 95% confidence intervals. The yields
obtained with this optimal treatment could be split into three
confidence interval groups that exhibited some overlap, but
with the Pectinex BE, Pectinex Ultra SP, Rohapect B5L, and
Vinozyme G preparations in the group with highest
anthocyanin yields (Table 5).
A similar ANOVA analysis on total phenol concentra-
tions showed that the highest levels of total phenols were
found with either treatment: P1, 210 min, 0.18% (E/S), 60
8C or treatment P2, 210 min, 0.18% (E/S), 60 8C. This result
applied for 8 and 7 of the 10 enzyme preparations tested,
respectively. In the next group, with the second highest
phenol level, the maximal phenols levels in the juices were
obtained with treatment, P1, 30 min, 0.18% (E/S), 60 8C,
and was valid for 8 of the 10 enzyme preparations. An
analysis of total phenols yields obtained from each of the 10
experiments treated according to treatment, P1, 30 min,
0.18% (E/S), 60 8C, but each with a different enzyme
preparation showed that there were no significant differ-
ences between the obtained yields from the 10 enzyme
preparations (Table 5).
The data signify that the degree of berry chrushing was
not as crucial for optimizing total phenols yields as for the
anthocyanins yields. Furthermore, the results indicate that
even though the reaction time did penetrate as significant for
phenols yields, i.e. the longer treatment, the better the
phenols yields in the juices, the relative increase with
extended treatment time was apparently not large.
When comparing the reaction parameters of the identi-
fied optimal treatments to the ones currently employed in
industrial black currant juice processing (Grassin &
Fauquembergue, 1996), the degree of crushing is finer, the
holding time is ~2–4 times shorter, the mashing temperature
is ~10 8C higher, and the enzyme dosage is 4–6 times
higher.
Control juice samples prepared after optimal maceration
treatment, i.e. using the most finely crushed mash (P1),
standing at 60 8C for 30 min, but just without any enzyme
510 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513

Table 5
Anthocyanin, total phenol, clarity and ascorbic acid yields in black currant juices obtained after similar optimal maceration treatments using the most finely
crushed mash (P1), 0.18% E/S, 30 min at 60 8C, but carried out with the 10 different enzyme preparations
Maceration enzyme (mg/l) (mg GAE/l)a (FNU) Yield (mg/kg)b Antioxidant activity atc
Ascorbic acid Total phenols Clarity Total phenolsc Anthocyaninsc 5 AM (min) 7.5 AM (min) 10 AM (min)
a b b b
Macer8k [FJ] 1120F96 6530F230 97 4547 2036 11 84 N276a
Pectinex Superpress 1200F104 6592F143 331 4358a 1917c 11b 51b,c N276a
Pectinex BE 1188F102 6267F63 143 4344a 2188a 1b,c 49b,c N276a
Pectinex Ultra SP-L 1328F114 6760F143 198 4441a 2082a,b 1b,c 42b,c 245a,b
Rapidase BE Super 1120F96 6463F59 133 4392a 1966b,c 0b,c 20c 189b
Rohapect B5L 1168F100 6502F35 499 4480a 2129a,b 0b,c 25c 126c
Rapidase EX Color 1372F118 6362F12 206 4394a 2038b 4b,c 43b,c 234a,b
Klerzyme Color 1220F106 6498F68 275 4480a 1982bc 0b,c 22c 134c
Rapidase Vino Super 1160F100 6351F35 83 4468a 2029b 0b,c 34b,c 166b,c
Vinozyme G 1316F114 6549F51 148 4521a 2113a,b 2b,c 55b,c 181b,c
Gallic acid – 203a N276a N276a

Antioxidant activities of black currant juices toward in vitro human LDL oxidation: antioxidant activities are given as the average net prolongation times of the
induction time for the conjugated diene hydroperoxide formation, i.e. subtracted the induction time for the control LDL oxidation without juice added. For each
test dose with each maceration enzyme n=3.
a
mg GAE/l juice; this concentration of phenols was used as a base for diluting samples to equimolar concentrations for the LDL assay.
b
mg/kg berry mash.
c
Results in the same column followed by different roman superscript letters are significantly different at Pb0.05.

addition, were analysed for concentrations of anthocyanins
and total phenols as well as juice yield. The anthocyanin
level was found to be 2120 mg/l (Table 3) while the
corresponding yields obtained with enzyme treatment with
each of the 10 different enzyme preparations at the same
optimal conditions varied between 2880 and 3170 mg/l.
Hence, a significant increase in anthocyanin yields was
obtained with enzyme treatment, irrespective of the partic-
ular pectinase preparation employed. With respect to the
level of total phenols, the result for the non-enzyme treated
control was 6100 mg/l (Table 3), while with enzyme
preparations it varied from 6270 to 6760 mg/l. Hence, the
gain in anthocyanin levels was apparently larger than the
increase in the total phenols. This optimization was
primarily focusing on the anthocyanin content and secondly
on the juice yield and total phenols. The combination of
parameters giving the highest anthocyanin yields was not
the exact same as the one(s) resulting in the highest
concentration of total phenols, as discussed above. The
data obtained here indicate that some of the anthocyanin
glucosides and some of the other phenolic substances in
black currants may be localized in—or at least tightly
associated to—the cell walls of the black currant skins.
Deposition of the anthocyanins within the skin cell walls
may account for the differences in anthocyanin yields seen
with different enzymatic maceration treatments. Although
knowledge on the existence of flavonoids, including
flavonoid glucosides, within plant cell walls is very sparse,
the proposition that flavonoids may be associated with the
cell wall components agree well with the few data available,
which however relate to location of flavonoids in spruce or
other needles, in various leaves or within petal cell wall
materials (Hutzler et al., 1998; Markham et al., 2000;
Strack, Heilemann, & Klinkott, 1988). Our data indicate that
the anthocyanin glucosides were more readily released from
the blackcurrant cell wall matrices than the other phenols.
However, the precise locations of anthocyanins and the
other phenolics compounds and their type of bonding or
possible physical entrapment in the lignin and plant cell wall
networks of fruit skins are largely unknown at present. Any
data on this issue would provide a significantly improved
foundation for further tailoring of enzymatic maceration
treatments for enhanced recovery of phytochemicals in fruit
juice processing.
When comparing the bulk juice yields obtained with
enzymatic maceration to that without enzyme addition,
enzyme treatments using the optimal maceration parameters
gave juice yields between 71.6% and 76.6% whereas the
juice yield without enzyme in the maceration step was
somewhat less, namely ~69% (Table 3). Thus, the differ-
ences in the final juice yield were not pronounced.
However, the pressing of the juice without enzyme added
in the maceration was much more difficult and protracted.
Furthermore, the turbidity of the non-enzyme treated juice
was so high, that it was beyond the Nephla reader’s range of
measurement at 8Brix 3 (Table 3).
Taken together, the data thus clearly demonstrated the
advantage of using enzyme preparations in the pre-press
step since both juice yields, anthocyanin levels, levels of
total phenols as well as the juice clarity were generally
improved. In addition, the pressing time was reduced.
Despite this positive influence of using enzyme preparations
their individual efficiencies did not vary significantly. Even
though some variations in the different enzyme prepara-
tions’ response to the four maceration factors were recorded,
none of the enzyme preparations consistently gave out-
standingly better responses than the others. However,
maceration time, maceration temperature, enzyme dose
and particle size could be optimized to enhance the effect
of the enzymatic maceration on the responses measured.
 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
The reason why the enzyme preparations did not differ
much in our experimental frame may have cause in the fact
that all the enzyme preparations were selected because of
their current use in the juice and wine industry. The enzymes
have thus all been developed for this industry and therefore
they work well at low pH values and their pectinolytic and
other plant polysaccharide degrading enzyme activities
work effectively to give high juice yields.
3.7. Profile of anthocyanins
The distribution of the four black currant anthocyanins in
the 10 juices made with treatment, P1, 30 min, 0.18% (E/S),
60 8C, was investigated by HPLC. No significant differ-
ences in the anthocyanin profiles were obtained with the
different enzyme preparations used for these macerations.
Hence, for all the 10 juices obtained from the optimal
enzymatic maceration with each of the 10 enzyme prepa-
rations, the percentage of the total anthocyanin content of
delphinidin-3-glucoside varied between 15% and 17%
(562F39 mg/l), between 46% and 48% (1634F80 mg/l)
for delphinidin-3-rutinoside, between 5% and 6% (177F15
mg/l) for cyanidin-3-glucoside, and between 31% and 32%
(1115F58 mg/l) for cyanidin-3-rutinoside. A very similar
anthocyanin distribution was previously obtained in black
currant berries and nectar (Iversen, 1999). The data thus
indicate that even though the anthocyanins yields were
almost doubled, the relative quantitative distribution of the
four major black currant anthocyanins did not change with
enzymatic maceration. The enzymatic maceration did thus
not selectively release (nor retain) any of the anthocyanin
glucosides in preference to the others. Provided that the
specific release of anthocyanin and phenolics correlated to
the degree of plant cell wall breakdown, as seen earlier with
black currant and wine pomace (Landbo & Meyer, 2001),
the lack of pronounced differences among the enzyme
preparations on the anthocyanins and total phenols levels
may be due to only low activities of the possible side
activities in the preparations at the very low pH of the black
currant mash.
On the other hand, the juice turbidities obtained with
the optimal treatment with the 10 different enzyme
preparations varied widely and ranged from 83 to 499
FNU (Table 5). This large span in turbidity indicated that
the 10 different enzyme preparations did in fact display
very different enzyme activities during maceration, even
though these differences did not result in substantial
differences in anthocyanins and phenols yields. As
discussed above, the differences in turbidity levels may
be both due to differences in the polysaccharide degrading
activity profile and due to differences in the temperature
and pH robustness among the enzyme activities present in
the different enzyme preparations.
The monosaccharide composition of black currant cell
wall material indicates that the cell wall polysaccharides in
black currants are structurally similar to those found in other
511
fruits such as grapes and apples (Hilz et al., 2003).
However, the press cake remaining from black currant juice
production were richer in glucose and the degree of
methylation of the present uronic acids was higher than
that in the alcohol insoluble solids fraction of untreated
black currant berries (Hilz et al., 2003).
The enzyme dosages employed here were much higher
than those currently employed in the industrial juice
processing as our aim was to evaluate the possible effects
of different, undeclared plant cell wall degrading side
activities, present in low concentrations in the different
preparations. However, a detailed elucidation of the
profiles of the side activities in each of the multi-
component pectinase preparations employed was beyond
the scope of the present work. Further studies on the
specific action of different plant cell wall degrading
enzyme activities on complex hemicellulosic and lignified
components as well as on the cutinized regions of fruit
skin cell wall at low pH are clearly warranted, however.
These type of studies are one of the subjects of our present
research within functional fruit juice processing and we
hope that the knowledge obtained can provide an improved
foundation to taylor targetted enzymatic hydrolysis in both
fruit juice and wine processing, where plant cell wall
degrading enzymes are widely used for fruit maceration
prior to juice extraction.
3.8. Antioxidant activities
The antioxidant activities of the juices resulting from
maceration with each of the 10 different enzyme prepara-
tions at the optimal maceration conditions (as described
above) were compared towards human LDL oxidation in
vitro at equimolar phenol concentrations of 5, 7.5 and 10
AM GAE (Table 5). Gallic acid was used as an antioxidant
control compound.
At 5 AM, only a modest net prolongation of induction
time for conjugated diene hydroperoxide formation was
recorded. The net prolongation varied from 0 to 11 min for
the 10 juices with the Macer 8 FJ and the Pectinex
Superpress macerated samples exhibiting the highest activ-
ities (Table 5). The control compound gallic acid inhibited
the oxidation for 203 min. When compared at 7.5 AM
addition levels, significant differences in antioxidant
potency among the 10 juice samples were found. The
recorded net delays in induction time varied from 20 to 84
min. The juice prepared with the Macer 8 FJ treatment
exerted the longest net prolongation of induction time, while
the juices made with Rapidase BE Super, Rohapect B5L,
and Klerzyme Color had the shortest. The rest, including the
Pectinex BE and Pectinex Superpress macerated juice
samples, exerted medium–high activities (Table 5). At 10
AM GAE, the juices delayed the formation of conjugated
dienes by 126–276 min. Three out of 10 juices (Macer 8 FJ,
Pectinex Superpress, and Pectinex BE treated) totally
inhibited the oxidation in the time interval measured (5 h).
512 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
The antioxidant control, gallic acid, blocked the oxidation at
both 7.5 and 10 AM.
HPLC determinations of ascorbic acid revealed that the
ascorbic acid concentrations ranged from 1120 to 1370 mg/l
averaging 1220F105 mg/l. However, the juice samples
containing the highest ascorbic acid levels were not those
exhibiting highest antioxidant activity on the LDL oxida-
tion. Even though the presence of ascorbic acid presumably
contributed to the antioxidant activity, the presence of
ascorbic acid was apparently not the factor being mainly
responsible for the antioxidant capacity of the juice
samples. Because the juices were diluted to the same
equimolar concentration, the differences in their antioxidant
potencies must be due to the differences in phenolic
composition, which thus in turn appeared to depend on
the kind of enzyme preparation used in the maceration.
Since the anthocyanin composition of the 10 juices was
investigated by HPLC and found to be practically similar in
all these 10 juice samples, it is unlikely that differences in
anthocyanin composition were responsible for the variation
in antioxidant potency. Neither did a thorough investigation
of the levels of the other phenolics in the black currant juice
samples, flavonols, hydroxycinnamates, and monomeric
flavan-3-ols by HPLC analyses (data not shown) reveal
any pronounced differences in the gross phenolic profiles
among these 10 juice samples. It is tempting to infer that
even subtle differences in phenolic compositions and
ascorbic acid levels may cause variations in synergistic
interactions among the antioxidant compounds and that this
is the reason for the differences in the recorded antioxidant
activities among the black currant juice samples. At present,
however, we find it more plausible to ascribe the differences
in the antioxidant potencies among the black currant juice
samples to differences in their levels of other compounds,
presumably high molecular weight polymeric flavanol
structures (trimer–pentamer proanthocyanidins), that
escaped our HPLC analysis. Polymeric dihydroflavonols
and polymeric proanthocyanidin structures are presumed to
be anthocyanin precursors in fruit tissues (Goodwin &
Mercer, 1990; Jaakola et al., 2002). Since black currants
contain high levels of anthocyanins, the presence of
proanthocyanidin precursors must be considered as likely.
Even though the HPLC method that we employed can
analyse dimeric flavanols, e.g. various procyanidins, larger
polyphenolic polymers escape reversed-phase liquid chro-
matography analysis (Kennedy & Waterhouse, 2000).
Provided that the high molecular weight dihydroflavonol
polymers (or proanthocyanidins) are able to exert antiox-
idant activity on human LDL in vitro, differences in
antioxidant activities of the differently pre-macerated juices
could result if the polymeric materials were extracted or
modified differently during the 10 different enzyme
macerations. Large polymeric proanthocyanidins may con-
tribute to the turbidity of black currant and other berry
juices. However, there was no correlation between anti-
oxidant activity (at 7.5 AM) and the turbidity (Table 5) of
the black currant juice samples. However, the idea that
turbidity causing proanthocyanidins may contribute to the
antioxidant activity of black currant juices cannot be
excluded, but further chemical knowledge on the compo-
sition of the turbidity causing substances are required before
any firm conclusions can be drawn. Even though our
current analytical set up for phenolics could not identify the
exact compositional differences in the black currant juices
to explain the variations in the antioxidant activities, the
finding that different enzymatic pretreatments affected the
antioxidant activity of the resulting juices agree well with
our previous data obtained on wine pomace and black
currant juice pomace extracts (Meyer, Jepsen, & Sbrensen
et al., 1998; Landbo & Meyer, 2001). Furthermore, the data
indicate that enzymatic maceration treatments in pre-press
berry juice processing may be optimized to maximise the
antioxidant potency of the phenolics extracted into the
juice. Additional research is obviously required to under-
stand how different enzymatic activities affect the release of
antioxidant phytochemicals from plant tissues and how they
act on various phenolic glucosides. Such knowledge might
provide new strategies for selective optimization of the
antioxidant potency of phenolics in processed foods and
beverages.
4. Conclusions
The juice yield, anthocyanin level, the level of total
phenols as well as the clarity of black currant juice were
improved by using pectinolytic enzyme preparations for pre-
press maceration. The 10 tested enzyme preparations turned
out to be almost equally good with respect to the main
responses measured on the juices, while variation in the
reaction variables maceration time, maceration temperature,
enzyme dosage, and extent of berry crushing lead to great
variation in the juice response parameters. Nevertheless, one
enzyme preparation, Pectinex BE, a cloned A. niger
preparation, consistently tended to be slightly better than
the others in giving high anthoycanins yields, high total
phenols yields, and low juice turbidity. It was therefore
possible on the basis of this work to recommend an optimal
enzymatic maceration treatment comprising use of 0.18% E/
S Pectinex BE, reacting for 30 min at 60 8C on a finely
crushed black currant mash. The juices were all potent
antioxidants on human LDL emphasizing their potential
health-promoting benefits in relation to atherogenesis and
risk of coronary heart disease. The demonstration in this
study that various pectinolytic preparations used in an
enzymatic pre-press treatment of black currants affected the
antioxidant potency of the resulting juices indicates that it
may be possible to tailor enzyme preparations for the
production of optimal antioxidant juice products. Clearly,
more research is warranted to understand the effects of
various enzyme activities on the antioxidant potential of
fruit phenolics.
 A.-K. Landbo, A.S. Meyer / Innovative Food Science and Emerging Technologies 5 (2004) 503–513
Acknowledgement
This research was a part of the bAnthocyanins Bio-
activities ProjectQ, QLRT-1999-00124, supported by the
European Commission under the Framework V Program.
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