Improving the Antioxidant Properties of Wild Raspberry

Juice by Enzymatic Treatments

SIMONA GAVRILAS1*, MICHAELA DINA STANESCU2
1
Aurel Vlaicu University from Arad, Faculty of Food Engineering, Tourism and Environmental Protection, 2-4 Elena Dragoi Str.,
310330, Arad, Romania
2
Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., Bucharest, 011061,
Romania

The research focused on finding the optimal parameters (time and enzyme quantity) to obtain higher
polyhydroxyphenol content in wild raspberry (Rubus Idaeus) juice and also to improve the juice yield. Due
to the absence of commercial enzyme products specially designed for berry juice production, enzymatic
products prepared for red and fragrant wines industry have been used. Thus, two pectinolytic commercial
products (Enovin Color, Enozym Vintage) were considered for improving the hydrolytic process of the fruit
cell walls and another product (Enozym Altair) for juice clarification. The results obtained showed an
improvement of the juice yield and an enhancement of total polyhydroxyphenol content. After the pectinolytic
treatment a two fold increase of polyhydroxyphenols may be observed compared to that in the untreated
juice. The effects of enzymatic treatments depend on the class of polyhydroxyphenols (total phenols,
flavonoids, anthocyanins) as well as on the quantity and type of the added commercial enzymes.
Key words: wild raspberry juice, commercial pectinolytic products, polyhydroxyphenols

Raspberry fruits have a high content of fibers, lignin and

a high quantity of pectins. Thus, enzymatic treatment
seems appropriate for an optimal juice production through
pectin degradation.
The raspberry ripening occurs with changes of the cell
wall. Polyglucanases, pectinmetilesterases, and galactosidase, present in the native fruits, [1] increase
during ripening stage. The study of the enzymatic processes
which take place during the soft fruits like raspberries
ripening shows on the first stage the increase of water
soluble fraction of pectins without a depolymerization
process, this last process occurring at the full ripening stage
[2].
A method for polyhydroxyphenol enrichment of juice
may be an enzymatic treatment in order to destroy the cell
walls in which these compounds are embedded. A high
yield extraction of the active principles from the cell walls,
especially of the polyhydroxyphenols, may be achieved by
using the synergetic effect of hydrolytic enzymes like:
cellulases, pectinases and hemicelullases [3].
Pectins have as a primary component D-galacturonic
acid. They are grouped into three main structures:
homogalacturonan (a linear polymer of D-galacturonic
acid, which in the case of raspberries is partially acetylated
[4]), rhamnogalacturonan I (a consecutive structure of
galacturonic acid and rhamnose disaccharide; the later
may be partially substituted by galactose, arabinose and
xylose) and rhamnogalacturonan II (a very complex
carbohydrate structure) [5].
The general trend of pectic substances is to form gels.
The gel structure appears as a three-dimensional network
in which are incorporated water and other various
compounds. An important factor for gel formation is the
interaction occurring between calcium ions and the
carboxyl groups of pectin [5]. This gel formation property
is very useful for production of jams, marmalades, jellies,
but represents a problem in the preparation of a clear juice.

The enzyme treatment destroys such networks.

Enzymes acting on the pectic substrates are hydrolases
and lyases which depolymerize the substrate. Acid
pectinases are generally used in the processes of hydrolysis
and clarification of fruit juices, nectars and pures [6, 7].
The advantages of using commercial pectinase
preparations consist in the shortening of the manufacturing
process with economic outcomes. The commercial
preparations contain all classes of pectinases, in various
proportions.
Modern medicine asserts that human aging is due to
imbalances that occur in the body, mostly due to the
formation of free radicals. Protection against chronic
diseases linked to aging processes involves the use of
products with antioxidant activity, compounds
sequestrating free radicals, such as polyhydroxyphenols
[8]. It is known that this type of compounds could influence
the antioxidant activity of the foods [9, 10]. Thus, the
improvement of polyhydroxyphenol content of food is of
great interest.
Experimental part
Materials and methods
Pectinolytic products
The pectinases were commercial products purchased
from Agrovin Products, Bucharest, Romania. The enzymes
have been obtained from Aspergillus niger fungus, GMO
free, grown in natural media. The enzymes were extracted
with water, purified concentrated and standardized [11].
For pectins degradation two comercial products have been
used. Both had in composition also a -glucanase. This
enzyme hydrolyzes -glucans formed in case of processing
fruits affected by Botrytis cinerea ; in such cases the
filtration process efficiency [12] and the organoleptic
proprieties of the juice being affected [13].
The commercial products used are the following:
Enovin Color (enzymatic product I) is a solid product,
appearing as yellowish white granules, free of mycotoxins

* email: [email protected]; Tel.: 0745255342

REV.CHIM.(Bucharest) 67 No.2 2016

http://www.revistadechimie.ro

219

[14]. It contains a mixture of pectinases having a complex

activity: polygalacturonase (PG) - 99.3 Ug -1 (polygalacturonic acid as substrate), pectin lyase (PL) - 4.0 Ug1
(polygalacturonic acid partially esterified as substrate)
and pectin esterase (PE) 3.3 Ug-1 (PE) (esterified pectin as
substrate). The -glucanase (-Gln) activity measured
using barley -glucan as substrate is 56.5 Ug-1. For all the
activity measurements the pH was 3.5 and the temperature
30oC [14].
Enozym Vintage (enzymatic product II) is the second
product used to break the pectic cell wall of the raspberry
fruit. Beside the pectinolytic activity this product presents
other hydrolytic activities which allow an easy release of
the tannins from the cell wall [15]. The enzymatic activity
was measured in the same conditions and using the same
substrates as previously mentioned. For this product the
activity values are: 546.6 Ug-1 (PG), 2.8 Ug-1 (PL), 7.3 Ug1
(PE) and 179.6 Ug-1 (-Gln) [15].
Enozym Altair (enzymatic product III) is used for
clarification. It is a granular creamy complex of enzymes
which may be used at low temperatures [11]. As a result
of enzymatic hydrolysis process the sedimentation takes
place with high speed, the volume of sediment obtained is
reduced, increasing the yields of further separations (by
centrifugation, filtration, etc.). It has in composition PL, PG
and a low quantity of pectin esterase and does not present
cinnamyl esterase activity. The conditions for the enzymatic
activity determinations are the same as those presented
above. The PG activity measured for the polygalacturonic
acid was 904.2 2 Ug-1. For the PL and PE a highly esterified
pectin was used as substrate, the activity values being 61.3
Ug-1 (PL) and 15I Ug-1 (PE) [11].
The raspberry juice
The wild raspberries ( Rubus Idaeus ) have been
collected from a mountain area. After the harvesting the
raspberries were frozen at -18oC. Six months later, it was
thawed and the raspberry nectar was obtained by passing
the raw material through a juicer. The nectar obtained had
a pH value of 3.2. The product obtained had a very high
content of pulp (fig. 1).
Fig. 1. Raspberry
product after the
mechanical process of
preparation

Enzymatic treatments
The resulted nectar, diluted in a ratio of 1:1, was treated
with solutions of the enzymatic products (I or II) in distilled
water, in concentrations ranging between 10-40 mg L-1
After the addition of I or II the samples were kept for 2h at
50 o C, since the optimal temperature suggested for
pectinases is 45-65oC [16]. At half of each period of heating
time, the clarification product III was added (1mL solution
of 10 mg L-1 concentration).
The samples were kept overnight at 4oC and, the next
day, after gravitational sedimentation, were centrifuged for
5 min at 3,600 rpm using a Hercule Z 200A device. The
liquid phase was separated and quantified.
Raspberry juice yield
The juice yield was determined by the measuring the
final volume of the clear juice acquired after the enzymatic
treatment.
220

Total polyphenol content (TPPC)

The total polyphenol content (TPPC) was determined
using the Folin-Ciocalteu method presented recently by
Khatiwora and al. [17]. An aliquot of 3 mL sample
(raspberry juice), 2 mL Na2CO3 20% and 0.5 mL FolinCiocalteu reagent was kept for 1 min. in boiling water. The
absorbance at 650 nm was measured. The TPPC was
expressed as catechol equivalent (CE, gL-1) based on a
calibration curve previously performed. All the results were
average values of 3 determinations.
Flavonoid content (FC)
For the determination of the flavonoids the aluminium
cloride method [18] was used. In a 10 mL volumetric flask,
1 mL juice sample, 0.3 mL NaNO2 5%, 4 mL distilled water,
were placed. After 5 min. 0.3 mL AlCl3 10% and in another
6 min. 2 mL NaOH 1 M were added and the flask filled up
with distilled water. The absorbance at 510 nm was
evaluated. The flavonoid content was expressed in
quercitine equivalents (QE, gL-1), based on a calibration
curve previously drawn. These determinations were also
made in triplicate.
Anthocyanin content (AC)
The anthocyanin content was determined by measuring
the absorbance (Abs) of prepared mixtures at 510 nm. A
10 mL flask was filled up with 1 mL juice sample and 9 mL
of a mixture of an extractive solution [2 mL HCl (=1.19
gmL-1) and 98 mL ethyl alcohol]. The samples were kept
24 h at 4C for extraction, centrifuged and the absorbance
of the solution was determined. The anthocyanin content
(AC, mg mL-1) was established using the relationship (1)
experimentally determined [19]:
AC=0.015Abs

(1)

where: AC=anthocyanin content (mg mL-1)

Abs=absorbance of the mixture containing the
sample;
All the spectrophotometric determinations were made
using a UV-Vis Split Beam Analytik Jena Specord R 200.
Results and discussions
The consumption of different type of berries is a subject
of interest due to their health benefits [20]. Among the
compounds involved into health improving polyhydroxyphenols play an important role [21]. By enzymatic
treatment, the quantity of these health benefic components
may be increased.
After the addition of the pectinolytic products, which
influence the fragmentation of the fruit cell walls and help
the release of the flush polyphenols, a process of separation
of raspberry pulp from the juice may also be observed.
Figure 2-4 presents the transformations of the juice when
the enzymatic product II was added, coupled with
clarification product III.
The samples from figure 2 have been worked up as
follows: samples 1 and 2 have no enzyme, 1 being kept at
21 and 2 at 50oC. A beginning of separation may be noticed

Fig. 2. Raspberry juic e untreated (1), heated at 50oC (2) and

treated with II (3-6) before applying the clarification product

http://www.revistadechimie.ro

REV.CHIM.(Bucharest) 67 No.2 2016

Fig. 3. Raspberry juic e

at 1 h after the
application of product
III in sample 3-6

in sample 2. For the samples 3-6 the quantity of added

enzymatic product II ranges between 10-40 mg L-1.
The product III is added in samples 3-6, at half of the
treatment time (1 h). After another hour, the increase in
juice clarity may be observed (fig. 3). On the contrary, it
may be noticed that the untreated samples do not change.
The samples were kept at room temperature for 24 h
since the beginning of the treatment and the
transformations may be seen in figure 4.
The untreated samples (1 and 2) show no or little
separation of the pulp from the solution. For the samples
treated with pectinolytic enzymes an increase in the degree
of separation accompanied by darkening of the color is
observed.
The yield in raspberry juice depends on the enzymatic
product used as well as the quantity added to the sample
of raspberry juice. In table 1 the juice volume enhancement
compared with the untreated sample is presented. Higher
yields in juice are obtained using the enzymatic product II
(Enozym Vintage). The best result is achieved using only
10 mg L-1 of that enzymatic product. In case of the product
I (Enovin Color) a good result is obtained at addition of 30
mg L-1 of the enzymatic product I, but the juice quantity
obtained is lower than in product II case. These results are
in agreement with the known enzymatic activity of the
two products, product II has a better PG and PL activities
compared with product I.

Fig. 1. Raspberry juice

treated with II and III
(3-6) after 24 h from the
enzymatic treatment;
untreated (1, 2)

The enzymatic treatment with pectinases improves the

juice yield, similar results being presented for other type of
berries treated with pectinases [22].
Besides the juice yield the polyhydroxyphenol content
was monitored as described in the experimental part. The
results are presented in the tables 2-4.
As expected, an increase in the amount of polyhydroxyphenols expressed as catechol is observed when the
amount of added enzyme increase. In terms of total
polyhydroxyphenolic content, it is found that a higher
efficiency offers product I compared with the enzymatic
product II [23].
Regarding the flavonoids content, presented in table 3,
both products enrich raspberry juice in this type of
polyhydroxyphenols with a beneficial effect for consumer
health [24, 25]. Products I and II have similar effect on the
flavonoid content [23].
After the enzymatic treatment a small increase in
antocyanins was observed [23] (table 4). Based on these
experimental results, it may be assumed that the change
in color (the juice being deep red after the treatments) is
due rather to a red elagotanin presence [26]. Besides,
according literature data, half of the antioxidant activity
manifested by the bioactive compounds presented in
raspberries is considered to be due to the elagotanins [27,
28]. A tanin which has a significant contribution to the
antioxidant activity manifested by the raspberries is
elagotanin sanguin H-6 [29] which has a deep red color.

Table 1
RASPBERRY JUICE VOLUME ENHANCEMENT
AFTER ENZYMATIC TREATMENT

Table 2
TOTAL POLYHYDROXYPHENOL CONTENT
OF RASPBERRY JUICE

Table 3
FLAVONOID CONTENT OF RASPBERRY JUICE

Table 4
ANTHOCYANIN CONTENT OF RASPBERRY JUICE

REV.CHIM.(Bucharest) 67 No.2 2016

http://www.revistadechimie.ro

221

All the measurements made on the samples kept for 4

h at 50oC did not show an improvement in juice volume or
polyhydroxyphenols quantities.
It worthwhile to mention that the samples kept in a
refrigerator, after 21 days from the start of the experiment,
behaved differently. The untreated samples were found
gelled due to the formation of the pectin gel, while such
transformation did not happen in the treated samples.
Conclusions
The experimental data obtained shows that the
pectinolytic treatments applied on the raspberry juice lead
the improvement of juice pulp separation. A larger quantity
of juice was obtained by adding the enzymatic product II
(10 mg L-1) accompanied by the clarification product.
Concerning the total polyhydroxyphenol content, in some
conditions, product I addition leads to a higher quantity of
such compounds. But, there are similar increases in
flavonoids and anthocyanins by using both products I and
II.
Taking in account the juice yield the use of product II is
recommended.
The color intensification of the juice by pectinolytic
treatment can be attributed to the elagotanins released
into solution by depolymerization of cell wall pectins.
The pectinases addition also stabilized the juice
preventing the gellation process.
The lack of pectinolytic treatment leads to a decrease
in quantity as well as in the nutritional quality of raspberry
juice.
References
1.LANG, C., DRNENBURG, H., Appl. Microbiol. Biotechnol., 53, 2000,
p. 366
2.VICENTE, A.R., ORTUGNO, C., POWELL, A.L.T., GREVE, L.C.,
LABAVITCH, J.M., J. Agric. Food Chem., 55, 2007, p. 4119
3.LAROZE, L., SOTO, C., ZUIGA, M.E., Electron. J. Biotechn., 13,
2010, http://dx.doi.org/10.2225/vol13-issue6-fulltext-12
4.STEWART, D., IANNETTA, P.P.M., DAVIES, H.V., Phytochem., 56, 2001,
p. 423
5.WILLATS, W.G.T., KNOX, P., MIKKELSEN, J.D., Trends Food Sci.
Technol., 17, 2006, p. 97
6.GUMMADI, S.N., MANOJ, N., KUMAR, D.S., Structural and Biochemical
Properties of Pectinases in Industrial Enzymes. Structure, Function

and Applications, POLAINA, J., MACCABE, A.P., editor., Springer

Publication, Dordrecht, Holand, 2007, pp. 99-116.
7.KASHYAP, D.R., VOHRA, P.K., CHOPRA, S., TEWARI, R., Bioresource
Technol., 77, 2001, p. 215
8.FERRARI, C.K.B., Biogerontolog., 5, 2004, p. 275
9.LUNGU, L., POPA, C.-V., SAVOIU, M., DANET, A.F., DINOIU, V., Rev.
Chim.(Bucharest), 61, nr. 10, 2010 p. 911
10.ESLAMI, B., NABAVI, S.F., NABAVI, S.M., EBRAHIMZADEH, M.A.,
Rev. Chim. (Bucharest), 62, nr. 12, 2011 p. 1216
11.Enozym Altair data sheet, Agrovin Products, EP 727, 2008, http://
www.agrovin.com/
12.KASHYAP, D.R., VOHRA, P.K., CHOPRA, S., TEWARI, R., Bioresource
Technol., 77, 2001, p. 215
13.KO, C.H., CHEN, S.S., Bioresource Technol., 99, 2008, p. 2293
14.**8 Enovin Color data sheet, Agrovin Products, EP 027, 2005, http:/
/www.agrovin.com/.
15.*** Enozym Vintage data sheet, Agrovin Products, 555, 2004, http:/
/www.agrovin.com/
16.KULKARNI, J.A., KOTHARI, M.N., VASEEM, BAIG M.M., Int. J. Adv.
Biotechnol. Res., 4, 2013, p. 324
17.KHATIWORA, E., ADSUL, V.B,, KULKARNI, M.M., DESHPANDE, N.R.,
KASHALKAR, R.V., Int. J. Chem. Tech. Res., 2, 2010, p. 1698
18.ZHISHEN, J., MENGCHENG, T., JIANMING, W., Food Chem., 64,
1999, p. 555
19.IANCULOV, I., MODORAN, D., Cercetri tiinifice, seria III: Procese
i Tehnologii Alimentare, Agroprint Publischer, Timioara, 1997.
20.BASU, A., RHONE, M., LYONS, T., Nutr. Rev., 68, 2010, p. 168
21. HAN, X., SHEN, T., LOU, H., Int. J. Mol. Sci., 8, 2007, p. 950
22.RAMADAN, M.F., MOERSEL, J.T., J. Sci. Food Agric., 87, 2007, p. 452
23.GAVRILA S,. PhD Thesis, 2014,Politehnica University of Bucharest.
24.YAO, L.H., JIANG, Y.M., SHI, J., TOMS-BARBER, A., DATTA, N.,
SINGANUSONG, R., CHEN, S.S., Plant Foods Hum. Nutr., 59, 2004, p.
113
25.MULVIHILL, E.E., HUFF, M.W., Can. J. Cardiol., 26, 2010, p. 17A
26.BUCHERT, J., KOPONEN, J.M., SUUTARINEN, M., MUSTRANTA, A.,
LILLE, M., TRRNEN, R., POUTANEN, K., J. Sci. Food Agric., 85,
2005,p. 2548
27.LEVAJ, B., DRAGOVI-UZELAC, V., DELONGA, K., KOVAEVI
GANI, K., BANOVI, M., BURSA KOVAEVI, D., Food Technol.
Biotechnol., 48, 2010, p. 538
28.WEBER, C., LIU, M., LI, X.Q., LIU, R.H., New York Fruit Quarterly,
9, 2001, p. 13
29.BEEKWILDER, J., JONKER, H., MEESTERS, P., HALL, R.D., VAN DER
MEER, I.M., RIC DE VOS, C.H., J. Agric. Food Chem., 53, 2005, p. 3313
Manuscript received: 29.01.2015

222

http://www.revistadechimie.ro

REV.CHIM.(Bucharest) 67 No.2 2016