Concentration_of_flavonoids_and_phenolic

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Journal of Food Engineering 96 (2010) 533–539
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Journal of Food Engineering
journal homepage: www.elsevier .com/ locate / j foodeng
Concentration of flavonoids and phenolic compounds in aqueous and ethanolic
propolis extracts through nanofiltration
Beatriz C.B.S. Mello a, José Carlos Cunha Petrus b, Miriam Dupas Hubinger a,*
a Department of Food Engineering, Faculty of Food Engineering, University of Campinas, P.O. Box 6121, Campinas, SP 13083-970, Brazil
b Department of Food Chemistry and Engineering, Federal University of Santa Catarina, P.O. Box 476, Florianópolis, SC 88040-900, Brazil
a r t i c l e i n f o
Article history:
Received 30 April 2009
Received in revised form 17 August 2009
Accepted 31 August 2009
Available online 10 September 2009
Keywords:
Propolis
Membrane concentration
Flavonoids
Phenolic compounds
Nanofiltration
0260-8774/$ - see front matter � 2009 Elsevier Ltd. A
doi:10.1016/j.jfoodeng.2009.08.040
* Corresponding author. Tel.: +55 19 3521 4036; fa
E-mail address: mhub@fea.unicamp.br (M.D. Hubi
a b s t r a c t
Propolis has a variable and complex chemical composition with high concentration of flavonoids and
phenolic compounds present in the extract. The extract varies with the solvent used in extraction. Etha-
nol extracts more phenolic acid and polar compounds than water. Before their use in industry, extracts
must be concentrated but the use of high temperatures can degrade some compounds. Membrane pro-
cesses is an option that allows concentration at low temperatures. Nanofiltration was carried out with
aqueous and ethanolic extracts and each extract results in two distinct fractions: permeate and retentate.
The capacity of the membrane to retain the compounds was verified by spectrophotometric analysis: for
aqueous solution, the membrane retained around 94% of the phenolic compounds and 99% of the flavo-
noids, while for the ethanolic solution these values were 53% and 90%, respectively. Ferulic acid retention
index was 1.00 and 0.88 to aqueous and ethanolic solutions, respectively. Thus, the nanofiltration process
showed high efficiency in the concentration of propolis extracts.
� 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Over the last few decades, interest in functional foods has been
growing fast, leading to the discovery of new functional compo-
nents or processes that can improve food processing, as well as
products that may help to retard aging or avoid diseases. In this
context, bee products have gained the attention of consumers and
researchers, due to their chemical compositions and functional
properties. Propolis is one of the bee products with functional prop-
erties, but it cannot be consumed as a food because it is a resinous
substance. It is prepared from the buds and exudates of certain
trees and plants. These substances are transformed by the addition
of wax and the enzyme glucosidase present in the bee saliva in or-
der to form propolis (Bankova et al., 2000; Park et al., 1998). The
product obtained is used by honeybees to protect the hives against
invaders and contamination, to seal holes and to maintain the tem-
perature. Some important characteristics have been reported for
this substance, such as anti-microbial and antioxidant effects, anes-
thetic properties and others. Due to these characteristics, which can
bring health benefits, propolis is considered a functional ingredient
and is used in food, beverages, cosmetics and medicine to improve
health and prevent diseases (Burdock, 1998; IFIC, 2009). There are
over 150 constituents in propolis, including polyphenols, terpe-
noids, steroids and amino acids. Flavonoids are one of the most
ll rights reserved.
x: +55 19 3521 4027.
nger).
important groups and can represent around 50% of the propolis
contents, depending on the region where it is collected, since its
characteristics is influenced by plants and weather (Krell, 1996;
Park et al., 1998). Kumazawa et al. (2004) tested the antioxidant
activity of propolis from various geographic origins and showed dif-
ferent activities for each sample. Other studies indicated that the
propolis from Europe and China contained many kinds of flavonoids
and phenolic acids, whereas the Brazilian samples had more terpe-
noids and prenylated derivatives of p-cumaric acid (Bankova et al.,
2000). Finally, each combination of compounds in the propolis of a
certain origin can represent specific characteristics in the final
product.
The most common propolis extracting process uses ethanol as
the solvent. However, this has some disadvantages such as the
strong residual flavor, adverse reactions and intolerance to alcohol
of some people (Konishi et al., 2004). Researchers and industry are
interested in producing a new type of extract with the same com-
pounds extracted by the ethanolic method, but without the disad-
vantages. Water has been tested as the solvent, but resulted in a
product containing less extracted compounds (Park et al., 1998).
Konishi et al. (2004) tested water with a combination of some
tensoactive compounds to replace part of the alcohol used in prop-
olis extraction and all the tests were efficient in extracting it, and
the product showed good anti-microbial activity.
Depending on the application, the solvent in propolis extracts
must be reduced or eliminated. The processes that are used today,
as lyophilization, vacuum distillation and evaporation, have some
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https://www.researchgate.net/publication/223329661_Antioxidant_activity_of_propolis_of_various_geographic_origins_Food_Chem?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/13633832_Review_of_the_Biological_Properties_and_Toxicity_of_Bee_Propolis_Propolis?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/41714552_Propolis_Recent_advances_in_chemical_and_plant_origin?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/296721907_Propolis_recent_advances_in_chemistry_and_plant_origin?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/296721907_Propolis_recent_advances_in_chemistry_and_plant_origin?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/296721907_Propolis_recent_advances_in_chemistry_and_plant_origin?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
534 B.C.B.S. Mello et al. / Journal of Food Engineering 96 (2010) 533–539
disadvantages like the use of high temperatures and high energy
consumption. Lyophilization requires large amounts of energy,
since the sample needs to be maintained at �20 �C for at least
24 h, and energy is also required for the sublimation of the solvent
used during preparation of the extract. Moreover, the method often
requires a previous stage of concentration, maintaining the prod-
uct at 70 �C until part of the solvent is evaporated.
Vacuum distillation requires great amounts of energy to gener-
ate the vacuum, and can lead to loss of compounds of low molec-
ular weight, which can be removed together with the solvent
evaporated in the system. Evaporation maintains the extract under
heating at 70 �C, until all the solvent is removed. This process, in
addition to thehigh demand for energy, can degrade the flavonoids
and phenolic compounds in propolis, due to the temperature used.
However, it is the process that gives greater ease of implementa-
tion in companies due to low cost on the equipment required com-
pared with the previous cases.
The use of membrane concentration processes has been grow-
ing due to certain advantages, such as: low temperatures, absence
of phase transition and low energy consumption (Matta et al.,
2004). This procedure is based on the principle of selective perme-
ation of the solute molecules through semi-permeable, polymeric
or inorganic membranes. The driving force for mass transfer across
the membrane in most membrane processes, such as a microfiltra-
tion, ultrafiltration, nanofiltration and reverse osmosis is mechan-
ical pressure (Maroulis and Saravacos, 2003). Nanofiltration is a
unit operation that permits many applications, such as solvent
recovery from filtered oil, exchange of solvents in the chemical
industry (Geens et al., 2006), concentration and purification of eth-
anolic extracts of xantophylls, which is important in both the phar-
maceutical and food industries (Tsui and Cheryan, 2007) and in
wine concentration (Banvolgyi et al., 2006), as well as in juice con-
centration (Vincze et al., 2006) in the food industry.
The objective of this study was to investigate the membrane
concentration of propolis extracts using water and ethanol as the
solvents, exclusively, verifying the quality of the concentrated
products in terms of the retention of flavonoids and phenolic com-
pounds during processing. The process was evaluated according to
the permeate flux, influence of temperature and pressure and con-
centration factor. The results obtained for each solution were com-
pared to verify the viability of developing a new propolis product,
based on water as the solvent.
2. Materials and methods
2.1. Propolis
Raw propolis was obtained from Apis mellifera beehives in the
State of São Paulo, Brazil, and was acquired in a single batch, in or-
der to minimize the variability associated with the vegetation used
for its production and the weather conditions. It was stored under
refrigeration (4 �C) until use in the preparation of extracts.
The propolis produced in this region is characterized as group
12 (Brazil has 12 different groups of propolis, with distinct charac-
teristics) and presents a great amount of soluble substances, anti-
microbial activity against Staphylococcus aureus and Streptococcus
mutans and greater anti-inflammatory activity than samples from
other parts of the country, which can be associated with the higher
concentrations of flavonoids and phenolic compounds found in this
group (Park et al., 2002).
The ethanolic propolis solution was prepared from crude prop-
olis previously comminuted in a bench blender with a 500 W mo-
tor, homogenized, weighed on a semi-analytical balance and mixed
with 80% ethanol. The mixture was kept at room temperature for
7 days and manually stirred once a day. After this period, the sam-
ple was centrifuged (Beckman – Allegra 25-R, Beckman Coulter,
German) at 8800 g for 20 min. The supernatant was filtered and
refrigerated for 3 h at 4 �C and then filtered again for wax removal.
Finally, the resulting extract was stored at room temperature in the
dark.
Preparation of the aqueous solutions followed the same proce-
dures, using deionized water. Each solution was prepared in a pro-
portion of 20% propolis and 80% solvent. Both extracts were
evaluated with respect to their flavonoid and phenolic compounds
contents, to be compared with the concentrated products.
2.2. Determination of total flavonoids
The total flavonoid content of the propolis solutions was deter-
mined by the aluminum complexation method (Marcucci et al.,
1998). In this procedure, the extracted solutions were diluted in
the proportion of 1:10 (0.5 mL) and mixed with 0.1 mL of 10% alu-
minum nitrate, 0.1 mL of 1 mol/L potassium acetate and 4.3 mL of
80% ethyl alcohol. The samples were kept at room temperature for
40 min and the absorbance read at 415 nm. Quercetin was used as
the standard to produce the calibration curve. The mean of three
readings was used and the total flavonoid content expressed in
mg of quercetin equivalents (mg/g).
2.3. Determination of the phenolic compounds
The polyphenols in the propolis solutions were determined by
the Folin–Ciocalteau colorimetric method (Kumazawa et al.,
2004). According to this procedure, the extracted solution was pre-
viously diluted in the proportion of 1:10 (0.5 mL) and then mixed
with 0.5 mL of the Folin–Ciocalteau reagent and 0.5 mL of 10%
Na2CO3. The absorbance was read at 760 nm after 1 h of incubation
at room temperature. Gallic acid was used as the standard to pro-
duce the calibration curve. The mean of three readings was used
and the total phenolic content expressed in mg of gallic acid equiv-
alents (mg/g).
2.4. HPLC determination
The compounds present in the initial extract, permeate and con-
centrated products, were determined by HPLC as described by Park
et al. (1998). Three hundred microliters of each solution were in-
jected into a liquid chromatograph (Shimadzu, Tokyo, Japan) con-
nected to a diode-array detector at 260 nm. The mobile phase
was water/acetic acid (19:1, v/v) (solvent A) and methanol (solvent
B), with a constant flow rate of 1 mL/min. The gradient started at
30% solvent B, passing to 60% at 45 min, 75% at 85 min, 90% at
95 min and back to 30% at 105 min. The column was maintained
at a constant temperature of 30 �C and the chromatograms pro-
cessed using the computer software Chromatography Workstation
(Shimatzu Corporation, Tokyo, Japan). The initial and concentrated
samples were diluted in 1.5 mL of distilled water and the permeate
sample was injected without dilution. The following authentic
standards of phenolic acids and flavonoids (Extrasynthese, Genay,
France) were examined: q-cumaric acid, ferulic acid, cinnamic
acid, gallic acid, quercetin, kaempferol, kaempferide, apigenin,
isorhamnetin, rhamnetin, sakuranetin, isosakuranetin, hesperidin,
hesperetin, pinocembrin, chrysin, acacetin, galangin, myricetin,
tectochrysin and artepillin C, as they correspond to the most usual
compounds present in propolis.
2.5. Membrane concentration
In this study, the propolis extracts were concentrated using a
tangential filtration system on a pilot scale, with a nanofiltration
membrane as seen in the schematic diagram shown in Fig. 1. The
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https://www.researchgate.net/publication/223329661_Antioxidant_activity_of_propolis_of_various_geographic_origins_Food_Chem?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
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Fig. 1. Schematic diagram of the nanofiltration unit.
0
5
10
15
20
25
30
0 10 20 30 40 50
Processing Time (min)
Pe
rm
ea
te
 F
lu
x 
(L
/h
.m
2 )
Aqueous solution
Ethanolic solution
Fig. 2. Permeate flux along the processing time (20 �C, 5 bar).
B.C.B.S. Mello et al. / Journal of Food Engineering 96 (2010) 533–539 535
experiments were performed on pilot equipment that permits the
batch circulation mode, which means that both permeate and con-
centrate could be carried back to the feed tank. The permeate was
totally removed just in a single experiment, where it was necessary
to obtain the concentrated product of the process. The nanofiltra-
tion module is equipped with a NF90 membrane (Osmonics, Min-
netonka, USA) which is composed of polyamide and polysulphone,
with 0.6 m2 of filtration area and 98% rejection of MgSO4 in a test
performed by manufacturing with a spiral module at 20 �C and
6.0 bar.
Approximately 5.0 L of each solution permeated through the
membrane over 30 min, this being the time necessary to complete
the concentration in an open system, which means that the perme-
ate was removed from the process. In the trials the permeate was
removed and the retentate re-circulated until a concentration fac-
tor of around four. The concentration factor is calculated according
to Eq. (1):
Fc ¼
Vf
Vc
� �
ð1Þ
where Vf is the total volume used in the feed, Vc is the volume col-
lected in the concentrate fraction and Fc is the concentration factor.
Other experiments were carried out at different temperatures
(20–45 �C) and pressures (2.0–5.0 bar), in order to evaluate the
influence of these parameters on the permeate flux and the con-
centrated product quality. In these experiments, both the permeate
and retentate were maintained under re-circulation in closed sys-
tems. The permeate flux was calculated according to the following
equation:
J ¼ Vp
t � Ap
� �
ð2Þ
where Vp is the permeate volume collected during the time interval
t and Ap is the membrane surface area of permeation.
The quality of the filtration process was measured according to
the quantity of flavonoids and phenolic compounds present in per-
meate, evaluated as described in Sections 2.2–2.4, and the effi-
ciency was measured according to the flux permeate rate and
retention index. This index measures the relation between the
amounts of the compound of interest in permeate and in the
concentrated solution, which demonstrates the ability of the
membrane to retain this compound under the experimental condi-
tions. The index is calculated according to Eq. (3), in which R is the
retention index, Cp is the concentration of the compound of inter-
est in the permeate, and Cr is the concentration of the same com-
pound in the retentate:
R ¼ 1� Cp
Cr
ð3Þ
It is important to know the rate of fouling that occurs in the
membrane process, and one way of measuring this is to compare
the permeate flux of the solution under study with the permeate
flux when water is used as feed solution, under different pressures.
Usually a variation in system pressure will cause a change directly
proportional to the permeate flux. The fouling influence was mea-
sured by comparison of the permeate flux of the aqueous propolis
extract with the flux of distilled water only, increasing the pressure
from 1.0 to 5.0 bar.
3. Results and discussion
The membrane process was carried out with the aqueous and
ethanolic solutions in a closed system, in which the retentate
and permeate streams being conducted back and mixed in a feed
tank isolated from the environment, to evaluate the variation in
permeate flux with time. The temperature was maintained at
20 �C and the pressure at 5.0 bar. The results are shown in Fig. 2.
10
15
20
25
30
35
40
45
Pe
rm
ea
te
 F
lu
x 
(L
/h
.m
2 )
536 B.C.B.S. Mello et al. / Journal of Food Engineering 96 (2010) 533–539
After stabilization of the process, the permeate flux began to de-
crease, after around 15 min of processing. The rate of decrease was
higher for the alcoholic extract than for the aqueous extract, evi-
dencing a greater rate of fouling with the alcohol solution. After
20 min of processing, the permeate flux tended to stabilize, that
is, concentration polarization already occurred and fouling did
not increase with time. The permeate flux in the stable region
was about 12.0 L/h m2 and 25.0 L/h m2 for alcoholic and aqueous
solutions, respectively. The difference between the permeate flux
of these solutions can be explained by their different compositions:
the alcohol extract contains more compounds of low molecular
weight, thus its concentration is more difficult to achieve, and this
reduces the flux rate. Some of these compounds form a kind of wax
which can cause more fouling in membrane.
Tsui and Cheryan (2007) used nanofiltration to purify alcoholic
corn extracts in the production of xanthophylls, and obtained a
permeate flux of around 10.0 L/h m2 when working at 27 bar and
50 �C. Hossain (2003) studied the membrane concentration of
anthocyanins from blackcurrant pomace extracts using ultrafiltra-
tion, obtaining a maximum permeate flux of 17.3 L/h m2 at 1.4 bar
and 18 �C. Using nanofiltration a permeate flux of 20 L/h m2 was
obtained at 20 bar and 50 �C in the concentration of red wine (Ban-
volgyi et al., 2006). The red wine concentration process is impor-
tant since it can be considered a similar process to the
concentration of alcoholic propolis, considering that they have
similar compounds in solution and use alcohol as the solvent.
The similarities between the processes allow a comparison be-
tween results.
Low pressures (around 6 bar) were used in the propolis concen-
tration process, when compared to other processes cited in the lit-
erature, but even so the values obtained for the permeate flux were
similar to those obtained in the other processes. Therefore this pro-
cess can be assumed to be viable, mainly because of the reduced
energy requirements necessary to generate the lower pressure.
The pressure adopted was not characteristic of nanofiltration pro-
cesses, but was sufficient to carry out this concentration procedure.
Fig. 3 shows the difference between the curve of the permeate
flux for the aqueous propolis extract and the curve of the permeate
flux for distilled water to measure the degree of fouling in the pro-
cess with the aqueous propolis solution.
The difference between the permeate fluxes of water and the
propolis solution shows the amount of fouling in the process, un-
der the same conditions of temperature and pressure. This param-
eter increased, reaching 32% at 5.0 bar. The procedure also
provided information on how the flux was affected by a pressure
variation, showing that the flux changed linearly with the pressure
in the region studied. In this pressure range of operation, the con-
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6
Pressure (bar)
Pe
rm
ea
te
 F
lu
x 
(L
/h
.m
2 )
Aqueous propolis
solution
Water
Fig. 3. Influence of pressure on the permeate fluxes of water and the aqueous
propolis solution (20 �C).
centrated products did not present significant variation among the
experiments.
By increasing the temperature from 20 to 45 �C and maintaining
the pressure at 6.0 bar it was possibleto determine the relation-
ship between the temperature and the permeability of the mem-
brane. Permeate flux increased proportionally, by around 8% per
degree of temperature, as shown in Fig. 4. This result may be
attributed to the effect of temperature on the viscosity of the solu-
tion. Also, the composition of the concentrated products obtained
at different temperatures showed no significant difference among
them, thus, a higher flux can be obtained by increasing the
temperature.
The initial solutions, permeates and concentrates obtained by
nanofiltration in open system were all subjected to a spectrophoto-
metric analysis as described in Sections 2.2 and 2.3. The results for
the aqueous solution indicated that this permeate only contained
small amounts of phenolic compounds and flavonoids, while the
permeate from the ethanolic solution showed greater amounts,
mostly of low molar weight phenolic compounds. Considering
the losses in the compounds of interest in the resulting permeate,
as compared to the initial solution, it can be seen that the aqueous
solution of propolis retained almost 99% of the flavonoids and 84%
of the phenolic compounds. However, for the ethanolic solution,
these values were 90% and 53% for the flavonoids and phenolic
compounds, respectively, as shown in Table 1. The lower retention
obtained can be explained by the occurrence of plasticization in
the case of ethanolic solution. This phenomenon can cause a mem-
brane swelling or dilation, which in turn can increase the mem-
brane diffusivity and solubility, causing loss of compounds in
process.
The results for the determination of flavonoids and phenolic
compounds carried out by spectrophotometric methods were ver-
ified by HPLC analysis, as described in Section 2.4. The substances
were identified by a comparison of their retention times and ultra-
violet spectra with those of standards in the literature. Chromato-
15 20 25 30 35 40 45 50
Temperature (°C)
Fig. 4. Influence of temperature on the permeate fluxes of aqueous propolis
solution (6.0 bar).
Table 1
Flavonoids and phenolic compounds after membrane concentration.
Solution Flavonoids
(mg/g)a
Total polyphenols
(mg/g)b
Ethanolic solution Initial 69.35 ± 0.38 98.74 ± 0.96
Concentrated 71.93 ± 0.21 105.08 ± 1.5
Permeated 6.95 ± 2.49 75.07 ± 2.1
Aqueous solution Initial 23.67 ± 2.14 36.57 ± 0.35
Concentrated 96.76 ± 2.35 104.74 ± 1.41
Permeated 0.003 ± 0.003 7.51 ± 1.61
Values are represented by mean ± S.D. at three experiments using VCF = 4,0.
a Quercetin equivalents.
b Gallic acid equivalents.
https://www.researchgate.net/publication/248515123_Membrane_processing_of_xanthophylls_in_ethanol_extracts_of_corn?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/244143834_Concentration_of_red_wine_by_nanofiltration?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/244143834_Concentration_of_red_wine_by_nanofiltration?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
https://www.researchgate.net/publication/288447093_Concentration_of_anthocyanin_pigments_in_blackcurrant_pomace_by_ultrafiltration?el=1_x_8&enrichId=rgreq-2d8f33d2-e659-433a-9e0f-6b92002ef93a&enrichSource=Y292ZXJQYWdlOzIyMjQxOTM2MjtBUzoxMDMyOTk3MjIyNTIzMDBAMTQwMTYzOTk4MjE1MA==
B.C.B.S. Mello et al. / Journal of Food Engineering 96 (2010) 533–539 537
grams were obtained from the initial aqueous extract, and from the
concentrated and permeated products, which are represented in
Fig. 5a–c, where the numbers 1–3 indicate the peaks identified in
the HPLC analysis.
Table 2 shows the results of the quantitative analysis for all
samples from the aqueous propolis solution. Comparing these data,
it can be seen that there were no losses of ferulic acid to the per-
meate and only 20% of the caffeic acid present in the initial solution
was lost to the permeate, this compound thus being the most
abundant in the concentrated extract, of the compounds identified.
All the aqueous solutions showed peaks located in the region
that represents a retention time of up to 20 min. This occurred
since the water, being a polar material, only extracts polar com-
Fig. 5. HPLC chromatograms of different propolis extracts. (a) Chromatogram of the initia
solution, (c) Chromatogram of the permeated aqueous propolis solution, (d) Chromatogr
ethanolic propolis solution and (f) Chromatogram of the permeated ethanolic propolis
cumaric acid (MW = 164, 16 g/mol); 3: ferulic acid (MW = 194, 18 g/mol).
pounds. The last peak identified in the permeated solution, proba-
bly does not represent an isolated compound but interference in
the system, since this compound was not present in the other
chromatograms.
Ferulic acid was not identified in the permeated solution, indi-
cating that no losses to the permeate occurred. The other peaks in
the chromatograms represented compounds that could not be
identified according to the standards in the literature. The perme-
ated solution showed a small amount of compounds in low con-
centration, allowing to verify the efficacy of the membrane
concentration process.
Park et al. (1998) analyzed an aqueous propolis solution pre-
pared in the laboratory, using HPLC, and obtained similar results
l aqueous propolis solution, (b) Chromatogram of the concentrated aqueous propolis
am of the initial ethanolic propolis solution, (e) Chromatogram of the concentrated
solution. The compounds identified are: 1: caffeic acid (MW = 180, 16 g/mol); 2:
Table 2
Some compounds detected in the HPLC analysis of the aqueous solution and their
concentrations (VCF = 4,0).
Solution Compound Peak number Concentration (lg/mL)
Initial Caffeic acid 1 1.04
Cumaric acid 2 1.50
Ferulic acid 3 0.92
Concentrated Caffeic acid 1 2.31
Cumaric acid 2 0.88
Ferulic acid 3 1.56
Permeated Caffeic acid 1 0.25
Cumaric acid 2 0.41
Table 4
Retention indexes of the compounds analyzed.
Compound Aqueous solution Ethanolic solution
Caffeic acid 0.89 0.75
Cumaric acid 0.56 0.71
Ferulic acid 1.00 0.88
538 B.C.B.S. Mello et al. / Journal of Food Engineering 96 (2010) 533–539
to those presented in Fig. 5a, reporting peaks with low retention
times that represent polar substances, and identifying the com-
pounds quercetin and pinocembrin. In their experiment, the pro-
portions of water and alcohol in the solvents for propolis
extraction were varied. Initial solutions contained 0–90% of alco-
hol, through which it was demonstrated that increasing the pro-
portion of alcohol in the solution also increased the amount of
extracted flavonoids and phenolic compounds in the propolis.
The chromatograms obtained for the ethanolic solutions are
presented in Fig. 5d–f.
It was possible to identify peaks in the ethanolic solution
throughout the process. Compounds with a retention time greater
than 20 min are apolar and were extracted by ethanol, this being
an advantage of the use of this solvent as compared to water,
which does not extract apolars. On analyzing the chromatogram
it can be seen that a considerable amount of cumaric acid was lost
to the permeate (peak number 2 in Fig. 5f). This acid belongs to the
phenolic acid class and has a low molar weight, which could ex-
plain the low retention capacity of the membrane for this com-
pound. The results of the spectrophotometric analysis shown in
Table 1 indicate a loss of phenolic compounds to the permeate,
accounting for the cumaric acid and other compounds not being
identified by the HPLC method.
Using the data given in Tables 2 and 3 the retention index could
be calculated from Eq. (2). These results can be observed in Table 4.
The retention indexes verified that the membrane process re-
tained the compounds of interest better when usingaqueous solu-
tions, since this resulted in smaller losses of the compounds
studied to the permeate, as compared to such losses when working
with the alcoholic solution. Cumaric acid was an exception, since
only 56% of this compound was not lost to permeate. Despite the
loss of compounds, the values obtained represent a high retention
index and verify that the nanofiltration process is appropriate for
propolis extracts.
During their study on the concentration of red wine by nanofil-
tration, Banvolgyi et al. (2006) obtained a retention index of 88%
for total acids, 50% for free sulfuric acids and 93% for the total ex-
tracts. Tsui and Cheryan (2007), working with the purification of
Table 3
Some compounds detected in the HPLC analysis of the ethanolic solution and their
concentrations (VCF = 4,0).
Solution Compound Peak number Concentration (lg/mL)
Initial Caffeic acid 1 1.10
Cumaric acid 2 2.89
Ferulic acid 3 3.04
Concentrated Caffeic acid 1 1.65
Cumaric acid 2 4.59
Ferulic acid 3 6.01
Permeated Caffeic acid 1 0.41
Cumaric acid 2 1.34
Ferulic acid 3 0.70
xantophylls by nanofiltration obtained a retention index of 90%
for total solids, 88% for proteins and 98% for xanthophylls, the ma-
jor compound of their study.
In the present study, the values obtained for the retention in-
dexes were very similar to those cited in the literature for similar
processes.
4. Conclusions
The results showed that nanofiltration can be considered as a
good alternative for concentrating propolis extracts, since the
membrane retained most of the flavonoids and phenolic com-
pounds, which are of major importance to propolis quality. Partic-
ularly in the case of the aqueous extract, it could be considered that
there was no loss of compounds to the permeate solution, since al-
most 100% of the major compounds were retained. In the experi-
ments with alcoholic propolis, the losses were considerable but
this is a consequence of the higher amount of compounds ex-
tracted by alcohol. However, the method can be used for alcoholic
solution since almost 90% of the flavonoids were retained. Applica-
tion of this technology could increase the use of propolis in many
industrial applications, it being feasible to use the aqueous extract
in new research projects and in the development of new products
with functional properties. Furthermore, this process allows re-
moval of the solvent from the extract, reducing the disadvantages
associated with using alcoholic extractions. It should be noted that
compared with other concentration methods, in the membrane
process the product is not submitted to high temperatures and
there is no change in the physical state of the solvent, which means
that the functional properties of the compounds of interest are pre-
served and the process as a whole is energy saving.
Acknowledgments
The authors wish to thank CNPq and CAPES/PROCAD for their
financial support of this research.
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