Food Hydrocolloids 44 (2015) 208e219

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Pilot plant preparation of light-coloured protein isolates from de-oiled

sunflower (Helianthus annuus L.) press cake by mild-acidic protein
extraction and polyphenol adsorption
Claudia Pickardt a, b, *, Peter Eisner b, Dietmar R. Kammerer a, c, Reinhold Carle a, d
a
Hohenheim University, Institute of Food Science and Biotechnology, Plant Foodstuff Technology, Garbenstrasse 25, 70599 Stuttgart, Germany
b
Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany
c
WALA Heilmittel GmbH, Department of Analytical Development & Research, Section Phytochemical Research, Dorfstraße 1, 73087 Bad Boll/Eckwa €lden,
Germany
d
King Abdulaziz University, Biological Science Department, Jeddah 21589, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to produce light-coloured protein isolates from sunflower press cake on a pilot
Received 30 October 2013 plant scale. Mild-acidic extraction at pH 6 prevented discolouration due to co-extracted phenolic com-
Accepted 9 September 2014 pounds and facilitated the adsorptive removal thereof using a styrene-divinylbenzene resin, coupled
Available online 2 October 2014
with ion exchange. To enhance protein solubility under acidic conditions, NaCl was added. Two con-
centrations (2 and 1.3 mol/L) were compared, previously identified as being most suitable for protein
Keywords:
extraction and polyphenol adsorption.
Sunflower protein isolate
Protein isolates recovered by precipitation accumulated globular helianthinin, while highly soluble
Adsorption
Chlorogenic acid
albumins were additionally recovered by ultrafiltration of the supernatants. Precipitated protein yields of
Functional properties 23e26% were obtained when 2 mol/L NaCl solution was used for extraction, whilst the lower salt level
De-oiled sunflower cake only gave protein yields of 15%. Albumin concentrates only marginally added to the overall yield with up
By-product valorisation to 5%.
The physicochemical and functional properties of the precipitated proteins obtained at the different
salt levels were comparable, being slightly inferior for the products obtained on a pilot plant compared to
laboratory scale. Generally, protein isolates obtained by isoelectric precipitation were of high purity and
light in colour, with protein contents of >94% and chlorogenic acid contents of <0.2%. Despite their poor
solubility, they had fair emulsifying and excellent foaming properties.
Altogether, sunflower protein isolates produced according to the novel process are promising food
ingredients. The study demonstrated the feasibility of the process on a pilot plant scale. Moreover, the
simultaneous recovery of phenolic compounds may enhance the economic viability of the overall
process.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction high protein content, the low amounts of antinutritive compounds

By-products of plant food processing can be valuable sources of
compounds with favourable technological and/or nutritional
properties (Schieber, Stintzing, & Carle, 2001). Press cakes and
meals originating from sunflower oil extraction are promising raw-
materials for food proteins due to their widespread availability, the
* Corresponding author. Fraunhofer Institute for Process Engineering and Pack-
aging (IVV), Giggenhauser Strasse 35, 85354 Freising, Germany. Tel.: þ49 8161 491
429; fax: þ49 8161 491 444.
E-mail addresses: claudia.pickardt@ivv.fraunhofer.de, claudia.pickardt@gmx.de
(C. Pickardt).
and freedom from toxic substances (Gonza lez-Pe
rez & Vereijken,
2007; Kausar, Ali, Javed, & Javed, 2004). The use of sunflower oil
by-products for the production of food proteins would answer the
needs of the food industry which is constantly searching for alter-
native food proteins to soy. However, such by-products have until
now only been used as low-value animal feed. This has mainly been
due to the poor sensory quality in terms of the dark colour, besides
bitter taste and astringency of sunflower by-products, which can be
ascribed to the presence of phenolic compounds (Gonz alez-Pe rez &
Vereijken, 2007). In contrast, the food industry prefers bland and
neutral functional ingredients that can be used for the full range of
food applications. However, with regard to their use as food

http://dx.doi.org/10.1016/j.foodhyd.2014.09.020
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
ingredients, isolated sunflower proteins have been shown to
exhibit functional properties partly similar to soy proteins,
depending on the isolation procedures used (Brueckner & Mieth,
1984; Gonza lez-Pe
rez & Vereijken, 2007). Overall, literature data
on the functional properties of sunflower proteins are contradictory
due to the diversity of methods and pre-treatments employed, as
well as the large variety of products investigated. Special attention
has been paid to emulsifying and foaming properties indicating
their potential use as emulsifiers and foaming agents, while their
gelation properties are poor compared to other protein sources.
Sunflower proteins have been shown to form stable emulsions
(Gonza lez-Pe rez et al., 2005), while slightly higher emulsifying
capacities were reported for an albumin-enriched isolate compared
to a globulin isolate (Canella, Castriotta, Bernardi, & Boni, 1985). The
emulsifying properties of sunflower proteins have been shown to
be slightly inferior, similar and even better than comparable soy
protein products in various studies (Brueckner & Mieth, 1984;
Gonz alez-Perez & Vereijken, 2007). Shchekoldina and Aider
(2012) reported that the emulsifying capacity of a sunflower pro-
tein isolate was similar to a soy protein isolate and better than that
of wheat gluten, skim milk powder and egg powder. Gonza lez-
Perez and Vereijken (2007) concluded that “the emulsifying
properties show very interesting perspectives to enhance the usage
of sunflower proteins as they seem to be at least comparable to
those of soy proteins”. Sunflower proteins have also been shown to
form stable foams, although literature reports are also contradic-
tory and quantitative comparison is virtually impossible due to the
different methods and products. High foaming activities in the
range of egg white have been reported (Kabirullah & Wills, 1982)
recommending sunflower proteins as whipping agents. Raymond,
Rakariyatham, and Azanza (1985) reported the foaming proper-
ties of a sunflower protein preparation to be superior to that of soy
proteins. Lower foaming activities but higher foam stabilities of
sunflower proteins compared to soy protein and overall better
foaming properties compared to skim milk powder and egg powder
were reported by Shchekoldina and Aider (2012). Lower foaming
activities but higher foam stabilities of helianthinin compared to
sunflower albumins were found by Gonza lez-Perez et al. (2005a).
Protein solubility has been investigated extensively since it is
considered a prerequisite for other functional properties (Saeed &
Cheryan, 1988; Gonza lez-Perez & Vereijken, 2007). Thus, correla-
tions between solubility and emulsifying activity have been re-
ported (Karayannidou et al., 2007; Minones Conde, Yust Escobar,
Pedroche Jimenez, Millan Rodriguez, & Rodriguez Patino, 2005)
and increased foaming activities in the presence of salt and after
hydrolysis were reported (Kabirullah & Wills, 1981; Karayannidou
et al., 2007) due to enhanced solubility. While sunflower albu-
mins are known to be readily soluble, most globulin isolates have
lower solubilities which are highly pH dependent (Canella et al.,
1985; Gonza lez-Pe
rez, Vereijken, Vereijken, van Koningsveld,
Gruppen, & Voragen, , 2005b; Kabirullah & Wills, 1982; Minones
Conde et al., 2005). However, the values vary significantly and
this has been ascribed to differences in the composition and pro-
cessing (Brueckner & Mieth, 1984; Gonza lez-Pe
rez et al., 2002;
Saeed & Cheryan, 1988) and also different conditions for the solu-
bility measurements themselves (Brueckner & Mieth, 1984;
Gonz alez-Perez et al., 2002). In particular, thermal or chemical
treatment during processing can lead to extensive denaturation
(Davin, Berot, & Petit, 1983; Kausar, Ali, Javed, & Khan, 2002).
Furthermore, the binding of phenolic compounds is believed to be
responsible for reduced solubility of sunflower proteins (Gonza lez-
Perez & Vereijken, 2007).
Sunflower seeds contain 1e4% phenolic compounds (Gonza lez-
Perez et al., 2002; Kausar et al., 2004; Weisz, Kammerer, & Carle,
2009), predominantly chlorogenic acids, which are likely to
209
impair the colour and functional properties of sunflower protein
preparations. Conventional protein extraction under alkaline con-
ditions, which is most suitable due to the solubility profile of
sunflower proteins, results in rapid oxidation of phenolic com-
pounds and their concomitant covalent bonding to protein side
chains (Cater, Gheyasuddin, & Mattil, 1972; Gonza lez-Perez &
Vereijken, 2007; Pawar, Patil, Sakhale, & Agarkar, 2001). For this
reason, over the past four decades, processes have been investi-
gated to isolate sunflower proteins devoid of phenolic acids.
Common strategies include the extraction of sunflower meal with
mixtures of organic solvents and/or water to lower the polyphenol
content prior to alkaline protein extraction, the exclusion of oxygen
using vacuum or inert gases, and the use of antioxidants during
alkaline protein extraction from polyphenol-rich material and the
subsequent removal of co-extracted phenolic acids from the pro-
tein extracts. These processes have been reviewed by Gonza lez-
Perez et al. (Gonza lez-Pe
rez et al., 2002; Gonza lez-Pe
rez &
Vereijken, 2007). However, none of these processes has to date
been transferred to industrial practice, most probably due to the
tedious and inefficient processing. The use of organic solvents may
impair the subsequent protein isolation by diminishing protein
solubility due to denaturation or residual solvent and, in addition,
requires special safety precautions. In contrast, pre-extraction of
phenolics with water appeared to be more appropriate for the
subsequent protein extraction. However, this measure caused high
loss of water-soluble albumins, and proved to be inefficient when
followed by alkaline extraction (Salgado, Drago, et al., 2012).
Moreover, only few studies have been performed with regard to
large-scale application of processes. This might be another reason
why the implementation of such processes has not been pursued.
Salgado and co-workers demonstrated the production of sunflower
protein concentrates with high solubility on a pilot plant scale,
using sunflower oil cake as the starting material (Salgado, Drago,
et al., 2012) and compared sunflower protein concentrates with
different contents of phenolic compounds (Salgado, Ortiz,
Petruccelli, & Mauri (2012) revealing different effects on func-
tional properties). However, this study was not focused on col-
ourless products. Moreover, only a low meal-to-solvent ratio of
1:15 was tested in the previous studies, whereas higher ratios
might be favourable with regard to industrial processing (Pickardt,
Hager, Eisner, Carle, & Kammerer, 2011). Davin et al., 1983 tested
various patented processes for the preparation of sunflower protein
isolates on a pilot plant scale and concluded that various qualities
could be produced with higher purities being associated with lower
yields. However, functional characterisation of the products has not
been performed. Taha, El-Nockrashy, & Shoeb (1981) investigated
the use of counter-current extraction and isoelectric precipitation
of sunflower proteins for the preparation of sunflower protein
isolates in high yield and purity in order to demonstrate suitable
conditions for industrial operation. However, this study was per-
formed on a laboratory scale, and gently pre-processed sunflower
meal was used.
An alternative process for the production of light-coloured
sunflower protein isolates has recently been proposed using
mild-acidic protein extraction and high salt concentrations to
enhance the protein solubility to exploitable levels (Pickardt et al.,
2009). This process was shown to hinder spontaneous oxidation of
co-extracted phenolic compounds, thus facilitating their subse-
quent removal and recovery by adsorption processes (Weisz,
Schneider, Schweiggert, Kammerer, & Carle, 2010). The simulta-
neous recovery of polyphenols for other uses may improve the
economic viability of the overall process (Weisz, Carle, &
Kammerer, 2013). Adsorption conditions have been optimised on
a laboratory scale to minimise the discolouration and the content of
chlorogenic acids in protein extracts. The combination of both
210 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219

processes has been successfully performed on a laboratory and

pilot plant scale (Pickardt et al., 2011; Weisz et al., 2010). However,
hitherto only optimal conditions for polyphenol adsorption have
been evaluated on a pilot plant scale (Weisz et al., 2009; Weisz
et al., 2010), while optimisation of the protein recovery has not
been considered thus far. In a previous laboratory study, protein
yields have been shown to increase at higher protein concentra-
tions and elevated salt levels (Pickardt et al., 2011, 2009).
The aims of the present study were to produce light-coloured
protein isolates from sunflower press cake by combining mild-
acidic protein extraction at elevated NaCl concentrations and
adsorptive removal of phenolic compounds using a styrene-
divinylbenzene copolymer resin. The feasibility of the overall pro-
cess should be demonstrated on a pilot plant scale. Furthermore,
two salt levels previously found to be optimal for the individual
process steps of protein extraction and polyphenol adsorption
should be compared in order to test their suitability for the com-
bined process providing evidence of potential process optimisation.
Therefore, the processes and products should be compared on a
laboratory and pilot plant scale in order to identify possible effects
of the different processing conditions. Relevant functional proper-
ties of the derived protein isolates were determined in order to
indicate their suitability as food ingredients. As the isoelectric
precipitate has been shown to solely consist of the globulin fraction
(Pickardt et al., 2011), the recovery of the water-soluble albumins
from the supernatants was included in the pilot plant experiments
in order to demonstrate ways allowing total protein recovery.
Fig. 1. Preparation of sunflower protein isolates (IP) and concentrates (UF) on a lab-
2. Materials and methods oratory (L) and pilot plant (P) scale. Individual parameters are listed in Table 1.

2.1. Materials

Cold-pressed sunflower cakes from industrial sunflower oil
€
processing were provided by Teutoburger Olmühle (Ibbenbüren,
Germany). These cakes were de-oiled with iso-hexane in a pilot
plant percolator (volume 1500 L, e&e Verfahrenstechnik, Ware-
ndorf, Germany) and flash-desolventised with hexane vapour at
400e500 mbar, prior to steam desolventisation at a maximum
product temperature of 60  C. Prior to protein extraction on a
laboratory scale, the de-oiled cake was milled in a pin mill (Alpine,
Augsburg, Germany) and passed through a sieve of 0.5 mm mesh
size. The de-oiled cake contained 38.1 ± 2.7 g protein per 100 g
(N  5.6), 7.1 ± 0.8 g ash per 100 g, 2.1 ± 0.5 g fat per 100 g,
2.1 ± 0.7 g chlorogenic acid (CGA) per 100 g, and 51 ± 2 g dietary
fibres and sugars per 100 g, relative to dry matter (dm, 90.3 ± 0.7 g
per 100 g). The methods used are listed below (Section 2.3.1).
Food grade NaCl was purchased from a local supermarket.
Methanol (Chromanorm for HPLC) was from VWR International
(Darmstadt, Germany), chlorogenic acid (98%) was obtained from
Sigma-Aldrich (Steinheim, Germany), ortho-phosphoric acid (85%)
from Merck (Darmstadt, Germany), and HPLC gradient grade water
from J.T. Baker (Deventer, Netherlands). All other reagents used
were of analytical grade. Reference products Supro 500E and Supro
EX33 were obtained from Protein Technologies International (now:
Danisco Dupont), and ProFam 464 from ADM Specialty Ingredients,
Koog aan de Zaan, The Netherlands.
2.2. Isolation of sunflower proteins by mild-acidic protein
extraction and polyphenol adsorption followed by isoelectric
precipitation or ultrafiltration
Protein isolation was performed according to the protocol
depicted in Fig. 1 with the parameters as detailed in Table 1. For
protein extraction, the de-oiled cake was immersed in salt solutions
having NaCl concentrations (cNaCl) of 1.3 and 2.0/2.1 mol/L,
respectively, as indicated in Table 1, and at a solid-to-liquid ratio of
1: 10 (1 kg de-oiled cake þ 9 kg NaCl solution). The exceptions were
IP-P1 and UF-P1, where a ratio of 1:15 was applied. These condi-
tions were chosen based on preliminary studies on protein
extraction (Pickardt et al., 2011) and polyphenol adsorption (Weisz
et al., 2010). Laboratory extraction was performed using a volume of
10e12 L, while pilot plant extraction was scaled up to 1500 (P1) and
1800 L (P2) using the equipment suggested by D'Agostina et al.
(2006). After extraction for 60 min at room temperature
(20 ± 3  C) and constant pH of 6.0 by adjusting with HCl or NaOH
(1 M on a laboratory scale and 3 M on a pilot plant scale), as
necessary, under gentle agitation (150 rpm on a laboratory scale
and 50 rpm on a pilot plant scale, the crude protein extracts were
recovered by centrifugation (a laboratory beaker centrifuge at
3300 g and a continuously running decanter (~1500 L/h) at approx.
3000 g on a pilot plant scale)). Phenolic compounds were removed
from the crude extracts by adsorption onto a styrene-
divinylbenzene resin Amberlite XAD 16HP (Rohm & Haas,
Chauny, France) as specified in Table 1. For the preparation of iso-
lates IP-L1 and IP-L2, 400 and 500 g of preconditioned adsorbent
resin, respectively, were added to 7 and 9 L of the crude protein
extract and gently agitated (~150 rpm) for 4 h at 20  C under a
nitrogen blanket in an agitator vessel as previously described
(Pickardt et al., 2011). Subsequently, the resin was removed using a
Miracloth filter (rayon-polyester, Merck, Darmstadt, Germany). For
the preparation of isolates IP-P1 and IP-P2 and concentrates UF-P1
and UF-P2 on a pilot plant scale (see Table 1), a two-step continuous
procedure was applied combining ion exchange and adsorption:
The protein extract was pumped through two cylindrical steel
vessels connected in series, packed with 90 L of an anion exchange
resin (Lewatit S 6328 A; Lanxess, Leverkusen, Germany) and the
aforementioned adsorbent resin, respectively, at a flow rate of 3 bed
volumes per hour as described by Weisz et al. (2010). Resin
 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
Table 1
Sample assignment and processing parameters for protein isolation.
Sample Characteristic processing steps Scale cNaCl (mol/L)
code during protein
extraction
IP-L1 Saline extraction, polyphenol adsorption, Lab 1.3
isoelectric precipitation
IP-P1 Saline extraction, polyphenol adsorption, Pilot 1.3
isoelectric precipitation
UF-P1 Saline extraction, polyphenol adsorption, Pilot 1.3
ultrafiltration of supernatant after
precipitation
IP-L2 Saline extraction polyphenol adsorption, Lab 2.0
isoelectric precipitation
IP-P2 Saline extraction, polyphenol adsorption, Pilot 2.1
isoelectric precipitation
UF-P2 Saline extraction, polyphenol adsorption, Pilot 2.1
ultrafiltration of supernatant after
precipitation
activation was performed using ethanol (60%, v/v) followed by
NaOH (2.5 mol/L) and deionised water (50  C) as described previ-
ously (Weisz et al., 2009). Prior to use, the resin was preconditioned
with NaCl solutions of the respective concentration.
To recover protein isolates (IP), the proteins were precipitated
by adding HCl (1e3 mol/L, depending on the required amounts) to
reach the pH values listed in Table 1. The protein curd obtained was
separated by centrifugation in a beaker centrifuge on a laboratory
scale and in a continuous plate separator (~500 L/h) on a pilot plant
scale and washed twice by mixing with water at ~ 15  C at a mass
ratio of 4e5: 1 as detailed in Table 1. The washed curd was sepa-
rated as described above, neutralised to pH 6.7e7.0 by adding NaOH
solution, homogenised using a colloid mill and spray dried in a Niro
atomizer (2 L/h) on a laboratory scale and in a GEA atomizer (25 L/
h) on a pilot plant scale, both from GEA (Mühlheim, Germany). Both
dryers were equipped with a mixing nozzle of 0.7 mm inner
diameter. They were operated at an inlet temperature of
170e180  C and outlet temperature of 75 ± 3  C. On a pilot plant
scale, the feed pipe was equipped with a heat exchanger, effecting a
short-duration pasteurisation at 55 ± 2  C.
To recover soluble proteins (UF), the supernatants after precip-
itate separation were subjected to ultrafiltration using a membrane
of 10 kDa cut-off as described by D'Agostina et al. (2006) at a
transmembrane pressure of 0.2 MPa and a volume concentration
ratio of 20. Co-solutes were separated by semi-continuous diafil-
tration using a diafiltration volume of 5, and the concentrated
protein solution was pasteurised inside the ultrafiltration vessel
before spray drying as described above.
Product and protein yields were calculated based on the dry
matter and protein contents of the isolates prior to drying. This
ruled out variable and disproportionally high losses associated with
batch spray drying, since optimization of the spray drying step was
not part of our study. Nevertheless, comparison with other studies
seems appropriate as most studies use freeze drying or air drying in
cups which do not cause significant losses either. While the product
yield represents the percentage of dry matter recovered as protein
isolate relative to the initial dry matter content of the de-oiled cake,
the protein yield represents the amount of protein in the protein
isolate relative to the protein content of the de-oiled cake.
2.3. Analytical methods
2.3.1. Proximate analysis of de-oiled press cake and protein isolates
The dry matter and ash contents were determined gravimetri-
cally at 105  C and 550  C, respectively, in a thermogravimetric
211
Polyphenol removal pH of Ultra- Washing procedure
precipitation filtration
Adsorber resin 3.5  1) 1/5, pH 4.0, 10  C
2) 1/5, pH 6.0, 10  C
Ion exchange þ adsorber resin 3.2  1) 1/4, pH 3.9, 15  C
2) 1/5, pH 4.1, 15  C
Ion exchange þ adsorber resin (3.2) þ Diafiltration vs. tap water
Adsorber resin 3.5  1) 1/4, pH 4.0, 10  C
2) 1/4, pH 6.0, 10  C
Ion exchange þ adsorber resin 3.3  1) 1/4, pH 3.5, 15  C
2) 1/5, pH 4.5, 15  C
Ion exchange þ adsorber resin (3.3) þ Diafiltration vs. tap water
system (TGA 601, Leco Instrumente, Mo €nchengladbach, Germany)
using standard methods (AOAC, 2005a). The protein content was
calculated from the nitrogen content as determined by the Dumas
combustion method (AOAC, 2005b), using a Protein/Nitrogen
Analyzer FP 528 (Leco, St. Joseph, MI, USA) and applying a con-
version factor of 5.6, which has been found to be most suitable for
sunflower proteins based on their amino acid profile as determined
in our laboratory (data not shown). Similar factors of 5.7 and 5.5, for
example, were used by Canella et al. (1985) and Zilic et al. (2010)
respectively. The lipid content was quantified by gas chromatog-
raphy after extraction with butanol and saponification (DGF, 2011).
The carbohydrate content (dietary fibres and sugars) was deter-
mined by the difference of total dry matter contents and those of all
other compounds.
2.3.2. Quantification of phenolic acids by HPLC-DAD
Phenolic acids were determined by HPLC with UV detection at
330 nm based on the method of Thiyam, Sto € ckmann, Zum Felde,
and Schwarz (2006). Only chlorogenic acid (CGA; 5-caffeoylquinic
acid) was quantified as lead substance due to its predominance in
sunflower seeds (Weisz et al., 2009). Aliquots of 1 g of the dry
samples were extracted in triplicate with 8 mL of 0.29 mol/L HCl,
sonicated for 1 min and centrifuged (20  C, 10 min, 20,000  g).
Extracts were filtered through folded Whatman filters and pooled
in volumetric flasks. The total volume was made up to 25 mL.
600 mL of the extract were mixed with 1400 mL of methanol in
2.0 mL Eppendorf tubes and centrifuged for 5 min at 12,100  g to
remove precipitated proteins. The samples were appropriately
diluted with 70% methanol (v/v), and filtered using syringe filters
(0.45 mm). HPLC analyses were carried out using an HPLC pump
P680, an automated sample injector ASI-100, a column oven ICS-
3000 DC, and a UV/VIS detector UVD170U operated by Chrome-
leon software (Dionex Softron, Germering, Germany). The column
used was a Synergi Fusion RP (250  4.6 mm) equipped with a
guard column cartridge with C18 polar embedded material of 5 mm
particle size (Phenomenex, Aschaffenburg, Germany), operated at
ambient temperature. Gradient elution was performed using water/
methanol (90/10, v/v) with 0.2% ortho-phosphoric acid as eluent A
and methanol with 0.1% ortho-phosphoric acid as eluent B. The
linear gradient programme was as follows: 10% B (0 min), 20% B
(7 min), 45% B (20 min), 70% B (25 min) and 100% B (28 min). The
injection volume was 20 mL, and all determinations were performed
in duplicate. For calibration, a chlorogenic acid stock solution of
2 mg/mL in 70% methanol was diluted with the same solvent to
obtain concentrations in the range of 0.02e0.5 mg/mL.
212 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
2.4. Gel electrophoresis (SDS capillary PAGE)
The protein molecular weight distribution was determined by
sodium dodecyl sulphate capillary polyacrylamide gel electropho-
resis (SDS capillary PAGE) as previously described (Pickardt et al.,
2011), using a capillary electrophoresis system HPCE 3D (Agilent,
Waldbronn, Germany) equipped with 3D-CE ChemStation software
(Rev. A.08.01) applying the CE-SDS protein kit 148-4160 (Bio-Rad
Laboratories, Munich, Germany). The BioRad protein size standard
148-2015, containing molecular weight markers ranging from 14 to
205 kDa (lysozyme (14,400 Da), trypsin inhibitor (21,500 Da), car-
bonic anhydrase (31,000 Da), ovalbumin (45,000 Da), BSA
(66,200 Da), phosphorylase b (97,000 Da), b-galactosidase
(116,000 Da), and myosin (200,000 Da)), was used for calibration
with benzoic acid as internal migration time reference, which was
detected after ~4.5 min. Dithiothreitol was added for reducing
disulphide bridges.
2.5. Physicochemical characterisation of the protein isolates
2.5.1. Colour
The colour of the dried powdered products was determined
using the CIE-L*a*b* colour system with a CR-300 Chroma Meter
(Minolta, Osaka, Japan) as the mean values of 8 measurements.
2.5.2. Protein solubility, emulsifying and foaming properties
Protein solubility at pH 7 was determined according to the
method of Morr et al. (1985) as the proportion of dissolved protein.
Protein solutions were prepared by dissolving 1.5e2.5 g sample in
50 mL of 0.1 mol/L NaCl adjusting the pH for 60 min under constant
magnetic stirring (~200 rpm) at ambient temperature and with
successive removal of undissolved matter by centrifugation
(20,000  g, 15 min). The content of dissolved proteins was ana-
lysed by the Dumas method (see Section 2.3.1).
The emulsifying capacity of the protein isolates was determined
as described by Yoshie-Stark, Wada, and Waesche (2008) by
detecting the sudden decrease in conductivity at the point of phase
inversion of the emulsion upon constant addition of oil (corn oil,
Mazola®, Unilever Deutschland, Hamburg, Germany) to a 1%
dispersion of the protein isolates or concentrates while dispersing
with an UltraTurrax at 24,000 rpm.
The foaming properties of the protein isolates and concentrates
were determined as described by D'Agostina et al. (2006) using
200 mL of 5% suspensions of the products in deionised water at pH
6.7e7.0 after whipping in a Hobart 50 N mixer for 8 min at high
speed (level 3) at room temperature. The foaming activity repre-
sents the foam overrun and is expressed as the percentage of initial
volume (D'Agostina et al., 2006). The foam drainage stability was
determined in a 250 mL measuring cylinder as the percentage of
foam volume remaining after 60 min.
2.6. Data evaluation
All analyses were performed in duplicate, and values are given
as mean ± mean deviation unless otherwise stated. Statistical dif-
ferences between mean values were analysed by pairwise t-test
(P ¼ 0.05) after running an F-test to check for equality of variances.
The protein isolates and concentrates were produced without
repetition on a pilot plant scale. Therefore, product characterisation
was based on one production batch, the big volumes ensuring
sufficient product homogeneity. The pilot plant was used in a
standard configuration that had been proved to provide adequate
reproducibility in several previous studies (e.g. Yoshie-Stark et al.,
2008; D'Agostina et al., 2006). Due to the high cost it was not
possible to repeat pilot plant runs in the present study, and the
parameters were not optimised for the specific process. However,
the aim of the pilot plant trials was to demonstrate the feasibility of
the process on a pilot plant scale and to recover protein isolates for
characterisation of their properties. The goal was not to determine
optimum process parameters for system configuration and plant
layout, nor to carry out precise quantification for an economic
assessment. Thus, yields are given only as an indicator to allow
general comparison with the laboratory runs performed at the
same salt concentration.
3. Results and discussion
Dark discolouration due to the oxidation and protein binding of
phenolic acids is a major drawback of sunflower proteins. As light
colour is a prerequisite for versatile industrial application of protein
isolates, the removal of chlorogenic acid derivatives was an
important aspect of the present study. Sunflower protein isolates
were produced from de-oiled press cake to demonstrate the
feasibility of the novel process that combines mild-acidic protein
extraction at pH 6 with adsorptive removal of phenolic compounds
from the protein crude extracts. The process scheme is depicted in
Fig. 1. Polyphenol removal was accomplished by a single-step
adsorption onto Amberlite resin on a laboratory scale and by
resin adsorption coupled with ion exchange on a pilot plant scale.
Furthermore, two different NaCl levels (1.3 mol/L and 2 mol/L,
respectively), which were previously found to be suitable for the
individual processing steps of protein extraction and polyphenol
adsorption, were used in order to compare their suitability for the
combined process (see Table 1): While the higher level was shown
to best favour protein extraction (Pickardt et al., 2009) and isolation
(Pickardt et al., 2011), the lower level had been demonstrated to be
more favourable for the adsorption process onto Amberlite resin
(Weisz et al., 2010).
3.1. Composition and purity of the products
3.1.1. Protein and ash contents
The major protein fractions were recovered from the extracts by
isoelectric precipitation (IP) followed by centrifugation. The
resulting supernatant was subjected to ultrafiltration on the pilot
plant scale, thereby yielding an additional product (UF, see Fig. 1).
The chemical compositions of the different samples are shown in
Table 2. Major differences in the protein contents of IP and UF were
found. In general, isoelectric precipitation yielded protein isolates
of high purity with protein contents ranging from 94.3 to 98.6% of
the dry matter, with slightly higher protein contents after extrac-
tion at the higher salt level as well as higher yields on a laboratory
scale compared to the pilot plant scale. Differences between the IP
isolates were generally insignificant except between IP-P1 and IP-
L2. Despite the high salt concentrations used during extraction,
the ash contents were low and ranged between 1.3% and 2.0%.
Significant differences were found between IP-L1 and IP-P1 which
could be ascribed to more efficient washing on a pilot plant scale
and between IP-P2 and IP-P1 probably due to higher initial salt
content during extraction.
In contrast, ultrafiltration yielded protein contents ranging from
65.8 to 68.7% (Table 2). Although the respective products are
considered to be protein isolates according to the underlying
isolation process, they did not fulfil the requirements of protein
isolates, because of their protein contents of <90%. Consequently,
they should be named protein concentrates. The divergent purities
were also reflected in their increased ash contents ranging from
3.3% to 21.6%, which was extremely high.
The protein contents of the precipitated proteins were in the
same range as reported for other sunflower protein and
 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
Table 2
Purity and colour of sunflower protein isolates (IP) and concentrates (UF) prepared on a laboratory (L) and pilot plant (P) scale.
Sample codea Dry matter (dm) % Protein (N  5.6) % dm
b
IP-L1 95.0 ± 1.0d 97.1 ± 0.5d,e
IP-P1b 94.7 ± 0.9d 94.3 ± 0.5d
UF-P1b 92.3 ± 0.9d 68.7 ± 0.3f
IP-L2c 92.7 ± 0.9d 98.6 ± 0.5e,g
IP-P2b 94.4 ± 0.9d 96.3 ± 0.5d,g
UF-P2b 91.0 ± 0.9d 65.8 ± 0.3h
CGA e chlorogenic acid, taken as lead substance for phenolic compounds.
deh: Values with the same online letters within one column are not significantly different (p ¼ 0.05).
a
For details refer to Table 1 and Fig. 1. dm e dry matter, n.d. not determined.
b
Mean of at least two determinations of one isolate ± mean deviation.
c
Average of four values (in each case two determinations on two different isolates) ± standard deviation, data from Pickardt et al. (2011).
helianthinin isolates (Canella et al., 1985; Gonza lez-Pe
rez et al.,
2002; Kabirullah & Wills, 1981). Similar protein contents were
also found in low-polyphenol protein isolates by Kabirullah and
Wills (1982), Pawar et al. (2001), Saeed and Cheryan (1988) and
Shchekoldina and Aider (2012). However, in their studies the ni-
trogen conversion factors used were higher than in our work. The
ash contents of the sunflower proteins were in the same range or
even lower than in the aforementioned studies.
The lower purities of the sunflower protein concentrates ob-
tained by ultrafiltration may be due to insufficient dilution of the
initial salt concentration during diafiltration, which is reflected in
the increased mineral contents of UF-P2 recovered from the higher
salt level compared to UF-P1 obtained from the lower salt level
extracts. These findings were accompanied by slight sweet and
bitter off-tastes for these products in sensory evaluation (data not
shown), indicating incomplete removal of co-extracted solutes. The
lower purities of protein isolates recovered by ultrafiltration
compared to precipitated proteins were also found by D'Agostina
et al. (2006), whereas Yoshie-Stark et al. (2008) and Xu and
Diosady (2002) reported opposite findings. Phytic acid interfer-
ence with proteins in the supernatant may be another reason for
the increased ash contents, since this compound was shown to be
extracted from sunflower seeds without being completely precip-
itated (Pickardt et al., 2011). The two salt concentrations in both
supernatants might have favoured phytate complexation during
ultrafiltration to different extents due to altered charge effects
(Saeed & Cheryan, 1988). In fact, ultrafiltration is a complex process
requiring further investigation in order to improve the yield and
purity of soluble proteins recovered from the supernatants.
3.1.2. Residual phenolic acid contents and colour of the protein
isolates
Chlorogenic acid (CGA) was determined as the lead substance
representing the majority of phenolic compounds in sunflower
seeds (Weisz et al., 2009). After the two-step adsorption, the re-
sidual CGA content was below 0.02 g/100 g dry matter (dm) for the
protein isolates obtained in the pilot plant experiment (Table 2).
This corresponds to CGA removal of 99% based on an initial CGA
content of 2.1 g/100 g of the raw material. In contrast, the residual
CGA content after single-step adsorption on a laboratory scale was
0.15% in IP-L2 (Pickardt et al., 2011), corresponding to a reduction of
~90%.
The CGA content was lower in the products obtained on a pilot
plant scale than on the laboratory scale. This was ascribed to
enhanced removal of monomeric phenolic acids by the ion ex-
change resin, whereas the adsorbent resin only lowered the con-
tents of polymeric phenolic compounds (Weisz et al., 2010). The
residual CGA content of 0.02 g/100 g in the protein isolates obtained
213
Ash % dm CGA % dm Colour parameters
L* a* b*
2.0 ± 0.1d n.d. 84.7 ± 0.8d 1.6 ± 0.0d,h 12.5 ± 0.4d,g
1.3 ± 0.0e 0.02 ± 0.01d,e 82.5 ± 0.8d,e 1.7 ± 0.1d,e 11.7 ± 0.4d,e
3.3 ± 0.1f n.d. 78.7 ± 0.8e,f 2.0 ± 0.1e,g 9.7 ± 0.3f
1.3 ± 0.2d,e,g 0.15 ± 0.03d 86.8 ± 0.9d 0.9 ± 0.0f 10.2 ± 0.3e,f
1.6 ± 0.0g <0.01e 75.1 ± 0.8f 2.4 ± 0.1g 14.2 ± 0.4g
21.6 ± 0.6h <0.01e 61.6 ± 0.6g 1.4 ± 0.0h 10.2 ± 0.3e,f
in the pilot plant experiments was below the threshold level of
0.05 g/100 g reported for the perception of discolouration by Saeed
and Cheryan (1988), while this limit was exceeded in IP-L2 which
contained 0.15 g CGA/100 g. However, all protein isolates recovered
by precipitation had very light colours (off-white to cream-
coloured, data not shown). These had high lightness values L* be-
tween 82.5 and 86.8 except for IP-P2 (L* ¼ 75.1), and low a* and b*
values, as specified in Table 2. a* values corresponding to a reddish
hue were very low ranging between 0.9 and 2.4 in all products, and
b* values corresponding to a faint yellow hue varied between 9.7
and 14.2. The UF concentrates had slightly darker (light grey to
beige) colours with L* values of 78.7 (UF-P1) and 61.6 (UF-P2) (as
shown in Table 2).
In general, the novel process efficiently removed CGA and pro-
duced light-coloured protein isolates. It was as effective in reducing
CGA contents and enhancing product colour as was exhaustive
polyphenol extraction with 80% methanol (Gonza lez-Perez et al.,
2002), extraction with acidic butanol prior to alkaline protein
extraction and precipitation (Pawar et al., 2001; Saeed & Cheryan,
1988), and protein precipitation with succinic acid (Shchekoldina
& Aider, 2012). Similarly, the use of absorber technology in the
processing of rape seed proteins resulted in light-coloured products
having low phenolic acid contents and better sensory acceptance
than products having higher phenolic contents (Xu & Diosady,
2002).
While the isolate IP-L2 produced at an increased salt level had a
lighter colour than IP-L1, the product IP-P2 obtained on a pilot plant
scale was significantly darker than IP-P1 (Table 2), although it had a
lower CGA content. However, both products recovered on a pilot
plant scale at the higher NaCl concentration (IP-P2 and UF-P2) were
significantly darker than the other products. Apparently, in the case
of P2, the polyphenol adsorption capacity was insufficient. This
appears to be in agreement with previous findings indicating that
the lower salt level was most suitable for polyphenol adsorption
due to the lower extract concentrations (Weisz et al., 2010),
although this was not confirmed in laboratory experiments (IP-L2).
In fact, in addition to the higher extract concentration, the amount
of extract used in P2 was greater than in P1 (1800 vs. 1500 L) while
applying the same amount of adsorbent resin. Hence, more detailed
adaptation would be needed to achieve the same efficiency as on a
laboratory scale. Moreover, the reduced adsorption efficiency in the
case of P2 was also aggravated by repeated use of the adsorbent
resin. To improve polyphenol adsorption, the amount of resin and
contact time may be increased, which has been shown to be as
effective as lowering the load of the protein extract on a laboratory
scale (Weisz et al., 2010). As another option, the use of an inter-
mediate salt level appears to be promising for optimising the pro-
tein yields while maintaining high adsorption capacity. For
214 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
example, 1.8 mol/L NaCl was reported to be most suitable for pro-
tein extraction by Shchekoldina and Aider (2012).
Comparing the precipitated isolates from different scales
(Table 2), the product colours did not strictly correlate with the
phenolic acid contents. This agrees with the observations of
Salgado, Drago, et al. (2012), and might be ascribed to the fact that
darkening already occurred during extraction and the subsequent
separation steps prior to phenolic acid removal. On a pilot plant
scale, the longer processing times of higher volumes allowed
enhanced polyphenol oxidation and this impaired the colour of the
final products. Moreover, contact with oxygen was not prevented
on a pilot plant scale, whereas laboratory extraction was performed
in an agitator vessel under a nitrogen blanket, and centrifugation
was carried out in sealed beakers. The two-step adsorption was
designed to remove monomeric CGA and oxidised derivatives, thus
improving the visual appearance of the proteins, provided that
sufficient amounts of resin are used.
3.2. Protein characterisation
The molecular weight distributions of the different protein
preparations are illustrated in Fig. 2. The results for the precipitated
proteins IP-P1 and IP-P2 were very similar, revealing the predom-
inance of molecular weight fractions of around 22e25, 32e33 and
Fig. 2. Molecular weight distribution of sunflower protein isolates (IP) and concentrates (UF) prepared on a pilot plant scale (P). SDS capillary gel electrophoresis under reducing
conditions; std. ¼ molecular weight standard; for other abbreviations see Table 1 and Fig. 1.
41e42 kDa, as well as minor fractions of ~15, 31, 35 and 50 kDa
under reducing conditions (Fig. 2). The electropherograms of pro-
tein isolates IP-L1 and IP-L2 obtained on a laboratory scale showed
identical patterns and were therefore omitted. The fractions with
molecular weights between 22 and 42 kDa represent basic and
acidic polypeptides derived from the helianthinin subunits by
reducing disulphide bonds (Kortt & Caldwell, 1990; Raymond,
Inquello, & Azanza, 1991; Zilic et al., 2010). The intact subunits
were detected under non-reducing conditions as the predominant
peaks corresponding to molecular weights of 56e58, 66e68, and
70e71 kDa with minor differences between the IP isolates (data not
shown), which is in accordance with literature data (Gonza lez-
rez et al., 2002; Gonz
Pe rez et al., 2002; Kortt & Caldwell,
alez-Pe
1990; Raymond et al., 1991). In contrast, the electropherograms of
the concentrates UF-P1 and UF-P2 obtained from the supernatants
represent the sunflower albumins characterised by fractions of
10e17 kDa with the main peak at ~12 kDa. Our findings are in
agreement with literature data (Gonz rez et al., 2005b;
alez-Pe
Pandya et al., 2000; Zilic et al., 2010). As previously reported
(Pickardt et al., 2011), an additional fraction was identified at
~23 kDa solely under reducing conditions, thus presumably rep-
resenting residual helianthinin subunits. Moreover, UF-P1 con-
tained considerable amounts of globulins represented by the peaks
of the subunits at 32e33 and 41e42 kDa detectable under reducing
 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219 215

conditions (Fig. 2, UF-P1), while UF-P2 only contained trace

amounts of these globulins. These results confirm the enrichment
of the globulin fraction in the precipitated isolates and the recovery
of albumins by ultrafiltration, with the higher salt level promoting
precipitation in agreement with our previous findings (Pickardt
et al., 2011).

3.3. Protein yields

The protein yields obtained by precipitation (IP) highly depen-

ded highly on the salt levels, ranging from 14.7 to 15.0% at the lower
salt level to 22.7 and 25.6% at the higher NaCl level on a laboratory
and pilot plant scale, respectively (Fig. 3). This corresponds to
product yields ranging from 6.2 to 10.6%. The protein distribution
throughout the process is illustrated in Fig. 4 taking the IP/UF-P2
run as an example: The protein yields and losses in all product
and by-product streams, respectively, are indicated following the
same trend for all experiments.
The protein yields on a pilot plant scale are only indicative due
to the single experiments. However, they are in the same range as
the laboratory scale yields analogously showing the influence of the
salt concentration, thus confirming the feasibility of the process. As
previously shown, higher salt levels resulted in higher protein
concentrations in the extracts (Pickardt et al., 2009), and thus,
higher efficiency of the precipitation step (Pickardt et al., 2011). In
contrast, the protein yields of the concentrates obtained by mem-
brane filtration (UF) were only 1.2% and 5.3%, corresponding to
product yields of 0.7% and 3.2% for UF-P1 and UF-P2 respectively. As
precipitation sufficed to recover the major protein fractions, only
small yields were achieved using the subsequent membrane
filtration (see Fig. 4).
The protein yields in the precipitated isolates of between 15.0
and 25.6% (Fig. 3) and the cumulative total protein yields (PI and
UF) of between 16.2 and 30.9% were similar to the yields reported
by Salgado, Drago, et al. (2012) of 12e14% and 26e29% with and
without precipitation respectively, after alkaline extraction from
de-oiled sunflower cake on a pilot plant scale with a pre-extraction
Fig. 4. Protein distribution in products and waste streams during pilot plant pro-
cessing at cNaCl ¼ 2 mol/L (P2). Protein yields (dark bars) and losses (light bars) of
individual processing steps as shown in Fig. 1 relative to initial sunflower cake protein.
adsorption incl. ion exchange, ultrafiltration incl. diafiltration.
step for polyphenol removal. Similar or slightly higher yields of
~30% were reported for phytate and/or polyphenol depleted pro-
tein isolates obtained by alkaline extraction and isoelectric pre-
cipitation after pre-extraction with acidic butanol (Saeed &
Cheryan, 1988) and water (Salgado, Molina Ortiz, Petruccelli, &
Mauri, 2011) or by extraction with salt solution after acetone
treatment (Prasad, 1990), while yields of 40% were reported after
alkaline extraction and acidic precipitation (Kabirullah & Wills,
1981). Alkaline extraction on a pilot plant scale (Davin et al.,
1983) gave protein yields ranging between 23 and 52%, with an
inverse correlation of yields and protein contents of the isolates. ln
contrast, Shchekoldina and Aider (2012) reported high protein
yields of up to 50% using alkaline extraction combined with 10%
NaCl (corresponding to 1.8 mol/L) from de-oiled sunflower cake
followed by acidic precipitation, whilst 60% of meal protein was
recovered by Gonza lez-Perez et al. (2002) in a total sunflower

Fig. 3. Comparison of sunflower protein isolates (IP) and concentrates (UF) after mild-acidic extraction at NaCl levels of cNaCl ¼ 1.3 (1) or 2.0/2.1 mol/L (2) prepared on a laboratory
(L) or pilot plant (P) scale. For sample details refer to Table 1 and Fig. 1. Values of functional properties are given as mean ± mean deviation. Protein yields are indicative and the
deviations of individual processing steps combined. Bars with the same superscript letter within one chart are not significantly different (p ¼ 0.05). *Foams of UF-P1 were not stable
after 60 min.
216 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
protein isolate after repeated alkaline extraction and ultrafiltration.
The lower yields in the present study compared to the latter studies
may partly be due to the mild-acidic vs. alkaline extraction which
might result in lower extraction yields despite the high salt levels
used (Pickardt et al., 2009). In fact, the extraction yield was only
about 57% for IP-L2 (Pickardt et al., 2011) and similar at pilot plant
scale (60% for IP-P2, see Fig. 4). To enhance extraction yields, a
second extraction step including counter-current extraction ap-
pears helpful. In contrast, the use of elevated temperatures to in-
crease protein extraction (Pickardt et al., 2009; Shchekoldina &
Aider, 2012) is not recommended due to enhanced polyphenol
binding and decreased adsorption efficiency (Pickardt et al., 2009;
Weisz et al., 2010). Moreover, protein yields also depend on the
quality of the starting material. In contrast to gently processed
experimental sunflower meal used by Gonza lez-Pe
rez et al. (2002)
and Kabirullah and Wills (1981), industrial press cakes were used in
the present study. Harsh treatments during industrial de-oiling
may induce partial denaturation of the proteins (Salgado et al.,
2011) affecting protein solubility (Davin et al., 1983; Kausar et al.,
2002; Raymond et al., 1985; Shchekoldina & Aider, 2012). Even
though low temperatures were used during solvent extraction, the
starting material used in the present study may have been affected
by such treatment.
Marked protein losses associated with further purification steps
used in the present study (see Fig. 4), are another reason for the
lower yields compared to the process proposed by Gonza lez-Perez
et al. (2002). Approximately 1/4 of the extracted protein was lost
upon precipitation (Pickardt et al., 2011) which could not be
completely recovered by ultrafiltration. High losses of low molec-
ular weight proteins in the permeate might be mitigated by using
membranes having a lower cut-off and adjusting the pH conditions,
respectively (Waesche, Mueller, & Knauf, 2001). Moreover, the
washing operations employed in the present study may account for
losses up to 17% of the precipitated proteins as shown in Fig. 4 for
IP-P1 and reported previously for IP-L2 (Pickardt et al., 2011).
Consequently, for industrial implementation, efficient water recy-
cling is required in order to recover these proteins.
Minor protein loss may also be ascribed to the adsorption pro-
cess (Fig. 4), but more to the washing of the batch than to protein
binding to the adsorber resin (Weisz et al., 2010).
3.4. Functional properties of protein isolates
To assess the suitability of the derived products for different
food applications, selected functional properties were determined.
Practical assays were applied which have been adapted to certain
food systems. Fig. 3 summarises the functional properties of the
protein isolates and concentrates that were produced on a labora-
tory and pilot plant scale. The protein solubility was deemed to be a
decisive parameter because it is associated with other functional
properties (Gonza lez-Perez & Vereijken, 2007; Kabirullah & Wills,
1981, 1982; Karayannidou et al., 2007; Salgado et al., 2011). The
emulsifying and foaming properties were also considered to be
promising for the sunflower protein isolates, whereas the gelling
properties were less notable, as revealed in preliminary tests and
literature data (Gonza lez-Perez & Vereijken, 2007). In our pre-
liminary studies, different sunflower protein isolates exhibited
weak gel strengths being far below that of soy protein isolates, and
high protein concentrations (>12%) and high gelation temperatures
(>95  C) were required for gel formation.
3.4.1. Protein solubility
Protein solubilities at pH 7 were generally low for the precipi-
tated proteins with values in the 7.3e8.4% range for IP-P and in the
10.3e13.0% range for IP-L. The pilot plant proteins revealed slightly,
however significantly lower solubility. In contrast, albumins
recovered by membrane filtration exhibited far better water solu-
bility (72.4 and 74.4% for UF-P1 and UF-P2, respectively).
General differences in the functional properties of the IP protein
isolates and UF concentrates may be ascribed to the predominance
of globulins and albumins in the protein preparations obtained by
precipitation and ultrafiltration respectively (see Section 3.2 and
Fig. 2). Sunflower albumins are known to be readily soluble,
whereas helianthinin solubility is generally low and pH dependent
(Canella et al., 1985; Gonza lez-Pe rez et al., 2005b; Kabirullah &
Wills, 1982). Therefore, the small but significant difference be-
tween IP-L1 and IP-P2 might be ascribed to the slightly higher ash
content (see Table 2), which is likely to interfere in the solubility
measurements (Pickardt et al., 2011).
However, our study revealed extremely poor solubility for the
protein isolates in water at pH 7, irrespective of the production scale
and NaCl level used. This was in agreement with earlier findings
showing solubility to be affected by the extraction and precipitation
conditions which was partly ascribed to denaturation (Pickardt
et al., 2011). Protein denaturation has been determined for IP-L2
at a denaturation enthalpy of 0.1 J/g protein (main peak at 99  C)
as compared to 3.1 J/g protein and 2.4 J/g protein for the respective
raw material and the protein isolate obtained by precipitation at pH
4.5, respectively (Pickardt et al., 2011). This confirms the denaturing
effect of low pH values. Similarly, denaturation enthalpy for IP-P2
was only 9% of the value measured for the press cake (data not
shown). Probably, denaturation of all IP products was high. Most
likely, harsher process conditions (longer times, stronger stirring
and mechanical treatment and local pH dips and slightly lower pH
values) caused severe denaturation of the proteins, especially on a
pilot plant scale, which is in accordance with previous findings
(Brueckner & Mieth, 1984). Furthermore, formation of firm aggre-
gates during precipitation and separation is possible, resulting in
lower solubility. This assumption may be supported by the poor
solubility of the globulin isolate even at low denaturation (Pickardt
et al., 2011). The low solubility of the globulin isolates might also be
ascribed to the intrinsic protein properties. These include the
higher proportion of hydrophobic amino acids compared to e.g. soy
proteins and the depletion of hydrophilic amino acids during the
isolation process (Brueckner & Mieth, 1984). Low protein solubil-
ities of about 24e30% were also observed for globulin isolates
(Kabirullah & Wills, 1982; Minones Conde et al., 2005; Yoshie-Stark
et al., 2008), whereas much higher values of 50% to > 80% have been
reported elsewhere (Gonza lez-Pe rez et al., 2002; Pawar et al., 2001;
Salgado et al., 2011). The different findings are mainly due to the
different methods used for the protein preparation and solubility
measurement, including different pH values which greatly affect
protein solubility (Pickardt et al., 2009).
Low protein solubility is not necessarily disadvantageous for
many applications of protein isolates. For applications requiring
high protein solubility, this feature may be modified by hydrolysis
or other measures (Kabirullah & Wills, 1981, 1982; Karayannidou
et al., 2007; Minones Conde et al., 2005).
3.4.2. Emulsifying capacity
The emulsifying capacities varied between 180e285 and
510e640 mL/g for IP and UF products, respectively, with signifi-
cantly lower values for the isolates obtained on a pilot plant scale.
Values around 200 mL/g represent a moderate emulsifying capac-
ity, while ~600 mL/g is quite high compared to 800e1000 mL/g for
sodium caseinate (data not shown) which serves as a reference.
Furthermore, the following values measured for soy protein isolates
in our laboratory may be taken as references: 645 mL/g for Supro
500E; 670 mL/g for Supro EX33, both from Dupont Protein Tech-
nologies; 715 mL/g ProFam 464, ADM. Therefore, the emulsifying
 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
activities of the albumin concentrates (UF) can be considered as
very high, while being fair for the helianthinin isolates (IP).
Our results are in agreement with literature reports confirming
the ability of both helianthinin and albumin preparations to form
stable emulsions (Gonza lez-Perez et al., 2005). However, literature
data on the functional properties of sunflower proteins are con-
tradictory, probably due to the diversity of methods and pre-
treatments used and the large variety of products investigated.
Due to the different methods used, quantitative comparison with
other studies is impossible. Using similar evaluation parameters,
Canella et al. (1985) also described slightly higher emulsifying ca-
pacities of 172 mL/g for an albumin-enriched isolate compared to
116 mL/g for a globulin isolate. Comparing our results to other
proteins produced in our laboratory using essentially the same
methods, emulsifying capacities of 400 and 693 mL/g have been
reported for rapeseed protein isolates obtained by precipitation
and ultrafiltration, respectively. Values of 495 and 800 mL/g were
measured for whole egg and egg white, respectively (Yoshie-Stark
et al., 2008). The emulsifying capacities of lupin protein isolates
were reported to range from 385 to 570 mL/g (D'Agostina et al.,
2006), thus, in a range similar to our samples.
The generally higher emulsifying activity of the albumin con-
centrates (UF) compared to the helianthinin isolates (IP) may be
ascribed to both the solubility and molecular size, with faster
diffusion of smaller molecules to the fluid interfaces (Minones
Conde et al., 2005) and greater molecular flexibility (Karayannidou
et al., 2007). As shown in Fig. 3, there is a clear correlation with
the solubility of both the different types of proteins, which in turn is
dependent on the intrinsic properties including the different amino
acid composition as discussed above (Section 3.4.1). The differences
between the precipitated isolates on the different production scales
seem to be correlated to the protein solubility which also was lower
on a pilot plant scale (Fig. 3). This correlation may be ascribed to the
fact that the functional behaviour is governed by the soluble portion
of the protein being able to unfold and interact at the oilewater-
interface, while the insoluble parts scarcely contribute to this
functionality (Gonza lez-Perez et al., 2005). Moreover, denaturation
of protein isolates at pilot plant scale due to harsher process con-
ditions as discussed above (Section 3.4.1) may decrease emulsifying
capacities, since the ability of the protein molecules to unfold and
align to the oilewater interface is lowered. Furthermore, formation
of firm aggregates during precipitation and separation may occur on
a pilot plant scale, thus limiting the formation of protein films at the
oilewater interfaces. Consequently, process conditions need to be
thoroughly adjusted for further process optimisation, with focus on
avoiding very low pH values for precipitation.
3.4.3. Foaming activity
The foaming capacity was very high for all precipitated samples
with overrun values between 1063 and 1299% with higher values at
the lower salt level, while differences between the production scales
were less pronounced. Significant differences were found comparing
IP-P1 and IP-P2. UF concentrates provided even higher foaming ca-
pacities of 1407e1500%, which were significantly higher as compared
to the IP of the respective salt concentration. However, these foams
rapidly collapsed, while IP foams were very stable keeping 85e100%
of their initial volume over 1 h (data not shown). This compares to
1100e1600% activity for egg white foam with a foam stability of 90%
(data not shown), which may serve as a reference.
Slightly higher values were generally found for the lower salt
level. These do not seem to be clearly related to solubility values,
which will be further discussed below. Comparing the composition
of the products, only small differences can be found which cannot
serve as an explanation either. Only the significantly darker colour
may indicate some bonding of phenolic compounds or further
217
impurities which might both affect functionality (Gonz rez
alez-Pe
et al., 2005a). In contrast, Salgado, Ortiz,et al. (2012) did not find
an influence of different phenolic contents on the foaming prop-
erties of sunflower protein concentrates.
Our results are in agreement with previous reports demon-
strating lower foaming activities but higher foam stabilities for
helianthinin compared to sunflower albumins (Gonz rez
alez-Pe
et al., 2005a). The high foaming activities in the range of egg
white and the high foam stability of the precipitated proteins are in
accordance with earlier reports (Kabirullah & Wills, 1982; Raymond
et al., 1985) which also recommended sunflower proteins as
whipping agents. Raymond et al. (1985) reported the foaming
properties of a sunflower protein preparation to be superior to that
of soy proteins. However, quantitative comparison is virtually
impossible due to the different methods and products, as
mentioned above (Section 3.4.2). Our preliminary studies showed
that the foaming properties are highly dependent on the protein
concentrations, whipping times and the foaming method. Using
similar methodology, slightly lower foaming activities of ~1000%
and stabilities >80% were reported for lupin proteins (D'Agostina
et al., 2006), which are known to have excellent foaming properties.
In contrast to the emulsifying properties, the foaming activities
did not show a clear correlation to the protein solubilities of the
protein isolates (Fig. 3). This is in agreement with previous reports
(Gonz rez et al., 2005a) assuming solubility to be a less
alez-Pe
important factor as only a small proportion of the protein might be
incorporated in the foam. Higher precipitation efficiency at
increased salt concentration and upon upscaling resulting in firmer
aggregates that are less prone to dissolution during whipping may
further impair the foaming activity. While the foaming activity may
be enhanced by the greater molecular flexibility of the smaller al-
bumins compared to the larger globulins, albumins were found to
be unable to stabilise the foams, probably due to their smaller
molecular size (Minones Conde et al., 2005). Higher molecular size
and increased viscosity of IP dispersions due to their lower solu-
bility may be another reason for the better stabilisation of foams by
globular proteins. This concurs with the findings of Kabirullah and
Wills (1981) who found hydrolysed sunflower protein isolates
having greatly enhanced solubility to have better foam volume but
decreased stability.
3.4.4. Comparison of different NaCl levels and different production
scales
Comparing the different NaCl levels, there are no clear-cut ef-
fects on solubility and emulsifying properties, while the foaming
activities were slightly higher for the lower salt level. Regarding the
different production scales, the protein solubilities and foaming
activities of the isolates from the pilot plant scale were only slightly
lower. However, the emulsifying capacities were markedly lower. It
should be noted that slightly different processing conditions, in
particular for the precipitation step, were used on the different
production scales. On a pilot plant scale, the acid used for fast pH
adjustment of high volumes was of higher concentration (3 mol/L
HCl), probably causing local pH dips as well as a generally slightly
lower pH level. Furthermore, a pasteurisation step was performed
on pilot plant scale, which was necessary to ensure product safety
for potential food applications. Therefore, greater denaturation of
the proteins on a pilot plant scale might have occurred, which is
likely to affect the functional properties even though protein sol-
ubility at pH 7 was only slightly decreased (Pickardt et al., 2011).
The inferior functionality of the protein isolates produced on a pilot
plant scale depicted in Fig. 3 is mainly ascribed to such process
variations. Consequently, in order to avoid protein denaturation,
the precipitation conditions must be carefully controlled in further
scale-up work.
218 C. Pickardt et al. / Food Hydrocolloids 44 (2015) 208e219
4. Conclusions
Sunflower protein isolates of light colour were produced from
de-oiled press cake by the novel process that combines mild-acidic
protein extraction at pH 6 with adsorptive removal of phenolic
compounds from the protein crude extracts. The adsorption of
phenolic compounds from the protein extracts improved the colour
of the protein isolates more effectively than hitherto proposed pre-
extraction processes confirming the general feasibility of the pro-
cess. Despite the slight processing differences which occurred
during scale-up to pilot plant scale, the overall properties of the
protein isolates were comparable to those produced on a laboratory
scale. This demonstrates the feasibility of scaling up the process.
However, process times and process conditions must be carefully
controlled in order to avoid extensive denaturation. Further pilot
plant trials are needed to carry out precise protein quantification
and an economic assessment. Additionally, the simultaneous re-
covery of polyphenols may improve the economic viability of the
overall process.
Comparing the different NaCl levels, the higher salt concentra-
tion was confirmed to enhance protein recovery by far, while the
lower level was slightly better with regard to the quality, especially
the colour, of the protein isolates. Therefore, higher salt concen-
trations should be applied in further scale-up optimisation, while
the amount of resin and time of adsorption must be customised in
order to compensate the impaired adsorption efficiency in higher
salt concentrations. Moreover, extraction at an intermediate salt
level appears to be a promising approach for optimising the protein
yield whilst maintaining high adsorption capacity.
Regarding product performance, our results show that sun-
flower protein isolates recovered by the novel process have
promising functional properties for their application in a variety of
food products, in particular as emulsifiers and whipping agents. In
preliminary studies, such protein ingredients have been success-
fully used in the production of vegan (egg-free) mayonnaise and
foamed dessert products as well as for the replacement of egg
white in marshmallow type confectionery products and sponge
cake formulations.
Acknowledgements
€
The authors thank Dr. Rass, Teutoburger Olmühle GmbH & Co.
KG (Ibbenbüren, Germany) for the provision of the sunflower press
cake. We are grateful to Prof. Dr. Endreb, Herbstreith & Fox KG
(Neuenbürg, Germany) for his gift of adsorbent resin to perform the
pilot plant experiments, and to Dr. Georg Weisz (Hohenheim Uni-
versity) for providing the protocol for the adsorption experiments.
The authors acknowledge the valuable assistance of Michael Schott
and Michael Frankl with the pilot plant experiments and thank
Sigrid Bergmann, Sigrid Gruppe, Elfriede Bischof, Maria Hillreiner,
Isaac Tenllado and Thomas Hager for their help with the physical
and chemical analyses and laboratory sample preparation.
This research project was supported by the German Ministry of
Economics and Technology (via AiF (Arbeitsgemeinschaft indus-
trieller Forschungsvereinigungen “Otto von Guericke” e.V.)) and the
FEI (Forschungskreis der Ern€ ahrungsindustrie e.V., Bonn), Project
AiF 14449 N.
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