Functional properties of protein isolates, globulin and albumin extracted from

Ginkgo biloba seeds

Qianchun Deng
a
, Lan Wang
b
, Fang Wei
a
, Bijun Xie
c
, FengHong Huang
a,
*
, Wen Huang
c
,
John Shi
d
, Qingde Huang
a
, Binqiang Tian
c
, Sophia Xue
d
a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
b
Institute for Farm Products Processing and Nuclear-Agricultural Technology, Hubei Academy of Agricultural Science, Wuhan 430064, China
c
Food Science and Technology College, Central China Agriculture University, Wuhan 430070, China
d
Guleph Food Research Center, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9
a r t i c l e i n f o
Article history:
Received 20 December 2009
Received in revised form 26 July 2010
Accepted 29 July 2010
Keywords:
Ginkgo seed protein isolate
Ginkgo seed globulin protein
Ginkgo seed albumin protein
Functional properties
a b s t r a c t
In this study, the functional properties of Ginkgo seed protein isolate (GPI), Ginkgo seed globulin protein
(GGP) and Ginkgo seed albumin protein (GAP) extracted from Ginkgo biloba seeds were investigated. The
protein contents of GPI, GGP and GAP were 91.0%, 93.4% and 87.8%, respectively in the samples in which
the sugar, polyphenol and crude bre were removed by the preparation procedure. For functional prop-
erties of Ginkgo seed proteins in the natural state, GAP showed the highest oil-absorption capacity
(9.3 ml/g), foaming capacity (67.8%), emulsifying capacity (65.4%) and emulsion stability (90.6%); while
GPI showed the highest water absorption capacity (1.93 ml/g), and GGP showed the highest foam
stability (55.5%). The differences of the chemical components, surface hydrophobicity, disulphide bond
(SS) and sulfhydryl group (SH) contents of GPI, GGP and GAP, which were correlated signicantly with
functional properties of Ginkgo seed proteins, were also investigated. The improved functional properties,
such as water absorption capacity, solubility, foaming properties and emulsifying properties of
Ginkgo seed proteins were observed in a pH range of 8.010.0 or sodium chloride concentration of
0.50.75 M.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Ginkgo biloba, cultivated in extratropical, warm and subtropical
zones, is one of the oldest species in the world, and 70% of Ginkgo
biloba is from China (Son & Kim, 1998). Recently, the medicinal and
health protection effects of extracts from Ginkgo biloba leaves have
attracted considerable attention. However, few of studies have
been conducted on Ginkgo seeds. As a traditional food and medi-
cine source, used for several thousand years, Ginkgo seeds could
be added to desserts, glazed fruit, beverages and tipple, also Ginkgo
biloba seed, as a traditional Chinese medicinal material, has been
recorded in the Compendium of Materia Medical (Tredici, 1991).
Hence, there is a growing interest in the utilisation of the ingredi-
ents extracted from Ginkgo biloba seeds.
There is 913% crude protein (dry basis, db) in Ginkgo biloba
seed, which is less than that in protein-rich vegetables. Consider-
ing the special status of Ginkgo in the vegetable kingdom, a lot of
research has been carried out on the molecular biology character-
istics of storage protein in Ginkgo biloba seed (Hager, Braun,
Czihal, Mller, & Bumlein, 1995). At the same time, Ginkgo bilo-
ba proteins with special biological activity have also been stud-
ied. Wang and Ng (2000) reported an antifungal protein with a
molecular weight of 13 kDa from Ginkgo seed. Albumin and
globulin, with free radical-scavenging activity, have been success-
fully separated from Ginkgo biloba seeds (Huang, Xie, Wang, Yang,
& Luo, 2004). The cited investigation indicated that Ginkgo
biloba seed proteins exhibited favourable bioactivity, and
could be applied potentially in the food industry as functional
additives.
Soybean, peanut and rapeseed proteins have been extensively
applied for special functionality in food processing (additive and
protein lm industry). With the increase of protein requirement,
some new sources have been developed, such as cashew nut pro-
tein concentrates and isolates (Semiu, Folake, Hans-Peter, Andrea,
& Samuel, 2009), and milkweed seed isolates (Mila, Roque, &
Wuc, 2009). It has now become desirable to obtain proteins pos-
sessing both ideal functionality and bioactivity, such as rapeseed
protein isolate, with free radical-scavenging activity (Yumiko,
Yoshiko, & Andreas, 2008), and Phyllanthus niruri L. protein isolates,
for protecting liver (Bhattacharjee & Sil, 2007).
0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.07.108
* Corresponding author. Tel.: +86 027 86827874; fax: +86 027 86815916.
E-mail address: chunn2@163.com (F. Huang).
Food Chemistry 124 (2011) 14581465
Contents lists available at ScienceDirect
Food Chemistry
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchem
Although the content of protein is not high in Ginkgo biloba
seed, the resource of Ginkgo seed is large in China. However, little
information about the functionality of Ginkgo biloba seed protein
has been reported, which might primarily affect its utility in food
products. This study aimed to evaluate and characterise different
Ginkgo seed proteins (Ginkgo seed protein isolate, Ginkgo seed
globulin protein and Ginkgo seed albumin protein), and to evaluate
their potential as food ingredients by studying their chemical com-
position and functional properties.
2. Materials and methods
2.1. Materials
Ginkgo biloba seeds were obtained from Taixin market (Jiang-
shu, China) in 2008. The seeds were cleaned and then stored in
polyethylene bags in the refrigerator at 10 C until used.
2.2. Preparations of Ginkgo seed protein isolate (GPI)
Ginkgo seed protein isolate (GPI) was obtained by an alkaline
dissolving and acid precipitating method (Chavan, McKenzie, &
Shahidi, 2001). Ginkgo biloba seeds were de-hulled manually and
freeze-dried (ALPHA 1-4 LD, Matin Christ, Germany). The seeds
were crushed and defatted by Soxhlet extractor with petroleum
ether for 12 h. The defatted our was air-dried at room tempera-
ture and ground again to pass through a 40 mesh sieve. Defatted
Ginkgo ours (DGF) were dispersed in distilled water (1:9, w/v),
and then adjusted to pH 9.0 using 1 M NaOH. The suspension
was stirred for 30 min at room temperature, and then centrifuged
at 4000g for 15 min. In order to obtain higher yields, the extraction
and centrifugation were repeated twice on the residue. The ex-
tracts were combined and acidied to pH 4.4, and then left to stand
for 30 min to separate into two layers. The precipitates were recov-
ered by centrifugation at 4000g for 15 min, then neutralised by
1.0 M NaOH to pH 7.0 and dialysed in distilled water for 24 h.
The neutralised precipitate (GPI) was freeze-dried, then milled
and sieved through 60 mesh.
2.3. Fractionation of defatted Ginkgo ours (DGF) and Ginkgo seed
protein isolate (GPI)
Fractionations of DGF and GPI were conducted according to the
method described by Chavan et al. (2001). Distilled water, 0.14 M
NaCl, 70% ethanol and 0.2 M NaOH were used consecutively to ex-
tract the protein. The sample (5 g) was extracted with distilled
water (90 ml) by stirring for 30 min, followed by centrifugation
at 4000g for 15 min at room temperature. The extraction was re-
peated three times for the precipitated protein. The extracts were
combined and freeze-dried as albumins. The residues were ex-
tracted consecutively by 0.14 M NaCl, 70% ethanol and 0.2 M
NaOH. The corresponding extracts were globulin, gliadin fraction
and alkali-dissolved protein, respectively. Protein content of the
supernatants was determined by the micro-Kjeldahl method
(N6.25). The different fractionations were expressed as percent-
ages of total protein. All determinations were conducted in
triplicate.
2.4. Preparation of Ginkgo seed albumin protein (GAP) and Ginkgo
seed globulin protein (GGP)
The Ginkgo seed albumin protein (GAP) and Ginkgo seed glob-
ulin protein (GGP) were prepared by ammonium sulphate fraction-
ation, followed by the salt-dissolving and salt-precipitating
methods described by Huang et al. (2004).
2.5. Chemical composition
Protein content was determined by the Bradford method. The
total carbohydrate content was measured using the phenol
sulphuric acid method. The polyphenol content was measured by
the FolinCiocalteau method. The moisture, crude protein, ash,
fat and bre were analysed using AOAC methods.
2.6. Measurement of surface hydrophobicity (S
0
), disulphide bond (SS)
and sulhydryl group (SH) contents
Surface hydrophobicity of soluble proteins was measured
according to the method described by Kato and Nakai (1980) by
FP-6500 uorescence spectrometer (Jasco, Japan) using 8-anilino-
1-naphthalene sulphonate (ANS). Protein was dissolved in 0.1 M,
pH 7.4, PBS buffer and centrifuged at 10,000g for 30 min. The
supernatant was collected and serially diluted with the same buf-
fer to obtain solutions with different protein concentrations. Sixty
microlitres of ANS solution (8 mM in 0.1 M, pH 7.4, PBS solution)
was added to 1.2 ml of protein solution. Fluorescence intensity
(FI) was measured at 380 nm (excitation) and 470 nm (emission).
The FI was calibrated by adjusting it to a value of 80 (1) with
15 ll ANS in 3 ml of methyl alcohol. The initial slope of the FI ver-
sus protein concentration plot (calculated by linear regression
analysis) was used as an index of protein S
0
.
Disulphide bonds and sulfhydryl groups were measured accord-
ing to the method described by Thannhauser, Konishi, and
Scheraga (1987). GPI (200 mg), GGP (400 mg) and GAP (800 mg)
were added together and thoroughly mixed for 1 h in a solution
(20 ml) of 8 M urea dissolved in 0.086 M Tris 0.09 M glycine
0.004 M EDTA (TrisGly, pH 8.0). The solution was then centri-
fuged at 10,000g for 10 min. After that, the supernatant was col-
lected and serially diluted with the same buffer to obtain three
solutions with different protein concentrations. Free SH content
(SH
F
) was determined as follows: 80 ll of Ellmans reagent
(4 mg/ml DNTB) was added to the supernatant (2 ml) and thor-
oughly mixed for 5 min. After that, the absorbance was measured
at 412 nm. Total SH content (SH
T
) was determined as follows:
2 ml of supernatant were solubilised in 0.024% b-mercaptoethanol.
After 2 h of incubation at room temperature, 3 ml of 12% trichloro-
acetic acid (TCA) were added and the samples centrifuged at
10,000g for 10 min. The pellets were thrice resuspended in 12%
TCA and nally dissolved in TrisglycineEDTA. The absorbance
was also measured at 412 nm.
Calculations
lmolSH=10
6
g protein
73:53A
412
C
D
lmolS S=10
6
g protein
SH
T
SH
F
2
where A
412
= the absorbance at 412 nm; C = the sample concentra-
tion in mg solids/ml; D = dilution factor; and 73.53 is derived from
10
6
/(1.36 10
4
); 1.36 10
4
is the molar absorptivity and 10
6
is for
conversions from the molar basis to the lM/ml basis and from mg
solids to g solids.
2.7. Functionality
2.7.1. Oil-absorption capacity (OAC)
Oil-absorption capacity (OAC) of the protein isolate was deter-
mined by mixing the protein (0.5 g) with bean oil (5 ml) for 1 h,
then centrifuging at 2200g for 30 min. After decantation, the sam-
ple was weighed and OAC was calculated as the percentage of oil
trapped by the protein isolate (Sze-Tao & Sathe, 2000).
Q. Deng et al. / Food Chemistry 124 (2011) 14581465 1459
2.7.2. Water absorption capacity (WAC)
WAC of the samples was determined by the method described
by Lopez, Falomir, and Olivares-Vasquez (1991). WAC of the pro-
tein isolate was measured at different pH values (pH 1.014.0
without adding NaCl) or NaCl concentrations (02 M NaCl dis-
solved in de-ionised water).
2.7.3. Protein solubility
Solubility of protein was analysed at different pH values (pH 1
14) according to the method described by Cepeda, 1998. Sample
(125 mg) was dissolved in 0.1 M NaOH (20 ml) by stirring for 1 h
at room temperature; 10 ml of solution were taken, and adjusted
to the desired pH by 1 M HCl. The solution was centrifuged at
4000g for 10 min. Protein contents of supernatants were analysed
using the Bradford method. Solubility of the protein was also ana-
lysed at different pH values (pH 114) and NaCl concentrations
(02 M).
2.7.4. Foaming capacity (FC) and foam stability (FS)
The tests on foaming capacity (FC) and stability (FS) were based
on the method described by Sze-Tao and Sathe (2000). Fifty micro-
litres of 10 mg protein/ml dispersions of samples in buffer were
stirred for 5 min. The blend was immediately transferred into a
100 ml graduated cylinder. The volume was recorded before and
after stirring. FC was expressed as the volume (%) increased due
to stirring. For the determination of FS, foam volume changes in
the graduated cylinder were recorded at 120 min of storage. FC
and FS of the protein were measured at different pH values (pH
1.014.0) and NaCl concentrations (02 M). Foam capacity and
foam stability were then calculated according to the following
formulae:
Foam capacity%

volume after whipping volume before whippingml

volume before whippingml
Foam stability%

volume after standing volume before whippingml

volume after whipping volume before whippingml
2.7.5. Emulsifying capacity (EC) and emulsion stability (ES)
EC and ES were determined according to the method reported
by Chau and Cheung, 1998 with minor modication. For emulsion
formation, 5 ml of 1% (w/v) untreated protein in de-ionised water
(pH 7.0.) and 5 ml of corn oil were homogenised at 15,000 rpm
for 5 min. The mixtures were centrifuged at 2000g for 5 min. EC
was calculated as the ratio of the height of emulsifying layer to
the height of total volume. After heating at 80 C for 30 min and
centrifuging at 2000g for 5 min, the ES of mixture was measured
as the ratio of the height of emulsifying layer after heating to the
initial height of emulsifying layer.
2.8. Statistical analysis
Results were expressed as the mean values standard deviation
(SD) of three separate determinations. The data were averages of
triplicate observations and were subjected to a one way analysis
of various (ANOVA), followed by Duncans multiple range test.
The data were subjected to correlation analysis, using SPSS soft-
ware (version 16.0, The Predictive Ana-223 lytics Company,
Chicago, USA).
3. Results and discussion
3.1. Protein fractionation of defatted Ginkgo our and Ginkgo seed
protein isolate
As shown in Fig. 1, Ginkgo seed protein isolate (GPI) and defat-
ted Ginkgo our (DGF) protein were mostly soluble protein,
accounting for 94.1% and 95.8%, respectively. The total contents
of albumin and globulin in GPI and DGF were 90.8% and 85.7%,
respectively, which are much higher than that in pumpkin seeds
(58.6%) (Giami, 2004) and moth bean (68.9%) (Sathe & Mahesh,
2007). Albumin and globulin were the main storage proteins and
generally accounted for 5075% of the total seed protein (Shastry
& John, 1991). The contents of prolamin in GPI and DFG were the
lowest, while the alkaline-dissolved protein in GPI was higher,
which may be due to the alkali extraction and acid precipitation
of Ginkgo seed protein isolate.
3.2. Proximate composition
The proximate compositions of defatted Ginkgo our (DGF),
Ginkgo seed protein isolate (GPI), Ginkgo seed albumin protein
(GAP) and Ginkgo seed globulin protein (GGP) are shown in Table
1. The chemical composition analysis results showed that carbohy-
drate was the dominant component (78.9 1.89%) in DGF samples,
followed by protein and ash with 11.6 0.26% and 3.32 0.16%,
respectively. The GPI prepared by the method described in Section
2.2, was 91.0 1.21% protein. The yield of GPI was 7.32 0.25 g per
100 g of DGF, which is less than the protein content (11.6 0.26%)
in DGF. This phenomenon could be ascribed to insoluble protein
and protein precipitation because of linking with other compo-
nents in Ginkgo seed (Chavan et al., 2001). Furthermore, GGP
and GAP, prepared according to Section 2.4, contained
93.4 1.26 and 87.7 1.31% protein, with yields of 3.80 0.13%
and 4.06 0.11% of DGF, respectively. Low contents of lipid (less
than 1.0%) and total sugar (less than 2.1%) and no crude bre were
found in Ginkgo protein, which indicated that the extraction meth-
ods can remove impurities from Ginkgo seeds. The total polyphe-
nol content in GPI was much less than that in DGF (P < 0.05), while
no polyphenol was found in GGP or GAP. The absence of polyphe-
nol could favour the sample quality, because the interaction be-
tween polyphenol and protein highlighted the colour of sample
and decreased the digestion of amino acid (Sanchez-Vioque,
Clement, Vioque, Bautista, & Millan, 1999). Total sugar content in
Ginkgo seeds protein (about 2%) was signicantly lower than in
Fig. 1. Distribution (%) of protein fractions in GPI and DGF.
1460 Q. Deng et al. / Food Chemistry 124 (2011) 14581465
DGF. Since the presence of sugar may have a negative impact on
the functional properties and bio-availability of protein, reduction
of the sugar content in the sample of protein is one of the goals of
sample preparation (Pedroche et al., 2004).
The results showed that the SH and SS contents of Ginkgo seed
proteins were similar to those of some vegetable proteins (Chung,
Lei, & Li-Chan, 2005; Meng & Ma, 2002). The SH and SS contents of
GAP were three times higher than those of GGP. The results of sur-
face hydrophobicity (S
0
) showed that GGP exhibited the highest
surface hydrophobicity (105), while GAP exhibited the lowest sur-
face hydrophobicity (23.5). Bigelow (1967) suggested that high
charge and low hydrophobicity of protein could promote the solu-
bility of protein, which was in accordance with the ndings that
the solubility of GAP was greater than those of GPI and GGP. The
surface hydrophobicity of Ginkgo seed proteins was found to be
similar to that of red bean proteins (Meng & Ma, 2002).
3.3. Functional properties
3.3.1. Oil-absorption capacity
The oil-absorption capacity (OAC) is of great importance, since
it affects the emulsifying capacity, a highly desirable characteris-
tics in products such as mayonnaise (Escamilla-Silva, Guzmn-
Maldonado, Cano-Medinal, & Gonzlez-Alatorre, 2003). OAC of
GPI was 2.95 ml/g, higher than that of chickpea protein isolate
(1.7 ml/g) (Lopez et al., 1991), sesame protein isolate (1.5 ml/g)
(Khalida, Babikerb, & EI-Tianay, 2003) and carinata protein isolate
(2.17 ml/g) (Pedroche et al., 2004), while OAC of GGP was 2.56
ml/g. The oil-absorption capacity of GAP was 9.3 ml/g, which was
higher than those of GPI and GGP. Similar results have been re-
ported showing that oil absorption capacities of American locust
bean albumin are higher than that of globulin (Lawal, Adebowale,
Ogunsanwo, Sosanwo, & Bankole, 2005). The differences of OAC
among Ginkgo seed proteins were possibly due to the different
conformational characteristics, surface hydrophobicity, lipophilic
groups and degeneration of these proteins. In the present study,
the OAC of GPI was comparable to that of commercial soy isolate
(3.29 ml/g), and winged bean protein concentrate (4.01 g/g), as
reported earlier (Sanchez-Vioque et al., 1999; Sathe, Deshpande,
& Salunkhe, 1982). It is suggested that the high oil-absorption
capacity of Ginkgo seed proteins could be utilised in food industry,
for ground meal formulation, meat substitutes and extenders,
doughnuts, baked goods and soups.
3.3.2. Protein solubility
Fig. 3 shows the effects of pH and sodium chloride concentra-
tion on the solubility of Ginkgo seed proteins (GPI, GGP and GAP)
in terms of nitrogen solubility. All the samples showed a similar
trend as U-shaped curves in the pH range 1.012.0 (Fig. 3a). The
solubilities of all the samples were decreased in the pH range
1.05.0, and then increased. The minimum solubilities of GPI
(42%), GGP (35%) and GAP (18%) were observed at pH 5.0. Similar
results were also observed in the proteins from different plants
(Bora, 2002; Lawal et al., 2005). Generally, solubility decreases as
the pH increases until it reaches the isoelectric point. The loss of
electrostatic repulsive forces provides benecial conditions for
the formation of protein aggregates; high bulk density and large
diameter of the aggregates results in precipitation of protein
(Singh, Kaur, & Sandhu, 2005); then the solubility increases with
further increase of pH. Electrostatic repulsive forces between the
positively charged proteins help to keep them apart and increase
protein-solvent interactions. The maximum protein solubilities of
GPI (91.3%) and GGP (66%) were observed at pH 12.0, while the
maximum solubility of GAP was 96% at pH 11.0.
The pH environment had a great effect on the solubility of Gink-
go seed protein, which showed that the natural structures of GPI,
Table 1
Physicochemical properties of different fractions in Gingko seed.
Chemical components DGF GPI GGP GAP
Proportion in the DGF (%) 7.32 0.25
a
4.02 0.13
b
3.45 0.21
c
Water content (%) 4.21 0.09
ab
4.35 0.11
b
3.80 0.13
a
4.06 0.11
ab
Protein (%) 11.6 0.26
a
91.0 1.21
bc
93.4 1.26
c
87.7 1.31
b
Lipid (%) 1.52 0.09
a
0.99 0.10
b
0.85 0.05
b
0.78 0.06
b
Fibre (%) 1.39 0.08 ND ND ND
Total sugar (%) 78.9 1.89
a
2.10 0.08
b
1.81 0.05
b
2.0 0.11
b
Reducing sugar (%) 0.23 0.02
ab
0.18 0.03
a
0.19 0.01
a
0.27 0.01
b
Ash (%) 3.32 0.16
c
2.24 0.09
b
0.72 0.02
a
3.20 0.22
c
Total acid (%) 0.26 0.05
b
0.11 0.01
a
0.07 0.01
a
0.23 0..03
b
Total polyphenol (lg/100 g) 3.5 0.22
b
1.7 0.17
a
ND ND
Free SH content (mol/10
6
g) 5.81 0.22
a
3.57 0.36
b
10.9 0.58
c
Total SH content (mol/10
6
g) 34.6 0.88
a
22.5 1.14
b
69.8 2.01
c
SS content (mol/10
6
g) 14.9 0.58
a
9.48 0.64
b
29.4 0.92
c
Surface hydrophobicity (S
0
) 89.3 105 23.5
Each value in the Table is the mean of three replications standard deviation. Means within a column followed by different letters are signicantly different (P < 0.05).
DGF, defatted gingko ours; GPI, Ginkgo seed protein isolates; GAP, Gingko seed albumin protein; GGP, Gingko seed globulin protein.
Fig. 2. Effects of pH and NaCl concentration on water absorption capacity of GPI, GGP and GAP.
Q. Deng et al. / Food Chemistry 124 (2011) 14581465 1461
GGP and GAP were little damaged; proteins with low solubility
over a broad range of pH are indicative of severe protein denatur-
ation and insolubilization (Kinsella, 1979; Nakai, 1983). The earlier
study on ginkgo seed protein showed that good solubility at alka-
line and acidic pH is an important characteristic for food formula-
tions (Idouraine, Yensen, & Weber, 1991).
Effects of sodium chloride on protein solubility of Ginkgo seed
proteins are presented in Fig. 3b. For Ginkgo seed proteins, the re-
sults showed that there was an increase in protein solubility as so-
dium chloride concentration increased up to 0.5 M. However,
beyond this concentration, the protein solubility of these three iso-
lates decreased. The maximum solubilities of GPI, GGP and GAP in
solution were 85%, 80% and 93%, respectively. Among Ginkgo seed
proteins, GAP showed the highest solubility at different sodium
chloride concentrations. Protein solubility is known to increase
with moderately increasing salt concentrations, due to the salt-
ing-in effect. However, the protein solubility does not increase at
a high salt concentration, as it is likely to undergo a salting-out
effect. The effective mechanisms of ionic strength on protein solu-
bility include electrostatic interactions, solvent effect, salting-in
and salting-out effect (Kinsella, 1979).
In comparison with other research reports, the solubility of GPI
(58%) in distilled water was higher than that of Brassica carinata
protein isolates (32%) (Pedroche et al., 2004), sesame protein iso-
late (33%) (Khalida et al., 2003) and cowpea protein isolate (32%)
(DiaelDin, Elfadil, & Abdullahi, 2004), which might be ascribed to
the high content of albumin in GPI. The solubility of GGP (32%)
was lower than that of African locust bean globulin (62%) (Lawal
et al., 2005) and red bean globulin (84%) (Meng & Ma, 2002). The
solubility of GAP (70%) was similar to that of African locust bean
albumin (65%). The surface hydrophobicity contributed to the dif-
ference of Ginkgo seed protein solubility.
3.3.3. Water absorption capacity
The effects of pH and sodium chloride concentration on water
absorption capacity (WAC) of three isolated proteins (GPI, GGP
and GAP) are presented in Fig. 2. The results show that water
absorption capacities (WAC) of GPI, GGP and GAP in distilled water
are 1.93, 1.51 and 0.41 ml/g, respectively, which are comparable to
cowpea protein isolate (DiaelDin et al., 2004), bitter lupin protein
isolate (El-Adawy, 2000), sesame protein isolate (Khalida et al.,
2003) and red bean globulin (Meng & Ma, 2002). But WAC of
GAP was very low, which might be because GAP possessed low
molecule mass, weak intermolecular interactions and weak inter-
action with water molecules.
The WAC values of GPI, GGP and GAP showed similar curves at
different pH values (Fig. 2a). WAC of Ginkgo seed proteins de-
creased with the increase of pH values in the range 14. The
Fig. 4. Effects of pH and NaCl concentration on foaming properties of GPI, GGP and GAP.
Fig. 3. Effects of pH and NaCl concentration on solubility of GPI, GGP and GAP.
1462 Q. Deng et al. / Food Chemistry 124 (2011) 14581465
minimum WAC values of GPI, GGP and GAP were observed at pH
4.0 (1.51, 1.4 and 0.4 ml/g, respectively). When pH was near the
isoelectric point, proteins aggregated with strong intermolecular
interactions, resulting in less interaction with water, which led to
a poor water absorption capacity. The results showed that the iso-
electric points were in the range of pH 4.04.5 for Ginkgo seed pro-
teins, which indicated that Ginkgo seed proteins are acidic in
nature. This was conrmed by the highest protein content and
yield obtained from the isoelectric precipitation method of Ginkgo
seed proteins production. WAC of Ginkgo seed proteins increased
with pH value when pH increased from 4.0 to 9.0; meanwhile,
the maximum WAC values were observed for GPI, GGP and GAP
(2.59, 1.99 and 0.52 ml/g, respectively). WAC of Ginkgo seed pro-
teins decreased moderately when pH was above 9.0. As shown in
Fig. 2b, WAC values of GPI, GGP and GAP increased with sodium
chloride concentration in the range 00.5 M, while WAC of Ginkgo
seed proteins decreased until the concentration of sodium chloride
was above 1.25 M. In the 0.5 M sodium chloride solution, the max-
imum WAC values of GPI, GGP and GAP were 2.55, 2.31 and
0.84 ml/g, which corresponded to 30%, 50% and 70% augmentation
when compared with the control. At low salt concentration, hy-
drated salt ions binding weakly to charged groups of protein did
not affect the hydration shell of charged groups of the protein; at
high salt concentration, much of the existing water was bound to
salt ions; meanwhile the intermolecular interactions of proteins
were strengthened and this caused dehydration of protein and
reduction in WAC (Lawal et al., 2005).
It was noteworthy that GAP exhibited less water absorption
capacity than did GPI or GGP (Fig. 2a and b), which might be due
to the higher solubility of GAP. As reported earlier, protein with
high solubility showed low water absorption capacity (El-Adawy,
2000).
3.3.4. Foaming properties
The effects of pH and salt concentration on FC and FS of three
isolated proteins (GPI, GGP and GAP) are shown in Fig. 4.
High protein solubility is a prerequisite to achieve better foam-
ing capacity and foam stability. As shown in Fig. 4a, the FC curves
of three isolates with different pH exhibited similar trends (analo-
gous to solubility), and NSI values of Ginkgo seed protein corre-
lated signicantly with FC (P < 0.01). GPI, GGP and GAP possessed
poor FC values of 7%, 11.4% and 16.8% at pH 4.0, respectively. The
highest FC values of GPI and GAP were at pH 9.0 (53.8% and
84.6%, respectively); while FC of GGP reached the highest value
(42.4%) at pH 8.0. The high FC at any given pH can be attributed
to the increased exibility of the protein, which diffuses more rap-
idly to the airwater interface to encapsulate air particles and then
enhances the foaming capacity (Chau, Cheung, & Wang, 1997).
As shown in Fig. 4b, a similar trend was observed for the FS val-
ues of GPI, GGP and GAP at different pH. The lowest FS of GPI, GGP
and GAP were at pH 4.0, and the highest at pH 9.0. For foam forma-
tion, reasonable solubility is desirable to enable surface adsorption.
Many proteins easily coagulate in the isoelectric region, resulting
in reduced lm stability (Mwanjala, Kharidah, Jamilah, & Yaakob,
2000). In pre-acidic and pre-alkali environments, the solubility
and surface-activity of proteins are strengthened as a result of
the higher net negative charge on the protein, resulting in better
foaming properties (DiaelDin et al., 2004). The decreased FC and
FS at pH > 10.0 might be due to the repulsion of peptides via ionic
repulsion. The pH alters the conguration of the protein molecules,
consequently alters the foaming capacity and stability of the foam
(Makri & Doxastakis, 2006). Comparisons of FC and FS of Ginkgo
seed proteins showed that the effects of pH on Ginkgo seed
proteins were immensely different, reecting differences in their
composition, conformation, structure, and interaction with other
compounds and their immediate environment.
The inuence of salt concentration on FC and FS of GPI, GGP and
GAP is presented in Fig. 4c and d. The results show that FC and FS
of GPI increased as the salt concentration increased from 0 to
0.75 M, and then decreased. The maximum expansion of GPI was
observed in 0.75 M sodium chloride, with the values of FC and
FS increased by 118% and 16.3%, respectively. Also, the initial in-
crease in salt concentration, up to 0.5 M, enhanced FC and FS of
GGP and GAP, after which further increase in salt concentration re-
duced FC and FS progressively. Initial increase in foaming proper-
ties might be attributed to increase in protein solubility at these
salt concentrations. Increased protein solubility facilitated forma-
tion of stable and viscous lms surrounding the vacuole and en-
hanced protein concentration at the airwater interface.
However, the ion-screening effect of high salt concentration
Fig. 5. Effects of pH and NaCl concentration on emulsifying properties of GPI, GGP and GAP.
Q. Deng et al. / Food Chemistry 124 (2011) 14581465 1463
improved the hydrophobic interactions of proteins and destroyed
the protein lm, which promoted occulation, aggregation and
precipitation of proteins (Lawal et al., 2005; Mwanjala et al.,
2000). For Ginkgo seed proteins, the sequence of FC at 02 M so-
dium chloride concentration was: GAP > GPI > GGP. By contrast,
the sequence of FS was: GGP > GPI > GAP.
3.3.5. Emulsifying properties
As shown in Fig. 5a, signicant reductions in the emulsion
capacities (EC) of GPI and GAP were observed at pH 5.0 in compar-
ison with other pH conditions studied, while a similar reduction
was also observed at pH 4.0 for GGP. The lowest EC values of
GPI, GAP and GGP were 52.5%, 59.2% and 48.9%, respectively. For
GPI and GGP, the highest EC was recorded at pH 9.0, with values
of 63.6% and 60.9%, respectively. The highest EC of GAP (68.2%)
was found at pH 10.0. The EC values of Ginkgo seed proteins de-
creased when the pH values were above 7.0. As pH changed in
the range 1.09.0, the relationship between EC and pH for Ginkgo
seed proteins was similar to that between nitrogen solubility and
pH. This was in agreement with the general correlation between
emulsion properties and nitrogen solubility given in previous re-
ports (Lawal et al., 2005). Dependence of EC on pH was expected,
as it is known that emulsion capacity of a total protein depends
upon the hydrophiliclipophilic balance, which is affected by pH
(Sathe et al., 1982). Halling (1981) suggested that pH exerted its ef-
fects on emulsication properties primarily by altering the charge
distribution on protein molecules. At extremes of the alkaline
range, the decrease in attractive hydrophobic forces, as a result
of the increased net charge on the protein, could increase exibility
and enable the protein to diffuse more rapidly to the airwater
interface, resulting in poor EC (Aluko & Yada, 1995). The EC of
GAP was the highest, followed by GPI and GGP in the tested pH
range. The differences in EC of Ginkgo seed proteins might be as-
cribed to differences in solubility and molecular structure of the
samples.
As shown in Fig. 5b, ES increased on increasing pH from 1.0 to
12.0. ES increased rapidly at the acidic pH stage and afterwards
slowly increased at the alkaline pH stage. The ES values of GPI,
GGP and GAP increased to 12.7%, 16.5% and 3.48%, respectively,
at pH 12.0 compared with those at pH 1.0. The low ES at low pH
might be attributed to increased interaction between the emulsi-
ed droplets, since net charge on the proteins was decreased by
the presence of the chloride ions (Chavan et al., 2001). As the pH
increased, the coulombic repulsion increased between neighbour-
ing droplets, coupled with increased hydration of the charged pro-
tein molecules. These factors resulted in reduction of interface
energy and combination of emulsion droplet, which might account
for the higher ES obtained (Chavan et al., 2001).
The results showed that EC of Ginkgo seed proteins linearly in-
creased in the range 00.5 M sodium chloride (Fig. 5c). In 0.5 M so-
dium chloride, the highest EC values of GPI, GGP and GAP were
observed (67.5%, 60.1% and 72.8%, respectively), which increased
by 21.8%, 14.9% and 11.3% compared with that at 0 M sodium chlo-
ride. With the increase of sodium chloride concentration, the ES of
Ginkgo seed proteins decreased (Fig. 5d). Meanwhile, the change of
ES was similar to that of EC, except that the ES was highest at
0.25 M sodium chloride. When the sodium chloride was 2 M, the
ES values of Ginkgo seed proteins were less than the controls.
There were many factors affecting the emulsifying properties of
protein, and the effective concentration of protein was positively
related to emulsion stability; while the sodium chloride had salt-
ing-in and salting-out effects on protein, the effective concentra-
tions of protein at both high and low sodium chloride
concentrations were completely opposite, resulting in similar ef-
fects on ES. The increase in ES resulting from a low sodiumchloride
concentration might have been achieved through formation of
charged layers around the fat globules, resulting in mutual repul-
sion and/or formation of a hydrated layer around the interfacial
material, factors which lower interfacial energy and retard droplet
coalescence (Aluko & Yada, 1995). As a result of addition of sodium
chloride, GAP showed the highest EC and ES, while GGP had the
lowest EC and ES. When the concentration of sodium chloride
was between 1.5 and 2.0 M, the ES of GGP was better than that
of GPI.
3.4. Correlation analysis between physicochemical properties and
different functional properties
Correlation analysis results between functional properties and
chemical components are shown in Table 2. The NSI of Ginkgo seed
protein was positively correlated with FC (r = 0.903), EA (r = 0.875)
and ES (r = 1), so available protein concentration in solution had a
signicant effect on its functional properties; the negative relation
between NSI and WAC (r = 0.537) was similar to the results ob-
tained by El-Adawy (2000). Hydrophobicity of protein has been
shown to be closely related to its functional properties, especially
solubility and emulsifying and foaming properties (Meng & Ma,
2002); surface hydrophobicity (S
0
) of Ginkgo seed protein was neg-
atively related to NSI (r = 0.894), FC (r = 0.990), EA (r = 0.999)
and ES (r = 0.838), which might be due to the decrease of solubil-
ity; S
0
was positively related to OAC (r = 0.777); there are probably
many lipophilic amino acids distributed in the surface of proteins
Table 2
Correlation analysis between physicochemical properties and different functional properties of GPI, GGP and GAP
a
.
WAC OAC NSI FC FS EA ES Protein Total sugar Ash Total acid Free SH Total SH SS
OAC 0.948
NSI 0.537 0.777
FC 0.847 0.972 0.903
FS 0.65 0.857 0.99 0.955
EA 0.878 0.984 0.875 0.998
*
0.935
ES 0.519 0.763 1
*
0.894 0.987 0.865
Protein 0.762 0.928
*
0.955 0.99 0.987 0.979 0.949
Total sugar 0.093 0.227 0.79 0.45 0.696 0.395 0.803 0.573
Ash 0.603 0.824 0.997 0.935 0.998
*
0.911 0.995 0.976 0.738
Total acid 0.871 0.982 0.882 0.999
*
-0.94 1
*
0.872 0.982 0.408 0.917
Free SH 0.84 0.968 0.909 1
**
0.959 0.997
*
0.9 0.992 0.462 0.939 0.988
*
Total SH 0.868 0.981 0.885 0.999
*
0.941 1
*
0.874 0.983 0.413 0.919 1
**
0.998
*
SS 0.86 0.977 0.892 1
*
0.947 0.999
*
0.883 -0.986 0.428 0.926 1
*
0.999
*
1
*
S
0
0.901 0.992
*
0.894 0.99 0.915 0.999
*
0.838 0.967 0.347 0.889 0.998
*
0.992 0.997
*
0.996
*
Correlation is signicant at the 0.05 level (1-tailed).
**
Correlation is signicant at the 0.01 level (1-tailed).
a
Pearson correlation coefcients.
1464 Q. Deng et al. / Food Chemistry 124 (2011) 14581465
with high S
0
, so the protein could absorb more oil. Free SH, total SH
and SS contents were all positively related to FC and EA s (P < 0.05);
the emulsion activity index of Rohu (Labeo rohita) proteins was
also positively related to total SH (Mohan, Ramachandran, &
Sankar, 2006); total SH and SS bonds in a protein possess high reac-
tivity (Friedman, 1994), which have great effects on its functional
properties.
4. Conclusions
The characteristics, functionality and behaviour of three pro-
teins isolated from Ginkgo biloba seed were investigated. The re-
sults showed that Ginkgo seed protein isolate (GPI), Ginkgo seed
globulin protein (GGP) and Ginkgo seed albumin protein (GAP)
had desired physicochemical properties. As compared with other
vegetable proteins, Ginkgo seed proteins exhibited better solubil-
ity, oil-holding capacity, foam stability and emulsifying properties,
but less water-holding and foaming capacity. GAP showed the best
oil-holding capacity, foaming capacity, emulsifying capacity, and
emulsion stability, while GPI had the best water absorption capac-
ity. GGP had the best foam stability, which was signicantly corre-
lated with the chemical components, surface hydrophobicity,
disulphide bond (SS) and sulfhydryl group (SH) contents. The
adjustment of pH or the addition of sodium chloride improved
the functionality, e.g. solubility, foaming properties and other
important properties of Ginkgo seed proteins. The results revealed
that protein isolates with suitable functional properties could be
produced from the Ginkgo seed as a good protein ingredient in
food systems.
Acknowledgements
The nancial support for this study, by the earmarked fund for
Modern Agro-industry Technology Research System, is gratefully
acknowledged.
References
Aluko, R. E., & Yada, R. Y. (1995). Structurefunction relationships of cowpea(Vigna
unguiculata) globulin isolate:inuence if pH and NaCl on physicochemical and
functional properties. Food Chemistry, 53, 259265.
Bhattacharjee, R., & Sil, P. C. (2007). Protein isolate from the herb, Phyllanthus niruri
L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride
induced liver damage via its antioxidant properties. Food and Chemical
Toxicology, 45(5), 817826.
Bigelow, C. C. (1967). On the average hydrophobicity of proteins and the relation
between it and protein structure. Journal of Theoretical Biology, 16, 187211.
Bora, S. P. (2002). Functional properties of native and succinylated lentil (Lens
culinaris) globulins. Food Chemistry, 77, 12711276.
Cepeda, E. (1998). Functional properties of Faba Bean protein our dried by spray
drying and freeze drying. Journal of Food Engineering, 36, 303310.
Chau, C. F., & Cheung, P. C. K. (1998). Functional properties of ours prepared from
three chinese indigenous legume seeds. Food Chemistry, 61(4), 429.
Chau, C. F., Cheung, K., & Wang, Y. S. (1997). Functional properties of protein isolates
from three indigenous legume seeds. Journal of Agricultural and Food Chemistry,
45, 25002503.
Chavan, U. D., McKenzie, D. B., & Shahidi, F. (2001). Functional properties of protein
isolates from beach pea (Lathyrus maritimus L.). Food Chemistry, 74, 177187.
Chung, M. W. Y., Lei, B., & Li-Chan, E. C. Y. (2005). Isolation and structural
characterization of the major protein fraction from NorMan axseed (Linum
usitatissimum L.). Food Chemistry, 90, 271279.
DiaelDin, M. R., Elfadil, E. B., & Abdullahi, H. E. (2004). Fractionation, solubility and
functional properties of cowpea (Vigna unguiculata) proteins as affected by pH
and/or salt concentration. Food Chemistry, 84, 207221.
El-Adawy, T. A. (2000). Functional properties and nutritional quality of acetylated
and succinylated mung bean protein isolate. Food Chemistry, 70, 8391.
Escamilla-Silva, E. M., Guzmn-Maldonado, S. H., Cano-Medinal, A., & Gonzlez-
Alatorre, G. (2003). Simplied process for the production of sesame protein
concentrate. Differential scanning calorimetry and nutritional, physicochemical
and functional properties. Journal of the Science of Food and Agriculture, 83(9),
972979.
Friedman, M. (1994). Improvement in the safety of foods by SH-containing amino
acids and peptides. A review. Journal of Agricultural and Food Chemistry, 42(1),
2030.
Giami, S. Y. (2004). Effect of fermentation on the seed proteins, nitrogenous
constituents, antinutrients and nutritional quality of uted pumpkin (Telfairia
occidentalis Hook). Food Chemistry, 88, 397404.
Hager, K. P., Braun, H., Czihal, A., Mller, B., & Bumlein, H. (1995). Evolution of seed
storage protein genes: Legumin genes of Ginkgo biloba. Journal of Molecular
Evolution (Historical Archive). Journal of Molecular Evolution, 41(4), 457466.
Halling, P. J. (1981). Protein stabilized foams and emulsions. Critical Reviews in Food
Science and Nutrition, 15, 155203.
Huang, W., Xie, B. J., Wang, Y., Yang, E. N., & Luo, R. (2004). Study on separation and
purication of protein from ginkgo seed and its antioxidant activity. Chinese
Academy of Agricultural Sciences, 37(10), 15371543.
Idouraine, A., Yensen, S. B., & Weber, C. W. (1991). Tepary bean our, albumin and
globulin fractions functional properties compared with soy protein isolate.
Journal of Food Science, 56, 13161318.
Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a uorescence probe
method and its corelation with surface properties of protein. Biochimica et
Biophysica Acta, 624, 1320.
Khalida, E. K., Babikerb, E. E., & EI-Tianay, A. H. (2003). Solubility and functional
properties of sesame seed proteins as inuenced by pH and/or salt
concentration. Food Chemistry, 82, 361366.
Kinsella, J. E. (1979). Functional properties of soy proteins. Journal of American Oil
Chemists Society, 56, 242257.
Lawal, O. S., Adebowale, K. O., Ogunsanwo, B. M., Sosanwo, O. A., & Bankole, S. A.
(2005). On the functional properties of globulin and albumin protein fractions
and ours of African locust bean (Parkia biglobossa). Food Chemistry, 92,
681691.
Lopez, O. P., Falomir, C. O., & Olivares-Vasquez, M. R. (1991). Chickpea protein
isolates: Physicochemical, functional and nutritional characterization. Journal of
Food Science, 56, 726729.
Makri, E. A., & Doxastakis, G. I. (2006). Emulsifying and foaming properties of
Phaseolus vulgaris and coccineus proteins. Food Chemistry, 98, 558568.
Meng, G. T., & Ma, C. Y. (2002). Characterization of globulin from Phaseolus angularis
(red bean). International Journal of Food Science and Technology, 37, 687695.
Mila, P. H., Roque, L. E., & Wuc, Y. V. (2009). Characterization of milkweed (Asclepias
spp.) seed proteins. Industrial crops and products, 29, 275280.
Mohan, M., Ramachandran, D., & Sankar, T. V. (2006). Functional properties of Rohu
(Labeo rohita) proteins during iced storage. Food Research International, 39,
847854.
Mwanjala, A. M., Kharidah, M., Jamilah, B., & Yaakob, B. C. B. (2000). Inuence of
altered solvent environment on the functionality of pigeonpea (Cajanus cajan)
and cowpea (Vigna unguiculata) protein isolates. Food Chemistry, 71, 157165.
Nakai, S. (1983). Structurefunction relationships of food proteins with an emphasis
on the importance of protein hydrophobicity. Journal of Agricultural and Food
Chemistry, 31, 676683.
Pedroche, J., Yust, M. M., Lqari, H., Giron-Calle, J., Alaiz, M., Vioque, J., et al. (2004).
Brassica carinata protein isolates: chemical composition, protein
characterization and improvement of functional properties by protein
hydrolysis. Food Chemistry, 88, 337346.
Sanchez-Vioque, R., Clement, A., Vioque, J., Bautista, J., & Millan, F. (1999). Protein
isolate from chickpea (Cicer arietinum L.): Chemical composition, functional
properties and protein characterization. Food Chemistry, 64, 237243.
Sathe, S. K., & Mahesh, V. (2007). Fractionation and biochemical characterization of
moth bean (Vigna aconitifolia L.) proteins. Food Science and Technology, 40(4),
600610.
Sathe, S. K., Deshpande, S. S., & Salunkhe, D. K. (1982). Functional properties of lupin
seed (Lupinus mutabilis) proteins and protein concentrates. Journal of Food
Science, 47, 491497.
Semiu, O. O., Folake, O. H., Hans-Peter, M., Andrea, S., & Samuel, O. A. (2009).
Functional properties of protein concentrates and isolates produced from
cashew (Anacardium occidentale L.) nut. Food Chemistry, 115(3), 852858.
Shastry, M., & John, E. (1991). Biochemical changes and in vitro protein digestibility
of the endosperm of germinating Dolichos lablab. Journal of the Science of Food
and Agriculture, 55, 529538.
Singh, N., Kaur, M., & Sandhu, K. S. (2005). Physicochemical and functional
properties of freeze-dried and oven dried corn gluten meals. Drying
Technology, 23, 114.
Son, Y., & Kim, H. (1998). Above-ground biomass and nutrient distribution in a 15-
year-old ginkgo (Ginkgo biloba) plantation in central Korea. Bioresource
Technology, 63, 173177.
Sze-Tao, K. W. C., & Sathe, S. K. (2000). Functional properties and in vitro
digestibility of almond. (Prunus dulcis L.) protein isolate. Food Chemistry, 69,
153160.
Thannhauser, T. W., Konishi, Y., & Scheraga, H. A. (1987). Analysis for disulde
bonds in peptides and proteins. Methods in Enzymology, 143, 115119.
Tredici, P. D. (1991). Ginkgos and people a thousand years of interaction. Arnoldia,
51, 215.
Wang, H., & Ng, T. B. (2000). Ginkbilobin, a novel antifungal protein from Ginkgo
biloba seeds with sequence similarity to embryo-abundant protein. Biochemical
and Biophysical Research Communications, 279(2), 407411.
Yumiko, Y., Yoshiko, W., & Andreas, W. (2008). Chemical composition, functional
properties, and bioactivities of rapeseed protein isolates. Food Chemistry, 107,
3239.
Q. Deng et al. / Food Chemistry 124 (2011) 14581465 1465