- Email: [email protected]
Physicochemical and functional properties of 11S globulin from chan (Hyptis suaveolens L. poit) seeds
Accepted Manuscript Physicochemical and functional properties of 11S globulin from chan (Hyptis suaveolens L. poit) seeds
Luis F. De la Cruz-Torres, Jaime D. Pérez-Martínez, Mayra Sánchez-Becerril, Jorge F. Toro-Vázquez, N. Alejandra Mancilla-Margalli, Juan A. Osuna-Castro, C.I. VillaVelázquezMendoza PII:
S0733-5210(16)30483-0
DOI:
10.1016/j.jcs.2017.06.017
Reference:
YJCRS 2390
To appear in:
Journal of Cereal Science
Received Date:
25 November 2016
Revised Date:
08 June 2017
Accepted Date:
27 June 2017
Please cite this article as: Luis F. De la Cruz-Torres, Jaime D. Pérez-Martínez, Mayra SánchezBecerril, Jorge F. Toro-Vázquez, N. Alejandra Mancilla-Margalli, Juan A. Osuna-Castro, C.I. VillaVelázquez-Mendoza, Physicochemical and functional properties of 11S globulin from chan ( Hyptis suaveolens L. poit) seeds, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.06.017
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Physicochemical and functional properties of 11S globulin from chan (Hyptis
2
suaveolens L. poit) seeds
3
Luis F. De la Cruz-Torresa, Jaime D. Pérez-Martínezb, Mayra Sánchez-Becerrilb,
4
Jorge F. Toro-Vázquezb, N. Alejandra Mancilla-Margallic, Juan A. Osuna-Castroa,*,
5
C.I. VillaVelázquez-Mendozad
6 7 8 9 10 11 12 13
aFacultad
de Ciencias Biológicas y Agropecuarias, Universidad de Colima, km 40
Autopista Colima-Manzanillo, C.P. 28100, Tecomán, Colima, México bFacultad
de Ciencias Químicas-CIEP, Universidad Autónoma de San Luis Potosí,
Manuel Nava 6, Zona Universitaria, C.P. 28210, San Luis Potosí, S. L. P., México cTecnológico
Nacional de México, Instituto Tecnológico de Tlajomulco, Carr. a San
Miguel Cuyutlán km 10, Tlajomulco de Zúñiga, Jalisco C.P. 45650, México dFacultad
de Ingeniería Civil, Universidad de Colima, Carr. Colima-Coquimatlán km 9, C.P. 28400, Coquimatlán, Colima, México
14
*Corresponding Author:
15
Email: [email protected]
16 17 18
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Abstract
21
The 11S globulin is one of the most abundant and important storage proteins of the
22
chan grain (Hs11S). For this reason, we investigated its physicochemical and
23
functional properties (surface tension, zeta potential and thermo-mechanics
24
analysis). A decrement in surface tension was registered with an increment of
25
Hs11S concentration, such results suggested a critical micelle concentration
26
(CMC) at 0.6 mg/mL and a surface activity until 55.5 mN/m. The Hs11S solutions
27
showed isoelectric points (pI) with a clear NaCl influence (i.e., without NaCl pI =
28
3.5, at 0.05 M NaCl pI = 3, at 0.5 M NaCl pI = 2.5) and zeta potential dependent
29
upon the pH. Differential scanning calorimetry and rheology studies revealed the
30
onset for the heat-induced gelation of Hs11S which occurred around 74‒76 °C,
31
developing strong gels with an elasticity of ~103 Pa, a denaturation temperature of
32
94.1 °C and an enthalpy of 266.9 kJ/mol. Consequently, the Hs11S thermal
33
denaturation is not a cooperative process. According to the dynamic light scattering
34
(DLS) data as a function of temperature, the Hs11S organizes in oligomeric forms
35
of trimer, tetramer, hexamer, octamer and dodecamer as well as aggregates (Rh
36
higher than 100 nm).
37
Keywords
38
11S Globulin, Functional properties, Heat-induced gelation, Thermodynamic
39
properties
40
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Introduction
42
Globulins (salt-soluble storage proteins) are the main Osborne protein fraction in
43
legumes and some cereals such as oats and rice, constituting around 70 ‒ 80 % of
44
the total protein (Boulter and Croy, 1997). The 11S globulins (legumins) are
45
hexameric proteins of 300 ‒ 450 kDa, composed by six non-covalently bonded
46
subunits of approximately 50 ‒ 70 kDa, with an acidic polypeptide and a basic
47
polypeptide of about 30 ‒ 40 kDa and ~20 kDa, respectively, and linked by
48
disulfide bonds (Boulter and Croy, 1997).
49
The 11S globulins from different sources such as amaranth, sunflower, soybean
50
and mungbean have been purified and physicochemical and functionally
51
characterized for their use in the food industry for the development of traditional
52
and new food products (González-Pérez and Vereijken, 2007; Kimura et al., 2008;
53
Tang and Sun, 2010; Carrazco-Peña et al., 2013). On the other hand, the
54
population growth and the emerging dietary preferences (e.g., vegans,
55
vegetarians) demand for novel food ingredients and plant-based products with
56
lower cost; therefore 11S globulins from non-conventional food crops are attractive
57
options (Jarpa-Parra et al., 2015).
58
Chan (Hyptis suaveolens L. Poit) is a dicotyledonous plant from the Lamiaceae
59
family, considered as a pseudocereal and its seeds were cultivated and highly
60
appreciated since pre-Colombian cultures due to its high nutritional and medicinal
61
characteristics (Aguirre et al., 2004). Hyptis suaveolens has been used as food and
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traditional medicine in America, Asia and Africa (Aguirre et al., 2012). Chan seeds
63
contain 13.9 % of protein on dry weight being globulins (39 %) the major fraction.
64
Bojórquez-Velázquez et al. (2016) reported a simple purification procedure and
65
biochemical characterization of 11S globulin from chan. This is a hexameric protein
66
of 300 kDa, composed by four different monomers between 53.5 ‒ 49.5 kDa with
67
the characteristic disulfide bond linking the acidic and basic polypeptides.
68
However, required information about the physicochemical and functional properties
69
of Hs11S in order to use in foods remains unknown.
70
Within this context, the objective of this investigation is to evaluate the following
71
physicochemical and functional properties of Hs11S: surface tension and zeta
72
potential as a function of pH and NaCl concentration, thermo-mechanical
73
properties and aggregation/de-aggregation process as a function of temperature
74
and ionic strength.
75 76
Materials and Methods
77
2.1 Purification of Hs11S
78
2.1.1 Protein extraction
79
Seeds were purchased from a local market at Colima, Mexico. Fractionation of
80
globulins was carried out according to Bojórquez-Velázquez et al. (2016). The
81
pellet obtained after albumin extraction was suspended in buffer A (0.1 M Tris-HCl,
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10 mM EDTA, 1.7 M NaCl, pH 7.5) 1:10 (w/v), stirred 2 h at 4 °C, and centrifuged
83
30 min at 13,800 g. Supernatant (crude globulins) was stored at -20 ºC.
84
2.1.2 Hs11S purification
85
10 mL of crude globulins were precipitated at 60 % of ammonium sulphate
86
saturation,
87
chromatography (SEC) using a Sephacryl S-300 HR column (GE Healthcare
88
Bioscience, NJ, USA). Proteins were eluted with buffer A at 1 mL/min flow rate.
89
Fractions of 2.5 mL were analyzed by absorbance at 280 nm, dialyzed, freeze-
90
dried, resolved in 12 % SDS-PAGE (Laemmli, 1970) and then stored at 4 °C for
91
further analysis.
92
2.1.4 Protein measurement
93
The protein content was determined by the Bradford method (Bradford, 1976)
94
using bovine serum albumin as standard.
95
2.2 Functional characterization Hs11S
96
2.2.1 Surface activity and CMC
97
A DuNouy tensiometer (CSC Scientific Company, INC USA) was used to measure
98
the surface tension of Hs11S solutions by the method of Tomczyńska-Mleko et al.
99
(2014) with some modifications. Protein was dissolved in HPLC-degree water at a
100
concentration range from 0.1 to 1 mg/mL (0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1.0
101
mg/mL). All measurements were carried out at 25 °C. The CMC of Hs11S was
resuspended
in
buffer
A
5
and
subjected
to
size-exclusion
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102
determined from the graph of interfacial tension vs. natural logarithm of protein
103
concentration.
104
2.2.2 Zeta potential and isoelectric point
105
The zeta potential (ζ-potential) of Hs11S was measured at 25 °C as a function of
106
pH using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, Worcestershire,
107
UK) according to the modified method of Withana-Gamage et al. (2013). The pH
108
adjustments were performed with 0.5 M and 0.005 M HCl and 0.1 NaOH aqueous
109
solutions using an MPT-2 autotitrator (Malvern Instruments. Worcestershire,
110
Ukraine). Protein dispersions of 0.02 % in deionized water were prepared. Three
111
different treatments with NaCl were tested (0.05 M, 0.5 M and without NaCl). The
112
pH value of zero ζ-potential was considered as the pI of the protein.
113
2.3 Hs11S physicochemical characterization
114
2.3.1 Heat-induced gelation by oscillatory rheology
115
The gelation of Hs11S was evaluated by oscillatory rheology following the method
116
proposed by Shevkani et al. (2015). Measurements were determined in a MCR 302
117
rheometer (Anton Paar, Stuttgart, Germany) with plate-plate measuring geometry
118
(25 mm in diameter, PP25/TG, Anton-Paar, Germany) equipped with the TruGapTM
119
system. The temperature control was performed with two peltier systems located at
120
the base of the lower plate and a peltier hood covering the measuring geometry (C-
121
PDT200, Anton-Paar, Germany). The Hs11S solutions were prepared at 20 %
122
(w/v) in buffer B (35 mM KH2PO4-K2HPO4, 0.1 M NaCl, pH 7.6). Samples were
123
heated at 1 °C/min from 20 to 90 °C, held at 90 °C for 3 min and then cooled from
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90 to 20 °C (5 °C/min). To prevent sample dehydration, the edge of the plate was
125
covered with low viscosity silicone. The elastic (G′) and loss (G″) moduli of the gel
126
were obtained as function of temperature (Shevkani et al., 2015). A frequency
127
sweep (1 ‒ 100 Hz) at constant strain of 0.002 % was performed on the gel
128
subjected to the thermal treatment previously described. For each sample solution
129
and the different stages of the test, measurements were done within the linear
130
viscoelastic region (LVR). The instrument uses the Rheoplus/32 v3.61, Anton Paar
131
(MCR 302) software.
132
2.3.2 Thermal characterization by differential scanning calorimetry (DSC)
133
Thermal denaturalization of Hs11S was established by differential scanning
134
calorimetry using a DSC discovery (TA Instruments, USA). Protein dispersion at 25
135
% (w/v) in buffer C (35 mM KH2PO4-K2HPO4, pH 7.6) was prepared and 15 mg of
136
mixture were weighed in T-ZeroTM pan, hermetically sealed and hydrated in the
137
same buffer for 2 h prior to the test. The sample pan was loaded in the calorimeter
138
and equilibrated for 5 min at 20 °C, heated from 20 to 150 °C at 5 °C/min. A sealed
139
empty pan was used as a reference. The analysis of the thermograms was carried
140
out according to Withana-Gamage et al. (2013) for the calculation of the
141
calorimetric van’t Hoff enthalpy change (ΔHvHTm) and the entropy change upon
142
(ΔSTm). The denaturation peak temperature (Tm), width at half peak height (ΔT1/2),
143
maximum heat capacity at denaturation (Cpmax), denaturation enthalpy (ΔHexpTm)
144
and heat capacity change (ΔCpN→U) were computed with the DSC Discovery
145
instrument software (TRIOS Q111 C53 V.52 2002).
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2.3.4 Assembly capacity by dynamic light scattering (DLS)
147
The effect of NaCl and temperature on Hs11S was studied measuring the
148
hydrodynamic radius (Rh) (molecular size) by DLS using a zetasizer Nano-ZS
149
(Malvern Instrument Ltd., Malvern, Worcestershire, UK) with a light source from
150
633 nm He–Ne laser. Measurements were performed at a scattering angle of 173°.
151
Protein dispersions at 0.2 mg/mL in buffer C with different NaCl concentrations (0.1
152
M, 0.4 M and without NaCl) were measured from 10 to 90 °C (sampling every 5
153
°C).
154
2.4 Statistical analysis
155
Assembly capacity was carried out ten times each experiment, while surface
156
tension and zeta potential experiments were conducted in quadruplicate and
157
calorimetric properties in triplicate. The data were expressed as the mean ±
158
standard error (SE). Gelation capacity was obtained in duplicate each experiment.
159 160
Results and discussion
161
3.1 Hs11S purification and SDS-PAGE analysis
162
Hs11S was purified by size-exclusion chromatography using a Sephacryl S-300.
163
This protein was eluted from 40 until 62 fraction number (Fig. 1a), and its purity
164
was corroborated by 12 % SDS-PAGE (Fig. 1b). In non-reducing conditions,
165
Hs11S showed four bands with estimated molecular weight of 50 kDa; meanwhile,
166
under reducing conditions, four acidic (α) subunits of around 32 kDa and four basic
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(β) subunits of approximately 20 kDa were shown. These data confirm the first
168
report of the Hs11S purification (Bojórquez-Velázquez et al., 2016). The purified
169
protein was used for the following analyses.
170
3.2 Functional characterization of Hs11S
171
3.2.1 Surface activity and CMC
172
As shown in Figure 2a, the surface tension of Hs11S aqueous solutions decreased
173
from 68 mN/m to 55.5 mN/m as protein concentration increased from 0.1 mg/mL to
174
0.6 mg/mL. This behavior has also been reported at low protein concentration of
175
whey protein isolates (Tomczyńska-Mleko et al., 2014) and seed storage proteins
176
like a soybean 7S globulin (β-conglycinin), 11S globulin (glycinin) and 11S globulin
177
from lentil (Pizones Ruiz-Henestrosa et al., 2014; Jarpa-Parra et al., 2015). The
178
surface tension decrement with increasing protein concentration is due to the
179
preferential accumulation of proteins at the air-water interface and the exposition of
180
hydrophobic residues (Jarpa-Parra et al., 2015); therefore, the adsorption capacity
181
is influenced by some intrinsic factors of the protein such as its molecular size,
182
structure, hydrophobicity and solubility (Pizones Ruiz-Henestrosa et al., 2014).
183
At Hs11S concentrations higher than 0.6 mg/mL the surface tension remained
184
constant, since the system achieved the formation of a fully packed monolayer and
185
no further accumulation of protein at the interface took place. Thus, the CMC for
186
the Hs11S occurred at 0.6 mg/mL and it was considered the minimum protein
187
concentration above which stable micelles are formed (Tomczyńska-Mleko et al.,
188
2014). The surface tension can be affected in addition by extrinsic variables such
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189
as temperature, pH, ionic strength, surfactant concentration, and preheating of the
190
sample (Tomczyńska-Mleko et al., 2014) therefore, the CMC might be affected
191
equally.
192
A CMC of 5 mg/mL in 11S globulin from lentil using the ring method (Jarpa-Parra
193
et al., 2015) and in whey protein isolate has been reported (Tomczyńska-Mleko et
194
al., 2014). Our results suggest that Hs11S could show higher emulsifying
195
properties than 11S globulin from lentil and whey protein isolate.
196
3.2.2 ζ-potential and isoelectric point of Hs11S
197
The ζ-potential curve of Hs11S without NaCl exhibited a progressive increase of
198
the ζ-potential from a negative (-34.5 mV) to a positive value (+18.7 mV) from pH
199
10 to pH 2 (Fig. 2b). This behavior showed that zeta potential is pH dependent,
200
typically reported for others 11S globulins from mungbean, pea and A. thaliana
201
(cruciferin) (Tang and Sun, 2010; Klassen and Nickerson, 2012; Whitana-Gamage
202
et al., 2013) and explained as changes in the electrostatic repulsion may be
203
attributed at a gradual protonation of carboxyl groups and de-protonation of amino
204
groups of the proteins when pH medium goes from alkaline to acidic values (Tang
205
and Sun, 2011; Klassen and Nickerson, 2012). It is noteworthy the high potential
206
stability exhibited for Hs11S in the pH range of 6.5 − 10, where the zeta potential is
207
kept in a narrow range between -30.3 mV to -34 mV (considering -30 mV as the
208
dividing value between stable and unstable colloidal systems) (Whitana-Gamage et
209
al., 2013).
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210
Influence of NaCl on ζ-potential was observed. At both NaCl concentrations tested
211
(0.05 M and 0.50 M), the stability in the colloidal system measured in protein
212
solution by ζ-potential values were lower than those without salt in overall pH range
213
from 2 to 10 (Fig. 2b). It may be attributed to chemical composition influencing the
214
conductivity of the solutions and therefore the ζ-potential. The conformation of
215
Hs11S in solution was influenced by the pH, which affects the extent of ionized
216
residues available for the interaction and charge neutralization by H3O+, Na+ and
217
Cl- ions (Salgin et al., 2012; Whitana-Gamage et al., 2013).
218
The effect of NaCl on the pI of Hs11S solution is clearly shown in Fig. 2b. The
219
Hs11S solution without NaCl has a pI around 3.5, while the presence of NaCl
220
modified this value to 3 and was 2.5 when concentrations were 0.05 and 0.5 M,
221
respectively. These differences in the pI by addition of NaCl might be attributed to
222
structural changes in the superficial properties which could modify the protein food
223
functionality (Salgin et al., 2012).
224
Witana-Gamage et al. (2013) reported similar pI for native cruciferin at pH of 3.8
225
while the calculated with amino acid composition of the subunits differed greatly,
226
indicating that theoretical values do not always agree with experimental values.
227
3.3 Physicochemical characterization of Hs11S
228
3.3.1 Gelation capacity
229
The storage modulus (G’) values during heating of Hs11S dispersion are presented
230
in Figure 3a, showing a progressive increase of G’ indicating the formation of a gel
231
network (Rao, 2007). The onset of Hs11S gelation occurred at 74 – 76 °C
11
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232
determined by extrapolation of the rapidly rising values of G’. Generally, the onset
233
temperatures for the main protein gels ranges from 65 to 80 °C (Chambal et al.,
234
2014). Hs11S denaturation peak temperature was around 94 °C, moreover, onset
235
thermal denaturation is around 76 °C (Fig. 4). These data suggest detectable
236
structural changes of Hs11S about 74 – 76 °C, finishing at 94 °C with thermal
237
denaturation of Hs11S. During cooling, values of G’ and G” increased indicating
238
formation of gel network (Fig. 3b). The increase in moduli during cooling may be
239
attributed to reversible formation of hydrogen bonds and Van der Waals
240
interactions between protein molecules within the gel primary network (Shevkani et
241
al., 2015). Gel reinforcement during cooling had been observed in pea legumin,
242
cruciferin and soybean glycinin (O’Kane et al., 2004; Withana-Gamage et al.,
243
2015).
244
Magnitudes of G’ and G” are influenced by frequency, temperature and strain for a
245
specific food. The loss tangent (tan δ = G”/G’) is the ratio of the dissipated energy
246
to the stored energy per cycle of deformation and it plays important roles in the
247
rheology of structural gels (Rao, 2007). The tan δ values of Hs11S in the LVR
248
showed a value of 0.22 (Fig. 3c), indicating that G’ was always higher than G” over
249
the whole frequency interval tested. The tan δ values lesser than 1 (0.22 in our
250
case) are associated with the formation of strong gels (Rao, 2007; Carrazco-Peña
251
et al., 2013). Figure 3c shows G’ and G” values as a function of frequency sweep
252
(Hertz) in the LVR, where Hs11S showed G’ values from 843.3 to 1338 Pa.
253
Carrazco-Peña et al. (2013) reported gelation of amarantin with G’ values of ~5 Pa
12
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254
at 21.8 % protein solution in 35 mM potassium phosphate (pH 7.6). The gelation
255
profiles of Hs11S could be useful to improve the texture of many food products.
256
3.3.2 Thermodynamic properties
257
Heat treatment is one of the most important processes in food industry. The Tm can
258
reflect the temperature needed to get the disruption of hydrogen bonds and Van
259
der Waals interactions stabilizing the tertiary and quaternary conformations of
260
proteins (Kimura et al., 2008; Tang & Sun, 2011). As depicted in Table 1, Tm of
261
Hs11S was 94.1 ± 1 °C, exhibits high thermostability at low ionic strength (μ =
262
0.08) than others 11S globulins like fava bean, pea and glycinin, whose
263
denaturation temperatures vary from 81 ‒ 85ºC (Kimura et al., 2008). Moreover,
264
Hs11S shows an endothermic peak, indicating a transition of protein denaturation
265
with experimental enthalpy (∆HexpTm) of 44.49 ± 6 kJ/mol (Table 1). Likewise, the
266
entropy (∆STm) was obtained from ∆HexpTm and Tm values, being of 0.12 ± 0.01
267
kJ/mol•K. This result indicates a lower degree of disordered conformation for
268
thermal unfolding of Hs11S than other cruciferin variants (0.26 ‒ 0.47 kJ/mol•K)
269
reported by Whitana-Gamage et al. (2013).
270
Variations in heat flow occurring after denaturation for the purified proteins can be
271
defined in terms of specific heat capacity change (∆pN→U) (Oates and Ledward,
272
1991). This thermodynamic parameter shows positive values for β-conglycinin,
273
glycinin and cruciferins (Danilenko et al., 1985 ; Oates and Ledward, 1991;
274
Whitana-Gamage, et al., 2013). The ∆pN→U value for Hs11S was 0.17 ± 0.0
13
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275
kJ/mol•K, with a maximum heat capacity of denaturation (Cpmax) of 0.50 ± 0.13
276
kJ/mol•K, obtained at Tm (Table 1).
277
The van’t Hoff enthalpy is dependent on fractions of the native and denatured
278
forms of the proteins, and is associated with the temperature dependence on the
279
excess heat capacities (native and denatured) and enthalpies in simple two-state
280
transition (Privalov and Potekhin, 1986). The ΔHvHTm can be used to describe the
281
transition mode or cooperative unfolding of 11S globulins. Comparison of the
282
directly measured ΔHexpTm with ΔHvHTm provides information of a cooperative unit
283
during the denaturation process (Tandang et al., 2004). A ratio ΔHvHTm/ΔHexpTm > 1
284
indicates that each subunit works as a cooperative unit in the unfolding process as
285
well as formation of an oligomer, whereas ΔHvHTm/ΔHexpTm < 1 indicates the
286
presence
287
cooperativeness (Privalov and Potekhin, 1986; Tandang et al., 2004; Whitana-
288
Gamage et al., 2013). The ΔHvHTm calculated for Hs11S was 260.5 ± 31 kJ/mol
289
(Table 1). The ratio ΔHvHTm/ΔHexpTm determined was 0.18 ± 0.04 (Table 1). This
290
data was corroborated with the high width at half peak height (ΔT1/2, 15.32 ± 0.66
291
°C). A low ΔT1/2 value indicates a highly cooperative unfolding process, and high
292
ΔT1/2 refers a not cooperative process. Moreover, the ratio ΔHvHTm/ΔHexpTm is
293
different to ~1, therefore, each monomer of Hs11S is not considered as a
294
cooperative unit in the denaturation process, indicating existence of possible
295
folding intermediates during the unfolding process (Privalov and Potekhin, 1986;
296
Whitana-Gamage et al., 2013).
of
folding
intermediates
during
14
denaturation,
indicating
not
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297
The native cruciferin and glycinin have been reported as a cooperative unit in the
298
denaturation process (ΔHvHTm/ΔHexpTm > 1) (Danilenko et al., 1985, Whitana-
299
Gamage et al., 2013), whereas some cruciferins from mutant Arabidopsis line
300
(triple-knockout line, CRU-) and modified procruciferins expressed in E. coli
301
showed a not cooperative behavior reporting ratios ΔHvHTm/ΔHexpTm <1 (Tandang et
302
al., 2004; Whitana-Gamage et al., 2013).
303
3.3.3 Assembly capacity and aggregation/de-aggregation of Hs11S by DLS
304
During industrial processes, seed storage proteins have structural changes, and
305
they can be studied and understood throughout the association/dissociation,
306
assembly, and aggregation/de-aggregation analysis. The Hs11S assembly was
307
dependent on temperature and ionic strength at pH 7.6 (Fig. 5a, b and c). At low
308
ionic strength (Fig. 5a, without NaCl) Hs11S presented a Rh of 6.77 nm (280 kDa)
309
at temperatures of 30, 40, 45, 50 and 60 °C, where the hexameric structure form
310
was predominant. At 25 and 55 °C, Hs11S showed an Rh of 5.85 (208.4 kDa)
311
indicating tetrameric form, and was found as trimer (Rh 5.05, 157 kDa) and
312
dodecamer (Rh 9.08, 562.2 kDa) at 35 °C. At 0.1 M NaCl, Hs11S was observed in
313
two assemblies: trimeric and hexameric quaternary structures from 20 until 60 °C
314
(Fig. 5b), meanwhile, at 65 °C it was showed as a dodecamer. On the other hand,
315
Hs11S at 0.4 M NaCl has three oligomeric states: trimer (25, 35, 40 and 45 °C),
316
hexamer (20, 30, 50, 60 and 65 °C) and octamer at 55 °C (Rh 7.85, 390.4 kDa).
317
Hs11S assembly was dependent on temperature and ionic strength. Bojórquez-
318
Velázquez et al. (2016) reported trimeric, tetrameric, hexameric and dodecameric
15
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319
forms on Hs11S assembly by DLS studies at different pH values at a range from
320
20 – 30 °C.
321
On the other hand, higher ionic strength contributes to the hexameric assembly of
322
the 11S form at 65 °C of our chan globulin (aggregates without NaCl, dodecamer
323
at 0.1 M NaCl, and hexamer at 0.4 M) showing greater stability with increment of
324
NaCl. Molina et al. (2004) studied the effect of NaCl and pH in the thermal stability
325
of helianthinin, showing hexameric form at µ = 0.5, but it converts to a 15S form
326
when the ionic strength falls below µ = 0.1, phenomena observed in Hs11S at 65
327
°C. As well, 7-8 (González-Pérez and Vereijken, 2007), which coincides with the
328
main Hs11S assemblies found in this work at pH 7.6.
329
In overall tests, the particle size distribution also exhibited peaks with high
330
molecular weights, indicating an Hs11S aggregation (Rh higher than 100 nm) (Fig.
331
5a, b and c). The protein aggregation/de-aggregation phenomena is one of the
332
most important parameters for functional properties in protein-based foods;
333
moreover these conditions influence their rheological properties (Boulet et al.,
334
2000; Ruan et al., 2014).
335
At higher temperatures than 70 °C, an increase in the Rh of Hs11S in overall tests,
336
was detected as aggregation (Fig. 5a, b and c). Physicothermally, the onset
337
denaturation temperature of Hs11S detected by DSC measurements was 80 °C
338
(Fig. 4) and gelation point by rheological behavior was around 7 °C (Fig. 3a).
339
However, a DLS study is a sensitive technical with real-time measuring, which
340
detects slight changes in aggregation phenomena of Hs11S at lower temperatures
16
ACCEPTED MANUSCRIPT
341
than 75 °C that are not detectable in DSC and rheology measurements (Pizones
342
Ruiz-Henestrosa et al., 2012).
343
Some proteins as globulins from soybean are widely used in food applications as
344
an ingredient in the formation of heat-induced gels (Ruan et al., 2014). Therefore,
345
Hs11S could be proposed as an attractive ingredient for the food industry due to its
346
physicochemical
347
investigation.
and
functional
characteristics
17
reported
in
the
present
ACCEPTED MANUSCRIPT
349
Conclusions
350
The functionality of Hs11S was characterized in addition to its physicothermal
351
properties. A decrement in surface tension was registered with increment of protein
352
concentration, showing a CMC at 0.6 mg/mL. Hs11S solutions showed ζ-potentials
353
and pIs with a clear NaCl and pH dependence. Also Hs11S presented a good heat
354
stability and high thermal denaturation. The onset for the heat-induced gelation of
355
Hs11S occurred around 74 – 76 °C, developing strong gels. Hs11S exhibited
356
assemblies in several oligomeric forms. Our results indicate that Hs11S might be
357
used as a potential heat-stable ingredient to improve the hardness in food systems.
358 359
Acknowledgements
360
We thank student Fabiola Blanco-García for technical support in DLS experiments.
361
We also thank Dr. Abel Moreno for the providing of zetasizer Nano-ZS used and
362
technical support in the DLS experiments.
363 364
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365
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Withana-Gamage, T.S., Hegedus, D.D., Qiu, X., McIntosh, T., Wanasundara, J.P.
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460
heat-induced gelation and pepsin susceptibility of cruciferin protein as affected by
461
subunit composition. Food Biophysics 10, 103-115.
22
462
TABLES
463
Table 1. Physicothermal properties of Hs11S by DSC measurements*. Tm (ºC) Hs11S
464
ΔT1/2 (°C)
ΔSTm ΔCpN→U (kJ/mol•K) (kJ/mol•K)
94.10 ± 1.0 15.32 ± 0.66 0.12 ± 0.01 0.169 ± 0.0
* Heating from 20 to 150 °C, at flow rate 5 ºC/min.
465
23
Cpmax (kJ/mol•K)
ΔHExpTm (kJ/mol)
0.502 ± 0.02 44.49 ± 6
ΔHVHTm (kJ/mol)
ΔHExpTm/ ΔHVHTm
260.5 ± 31 0.18 ± 0.04
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FIGURE CAPTIONS
467
Figure 1. Purification of Hs11S.
468
a) Elution profile of Hs11S applied onto Sephacryl S-300.
469 470 471 472
b) SDS−PAGE profile of Hs11S. Line MW = molecular weight marker, line 1 and 2 = pure Hs11S in non-reducing conditions (4 µg and 16 µg, respectively), line 3 and 4 = Hs11S in reducing conditions using 2-mercaptoethanol (4 µg and 16 µg respectively). α and β are the acidic and basic polypeptides, respectively.
473
Figure 2. Functional characterization of Hs11S.
474 475
a) Critical micelle concentration measured by surface tension in HPLC-degree water at 25 ºC.
476 477
b) Effect of NaCl concentrations on zeta potential as function of pH. Zeta potential was measured at pH 2 - 10 with different NaCl concentrations.
478
Figure 3. Profile of Hs11S [20 % (w/v) in buffer B] gelation by oscillatory rheology.
479
a) Heating rate from 20 to 90 ºC, at a flow rate of 1 ºC/min.
480
b) Cooling rate from 90 to 20 ºC, at a flow rate of 5 ºC/min.
481
c) Frequency sweep in linear viscoelastic region (1 - 100 Hz) at 20 ºC.
482 483
Figure 4. Differential scanning calorimetry profile of Hs11S. Heating from 20 to 150 °C, at a flow rate 5 °C/min.
484 485 486
Figure 5. Effect of temperature and NaCl concentrations on assembly capacity and aggregation/de-aggregation of Hs11S in buffer C by DLS. A) without NaCl, B) 0.1 M NaCl, C) 0.4 M NaCl.
487
24
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488 489 490
FIGURES Figure 1
491 492 493 494 495 496 497 498 499 500 501
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Figure 2
503 504 505 506 507 508 509 510 511 512 513 514 515
26
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Figure 3
517 518
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Figure 4
520 521 522 523 524 525 526 527 528 529
28
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530 531
Figure 5
532 533
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1) Hs11S had a surface activity until 55.5 mN/m and a CMC value of 0.6 mg/mL. 2) Different isoelectric points (2, 3 and 3.5) were revealed in Hs11S with NaCl variations. 3) The denaturation temperature of the Hs11S was 94.1 °C, with an enthalpy of 44.49 kJ/mol. 4) Hs11S showed gelation temperature around 74–76°C and strong gels (~1000 Pa). 5) Hs11S exhibited assemblies in several oligomeric forms, predominating trimer, tetramer, hexamer and aggregates.
Luis F. De la Cruz-Torres, Jaime D. Pérez-Martínez, Mayra Sánchez-Becerril, Jorge F. Toro-Vázquez, N. Alejandra Mancilla-Margalli, Juan A. Osuna-Castro, C.I. VillaVelázquezMendoza PII:
S0733-5210(16)30483-0
DOI:
10.1016/j.jcs.2017.06.017
Reference:
YJCRS 2390
To appear in:
Journal of Cereal Science
Received Date:
25 November 2016
Revised Date:
08 June 2017
Accepted Date:
27 June 2017
Please cite this article as: Luis F. De la Cruz-Torres, Jaime D. Pérez-Martínez, Mayra SánchezBecerril, Jorge F. Toro-Vázquez, N. Alejandra Mancilla-Margalli, Juan A. Osuna-Castro, C.I. VillaVelázquez-Mendoza, Physicochemical and functional properties of 11S globulin from chan ( Hyptis suaveolens L. poit) seeds, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.06.017
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ACCEPTED MANUSCRIPT
1
Physicochemical and functional properties of 11S globulin from chan (Hyptis
2
suaveolens L. poit) seeds
3
Luis F. De la Cruz-Torresa, Jaime D. Pérez-Martínezb, Mayra Sánchez-Becerrilb,
4
Jorge F. Toro-Vázquezb, N. Alejandra Mancilla-Margallic, Juan A. Osuna-Castroa,*,
5
C.I. VillaVelázquez-Mendozad
6 7 8 9 10 11 12 13
aFacultad
de Ciencias Biológicas y Agropecuarias, Universidad de Colima, km 40
Autopista Colima-Manzanillo, C.P. 28100, Tecomán, Colima, México bFacultad
de Ciencias Químicas-CIEP, Universidad Autónoma de San Luis Potosí,
Manuel Nava 6, Zona Universitaria, C.P. 28210, San Luis Potosí, S. L. P., México cTecnológico
Nacional de México, Instituto Tecnológico de Tlajomulco, Carr. a San
Miguel Cuyutlán km 10, Tlajomulco de Zúñiga, Jalisco C.P. 45650, México dFacultad
de Ingeniería Civil, Universidad de Colima, Carr. Colima-Coquimatlán km 9, C.P. 28400, Coquimatlán, Colima, México
14
*Corresponding Author:
15
Email: [email protected]
16 17 18
1
ACCEPTED MANUSCRIPT
19 20
Abstract
21
The 11S globulin is one of the most abundant and important storage proteins of the
22
chan grain (Hs11S). For this reason, we investigated its physicochemical and
23
functional properties (surface tension, zeta potential and thermo-mechanics
24
analysis). A decrement in surface tension was registered with an increment of
25
Hs11S concentration, such results suggested a critical micelle concentration
26
(CMC) at 0.6 mg/mL and a surface activity until 55.5 mN/m. The Hs11S solutions
27
showed isoelectric points (pI) with a clear NaCl influence (i.e., without NaCl pI =
28
3.5, at 0.05 M NaCl pI = 3, at 0.5 M NaCl pI = 2.5) and zeta potential dependent
29
upon the pH. Differential scanning calorimetry and rheology studies revealed the
30
onset for the heat-induced gelation of Hs11S which occurred around 74‒76 °C,
31
developing strong gels with an elasticity of ~103 Pa, a denaturation temperature of
32
94.1 °C and an enthalpy of 266.9 kJ/mol. Consequently, the Hs11S thermal
33
denaturation is not a cooperative process. According to the dynamic light scattering
34
(DLS) data as a function of temperature, the Hs11S organizes in oligomeric forms
35
of trimer, tetramer, hexamer, octamer and dodecamer as well as aggregates (Rh
36
higher than 100 nm).
37
Keywords
38
11S Globulin, Functional properties, Heat-induced gelation, Thermodynamic
39
properties
40
2
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41
Introduction
42
Globulins (salt-soluble storage proteins) are the main Osborne protein fraction in
43
legumes and some cereals such as oats and rice, constituting around 70 ‒ 80 % of
44
the total protein (Boulter and Croy, 1997). The 11S globulins (legumins) are
45
hexameric proteins of 300 ‒ 450 kDa, composed by six non-covalently bonded
46
subunits of approximately 50 ‒ 70 kDa, with an acidic polypeptide and a basic
47
polypeptide of about 30 ‒ 40 kDa and ~20 kDa, respectively, and linked by
48
disulfide bonds (Boulter and Croy, 1997).
49
The 11S globulins from different sources such as amaranth, sunflower, soybean
50
and mungbean have been purified and physicochemical and functionally
51
characterized for their use in the food industry for the development of traditional
52
and new food products (González-Pérez and Vereijken, 2007; Kimura et al., 2008;
53
Tang and Sun, 2010; Carrazco-Peña et al., 2013). On the other hand, the
54
population growth and the emerging dietary preferences (e.g., vegans,
55
vegetarians) demand for novel food ingredients and plant-based products with
56
lower cost; therefore 11S globulins from non-conventional food crops are attractive
57
options (Jarpa-Parra et al., 2015).
58
Chan (Hyptis suaveolens L. Poit) is a dicotyledonous plant from the Lamiaceae
59
family, considered as a pseudocereal and its seeds were cultivated and highly
60
appreciated since pre-Colombian cultures due to its high nutritional and medicinal
61
characteristics (Aguirre et al., 2004). Hyptis suaveolens has been used as food and
3
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62
traditional medicine in America, Asia and Africa (Aguirre et al., 2012). Chan seeds
63
contain 13.9 % of protein on dry weight being globulins (39 %) the major fraction.
64
Bojórquez-Velázquez et al. (2016) reported a simple purification procedure and
65
biochemical characterization of 11S globulin from chan. This is a hexameric protein
66
of 300 kDa, composed by four different monomers between 53.5 ‒ 49.5 kDa with
67
the characteristic disulfide bond linking the acidic and basic polypeptides.
68
However, required information about the physicochemical and functional properties
69
of Hs11S in order to use in foods remains unknown.
70
Within this context, the objective of this investigation is to evaluate the following
71
physicochemical and functional properties of Hs11S: surface tension and zeta
72
potential as a function of pH and NaCl concentration, thermo-mechanical
73
properties and aggregation/de-aggregation process as a function of temperature
74
and ionic strength.
75 76
Materials and Methods
77
2.1 Purification of Hs11S
78
2.1.1 Protein extraction
79
Seeds were purchased from a local market at Colima, Mexico. Fractionation of
80
globulins was carried out according to Bojórquez-Velázquez et al. (2016). The
81
pellet obtained after albumin extraction was suspended in buffer A (0.1 M Tris-HCl,
4
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82
10 mM EDTA, 1.7 M NaCl, pH 7.5) 1:10 (w/v), stirred 2 h at 4 °C, and centrifuged
83
30 min at 13,800 g. Supernatant (crude globulins) was stored at -20 ºC.
84
2.1.2 Hs11S purification
85
10 mL of crude globulins were precipitated at 60 % of ammonium sulphate
86
saturation,
87
chromatography (SEC) using a Sephacryl S-300 HR column (GE Healthcare
88
Bioscience, NJ, USA). Proteins were eluted with buffer A at 1 mL/min flow rate.
89
Fractions of 2.5 mL were analyzed by absorbance at 280 nm, dialyzed, freeze-
90
dried, resolved in 12 % SDS-PAGE (Laemmli, 1970) and then stored at 4 °C for
91
further analysis.
92
2.1.4 Protein measurement
93
The protein content was determined by the Bradford method (Bradford, 1976)
94
using bovine serum albumin as standard.
95
2.2 Functional characterization Hs11S
96
2.2.1 Surface activity and CMC
97
A DuNouy tensiometer (CSC Scientific Company, INC USA) was used to measure
98
the surface tension of Hs11S solutions by the method of Tomczyńska-Mleko et al.
99
(2014) with some modifications. Protein was dissolved in HPLC-degree water at a
100
concentration range from 0.1 to 1 mg/mL (0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1.0
101
mg/mL). All measurements were carried out at 25 °C. The CMC of Hs11S was
resuspended
in
buffer
A
5
and
subjected
to
size-exclusion
ACCEPTED MANUSCRIPT
102
determined from the graph of interfacial tension vs. natural logarithm of protein
103
concentration.
104
2.2.2 Zeta potential and isoelectric point
105
The zeta potential (ζ-potential) of Hs11S was measured at 25 °C as a function of
106
pH using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, Worcestershire,
107
UK) according to the modified method of Withana-Gamage et al. (2013). The pH
108
adjustments were performed with 0.5 M and 0.005 M HCl and 0.1 NaOH aqueous
109
solutions using an MPT-2 autotitrator (Malvern Instruments. Worcestershire,
110
Ukraine). Protein dispersions of 0.02 % in deionized water were prepared. Three
111
different treatments with NaCl were tested (0.05 M, 0.5 M and without NaCl). The
112
pH value of zero ζ-potential was considered as the pI of the protein.
113
2.3 Hs11S physicochemical characterization
114
2.3.1 Heat-induced gelation by oscillatory rheology
115
The gelation of Hs11S was evaluated by oscillatory rheology following the method
116
proposed by Shevkani et al. (2015). Measurements were determined in a MCR 302
117
rheometer (Anton Paar, Stuttgart, Germany) with plate-plate measuring geometry
118
(25 mm in diameter, PP25/TG, Anton-Paar, Germany) equipped with the TruGapTM
119
system. The temperature control was performed with two peltier systems located at
120
the base of the lower plate and a peltier hood covering the measuring geometry (C-
121
PDT200, Anton-Paar, Germany). The Hs11S solutions were prepared at 20 %
122
(w/v) in buffer B (35 mM KH2PO4-K2HPO4, 0.1 M NaCl, pH 7.6). Samples were
123
heated at 1 °C/min from 20 to 90 °C, held at 90 °C for 3 min and then cooled from
6
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90 to 20 °C (5 °C/min). To prevent sample dehydration, the edge of the plate was
125
covered with low viscosity silicone. The elastic (G′) and loss (G″) moduli of the gel
126
were obtained as function of temperature (Shevkani et al., 2015). A frequency
127
sweep (1 ‒ 100 Hz) at constant strain of 0.002 % was performed on the gel
128
subjected to the thermal treatment previously described. For each sample solution
129
and the different stages of the test, measurements were done within the linear
130
viscoelastic region (LVR). The instrument uses the Rheoplus/32 v3.61, Anton Paar
131
(MCR 302) software.
132
2.3.2 Thermal characterization by differential scanning calorimetry (DSC)
133
Thermal denaturalization of Hs11S was established by differential scanning
134
calorimetry using a DSC discovery (TA Instruments, USA). Protein dispersion at 25
135
% (w/v) in buffer C (35 mM KH2PO4-K2HPO4, pH 7.6) was prepared and 15 mg of
136
mixture were weighed in T-ZeroTM pan, hermetically sealed and hydrated in the
137
same buffer for 2 h prior to the test. The sample pan was loaded in the calorimeter
138
and equilibrated for 5 min at 20 °C, heated from 20 to 150 °C at 5 °C/min. A sealed
139
empty pan was used as a reference. The analysis of the thermograms was carried
140
out according to Withana-Gamage et al. (2013) for the calculation of the
141
calorimetric van’t Hoff enthalpy change (ΔHvHTm) and the entropy change upon
142
(ΔSTm). The denaturation peak temperature (Tm), width at half peak height (ΔT1/2),
143
maximum heat capacity at denaturation (Cpmax), denaturation enthalpy (ΔHexpTm)
144
and heat capacity change (ΔCpN→U) were computed with the DSC Discovery
145
instrument software (TRIOS Q111 C53 V.52 2002).
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2.3.4 Assembly capacity by dynamic light scattering (DLS)
147
The effect of NaCl and temperature on Hs11S was studied measuring the
148
hydrodynamic radius (Rh) (molecular size) by DLS using a zetasizer Nano-ZS
149
(Malvern Instrument Ltd., Malvern, Worcestershire, UK) with a light source from
150
633 nm He–Ne laser. Measurements were performed at a scattering angle of 173°.
151
Protein dispersions at 0.2 mg/mL in buffer C with different NaCl concentrations (0.1
152
M, 0.4 M and without NaCl) were measured from 10 to 90 °C (sampling every 5
153
°C).
154
2.4 Statistical analysis
155
Assembly capacity was carried out ten times each experiment, while surface
156
tension and zeta potential experiments were conducted in quadruplicate and
157
calorimetric properties in triplicate. The data were expressed as the mean ±
158
standard error (SE). Gelation capacity was obtained in duplicate each experiment.
159 160
Results and discussion
161
3.1 Hs11S purification and SDS-PAGE analysis
162
Hs11S was purified by size-exclusion chromatography using a Sephacryl S-300.
163
This protein was eluted from 40 until 62 fraction number (Fig. 1a), and its purity
164
was corroborated by 12 % SDS-PAGE (Fig. 1b). In non-reducing conditions,
165
Hs11S showed four bands with estimated molecular weight of 50 kDa; meanwhile,
166
under reducing conditions, four acidic (α) subunits of around 32 kDa and four basic
8
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167
(β) subunits of approximately 20 kDa were shown. These data confirm the first
168
report of the Hs11S purification (Bojórquez-Velázquez et al., 2016). The purified
169
protein was used for the following analyses.
170
3.2 Functional characterization of Hs11S
171
3.2.1 Surface activity and CMC
172
As shown in Figure 2a, the surface tension of Hs11S aqueous solutions decreased
173
from 68 mN/m to 55.5 mN/m as protein concentration increased from 0.1 mg/mL to
174
0.6 mg/mL. This behavior has also been reported at low protein concentration of
175
whey protein isolates (Tomczyńska-Mleko et al., 2014) and seed storage proteins
176
like a soybean 7S globulin (β-conglycinin), 11S globulin (glycinin) and 11S globulin
177
from lentil (Pizones Ruiz-Henestrosa et al., 2014; Jarpa-Parra et al., 2015). The
178
surface tension decrement with increasing protein concentration is due to the
179
preferential accumulation of proteins at the air-water interface and the exposition of
180
hydrophobic residues (Jarpa-Parra et al., 2015); therefore, the adsorption capacity
181
is influenced by some intrinsic factors of the protein such as its molecular size,
182
structure, hydrophobicity and solubility (Pizones Ruiz-Henestrosa et al., 2014).
183
At Hs11S concentrations higher than 0.6 mg/mL the surface tension remained
184
constant, since the system achieved the formation of a fully packed monolayer and
185
no further accumulation of protein at the interface took place. Thus, the CMC for
186
the Hs11S occurred at 0.6 mg/mL and it was considered the minimum protein
187
concentration above which stable micelles are formed (Tomczyńska-Mleko et al.,
188
2014). The surface tension can be affected in addition by extrinsic variables such
9
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189
as temperature, pH, ionic strength, surfactant concentration, and preheating of the
190
sample (Tomczyńska-Mleko et al., 2014) therefore, the CMC might be affected
191
equally.
192
A CMC of 5 mg/mL in 11S globulin from lentil using the ring method (Jarpa-Parra
193
et al., 2015) and in whey protein isolate has been reported (Tomczyńska-Mleko et
194
al., 2014). Our results suggest that Hs11S could show higher emulsifying
195
properties than 11S globulin from lentil and whey protein isolate.
196
3.2.2 ζ-potential and isoelectric point of Hs11S
197
The ζ-potential curve of Hs11S without NaCl exhibited a progressive increase of
198
the ζ-potential from a negative (-34.5 mV) to a positive value (+18.7 mV) from pH
199
10 to pH 2 (Fig. 2b). This behavior showed that zeta potential is pH dependent,
200
typically reported for others 11S globulins from mungbean, pea and A. thaliana
201
(cruciferin) (Tang and Sun, 2010; Klassen and Nickerson, 2012; Whitana-Gamage
202
et al., 2013) and explained as changes in the electrostatic repulsion may be
203
attributed at a gradual protonation of carboxyl groups and de-protonation of amino
204
groups of the proteins when pH medium goes from alkaline to acidic values (Tang
205
and Sun, 2011; Klassen and Nickerson, 2012). It is noteworthy the high potential
206
stability exhibited for Hs11S in the pH range of 6.5 − 10, where the zeta potential is
207
kept in a narrow range between -30.3 mV to -34 mV (considering -30 mV as the
208
dividing value between stable and unstable colloidal systems) (Whitana-Gamage et
209
al., 2013).
10
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210
Influence of NaCl on ζ-potential was observed. At both NaCl concentrations tested
211
(0.05 M and 0.50 M), the stability in the colloidal system measured in protein
212
solution by ζ-potential values were lower than those without salt in overall pH range
213
from 2 to 10 (Fig. 2b). It may be attributed to chemical composition influencing the
214
conductivity of the solutions and therefore the ζ-potential. The conformation of
215
Hs11S in solution was influenced by the pH, which affects the extent of ionized
216
residues available for the interaction and charge neutralization by H3O+, Na+ and
217
Cl- ions (Salgin et al., 2012; Whitana-Gamage et al., 2013).
218
The effect of NaCl on the pI of Hs11S solution is clearly shown in Fig. 2b. The
219
Hs11S solution without NaCl has a pI around 3.5, while the presence of NaCl
220
modified this value to 3 and was 2.5 when concentrations were 0.05 and 0.5 M,
221
respectively. These differences in the pI by addition of NaCl might be attributed to
222
structural changes in the superficial properties which could modify the protein food
223
functionality (Salgin et al., 2012).
224
Witana-Gamage et al. (2013) reported similar pI for native cruciferin at pH of 3.8
225
while the calculated with amino acid composition of the subunits differed greatly,
226
indicating that theoretical values do not always agree with experimental values.
227
3.3 Physicochemical characterization of Hs11S
228
3.3.1 Gelation capacity
229
The storage modulus (G’) values during heating of Hs11S dispersion are presented
230
in Figure 3a, showing a progressive increase of G’ indicating the formation of a gel
231
network (Rao, 2007). The onset of Hs11S gelation occurred at 74 – 76 °C
11
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232
determined by extrapolation of the rapidly rising values of G’. Generally, the onset
233
temperatures for the main protein gels ranges from 65 to 80 °C (Chambal et al.,
234
2014). Hs11S denaturation peak temperature was around 94 °C, moreover, onset
235
thermal denaturation is around 76 °C (Fig. 4). These data suggest detectable
236
structural changes of Hs11S about 74 – 76 °C, finishing at 94 °C with thermal
237
denaturation of Hs11S. During cooling, values of G’ and G” increased indicating
238
formation of gel network (Fig. 3b). The increase in moduli during cooling may be
239
attributed to reversible formation of hydrogen bonds and Van der Waals
240
interactions between protein molecules within the gel primary network (Shevkani et
241
al., 2015). Gel reinforcement during cooling had been observed in pea legumin,
242
cruciferin and soybean glycinin (O’Kane et al., 2004; Withana-Gamage et al.,
243
2015).
244
Magnitudes of G’ and G” are influenced by frequency, temperature and strain for a
245
specific food. The loss tangent (tan δ = G”/G’) is the ratio of the dissipated energy
246
to the stored energy per cycle of deformation and it plays important roles in the
247
rheology of structural gels (Rao, 2007). The tan δ values of Hs11S in the LVR
248
showed a value of 0.22 (Fig. 3c), indicating that G’ was always higher than G” over
249
the whole frequency interval tested. The tan δ values lesser than 1 (0.22 in our
250
case) are associated with the formation of strong gels (Rao, 2007; Carrazco-Peña
251
et al., 2013). Figure 3c shows G’ and G” values as a function of frequency sweep
252
(Hertz) in the LVR, where Hs11S showed G’ values from 843.3 to 1338 Pa.
253
Carrazco-Peña et al. (2013) reported gelation of amarantin with G’ values of ~5 Pa
12
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254
at 21.8 % protein solution in 35 mM potassium phosphate (pH 7.6). The gelation
255
profiles of Hs11S could be useful to improve the texture of many food products.
256
3.3.2 Thermodynamic properties
257
Heat treatment is one of the most important processes in food industry. The Tm can
258
reflect the temperature needed to get the disruption of hydrogen bonds and Van
259
der Waals interactions stabilizing the tertiary and quaternary conformations of
260
proteins (Kimura et al., 2008; Tang & Sun, 2011). As depicted in Table 1, Tm of
261
Hs11S was 94.1 ± 1 °C, exhibits high thermostability at low ionic strength (μ =
262
0.08) than others 11S globulins like fava bean, pea and glycinin, whose
263
denaturation temperatures vary from 81 ‒ 85ºC (Kimura et al., 2008). Moreover,
264
Hs11S shows an endothermic peak, indicating a transition of protein denaturation
265
with experimental enthalpy (∆HexpTm) of 44.49 ± 6 kJ/mol (Table 1). Likewise, the
266
entropy (∆STm) was obtained from ∆HexpTm and Tm values, being of 0.12 ± 0.01
267
kJ/mol•K. This result indicates a lower degree of disordered conformation for
268
thermal unfolding of Hs11S than other cruciferin variants (0.26 ‒ 0.47 kJ/mol•K)
269
reported by Whitana-Gamage et al. (2013).
270
Variations in heat flow occurring after denaturation for the purified proteins can be
271
defined in terms of specific heat capacity change (∆pN→U) (Oates and Ledward,
272
1991). This thermodynamic parameter shows positive values for β-conglycinin,
273
glycinin and cruciferins (Danilenko et al., 1985 ; Oates and Ledward, 1991;
274
Whitana-Gamage, et al., 2013). The ∆pN→U value for Hs11S was 0.17 ± 0.0
13
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275
kJ/mol•K, with a maximum heat capacity of denaturation (Cpmax) of 0.50 ± 0.13
276
kJ/mol•K, obtained at Tm (Table 1).
277
The van’t Hoff enthalpy is dependent on fractions of the native and denatured
278
forms of the proteins, and is associated with the temperature dependence on the
279
excess heat capacities (native and denatured) and enthalpies in simple two-state
280
transition (Privalov and Potekhin, 1986). The ΔHvHTm can be used to describe the
281
transition mode or cooperative unfolding of 11S globulins. Comparison of the
282
directly measured ΔHexpTm with ΔHvHTm provides information of a cooperative unit
283
during the denaturation process (Tandang et al., 2004). A ratio ΔHvHTm/ΔHexpTm > 1
284
indicates that each subunit works as a cooperative unit in the unfolding process as
285
well as formation of an oligomer, whereas ΔHvHTm/ΔHexpTm < 1 indicates the
286
presence
287
cooperativeness (Privalov and Potekhin, 1986; Tandang et al., 2004; Whitana-
288
Gamage et al., 2013). The ΔHvHTm calculated for Hs11S was 260.5 ± 31 kJ/mol
289
(Table 1). The ratio ΔHvHTm/ΔHexpTm determined was 0.18 ± 0.04 (Table 1). This
290
data was corroborated with the high width at half peak height (ΔT1/2, 15.32 ± 0.66
291
°C). A low ΔT1/2 value indicates a highly cooperative unfolding process, and high
292
ΔT1/2 refers a not cooperative process. Moreover, the ratio ΔHvHTm/ΔHexpTm is
293
different to ~1, therefore, each monomer of Hs11S is not considered as a
294
cooperative unit in the denaturation process, indicating existence of possible
295
folding intermediates during the unfolding process (Privalov and Potekhin, 1986;
296
Whitana-Gamage et al., 2013).
of
folding
intermediates
during
14
denaturation,
indicating
not
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297
The native cruciferin and glycinin have been reported as a cooperative unit in the
298
denaturation process (ΔHvHTm/ΔHexpTm > 1) (Danilenko et al., 1985, Whitana-
299
Gamage et al., 2013), whereas some cruciferins from mutant Arabidopsis line
300
(triple-knockout line, CRU-) and modified procruciferins expressed in E. coli
301
showed a not cooperative behavior reporting ratios ΔHvHTm/ΔHexpTm <1 (Tandang et
302
al., 2004; Whitana-Gamage et al., 2013).
303
3.3.3 Assembly capacity and aggregation/de-aggregation of Hs11S by DLS
304
During industrial processes, seed storage proteins have structural changes, and
305
they can be studied and understood throughout the association/dissociation,
306
assembly, and aggregation/de-aggregation analysis. The Hs11S assembly was
307
dependent on temperature and ionic strength at pH 7.6 (Fig. 5a, b and c). At low
308
ionic strength (Fig. 5a, without NaCl) Hs11S presented a Rh of 6.77 nm (280 kDa)
309
at temperatures of 30, 40, 45, 50 and 60 °C, where the hexameric structure form
310
was predominant. At 25 and 55 °C, Hs11S showed an Rh of 5.85 (208.4 kDa)
311
indicating tetrameric form, and was found as trimer (Rh 5.05, 157 kDa) and
312
dodecamer (Rh 9.08, 562.2 kDa) at 35 °C. At 0.1 M NaCl, Hs11S was observed in
313
two assemblies: trimeric and hexameric quaternary structures from 20 until 60 °C
314
(Fig. 5b), meanwhile, at 65 °C it was showed as a dodecamer. On the other hand,
315
Hs11S at 0.4 M NaCl has three oligomeric states: trimer (25, 35, 40 and 45 °C),
316
hexamer (20, 30, 50, 60 and 65 °C) and octamer at 55 °C (Rh 7.85, 390.4 kDa).
317
Hs11S assembly was dependent on temperature and ionic strength. Bojórquez-
318
Velázquez et al. (2016) reported trimeric, tetrameric, hexameric and dodecameric
15
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319
forms on Hs11S assembly by DLS studies at different pH values at a range from
320
20 – 30 °C.
321
On the other hand, higher ionic strength contributes to the hexameric assembly of
322
the 11S form at 65 °C of our chan globulin (aggregates without NaCl, dodecamer
323
at 0.1 M NaCl, and hexamer at 0.4 M) showing greater stability with increment of
324
NaCl. Molina et al. (2004) studied the effect of NaCl and pH in the thermal stability
325
of helianthinin, showing hexameric form at µ = 0.5, but it converts to a 15S form
326
when the ionic strength falls below µ = 0.1, phenomena observed in Hs11S at 65
327
°C. As well, 7-8 (González-Pérez and Vereijken, 2007), which coincides with the
328
main Hs11S assemblies found in this work at pH 7.6.
329
In overall tests, the particle size distribution also exhibited peaks with high
330
molecular weights, indicating an Hs11S aggregation (Rh higher than 100 nm) (Fig.
331
5a, b and c). The protein aggregation/de-aggregation phenomena is one of the
332
most important parameters for functional properties in protein-based foods;
333
moreover these conditions influence their rheological properties (Boulet et al.,
334
2000; Ruan et al., 2014).
335
At higher temperatures than 70 °C, an increase in the Rh of Hs11S in overall tests,
336
was detected as aggregation (Fig. 5a, b and c). Physicothermally, the onset
337
denaturation temperature of Hs11S detected by DSC measurements was 80 °C
338
(Fig. 4) and gelation point by rheological behavior was around 7 °C (Fig. 3a).
339
However, a DLS study is a sensitive technical with real-time measuring, which
340
detects slight changes in aggregation phenomena of Hs11S at lower temperatures
16
ACCEPTED MANUSCRIPT
341
than 75 °C that are not detectable in DSC and rheology measurements (Pizones
342
Ruiz-Henestrosa et al., 2012).
343
Some proteins as globulins from soybean are widely used in food applications as
344
an ingredient in the formation of heat-induced gels (Ruan et al., 2014). Therefore,
345
Hs11S could be proposed as an attractive ingredient for the food industry due to its
346
physicochemical
347
investigation.
and
functional
characteristics
17
reported
in
the
present
ACCEPTED MANUSCRIPT
349
Conclusions
350
The functionality of Hs11S was characterized in addition to its physicothermal
351
properties. A decrement in surface tension was registered with increment of protein
352
concentration, showing a CMC at 0.6 mg/mL. Hs11S solutions showed ζ-potentials
353
and pIs with a clear NaCl and pH dependence. Also Hs11S presented a good heat
354
stability and high thermal denaturation. The onset for the heat-induced gelation of
355
Hs11S occurred around 74 – 76 °C, developing strong gels. Hs11S exhibited
356
assemblies in several oligomeric forms. Our results indicate that Hs11S might be
357
used as a potential heat-stable ingredient to improve the hardness in food systems.
358 359
Acknowledgements
360
We thank student Fabiola Blanco-García for technical support in DLS experiments.
361
We also thank Dr. Abel Moreno for the providing of zetasizer Nano-ZS used and
362
technical support in the DLS experiments.
363 364
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365
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460
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461
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22
462
TABLES
463
Table 1. Physicothermal properties of Hs11S by DSC measurements*. Tm (ºC) Hs11S
464
ΔT1/2 (°C)
ΔSTm ΔCpN→U (kJ/mol•K) (kJ/mol•K)
94.10 ± 1.0 15.32 ± 0.66 0.12 ± 0.01 0.169 ± 0.0
* Heating from 20 to 150 °C, at flow rate 5 ºC/min.
465
23
Cpmax (kJ/mol•K)
ΔHExpTm (kJ/mol)
0.502 ± 0.02 44.49 ± 6
ΔHVHTm (kJ/mol)
ΔHExpTm/ ΔHVHTm
260.5 ± 31 0.18 ± 0.04
ACCEPTED MANUSCRIPT
466
FIGURE CAPTIONS
467
Figure 1. Purification of Hs11S.
468
a) Elution profile of Hs11S applied onto Sephacryl S-300.
469 470 471 472
b) SDS−PAGE profile of Hs11S. Line MW = molecular weight marker, line 1 and 2 = pure Hs11S in non-reducing conditions (4 µg and 16 µg, respectively), line 3 and 4 = Hs11S in reducing conditions using 2-mercaptoethanol (4 µg and 16 µg respectively). α and β are the acidic and basic polypeptides, respectively.
473
Figure 2. Functional characterization of Hs11S.
474 475
a) Critical micelle concentration measured by surface tension in HPLC-degree water at 25 ºC.
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b) Effect of NaCl concentrations on zeta potential as function of pH. Zeta potential was measured at pH 2 - 10 with different NaCl concentrations.
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Figure 3. Profile of Hs11S [20 % (w/v) in buffer B] gelation by oscillatory rheology.
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a) Heating rate from 20 to 90 ºC, at a flow rate of 1 ºC/min.
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b) Cooling rate from 90 to 20 ºC, at a flow rate of 5 ºC/min.
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c) Frequency sweep in linear viscoelastic region (1 - 100 Hz) at 20 ºC.
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Figure 4. Differential scanning calorimetry profile of Hs11S. Heating from 20 to 150 °C, at a flow rate 5 °C/min.
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Figure 5. Effect of temperature and NaCl concentrations on assembly capacity and aggregation/de-aggregation of Hs11S in buffer C by DLS. A) without NaCl, B) 0.1 M NaCl, C) 0.4 M NaCl.
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FIGURES Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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1) Hs11S had a surface activity until 55.5 mN/m and a CMC value of 0.6 mg/mL. 2) Different isoelectric points (2, 3 and 3.5) were revealed in Hs11S with NaCl variations. 3) The denaturation temperature of the Hs11S was 94.1 °C, with an enthalpy of 44.49 kJ/mol. 4) Hs11S showed gelation temperature around 74–76°C and strong gels (~1000 Pa). 5) Hs11S exhibited assemblies in several oligomeric forms, predominating trimer, tetramer, hexamer and aggregates.