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Determination of molecular driving forces involved in heat-induced corn germ proteins gelation
Journal of Cereal Science 66 (2015) 24e30
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Determination of molecular driving forces involved in heat-induced corn germ proteins gelation Xiang Dong Sun a, *, Yu Lan c, Dan Shi c, Shu Wen Lu b, Hui Liao a, Rui Ying Zhang a, Xin Miao Yao b, Ying Lei Zhang b, Ping Su a, Hong Shan a a b c
Quality & Safety Institute of Agricultural Products, Heilongjiang Academy of Agricultural Science, Harbin 150086, China Food Processing Institute, Heilongjiang Academy of Agricultural Science, Harbin 150086, China Food Science Department, East University of Heilongjiang, Harbin 150086, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 May 2015 Received in revised form 9 September 2015 Accepted 16 September 2015 Available online 24 September 2015
Molecular forces involved in corn germ proteins (CGP) gelation were investigated using a rheometer. With the inclusion of urea and guanidine hydrochloride (GuHCl), the storage moduli (G0 ) of CGP gels tends to decrease. This is an indication that hydrophobic interactions and hydrogen bonds are present. The involvement of propylene glycol (PG) provides evidence that hydrogen bonds and electrostatic interactions are present. The impact of thiocyanate (NaSCN) and sodium sulfate (Na2SO4) further proves the involvement of hydrogen bonds. The effects of 2-mercaptoethanol (2-ME) and N-ethylmaleimide (NEM) reveal that involvement of disulfide bonds contribute to CGP gel stiffness. Reheating and recooling tests revealed that during the initial cooling phase the gel is thermally reversible and higher levels of G0 suggested more hydrogen bonds are formed when recooled. It can be deduced that hydrogen bonds play a greater role than hydrophobic interactions from the increasing G0 during cooling and recooling. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Corn germ protein Storage modulus Molecular forces Gelation Reagents
1. Introduction Besides the predominant constituent oil, corn germ contains 18e22% proteins, and these proteins account for around 29% of the total proteins in corn seeds (Watson, 1987). Water-soluble albumins plus saline-soluble globulins account for more than 60% of corn germ proteins (CGP) (Wilson, 1987). These proteins are proven to possess a good balance of amino acids, which presents a superior nutritional value to endosperm protein (Wilson, 1987) and have better functional properties than corn endosperm proteins. Corn germ is generally used to extract oil through solvent extraction and high temperature processing, which results in serious denaturation of the residual protein; these denatured proteins almost completely lose their functional properties (Kinsella, 1979), and thus are mainly used as animal feed. Due to increasing demand for high quality proteins with good functional characteristics in the food industry, researchers have been attempting to recover proteins from various plant sources. Corm germ proteins seem to be a good alternative for soy proteins
* Corresponding author. E-mail address: [email protected] (X.D. Sun). http://dx.doi.org/10.1016/j.jcs.2015.09.007 0733-5210/© 2015 Elsevier Ltd. All rights reserved.
and some workers have published their works on the extraction procedure and functional properties evaluation of corn germ proteins (Mila and Hojilla-Evangelista, 2012). The mechanism of protein gelation is described as a threedimensional network formation through the cross-linking of its polypeptide chains. Cross linking of protein polypeptide chains is driven by different molecular forces which may include disulphide bonds, hydrophobic interactions, hydrogen bonds, ionic interactions, or a combination of the all. O'Riordan et al. (1988) indicated that when electrostatic interactions are involved in whole plasma protein gel formation, gel strength is affected by salts and pH. The molecular forces that exist in the gel network are dependent on the protein and its structure, which can be influenced by the method used for protein extraction (Utsumi and Kinsella, 1985). Urea and guanidine hydrochloride (GuHCl) can destabilize hydrogen bonds and hydrophobic interactions in proteins. A protein molecule can be denatured by urea through preferential adsorption (Wallqvist et al., 1998). Some workers proposed that Sulfhydryl/disulfide interchanges are involved in soy protein gelation due to the reaction of the gel to certain reagents: b-mercaptoethanol (b-ME or 2-ME) (Utsumi and Kinsella, 1985), and Nethylmaleimide (NEM) (Utsumi and Kinsella, 1985; Wang and
X.D. Sun et al. / Journal of Cereal Science 66 (2015) 24e30
Damodaran, 1990). Hofmeister (1888) observed that the solubility of proteins in water could be affected to a certain degree by salts. Generally, salts may either reduce or enhance the hydrophobicity of a solute in water. The “Hofmeister series” is a series of ions ranked in the sequence of how strongly they influence the hydrophobicity. Case et al. (1992) reported that Na2SO4 facilitated the gelation of konjac glucomannan, whereas NaSCN and NaNO3 suppressed it. We hypothesize these salts also have the same effects in CGP gelation thus providing evidence of hydrophobic interaction. The effects of salts, pH, reducing agents and dissociating agents may be employed to determine the involvement of different interaction forces in forming the structure of protein gels (Utsumi and Kinsella, 1985). Techniques employed to identify various molecular forces are summarized in Table 1. Plant protein concentrate is one type of protein product on the market. We intend to explore the molecular forces involved in this particular product and confirm the involvement of these forces since no study has been conducted to investigate such forces involved in CGP concentrate. This is fundamental for the future development and application of CGP concentrate in foods. The objective of this work was to investigate the impacts of salts, chemicals that target non-covalent and covalent interactions, and reheating and recooling on gelation properties of CGP gels to better understand the importance of different molecular forces involved in network formation, particularly gels formed by CGP concentrate. This will enable us to elucidate the driving molecular forces presented in CGP concentrate gel network formation and maintenance. 2. Materials and methods 2.1. Commercial corn germ and corn germ protein concentrate Commercial corn germ was purchased from a local corn milling plant and the corn germ was separated via a dry milling process. CGP was extracted following a typical isoelectric precipitation procedure. The corn germ was first ground to flour by a grinder (Philips Electronics, HR2168, China); then passed through a 80 mesh screen, sealed in plastic bags and stored in a freezer for the further extraction. A sample of 1000 g corn germ flour was suspended in 3000 mL of 0.1 M NaOH solution (pH 9.3) and stirred for 30 min to enhance solubility of the globular protein in corn germ, followed by centrifugation at 3000 rpm for 30 min. The suspension was collected and the pH value was adjusted to 4.5 (Nielsen et al., 1973) using 2 M HCl to reach the isoelectric point of corn germ proteins. The suspension was then centrifuged at 4000 rpm for 30 min, followed by sediment collection and freeze drying. The dry powder was finally defatted for 2 h with 10-fold petroleum ether
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two times. The recovered CGP contained 55.6% (w/w) of protein as determined by the Kjeldahl method, and an N to protein conversion factor of 6.25 was employed (AACC, 1982). GuHCl and b-mercaptoethanol (Nuotai Chemical Co. Ltd., Shanghai, China), PG (Jusheng Technology Co. Ltd., Hubei, China), Na2SO4 and NaSCN (Ziyi Chemical Co. Ltd., Shanghai, China), and N-ethylmaleimide (Klamar Shanghai Puzhen Biotech. Co. Ltd., Shanghai, China) were of analytical grade. 2.2. Determination of crude fat, ash, total starch, and total dietary fiber Crude fat (method 960.39) and ash (method 920.153) of commercial corn germ and CGP were determined in duplicate using AOAC (1990) procedures. The Megazyme (Megazyme International Ltd., Wicklow, Ireland) total starch analysis kit (K-TSTA/05/06) and total dietary fiber kit (K-TDFR/12/05) were used for determining the total starch and dietary fiber contents, respectively. 2.3. Rheology The CGP was mixed with 0.3 M NaCl to obtain a suspension with protein concentration of 15% (w/v) as a good gel can form at this salt concentration. The samples were mixed by a stirring rod for 1 min to achieve complete suspension. The samples were then loaded into a TA Discover HR-1 rheometer (TA Instruments, Newcastle, Del. USA) equipped with a 4 cm diameter parallel plate geometry. Rheological properties were then determined as previously described by Sun and Arntfield (2012). Approximately 1 mL of the CGP suspension was loaded to the lower plate of the parallel plate geometry of the rheometer for each test. Then, the upper plate was lowered to reach a gap width of 1.00 mm and a thin layer of light mineral oil was applied to the well of the upper plate geometry. A solvent trap cover was used to prevent sample drying during heating and cooling. A watersaturated atmosphere can be maintained at the sample's surface through this method. The following heating and cooling procedures were employed. First, samples were equilibrated at 25 C for 2 min and then heated and cooled over a temperature range of 25e95e25 C at a controlled rate (4 C/min). Rheological data was collected every 30 s during heating and cooling. This was followed by a frequency sweep over a range of 0.01e10 Hz at 25 C. Data during gel formation was collected along with data of the final gel. The values of storage modulus (G0 ) and loss modulus (G00 ) were collected as a function of frequency. The tan delta (tan d ¼ G00 /G0 ), a measure of the energy lost compared to the energy stored in a single deformation cycle, was also determined. The input amplitude strain of 0.02, a value found to be in the linear viscoelastic
Table 1 Impact of different reagents on molecular driving forces exist in proteins. Non-covalent bonds Electrostatic interaction NaCl Na2SO4 NaSCN GuHCl 2-ME NEM PG Urea
Covalent bond Hydrophobic interaction
Hydrogen bond
Disrupt
Disrupt Promote Disrupt Disrupt
Disrupt
Kauzmann (1959)
Disrupt Disrupt Promote
Disrupt Disrupt
Promote Disrupt
References
Disulfide bond
Tanford (1968) Wall and Huebner (1981) Creighton (1993) Tanford (1962) Gordon and Jencks (1963)
Abbreviations: Sodium chloride, NaCl; Sodium sulfate, Na2SO4; Thiocyanate, NaSCN; Guanidine hydrochloride, GuHCl; b-mercaptoethanol, b-ME or 2-ME; N-ethylmaleimide, NEM; Propylene glycol, PG.
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region for heat-induced protein networks in preliminary experimentation was used for the dynamic analysis. Samples were run at least in duplicate. 2.4. Statistical analysis One-way ANOVA was employed to analyze significant differences for all data using Tukey's test by GraphPad InStat software version 3.06 with a minimum significance at 5% level (P < 0.05), (GraphPad Software Inc. La Jolla, CA, USA). 3. Results and discussion 3.1. Proximate composition of commercial corn germ and CGP The proximate composition of the commercial corn germ and CGP concentrate are presented in Table 2. Compared to the commercial corn germ, the protein content of CGP concentrate has increased from 19.85 to 55.61%, while the fat, starch, and total dietary fiber contents have decreased from 37.54, 22.18, and 11.15% to 16.73, 10.56, and 4.82%, respectively. Almost no change for the ash content is found between commercial corn germ and CGP concentrate. It is expected that the remaining oil, starch, and dietary fiber may greatly impact the gelation characteristics of CGP through interfering with the formation of the three-dimensional gel network and this detrimental effect will likely result in reduced gel stiffness. 3.2. Impacts of different chemicals on non-covalent bonds Most chaotropic agents such as detergents, urea, or GuHCl tend to disrupt non-covalent molecular forces include electrostatic interactions, hydrogen bonds, and hydrophobic interactions (Sood and Slattery, 2003). To investigate which non-covalent bonds are involved in CGP gel formation, CGP was dispersed into these chemical solutions (GuHCl, PG, Na2SO4, NaSCN, and Urea) containing 0.3 M NaCl before heat treatment. 3.2.1. Impact of GuHCl GuHCl is believed to be a strong ionic denaturing agent (Tanford, 1968) which inhibits hydrogen and ionic bonds and disrupts hydrophobic interactions. Tanford (1968) indicated that GuHCl might result in the most extensively unfolded state, enabling the protein molecules to behave as random coils. Compared to urea, GuHCl is a more effective denaturant, which unfolds proteins at around two to three times lower concentrations (Greene and Pace, 1974). Furthermore, GuHCl is very stable, while urea may gradually decompose into ammonia and cyanate. No significant difference was observed for G0 values among various GuHCl concentrations (Fig. 1a). The inclusion of GuHCl to the CGP dispersion was supposed to disrupt hydrophobic interactions and hydrogen bonds but the result obtained seemed to suggest that hydrophobic interactions and hydrogen bonds are not major molecular forces for CGP gel formation. However, a tendency of decreasing in the G0 values with the addition of GuHCl was observed. Gelation was inhibited at a concentration of 1.0 M GuHCl since tan d was significantly increased; this evidence supports the
Fig. 1. Effect of different concentration reagents on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. GuHCl. b. PG. c. urea. Error bars represent standard deviation of 2 replicates.
assumption that hydrogen bonds and hydrophobic interactions are present in CGP gel formation. 3.2.2. Impact of propylene glycol (PG) PG is generally employed to enhance hydrogen bonds and electrostatic interactions while disrupting hydrophobic forces by reducing the dielectric constant of the solvent, and sufficiently lowering the energy barrier to proteineprotein interaction to further facilitate structure formation (Utsumi and Kinsella, 1985).
Table 2 Components of commercial corn germ and CGP.
Commercial corn germ CGP concentrate
Protein %
Oil %
Starch %
Total dietary fiber %
Ash %
19.85 ± 1.37 55.61 ± 1.93
37.54 ± 1.62 16.73 ± 1.59
22.18 ± 2.67 10.56 ± 1.02
11.15 ± 2.03 4.82 ± 0.57
4.61 ± 0.87 4.56 ± 0.26
X.D. Sun et al. / Journal of Cereal Science 66 (2015) 24e30
Our results indicated that with increased concentration of PG up to 10% (v/v), the gel stiffness of CGP was almost not affected, but with concentration above 10%, the stiffness significantly decreased. The relative elasticity (tan d) was seen to gradually increase (Fig. 1b). This suggests that rather than hydrogen bonds and electrostatic interaction, hydrophobic interaction plays a more important role in determining CGP gel stiffness. Utsumi and Kinsella (1985) proposed that for molecular forces present in soy protein gel formation and maintenance, mostly hydrogen bonds in 7S globulin gels and hydrogen bonds and hydrophobic interactions in soy protein isolate gels. Our results for CGP gels are essentially in agreement with their findings. 3.2.3. Impact of urea Through hydrogen bonds, urea can bind to amide groups and decrease the hydrophobic effect through dehydration of the protein molecules. It was also indicated that in the urea induced protein denaturation, hydrophobic and hydrophilic groups were included (Zou et al., 1998). The denaturing effect of urea was also believed to be induced by a dehydration of peptide bonds which also weakened hydrophobic interactions (Walstra, 2003). In Fig. 1c, no significant difference was observed for G0 values between 0 M and 2 M urea, but decreased dramatically at 5 M. Interestingly, G0 increased a little when urea concentration was raised to 8 M, possibly due to the severe denaturation of CGP which resulted in further disruption of hydrophobic interactions and hydrogen bonds. This would have increased the exposure of previously buried functional groups such as eSH that enable the formation of disulfide bonds. Urea denatured CGP and prevented its network formation by breaking down hydrogen bonds and hydrophobic interactions. The decrease in G0 value of CGP gel further confirms the presence of hydrophobic interactions and/or hydrogen bonds in CGP gel formation. Being a strong denaturing agent, Tanford (1968) pointed out that urea can result in an extensively unfolded state, where the protein molecule acts as a random coil. Tanford (1968) further defined an “intermediate” state between native and urea denatured states and indicated that the denatured states obtained by other denaturants belong to this intermediate state. The above tests confirm that hydrophobic interactions, hydrogen bonds, and electrostatic interactions are all present in CGP network formation and contribute to the overall balance of attractive and repulsive forces for CGP gel network maintenance. 3.2.4. Impact of Na2SO4 and NaSCN Kosmotropes or lyotropes are ions that exhibit strong interactions with water and generally stabilize macromolecules such as proteins. This stabilization effect is brought about by increasing the order of water and its surface tension (Uedaira and Uedaira, 2001). Sodium sulfate (Na2SO4) is a typical kosmotrope which promotes hydrogen bonding formation thereby stabilizing protein structure. Chaotropes are well known ions that exhibit weak interactions with water thereby destabilizing protein molecules. These compounds disrupt hydrogen-bonded network of water and reduce its surface tension, thus increase more structural freedom and denaturation of proteins (Uedaira and Uedaira, 2001). NaSCN is a typical chaotrope which disrupts hydrogen bonding and destabilizes protein molecules. The impacts of salts on protein structure include three mechanisms: direct ionemacromolecule interactions, non-specific charge neutralization, and electrostatic effects (Zhang and Cremer, 2006). The ion specific effects are believed to arise from changes in the hydrophobic core of the protein (Zhang and Cremer, 2006). By interacting with charged groups on the proteins, salts are considered to mainly impact electrostatic interactions at low ionic
27
strengths. However, when at higher concentrations, the ion specific effects become prominent (Damodaran and Kinsella, 1981). At the above higher concentrations, NaSCN behaves as a destabilizing salt while NaCl shows effects of stabilizing protein structure (Damodaran and Kinsella, 1981). Melander and Horv ath (1977) proposed that a protein's molar surface tension increment (s) determines the property of a salt that affects hydrophobic interactions in this protein independent of the salt concentration. They also indicated that the basis of a natural lyotropic series was formed by incrementing the tension of s. NaSCN shows a strong ability to bind to proteins and is believed to be a destabilizing salt within this series. The SCN enhances the globular proteins' unfolding at lower temperatures, both by changing the protein's charge and creating an excessive negative charge (Melander and Horv ath, 1977). In contrast, SO4 2 anions are believed to behave as protein structure stabilizers. With addition of 0.3 M Na2SO4 and NaSCN, improved structure development was obtained for the rheological data of the CGP in comparison to the 0.3 M NaCl control (Fig. 2a and b). Poor solubility (data not presented in this report) for the 0.3 M NaCl sample might account for the weak network formation. Compared to the 0.3 M Na2SO4 sample, the SCN anion showed the greater increase in G0 values than SO4 2 . The high G0 value with SCN is probably caused by reactive groups' exposure plus electrostatic shielding effects and these effects may induce minimized charge repulsion within the protein. Although these salts influenced gel stiffness differently, they also impacted the relative elasticity of the networks differently as the tan d value was significantly affected by Na2SO4 (Fig. 2a), whereas it was not dramatically influenced by NaSCN (Fig. 2b).
Fig. 2. Effects of different concentration sodium salts on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. Na2SO4. b. NaSCN. Error bars represent standard deviation of 2 replicates.
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As NaSCN is a chaotropic salt which promotes protein solubility and destabilizes proteins in solution, its capacity to promote a stiff network should be investigated. Hincha (1998) conducted research on chaotropic anions on the release of insoluble membrane proteins, and found that for the dissociation of proteins from their binding sites on the membrane, the inclusion of chaotropic salts decreased the energy barrier. In our study, this decrease in the energy at the relatively low NaSCN concentration resulted in strengthened gel stiffness as seen for 0.3 M NaSCN. To investigate the effect of NaSCN at higher concentrations, 1 M and 3 M NaSCN were employed and the gel stiffness (G0 ) was not significantly reduced, and neither was the relative elasticity (tan d). Therefore, gel development is not dramatically inhibited with the inclusion of more concentrated NaSCN (Fig. 2b). Some workers (Damodaran and Kinsella, 1981) indicated that the binding of SCN to proteins caused a change in overall protein charges at higher SCN concentrations, therefore destabilizing the protein's structure and inhibiting the gel formation. Our findings are inconsistent with their conclusion, possibly because of a lower protein content in our CGP concentrate sample. Impurities such as starch, oil, and fiber may also have interfered with CGP gelation characteristics with the inclusion of higher concentrations of NaSCN.
with the addition of 0.1 M 2-ME. In addition, higher tan d values with the inclusion of 2-ME (Fig. 3a) further provided evidence that disulfide bonds are involved in CGP gel formation. However, no significant difference for G0 and tan d were found among different levels of 2-ME. This indicates 0.1 M 2-ME is sufficient to disrupt disulfide bonds in CGP.
3.3. Impact on covalent bond
Heating and cooling curves provide dynamic procedure of protein structure development during processing and the details may reveal specific protein molecular interactions at certain stage. Fig. 4 showed that CGP structure development starts at about 85 C during the heating stage. Since hydrophobic interactions are endothermic, it is generally inferred that hydrophobic interactions are strengthened at high temperatures; while at low temperatures, they are weakened (contrary to that for hydrogen bonds). Consequently, hydrophobic interactions probably played a greater role than hydrogen bonds in CGP structure formation at high temperatures. To investigate the contribution of hydrogen bonds to the formation of corn germ protein gel, a reheating and recooling process is a typical approach to apply. In this research results showed that G0 gradually dropped as the temperature rose to 95 C during the reheating period, with a rate of decrease identical to the rate of increase in G0 during the cooling phase, and approached a higher level compared to the CGP dispersion which was heated to 95 C (Fig. 4). This phenomenon is an indication that the gel formation at the cooling stage is thermally reversible. Because it is generally believed that with increasing temperature, hydrogen bonds are weakened; it can be deduced that the evidence for the contribution of hydrogen bonds to gel stiffening was provided by the thermal reversibility of the curves (Fig. 4). It was noted that there is a separation for the cooling curve and reheating curve at temperatures higher than 87 C; this is probably an indication of more hydrophobic interactions being formed, because they are favored at high temperatures. At the final recooling stage, it is believed that hydrogen bonds again play an important role in gel stiffening. Since low temperatures are beneficial to the formation of hydrogen bonds, the steady increase in G0 at the recooling stage can be once again ascribed to this molecular interaction. Surprisingly, G0 curve reached an even higher level compared to the levels achieved during the initial cooling phase. This is probably because through the recooling process, all the hydrogen bonds disrupted at the reheating stage can be recovered. In addition, it is likely some new hydrogen bonds also formed. Considering the fact that at lower temperatures hydrogen bonds are enforced while hydrophobic interactions are weakened, it appears that during cooling and recooling periods hydrogen bonds play a more important role than hydrophobic interactions due to
3.3.1. Impact of 2-mercaptoethanol (2-ME) 2-ME can disrupt disulfide bonds in proteins through competing for sulfhydryl groups (Wang and Damodaran, 1990). Consequently, the impact of the inclusion 2-ME reveals the role of disulfide bonds to CGP gel network formation and maintenance. Results showed that 2-ME had a significant effect on G0 value for the CGP gels from the statistical point of view, since G0 value dramatically decreased
Fig. 3. Effect of different concentration reducing agents on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. 2-ME. b. NEM. Error bars represent standard deviation of 2 replicates.
3.3.2. Impact of NEM Through reaction with sulfhydryl groups to form a stable alkyl derivative, NEM can prevent the formation of disulfide bonds between protein molecules (Creighton, 1993). Our results indicated that inclusion of NEM had an significant impact on gel characteristics; G0 value dramatically decreased with the inclusion of 10, 20, and 40 mM NEM, while tan d value did not significantly increase (Fig. 3b). The result was similar to that of with 2-ME and further supports the finding that disulfide bonds were presented in CGP gel formation. It was noted that no significant difference exists for G0 and tan d among samples with different levels of NEM. This is probably an indication that 10 mM NEM is sufficient to cleave the disulfide bonds in CGP gel. 3.4. Impact of reheating and recooling
X.D. Sun et al. / Journal of Cereal Science 66 (2015) 24e30
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Fig. 4. Effect of reheating and recooling on gelation properties of G0 of 15% (w/v) CGP dispersed in 0.3 M NaCl. Enlarged temperature scale region (75e100 C) for gel formation in the heating stage is presented in the top right corner.
the gradual increase in G0 at the stage of CGP gel formation. The results of heating CGP with the inclusion of 2-ME suggested that disulfide bonds play a major part in the gelation process; inclusion of NEM further provided evidence that disulfide bonds are involved and have a great effect on CGP gel formation. ger and Arntfield (1993) proposed that if a gel is unable to melt Le by heating treatment this is typically an indication of the involvement of covalent bonds. This situation arose when CGP gel was reheated to 95 C (Fig. 4); the gel was unable to completely melt and a weak gel structure was maintained. Therefore, disulfide bonds are included in the formation and stabilization of the CGP gel structure. 4. Conclusion Our results indicated that for the CGP recovered by isoelectric precipitation, non-covalent bonds including hydrogen bonds, electrostatic, and hydrophobic interactions were involved for the gel structure generation during gel network formation through inclusion of GuHCl, PG, Na2SO4, NaSCN, and Urea. Disulfide bonds prevention or reduction through inclusion of 2-ME and NEM proved that they play a major role in maintaining gel stiffness. Incomplete melting of the gel upon reheating provides further evidence that disulfide bonds are involved. This role is major and under normal heating conditions influences gel stiffness and elasticity. Non-covalent bonds only played a minor role in CGP gel formation and it appeared that hydrogen bonds contributed more to gel structure than hydrophobic interactions since at the cooling stage hydrogen bonds account for the gel stiffness. Acknowledgment Financial support from the Foundation for Excellent Academic Leaders of Harbin (2013RFXYJ049) and National Quality and Safety Risk Assessment Project for Food and Oil Crop Products (GJFP2014006) is greatly acknowledged.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Determination of molecular driving forces involved in heat-induced corn germ proteins gelation Xiang Dong Sun a, *, Yu Lan c, Dan Shi c, Shu Wen Lu b, Hui Liao a, Rui Ying Zhang a, Xin Miao Yao b, Ying Lei Zhang b, Ping Su a, Hong Shan a a b c
Quality & Safety Institute of Agricultural Products, Heilongjiang Academy of Agricultural Science, Harbin 150086, China Food Processing Institute, Heilongjiang Academy of Agricultural Science, Harbin 150086, China Food Science Department, East University of Heilongjiang, Harbin 150086, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 May 2015 Received in revised form 9 September 2015 Accepted 16 September 2015 Available online 24 September 2015
Molecular forces involved in corn germ proteins (CGP) gelation were investigated using a rheometer. With the inclusion of urea and guanidine hydrochloride (GuHCl), the storage moduli (G0 ) of CGP gels tends to decrease. This is an indication that hydrophobic interactions and hydrogen bonds are present. The involvement of propylene glycol (PG) provides evidence that hydrogen bonds and electrostatic interactions are present. The impact of thiocyanate (NaSCN) and sodium sulfate (Na2SO4) further proves the involvement of hydrogen bonds. The effects of 2-mercaptoethanol (2-ME) and N-ethylmaleimide (NEM) reveal that involvement of disulfide bonds contribute to CGP gel stiffness. Reheating and recooling tests revealed that during the initial cooling phase the gel is thermally reversible and higher levels of G0 suggested more hydrogen bonds are formed when recooled. It can be deduced that hydrogen bonds play a greater role than hydrophobic interactions from the increasing G0 during cooling and recooling. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Corn germ protein Storage modulus Molecular forces Gelation Reagents
1. Introduction Besides the predominant constituent oil, corn germ contains 18e22% proteins, and these proteins account for around 29% of the total proteins in corn seeds (Watson, 1987). Water-soluble albumins plus saline-soluble globulins account for more than 60% of corn germ proteins (CGP) (Wilson, 1987). These proteins are proven to possess a good balance of amino acids, which presents a superior nutritional value to endosperm protein (Wilson, 1987) and have better functional properties than corn endosperm proteins. Corn germ is generally used to extract oil through solvent extraction and high temperature processing, which results in serious denaturation of the residual protein; these denatured proteins almost completely lose their functional properties (Kinsella, 1979), and thus are mainly used as animal feed. Due to increasing demand for high quality proteins with good functional characteristics in the food industry, researchers have been attempting to recover proteins from various plant sources. Corm germ proteins seem to be a good alternative for soy proteins
* Corresponding author. E-mail address: [email protected] (X.D. Sun). http://dx.doi.org/10.1016/j.jcs.2015.09.007 0733-5210/© 2015 Elsevier Ltd. All rights reserved.
and some workers have published their works on the extraction procedure and functional properties evaluation of corn germ proteins (Mila and Hojilla-Evangelista, 2012). The mechanism of protein gelation is described as a threedimensional network formation through the cross-linking of its polypeptide chains. Cross linking of protein polypeptide chains is driven by different molecular forces which may include disulphide bonds, hydrophobic interactions, hydrogen bonds, ionic interactions, or a combination of the all. O'Riordan et al. (1988) indicated that when electrostatic interactions are involved in whole plasma protein gel formation, gel strength is affected by salts and pH. The molecular forces that exist in the gel network are dependent on the protein and its structure, which can be influenced by the method used for protein extraction (Utsumi and Kinsella, 1985). Urea and guanidine hydrochloride (GuHCl) can destabilize hydrogen bonds and hydrophobic interactions in proteins. A protein molecule can be denatured by urea through preferential adsorption (Wallqvist et al., 1998). Some workers proposed that Sulfhydryl/disulfide interchanges are involved in soy protein gelation due to the reaction of the gel to certain reagents: b-mercaptoethanol (b-ME or 2-ME) (Utsumi and Kinsella, 1985), and Nethylmaleimide (NEM) (Utsumi and Kinsella, 1985; Wang and
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Damodaran, 1990). Hofmeister (1888) observed that the solubility of proteins in water could be affected to a certain degree by salts. Generally, salts may either reduce or enhance the hydrophobicity of a solute in water. The “Hofmeister series” is a series of ions ranked in the sequence of how strongly they influence the hydrophobicity. Case et al. (1992) reported that Na2SO4 facilitated the gelation of konjac glucomannan, whereas NaSCN and NaNO3 suppressed it. We hypothesize these salts also have the same effects in CGP gelation thus providing evidence of hydrophobic interaction. The effects of salts, pH, reducing agents and dissociating agents may be employed to determine the involvement of different interaction forces in forming the structure of protein gels (Utsumi and Kinsella, 1985). Techniques employed to identify various molecular forces are summarized in Table 1. Plant protein concentrate is one type of protein product on the market. We intend to explore the molecular forces involved in this particular product and confirm the involvement of these forces since no study has been conducted to investigate such forces involved in CGP concentrate. This is fundamental for the future development and application of CGP concentrate in foods. The objective of this work was to investigate the impacts of salts, chemicals that target non-covalent and covalent interactions, and reheating and recooling on gelation properties of CGP gels to better understand the importance of different molecular forces involved in network formation, particularly gels formed by CGP concentrate. This will enable us to elucidate the driving molecular forces presented in CGP concentrate gel network formation and maintenance. 2. Materials and methods 2.1. Commercial corn germ and corn germ protein concentrate Commercial corn germ was purchased from a local corn milling plant and the corn germ was separated via a dry milling process. CGP was extracted following a typical isoelectric precipitation procedure. The corn germ was first ground to flour by a grinder (Philips Electronics, HR2168, China); then passed through a 80 mesh screen, sealed in plastic bags and stored in a freezer for the further extraction. A sample of 1000 g corn germ flour was suspended in 3000 mL of 0.1 M NaOH solution (pH 9.3) and stirred for 30 min to enhance solubility of the globular protein in corn germ, followed by centrifugation at 3000 rpm for 30 min. The suspension was collected and the pH value was adjusted to 4.5 (Nielsen et al., 1973) using 2 M HCl to reach the isoelectric point of corn germ proteins. The suspension was then centrifuged at 4000 rpm for 30 min, followed by sediment collection and freeze drying. The dry powder was finally defatted for 2 h with 10-fold petroleum ether
25
two times. The recovered CGP contained 55.6% (w/w) of protein as determined by the Kjeldahl method, and an N to protein conversion factor of 6.25 was employed (AACC, 1982). GuHCl and b-mercaptoethanol (Nuotai Chemical Co. Ltd., Shanghai, China), PG (Jusheng Technology Co. Ltd., Hubei, China), Na2SO4 and NaSCN (Ziyi Chemical Co. Ltd., Shanghai, China), and N-ethylmaleimide (Klamar Shanghai Puzhen Biotech. Co. Ltd., Shanghai, China) were of analytical grade. 2.2. Determination of crude fat, ash, total starch, and total dietary fiber Crude fat (method 960.39) and ash (method 920.153) of commercial corn germ and CGP were determined in duplicate using AOAC (1990) procedures. The Megazyme (Megazyme International Ltd., Wicklow, Ireland) total starch analysis kit (K-TSTA/05/06) and total dietary fiber kit (K-TDFR/12/05) were used for determining the total starch and dietary fiber contents, respectively. 2.3. Rheology The CGP was mixed with 0.3 M NaCl to obtain a suspension with protein concentration of 15% (w/v) as a good gel can form at this salt concentration. The samples were mixed by a stirring rod for 1 min to achieve complete suspension. The samples were then loaded into a TA Discover HR-1 rheometer (TA Instruments, Newcastle, Del. USA) equipped with a 4 cm diameter parallel plate geometry. Rheological properties were then determined as previously described by Sun and Arntfield (2012). Approximately 1 mL of the CGP suspension was loaded to the lower plate of the parallel plate geometry of the rheometer for each test. Then, the upper plate was lowered to reach a gap width of 1.00 mm and a thin layer of light mineral oil was applied to the well of the upper plate geometry. A solvent trap cover was used to prevent sample drying during heating and cooling. A watersaturated atmosphere can be maintained at the sample's surface through this method. The following heating and cooling procedures were employed. First, samples were equilibrated at 25 C for 2 min and then heated and cooled over a temperature range of 25e95e25 C at a controlled rate (4 C/min). Rheological data was collected every 30 s during heating and cooling. This was followed by a frequency sweep over a range of 0.01e10 Hz at 25 C. Data during gel formation was collected along with data of the final gel. The values of storage modulus (G0 ) and loss modulus (G00 ) were collected as a function of frequency. The tan delta (tan d ¼ G00 /G0 ), a measure of the energy lost compared to the energy stored in a single deformation cycle, was also determined. The input amplitude strain of 0.02, a value found to be in the linear viscoelastic
Table 1 Impact of different reagents on molecular driving forces exist in proteins. Non-covalent bonds Electrostatic interaction NaCl Na2SO4 NaSCN GuHCl 2-ME NEM PG Urea
Covalent bond Hydrophobic interaction
Hydrogen bond
Disrupt
Disrupt Promote Disrupt Disrupt
Disrupt
Kauzmann (1959)
Disrupt Disrupt Promote
Disrupt Disrupt
Promote Disrupt
References
Disulfide bond
Tanford (1968) Wall and Huebner (1981) Creighton (1993) Tanford (1962) Gordon and Jencks (1963)
Abbreviations: Sodium chloride, NaCl; Sodium sulfate, Na2SO4; Thiocyanate, NaSCN; Guanidine hydrochloride, GuHCl; b-mercaptoethanol, b-ME or 2-ME; N-ethylmaleimide, NEM; Propylene glycol, PG.
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region for heat-induced protein networks in preliminary experimentation was used for the dynamic analysis. Samples were run at least in duplicate. 2.4. Statistical analysis One-way ANOVA was employed to analyze significant differences for all data using Tukey's test by GraphPad InStat software version 3.06 with a minimum significance at 5% level (P < 0.05), (GraphPad Software Inc. La Jolla, CA, USA). 3. Results and discussion 3.1. Proximate composition of commercial corn germ and CGP The proximate composition of the commercial corn germ and CGP concentrate are presented in Table 2. Compared to the commercial corn germ, the protein content of CGP concentrate has increased from 19.85 to 55.61%, while the fat, starch, and total dietary fiber contents have decreased from 37.54, 22.18, and 11.15% to 16.73, 10.56, and 4.82%, respectively. Almost no change for the ash content is found between commercial corn germ and CGP concentrate. It is expected that the remaining oil, starch, and dietary fiber may greatly impact the gelation characteristics of CGP through interfering with the formation of the three-dimensional gel network and this detrimental effect will likely result in reduced gel stiffness. 3.2. Impacts of different chemicals on non-covalent bonds Most chaotropic agents such as detergents, urea, or GuHCl tend to disrupt non-covalent molecular forces include electrostatic interactions, hydrogen bonds, and hydrophobic interactions (Sood and Slattery, 2003). To investigate which non-covalent bonds are involved in CGP gel formation, CGP was dispersed into these chemical solutions (GuHCl, PG, Na2SO4, NaSCN, and Urea) containing 0.3 M NaCl before heat treatment. 3.2.1. Impact of GuHCl GuHCl is believed to be a strong ionic denaturing agent (Tanford, 1968) which inhibits hydrogen and ionic bonds and disrupts hydrophobic interactions. Tanford (1968) indicated that GuHCl might result in the most extensively unfolded state, enabling the protein molecules to behave as random coils. Compared to urea, GuHCl is a more effective denaturant, which unfolds proteins at around two to three times lower concentrations (Greene and Pace, 1974). Furthermore, GuHCl is very stable, while urea may gradually decompose into ammonia and cyanate. No significant difference was observed for G0 values among various GuHCl concentrations (Fig. 1a). The inclusion of GuHCl to the CGP dispersion was supposed to disrupt hydrophobic interactions and hydrogen bonds but the result obtained seemed to suggest that hydrophobic interactions and hydrogen bonds are not major molecular forces for CGP gel formation. However, a tendency of decreasing in the G0 values with the addition of GuHCl was observed. Gelation was inhibited at a concentration of 1.0 M GuHCl since tan d was significantly increased; this evidence supports the
Fig. 1. Effect of different concentration reagents on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. GuHCl. b. PG. c. urea. Error bars represent standard deviation of 2 replicates.
assumption that hydrogen bonds and hydrophobic interactions are present in CGP gel formation. 3.2.2. Impact of propylene glycol (PG) PG is generally employed to enhance hydrogen bonds and electrostatic interactions while disrupting hydrophobic forces by reducing the dielectric constant of the solvent, and sufficiently lowering the energy barrier to proteineprotein interaction to further facilitate structure formation (Utsumi and Kinsella, 1985).
Table 2 Components of commercial corn germ and CGP.
Commercial corn germ CGP concentrate
Protein %
Oil %
Starch %
Total dietary fiber %
Ash %
19.85 ± 1.37 55.61 ± 1.93
37.54 ± 1.62 16.73 ± 1.59
22.18 ± 2.67 10.56 ± 1.02
11.15 ± 2.03 4.82 ± 0.57
4.61 ± 0.87 4.56 ± 0.26
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Our results indicated that with increased concentration of PG up to 10% (v/v), the gel stiffness of CGP was almost not affected, but with concentration above 10%, the stiffness significantly decreased. The relative elasticity (tan d) was seen to gradually increase (Fig. 1b). This suggests that rather than hydrogen bonds and electrostatic interaction, hydrophobic interaction plays a more important role in determining CGP gel stiffness. Utsumi and Kinsella (1985) proposed that for molecular forces present in soy protein gel formation and maintenance, mostly hydrogen bonds in 7S globulin gels and hydrogen bonds and hydrophobic interactions in soy protein isolate gels. Our results for CGP gels are essentially in agreement with their findings. 3.2.3. Impact of urea Through hydrogen bonds, urea can bind to amide groups and decrease the hydrophobic effect through dehydration of the protein molecules. It was also indicated that in the urea induced protein denaturation, hydrophobic and hydrophilic groups were included (Zou et al., 1998). The denaturing effect of urea was also believed to be induced by a dehydration of peptide bonds which also weakened hydrophobic interactions (Walstra, 2003). In Fig. 1c, no significant difference was observed for G0 values between 0 M and 2 M urea, but decreased dramatically at 5 M. Interestingly, G0 increased a little when urea concentration was raised to 8 M, possibly due to the severe denaturation of CGP which resulted in further disruption of hydrophobic interactions and hydrogen bonds. This would have increased the exposure of previously buried functional groups such as eSH that enable the formation of disulfide bonds. Urea denatured CGP and prevented its network formation by breaking down hydrogen bonds and hydrophobic interactions. The decrease in G0 value of CGP gel further confirms the presence of hydrophobic interactions and/or hydrogen bonds in CGP gel formation. Being a strong denaturing agent, Tanford (1968) pointed out that urea can result in an extensively unfolded state, where the protein molecule acts as a random coil. Tanford (1968) further defined an “intermediate” state between native and urea denatured states and indicated that the denatured states obtained by other denaturants belong to this intermediate state. The above tests confirm that hydrophobic interactions, hydrogen bonds, and electrostatic interactions are all present in CGP network formation and contribute to the overall balance of attractive and repulsive forces for CGP gel network maintenance. 3.2.4. Impact of Na2SO4 and NaSCN Kosmotropes or lyotropes are ions that exhibit strong interactions with water and generally stabilize macromolecules such as proteins. This stabilization effect is brought about by increasing the order of water and its surface tension (Uedaira and Uedaira, 2001). Sodium sulfate (Na2SO4) is a typical kosmotrope which promotes hydrogen bonding formation thereby stabilizing protein structure. Chaotropes are well known ions that exhibit weak interactions with water thereby destabilizing protein molecules. These compounds disrupt hydrogen-bonded network of water and reduce its surface tension, thus increase more structural freedom and denaturation of proteins (Uedaira and Uedaira, 2001). NaSCN is a typical chaotrope which disrupts hydrogen bonding and destabilizes protein molecules. The impacts of salts on protein structure include three mechanisms: direct ionemacromolecule interactions, non-specific charge neutralization, and electrostatic effects (Zhang and Cremer, 2006). The ion specific effects are believed to arise from changes in the hydrophobic core of the protein (Zhang and Cremer, 2006). By interacting with charged groups on the proteins, salts are considered to mainly impact electrostatic interactions at low ionic
27
strengths. However, when at higher concentrations, the ion specific effects become prominent (Damodaran and Kinsella, 1981). At the above higher concentrations, NaSCN behaves as a destabilizing salt while NaCl shows effects of stabilizing protein structure (Damodaran and Kinsella, 1981). Melander and Horv ath (1977) proposed that a protein's molar surface tension increment (s) determines the property of a salt that affects hydrophobic interactions in this protein independent of the salt concentration. They also indicated that the basis of a natural lyotropic series was formed by incrementing the tension of s. NaSCN shows a strong ability to bind to proteins and is believed to be a destabilizing salt within this series. The SCN enhances the globular proteins' unfolding at lower temperatures, both by changing the protein's charge and creating an excessive negative charge (Melander and Horv ath, 1977). In contrast, SO4 2 anions are believed to behave as protein structure stabilizers. With addition of 0.3 M Na2SO4 and NaSCN, improved structure development was obtained for the rheological data of the CGP in comparison to the 0.3 M NaCl control (Fig. 2a and b). Poor solubility (data not presented in this report) for the 0.3 M NaCl sample might account for the weak network formation. Compared to the 0.3 M Na2SO4 sample, the SCN anion showed the greater increase in G0 values than SO4 2 . The high G0 value with SCN is probably caused by reactive groups' exposure plus electrostatic shielding effects and these effects may induce minimized charge repulsion within the protein. Although these salts influenced gel stiffness differently, they also impacted the relative elasticity of the networks differently as the tan d value was significantly affected by Na2SO4 (Fig. 2a), whereas it was not dramatically influenced by NaSCN (Fig. 2b).
Fig. 2. Effects of different concentration sodium salts on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. Na2SO4. b. NaSCN. Error bars represent standard deviation of 2 replicates.
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As NaSCN is a chaotropic salt which promotes protein solubility and destabilizes proteins in solution, its capacity to promote a stiff network should be investigated. Hincha (1998) conducted research on chaotropic anions on the release of insoluble membrane proteins, and found that for the dissociation of proteins from their binding sites on the membrane, the inclusion of chaotropic salts decreased the energy barrier. In our study, this decrease in the energy at the relatively low NaSCN concentration resulted in strengthened gel stiffness as seen for 0.3 M NaSCN. To investigate the effect of NaSCN at higher concentrations, 1 M and 3 M NaSCN were employed and the gel stiffness (G0 ) was not significantly reduced, and neither was the relative elasticity (tan d). Therefore, gel development is not dramatically inhibited with the inclusion of more concentrated NaSCN (Fig. 2b). Some workers (Damodaran and Kinsella, 1981) indicated that the binding of SCN to proteins caused a change in overall protein charges at higher SCN concentrations, therefore destabilizing the protein's structure and inhibiting the gel formation. Our findings are inconsistent with their conclusion, possibly because of a lower protein content in our CGP concentrate sample. Impurities such as starch, oil, and fiber may also have interfered with CGP gelation characteristics with the inclusion of higher concentrations of NaSCN.
with the addition of 0.1 M 2-ME. In addition, higher tan d values with the inclusion of 2-ME (Fig. 3a) further provided evidence that disulfide bonds are involved in CGP gel formation. However, no significant difference for G0 and tan d were found among different levels of 2-ME. This indicates 0.1 M 2-ME is sufficient to disrupt disulfide bonds in CGP.
3.3. Impact on covalent bond
Heating and cooling curves provide dynamic procedure of protein structure development during processing and the details may reveal specific protein molecular interactions at certain stage. Fig. 4 showed that CGP structure development starts at about 85 C during the heating stage. Since hydrophobic interactions are endothermic, it is generally inferred that hydrophobic interactions are strengthened at high temperatures; while at low temperatures, they are weakened (contrary to that for hydrogen bonds). Consequently, hydrophobic interactions probably played a greater role than hydrogen bonds in CGP structure formation at high temperatures. To investigate the contribution of hydrogen bonds to the formation of corn germ protein gel, a reheating and recooling process is a typical approach to apply. In this research results showed that G0 gradually dropped as the temperature rose to 95 C during the reheating period, with a rate of decrease identical to the rate of increase in G0 during the cooling phase, and approached a higher level compared to the CGP dispersion which was heated to 95 C (Fig. 4). This phenomenon is an indication that the gel formation at the cooling stage is thermally reversible. Because it is generally believed that with increasing temperature, hydrogen bonds are weakened; it can be deduced that the evidence for the contribution of hydrogen bonds to gel stiffening was provided by the thermal reversibility of the curves (Fig. 4). It was noted that there is a separation for the cooling curve and reheating curve at temperatures higher than 87 C; this is probably an indication of more hydrophobic interactions being formed, because they are favored at high temperatures. At the final recooling stage, it is believed that hydrogen bonds again play an important role in gel stiffening. Since low temperatures are beneficial to the formation of hydrogen bonds, the steady increase in G0 at the recooling stage can be once again ascribed to this molecular interaction. Surprisingly, G0 curve reached an even higher level compared to the levels achieved during the initial cooling phase. This is probably because through the recooling process, all the hydrogen bonds disrupted at the reheating stage can be recovered. In addition, it is likely some new hydrogen bonds also formed. Considering the fact that at lower temperatures hydrogen bonds are enforced while hydrophobic interactions are weakened, it appears that during cooling and recooling periods hydrogen bonds play a more important role than hydrophobic interactions due to
3.3.1. Impact of 2-mercaptoethanol (2-ME) 2-ME can disrupt disulfide bonds in proteins through competing for sulfhydryl groups (Wang and Damodaran, 1990). Consequently, the impact of the inclusion 2-ME reveals the role of disulfide bonds to CGP gel network formation and maintenance. Results showed that 2-ME had a significant effect on G0 value for the CGP gels from the statistical point of view, since G0 value dramatically decreased
Fig. 3. Effect of different concentration reducing agents on gelation properties of 15% CGP dispersion contain 0.3 M NaCl. a. 2-ME. b. NEM. Error bars represent standard deviation of 2 replicates.
3.3.2. Impact of NEM Through reaction with sulfhydryl groups to form a stable alkyl derivative, NEM can prevent the formation of disulfide bonds between protein molecules (Creighton, 1993). Our results indicated that inclusion of NEM had an significant impact on gel characteristics; G0 value dramatically decreased with the inclusion of 10, 20, and 40 mM NEM, while tan d value did not significantly increase (Fig. 3b). The result was similar to that of with 2-ME and further supports the finding that disulfide bonds were presented in CGP gel formation. It was noted that no significant difference exists for G0 and tan d among samples with different levels of NEM. This is probably an indication that 10 mM NEM is sufficient to cleave the disulfide bonds in CGP gel. 3.4. Impact of reheating and recooling
X.D. Sun et al. / Journal of Cereal Science 66 (2015) 24e30
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Fig. 4. Effect of reheating and recooling on gelation properties of G0 of 15% (w/v) CGP dispersed in 0.3 M NaCl. Enlarged temperature scale region (75e100 C) for gel formation in the heating stage is presented in the top right corner.
the gradual increase in G0 at the stage of CGP gel formation. The results of heating CGP with the inclusion of 2-ME suggested that disulfide bonds play a major part in the gelation process; inclusion of NEM further provided evidence that disulfide bonds are involved and have a great effect on CGP gel formation. ger and Arntfield (1993) proposed that if a gel is unable to melt Le by heating treatment this is typically an indication of the involvement of covalent bonds. This situation arose when CGP gel was reheated to 95 C (Fig. 4); the gel was unable to completely melt and a weak gel structure was maintained. Therefore, disulfide bonds are included in the formation and stabilization of the CGP gel structure. 4. Conclusion Our results indicated that for the CGP recovered by isoelectric precipitation, non-covalent bonds including hydrogen bonds, electrostatic, and hydrophobic interactions were involved for the gel structure generation during gel network formation through inclusion of GuHCl, PG, Na2SO4, NaSCN, and Urea. Disulfide bonds prevention or reduction through inclusion of 2-ME and NEM proved that they play a major role in maintaining gel stiffness. Incomplete melting of the gel upon reheating provides further evidence that disulfide bonds are involved. This role is major and under normal heating conditions influences gel stiffness and elasticity. Non-covalent bonds only played a minor role in CGP gel formation and it appeared that hydrogen bonds contributed more to gel structure than hydrophobic interactions since at the cooling stage hydrogen bonds account for the gel stiffness. Acknowledgment Financial support from the Foundation for Excellent Academic Leaders of Harbin (2013RFXYJ049) and National Quality and Safety Risk Assessment Project for Food and Oil Crop Products (GJFP2014006) is greatly acknowledged.
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