Effect of Extrusion Temperature on Functional and Structural Properties of Extruded Rice Protein and Glucose Conjugates

HUO Jinjie, XU Yan, WANG Na, WANG Peng, ZHU Minpeng, ZHANG Yifan, GAO Yuzhe,*, XIAO Zhigang,*

(1. College of Grain Science and Technology, Shenyang Normal University, Shenyang 110034, China;2. College of Light Industry, Liaoning University, Shenyang 110036, China)

Abstract: In order to develop a rapid and efficient method for the industrial production of rice protein-glucose conjugates,rice protein was extruded and then subjected to the Maillard reaction with glucose. Herein, rice protein was extruded at different temperatures (80, 90, 100, 110, 120 and 130 ℃) and then conjugated with glucose for 30 minutes at pH 10.5. We analyzed the effect of different extrusion temperatures on the functional properties (solubility, emulsification activity index(EAI) and emulsion stability index (ESI)) and structural properties of extruded rice protein and glucose (ERPG) conjugates and characterized ERPG conjugates using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy(FTIR) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Compared to native rice protein and glucose (NRPG) conjugate, the degree of glycosylation (DG) of ERPG conjugate prepared with rice protein extruded at 90 ℃was the highest, and the solubility of ERPG conjugates prepared with rice protein extruded at 90–120 ℃ decreased. The EAI, ESI and surface hydrophobicity of ERPG conjugates prepared with rice protein extruded at 80–90 ℃ increased, while those of ERPG conjugates prepared with rice protein extruded at 100–130 ℃ slowly decreased. The FTIR results showed that ERPG conjugate had higher contents of α-helix, β-turn and random coil and a lower content of β-sheet than NRPG.The SDS-PAGE indicated that the protein was aggregated into larger particles by extrusion. Under SEM, ERPG conjugates exhibited more disordered structure with irregular fragments.

Keywords: extrusion; rice protein; glucose; glycation; functional properties; structural properties

Rice protein has a high biological value and low hypoallergenic characteristics and contains a favorable balance of amino acids. However, compared to other plant proteins, the application of rice protein in foods industry has been limited due to glutelins’ content is over 80%, which lead to poor functional properties[1-2]. To modify the functional and physical properties of proteins such as soybean protein,whey protein, lactalbumin, many physical, chemical,thermo mechanical and enzymatic modifications have been applied[3-8]. The use of chemical and enzymatic methods has still been limited due to harmful reagents to human health respectively; physical modification has a huge space for development. Li Weiwei et al.[9]reported that protein denaturation occurs under heating conditions, and molecular structure unfolds to improve emulsification. Shepherd reported the functional properties of protein-saccharide conjugates combine improved, thus the Maillard reactions has been an effective way to improve the functional properties of proteins[10]. Maillard reaction could be conducted via dry or wet heating. Dry-heating takes long time to form the proteinsaccharide grafts, whereas wet heating takes less time, applied to protein and mono-oligosaccharides aggregation[11-12], and conform to the requirement of industrial production.

Microwave, enzyme hydrolysis, ultrasound et al. are applied to Maillard reaction of protein and saccharide in order to improve the functional property of conjugates[13-16].However, the above methods are not easy to control and are not suitable for industrial continuous production. Therefore,seeking a rapid, effective method suitable for industrial continuous production is very important. Extrusion cooking is a high temperature short time process in which protein material expose to high temperature, high pressure and mechanical shear, resulting in protein denaturation, partial unfolding and aggregation[17-20]. The heating of proteins during extrusion could change the functional properties, such as emulsifying activities, solubility, and foamability, and the components of protein.

Based on preliminary tests, the wet-heating glycation condition of rice protein and glucose conjugate was fixed.In this paper, we analyze the effect of different extrusion temperature (in the lower temperature range) on functional and structural properties of the extruded rice protein and glucose (ERPG) conjugates, and also examine the mechanism about the glycation of the modified rice protein extruded at 80, 90, 100, 110, 120 and 130 ℃ by controlled wet heating condition at pH 10.5, 90 ℃, and reaction time 30 min.

1Materials and Methods

1.1 Materials

Rice protein (82.50% dry protein basis) was supplied by Jiangxi Jinnong Biological Technology (Jiangsu Province, China). The protein content was determined by the methods of GB 5009.5-2016National food safety standard Determination of protein in foods(The value of 5.95 was used as a protein conversion factor). Glucose was provided by Tianjin Dongli Tianda Chemical Reagent Factory;1-anilinonaphthalene-8-sulfonic acid was provided by Sigma Co. Ltd., (USA);o-phthaldialdehyde (OPA) was provided by Sinopharm Shanghai Chemical Reagent Co. Ltd., (Shanghai China); standard protein sample (electrophoresis pure) was provided by Beijing Solaibao Technology Co. Ltd.; other reagents were analytically pure.

1.2 Instruments and equipment

DS56-III Twin-screw extruder was produced by Jinan Saixin Extrusion Machinery Co. Ltd. (Shandong Province, China); HH-4 digital display agitation water bath was produced by Changzhou Saipu Experimental Instrument Factory (Jiangsu Province, China); UV-2401PC spectrophotometer was produced by Shimadzu Co. Ltd.(Japan); F-4500 fluorimeter was produced by Hitachi Co.Ltd. (Japan); Nicolet Nexus 470 Fourier transform infrared spectroscopy (FTIR) spectroscopy was produced by Thermo Electron Co. Ltd. (USA); S-3400 scanning electron microscopy(SEM) was produced by Hitachi, Co. Ltd. (Japan).

1.3 Methods

1.3.1 Preparation of extruded rice protein

Extrusion experiments were performed using a DS 56-III twin-screw extruder. According to the experimental design,the rice protein was fed to the extruder at a constant speed of 10 kg/h in raw material zone, water was added to the rice protein to reach 35% of water content, the screw speed was 180 r/min. The barrel diameter of 400 mm and a barrel length to diameter (L/D) ratio of 16:1, the diameter of extruder die was 4.0 mm.

The melt zone temperature was controlled at 80, 90, 100,110, 120, and 130 ℃, respectively. For all extrudate samples:drying at room temperature, then crushed through 100 mesh and set aside.

1.3.2 Preparation of rice protein and glucose conjugates

Rice protein (1 g/100 mL) and glucose (3 g/100 mL)were dispersed and adjusted to pH 10.5 with 0.1 mol/L HCl.The samples were heated in a water bath at 90 ℃ for 30 min,then placed the beakers in an ice-bath immediately, adjusted the pH to 7 and dialyzed 24 h, ERPG conjugates and native rice protein and glucose (NRPG) were achieved via freezedrying. The samples at various levels of extrusion temperature(80, 90, 100, 110, 120, 130 ℃) and glucose conjugates were analyzed, the control samples were the NRPG conjugates under the same glycation treatment.

1.3.3 Measurement of the degree of glycosylation (DG)

The DG was calculated based on the loss of amino groups. The modified OPA method was used to determine the quantity of available amino groups[21]. The OPA reagent was freshly prepared just before use which was prepared by mixing 40 mg OPA, 1 mL of methanol, 25 mL of 0.1 mol/L sodium borate buffer (pH 9.75), 100 μLβ-mercaptoethanol, and 2.5 mL of 20 g/100 mL sodium dodecyl sulfate (SDS). The mixture was thoroughly mixed and then diluted to 50 mL with deionized water[22].

Subsequently, 200 μL 0.4% sample solution was added to 4 mL of OPA reagent and the mixture was incubated at 35 ℃ for 2 min. The absorbance value was immediately measured at 340 nm. A calibration curve was obtained by using 0.25–2 mmol/L lysine as a standard. DG was determined as:

WhereC0andCtare the absorbance values before and after DG with glucose respectively.

1.3.4 Measurement of solubility

The solubility of NRPG and ERPG conjugates were determined by the Bradford method[23], Bovine serum protein was used as the standard curve. According to the protein content in the sample quantity and protein content in solution to calculate solubility, and it was expressed by nitrogen solubility index (NSI).

1.3.5 Measurement of emulsifying activity and stability

The emulsifying properties of NRPG and ERPG conjugates were determined by the method of Pearce and Kinsella with slight modifications[24]. The sample was diluted with buffer (0.1 mol/L sodium phosphate buffer, pH 7) to reach a protein concentration of 0.1 g/100 mL. Then the protein sample solution and soy oil were mixed in a volume ratio of 25:8, and the mixture was homogenized (10 000 r/min) for 1 min. Afterwards, 100 μL sample was taken from the bottom of the test tube at 0 and 10 min and diluted with 10 mL of 0.1% sodium dodecyl sulphate solution respectively.Immediately, the absorbance of the diluted emulsion was determined at 500 nm. The emulsifying activity index (EAI)and the emulsion stability index (ESI) were calculated by the equation as follows:

WhereA0andA10are the absorbance at 0 min and 10 min respectively;Nis the dilution factor;φis the oil phase fraction in the system;Cis the protein concentration; Δtis the time difference between two absorbance measurements.

1.3.6 Measurement of hydrophobicity

The hydrophobicity of NRPG and ERPG conjugates were determined according to the ANS method[25]. A freezedried sample was dissolved in 0.01 mol/L phosphate buffer(pH 7) at different concentrations (0.006 25, 0.012 5, 0.025,0.05, and 0.10 g/100 mL. Then 20 μL 1-anilinonaphthalene-8-sulfonic acid (ANS) (8 mmol/L) was added to 4 mL protein solution, and the fluorescence intensity of the sample was measured after mixing thoroughly and standing for 15 min at room temperature in dark environment. The excitation wavelength was set to 390 nm and emission wavelength was 470 nm. The hydrophobicity index was the initial slope of the plot of the uorescence intensity as a function of the protein concentration, which was calculated by linear regression(R2＞ 0.95 in all cases). All samples were analyzed in triplicate.

1.3.7 Measurement of fluorescence spectroscopy

The samples were dissolved in phosphate buffer solution(10 mmol/L, pH 7.0), then centrifuged at 7 000 ×gfor 20 min. Fluorescence was measured by exciting the protein at 347 nm and recording the emission spectra in the wavelength range of 350–500 nm with a slit width of 5.0 nm[26].

1.3.8 Measurement of FTIR

Approximately 1 mg sample was mixed with 100 mg potassium bromide and ground in an agate mortar for 15 min,then tablets were pressed under 10 kg pressure[27]. Condition of scanning: spectral sweep range was 400–4 000 cm-1,resolution was 4 cm-1, signal scanning was accumulated for 64 times, the spectra were processed using Peakfit (Version 4.12) software.

1.3.9 Measurement of sodium dodecyl sulphatepolyacrylamide gel electrophoresis(SDS-PAGE)

The NRPG and ERPG conjugates were analyzed with SDS-PAGE by Laemmli methods with modifications slightly[28], on 12 g/100 mL acrylamide separating gel and 5 g/100 mL acrylamide stacking gel. Samples were prepared in 0.1 mol/L, Tris-HCl of pH 6.8, containing 4 g/100 mL SDS and 0.2% (V/V) bromophenol blue, 20% (V/V)glycerol. 1 mol/L dithiothreitol (DTT), containing 1.546 g DTT, 0.01 mol/L (pH 5.2) sodium acetate buffer, and heated for 5 min in boiling water before electrophoresis. After the electrophoresis, the gel sheets were stained for protein with Coomassie brilliant blue R-250. The protein stain was destained with 10% acetic acid (V/V) containing 10%methanol (V/V).

1.3.10 Measurement of SEM

The micromorphology of the NRPG and ERPG conjugates were examined and the acceleration voltage was 5 kV. The samples were ground slightly and coated with gold to a thickness of 15 nm using an ion sputter.

1.4 Statistical analysis

All treatments were performed in triplicate. Statistical analysis was analyzed using IBM SPSS Statistics 22 software. Results were subjected to a One-way AVONA analysis of variance, followed by Duncan’s test. Significance was defined at the 5% level. The OriginPro.8.5 software was applied to plot.

2Results and Analysis

2.1 The properties of NRPG and ERPG conjugates

Table 1 Properties of NRPG and ERPG conjugates

The properties of NRPG and ERPG conjugates are presented in Table 1. Evaluation results showed that ERPG conjugates (80–130 ℃) prepared in the present study, the DG were 17.43%–31.11% (Table 1). The DG of ERPG-90 ℃increased 30.7% compared to NRPG. It was due to the fact that the structure of rice protein significantly changed after extrusion, the molecule unfolded could accelerate the contact reaction of protein and glucose[29], but at higher extruded temperature (100–130 ℃), the DG of ERPG decreased, it might be because that higher extrusion temperature caused rice protein aggregated, which was not conducive to the reaction with glucose.

Compared with NRPG conjugates, the solubility of ERPG conjugates decreased, especially at 120 ℃, the solubility of ERPG conjugate decreased 24.51%. It was due to the fact that during extrusion, the high pressure and shear force caused the rice protein molecule structure destroyed,then protein molecules occurred chemical cross-linking reactions, the hydrophobic grouping buried into interior,the solubility decreased, after the glycation the hydroxyl was introduced, hydrophily increased, which led to poor solubility[30-31].

However, the EAI increased with increasing extrusion temperature and reached maximum (15.41 ± 0.11) m2/g at 90 ℃. It was possible that mechanical forces disrupt the intermolecular interactions and expose the hydrophobic groups beneath the surface of the protein[32]. Besides,extrusion process could change the rice protein’s structure,including unfolded, exposed functional groups (hydrophobic groups) et al[33]. Therefore, the emulsifying capacity after extrusion and glycation would be increased due to exposing the inside hydrophobic groups.

The ESI increased with extrusion temperature range 80 to 100 ℃, reach (29.52 ± 0.11) min, and decreased with extrusion temperature range 110 to 130 ℃, compared to NRPG conjugate. This phenomenon might be related to the formation of larger particles when the extrusion temperature raised to 110 ℃.

2.2 Surface hydrophobicity of NRPG and ERPG conjugates

Fig. 1 Hydrophobicity (H0) of NRPG and ERPG conjugates

Compared with the NRPG, theH0of ERPG conjugates decreased significantly (Fig. 1) (P＜ 0.05), the results might be attributed to the protein-protein interactions inside the extruder, and rice protein contains large hydrophily amino acids, hydrophily amino acids aggregates under extrusion condition, rice protein and glucose covalent cross-linking, the hydrophilic of ERPG conjugates increased, thus hydrophobic index decreased accordingly[34-35].

TheH0of ERPG conjugates increased from 80 to 90 ℃, and the ERPG-90 ℃ had the highestH0(553.9), since the hydrophobic group was exposed, adsorb rapidly at the oil/water interface, it exhibited much improved emulsifying capability. However, when the temperature exceeds 90 ℃,the protein aggregation degree became larger, leading to the decrease of surface hydrophobicity. Li et al.[36]reported that glycation with saccharide dramatically reduced the surface hydrophobicity of rice protein, it also consistent to our experiment results. Therefore, under the condition of appropriate extruded, rice protein denatured, conducive to the grafting reaction of glucose, it improved the rice protein properties such as surface hydrophobicity, solubility and emulsification.

2.3 Fluorescence analysis of NRPG and ERPG conjugates

The strongest fluorescence intensity of NRPG and ERPG conjugates was found at an emission of 420 nm with an excitation of 347 nm (Fig. 2). The results suggested the formation of Maillard reaction fluorescent products.However, the fluorescence intensity of extruded rice protein at extrusion temperature (80–90 ℃) and glucose conjugates increased, while the fluorescence intensity of extruded rice protein at extrusion temperature at (110–130 ℃) and glucose conjugates reduced.

Fig. 2 Fluorescence spectra of NRPG and ERPG conjugates

Low temperature extrusion could disrupt hydrophobic interactions within protein molecules, resulted in an increase in hydrophobic areas exposed to the molecular surface, and accelerated the Maillard reaction process of rice protein and glucose. When the fluorescence was related to functional properties of NRPG and ERPG conjugates (Table 1), it was found that the glycation reaction benefits the emulsifying capacity of hydrophobic protein. The increment of DG might attribute to extrusion destroyed the structure of rice protein,and accelerated Maillard reaction process.

2.4 FTIR of NRPG and ERPG conjugates

Fig. 3 FTIR spectra of NRPG and ERPG conjugates

Table 2 Secondary structure contents of NRPG and ERPG conjugates%

FTIR analysis was used to reveal the structural characteristics of the native/extrusion modified conjugates.The obtained results of extrusion modified (extrusion temperature 80–130 ℃) and NRPG conjugates given in Fig. 3,indicated that the ERPG conjugates differed from NRPG of structural characteristics at around 1 027 cm-1, while the varied temperature extrusion modified rice protein and glucose conjugates showed a similar pattern in this region.The absorbance of –C–O stretching and –OH deformation vibrations (1 050–1 150 cm-1) and free –OH form(3 643–3 630 cm-1) in rice protein grafts by extrusion temperature increased compared to NRPG conjugates, which also indicated that saccharide molecules were linked to the protein by the covalent bonds[37]. The enhanced absorbance at 1 070 cm-1suggested that the ERPG contained more –OH than the NRPG.

The contents of the secondary structures on NRPG and ERPG conjugates analyzed by second derivative deconvolution IR spectra and quantitative information of fitting (Table 2). Compared with NRPG conjugates, ERPG showed a decrease in theβ-sheet proportion and an increase inα-helix,β-turn, and random coil. The proportions ofα-helix,β-sheet,β-turn, and random coil were changed following varied temperature extrusion process. The content ofβ-sheet of ERPG conjugates were obvious diminished with the extrusion temperature increased to 120 ℃, but increased when extrusion temperature 130 ℃. The content ofα-helix was increased from 18.8% to 20.6% range from 80 to 120 ℃, and decreased to 17.6% at 120 ℃. The contents ofβ-turn and random coil of ERPG-90 ℃ exhibited about 22.8% and 22.4% increments respectively compared with NRPG conjugates.

These results showed that the degree of glycation reaction due to the presence of the free amino group in the extrusion process of ERPG. The process of extrusion and glycation, could cause the interaction of biopolymers and the heat denaturation of proteins and affect the secondary structure of proteins. The interactions glycation between ERPG, could change the properties of rice protein[38].

2.5 SDS-PAGE of NRPG and ERPG conjugates

Fig. 4 SDS-PAGE profiles of NRPG and ERPG conjugates

The SDS-PAGE of extrusion modified (extrusion temperature 80–130 ℃) and NRPG conjugates are shown in Fig. 4. The bands of every subunit in rice protein corresponding to the molecular weight of 80–82, 51.0, 37.0,22.0 kDa[39-40].

The protein band of SDS-PAGE for ERPG showed a slight difference from NRPG, several protein polymers near 22 kDa became narrow and 37 kDa became widen (Fig. 4).In ERPG conjugates, as extrusion temperature increased,bands in reactants were visible in the region (37.0 kDa) and(22.0 kDa) and gradually shaded, meaning that the protein was polymerized into larger particles by extrusion, and identified the formation of high molecular weight products in both NRPG and ERPG conjugates. This might be due to extrusion modified rice protein expose more available lysine residues, benefit for the glycation of rice protein and glucose[41].

2.6 SEM of NRPG and ERPG conjugates

Fig. 5 Microstructure of NRPG and ERPG conjugates

The microstructures of extrusion modified (extrusion temperature 80–120 ℃) and NRPG conjugates were shown in Fig. 5. Compare to NRPG conjugates, the ERPG exhibited more disordered structure and irregular fragments. It might be because partial molecule structure unfolding of rice proteins: functional groups (such as hydrophobic groups)were exposed, and they immediately interacted with each other, which led to protein aggregation and form network structure[42].

Samples of ERPG-80 ℃ and ERPG-90 ℃ were more aggregated than other samples, ERPG-80 were characterized by minor surface heterogeneities and cavities, of which showed compact structures. ERPG-90 ℃ showed crosslinking reactions (Maillard reaction) occurred between glucose and rice protein during extrusion lead to more compact structures,this might be caused by the introduction of sugar molecules,which made the original rigid structure of rice protein disappear, peptide chain stretching and molecule stretching,so the rice protein with high hydrophobicity was more conducive to sugar graft reaction with sugar molecules after extrusion.

3Conclusions

Compared to NRPG, the DG of ERPG-90 ℃ increased 30.7%, the solubility decreased 6.77%, the EAI and ESI increased 34.0% and 31.88%, the surface hydrophobicity decreased 45.18% respectively compared with NRPG. The extrusion modification changed the secondary and tertiary structures of rice protein. ERPG with a higher content ofα-helix,β-turn and unordered coil, and a lower content ofβ-sheet structure. Otherwise, Bands in reactants were visible in the region (37.0 kDa) and (22.0 kDa) and gradually shaded with the raise of temperature, the protein was polymerized into larger particles by extrusion. SEM indicated that the ERPG exhibited more disordered structure and irregular fragments.

In conclusion, wet heating Maillard reaction between extrusion modified rice protein and glucose could be a promising way to improve functional properties of rice protein. Further investigations will be conducted to achieve more improvements in protein physic-chemical properties and elucidate the Maillard reaction mechanism.