1. Introduction

Rice is the main grain in China and some other countries in the world, it is not only an important staple food for millions of people, but also a significant material for many industries such as syrup, organic acids and antibiotics fermentation.At the same time, rice protein, which has a reasonable amino acid composition and hypoallergenic properties, is considered as a high-quality plant protein that may be useful in infant formulations^[1-2], and its important health care function, such as application in hyperlipidemia, cholesterol-lowering, anti-leukaemia, antihypertensive, has also been reported^[3-5].

Thermo-denatured rice protein (TDRP) powder containing 50% protein was a by-product from the process of rice syrup production.The by-product yields are about 1/7 of the rice material used in those production processes, which suggests a significant source of plant protein.But the rice protein has been heated at above 92 ℃ in the process and results in serious thermal denaturation, lots of conventional technologies and methods on solubility and structural characterization of protein cannot perform effectively, and only a few related researches have been reported.Therefore, it is of significance to find a way to efficiently utilize this protein source.

Many researches have shown that high-pressure (HP) treatment can influence the three-dimensional structure of protein, and bring about reversible or irreversible changes of protein conformation.The degree of the changes induced by HP depends not only upon the level and duration time of pressure, but also the type of protein, pH, and temperature^[6].The enzymatic hydrolysis of HP treated protein has different phenomenon from that of ordinary protein due to the change of protein structure.For example, β-lactoglobulin (β-Lg) of milk is not easy to be hydrolyzed by pepsin at atmospheric pressure, but it is easily enzymatic hydrolyzed at 200 MPa, and there is no whole β-Lg molecular existing in the solution hydrolyzed with pepsin for 10 min at 400 MPa^[7-8].Most of the peptide products of HP treated (600 MPa) β-Lg hydrolyzed by pepsin for 1 min were less than 1 500 u in size^[9].

This study aimed to investigate the changes in the solubility, molecular weight distribution and conformation of rice protein during its enzymolysis after 100 MPa HP treatment.

2. Materials and Methods

2.1 Materials

The thermo-denatured rice protein (TDRP) was donated by Hanke Bioengineering Ltd., China, it was a by-product from the process of rice syrup production.Protein content of the sample was determined by Kjeldahl method as 61.98%.Alcalase 2.4L (2.4 AU/g) were purchased from Novozymes, Denmark.Molecular weight (MW) markers were rabbit phosphorylase b (97.4 ku), bovine serum albumin (66.2 ku), rabbit actin (43 ku), carbonic bovine carbonic anhydrase (31 ku), trypsin inhibitor (20.1 ku), and hen egg white lysozyme (14.4 ku).All other chemicals were of analytical grade.

2.2 High-Pressure Processing

The TDRP was suspended as a 5% (mass concentration) solution in 30 mL distilled water adjusted to pH 8.0 with 1 mol/L NaOH.The solutions were conditioned vacuum in a double polyethylene bag and subjected to 100 MPa high-pressure treatment for 10 min.The high-pressure processing of rice protein samples was carried out in a 5 L vessel (UHP900×2-Z, Wentian Ltd., Baotou, China) equipped with temperature and pressure regulator device, and oil was used as a fluid of low compressibility.Temperature of medium in the vessel was settled at 25 ℃.The loading speed was 10 MPa/s, and after hold at 100 MPa for 10 min, the pressure was quickly released (300 MPa/s) to atmospheric pressure.Each sample was replicated 3 times.And then, the HP treated substrate was utilized for proteolysis at atmospheric pressure.

2.3 Enzymatic Hydrolysis of Rice Proteins

Hydrolysis was performed at atmospheric pressure (0.1 MPa) for 30, 60, 90, 120 min with 15 μL Alcalase solution, using HP treated and untreated (control) substrate, respectively.The temperature of reaction was 50 ℃.The pH was kept at 8.0 by adding 1 mol/L NaOH, according to pH-stat technique^[10].The same reactions were performed without any enzyme addition to prepare the blanks of the experiment.The derived hydrolysates were heated (95 ℃, 15 min) and centrifuged at 3 500 r/min for 10 min at 25 ℃.The soluble and insoluble rice protein were freeze-dried (LYOVACGT2 Vacuum Freeze-dried Instrument, SRK-SYSTEM TECHNIK Company, Germany).The crude protein content of both preparations was determined by micro-Kjeldahl method according to Petrucelli and A on^[11].The coefficient of protein-nitrogen was 5.95.

2.4 SE-HPLC

The hydrolysates were filtered (0.45 μm) and loaded on a TSK-GEL G2000SWXL (Tosoh, Japan) steel column (7.8 mm×300 mm) connected to a LC600 System (LabTech, Beijing, China).Separation was conducted at 30 ℃ with a flow rate of 1.0 mL/min, the mobile phase was acetonitrile/water/TFA (45/55/0.1).The eluted proteins were detected at 215 nm.

2.5 FTIR Spectroscopy

Infrared spectra of insoluble rice protein were recorded by a Nicolet 5700 FTIR spectrometer (Thermo Company, USA) equipped with a DTGS detector and a Ge/KBr beamsplitter.Samples were mixed with KBr, and tablet pressed.The tablet was contained in Golden Gate Diamond ATR (Attenuated Total Teflectance) system.A total of 32 scans were averaged at 4 cm^-1 resolution.Deconvolution of infrared spectra was performed using Peakfit version 4.12 (Seasolve Software Inc.) according to Byler et al.^[11].The half width used for deconvolution was 10 cm^-1 and the filter 60%.To ensure that the spectra were not over-deconvoluted, the acquired spectra were judged by evaluating the second derivative spectra, comparing the number and position of bands with the deconvolution spectra.Band assignment in the amide Ⅰ region (1 600-1 700 cm^-1) was determined according to Ref.[11].Quantitative analysis of secondary structure components was performed using Gaussian peaks and curve fitting models.

2.6 Scanning Electron Microscopy (SEM)

The 100 MPa treated and control samples were fractured with a blade, the fragments were mounted on aluminum stubs, and gold-coated in vacuum.The surface morpha of both samples were observed with an SEM (JSM-6490LV, Japan) at an accelerating voltage of 10 kV and 10 mm working distance.

3. Results and Discussion

3.1 Protein Contents of Hydrolysates

The solubility and degree of hydrolysis of rice protein were determined to evaluate the extraction efficiency under different conditions, as shown in Fig. 1.Both at control and 100 MPa, a remarkable increase of rice protein solubility was observed during the extracting time (from 0 to 30 min).Beyond that time range, there was little difference in the solubility (Fig. 1(a)), which was typical for hydrolysis curves of plant protein.The solubility values of 100 MPa treated samples varied from 16.05% to 75.33% after 120 min of hydrolysis, but that of control samples were just from 12.03% to 58.9%.Samples treated by HP gave the highest solubility value and were more efficient in rice protein hydrolysis than control samples.

Figure  1.  Enzymatic hydrolysis of rice protein with different substrates at 5% rice protein concentration (enzyme/substrate mass ratio=1:100)

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But as to degree of hydrolysis (DH), it increased at a rapid rate during the initial 30 min, and went up quickly as well thereafter, especially for the control samples (Fig. 1(b)).This result did not accord with the tendency of solubility.The reason might be that soluble rice protein macromolecules degraded into small molecular-weight peptides.HP treatment changed the three-dimensional structure and the enzymatic hydrolysis feature, leading to more protein dissolving.

3.2 Gel Filtration Chromatography of Soluble Protein

The effect of pressurization on the molecular weight distribution of rice protein was investigated by SE-HPLC, as shown in Fig. 2.Samples of control with different hydrolysis time showed similar patterns of two major peaks (fraction over 60%) whose corresponding MW were 4.4 and 2.0 ku, respectively.The fraction of 4.4 and 2.0 ku proteins increased with the hydrolysis time, which was consistent with the hydrolysis degree.At 100 MPa, the fraction of 57-105 ku proteins was 20% in the blank group, which might be caused by the cross-link between the subunits of rice protein in the process of syrup production.As the hydrolysis went along, the fraction of 57-105 ku decreased, and the fraction of 4.4 and 2.0 ku increased.Furthermore, HP treatment changed the structure of rice protein, leading to the change of enzymatic mechanism and benefit to solubility.

Figure  2.  Profiles of SE-HPLC of soluble rice protein with different hydrolysis time

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3.3 Spectral Assignment

Fig. 3 shows the deconvoluted infrared spectra (1 600-1 700 cm^-1) of insoluble rice protein after hydrolysis.Eight major bands were observed in the amide Ⅰ region.From the infrared spectra, the secondary structure composition of rice protein both at control and 100 MPa, estimated by integrating the major bands, was found to be predominantly β-sheets and turns (over 60%), followed by α-helices (about 20%) and random coils (about 20%), as shown in Fig. 4.A previous FTIR study showed that prolamin and glutelin exhibited α-helices and random coils as the major secondary structures^[13].However, many seed globulins from monocotyledonous and dicotyledonous plants were found to possess low levels of α-helix and high levels of β-sheet secondary structure^[14].Although rice protein both at control and 100 MPa exhibited similar FTIR spectral characteristics (mainly β-sheets and turns), an increase in the intensity of 1 644 and 1 691 cm^-1 band and decrease in the intensity of 1 620 cm^-1 suggest a transition from ordered conformation to random coils and from aggregated strands to β-sheet.The results indicate a change in hydrogen bonding or conformation after the treatment of 100 MPa.

Figure  3.  Stacked plot of deconvoluted infrared spectra of insoluble rice protein after hydrolysis

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Figure  4.  Integrated intensity of several infrared spectral bands of insoluble rice protein after hydrolysis (Error bars represent standard deviations of means)

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Over time, the intensity of bands attributed to side chain vibration (1 609 cm^-1), aggregated strands (1 620 cm^-1) and β-strand (1 632 cm^-1) decreased remarkably, while the intensity of α-helix (1 656 cm^-1), β-turns (1 667 cm^-1), β-sheet (1 679 cm^-1) increased significantly at control.The reason might be that HP changed the granular, lamellar structure to swollen and deformed, which benefited the enzymatic hydrolysis.At the same time, we found that even β-sheets and turns were main components of rice protein structure, the solubility was still poor.The relationship between the secondary structure and solubility will be undertaken in the next phase.

3.4 SEM Analysis

The structures of control and 100 MPa treated samples were also examined under scanning electron microscope (Fig. 5), which contributed to understand the structure of surface and analyze the molecule structure.Changes in the shape and surface of rice protein induced by 100 MPa treatment were clearly visible.At control, the rice protein presented as protein body granules and was smooth.But the protein body granule changed after treated by 100 MPa.Most granules appeared swollen and deformed.This disaggregation of protein body into small amorphous granules may be a major cause of improvement in the digestibility of 100 MPa treated rice protein.

Figure  5.  SEM profiles of rice protein

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4. Conclusions

It was confirmed in this work that enzymatic hydrolysis could significantly improve the extraction efficiency of rice protein.HP treatment showed an enhancement effect on the enzymatic hydrolysis of rice protein to some extent.Compared with control, there were 105 ku proteins existing at 100 MPa, which was different from the basic subunit of rice protein, and might be caused by aggregation.The subunits of rice protein with different processing were not markedly different.Although β-sheets and turns were the major secondary structures of rice protein, the solubility was still poor.The results indicate a change in hydrogen bonding or conformation after the treatment of 100 MPa HP, leading to the enhancement effect on the enzymatic hydrolysis.