Solubility, structural properties, and immunomodulatory activities of rice dreg protein modified with sodium alginate under microwave heating

Abstract This research aims to investigate the solubility, structural properties, and immunomodulatory of rice dreg protein (RDP) modified with sodium alginate. The modification was done by wet heating assisted with microwave treatment. The solubility, emulsifying properties at pH 2–12, amino acid composition, molecular weight distribution, circular dichroism (CD) spectroscopy, and FTIR spectra of modified RDP were analyzed and discussed. Results showed that Maillard reaction could significantly enhance the solubility and emulsifying capacity of RDP. Further, an animal model for cyclophosphamide‐induced immunodeficiency was designed to evaluate the immunomodulatory effect of modified RDP. It is therefore suggested that modified RDP could improve the immunomodulatory effect of immunosuppressed mice, and the immunomodulation was concentration dependent, being generally enhanced by increased concentrations. This research revealed that glycosylation modification of RDP through Maillard reaction by wet heating assisted with microwave treatment may be successfully applied to improve the physicochemical properties and bioactive benefits of the final product.

methods (i.e., acylation, glycosylation, and covalent cross-linking effect.), and enzyme methods have been investigated to improve the technical properties of proteins in the food matrix. Li et al. (2016) investigated the effects of combined treatment of ultrasound and alkaline pretreatments on the structural characteristics of rice protein. The results showed that ultrasound decreased ahelices but increased β-sheets and the content of nonhydrophilic amino acids. Zhao et al. (2012) investigated the effects of enzyme type on the size and amino acids of RDP. They reported that the combined treatment with alkaline protease and neutrase enzymes increased the solubility of RDP, but also increased the content of bitter amino acids such as arginine and leucine. Although these technologies successfully improved the solubility of RDP, some undesirable substances, such as bitter hydrolysates produced by chemical or biochemical modifications, could lead to commercial value loss.
Maillard reaction is a nonenzymatic reaction of reducing sugars and available amino acids of proteins without additional extraneous chemicals and has been proven to be very important in producing flavor, aroma, and functional foods. The two types of Maillard reactions are caused by dry heating and wet heating. Dry heating takes several days to form protein-polysaccharide conjugates, making it slower than wet heating. Moreover, the products have a dark brown color, which is not desirable for many functional foods, and the reaction process of dry heating is difficult to control. Therefore, improved RDP modification technologies should be developed for functional foods. Sodium alginate, an important cell wall component isolated from brown marine algae (Phaeophyceae), is composed of polymeric sequences of (1-4) linked β-D-mannuronate (M) and α-L-guluronate (G) residues and has a high affinity for cationic protein molecules (Bokkhim, Bansal, Grøndahl, & Bhandari, 2015;Fioramonti, Perez, Aríngoli, Rubiolo, & Santiago, 2014). Sodium alginate has been extensively used to improve water holding, gelling, and emulsifying properties of food proteins based on the interactions between protein and polysaccharide (Yao et al., 2018).
Usually, plant peptides contain 2-20 amino acid residues and may show a wide range of biological activities such as antioxidant, antihypertensive, and immunoregulation. (Ferri et al., 2017;Shen et al., 2016). In the reported studies, the constituents of rice or byproducts of rice milling such as proteins and peptides have been studied to a certain health-related bioactivity such as antityrosinase, anti-inflammatory, antihypertensive, and antioxidant properties . Up to now, there are no related reports that rice protein-polysaccharide conjugates with antioxidants could exhibit immunoregulatory activities.
Therefore, the objective of this study was to investigate the effects of glycosylation modification of RDP with sodium alginate through Maillard reaction by wet heating in addition to microwave heating. The effects of glycosylation on the solubility, emulsifying properties, structure, and immunomodulatory activity of modified RDP were investigated. The findings of this research would be beneficial for developing novel techno-functional additives as addedvalue food ingredients.

| Preparation of RDP
According to the methods of Song and Zhang (2017), RDP was prepared with the following steps. Firstly, the rice dregs were pretreated with 650 U/g lipase (enzymatic activity 10,000 U/g, Jiangsu Ruiyang Group Co., Wuxi, China) at 50ºC with pH 9.0 and hydrolysis time 90 min. Secondly, to improve the purity of protein, the effects of freeze-thawing pretreatment, enzymatic hydrolysis, ultrasound, and their combinations on the content of protein were investigated according to the methods of Song and Zhang (2017).

| Preparation of products with Maillard reaction
RDP was weighed and dispersed in 2% (w/w) deionized water. The pH of the solution was adjusted to 12.0, magnetically stirred for 30 min at 50ºC, then cooled down to room temperature. Sodium alginate was added into the dispersion at a rate of RDP 1.88:1 (w/w), and the pH was reduced to 10.2 with 1 mol/L HCL or 1 mol/L NaOH, magnetically stirred for 10 min. Subsequently, 50 ml of a mixed solution of RDP and sodium alginate was transferred to a 100-ml flask and placed in an experimental microwave oven with a reflux device.
The mixed solution was treated by intermittent microwave heating by the methods reported in our previous paper (Meng, Zhang, Song, & Zhang 2018). At the end of the reaction, the flask was immediately cooled down in an ice bath for 5 min.

| Solubility measurement
The solubility of RDP modified with sodium alginate through the heat treatment was determined following the reports of Sun, Yu, Zeng, Yang, and Jia (2011). products with Maillard reaction (PMR) was dispersed in deionized water (1 mg/ml), and then, the pH was adjusted to 2, 4, 6, 8, 10, and 12 using 2 mol/L solutions of HCl and NaOH. Then, the solution was magnetically stirred for 30 min at room temperature and centrifuged for 15 min at 4,500 r/min. The absorbance of the supernatants at 280 nm was determined to reflect the protein content, and the solubility of protein in the supernatants was calculated as a percentage of the total protein content.

| Emulsifying stability measurement
As the methods described by Li et al. (2013), the emulsifying stability of PMR was determined with slight modifications. The PMR sample was diluted with pH 8.0 0.05 mol/L PBS to 0.4% protein concentration. Briefly, 9 ml of the sample solution was mixed with 3 ml of soy oil and kept continuous agitation. Then, the coarse emulsion was homogenized for 5 min at 10,000 g using with a homogenizer to achieve a good emulsion level. The emulsion was, respectively, sampled at 0 and 10 min, and the dilution solution was immediately prepared by adding 5 ml of 0.1% sodium dodecyl sulfate (SDS). Once the emulsion formed (0 min), the absorbance at 500 nm of the diluted emulsion was immediately determined and measured. Then, the emulsion stability (ES) was calculated as follows: where A 0 is the absorbance of the emulsion (0 min), and A 10 is the absorbance of the emulsion (10 min).

| Amino acid analysis
The methods reported by Li et al. (2016) with slight modifications were used to determine the amino acid composition of PMR. Firstly, 6 mol/L HCl was used to hydrolyze the samples at 110ºC. Then, the hydrolysate was filtered and set to 50 ml with deionized water.
The determination of the amino acid content was carried out by the methods described by Li et al. (2013).

| Molecular weight distribution
The molecular weight distribution of PMR was analyzed with highperformance gel permeation chromatography (HPGPC) with a TSK Gel G4000 PWXL column was used to analyze (Li et al., 2013).
Before injection, the supernatant was collected and filtered (0.2-mm Whatman filter) after centrifuging for 10 min at 1,000 g. The mobile phase contained PBS buffer (0.05 mol/L, pH 8.5) with a flow rate of 1 ml/min at 20ºC. Detection was determined at a wavelength of 230 nm.

| Circular dichroism spectrum
Briefly, the samples were mixed with PBS buffer (10 mmol/L, pH 7.2) to prepare 0.1 mg/ml solution, and then, the solution was centrifuged for 10 min at 10,000 r/min. A quartz cuvette (1 mm optical path length) was supplemented with nitrogen flux at room temperature (25 ± 1ºC), and the data of circular dichroism (CD) spectra were acquired from 190 to 250 nm. The average spectra data of three scans with PBS (0.01 mol/L, pH 8.0) were used as blank.

| FTIR spectra measurement
The FTRI spectra were determined using a Thermo Nicolet iS 50 FTIR spectrometer with full-band scanning (4,000-400 cm −1 ) according to the method of Ji et al. (2018). One milligram of the samples was mixed with 150 mg of KBr and ground gently with an agate pestle and mortar under an infrared lamp. Next, the sample powder was pressed into a 13-mm-diameter disk by applying 15 tons of pressure for 30 s. Then, the spectrum intensities of the secondary structure of PMR were analyzed using Omnic software (version 8.0, Thermo Nicolet Inc., Waltham, MA, USA).

| Animal experiments
Healthy male BALB/c mice (18-20 g) after eight weeks of the birth were purchased from Laboratory Animal Center which is located at Soochow University, then transferred to Jiangnan University.
All animal experiments were permitted by the Ethics Committee of Jiangnan University. According to the methods described by Shen, Mao, Chen, Meng, and Ji (2015), mice were randomized to five groups, each with 10 mice: blank group, model group, low-dose PMR of 80 mg kg −1 day −1 , mid-dose PMR of 160 mg kg −1 day −1 , and highdose PMR of 320 mg kg −1 d −1 . After fed adaptively for 4 days, the blank group of healthy mice were given physiological saline solution once daily for 14 consecutive days. In the first 3 days, all other mice were intraperitoneally injected 100 mg kg −1 d −1 cyclophosphamide (CY). Then, the model mice were gavaged with physiological saline solution once daily from days 4 to 14. Other three groups of mice, respectively, were intraperitoneally injected with PMR at dose of 80, 160, and 320 mg/kg body weight.

| Immune organ indexes
After 24 hr of the last drug administration, the mice were decapitated, and then, the spleen and thymus were immediately weighed after excision from the mice. The thymus and spleen indices were calculated by the following equation:

| Lymphocyte proliferation assay
The spleens were removed from the mice, washed with 0.1 mol/L PBS buffer solution (4ºC), and gently grated and filtered with a 40-μm nylon cell strainer to obtain single-cell suspensions. Then, the singlecell suspensions were treated with the lymphocyte separation medium to separate erythrocytes and lymphocytes. Lymphocytes were washed with PBS and resuspended to a final density of 1 × 10 6 cells/ mL in RPMI 1640 medium supplemented with newborn bovine serum (10%), 1% glutamine (200 mmol/L), penicillin (100 U/μL), streptomycin (100 μg/L), and 2-mercaptoethanol (5 × 10 −5 mol/L). In a 96-well plate containing concanavalin A (5 μg/ml), the spleen cells were cultured for 72 hr in 5% CO 2 atmosphere at 37ºC and further incubated for 4 hr in MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, 5 g/L). Afterward, the plate was centrifuged for 15 min at 200×g to remove the supernatant. Subsequently, the plate was shaken after the addition of 150 μl dimethyl sulfoxide until all crystals dissolved. The absorbance of samples was measured at 570 nm.

| Phagocytic index
According to the methods of Shen et al. (2015), after intravenous injection with diluted India ink (100 ml/kg), the blood samples from the retinal venous plexuses were collected at 2 min and 10 min, respectively. Then, 20 µl of the blood samples from each mouse was added into 2 ml 0.1 Na 2 CO 3 . Subsequently, the absorbance of the mixed solution was measured at 600 nm with 0.1% Na 2 CO 3 as the blank. The liver and the spleen were weighed, and the phagocytic index was calculated as follows:

| Statistical analysis
All analysis was made by one-way analysis of variance (ANOVA) test using SPSS 22.0. The animal experiment results were expressed as the mean ± SD (n = 10), and other experimental results were expressed as the mean ± SD (n = 3). Significant differences were considered statistically at the level of 0.05.

| Solubility of PMR
The solubility of rice dreg, RDP, and PMR at different pH is presented in Figure 1a. The solubility was increased further (p < 0.05) by the modifications of sodium alginate under microwave radiation when Solubility of rice dreg, RDP, and PMR and emulsifying stability at different pH. a-c The different lowercase superscript letters in (a) mean significant differences between RDP, PMR, and rice dreg (p < 0.05); A-C the different lowercase superscript letters in bars mean significant differences at different pH value (p < 0.05). a-e The different lowercase superscript letters in (b) mean significant differences at different pH vlue (p < 0.05) compared to the unmodified RDP and rice dreg. Under the same Maillard reaction conditions, PMR at pH greater than 10 showed the highest solubility. Similarly, PMR at pH 2 showed higher solubility than that of samples with pH 4-6. In addition, the solubility of PMR increased from 39.78% to 90.97% between pH 6 and pH 12, but it decreased from 88.03% to 39.19% between pH 2 and pH 4. It has been reported that the solubility of PMR increased to a higher level than that of the mixture of protein and polysaccharides untreated by means of heat or protein at the same pH (de Oliveira, Coimbra, de Oliveira, Zuniga, & Rojas, 2016). Du et al. (2013) and Mishra, Mann, and Joshi (2001) proposed that the hydration of protein with the con-

| Emulsifying stability
The emulsifying stability of rice protein samples from rice dregs was studied under different pH conditions, as shown in Figure 1b. PMR had higher emulsifying stability at pH 2-12 and was, on average, about 10% higher than that of RDP. The emulsifying stability of PMR decreased gradually from pH 2 to pH 6 and increased between pH 6 and pH 12. PMR and RDP produced maximum emulsifying stability of 75.43% and 54.77%, respectively, at pH 12. In other words, the change of emulsifying stability under different pH conditions indicated an improved emulsifying capacity and a positive correlation between the emulsifying stability and solubility of PMR. As previously proposed by Cao, Wen, Li, and Gu (2009), the emulsifying stability of RDPs was influenced by protein solubility and surface hydrophobicity, and PMR showed better emulsifying stability than the blank samples. In fact, the grafting reaction of sodium alginate to RDP probably causes to aggregation of some unfolded protein molecules and inhibits the interaction between unfolded protein molecules. Therefore, PMR has better emulsify effects than the mixture of RDP and sodium alginate (Pirestani, Nasirpour, Keramat, Desobry, & Jasniewski, 2017). In addition, the emulsion stability of PMR was confirmed by the formation of reducing droplet size in terms of the steric effects related to sodium alginate around the oil droplets.

| Amino acid analysis
The analysis of amino acids of RDP and PMR is shown in

| Molecular weight
To further study the effect of Millard reaction on the formation of PMR, HPGPC was used to detect molecular weight distribution at 280 nm using UV detector. Chromatographs of PMR, RDP, and rice dreg revealed a significant difference among the molecular weight distributions ( Table 2). The high-molecular-weight groups increased with the formation of the RDP-sodium alginate conjugation, but the proportion of low-molecular-weight species decreased.
The relative proportions of fractions larger than 5,000 Da between PMR, RDP, and rice dreg were 54.15%, 9.55%, and 52.83%, respectively. Moreover, the proportion of fractions larger than 5,000 Da in PMR increased by 4.53 times as that of RDP. Inversely, the proportion of fractions less than 180 Da decreased markedly from 35.65% to 9.90%. It was probably due to the reaction between the amino groups of the peptides from RDP aggregates and the carbonyl groups of sodium alginate (Pirestani et al., 2017). According to the data in Table 2, the proportions of fractions between 180

| Circular dichroism spectroscopy
CD spectra in far-UV region (190-250 nm) are often used as an indicator of the changes of proteins secondary structure such as αhelix, β-fold, β-turn, and random coil. Therefore, CD spectroscopy was applied to characterize the spatial conformation of the Maillard reaction products between RDP and sodium alginate. Table 3 shows the secondary structure of PMR. Based on the data of proportion of β-pleated sheets (from 15.63% to 4.54%) and β-turns (from 17.16% to 2.37%), but a significant increase was observed in α-helices (from 1.42% to 4.24%) and random coils (from 65.58% to 88.95%), indicating that random coils accounted for the dominated secondary structure of PMR. The underlying cause may be that the introduction of more hydroxyl groups from sodium alginate to RDP resulted in the development of hydrogen bonds between RDP and sodium alginate molecules, which could inhibit neighboring proteins from the interaction with each other and therefore led to an increase in α-helix and a decrease in β-turn and β-pleated sheet (Du et al., 2013). Meanwhile, it was proposed that the conjugation of RDP with sodium alginate may lead to unfolding and conformational flexibility and changed spatial structure of proteins, which may alter their functional properties.

| FTIR spectra
The FTIR spectra of PMR and RDP are shown in Figure

| Immune activities of PMR
The thymus plays a vital role in the immune regulation on immunodeficient mice, maintaining T-cell differentiation and maturation.

| CON CLUS ION
The glycosylation modification of RDP with sodium alginate by wet heating assisted with microwave treatment is an effective method.
The solubility of PMR was significantly increased by grafting RDP with sodium alginate. Meanwhile, under the same grafted conditions, the emulsifying capacity of PMR was also improved, when compared to that of RDP. Based on structural analysis of PMR, it was concluded that the amino groups of RDP can be reacted with the reducing-end carbonyl groups of sodium alginate, leading to unfolding and conformational flexibility of RDP and spatial structure changes and consequently improved solubility and emulsifying capacity of RDP grafted with sodium alginate. Furthermore, it can be concluded from the results that RDP grafted with sodium alginate had strong immunomodulatory properties. Therefore, the glycosylation modification of protein with polysaccharide is a promising and effective way to develop novel techno-functional additives in a wide range of food industry, cosmetic, and pharmaceutical formulations as added-value food ingredients.

ACK N OWLED G M ENTS
This research was financially supported by the National Natural

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

E TH I C A L S TATEM ENTS
All animals were housed and cared for in accordance with the Chinese Pharmacological Society Guidelines for Animal Use.