1. Introduction
Rice is one of the most widely grown and consumed cereal crops in the world [
1]. In addition to being used as whole grains, rice, especially broken rice, is also commonly processed into rice flour (RF), which is frequently included in cakes, noodles, pasta, and other RF-based foods [
2,
3]. Starch and protein, which account for 80–90% and 5–10%, respectively, of the dry weight of RF, determine the processing method and quality of the final products. Rice protein has a reasonable amino acid composition, high digestibility, and nutritional value when compared with the protein from other major cereals, such as wheat and corn [
4]. Furthermore, rice protein is shown to have excellent physiological functions, such as inflammation-reducing, anticancer, and antioxidant [
5]. However, the protein content of RF is comparatively low, and to get enough protein, too much rice and easily digested starch are consumed, which may cause obesity and increase the risk of type II diabetes. Therefore, it is desirable to increase rice protein content in RF.
According to the origin of the protein, the reinforcement can be divided into either exogenous reconstitution or endogenous enrichment. At present, exogenous reconstitution is the main way, and protein concentrates/isolates, such as rice protein [
6], whey proteins [
7], wheat proteins [
8], etc., are used to reconstitute RF or rice starch (RS). In the case of rice proteins, the concentrates or isolates are mainly prepared by the alkaline method, which negatively affects the quality of the reconstitution due to some drawbacks, such as dark color and toxic compounds such as lysinoalanine [
4]. In endogenous enrichment, the starch is enzymatically degraded [
9] or physically separated [
10] to increase the relative content of protein in RF. Due to expensive devices and tedious procedures, physical enrichments, such as microfluidization [
10], supercritical fluid extrusion [
11], and ultrasound [
12,
13], are of limited value. For starch degradation, RF is commonly gelatinized at a high temperature and then hydrolyzed by thermostable α-amylase and amyloglucosidase [
14,
15]. However, because the starch granule structure is destroyed during the gelatinization, a series of physicochemical properties are lost, particularly pasting behaviors and endothermic characteristics, which reduces the application in food processing. Furthermore, the enriched protein is denatured [
16], and side effects such as Maillard reactions [
17] lower the nutritional value. In addition, genetic breeding and nitrogen-rich cultivation are commonly used ways to increase the protein content in RF. At present, a commercial high-protein rice variety named Frontière (average protein content is 11%), is available and has been used in the preparation of gluten-free muffins, bread, and cupcakes [
18,
19,
20].
However, with starch-degrading enzymes developing, hydrolysis of raw starch at moderate temperatures could avoid the starch gelatinization step. The key enzymes in this heterogeneous hydrolysis are called granular starch hydrolyzing enzymes (GSHE) or raw starch degrading enzymes (RSDE), including α-amylase, β-amylase, and amyloglucosidase [
21,
22]. There are many advantages to using GSHE, such as low energy consumption, cost savings, and the absence of side reactions. At present, GSHE, especially from commercial sources, has mostly been explored and applied in the field of fuel ethanol [
23] and enzymatically modified starch [
22,
24]. While numerous studies report on the effects of enzymatic modification on the structural and physicochemical properties of starch, detailed insight into the mechanisms of degradation for different enzymes is lagging. Nevertheless, the previous application of 4-α-glucanotransferase to multicomponent food raw materials such as RF [
25] provides an example and inspiration for us to improve the endogenous rice protein content in RF.
In RF, conceivably, the presence of non-starch components (protein and fat) will restrict the efficiency of starch hydrolysis by the GSHE, the key factor determining the extent of elevation of the endogenous rice protein content. Therefore, it is important to understand the mechanism of GSHE to improve the efficiency of enzymatic hydrolysis. Rice starch (RS) is the major component in RF and the substrate of GSHE. In RF, RS displays a multi-scale structure mainly including six levels: individual branches (level 1), amylopectin and amylose (level 2), semi-crystalline lamellas (level 3), growth rings (level 4), starch granules (level 5), and whole-grain structure (level 6) [
26]. Enzymatic hydrolysis alters the multi-scale structure of RS and simultaneously improves the relative protein content, eventually influencing the physicochemical properties of RF. In the food field, processability can be defined as the behavior and interaction between food components during the processing stages, such as transportation, mixing, stirring, extrusion, and various heat treatments, which can be reflected by physicochemical properties [
27,
28,
29]. In order to improve protein content under the premise of maintaining the processing properties, GSHE was selected to modify RS and RF in this study. During the process, the structural and physicochemical properties of the RF and RS prepared from RF were determined to propose a hydrolytic mechanism and create a new RF raw material for improving traditional or developing new RF-based foods.
3. Materials and Methods
3.1. Materials
Dry-milled Indica RF, made in Taizhou, Jiangsu Province (purchased online) was passed through a 100-mesh sieve for later use. The experimental enzyme mixture (Novozyme 5009) was generously provided by Novozymes Co. Ltd. (Copenhagen, Denmark), including fungal α-amylase (EC 3.2.1.1, GH 13) and amyloglucosidase (EC 3.2.1.3, GH 15) and named GSHE in this work. The activity (8111 U/mL) was assayed at 55 °C with 1.0% (
w/
v) RS suspension as substrate in 50 mM sodium acetate, pH 5.0, using the 3,5-dinitrosalicylic acid (DNS) method to quantify released reducing sugars [
57] and glucose as standard. One U was defined as the amount of enzyme forming 1.0 mg of reducing sugar per mL under the above conditions. Total starch assay kit (K-TSTA), amylose/amylopectin assay kit (K-AMYL), and isoamylase (E-ISAMY, 200 U/mL) were purchased from Megazyme Co. Ltd. (Wicklow, Ireland). All other reagents, solvents, and chemicals were reagent-grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
3.2. RS Isolation
RS was isolated following the method of Liu et al. [
58] with slight modification. RF was mixed with 0.2% (
w/
v) NaOH 1:4 (
w/
v), stirred continuously at room temperature for 4 h, and centrifuged (5000 rpm, 10 min). The yellow protein layer on top of the precipitate was discarded, and the residue was washed 3 times using 0.2% (
w/
v) NaOH until the yellow layer disappeared. The slurry was neutralized to pH 7 by 1 M HCl and washed 3 times with deionized water. The final starch sediment was dried at 45 °C for 48 h, ground in a mortar, and passed through a 100-mesh sieve.
3.3. Preparation of Hydrolyzed RF and RS by GSHE
RS (20.0 g, dry basis) and RF (25.5 g, dry basis, equivalent to 20.0 g RS dry basis) were suspended in 100 mL 50 mM sodium acetate, pH 5.0, and preheated in a water bath shaker (55 °C, 30 min). GSHE (50 U/g dry starch) was added to the RF and RS slurries and incubated for 1, 6, and 24 h (55 °C, continuous shaking at 160 rpm). The reaction was terminated by placing the slurries in an ice bath to cool instantly and rapidly adding 3 mL 1 M NaOH. After 15 min with intermittent shaking, pH was adjusted back to 5.0 by adding 3 mL 1 M HCl, and the slurry was centrifugated (5000 rpm, 10 min). The supernatant was collected to analyze the degree of hydrolysis (DH), and the sediment was washed 3 times using abundant deionized water. Finally, the hydrolyzed RS and RF were dried (45 °C, 48 h), ground in a mortar, and passed through 100-mesh sieves. The samples were named RS-1, RS-6, RS-24, RF-1, RF-6, and RF-24. Native samples without any treatment were named RS-N and RF-N.
Furthermore, to investigate the hydrolytic mechanism of GSHE in detail, RS was also hydrolyzed by GSHE under the above process for 3 and 9 h, and the obtained samples were named RS-3 and RS-9. RS subjected to the above process was used as the control, named RS-C, but without the addition of GSHE (only continuously shaken at 55 °C for 24 h as annealing treatment).
3.4. Determination of Total Starch, Total Protein, Amylose Contents, DH, and the Released Oligosaccharides
The total starch and amylose content were determined using a total starch assay kit (K-TSTA) and an amylose/amylopectin assay kit (K-AMYL), respectively. The total protein content was determined by an automatic Kjeldahl nitrogen analyzer (K1160, Hanon Advanced Technology Group Co., Ltd., Jinan, China) using the factor 5.95 to convert nitrogen content to crude protein content.
DH was determined by the amount of reducing sugar in the supernatant measured by the DNS method [
57] using the equation below. Briefly, the supernatant was suitably diluted by deionized water, and 0.5 mL was mixed with 0.5 mL DNS reagent, boiled for 5 min, and transferred to an ice-water bath to rapidly cool down to R.T. The absorbance was measured at 540 nm using a microplate reader (SPECTRA MAX 190, Molecular Devices, San Jose, CA, USA). The standard curve was made by replacing the diluted supernatant with the standard glucose solution (0.0–0.5 mg/mL).
where Tr is the weight of reducing sugar in the supernatant expressed as glucose, 0.9 is the glucose-to-starch conversion factor, and Ms is the weight of dry starch.
The relative composition and content of released oligosaccharides were determined by high-performance liquid chromatography (HPLC, Waters e2695, Water, Milford, MA, USA) equipped with X-Bridge BEH Amide column (250 mm × 4.6 mm). The supernatant of hydrolyzed starch was diluted with ultrapure water, and the same volume of pure acetonitrile was added and centrifugated (10000× g, 30 min). Acetonitrile solution (65%, w/w) was used as the mobile phase with 0.8 mL/min flow rate, the column temperature was 30 °C, and the loading volume was 50 μL. Glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltospentaose (G5), maltohexaose (G6), and maltoheptaose (G7) were used as the standard.
3.5. Particle Size Distribution
Particle size distribution of all samples was determined using the laser particle size analyzer (BT-9300 ST, Dandong Baxter Instrument Co., Ltd., Dandong, China). Deionized water was used as a dispersant, and the sample flour was added gradually to the sample pool. The sample was completely dispersed by ultrasound 3 times for 3 s each time. The measurement was carried out under the conditions of a pump speed of 1600 rpm and a shielding range of 10–15%. The refractive indexes of water and samples were 1.52 and 1.33, respectively [
59]. The measurement was carried out in triplicate, and d
50, D
[4,3], D
[3,2], and the span factor were recorded.
where the d
10, d
50, and d
90 mean of the cumulative particle diameters represent 10%, 50%, and 90% of the entire range of size.
3.6. Scanning Electron Microscopy (SEM)
The micromorphology was captured by scanning electron microscope (SEM, SU8100, Hitachi High-Tech, Tokyo, Japan). Flour adhered to the conductive tape, and gold was sprayed onto the flour in a vacuum. The final observation was conducted at ×3000 magnification operating at a low accelerating voltage of 15 kV and irradiation voltage of 1 kV.
3.7. XRD
XRD was conducted by using a diffractometer (D2 PHASER, Bruker AXS, Karlsruhe, Germany) equipped with Cu-Kα radiation and operated with scanning from 4–40° (4.79 °/min) at 0.04° with a count time of 0.5 s. RC was calculated by using Jade 6.0 software (Material Date, Inc., Livermore, CA, USA) as previously described [
60].
3.8. FTIR
FTIR spectra were obtained by an FTIR spectrometer (IS10, Thermo Nicolet Inc., Waltham, MA, USA) equipped with an ATR accessory in the region of 400–4000 cm−1 with a resolution of 4 cm−1 by 64 scans. The air background spectrum was deducted from each tested sample. The region of 1200−800 cm−1 was chosen to analyze the short-range order of samples with OMNIC 8.0, the half-bandwidth, and enhancement factor were set as 19 cm−1 and 1.9, respectively. The ratios between intensities at 1047 and 1022 cm−1 (R1047/1022) and 995 and 1022 cm−1 (R995/1015) over deconvoluted spectra were calculated.
3.9. HPAEC
The branch chain length distribution of RS samples was determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, ICS-5000+, Thermo Fisher Scientific, Waltham, MA, USA). Starch (10 mg) was suspended in 5 mL 50 mM sodium acetate, pH 4.5, and boiled for 30 min to be completely gelatinized. The gelatinized starch was fully debranched by 2 U isoamylase (40 °C, 12 h). After inactivation by boiling for 10 min and centrifugation (10,000×
g, 10 min), the supernatant was filtered (0.22 μm membrane filter), and 20 μL was injected onto a CarboPac PA200 column at 30 °C, eluted at a flow rate of 0.4 mL/min using isocratic 150 mM NaOH and a linear gradient of 0–400 mM sodium acetate as mobile phase [
49].
3.10. DSC
The thermal property of the samples was measured by a calorimeter (DSC7000, HITACHI, Japan). First, 3 mg of sample powder and 6 mg of deionized water were added to an aluminum pan. After a slight shaking, sample pans were sealed and equilibrated overnight at 4 °C. The temperature was linearly increased from 30 to 100 °C° at 10 °C/min. An empty aluminum pan was used as a reference. To, Tp, Tc, and ΔH were reported for each endothermic peak.
3.11. RVA
The pasting properties of samples were determined using a rapid viscosity analyzer (RVA-Starch Master2, Perten Instruments, Stockholm, Sweden). Deionized water was added to each sample (3.0 g on a 14% moisture basis) to a total weight of 28 g, and the analysis was conducted as previously described [
60]. Viscosity parameters, including PV, TV, BV, FV, and SV, were obtained through the built-in software.
3.12. Rheological Measurement
The rheological characterization was done using a rheometer (Discovery hybrid rheometer, TA Instruments, New Castle, DE, USA) with a parallel-plate system (40 mm diameter) and a working gap of 1000 mm. After the gelatinization of samples by RVA (described in
Section 3.11), the paste was rapidly cooled to 25 °C for 10 min. The paste was subjected to a frequency sweep test from 0.1 to 100 rad/s at a target strain of 1% (in the linear viscoelastic region) at 25 °C, and G’, G’’, and Tan (θ) were determined.
3.13. Statistic Analysis
Experimental results are reported as the average of triplicate measurements. The statistical significance was assessed by Tukey’s test using OriginPro 2022 b (OriginLab, Northampton, MA, USA). A p-value <0.05 was considered to be statistically significant throughout the study.
4. Conclusions
By controlling the reaction time of GSHE to RF, significant endogenous enrichment of the rice protein was achieved, while appropriate processing properties of RF were maintained. After the 24 h hydrolysis, the relative rice protein content improved from 8.52% to 13.17%. The results of the microstructure, crystal structure, and molecular structure of the RF indicated that the GSHE treatment rapidly increased the very short chains of amylopectin through the early-stage pinhole erosion, which then changed to pit erosion, further hydrolyzing the very short chains and increasing the relative content of the middle and long chains. In the whole process, the crystalline and amorphous regions were hydrolyzed, causing a slight increase in relative crystallinity. This hydrolytic mechanism led to a sharp decrease in viscosity during pasting after 1 h of hydrolysis followed by a slow increase, similar to the change in the viscoelastic properties of the pastes, but without significantly changing the thermal properties, which confirms that the physicochemical properties of RF can be properly preserved. The study brings more insights into structure–function relationships during the GSHE hydrolysis and creates a new RF raw material with high protein content by endogenous enrichment for application in various RF-based food, such as rice noodles and reconstituted rice, especially for gluten-free food, such as muffins, cakes, and bread.