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. 2020 Jul 23;58(4):1368–1377. doi: 10.1007/s13197-020-04648-3

Heat-induced changes in the physicochemical properties and in vitro digestibility of rice protein fractions

Kunlun Liu 1,✉, Jiabao Zheng 1, Fusheng Chen 1
PMCID: PMC7925720  PMID: 33746265

Abstract

The effects of heat treatment on protein interaction, surface hydrophobicity, protein profile, amino acid composition, and in vitro digestibility of individual rice protein fractions were investigated. Heat treatment at 100 °C for 20 min had no negative effect on essential amino acids in rice protein. Surface hydrophobicity increased significantly with the increased heat treatment temperature. Moreover, free-thiol content decreased significantly with increased temperature and time extension. Hydrophobic interactions contributed to the heat-induced interaction of glutelin and prolamin. Intramolecular disulfide linkages participated in the heat-induced interaction of all rice-protein fractions. Heat treatment had no effects on the in vitro digestibility of glutelin, globulin, and albumin. Thus, the heat-induced interactions of glutelin, globulin, and albumin were not related to their digestibility. By contrast, the formation of intramolecular disulfide bonds and hydrophobic interactions in prolamin may reduce its digestibility by strengthening protein bodies-Is.

Keywords: Heat treatment, Rice protein, Protein interactions, Protein digestibility

Introduction

Rice (Oryza sativa L.) is the predominant staple food in many regions of the world and provides 20% of the world’s dietary energy supply (FAO 2004). Rice protein is the second most abundant component of rice, accounting for approximately 6.3–7.1% of rice weight (milled rice). The Osborne extraction method has shown that rice protein is composed of glutelin (about 80%), globulin (about 12%), albumin (about 5%), and prolamin (about 3%), which dissolve in alkaline solution, salt solution, water, and alcohol, respectively. Rice protein exists as protein bodies, including protein bodies-I (PB-Is) and protein bodies-II (PB-IIs). PB-Is have a lamellar structure, are spherical in shape, and are rich in prolamin; PB-IIs have a crystalline structure, showing the irregular shape and are rich in glutelin (Amagliani et al. 2017). Moreover, the protein digestibility and biological value of rice were higher than those of the other major cereal (i.e., wheat, corn, and barley). Rice protein is generally regarded as hypoallergenic (Helm and Burks 1996), with several studies suggesting that rice proteins (in many cases, rice protein-based hydrolysates and specific peptide fractions) have anti-oxidative (Liu et al. 2015; Phongthai et al. 2018), anti-hypertensive (Hiroko et al. 2009), anticancer (Arvind Kannan et al. 2009, 2010), and anti-obesity properties (Arvind Kannan et al. 2012; T. Yang et al. 2012a, b).

However, like to some major cereals, such as sorghum (Hamaker et al. 1987), maize (Emmambux and Taylor 2009), and proso millet (Gulati et al. 2017), protein digestibility of rice also decreased subject to heat treatment. Bradbury et al. (1984) found that in vitro protein digestibility of brown rice and milled rice were decreased by 6.8% and 11.4%, respectively, after cooking. Moreover, Collier et al. (1998) found that the cooked rice diet produced more fecal protein particles in both mice and sheep, which have mono- and poly-gastric digestive systems, respectively, after they were fed with cooked and uncooked rice endosperm protein. According to previous studies, the decline in the protein digestibility of rice subjected to heat treatment was ascribed to the indigestibility of prolamin and disulfide bond crosslinking Bradbury et al. (1984). suggested that the formation of the cysteine-rich core (rather than isopeptide crosslinking) during cooking can make rice protein resistant to proteases. In addition, Kubota et al. (2014) reported that rice prolamin is not indigestible by nature but is rendered indigestible by cooking. They also reported that alkali extraction could improve the digestibility and bioavailability of rice protein by changing the particle structures of PB-Is where prolamin accumulates (Kubota et al. 2010). By contrast, Sagum and Arcot (2000) reported heat processes significantly increased the protein digestibility of rice by boiling and pressure cooking. This result may be attributed to the inactivation of proteinase inhibitors and the denaturation of the protein structure. In general, the factors, which are likely to affect cereal protein digestibility, can be categorized into exogenous and endogenous factors (Duodu et al. 2003). The former includes organizational structure, antinutrients (protease inhibitors, amylase, and phytic acid), starch, and non-starch macromolecules, while the latter includes protein crosslinking (disulfide crosslinking, hydrogen bonds, and isopeptide crosslinking), hydrophobicity, and protein secondary structure. However, the effects of heat-induced interactions and physicochemical changes on the protein digestibility of rice-protein fractions have not been reported.

This study focused on individual rice-protein fractions to (1) investigate the effect of heat treatment on the heat-induced interactions and surface hydrophobicity of individual rice-protein fractions; (2) evaluate the effect of heat treatment on amino acid composition and protein digestibility; and (3) identify the relationships among heat-induced interactions, surface hydrophobicity, and protein digestibility.

Materials and methods

Protein sources

Brown rice grains of japonica cultivar Xinfeng 2 (X2) were used in this study. The brown rice X2 was milled with a laboratory scale milling machine to obtain the milled rice samples (degree of milling = 9%). Then, the milled rice samples were ground into powder. Rice protein was extracted followed the method reported by Ju et al. (2010) A 100-g rice flour was extracted with 400 mL deionized water, 400 mL 5% NaCl, 400 mL 0.2 M NaOH, and 300 mL 70% ethanol at 20 °C in sequence for 4 h and centrifuged at 3000 × g for 30 min to obtain albumin, globulin, glutelin, and prolamin, respectively, in the supernatant. Each extraction was repeated 2 times. The supernatant from each extracted process was adjusted to pH 4.1, 4.3, 4.8, and 5.0 with 0.1 M HCl to precipitate the proteins. The precipitated protein was collected after centrifuging at 3000 × g for 30 min and washed twice with distilled water. Then the pH value of protein was brought up to 7.0, freeze-dried, and stored at − 20 °C.

Heat treatment

The rice protein powder was suspended at 5% (w/v) in deionized water and stirred at 25 °C for 2 h in a 100 mL covered conical flask. Each rice protein suspension was placed in a digital water bath oscillator and heated at 80 °C and 100 °C for 5, 10, and 20 min, respectively. The heated rice proteins were cooled in ice and freeze-dried for further analysis.

Determination of changes in protein interactions

The changes in protein interactions were evaluated by comparison of protein solubility methods using different solvent systems, as reported by Liu and Hsieh (2008). A total of six reagents were selected: phosphate buffer, urea, dithiothreitol (DTT), thiourea, Triton X-100, and 3-[3-(cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Phosphate buffer was used to extract the protein in a native state. Both urea and thiourea were used to break non-covalent interactions. Urea is more efficient than thiourea in breaking hydrogen bonds, whereas thiourea is more efficient than urea in breaking hydrophobic interactions. Triton X-100 and CHAPS also disrupt hydrophobic bonds. Uses of these two reagents could help differentiate the relative importance among non-covalent interactions. DTT, a common protein reducing agent, was used to break disulfide bonds. Moreover, the solution containing all of the above reagents is known as isoelectric focus (IEF) buffer. This buffer is commonly used in the proteomic study and known to break up all non-covalent bonds as well as disulfide bonds. The protein–protein interactions were observed by subtracting a type of reagent (such as the reagents breaking hydrogen bond, hydrophobicity interaction, and disulfide bond, respectively) from the IEF buffer. Therefore, the selective reagents were a combination of five extracting solutions as follows: (1) 0.1 M phosphate buffer + 8 M urea + 0.05 M DTT + 2 M thiourea + 2% Triton X-100 + 2% CHAPS; (2) 0.1 M phosphate buffer + 8 M urea + 0.05 M DTT; (3) 0.1 M phosphate buffer + 0.05 M DTT + 2 M thiourea + 2% Triton X-100 + 2% CHAPS; (4) 0.1 M phosphate buffer + 8 M urea + 2 M thiourea + 2% Triton X-100 + 2% CHAPS; and (5) 0.1 M phosphate buffer.

Each protein sample was homogenized with 10 mL extracting solution by for 2 min and shook for 2 h at 25 °C. Then, the homogenates were centrifuged for 20 min at 10,000 × g. The protein concentration in the supernatants was determined using the Bradford method. The total protein content of protein samples was measured by the Automatic Kieldahl apparatus, in accordance with the AACC method 46–13 (AACC 2000). The protein solubility was calculated as the protein in the supernatant to the protein in samples. Each measurement was performed in triplicate.

Determination of free-thiol content

The free-thiol content was determined according to the method of flour and gluten described by Beveridge et al. (1974). Each measurement was performed in triplicate.

Determination of surface hydrophobicity

Surface hydrophobicity (H0) was determined according to the method given by Zhao et al. (2012) using 8 mM 1-anilino-8-naphthalene sulfonate as the hydrophobic fluorescence probes. Protein solutions with concentrations between 0.005 and 0.025% were prepared with 10 mM phosphate buffer (pH 7.0). Each measurement was performed in triplicate.

Determination of in vitro protein digestibility

Protein samples were digested under simulated gastric and gastrointestinal conditions as a modified method described by Deng et al. (2015). In the simulated gastric procedure, 0.1 g of protein sample was mixed with 17 mL of HCl (0.1 M) and 2.5 mL of pepsin solution (2.5 mg/mL in 0.1 M HCl). The mixture was blended using a vortex mixer for 2 min and shook in a digital water bath oscillator for 2 h at 37 °C. In the simulated gastrointestinal procedure, which was based on simulated gastric digestion, 0.5 M NaOH was added to adjust the pH of the mixture to 8.0. Then, 2.5 mL of pancreatic solution (2.5 mg/mL in 0.1 M phosphate buffer, pH 8.0) was added to initiate the 2 h simulated gastrointestinal digestion at 37 °C. Then, 10 mL of 100 g/L trichloroacetic acid was added to stop gastric and gastrointestinal digestion. The mixture was centrifuged at 9000 × g for 15 min, and 10 mL supernatant was collected for Kjeldahl measurement. The protein digestibility was calculated as the protein in the supernatant to the protein in samples. Each experiment was performed in triplicate.

Amino acid composition analysis

Amino acid composition was measured with an amino acid analyzer (S433D, Sykam, Germany) according to the method given by Liu et al. (2017). The results were calculated by external standard method and integration of the peak area. Each measurement was performed in twice.

Reducing and non-reducing SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the discontinuous system (12% separating/5% stacking gel) according to the method of Liu et al. (2011) Molecular weight (MW) markers in ranging of 14.4–97.4 kDa were applied on each well.

Statistical analysis

Values were expressed as means ± standard deviations. The significant difference was determined at the P < 0.05 level for Duncan's multiple range test by using SPSS software (version 16.0, Chicago, USA).

Results and discussion

Chemical interaction analysis of heat-induced rice protein fractions

In this study, protein interactions were evaluated by the comparing protein solubility methods by using different solvent systems, as reported by Liu et al. (2017). Meanwhile, the quantitative analysis of free-thiol content (Fig. 1, 2) and surface hydrophobicity (Fig. 3) was performed to validate the results of protein solubility assay.

Fig. 1.

Fig. 1

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Effect of heat treatment on the solubility of rice glutelin (a), globulin (b), albumin (c), and prolamin (d). Treatments of I, II, III, IV, V, VI, and VII represent 0 min, 80 °C for 5 min, 80 °C for 10 min, 80 °C for 20 min, 100 °C for 5 min, 100 °C for 10 min, and 100 °C for 20 min, respectively

Fig. 2.

Fig. 2

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Effect of heat treatment on the free-thiol content of rice glutelin (a), globulin (b), albumin (c), and prolamin (d). Treatments of I, II, III, IV, V, VI, and VII represent 0 min, 80 °C for 5 min, 80 °C for 10 min, 80 °C for 20 min, 100 °C for 5 min, 100 °C for 10 min, and 100 °C for 20 min, respectively

Fig. 3.

Fig. 3

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Effect of heat treatment on the surface hydrophobicity of rice glutelin (a), globulin (b), albumin (c), and prolamin (d). Treatments of I, II, III, IV, V, VI, and VII represent 0 min, 80 °C for 5 min, 80 °C for 10 min, 80 °C for 20 min, 100 °C for 5 min, 100 °C for 10 min, and 100 °C for 20 min, respectively

The protein solubility of rice glutelin is shown in Fig. 1a. Among the five selected extracting solutions, the sharpest decrease in protein solubility was observed between IEF buffer and solvent without thiourea, Triton, and CHAPS. Glutelin solubility also decreased when DTT and urea were removed, respectively. These results suggested that both disulfide bonds and non-covalent interactions involved in maintaining of rice glutelin, especially hydrophobic interactions were abundant in rice glutelin. In addition, glutelin solubility in phosphate buffer significantly decreased (P < 0.05) after heat treatments of 80 °C for 20 min and 100 °C for 5, 10, and 20 min. Thus, parts of rice glutelin were denatured after heat treatment. Similar to this result, surface hydrophobicity of glutelin also significantly decreased subjected to these heat treatments (Fig. 3a). Moreover, denaturation was accelerated with increased temperature from 80 to 100 °C, and it was expanded after prolonging the duration of heat treatment from 5 to 20 min. These results may be due to the increasing rate of denaturation because the rise in temperature was much higher than the increase in common chemical reaction velocity due to rising temperature. A slight decrease in glutelin solubility in the solvent without thiourea, CHAPS, and Triton was found between heat-induced and unheated glutelin. Significant decreases of 10.3% and 11.8% were observed at 80 °C for 10 min and 100 °C for 20 min, respectively (Fig. 1a). Similar results were observed in the solvent without DTT. The solubility of glutelin slightly decreased with increasing temperature and heating time. Similar to this observation, free-thiol content decreased significantly after heat treatments, as shown in Fig. 2a. Visschers and Jongh (2005) reported that cysteine residues, which are inaccessible in the native conformation, become available and may react to form disulfide bonds cross-linking during heat-induced denaturation. Therefore, disulfide bridges and non-covalent interactions were both responsible for the structure of glutelin. Disulfide bridges and hydrophobic interactions played important roles in heat-induced rice glutelin interaction.

The protein solubility of rice globulin is shown in Fig. 1b. The globulin solubility in phosphate buffer was significantly higher than three other rice protein factions because it can easily dissolve in salt solution due to its net electrical charge (Hamada 1997). The solubility of untreated globulin decreased when the reagent was removed, which can break non-covalent bonds. Thus, non-covalent interactions were both responsible for the structure of globulin. However, hydrophobic interactions and hydrogen bonds were not correlated with heat-induced protein interaction. By contrast, disulfide bonds played roles in heat-induced interactions of globulin, because a significant decrease of 38.1% occurred only when DTT was removed after 20 min of heat treatment at 100 °C. Meanwhile, a sharp decrease of free-thiol content in globulin was observed after 20 min of heat treatment 100 °C. This result may be the reason for the significant reduction in phosphate buffer that occurred only for globulin which was heated at 100 °C for 20 min. Therefore, non-covalent interactions were responsible for the structure of glutelin. However, disulfide bridges played important roles in the heat-induced rice glutelin interaction.

As shown in Fig. 1c, the solubility of albumin in phosphate buffer decreased significantly after 20 min of heat treatment at 100 °C. Moreover, surface hydrophobicity of albumin increased significantly after heat treatment (Fig. 3c). These results indicated that parts of albumin unfolded, and hydrophobic patches were exposed after heat treatment at 80 °C or 100 °C. The solubility of albumin subjected to heat treatment at 80 °C for 20 min significantly decreased when urea or thiourea, Triton, and CHAPS were subtracted from IEF buffer. It indicated that hydrophobic interactions were responsible for the structure of albumin. However, a heat-induced decrease in albumin solubility occurred only in the solvent after removing DTT from IEF buffer. The decrease in free-thiol content after heat treatments also supported this result (Fig. 2c). Although unheated albumin have sufficient net charge and lack of any extensive disulfide cross-linking or aggregation, the heat-induced protein interactions of albumin may be related only to disulfide bonds (Hamada 1997).

As shown in Fig. 1d, non-covalent bonds and disulfide linkages were involved in maintaining the structure of rice prolamin because the solubility of unheated prolamin sharply decreased when any type of reagent was subtracted from IEF buffer. Moreover, prolamin solubility significantly decreased in the solvent without DTT after heat treatment.The reduction resulting from heat treatment at 100 °C was significantly higher than reduction in heat treatment at 80 °C. Besides, a slight decrease was also observed in prolamin solubility in the solvent which removed DTT from IEF buffer after heat treatment at 100 °C for 20 min. Thus, both disulfide bond and hydrophobic interaction played roles in heat-induced interactions of rice prolamin. The formation of disulfide bond (–SH converted to S–S) may be the predominant interaction in this reaction. Similar to these results, Kubota et al. (2014) found that the hydrophobic interactions and disulfide linkages in PB-Is increased after rice was cooked.

In general, according to the changes in rice protein solubility and surface hydrophobicity, heat treatment at 80 °C for 20 min was enough to denature rice protein, especially albumin, which has the lowest denaturation temperature (73.3 °C) (Ju et al. 2010). In addition, disulfide bonds may not be the predominant chemical bonds in rice protein structure. However, it played the most important role in heat-induced protein interactions of rice protein.

Analysis of protein profile in heat-induced rice protein

The rice protein profiles obtained via reduced and non-reduced SDS-PAGE are shown in Fig. 4 to further indicate the roles of disulfide bonds during heat treatment of rice protein. In general, no significant change was observed on rice protein polypeptide subunits after heat treatments in the reduced condition. By contrast, the intensity of the main polypeptide subunits decreased after heat treatments in non-reduced condition.

Fig. 4.

Fig. 4

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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis patterns of rice glutelin (a), globulin (b), albumin (c), and prolamin (d). (MW: molecular weight markers; 1: unheated protein in reduced condition; 2: unheated rice protein in non-reduced condition; 3: 80 °C for 20 min heated rice protein in reduced condition; 4: 80 °C for 20 min heated rice protein in non-reduced condition; 5: 100 °C for 20 min heated rice protein in reduced condition; 6: 100 °C for 20 min heated rice protein in non-reduced condition)

As shown in Fig. 4a, in non-reduced condition, two major polypeptide subunits of rice glutelin with MWs of 30–40 (α or acidic) and 19–23 kDa (β or basic), glutelin precursor with MWs of 51 kDa, and some cross-contamination polypeptide subunits, such as globulin with MWs of 25 kDa and prolamin with MWs of 13 and 16 kDa, all decreased after heat treatment. Similar to the results of Amagliani et al. (2017) two major polypeptide subunits of rice globulin with MWs of 19–25 and 13 kDa were observed in reducing condition. And the subunit in the range of 19–25 kDa was predominant. In non-reduced conditions, a sharp decline in subunit intensity appeared after heat treatment at 100 °C for 20 min. Except for the predominant subunits with MWs of 19–25 kDa, all polypeptide subunits disappeared, thereby suggesting that disulfide bonds increased significantly after heat treatment at 100 °C for 20 min. This result was in agreement with the significant decrease in free-thiol content (Fig. 2b) and globulin solubility in phosphate buffer (Fig. 1b).

In this study, the protein profile of rice albumin was heterogeneous (Fig. 4c), which was resolved into a wide range of bonds with MW in the range of 13–110 kDa. The intensity of all subunits in rice albumin decreased after heat treatment in non-reduced conditions. This result showed that disulfide bridges were involved in heat-induced interactions of albumin, which may decrease the solubility of albumin.

In addition to the most predominant 13 kDa subunit of prolamin, two other subunits of approximately 18 and 31–32 kDa presented in this study. Similar results were reported by Amagliani et al. (2017), and this finding was most likely due to cross-contamination with glutelin, which accounts for 79–83% of milled rice protein, during prolamin extraction. In non-reduced conditions, the intensity of all bonds decreased significantly after heat treatment. The reduction caused by heating at 100 °C for 20 min was more than that induced by heating at 80 °C for 20 min.

In addition, protein profiles of four rice-protein fractions all displayed the formation of intramolecular disulfide bonds because no new polypeptide subunit in non-reduced condition after heat treatment. Thus, the solubility of almost all subunits decreased under heat treatment due to the formation of intramolecular disulfide linkages. In addition, under high temperature conditions, the rice protein was more likely to create disulfide bonds due to the availability of cysteine residues or restrain the unfavorable exposure of hydrophobic patches.

Analysis of amino acid composition

Changes in the amino acid composition of rice protein after heat treatment at 100 °C for 20 min are shown in Table 1. The highest total amino acid (TAA) was observed in prolamin. By contrast, albumin had the lowest TAA, which may be due to the contamination of other water-soluble compounds in the extraction process of rice albumin. Lysine is recognized as the first limiting amino acid among cereal proteins (Young 1994). In this study, the highest lysine content was found in albumin, followed by prolamin, glutelin, and globulin. This result did not correspond with the findings of Juliano (1985) who reported that prolamin has the lowest lysine content. This result may be due to the high TAA content in rice prolamin and cross-contamination with glutelin.

Table 1.

Changes in amino acid composition of rice-protein fractions after heat treatment at 100 °C for 20 min (g/100 g)

Amino acid Glutelin Globulin Albumin Prolamin
Unheated Heated Unheated Heated Unheated Heated Unheated Heated
Asp 15.7 ± 1.5a 8.90 ± 0.18b 10.5 ± 0.4a 4.66 ± 0.11b 6.97 ± 0.33a 7.78 ± 0.29a 9.34 ± 0.37a 9.49 ± 0.39a
Thr 1.91 ± 0.02a 1.61 ± 0.06b 1.12 ± 0.11a 1.19 ± 0.03a 1.75 ± 0.08a 1.74 ± 0.07a 2.06 ± 0.04a 2.15 ± 0.23a
Ser 3.80 ± 0.15a 3.73 ± 0.20a 5.22 ± 0.24a 5.32 ± 0.22a 3.06 ± 0.07a 3.20 ± 0.18a 3.76 ± 0.10a 4.03 ± 0.27a
Glu 12.1 ± 0.3a 17.0 ± 0.2b 14.6 ± 0.2a 22.7 ± 0.2b 8.42 ± 0.29a 12.3 ± 1.24b 12.2 ± 0.3a 17.7 ± 1.2a
Gly 3.57 ± 0.18a 3.73 ± 0.03a 4.38 ± 0.16a 4.42 ± 0.04a 3.89 ± 0.04a 4.17 ± 0.11a 4.15 ± 0.12a 3.79 ± 0.20a
Ala 4.28 ± 0.21a 4.68 ± 0.08a 3.83 ± 0.25a 3.94 ± 0.11a 4.88 ± 0.04a 4.64 ± 0.15a 5.76 ± 0.12a 4.85 ± 0.22b
Val 4.53 ± 0.28a 5.20 ± 0.31a 3.79 ± 0.12a 3.43 ± 0.34a 4.50 ± 0.03a 5.19 ± 0.09b 5.76 ± 0.13a 6.31 ± 0.31a
Met 0.89 ± 0.04a 0.70 ± 0.09a 2.97 ± 0.04a 2.29 ± 0.34a 2.01 ± 0.10a 1.53 ± 0.14a 1.50 ± 0.20a 1.25 ± 0.17a
Ile 3.72 ± 0.16a 4.03 ± 0.02a 1.69 ± 0.03a 2.08 ± 0.25a 3.23 ± 0.04a 3.80 ± 0.06b 4.33 ± 0.24a 4.38 ± 0.29a
Leu 6.42 ± 0.29a 7.50 ± 0.50a 5.55 ± 0.02a 5.85 ± 0.22a 5.55 ± 0.03a 5.60 ± 0.00a 8.20 ± 0.08a 7.25 ± 0.03b
Tyr 3.65 ± 0.21a 4.03 ± 0.07a 4.99 ± 0.09a 5.46 ± 0.10b 2.61 ± 0.23a 2.45 ± 0.06a 3.71 ± 0.05a 3.58 ± 0.03a
Phe 5.34 ± 0.25a 6.03 ± 0.48a 3.25 ± 0.21a 3.67 ± 0.34a 3.63 ± 0.30a 4.09 ± 0.02a 5.86 ± 0.64a 5.48 ± 0.18a
His 2.87 ± 0.18a 3.86 ± 0.03b 2.55 ± 0.13a 3.82 ± 0.14b 2.75 ± 0.13a 3.53 ± 0.05b 3.65 ± 0.17a 4.12 ± 0.10a
Lys 2.69 ± 0.20a 3.29 ± 0.01a 1.18 ± 0.01a 1.56 ± 0.11b 4.21 ± 0.19a 4.52 ± 0.16a 3.74 ± 0.12a 3.74 ± 0.03a
Arg 8.42 ± 0.36a 7.81 ± 0.12a 12.0 ± 0.2a 11.31 ± 0.3a 8.05 ± 0.67a 5.32 ± 0.49b 10.1 ± 0.0a 6.55 ± 0.31b
Pro 4.31 ± 0.03a 4.00 ± 0.11a 4.70 ± 0.14a 3.09 ± 0.11b 7.03 ± 0.18a 3.72 ± 0.35b 7.03 ± 0.76a 4.52 ± 0.34b
Cys 0.41 ± 0.12a 0.01 ± 0.02b 1.14 ± 0.09a 0.03 ± 0.00b 0.90 ± 0.13a 0.00 ± 0.00b 0.64 ± 0.17a 0.01 ± 0.00b
TAA 84.6 86.1 83.5 84.8 73.4 73.6 91.8 89.2
EAA 25.5 28.3 19.5 20.1 24.9 26.5 31.5 30.6
NEAA 59.1 58.1 63.9 64.7 48.6 47.1 60.3 58.7
E/T (%) 30.2 32.8 23.4 23.7 33.9 36.0 34.3 34.3
E/N (%) 43.2 48.7 30.6 31.0 51.2 56.2 52.1 52.1
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Means followed by different small cases for the same amino acid and the same rice protein fractions are significantly different (P < 0.05). TAA: total amino acids, EAA: essential amino acids, NEAA: non-essential amino acids.E/T (\% ) = EAA/TAA; E/N (\% ) = EAA/NEAA

Glutamic acid and histidine contents of rice glutelin, globulin, albumin, and prolamin increased significantly (P < 0.05) after heat treatment at 100 °C for 20 min. Notably, lysine content increased slightly except in rice prolamin. However, heat treatment at 100 °C for 20 min led to a sharp decrease in cysteine, although cysteine and methionine were partially destroyed by hydrochloric acid during hydrolysis. The reduction in the cysteine content of rice glutelin, globulin, albumin, and prolamin reached 97.4%, 97.6%, 100%, and 98.2%, respectively. Exposure of cysteine residues during heat-induced protein unfolding caused almost all cysteines to be oxidized or exchanged after heat treatment at 100 °C for 20 min. Moreover, a sharp decline was observed in proline. However, the decrease was not significant (P > 0.05) in rice glutelin. Meanwhile, the aspartic acid content of glutelin and globulin, as well as the arginine content of albumin and prolamin, decreased significantly after heat treatment.

Serine, glycine, alanine, and tyrosine were stabilized during heat treatment. In addition, the alanine content in prolamin decreased by 15.8%, and tyrosine in globulin increased by 8.6%. Except lysine, the essential amino acid contents of threonine, valine, methionine, isoleucine, leucine, and phenylalanine were almost unchanged. This result was also observed on the relatively stable levels of essential amino acids (EAA), the ratio of EAA to total amino acids (E/T), and the ration of EAA to non-EAA (E/N), as shown in Table 1.

In vitro digestibility of heat-induced rice-protein fractions

The gastric and gastrointestinal rice protein digestibilities are shown in Fig. 5. In general, no significant changes were observed in the digestibilities of four rice-protein fractions after heating at 80 °C. The gastric digestibility of glutelin significantly decreased (6.3%) after heat treatment at 100 °C for 10 min. Both the gastric and gastrointestinal digestibilities of glutelin decreased (5.5% and 6.3%, respectively) after heating at 100 °C for 20 min (Fig. 5a). Meanwhile, gastric digestibility of prolamin also significantly decreased (6.3%) after 20 min of heat treatment at 100 °C (Fig. 5d). Thus, heat-induced disulfide bonds of albumin and globulin had no effect on their protein digestibilities. Xia et al. (2012) reported all polypeptide subunits of rice protein, except prolamin, can be hydrolyzed by pepsin. And similar result was also observed in our preliminary test, all rice protein polypeptide subunits, except the 13 kDa prolamin polypeptide subunit, before and after heat treatment can be digested after in vitro pepsin digestion. This result indicated that the significant decrease in glutelin digestibility after heat treatment at 100 °C was caused by cross-contamination with prolamin, which was found at the glutelin profile shown in Fig. 4a. In addition, the decrease (5.5–6.3%) in protein digestibility in this study were lower than the result of previous research (Bradbury et al. 1984). As a result of alkali treatment, which can weaken and degrade the structure of PB-Is, the release and digestion of prolamin can be facilitated (Kubota, et al. 2010; Yang et al. 2012a, b). This result may lead to the decrease in reduction in protein digestibility. Therefore, the decline in rice protein digestibility was highly correlated with PB-Is. Kubuta et al. (2014) demonstrated that prolamin digestibility can be increased once hydrophobic interactions and disulfide bonds are broken. Thus, considering the formation of intramolecular disulfide bonds and hydrophobic interactions in prolamin subjected to heat treatment in this study, we can hypothesize that heat-induced formation of intramolecular disulfide linkages and hydrophobic interactions can strengthen PB-Is, which may decrease the digestibility of rice prolamin.

Fig. 5.

Fig. 5

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Effect of heat treatments on digestibilities of rice glutelin (a), globulin (b), albumin (c), and prolamin (d). Treatments of I, II, III, IV, V, VI, and VII represent 0 min, 80 °C for 5 min, 80 °C for 10 min, 80 °C for 20 min, 100 °C for 5 min, 100 °C for 10 min, and 100 °C for 20 min, respectively. Different lowercase letters and lowercase letters with single quotation were used to indicate significant differences (P < 0.05) between various treatments

Conclusion

Heat treatment at 100 °C for 20 min showed no negative effect on essential amino acids. High temperature and prolonged heating time were conducive to the generation of disulfide bonds and the increase in surface hydrophobicity as a result of the accessible and available cysteine residues and hydrophobic groups after heat treatment. Disulfide linkages contributed to heat-induced interaction for all rice protein fractions. Hydrophobic interactions were responsible for heat-induced interaction of glutelin and prolamin. However, the protein interactions of glutelin, globulin, and albumin subjected to heat treatment did not affect their digestibility. By contrast, the heat-induced formation of intramolecular disulfide bonds and hydrophobic interactions in prolamin may reduce its digestibility by strengthening PB-Is. This study provided a theoretical basis for the changes in nutritional properties of rice protein and corresponding molecular mechanisms during domestic cooking and commercial heat treatments.

Acknowledgements

This study was supported by the Engineering Technology Research Center for Grain & Oil Food, State Administration of Grain (GA2018005), the Natural Science Research Project of Henan Educational Department (19zx013), the Talent Projects from Henan University of Technology (2018RCJH05), and the Science and Technology Project of Henan Province (192102110208).

Footnotes

Publisher's Note

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