Rice Bran Protein Hydrolysates Research Articles

Effects of rice bran stabilization procedures and proteases on rice bran protein hydrolysates

Rice bran stabilization is typically performed after rice milling to improve storage performance. In the present study, we investigated the effect of extrusion (EX) and superheated steam (SS) on the physicochemical and functional characteristics of rice bran protein hydrolysates (RBPHs) obtained with acid, alkaline, and neutral proteases (AP, ALP, and NP). The deactivation parameters were optimized as EX (140 °C, water content: 25%) and SS (130 °C, 5 min). After stabilization, the enzymatic hydrolysis of proteins was inhibited. The untreated RBPHs obtained with ALP exhibited the highest degree of hydrolysis (15.93%), while RBPH-EX-NP presented the lowest (10.75%). EX reduced the solubility of RBPHs, while SS improved the solubility at pH 2–4. Both stabilizations increased the β-sheet and β-turn contents of RBPHs while reducing the α-helix and random coil contents. The antioxidant activity of RBPHs decreased, while their emulsifying activity and ferrous chelating ability increased. This study could contribute to insight into the effects of industrial stabilization on RBPHs.

Evaluation of antioxidant and ACE inhibitory peptides in rice bran protein hydrolysate using non‐targeted UHPLC‐QTOF‐IMS profiling, MALDI‐TOF‐TOF‐MS/MS, and molecular docking study

SummaryIn the present study, rice bran protein (RBP) from the PB1121 variety was hydrolysed using trypsin and subjected to non‐targeted IMS profiling to identify antioxidant and antihypertensive amino acids in the resulting RBP hydrolysates. The hydrolysate was then separated into three fractions using ultrafiltration, and each fraction was tested for antioxidant and ACE inhibitory activities. The ACE activity was assessed using the tripeptide hippuryl‐histidyl‐leucine (HHL) as a model peptide via HPLC‐DAD. The fraction with the molecular weight (>3000 Da) displayed the strongest antioxidant and ACE inhibitory activity. This fraction was further divided into five fractions using gel Sephadex G‐25, and the greatest levels of antioxidant and ACE inhibitory activity were found in fraction F3B. Fraction F3B was then fractionated using HPLC, and the fraction with ACE inhibitory activity (IC50 of 43 mg mL−1) was analysed using MALDI‐TOF‐TOF mass spectrometry to determine its exact molecular mass and amino acid sequence. The amino acid sequence FMKSK (phe‐met‐lys‐ser‐lys) with a Mw of 655.376 Da was detected, and the molecular docking investigation revealed that FMKSK suppresses ACE by forming strong hydrogen bonds with the active pockets of human ACE. These findings suggest that rice bran contains bioactive peptides with antioxidant and ACE inhibitory effects, making it a promising raw material for the production of beneficial products.

Lipolytic behavior and bioaccessibility of curcumin nanoemulsions stabilized by rice bran protein hydrolysate

This study aimed to use rice bran protein hydrolysate (RBPH)-stabilized curcumin nanoemulsions, compared with rice bran protein (RBP), to investigate digestion in the oral and gastrointestinal tracts and to investigate the effect of each digestion factor (pepsin, bile salts, pancreatic lipase) on the lipolysis rate and curcumin bioaccessibility of oil-in-water emulsions. After the enzymatic hydrolysis with the 3% degree of hydrolysis, the protein molecules tended to be looser and disordered. The droplet size analysis results indicated that during oral digestion the RBPH nanoemulsion showed extensive flocculation from 216 ± 2 nm to 1208 ± 81 nm, while 259 ± 2 nm to 383 ± 4 nm for the RBP nanoemulsion. During gastric digestion, the conformation of the protein emulsifier, especially RBPH, was destroyed by pepsin according to SDS-PAGE. During the simulated small intestine digestion, the RBPH emulsion obtained higher lipolysis rate (107 ± 2%) and bioaccessibility of curcumin (42.4 ± 0.1%). Moreover, the influence degrees of different factors on the in vitro digestion were obtained as follows: pancreatic lipase (oil digestion) > bile salt (micellization) > pepsin (emulsifier digestion). These studies contributed to the construction of an effective delivery system for bioactive compounds based on rice bran protein.

Two Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from Rice (Oryza sativa L.) Bran Protein.

To realize the high-value utilization of rice byproducts, the rice bran protein hydrolysate was separated and purified by ultrafiltration and reversed-phase high-performance liquid chromatography (RP-HPLC), then the sequences of peptides were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and their molecular docking analysis and activities in vitro and in the cell were carried out. Two novel peptides FDGSPVGY (840.3654 Da) and VFDGVLRPGQ (1086.582 Da) were obtained with IC50 values of 0.079 mg/mL (94.05 μM) and 0.093 mg/mL (85.59 μM) on angiotensin I-converting enzyme (ACE) inhibitory activity in vitro, respectively. Molecular docking results showed that two peptides interacted with ACE receptor protein through hydrogen bonding, hydrophobic interactions, etc. Through the EA.hy926 cells, it was found that FDGSPVGY and VFDGVLRPGQ could promote the release of nitric oxide (NO) and reduce the content of ET-1 to achieve the effect of antihypertension. In conclusion, the peptides from rice bran protein exhibited significant antihypertension activity and may be expected to realize the high-value utilization of rice byproducts.

Impact of gamma irradiation pre-treatment before subcritical water extraction on recovery yields and antioxidant properties of rice bran extract

Gamma irradiation pre-treatment of defatted rice bran was carried out before protein extraction. The extracted content included rice bran protein (RBP), rice bran protein hydrolysate (RBPH) and rice bran hydrolysate (RBH). The extraction yields and protein recovery increased with irradiation for all extracts, especially RBH, of which the yield and protein recovery increased by 21.2% and 13.8%, respectively. The total phenolic content of RBP and RBH was found to be increased in the irradiation-processed extracts. Gamma irradiation could change the structure of rice bran, increasing the release of bioactive compounds. Antioxidant activities (ABTS, DPPH, FRAP and MIC assays) increased significantly in irradiation-processed extracts. FTIR spectra confirmed that the extracts consisted of protein. The FTIR spectra of the chemical functional groups differed between RBP and RBH. In summary, gamma irradiation pre-treatment increased the extraction yield and total phenolic content and enhanced the antioxidant activity.

Physicochemical and antioxidant properties of rice bran protein hydrolysates obtained from different proteases

Physicochemical and antioxidant properties of rice bran protein hydrolysates obtained from different proteases

Supplementation with rice bran hydrolysates reduces oxidative stress and improves lipid profiles in adult dogs.

Oxidative stress is defined as an imbalance between reactive oxygen species (ROS) production and antioxidant defense mechanisms of the body. An overproduction of ROS leads to lipid and protein oxidation, injuring the cells both in normal and pathological conditions. Rice bran protein hydrolysates (RBH) has potent antioxidant, anti-inflammatory, anti-angiotensin converting enzyme (ACE) and hypolipidemic effects. Little is known, however, about the effects of RBH in dogs. The present study evaluated the antioxidative, anti-ACE and metabolic effects of RBH in adult dogs. Eighteen adult dogs were divided into 2 groups: control (n=7) and RBH supplemented groups (n=11), received a diet with the same nutritional compositions. The RBH supplemented group was fed with RBH 500 mg/kg body weight (BW) mixed with food for 30 days. BW, blood glucose, lipid profiles, liver enzymes, electrocardiography (ECG), plasma ACE activity, oxidative stress and antioxidant biomarkers were determined on day 0 and day 30 of supplementation periods. Results showed that RBH decreased oxidative stress and increased antioxidant biomarkers by significantly reducing plasma malondialdehyde (MDA) and protein carbonyl, enhanced blood glutathione (GSH) and improved the GSH redox ratio. Moreover, decreased LDL-C and increased HDL-C levels were found after RBH supplementation whereas BW, blood glucose, liver enzymes, plasma ACE activity, plasma catalase (CAT) and superoxide dismutase (SOD) activity and cardiac function were not significantly changed. These results suggest that RBH may help to lower the risk of oxidative stress and dyslipidemia in adult dogs.

Identification, in silico selection, and mechanism study of novel antioxidant peptides derived from the rice bran protein hydrolysates

The work aimed to assess the antioxidant ability and obtain a new antioxidant peptide from rice bran protein. Rice bran protein was hydrolyzed by Alcalase, Neutral, Pepsin, Chymotrypsin, and Trypsin, separately. Trypsin hydrolysate (T-RBPH) showed high Fe2+ chelating activity (IC50, 2.271 ± 0.007 mg/mL), DPPH and hydroxyl radical scavenging ability (IC50, 0.191 ± 0.006 and 1.038 ± 0.034 mg/mL). Moreover, T-RBPH could alleviate the H2O2-induced oxidative damage in Caco-2. The T-RBPH was purified and identified by UF, GF, FPLC, and LC-MS/MS. Finally, 9-amino acid peptide-AFDEGPWPK with low molecular weight (1045.48 Da), high antioxidant activity, good safety, and solubility was screened by in silico method and chemical oxidation determination, and its interaction with Keap1 was also demonstrated. The ORAC and DPPH radical scavenging ability of AFDEGPWPK were 44.16 ± 0.79 and 28.38 ± 0.14 μmol TE/mM. Moreover, the Molecular docking and Western blot (WB) results showed that AFDEGPWPK could enter the binding pocket in the Kelch domain and activate Keap1/Nrf2/HO-1 pathway.

Physicochemical and antioxidative characteristics of rice bran protein extracted using subcritical water as a pretreatment and stability in a functional drink model during storage

Industrial hexane-defatted rice bran, a waste byproduct from the rice bran oil industry, was treated with subcritical water conditions followed by alkaline extraction and enzymatic hydrolysis. The extracted content included rice bran protein (RBP), rice bran protein hydrolysate (RBPH), and rice bran hydrolysate (RBH). The highest protein content, protein recovery, total phenolic content, and antioxidant activity, determined by ABTS, DPPH, FRAP, and MIC were seen in RBH. The essential amino acids index of RBH was 48%. FTIR spectra confirmed RBH consisted of protein and phenolic hydroxyl group. RBH was selected for application in the development of a functional drink, and quality was determined during storage. A functional drink supplemented with RBH contained quality protein, with high antioxidant activity and stability during storage. These results show that RBH extract has potential as a novel protein and antioxidant source that could be used in the development of functional food and drinks.

Optimization of enzymatic hydrolysis by alcalase and flavourzyme to enhance the antioxidant properties of jasmine rice bran protein hydrolysate

This study aimed to optimize the hydrolysis conditions for producing jasmine rice bran protein hydrolysate (JBH) using response surface methodology (RSM). The independent variables were the ratio of flavourzyme to alcalase (Fl:Al; 0: 100 to 15: 85; 2.84% enzyme concentration) and hydrolysis time (60–540 min). The optimum hydrolysate was obtained at an Fl:Al ratio of 9.81: 90.19 for 60 min, since it enabled high amounts of protein, high antioxidant activity and more low molecular weight proteins. The experimental values obtained were a degree of hydrolysis (DH) of 7.18%, a protein content of 41.73%, an IC50 for DPPH of 6.59 mg/mL, an IC50 for ABTS of 0.99 mg/mL, FRAP of 724.81 mmol FeSO4/100 g, and 322.35 and 479.05 mAU*s for peptides with a molecular weight of < 3 and 3–5 kDa, respectively. Using a mixture of enzymes revealed the potential of mixed enzymes to produce JBH containing more small peptides and high antioxidant activity.

Fabrication of emulsions prepared by rice bran protein hydrolysate and ferulic acid covalent conjugate: Focus on ultrasonic emulsification

The aim of the paper was to investigate the effect of ultrasonic emulsification treatment on the fabrication mechanism and stability of the emulsion. The covalent conjugate made with rice bran protein hydrolysate (RBPH) and ferulic acid (FA) was used as the emulsifier. The effects of high intensity ultrasound (HIU) power with different level (0 W, 150 W, 300 W, 450 W and 600 W) on the stability of emulsion were evaluated. The results showed that ultrasonic emulsification can significantly improve the stability of the emulsions (p < 0.05). The emulsion gained better stability and emulsifying property at 300 W. It was able to fabricate emulsion with smaller particle size, more uniform distribution and higher interfacial protein content. It was confirmed by fluorescent microscopy and cryo-scanning electron microscopy (cryo-SEM) furtherly. And it was also proved that the emulsion treated by proper HIU treatment at 300 W had better storage stability. Excessive HIU treatment (450 W, 600 W) had negative effects on the stability of emulsion. The stability of emulsion (300 W) against different environmental stresses was further explored, which established a theoretical basis for the industrial application of emulsion in food industry.

Effects of High Hydrostatic Pressure Pretreatment on the Functional and Structural Properties of Rice Bran Protein Hydrolysates.

Rice bran protein (RBP) hydrolysis was conducted after high hydrostatic pressure (HHP) pretreatment. The structural and functional properties of HHP-pretreated rice bran protein hydrolysates (RBPH) were investigated. HHP pretreatments were conducted at 100, 200, and 300 MPa; then, enzymatic hydrolysis at atmospheric pressure was performed using trypsin. An RBPH sample that had not been pretreated by HHP was used as a control. Free sulfhydryl (SH) content, SDS-PAGE profiles, high-performance size exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectrum, scanning electron microscopy (SEM), intrinsic fluorescence spectrum, solubility, and emulsifying and foaming properties were evaluated. Changes in particle size and ζ-potential were monitored. Compared with the control, the results of solubility, the emulsifying activity index (EAI) and the emulsifying stability index (ESI) increased significantly (p < 0.05) at 200 MPa. The content of free SH increased significantly (p < 0.05) at 100 MPa. FTIR spectrum and fluorescence analysis confirmed the changes in the secondary and tertiary structures. The experimental results indicated that the structural and functional properties of HHP-pretreated RBPH improved.

Covalent Interaction between High Hydrostatic Pressure-Pretreated Rice Bran Protein Hydrolysates and Ferulic Acid: Focus on Antioxidant Activities and Emulsifying Properties

Rice bran protein hydrolysates (RBPH) pretreated with high hydrostatic pressure (HHP) covalently interacted with ferulic acid (FA) (0.5 to 2.5 mg/mL) under alkaline conditions. The structural and functional properties of the conjugates were investigated. The results revealed that the FA binding equivalent on RBPH increased from 6.03 to 207.64 nmol/mg. FTIR spectral analysis indicated that the content of α-helix increased, whereas the contents of β-sheet, β-turn, and random coil decreased. The surface hydrophobicity (H0) of RBPH increased, the fluorescence intensity decreased, and the tertiary structure changed because of covalent interactions between RBPH and FA. The emulsifying activity index of RBPH-FA (1.5 mg/mL) was 35.10% higher than that of the control, whereas FA concentrations higher than 1.5 mg/mL had a negative effect on emulsifying properties. RBPH-FA (2.5 mg/mL) exhibited the strongest antioxidant activity. This study provides a new reference for the application of RBPH-FA conjugates in food processing.

Pyrazine yield and functional properties of rice bran protein hydrolysate formed by the Maillard reaction at varying pH.

Interest in plant-based protein sources has grown rapidly. Rice bran contains excellent quality protein, but it is still rare in the market, due to its poor functional properties and undesirable odors. Therefore, we studied the Maillard reaction at different pHs on the formation of pyrazines and improved functional properties of rice bran protein hydrolysate. Protein from rice bran was extracted and hydrolyzed, using alcalase, at 55°C for 4h. Fructose was added, and the pH of the hydrolysate was adjusted to pHs between 7.0 and 10.0, before spray drying. Five pyrazines were detected in the hydrolysate powder, with the highest yield at pH 9 ( p < 0.05). The highest odor active value came from 2-ethyl-3,5-dimethylpyrazine (26.26), which contributed coffee,nutty and caramel aromas, followed by 2,5-dimethylpyrazine (9.2) and 2-ethyl-5-methylpyrazine (5.0). Increased pH led to a darker color (L* = 58.5) and redder (a* = 11.7) and yellower (b* = 13.8) powder. Key functional properties-solubility, water and oil binding capacity and emulsion stability index and foaming capacity-were increased with pH ( p < 0.05). The optimum pH for pyrazine formation and improved properties of enzymatic rice bran protein hydrolysate was pH 9.0.

Effects of thermal processing on antioxidant activities, amino acid composition and protein molecular weight distributions of jasmine rice bran protein hydrolysate

AbstractThis research aimed to evaluate the effects of thermal processing on the amino acid composition, molecular weight distribution (MW) and antioxidant activities of Jasmine rice bran protein hydrolysates (JBH). JBH was prepared by enzymatic hydrolysate (alcalase and flavourzyme at a ratio of 9.81:90.19, 2.84% w/w) for 60 min. JBH samples were treated as follows: non‐heat (NJBH), pasteurisation (72ºC, 15 min; PJBH) and sterilisation (121ºC, 15 min; SJBH). For SJBH treatment, the browning index and fluorescence intensity increased (P &lt; 0.05). FTIR showed that thermal process changed the intensities and location of some bands. Thermal processing affected MW by decreasing it from high MW (&gt;10 kDa) to medium (3–10 kDa) and low MW (&lt;3 kDa). Val, Met, Ile and Leu contents increased after thermal processing. Compared with NJBH, the IC50 values of ABTS and FRAP of SJBH were not significantly different, while the IC50 of DPPH increased (P ≥ 0.05).

Antioxidant and antihypertensive protein hydrolysates from rice bran: optimization of microwave assisted extraction

In this study, the production of antihypertensive and antioxidant hydrolysates by microwave assisted enzymatic hydrolysis was optimized for the proteins extracted from the rice bran. The central composite design was used for optimization and the independent parameters were solid/liquid ratio, power, and time. Optimum protein extraction parameters were obtained as; 0.42 solid/liquid ratio, 100.68 W power, 100.68 s. During alcalase and pepsin hydrolysis, solid/liquid ratio and time were selected independent factors, the results of angiotensin-converting enzyme (ACE) inhibitory activity, degree of hydrolysis (DH), and DPPH radical scavenging activity were used as responses. Optimum hydrolysis parameters were determined. The DH, DPPH radical scavenging activity, ACE inhibitory activity, values were 40.60%, 36.26%, and 59.16% respectively. The rice bran protein hydrolysates was fractionated as < 1 kDa, 1–5 kDa, and 5–10 kDa by ultrafiltration. The highest ACE inhibition activity, DPPH, and OH radical scavenging activity and Trolox equivalent antioxidant activity values were respectively obtained for 5–10 kDa fraction as 76.66%, 13.25%, 5.03%, and 8.82 µg trolox/mg sample. The study may provide a new approach for obtaining biofunctional protein hydrolysates from rice bran emerging relatively high volumes as a by-product during paddy processing.

Production of milk analogues from rice bran protein hydrolysate using the subcritical water technique

Abstract Subcritical water treatment is a useful technique for the extraction of active compounds from biomass materials due to its short processing time, low cost, and environmental sustainability. Defatted rice bran is a by-product of rice bran oil, which still contains protein. In this study, rice bran protein was extracted using subcritical water treatment at temperatures of 120, 140, 160, and 180 °C and reaction times of 30, 45, and 60 minutes to determine the effects of following parameters on the production of milk analogues. At higher temperatures, the protein content and emulsifying activity index (EAI) increased, and the nitrogen solubility index (NSI) ranged from 87-100%. The emulsifying stability index (ESI) reduced when the reaction time was increased, while the molecular weight of the protein was less than 50 kDa. These temperatures and reaction times were shown to have direct impacts on both protein extraction and the molecular size of rice bran protein, which significantly increase the production of milk analogues. The results showed that milk analogues appeared to be more viscous than cow’s milk, as evidenced by the shear thinning. In sensory evaluation, the color, texture, taste, and acceptability of cow’s milk scored higher in comparison to milk analogues. All of the sensory parameters analyzed, except odor, were significantly different (P ≤ 0.05).

Enzymatic modification of rice bran protein: Impact on structural, antioxidant and functional properties

Rice bran protein concentrate (RBPC) was hydrolysed by a commercial food grade enzyme-papain to get ~15%, 25% and 32% degree of hydrolysis (DH) to investigate its effect on structural, antioxidant and functional properties as well as in-vitro digestibility. Bulk density and structural flexibility (α-helix and random coils) of rice bran protein hydrolysates (RBPHs) increased with an increase in DH. However, surface hydrophobicity (SHO), total β-sheets, β-turns, and thermal properties decreased with increasing DH. RBPHs possessed improved antioxidant activity as compared to native protein, except metal chelating power. RBPHs had higher solubility and digestibility. Thermal properties were positively correlated with β-sheets content while inversely related to α-helix content. An inverse correlation was observed in protein solubility and SHO. Emulsion activity index and foaming stability were positively related to SHO. Protein digestibility was negatively correlated to the β-sheets, β-turns, and SHO. The results indicated that secondary structure and SHO are the primary factors in affecting the thermal and functional properties as well as digestibility. It is thus concluded that the enzymatic hydrolysis might be applicable for RBPC to increase its potential bioactivity but reserve partial interfacial properties.

Physicochemical and Mechanical Properties of Rice Bran Protein Hydrolysate-Loaded Films

The objective of this study was to apply rice bran protein hydrolysates (RBH) as bioactive additives of gelatin/Eudragit® NE 30D film and characterize the physicochemical and mechanical properties of its. The RBH was obtained by extraction with 2% sodium chloride (RBH-NaCl) and 0.1 N sodium hydroxide (RBH-NaOH) followed by digestion with Alcalase®. Then, RBH was incorporated in gelatin/Eudragit® NE 30D film. Effect of RBHs on film thickness, moisture content, pH, Young's modulus, tensile strength and the elongation at break were investigated. The RBH-NaCl enriched film showed non-homogeneous mixture and reduced moisture content, tensile strength and the elongation at break (1.8 – 2 folds). However, the RBH-NaOH enriched film exhibited a few non-homogeneous mixture and the Young's modulus was slightly decreased. The pH value was increased in the range of 6.77 – 6.88. Our results provide insight for the potential to develop RBH containing films as topical products.

Enzymatic preparation and antioxidative activity of hydrolysate from Rice bran protein

As a protein-rich resource, rice bran protein has not been developed and utilized effectively. In this study, the optimal condition to prepare hydrolysate from rice bran protein was determined by screening protease and conducting single factor experiments along with response surface methodology. Optimal hydrolysate was prepared to further investigate its chemical (DPPH, ABTS, Fe2+) and cellular antioxidative activity. Finally, its amino acid composition was explored. In the results, alcalase was selected and other parameters were: pH 8.5, enzyme-to-substrate ratio 0.38%, and substrate concentration 6%. It was proved that the hydrolysate was an excellent DPPH (EC50 = 5.647) and ABTS (EC50 = 0.195) scavenger as well as good Fe2+ (EC50 = 1.765) chelator, showing directly proportional relationships with dose. In cellular antioxidant activity (CAA) assay, the CAA value reached 31.76% at the concentration of 1 mg/mL and the increasing use of rice bran protein hydrolysate significantly improved CAA. Amino acid composition analysis showed that the hydrolysate was rich in glutamic acid (13.02%), aspartic acid (6.68%), arginine (3.34%) and many kinds of hydrophobic amino acids which contribute to the antioxidant property. This research revealed the potential application of rice bran protein hydrolysate in natural antioxidants.