Ameliorative potential of rice protein hydrolysates prepared through high-pressure processing against diabetic cardiomyopathy

Abstract

Various functional foods, including meal replacements and nutritional supplements, are available to consistently regulate blood glucose levels in patients with type 2 diabetes mellitus (T2DM). This study proposes the development of high-pressure processing (HPP) treatment for rice protein (RP) flour to produce rice protein peptides (RPP). This dietary intervention aims to stabilize glycemic levels and mitigate myocardial damage caused by oxidative stress (OS) in patients with diabetic cardiomyopathy (DCM). This study demonstrated that the protein, branched-chain amino acid (BCAA), and peptide contents in RPP increased significantly following HPP treatments ranging from 200 to 600 MPa. Additionally, the study found that RPP effectively regulated blood glucose levels (621.85–181.73 mg dL⁻¹) in rats with DCM, attenuated myocardial injury caused by OS, and prevented the development of myocardial infarction (MI) indices and cardiac oxidative-inflammatory parameters in an animal model. Notably, RPP may effectively inhibit nuclear factor (NF)-κB expression and activity, thereby reducing myocardial cellular pyroptosis. Consequently, the findings of this study contribute to the advancement and commercialization of RPP as a potential health food product, with the dual benefits of regulating blood glucose levels and providing antioxidant properties. Furthermore, this study offers novel perspectives for preventing and alleviating heart failure in DCM, thus guiding future research.

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1. Introduction

The metabolic disorders commonly observed in individuals with T2DM exert a significant impact on multiple organ systems, thereby contributing to potentially severe complications that have been acknowledged as a major global public health threat.^1,2 It is notable that patients with T2DM, in the absence of other heart diseases, develop peculiar cardiovascular complications, mainly resulting in myocardial dysfunction, known as diabetic cardiomyopathy (DCM), an extensively recognized clinical entity.^3–5 Nevertheless, patients with T2DM who experienced suboptimal glycemic control and concurrent cardiovascular risk factors were at an elevated risk of developing DCM, which was observed in up to 30% of individuals with T2DM.⁵ Additionally, a higher susceptibility to DCM has been documented among elderly patients with T2DM.⁶ Advancement of these lesions is associated with mitochondrial dysfunction due to excessive accumulation of lipids in the myocardium, increased production of reactive oxygen species (ROS), and elevated glucose levels. Namely, it contributes to developing advanced glycation end-products that promote OS and sustained inflammation, leading to myocardial injury.^3,5,7–10 Moreover, it has been recently demonstrated that cardiomyocyte pyroptosis is linked to the aberrant activation of the NF-κB signaling pathway in cardiovascular diseases, indicating that intervention in the expression of this pathway provides an apparent therapeutic target.¹¹ This condition frequently lacks symptoms during the initial stages of DCM and frequently coincide with other diabetic complications, complicating the process of establishing a definitive diagnosis complex and prolonging the subclinical period.^9,12 However, the most appropriate approach for identifying DCM involves discerning the left ventricle's structure and detecting functional changes through echocardiography, excluding other conditions that may contribute to heart failure.^9,12,13 Unfortunately, the specific mechanisms have not been elucidated, and practical agents or inhibitors to inhibit this pathway are lacking.¹¹ According to those mentioned above, these multifactorial pathological mechanisms play a pivotal role in the development of DCM, all intricately interconnected with controlling blood glucose levels.^2,4,8,10,14 Nevertheless, it has been suggested that in addition to improving blood glucose levels, it is imperative to augment the antioxidant system within the body sufficiently to mitigate cardiac OS in rats with DCM.¹⁵

The HPP technology is a commercially successful non-thermal sterilization method with diverse applications, including inactivating pathogenic microorganisms and preserving natural attributes such as shape, color, aroma, flavor, and nutrients in food products.^16–18 This technology has demonstrated the ability to induce degradation of the cellular walls in plants or animals, resulting in enhanced permeability and improved intracellular mass transfer mechanisms.^19–21 Specifically, it enhances dietary bioactive substances (such as polyphenols, peptides, free amino acids (AA), γ-aminobutyric acid, etc.) and starch (molecular/crystalline arrangement and functionalities).^17,18,22,23 In addition, the bioactive peptides, comprising a range of several to fewer than 20 AA, can be directly absorbed by the human body to provide physiological functions such as antioxidant, anti-inflammatory, anti-cancer, antibacterial, intestine-modulatory, immunomodulatory, metal-chelating, and blood pressure-lowering.^24,25 Therefore, the health benefits of functional foods conferred by these specific protein fragments’ bioactive peptides (antioxidant peptides) can maintain human health, food safety, and quality by reducing lipid peroxidation and OS.¹⁶ This was caused by free radicals produced during oxidative reactions in the body and foods.¹⁶ However, the presence of bitterness peptides resulting from enzymatic hydrolysis and deamidation poses concerns regarding food safety.^26,27 This ultimately restricts the nutritional and application benefits of RP. Nevertheless, exploring alternative methods to enhance RP solubility could potentially overcome these challenges that are difficult to address simultaneously.²⁷

In previous times, RP's relatively limited protein content and solubility have resulted in its lack of appeal, namely being a by-product of starch extraction, which has traditionally been regarded as having limited value.²⁶ However, the suitability of RP as a protein for preventing obesity and DM has been demonstrated, with its positive impact on body performance and health being attributed to a specific protein fragment.^28–31 The global market for RP witnessed a stable growth, expanding from USD 132.99 million in 2024 to USD 249.83 million in 2032, reflecting a robust Compound Annual Growth Rate (CAGR) of 8.2%.³² The key advantages of RP products lie in their hypoallergenic and nutritional properties, endowing them with a competitive edge over other plant-based protein alternatives.^33–37 Notably, most RP is obtained through hydrolysis using alkaline or proteolytic enzymes. According to reports, the structure of RP was altered (such as indigestible prolamin) by alkaline extraction to enhance digestibility (in vivo) compared to rice flour and starch-degraded RP, thereby potentially improving the nutritional value of high-starch base foods, particularly for elderly individuals.^38–40 Consequently, incorporating RP could be beneficial with higher bioavailability attributed to alkaline-extracted RP.^38,41 In addition, it has been indicated that proteins with limited digestibility could be characterized as prolamins, which are lipid-rich core proteins deficient in lysine but abundant in cysteine.³⁶ Furthermore, ingesting essential AA derived from proteins has been shown to markedly augment the relative abundance of the gut microbiota (Alistipes spp.), thereby facilitating the performance of muscle function in elderly individuals.^42,43 In addition, a clinical study (n = 50) showed that dietary supplementation with highly bioavailable purified rice endothelial protein in maintenance hemodialysis patients led to a statistically significant 0.07 g kg⁻¹ day⁻¹ increase in urea kinetic-based normalized protein catabolic rate.⁴⁰ The authors highlighted that this effect was achieved while maintaining stable serum phosphorus and potassium concentrations, mitigating the risk factors associated with elevated protein intake and mortality. In particular, proteins and protein hydrolysates exhibit potential as functional food ingredients through biological activities.^37,44–47 These bioactive peptides derived from diverse sources exhibit the potential to reduce OS, modulate immune responses, mitigate inflammation, and enhance intestinal barrier integrity to fortify, uphold, or restore various physiological homeostatic processes.^46,47 However, these biological activities are intricately associated with the AA composition, sequence, molecular weight, and hydrophobicity of peptides; specifically, peptides characterized by low molecular weight and pronounced hydrophobicity may exhibit enhanced anti-inflammatory properties.^47–52

Based on the above, there have been many explorations of HPP achieving increased bioactivity content in various food materials and realizing the potential of having the desired functional food components. However, more information is needed on advanced practical applications for products and efficacy validation. Concurrently, developing a robust and pragmatic food matrix was deemed essential to enhance the assessment of our nutritious foods’ functionality, nutrient composition, bioaccessibility, bioavailability, etc., while exploring the interrelationships among individual food components in an in vivo model.⁵³

Therefore, this study assessed the practical applicability of RPP by utilizing a previously successful functional food carrier in an established animal model of DCM.² Specifically, this study evaluated the impact of HPP-treated RPP on the physiological parameters of DCM rats, encompassing growth performance, serum biochemistry, MI indices, and cardiac oxide-inflammatory markers. This study aimed to investigate the health-promoting efficacy of RPP as a dietary supplement for five consecutive months in rats with DCM to gain a more comprehensive understanding of RPP.

2. Materials and methods

2.1. Materials

The 80% RP flour [made from Vietnamese rice (cóm tấm; Oryza sativa L. subsp. indica Kato), >80% crude protein content and lower trace mineral (phosphorus and potassium) contents] was offered as a gift from Vedan Vietnam Enterprise Co., Ltd (Long Thanh, Vietnam). The corn flour (CF) and food-grade ingredients, such as cellulose, lard, and casein, were procured from a local market (Taichung, Taiwan). The chemicals used in this study were procured from Sigma-Aldrich® (Merck KGaA, Darmstadt, Germany) and used as is unless explicitly stated otherwise.

2.2. Preparation of the extruded products

2.2.1. Treatment of rice protein flour with HPP. The condition setting of HPP was established based on the previous research findings of this team.²¹ This study used experimental HPP equipment (Bao Tou KeFa High-Pressure Technology Co. Ltd, Baotou, China) with a chamber volume of 6.2 L and pure water as the hydraulic medium. Specifications include a maximum operating pressure of 800 MPa, a ramping rate of 45 MPa s⁻¹, and an increase in temperature during operation, with the temperature increasing by 3 °C for each 100 MPa increase in pressure. The specific conditions of the HPP treatment of RPP flour in this study were as follows: the initial temperature was 18 °C, and the pressurization was applied at 200, 400, and 600 MPa for 5 min. In addition, the processing time excluded the period of pressurization and depressurization, and the pressure could be released within 10 s after the HPP treatment. The HPP-treated RPP flour was analyzed for physicochemical properties (as in section 2.3) before extruded product preparation.

2.2.2. Extrusion processing formulation and conditions for HPP-treated RPP flour incorporation. The formulation of HPP-treated RPP flour and the extrusion process conditions followed previous publications of this team with minor modifications.² The control diet consisted of 55% corn starch, with other formulations detailed in Table 1. In contrast, the experimental groups (HPP 200, 400, and 600) were supplemented with 30% HPP-treated RPP flour and 25% corn starch, respectively. The extrusion process employed in this study utilized a co-rotating twin-screw extruder (BC-45, Clextral^©, Firminy, France). The procedure was conducted with a barrel temperature set at 150 °C, a screw speed of 175 revolutions per min (rpm), and 20% moisture content. The screw shaft utilized in this process measured 750 mm in length and consisted of three barrels. The second and third barrels were equipped with an electromagnetic induction heating and cooling system to regulate each respective temperature precisely. Specifically, the experiment involved setting the desired temperature for the third barrel while adjusting the second to half of that temperature, while the first barrel was kept at 25 °C. Meanwhile, the mold utilized in this procedure featured two evenly distributed apertures measuring 6 mm in diameter.

Table 1 The specific formulation for an extruded snack (ES) incorporates high-pressure processed (HPP) 200, 400, and 600 MPa-treated rice protein peptide (RPP) flour

Group
Composition (%)	Control and diabetes (DM)	RPP
		200	400	600
Casein	16
Lard	8
DL-Methionine	0.2
AIN-76 mineral mix	1
AIN-76 vitamin mix	4
Cholesterol	0.5
Cholic acid	0.1
Choline chloride	0.2
Cellulose	5
Corn starch	25
RPP flour	30
Rice bran	10

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2.3. Physicochemical properties of the HPP-treated RPP flour

2.3.1. Protein content. The protein content of the samples was determined following the standard method (984.13) via AOAC.⁵⁴

2.3.2. Branched-chain amino acid (BCAA) content. This study was performed to determine the content of BCAA (including leucine, isoleucine, and valine) in the samples using a BCAA assay kit (MAK562, Merck KGaA) for measurements.

2.3.3. Peptide content. The peptide content was determined according to the method described by Dinnella et al.⁵⁵ with some modifications. The O-phthaldialdehyde reagent was prepared by combining 25 mL of a 100 mM sodium tetraborate (Na₂B₄O₇) solution, 2.5 mL of a 20% sodium dodecyl sulfate (SDS) solution, 1 mL of OPA (40 mg of O-phthaldialdehyde/1 mL MeOH), and 100 μL of mercaptoethanol, followed by the addition of distilled water to make a final volume of 50 mL. Next, 2 mL of the reagent was added to 5 mL of the sample and mixed thoroughly at 25 °C for a period of 8 min. The absorbance value at 340 nm was then measured using a spectrophotometer (U-2001, Hitachi, Ltd, Tokyo, Japan). The standard curve was generated using L-leucine (0–50 mg mL⁻¹) and subsequently utilized for interpolation to determine the sample's peptide amount (mM).

2.3.4. Water solubility index (WSI) and water holding capacity (WHC). The WSI and WHC of the RPP were measured following Huang et al.⁵⁶ and Guo et al.,⁵⁷ respectively. The 2.5 g sample was added to 30 mL of distilled water while stirring gently with a glass rod to prevent agglomeration. The reaction mixture was then placed in an oscillating water bath set at 30 °C and 120 rpm for 30 min, with intermittent stirring using a glass rod to avoid agglomeration. Following centrifugation at 1000g for 10 min, the supernatant was transferred to a beaker (W₁), which was subsequently weighed and dried at 105 °C until a constant weight (W₂) was reached. Subsequently, the precipitate was weighed (W₃). The WSI and WAI were calculated using the following equations corresponding to each code:

where W₁ represents the supernatant weight; W₂ represents the constant weight; and W₃ represents the precipitate weight.

2.3.5. Sulfhydryl (SH) and disulfide bond (S–S) contents. The determination of SH and S–S contents in the RP was performed following the methods described by Guo et al.⁵⁸ and Ou et al.,⁵⁹ with minor modifications. The RP flour (1 g) was mixed with 10 mL of 0.1 M phosphate buffer (PBS; pH 7.0) to form a suspension solution, which was freshly prepared prior to each assay.

To 2 mL of the above suspension was added 8 mL of 0.1 M PBS (pH 8.0), and the pH of the solution was adjusted to 8.0 with 1 M NaOH. Next, 1 mL of 2 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent) was added and allowed to stand for 40 min. Subsequently, 0.1 M PBS (pH 7.0) was added until the final volume was 11 mL and centrifuged (3000g) for 10 min. The supernatant was collected and filtered through a 0.45 μm membrane before the absorbance at 412 nm was measured, and then the SH content in RP was calculated using the following formula:

where ABS 412 nm indicates the absorbance of RPP at 412 nm; V_t indicates the final reaction volume (L); 10⁶ indicates the unit conversion factor (μmol mol⁻¹); ε indicates the molar extinction coefficient of DTNB (13 600 M⁻¹ cm⁻¹); b indicates the optical range of the cuvette (1 cm); and V indicates the volume of the sample (L).

Moreover, the S–S bond contents were determined using cysteine. Briefly, 1 mL of the above suspension was added to 50 μL of 0.1 M ethylenediaminetetraacetic acid (EDTA) solution and 3 mL of 0.1 M PBS (pH 8.0), followed by 2 μL of octanol and 0.48 g of urea, and then kept for dissolution. Next, 1 mL of NaBH₄ solution (2.5%) was added and reacted in a water bath at 40 °C for 30 min. Afterward, 1 mL of an acidifier (pH 2.7; 10.4 g of NaH₂PO₄·H₂O dissolved in 100 mL of 0.1 M HCl) and 2 mL of acetone were added, followed by blowing with N₂ gas for 6–8 min at 25 °C (to remove excess NaBH₄). Then, 1 mL of DTNB solution (2 mM) was added, allowed to stand for 40 min, and centrifuged for 10 min (3000g);then the supernatant was collected for filtration using a 0.45 μm membrane. The absorbance of the filtrate was measured at 412 nm, and the S–S bond contents in RP were calculated using the above formula.

2.3.6. Free radical scavenging capacities.
2.3.6.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH). The methodology described by Wang et al.⁶⁰ was employed to determine the DPPH radical scavenging capacity of the sample. The sample (1.5 mg mL⁻¹) was mixed with a 150 μM DPPH reagent dissolved in an 80% (v/v) methanol solution. The mixture was subsequently shielded from any light exposure for 90 min, followed by conducting absorbance measurements at 517 nm using a microplate reader (Emax, Molecular Devices, LLC, San Jose, CA, USA). The DPPH radical scavenging capacity of the sample was calculated by employing the following equation:
where ABS 517 nm indicates the absorbance of RPP at 517 nm.
2.3.6.2. Oxygen radical absorbance capacity (ORAC). The ORAC of the RPP for this study was determined following the description of Asma et al.,⁶¹ with some modifications. ORAC has been extensively employed in the evaluation of the antioxidative potential of food items, utilizing the AAPH [2,2′-azobis(2-amidinopropane) dihydrochloride] radical to induce the formation of peroxyl radicals through heating in the presence of ample oxygen and expedite fluorescence degradation.⁶² The fluorescence change can serve as an indicator for assessing the antioxidant capacity of the samples. The sample in a 96-well plate was incubated in a water bath (37 °C) for 5 min, followed by the sequential addition of 50 μL of fluorescein sodium salt (78 nM) to each well, along with 50 μL of the sample and 25 μL of AAPH (221 mM), which were shaken for 10 s. The fluorescence values were recorded every 5 min for 90 min, with excitation at 485 nm and emission at 520 nm. The fluorescence value and time were used to generate a plot, from which the area under the sample and blank curves was derived. The equation provided below calculates the Net AUC by determining the area under the net curves. The standard substance 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was employed, and linear regression was performed to establish the standard curves for determining the ORAC of the sample.

2.3.7. Fourier transform infrared (FTIR) spectroscopy. The sample group structures were analyzed according to the methodology described by Lin et al.⁶³ with slight modifications. FTIR spectroscopy (Nicolet 380, Thermo Fisher Scientific Inc., Waltham, USA) was employed to analyze the sample (1 g) through wavelength scanning. The operational parameters included a wavelength range of 4000–400 cm⁻¹, a resolution set at 4 cm⁻¹, and a scanning frequency of 64 times.

2.3.8. X-ray diffraction (XRD). The sample's crystalline structure was determined following the method described by Cheng et al.⁶⁴ with slight modifications. The XRD pattern (X'Pert³ MRD, Malvern Panalytical Ltd, Malvern, UK) of a 100 mg sample was determined. The analysis was conducted under the following conditions: an operating current of 20 mA and voltage of 40 kV, with a scanning speed of 2° min⁻¹, while the scanning angle (2θ) ranged from 3° to 80°.

2.3.9. Scanning electron microscopy (SEM). 0.5 g of the sample was affixed onto an aluminum holder and underwent a 90 s gold coating process (Ion Sputter Coater, JBS-ES 150 model, Topon Co., Ltd, Tokyo, Japan) in a vacuum environment. Subsequently, the sample was examined and imaged using a scanning electron microscope (ABT-150S, Topon Co., Ltd, Tokyo, Japan) operating at an accelerating voltage of 10 kV and a magnification of 4000 times.

2.4. Experimental animals

The design and operations of the animal experiments in this study were performed with minor modifications based on our team's descriptions of the published study by Cheng et al.² This study utilized male Wistar rats aged six weeks (body weight (BW) 200 ± 20 g) purchased from LASCO Co., Ltd (Taipei, Taiwan). Upon arrival at the laboratory, the animals were allowed 1 week of acclimatization before being randomly divided into groups (n = 6) (Fig. 1). These groups included the control group (standard diet, without induction or treatment), the DCM group (streptozotocin (STZ) induction for T2DCM on a standard diet), and the HPP treatment 200, 400, and 600 groups. Throughout the experiment, a controlled environment (pathogen-free; temperature and humidity were maintained at 25 ± 2 °C and 65 ± 5%; the dark and light periods were from 07:00 to 19:00 and 19:00 to 07:00) was provided for all rats, which were housed in cages made of plastic measuring 30 [L] × 20 [W] × 10 [H] cm³, with two rats per cage. The dietary formulas for each group were prepared according to the protocol described in section 2.2.2 and the diet was provided ad libitum, with daily renewal. All groups (except for the control group) were induced with T2DM, which underwent a 12 h fast before the injection of STZ (Millipore®, Merck KGaA). The STZ solution (40 mg kg⁻¹) was prepared in a light-protected container (maintained in an ice bath) using ice-cold 0.01 mM citric acid buffer (pH 4.5), which was administered intravenously. The induction sessions were conducted consecutively for seven sessions with three-day intervals. On the last day of the experiment, all animals were subjected to a 24 h fasting period, during which their BWs and blood glucose levels were measured. Subsequently, euthanasia was performed by intraperitoneal injection of sodium pentobarbital (150 mg per kg BW). Afterward, a cardiac blood sample was obtained through puncture and subjected to centrifugation (at 4 °C, 12 000g) for 10 min, while the resulting serum was utilized for the subsequent biochemical analysis. The excised cardiac tissue was rinsed with a 0.9% NaCl solution and weighed. Additionally, one-third of the cardiac tissue was preserved in 10% formalin-buffered solution. The other cardiac tissue was homogenized in phosphate-buffered saline, followed by centrifugation (3000g) for 30 min. These steps were performed to facilitate the subsequent correlative analyses (section 2.5). All animal experiments complied with the regulations outlined by the Committee for Control and Supervision of Experiments on Animals and the National Institutes of Health, ensuring proper care and use of experimental animals. The protocol under code 20230807 A001 has been approved by the Committee on Animal Research at Providence University.

Fig. 1 Grouping and induction of type 2 diabetes mellitus (T2DM) in experimental animals were conducted, followed by the evaluation of the effects of rice protein peptides on ameliorating diabetic cardiomyopathy through supplementation with extruded snacks (ES) manufactured from high-pressure-processed (HPP) rice protein (RP) flour for a consecutive period of five months.

2.5. Analysis of serum biochemistry and determination of factors related to cardiac oxidative inflammation

2.5.1. Serum biochemical parameters. The serum biochemical parameters [including triglycerides (TG, mg dL⁻¹), total cholesterol (TC, mg dL⁻¹), aspartate aminotransferase (AST, unit (U) per L), and lactate dehydrogenase (LDH, U L⁻¹)] were analyzed according to the protocol described by Chou et al.⁶⁵ while using a synchro system (LX-20, Beckman Coulter Inc., Brea, CA, USA).

2.5.2. Myocardial infarction (MI) indices. The cardiac troponin (TnT) I ELISA kit (ab200016, sensitivity: 4.4 pg mL⁻¹, range: 31.3–4000 pg mL⁻¹, Abcam Plc., Cambridge, UK) and the creatine kinase MB (CK-MB) ELISA kit (ab285231, sensitivity: 46.825 pg mL⁻¹, range: 78.125–5000 pg mL⁻¹, Abcam Plc.) were employed to measure the levels of MI indices following the standard operating procedures (SOPs) provided by the manufacturer.

2.5.3. Cardiac oxidative stress (OS) and oxide-inflammatory parameters. The supernatant obtained from the cardiac homogenate was utilized for determining the OS parameters in the heart, which included measuring malondialdehyde (MDA) levels using an ELISA kit (CSB-E08558r, sensitivity: 7.81 pmol mL⁻¹, range: 31.25–2000 pmol mL⁻¹, Shanghai Gaochuang Chemical Technology Co., Ltd), determining catalase (CAT) activity through the ab118184 kit (range: 1–1000 µg mL⁻¹, Abcam Plc.), quantifying glutathione (GSH) peroxidase activity with the glutathione peroxidase (GPx) assay kit (ab102530, sensitivity: 0.5 mU mL⁻¹, Abcam Plc.), evaluating superoxide dismutase (SOD) activity using the ab65354 kit (Abcam Plc.), and measuring caspase-3 levels (ab281085, within the reference range of 23–1500 pg mL⁻¹, Abcam Plc.).

The other parameters employed in this study were determined following the procedures outlined by Cheng et al.² and employing specialized ELISA kits (Abcam Plc.). According to the manufacturer's instructions, these factors in cardiac homogenates were quantified. Specifically, the determined immune-related factors were interleukin (IL)-6 (ab234570, sensitivity: 43 pg mL⁻¹, range: 125–8000 pg mL⁻¹), IL-1β (ab255730, sensitivity: 26.58 pg mL⁻¹, range: 54.69–3500 pg mL⁻¹), tumor necrosis factor (TNF-α; ab236712, sensitivity: 1.04 pg mL⁻¹, range: 18.75–1200 pg mL⁻¹), and NF-κB (ab100785 with a sensitivity of 25 pg mL⁻¹ and a range of 82.3–20 000 pg mL⁻¹).

2.6. Statistical analysis

The data were presented as mean ± standard deviation (SD). The RP extruded products underwent triplicate measurements (n = 3), while the animal experiments were conducted with 6 replicates per group (n = 6). In addition, the data were graphed using GraphPad Prism software (version 9, GraphPad Software, LLC, Boston, MA, USA) and subjected to one-way analysis of variance, followed by Newman–Keuls post-hoc analyses. A significance level of p < 0.05 was deemed statistically significant.

3. Results and discussion

3.1. Effects of different HPP treatments on the compositions of rice protein peptide (RPP) flour

The present study showed that the protein, BCAA, and peptide contents of all RPP groups increased in response to increasing pressure (Fig. 2), with significant variations observed among the different groups (p < 0.05). Specifically, 600 MPa provides satisfactory performance for these substances. This also signified that treatment with HPP can provide satisfactory reinforcement at the level where RP already contains high levels of arginine and BCAA levels.³⁶ This observed phenomenon can be ascribed to excessively high pressures (>400 MPa), which enhance the diffusion and collision rates of proteins or peptides, expedite protein–protein or peptide–peptide aggregation, and reduce the hydrolysis rate, thereby facilitating the production of bioactive peptides.^{18,25,44,66,67} However, the hydrolysates obtained consisted of intricate peptide mixtures, some of which may exhibit limited efficacy in vivo.^44,66 Conversely, treatment with HPP facilitated the protein conformation and molecular chain extension change. Then, the exposure to specific or new restriction sites enabled the subsequent digestive enzymes to selectively recognize and hydrolyze AA sequences to generate peptides possessing distinct active motifs.^{16,25,37,44,66,68} Therefore, this study showed that the application of HPP effectively increased the protein, BCAA, and peptide contents in RPP flour.

Fig. 2 Effect of different high-pressure processing (HPP) treatments on the compositions of rice protein (RP) flour: (A) protein content, (B) branched-chain amino acid (BCAA) content, and (C) peptide content.

3.2. Effects of different HPP treatments on the physicochemical properties of rice protein peptide (RPP) flour

It is well known that the WSI of proteins forms the base for essential functional properties in food systems, including WHC, emulsification, foaming, oil-holding capacity, and gelation.^27,69 It serves as a reliable indicator of the potential applications of proteins while effectively enhancing the solubility of RP, facilitating its utilization in a broader range of fields.²⁷ However, the abundance of glutelin in RP leads to its poor solubility in aqueous solution, particularly with high concentrations, whereas the aggregation and cross-linking of disulfide bonds provide the primary constraints on its insolubility.⁶⁹ The findings of this study showed that the WSI and WHC increased as the pressure increased in all RPP groups (Fig. 3A and B), which were significantly different from each other (p < 0.05). The above phenomenon may be attributed to the HPP treatment that makes the RPP flour particles and forms a dense 3-dimensional protein network structure, while a few starch gelatinizations occurred that could effectively cover the water, increasing the WHC.²¹ Conversely, fluctuations in temperature can lead to denaturation or aggregation of proteins, thereby diminishing part of the protein's hydration capacity,¹⁷ while the alteration may subsequently result in an elevated WSI. Another plausible explanation for the increase in solubility is that external forces (HPP, extrusion, heat treatments, ultrasonics, micro-fluidics, etc.) induce a higher exposure of the hydrophilic groups of the RP, thereby facilitating enhanced interaction between proteins and water molecules.^27,69 It is worth mentioning that studies have reported that the non-covalent reactions (primarily hydrogen bonding) of protein–protein (RP-cod protein) with an alkaline solution trigger the sheet helix transition and enhance functional properties like solubility (more than 90% (w/v)), gelling, and emulsification.^70,71 These observed phenomena have also been attributed to the exposure of hydrophilic sequences and the burial of hydrophobic regions.⁷¹ Interestingly, Abd Rahim et al.⁷² have reported that the salting out of ions at pH 7 disrupts the protein–water interaction, accumulating protein molecules while leading to enhanced protein–protein interactions and reduced solubility. Therefore, this study indicated that the 600 MPa-treated HPP provided improved RPP flour with satisfactory WSI and WHC.

Fig. 3 Effects of different high-pressure processing (HPP) treatments on the physicochemical properties of rice protein (RP) flour: (A) water solubility index (WSI), (B) water holding capacity (WHC), (C) sulfhydryl content, (D) S–S content, (E) DPPH radical scavenging capacity, (F) oxygen radical absorbance capacity (ORAC), (G) Fourier transform infrared (FTIR) spectroscopy, (H) X-ray diffraction (XRD), and (I) scanning electron microscopy (SEM).

This study showed that the free SH content in RP flour from all HPP-treated groups increased with pressure (Fig. 3C), and there were significant differences (p < 0.05). Conversely, the S–S content showed the opposite trend (Fig. 3D), with significant differences among the groups (p < 0.05). Notably, it has been suggested that the poor solubility of RP may be attributed to the presence of intermolecular S–S bonds (glutelins and globulins) and the relatively high molecular weight.³⁶ In comparison, the issue can be addressed by reducing these factors to enhance the extractability and bioavailability of RP.³⁶ Therefore, this study's results indicated that HPP treatment of RPP can effectively dissociate and refold the protein structure and contribute to applications for deeper processing.

Regarding the free radical scavenging capacities, both DPPH and ORAC of the HPP-treated groups in this study exhibited a positive correlation with the pressure (Fig. 3E and F), and there were significant differences between each group (p < 0.05). These phenomena were attributed to releasing antioxidant peptides (or AA) from RPP, thereby providing high activity against oxidants and exhibiting antioxidant capacity.^73–75 The enzymatic hydrolysis of RP was also found to yield antioxidant peptides or AA, thereby enhancing its antioxidant capacity, including DPPH and ABTS scavenging activity, reducing capacity, and ORAC.^74–76 It is worth mentioning that Liu et al.⁷⁷ also reported that the antioxidant ability of RP is related to the process conditions of alkaline hydrolysis, which involves the AA composition. Therefore, this study's results indicated that HPP treatment could remarkably improve the antioxidant ability of RP flour.

The molecular structure of the HPP-treated RPP in this study, determined by FTIR, showed absorption peaks ranging from 1000–1300 cm⁻¹ in the infrared spectrum (Fig. 3G). These were attributed to functional groups such as C–C, C–O, and C–H stretching vibrations and C–OH bending vibrations.^78,79 The peak marked absorption between 1010 and 1095 cm⁻¹ was detected in all groups, indicating the presence of pyranose rings, whereas the peaks of carboxyl (COOCH₃) and free carboxyl (COO–) group asymmetric and symmetric stretching were measured at 1600–1630 cm⁻¹.^79–81 The structural components, α-helix, β-sheet, β-turn, and aggregated strands of RPP, were exhibited at 1648–1660, 1626–1640, 1662–1684, and 1610–1628 cm⁻¹, respectively.^72,82,83 In addition, the peak ranges at 1600–1700 and 1500–1600 cm⁻¹ were attributed to amides I and II, respectively, which included C=O stretching vibrations and N–H bending vibrations, namely, protein secondary structure conformation.⁶⁹ The presence of bound water was indicated by the peak observed at 1650 cm⁻¹.⁷⁹ Several weaker wave peaks at 1965 and 2150–2200 cm⁻¹ belonged to the C–O bound folded state of the protein and the triple bond of alkyne stretching, respectively.⁷² The vibration at 2850 cm⁻¹ corresponded to the helical structures formed by AA (lysine and arginine) and the turn structure of proline.^72,84 Moreover, the peak profiles of 2800–3000 (the symmetric stretching mode of C–H bonds in the aliphatic side chain and aldehyde functional groups, including methyl and methylene) and 3100–3500 cm⁻¹ (the vibration of amide A; hydroxyl groups) corresponded to the hydrophobicity and hydrophilicity of RPP, respectively.^69,72,78,81 Yet, there was no evident change in the peak profiles of all groups, whereas the light spectrum increased with the increased stress of HPP, which agreed with the above results of the WSI and WHC.

This study showed that the diffraction peaks at 15°, 17°, 18° and 23° (2θ) for all groups were typically characteristic of the A-type crystalline structure of rice starch (Fig. 3H), which was consistent with the results reported by Chi et al.⁸⁵ and Lu et al.³¹ However, increased HPP stress led to observing minor peaks at 20°–21° in the groups exposed to 400 and 600 MPa, indicating the formation of V-type crystallites. The observed phenomenon could be attributed to HPP and heat exposure, and the partial disruption of non-covalent bonds, including hydrogen and ionic bonds, within the intermolecular chains of few starch molecules in RPP.⁸⁶ These disruptions alter the orientation and arrangement of starch molecules during crystallization, thereby perturbing the crystalline structure.^85–87 Furthermore, this phenomenon also facilitates the endogenous assembly of lipid–amylose in the RPP, forming complex V-type crystalline structures.^85,87 It is worth mentioning that Zhang et al.⁸⁸ reported higher crystallinity of RP-free rice starch through gelatinization, which decreased with the incorporated RP contents. Lu et al.³¹ also reported that the complexation of rice starch with RP also caused a pronounced decrease in the relative crystalline value, namely, altering the rice starch's crystalline structure.

The present study showed that the RPP particles in all samples exhibited comparable characteristics, featuring smooth and continuous lamellar surfaces with irregular porosity and grooves (Fig. 3I).^73,89 This observation suggests that HPP does not remarkably impact the shape of the protein particles; instead, higher stresses appear to result in a transition from loose grooves to tighter ones. This observed phenomenon can be attributed to the spray drying process of RP during manufacturing, wherein the elevated temperature of the hot air accelerates water evaporation by increasing the thermal energy transferred to protein-bound water (or droplets).^73,90 In addition, water vapor moves from the center of the protein particle to its surface, preventing the internal pressure from distorting or expanding to form inward collapsing blowholes, namely, hollow craters,^73,91 as observed by SEM. Moreover, it has been reported that HPP is unaffected by the presently achievable stress range for the primary structure of proteins, whereas the secondary structure may undergo minimal changes.^37,44 Conversely, the compression affects tertiary and quaternary structures by disrupting non-covalent bonds such as hydrogen bonds or the exposure of hydrophobic groups.³⁷ However, it does not impact covalent bonds, thus preserving the primary chemical integrity of flavoring compounds and colors in foodstuffs without altering their original flavor and nutritional properties, while the HPP finds extensive applications.^37,92

3.3. Effects of HPP-treated RPP on the growth performance of the experimental animals

This study showed that the BW in all rats at 1–2 weeks was elevated in all four groups of induced DCM compared to the control group (Fig. 4A). This phenomenon was hypothesized to be related to the initial overeating of DM, which increased the BW. Unsurprisingly, the BW performance of the control group increased to the highest level as time increased to the end of the experiment. While the BW of the RPP-supplemented rats in the three groups was substantially higher than that of the DCM group, albeit lower than that of the control group (Fig. 4B), all groups exhibited a significantly different performance (p < 0.05). These phenomena mentioned above revealed that RPP was effective in controlling the BW with a decreased risk of obesity in rats, and the trends agreed with the results reported by Liu et al.⁹³ Concurrently, the authors also noted that the influence of age on experimental animals did not limit the effects of RP. This study also suggested that the growth and metabolism of the three experimental animal groups fed RPP-prepared ES remained unaffected.^94,95

Fig. 4 Effects of HPP-treated rice protein peptide (RPP) prepared extruded snack (ES) supplementation for five months on the growth performance of the experimental animals: (A) weight change, (B) body weight (BW) difference, (C) heart weight, (D) heart/BW ratio and (E) blood glucose level.

In addition, the heart weights of rats in each group were also consistently associated with the above trend of BW changes (Fig. 4C), where the DM group was the lowest, and there were significant differences among all groups (p < 0.05). The heart weight to BW ratio was the highest in the DCM group (Fig. 4D), whereas there were significant differences between the groups (p < 0.05), but the three RPP groups’ ratios were closer to the control group. The results showed that continuous supplementation of RPP effectively reduced the heart-to-body weight ratio in DCM rats, indicating a potential reversal of cardiac inflammation or attenuation of symptoms.^2,96 Consequently, this intervention facilitated the restoration of impaired cardiac cells caused by DCM, thereby enhancing cardiac pump efficiency.² Li et al.⁹⁷ also revealed that obese T2DM mice exhibited pronounced myocardial lipotoxicity, characterized by enhanced uptake of free fatty acids and accumulation of ROS, glycogen, and lipid droplets. It is worth mentioning that excessive accumulation of free fatty acids in non-adipocytes can lead to glucose and lipid metabolic disorders, namely lipotoxicity and insulin resistance.^97,98 However, clinical evidence has demonstrated myocardial lipid accumulation in patients’ hearts with insulin resistance and hyperglycemia.¹³

In terms of blood glucose levels, the results of this study showed that the DCM group exhibited the maximum (Fig. 4E). This also suggested that insulin utilization was impaired in the rats in the DCM group.⁹⁹ Namely, the pattern of induction of DCM in this study was successfully established. In contrast, the blood glucose levels of the RPP groups decreased with increased HPP pressure, similar to the control group, whereas there were significant differences between all groups (p < 0.05). These phenomena also implied that continuous supplementation of high-pressure (600 MPa) treatment RPP of ES for five months facilitated the restoration of blood glucose levels in T2DM rats. Specifically, it alleviates impaired insulin utilization in rats.⁹⁹ Notably, Higuchi et al.²⁸ reported that rice endosperm protein administration was associated with markedly decreased fasting glucose, plasma insulin, and homeostatic model assessment of insulin resistance in adult mice fed a high-fat diet for 12 weeks. The authors also showed that the protein source provided to juvenile mice for six weeks substantially influenced the susceptibility to obesity and associated diseases induced by a high-fat diet during adulthood. It has also been reported that sustained hyperglycemia stimulates the production of ROS, consequently promoting the secretion of pro-inflammatory cytokines in DCM.¹ Therefore, this study's results revealed that the elevated pressure of high-pressure (600 MPa) HPP-treated RPP of ES positively restored growth performance and blood glucose levels in DCM rats.

3.4. Effects of HPP-treated RPP on the serum biochemical parameters and myocardial infarction (MI) indices of the experimental animals

This study showed that the TG, TC, AST, and LDH levels in the three RPP groups were lower than those in the DCM group (Fig. 5A–D), while the values were closer to the control group. In particular, the RPP600 group exhibited more favorable results, and there were significant differences between the groups (p < 0.05). This study showed abnormalities in serum biochemical parameters in the DCM group, the basic module of metabolic disorders in rats.¹⁰⁰ According to the report, apart from TC and TG, there is also a concomitant elevation in the low-density lipoprotein-cholesterol level.¹⁰⁰

Fig. 5 Effects of HPP-treated rice protein peptide (RPP) prepared extruded snack (ES) supplementation for five months on the serum biochemical parameters [(A) triglycerides (TG), (B) total cholesterol (TC), (C) aspartate aminotransferase (AST), and (D) lactate dehydrogenase (LDH)] and myocardial infarction indices [MI; (E) troponin and (F) creatine kinase-MB (CK-MB)] of the experimental animals.

Yang et al.⁹⁴ provided evidence that the administration of RP reduced TG and cholesterol levels. This outcome was correlated with decreased protein digestibility and increased fecal excretion of neutral sterols. Notably, this mechanism was facilitated by converting cholesterol into bile acids within the body through enterohepatic circulation.⁹⁴ Interestingly, Liu et al.⁹³ also reported that continuous supplementation of RP for two weeks exhibited the potential to reduce TG levels in both plasma and liver among juvenile and adult rats. However, the results of a four-week clinical trial using rice endosperm protein, as Nakayama et al.¹⁰¹ reported, did not demonstrate any significant positive effects on subjective mood states and the insomnia severity index. The lack of efficacy may be attributed to the relatively short intervention period.¹⁰¹

This study also showed that AST was the highest in the DCM group, meaning that liver function and myocardial cells were impaired, but RPP supplementation effectively inhibited liver and myocardial cell injury in DCM rats. In contrast, AST exhibited a decrease in the three RPP groups, which agreed with the results reported by Gilani et al.¹⁰⁰ and Wang et al.⁹⁹ AST is distributed in cardiac, liver, and skeletal muscle cells.⁹⁹ In addition, hepatic enzymes in the bloodstream, originating from the liver cytosol, indicate hepatocyte damage.¹⁰⁰ Kalpanadevi et al.¹⁰² also reported that enzymes such as AST, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) primarily aid in the breakdown of proteins by the liver, which were relatively high in rats supplemented with rice bran protein. In contrast, AST and ALT were typically elevated in response to acute hepatotoxicity, yet tended to decline with prolonged intoxication.¹⁰² Notably, LDH, TG, cholesterol, and CK play critical roles in understanding cardiac function, where LDH catalyzes the conversion of lactate to pyruvate, an important step in cellular energy production.^102,103 Creatine kinase serves as an essential enzyme modulator in the production and utilization of high-energy phosphates in the contractile tissues.^102,103 Kalpanadevi et al.¹⁰² reported that rats supplemented with rice bran protein exhibited markedly increased levels of LDH and enzymes such as creatine kinase and decreased cholesterol levels. Conversely, elevated cholesterol, CK, and LDH levels indicated that the control rats were more susceptible to cardiac dysfunction in the presence of protein malnutrition,¹⁰² which was comparable to the results observed in the present study. Therefore, the ameliorative effect of bioactive substances on cardiotoxicity was evaluated as a decrease in the serum levels of creatine phosphokinase, CK-MB, and LDH, which also included a reduction in the level of MDA in the OS index.¹⁰⁴

Moreover, the same trends described above were observed for MI indices (TnT and CK-MB) in DCM rats of three RPP groups following continuous HPP-treated RPP flour-produced ES supplementation for five months (Fig. 5E and F). Specifically, the RPP 600 MPa group exhibited satisfactory performance in recovering the TnT content, with significant differences among the groups (p < 0.05). In contrast, the CK-MB activities of the three RPP groups were not significantly different from each other, and they were lower than that of the DCM group (p < 0.05). These phenomena were attributed to the structural and property changes in RP flour post-HPP treatment and the recognition of specific AA sequences during hydrolysis by enzymes in the rat's digestive tract, producing peptides with potent bioactivities (such as antioxidant capacities).²⁵ However, the findings of this study indicated that the bioactivity of RPP played a crucial role in eliminating MI indices and alleviating irreversible myocardial damage induced by DCM. Thereby, the OS risk of heart failure was reduced, which was associated with elevated levels of TnT and CK-MB.^2,105 Hence, this study suggested that ES produced from HPP-treated RPP flour supplemented for five consecutive months contributed to the recovery of serum biochemical parameters and MI indices in patients with DCM, contributing to health promotion.

3.5. Effects of HPP-treated RPP on the cardiac oxide-inflammatory parameters of the experimental animals

This study showed a positive correlation between the reduction of MDA content in rat serum in the three RPP groups and the pressure of HPP used (Fig. 6A), with significant differences among all groups (p < 0.05). Namely, the RPP600 group exhibited the best effect in reducing the MDA content, which was closer to the value of the control group. This finding also correlated with the antioxidant ability of HPP-treated RPP flour, as described above, whereas the highest MDA content, as expected, was found in the DCM group. It has been reported that these advances and related issues regarding DCM were attributable to an increase in MDA caused by OS, while a decrease in MDA levels could be attributed to the contribution of antioxidant enzymes (SOD and CAT).^100,106 Specifically, it also confirmed that HPP-treated RPP-prepared ES exhibited antioxidant ability similar to the results reported by Gilani et al.¹⁰⁰ and this was also consistent with the results of this study mentioned in section 3.2. This also implied that the RPP (peptide or AA) of the ES in this study still had antioxidant capacity after being metabolized by rat digestion, and the LMW of the hydrolysate (<10 kDa) following digestion also proved to exert more excellent antioxidant activity.¹⁰⁷ The limitations of the present study lie in the absence of further confirmation regarding the distribution of undigested RPP and intestinal flora in the rat colon. This hinders our ability to elucidate both beneficial and adverse health effects resulting from the products derived from fermentative hydrolysis of these proteins (peptide or AA).¹⁰⁸

Fig. 6 Effects of HPP-treated rice protein peptide (RPP) prepared extruded snack (ES) supplementation for five months on the cardiac oxide-inflammatory parameters [(A) malondialdehyde (MDA), (B) catalase (CAT), (C) glutathione peroxidase (GPx), (D) glutathione (GSH), (E) superoxide dismutase (SOD), (F) caspase-3, (G) interleukin (IL)-6, (H) IL-1β, (I) tumor necrosis factor (TNF)-α, and (J) nuclear factor (NF)-κB] of the experimental animals.

In terms of several cardiac oxide-inflammatory parameters (including CAT, GPx, GSH, and SOD), this study showed that all three RPP groups were significantly improved compared to the DCM group while they were closer to the control group (p < 0.05) (Fig. 6B–E). In particular, the 600 MPa group showed a satisfactory improvement. These findings indicated that in the DCM group, the continuous elevation of OS and the subsequent inflammatory response exhausted the endogenous antioxidants (such as SOD and GSH-Px) in the heart, thereby leading to cardiomyocyte damage.¹

However, replenishing exogenous antioxidants through five consecutive months of supplementation with RPP-prepared ES effectively mitigated OS damage to cardiomyocytes, enhancing their antioxidant capabilities. This was similar to our previous report on the improvement and benefit of supplementation with extruded djulis snacks on DCM.² Furthermore, dietary supplementation with RP has been reported to enhance endogenous antioxidant capacity (up-regulation of expression levels of SOD, GPx, etc.) in both juvenile and adult rats, while reducing the hepatic accumulation of MDA, carbonylated proteins, and ROS, leading to a decrease in the damage caused by OS.¹⁰⁹ It has also been reported that bioactive peptides obtained from the enzymatic hydrolysis of whey proteins can also exert antioxidant effects in Caco-2 cell lines by enhancing the levels of both GSH and SOD, thereby safeguarding intestinal cells against OS.^110,111 Therefore, the findings revealed the beneficial levels of all related antioxidant status. Thus, modifying the ES formula composition by incorporating the RPP reduced OS risk due to DCM, which aligns with the extruded djulis snack results previously reported by this team.²

Well known as a critical executive molecule in apoptosis, caspase-3 is activated in the early stages and cleaves the corresponding cytoplasmic and cytosolic substrates, leading to apoptosis. Notably, the activity of caspase-3 is dramatically reduced in the late stages of apoptosis and dead cells.¹¹² This study showed that all three supplemental RPP groups exhibited a substantial reduction in caspase-3 levels compared to the DCM group (Fig. 6F), and there were significant differences among the groups (p < 0.05). Specifically, the decrease in the RPP600 group was closest to the control group. These phenomena can be attributed to the antioxidant ability linked to HPP-treated RPP flour, which mitigates OS in the myocardial tissue cells from T2DM-induced inflammation.

Conversely, for other cardiac oxide-inflammatory parameters such as IL-6, IL-1β, TNF-α, and NF-κB, this study showed that the three groups exhibited statistically significantly lower levels of these inflammatory parameters in the RPP group than in the DCM group (p < 0.05) (Fig. 6G–J). Specifically, the RPP600 group exhibited the greatest reduction, close to that of the control group. These results showed that by reducing the production of cardiac oxide-inflammatory parameters were alleviated, namely decreased levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) in the cardiac tissues of the DCM group.^1,2

Subsequently, the aberrant accumulation of varying growth factors activated the NF-κB signaling pathway in cardiovascular disease, eventually contributing to cardiomyocyte pyroptosis.^7,11 However, continuous supplementation of ES for 5 months in the three RPP groups exhibited a restraining effect on the aforementioned inflammatory cytokines and facilitated a healthy recovery of the myocardium.^1,2 It is noteworthy that excessive free radical production has been reported to promote NF-κB triggering, which in turn contributes to gene transcription of encrypting inflammatory proteins.¹⁰⁰ Wang et al.¹¹³ also reported that feeding RP for two weeks effectively down-regulated the hepatic protein expression and mRNA levels of NF-B1 in juvenile and adult rats, thereby inhibiting the activation of the NF-B metabolic pathway. The same authors also stated that RP supplementation for two weeks showed a decrease in the mRNA levels of pro-inflammatory cytokines [IL-1, IL-6, TNF-α, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and monocyte chemoattractant protein-1 (MCP-1)]. In addition, Wen et al.¹¹⁴ showed that trypsin-derived RP hydrolysates inhibited LPS-induced inflammatory responses and suppressed the NF-κB signaling pathway in RAW264.7 macrophages by inhibiting nitric oxide (NO) release and decreasing IL-1β, IL-6, TNF-α, and iNOS expression in a dose-dependent manner. However, increased NF-κB levels were also observed in the DCM group of this study, but effective mitigation of this phenomenon was also found in the three RPP groups by supplementation with RPP-prepared ES.

Moreover, it has been reported that rice endosperm protein administration also decreased the serum levels of IL-1β, IL-6, leptin, and lipopolysaccharide-binding protein in high-fat diet-induced obese C57BL/6J male mice (12 week old adults).²⁸ The same authors also stated that in the serum, kidneys, and liver of the fed mice (6 week old juvenile), it was observed that RP intake contributed to lowering the levels of TNF-α and monocyte chemoattractant protein-1 apart from the biomarkers mentioned above. In addition, Ge et al.⁴⁵ reported that egg white peptide supplementation ameliorated colonic inflammatory symptoms and modulated inflammatory factors, such as IL-6, in mice with ulcerative colitis attributed to altered gut flora. Notably, all of these changes in inflammatory parameters were also involved in the composition of the intestinal microbiota and variations in the levels of SCFAs.⁴⁷ Galland et al.¹¹¹ also reported that LMW (<5 kDa) peptides derived from whey protein isolate through digestive hydrolysis contributed to the attenuation of inflammation in astrocytes and microglia while enhancing IL-6 and TNF-α mRNA expression, thereby reducing the levels of these cytokines. Interestingly, a similar phenomenon has been observed in the Parkinson's disease model, wherein microglia exhibit heightened activation and release inflammatory molecules (such as IL-1β, IL-6, TNF-α, IL-18, and chemokines), thereby inducing neuronal damage.¹¹²

Protein digestion in the gastrointestinal tract involves complex mechanisms, while proteins are broken down and AA is released into the stomach. Then, partially digested proteins enter the small intestine along with other gastric contents for absorption, where they undergo hydrolysis by additional proteolytic enzymes to yield oligopeptides and free AA.¹¹⁵ Further investigation is necessary to comprehensively analyze the peptide and AA composition and the distribution of hydrolyzed substances during digestion to address the current limitations and ensure a more comprehensive understanding of these factors. However, the above results suggested that continuous supplementation of ES prepared from HPP-treated RPP flour may be a promising approach for mitigating DCM-induced cardiac oxidative-inflammatory parameters. Therefore, it also implies that patients can supplement or incorporate foods prepared with HPP-treated RPP flour in their daily diets to mitigate cardiac oxide inflammation caused by DCM, apart from receiving conventional therapeutic medications. Moreover, integrating HPP-RPP ES into their dietary regimen during hypoglycemia episodes can enhance general well-being.

4. Conclusions

This study revealed that HPP treatment for RPP enhanced the physicochemical properties. Incorporating RPP of ES formulation also proved that RPP ES's glucose modulation and antioxidant potential alleviated the OS in DCM and performed satisfactorily to improve cardiac oxide-inflammatory parameters. Specifically, five consecutive months of dietary supplementation effectively ameliorated OS-related myocardial damage in DCM rats. However, the detailed mechanism underlying the RPP-mediated NF-κB metabolic pathway and the extent of involvement of the gut microbiota remain unclear, necessitating further investigation. Another limitation of the present study was the validation phase of the animal model. It was necessary to examine whether consistent effects were maintained through human clinical trials before sufficient information could be obtained about the actual contribution of RPP-prepared ES. Therefore, this study offers valuable insights and information regarding the utilization of HPP for processing, enhancement of bioactive functionality, and integration with existing technologies.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.

Data availability

The data that support the findings of this study are available from the corresponding author, Po-Hsien Li, upon reasonable request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5fo01477h

Acknowledgements

This research was financially supported by the Taichung Veterans General Hospital/Providence University Joint Research Program (TCVGH-PU-1128102), Taiwan.

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Footnote

† These authors contributed equally to this work.

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