Degree Of Hydrolysis Research Articles

Wheat bran extract as a source of amino compounds and sugar for biotech processes

AbstractWheat bran is an abundant source of starch fragments, peptides and amino acids which represent a valuable nutrient source for bacterial production of natural products. The commercial protease Corolase® 7089 efficiently improves extraction of N‐compounds from wheat bran. Here we show the effect of combined applications with the commercial α‐amylase Termamyl® 120. Wheat bran was extracted with at a liquid/solid ratio of 12.3. The same product yield of 24% was obtained after simultaneous enzyme application for 1 h and after serial application (first amylase) with a total treatment time of 7.75 h. Crude protein concentration in the product was 23% and 22%, respectively. Measurements of the sugar concentration in the extraction liquid indicate that the degree of starch hydrolysis was decreased in the presence of protease. In a stirred pilot‐scale reactor a product yield of 32% was achieved.

Application of experimental design as a statistical approach to recover bioactive peptides from different food sources.

Bioactive peptides (BAPs) derived from samples of animals and plants have been widely recommended and consumed for their beneficial properties to human health and to control several diseases. This work presents the applications of experimental designs (DoE) used to perform factor screening and/or optimization focused on finding the ideal hydrolysis condition to obtain BAPs with specific biological activities. The collection and discussion of articles revealed that Box Behnken Desing and Central Composite Design were the most used. The main parameters evaluated were pH, time, temperature and enzyme/substrate ratio. Among vegetable protein sources, soy was the most used in the generation of BAPs, and among animal proteins, milk and shrimp stood out as the most explored sources. The degree of hydrolysis and antioxidant activity were the most investigated responses in obtaining BAPs. This review brings new information that helps researchers apply these DoE to obtain high-quality BAPs with the desired biological activities.

Reduction of maillard browning in spray dried low-lactose milk powders due to protein polysaccharide interactions

Lactose hydrolysed concentrated milk was prepared using β-galactosidase enzyme (4.76U/mL) with a reaction period of 12 h at 4 °C. Addition of polysaccharides (5 % maltodextrin/β-cyclodextrin) to concentrated milk either before or after lactose hydrolysis did not result in significant differences (p > 0.05) in degree of hydrolysis (% DH) of lactose and residual lactose content (%). Three different inlet temperatures (165 °C, 175 °C and 185 °C) were used for the preparation of powders which were later characterised based on physico-chemical and maillard browning characteristics. Moisture content, solubility and available lysine content of the powders decreased significantly, whereas, browning parameters i.e., browning index, 5-hydroxymethylfurfural, furosine content increased significantly (p < 0.05) with an increase in inlet air temperature. The powder was finally prepared with 5 % polysaccharide and an inlet air temperature of 185 °C which reduced maillard browning. Protein-polysaccharide interactions were identified using Fourier Transform infrared spectroscopy, fluorescence spectroscopy and determination of free amino groups in the powder samples. Maltodextrin and β-cyclodextrin containing powder samples exhibited lower free amino groups and higher degree of graft value as compared to control sample which indicated protein-polysaccharide interactions. Results obtained from Fourier Transform infrared spectroscopy also confirmed strong protein-polysaccharide interactions, moreover a significant decrease in fluorescence intensity was also observed in the powder samples. These interactions between the proteins and polysaccharides reduced the maillard browning in powders.

Protein hydrolysates prepared by Alcalase using ultrasound and microwave pretreated almond meal and their characterization.

The study aimed to optimize ultrasonic (US: 40kHz/200W for 10, 20, 30, 40, and 50min), and microwave (MW: 160W for 45, 90, 125, 180, and 225s) pretreatment conditions on protein extraction yield and degree of protein hydrolysis (DH) from almond de-oiled meal, an industrial by-product. First order model was used to describe the kinetics of almond protein hydrolysates obtained with Alcalase. The highest DH, 10.95% was recorded for the US-50min and 8.87% for MW-45s; while it was 5.76% for the untreated/control sample. At these optimized pretreatment conditions, a 1.16- and 1.18-fold increment in protein recovery was observed for theUS and MW pretreatments, respectively in comparison to the conventional alkaline extraction. The molecular weight distribution recorded for pretreated samples disclosed a significant reduction in the band thickness in comparison with control. Both the pretreatments resulted in a significant increase (P < 0.05) in the antioxidant activity, and TCA solubility index when compared with the control. Results evinced that US and/or MW pretreatments before enzymatic hydrolysis can be a promising approach for the valorization of almond meal for its subsequent useas an ingredient for functional foods/nutraceuticals which otherwise fetches low value as an animal feed.

Structural characteristics, functional properties and nutritional value of walnut protein by limited enzymatic hydrolysis

This study investigates the effects of limited enzymatic hydrolysis on the structural and functional characteristics, as well as nutritional value of walnut protein (WP). WP was hydrolyzed using papain to obtain varying degrees of hydrolysis (DHs) at 6, 8.33, 10 and 11.4%. The results indicate significant changes in the structural properties of WP due to papain hydrolysis. These changes primarily included a decrease in molecular weight, increased flexibility in secondary structure, reduced intrinsic fluorescence intensity, and a rise in H0 correlating with the DH increase. Accordingly, hydrolyzed WP displayed better functional properties and higher nutritional value. Notably, WP with a DH of 11.4% exhibited superior emulsification properties and the highest antioxidant abilities. In conclusion, the use of limited enzymatic hydrolysis with papain appears to be an effective approach for expanding the application of WP.

In silico bioactivity analysis of peptide fractions derived from brewer's spent grain hydrolysates

SummaryBrewer's spent grain is an agro‐industrial waste of barley malt from the manufacture of beer, which is rich in protein. Thus, this by‐product of the beer industry could be utilised to obtain bioactive peptides through enzymatic hydrolysis. The present study aimed to evaluate potential bioactivities (anti‐inflammatory, antidiabetic, antioxidant, antimicrobial) of peptide sequences identified in 5‐kDa peptide fractions derived from brewer's spent grain hydrolysates. Two experimental hydrolysates (run 5 and run 21) were selected based on their higher degree of hydrolysis (&gt;55%), which were subjected to peptide sequence identification and used for in silico analysis. A total of fifty peptide sequences were identified between both runs. In silico analysis identified seventeen and thirteen multifunctional peptides in runs 5 and 21, respectively, which showed between two and four potential bioactivities. Interestingly, one peptide sequence (DLPPER) showed four potential bioactivities and, according to the Docking analysis, showed better Vina scores towards the protein's targets DPP‐IV (antidiabetic) and glucocorticoid receptor (anti‐inflammatory). These results demonstrate that peptides from brewer's spent grain hydrolysates may contribute to improving human health by offering potential protection against some chronic‐related diseases; however, further follow‐up in vitro and in vivo studies are needed to evaluate its actual capability.

Protein hydrolysates from buffalo cheese whey: production and characterisation for food supplementation

SummaryThe objective of this study was to obtain protein hydrolysates from buffalo cheese whey proteins and characterise them. Degrees of hydrolysis ranged between 10% and 16% depending on processing time. The hydrolysate obtained after 12 h showed a low water activity value (&lt;0.4), and the lowest moisture content (4.88% w/w), besides demonstrating better solubility, and enhanced emulsion stability. The hydrolysate obtained after an 18‐h process exhibited higher foam production capacity. In vitro, protein digestibility values of the hydrolysates ranged from 25% to 27% (w/w). Prolonging the hydrolysis (18 h) of the buffalo whey proteins with Flavourzyme resulted in a greater release of amino acids. The hydrolysate obtained after a 12‐h process exhibited the best techno‐functional characteristics (moisture, oil holding capacity, and emulsion stability), while the 18‐h showed the highest levels of branched‐chain amino acids. The characteristics of these hydrolysates enable their use in the production of supplements, providing another strategy for bubaline whey utilisation.

Antioxidant and angiotensin converting enzyme inhibitory activities of red lionfish (Pterois volitans L.) muscle protein hydrolysates obtained using pepsin-pancreatin system

Despite being an invasive species and representing a threat in the area, red lionfish (Pterois volitans L.) meat is valued for its pleasant taste and high protein content. The objective of the present work was to evaluate the angiotensin converting enzyme inhibition (ACE-I) and the antioxidant activities of red lionfish muscle protein hydrolysates in vitro. Hydrolysates were obtained using the pepsin-pancreatin system, and their degree of hydrolysis (DH), electrophoretic, and amino acid profile were determined. Subsequently, their ACE-I and ABTS+ radical scavenging activity were evaluated, selecting those with the highest response. The highest DH (66.1%) was found in the hydrolysate obtained at 120 min of reaction time (H120); electrophoresis revealed the action of the enzymes on the muscle proteins. The highest bioactivities occurred during hydrolysis with pepsin for 60 min (H60), obtaining the highest ACE-I of 68.8% (evaluated with 500 µg protein) with a DH of 16.8%, ACE-I related amino acid content of 33.6%, and hydrophobic amino acid content of 42.9%. With the 30 min hydrolysates (H30), the highest Trolox equivalent antioxidant capacity of 60.3 mM/mg protein was obtained with 18.4% DH and 22.1% radical scavenger amino acids; its comparison with H120 proved that higher DH did not produce a greater response in ABTS+ radical scavenging activity, DPPH, reducing power, and copper chelation assays; with β-carotene bleaching being the exception. Therefore, it can be concluded that red lionfish muscle hydrolysates could be a promising source of peptides with antihypertensive and antioxidant properties, and can be suitable as functional ingredients.

Novel strategy for depolymerization of avermectin fermentation residue to value-added amino acid product.

Avermectin fermentation residue (AFR) is rich in proteins, which can be depolymerized to value-added amino acids for in-plant reuse. The hydrochloric acid (HCl) hydrolysis is performed and investigated under different conditions, including HCl concentration, solid-liquid ratio, temperature, and time. The hydrolysis degree (HD) of 67.7% can be achieved. The empirical correlation of HD is established with a good practicability to control the HD and predict the experimental conditions. Solid-liquid reaction is confirmed to be dominant during the hydrolysis process. There are 17 kinds of amino acids in the hydrolysate, benefiting the reuse. Avermectin is not detected in the hydrolysate and AFR, and the mass of AFR is reduced by 53.8wt%. This work provides a novel strategy for the environmentally friendly treatment and meanwhile the resource recovery of AFR.

Ultrasound viscous reduction effects on the proteolysis of soy protein isolate at a limited degree of hydrolysis: Changes in the functional characteristics and protein structure

High-concentration soy protein isolate was subjected to ultrasonication for viscosity reduction to assist the process of limited enzymatic hydrolysis. Ultrasonication (20 kHz, 10 min, 160 W/L) effectively reduced the viscosity of soy protein isolate at a comparatively high concentration of 14 % (w/v) and promoted the limited enzymatic hydrolysis (controlled degree of hydrolysis of 12 %) with a higher peptide yield than that of the conventional method. The correlations between substrate viscosity and peptide yield, as well as the viscosities of the resulting hydrolysates, were studied. The findings revealed positive correlations between the viscosities of the substrate and hydrolysate, underscoring the potential impact of altering substrate viscosity on the final product. Furthermore, the utilization of ultrasonic viscosity reduction-assisted proteolysis has shown its capability to improve the functional and physicochemical properties, as well as the protein structure of the hydrolysate, while maintaining the same level of hydrolysis. It is worth noting that there were significant alterations in particle size (decrease), β-sheet content (increase), β-turn content (increase), and random coil content (increase). Interestingly, ultrasonication unexpectedly impeded the degradation of molecular mass in proteins during proteolysis, while increasing the hydrophobic properties of the hydrolysate. These findings aligned with the observed reduction in bitterness and improvement in emulsifying properties and water-holding capacity.

Enhancing the functional properties of peanut protein hydrolysates: covalent modification with chlorogenic acid

SummaryThis study aimed to enhance the functional properties of peanut protein hydrolysates (PPHs) by coupling limited enzymatic hydrolysis with chlorogenic acid (CGA) modification, thus offering novel insights into the potential food industry applications of peanut proteins. Initially, peanut protein isolate was hydrolysed using trypsin. Subsequently, covalent conjugation with CGA was achieved via free‐radical grafting. The moderately hydrolysed 4% PPHs had the highest polyphenol‐binding capacity. Measurement of the PPHs‐CGA complex revealed that free amino, thiol, and tryptophan contents were reduced. The amino acid content results also showed that cysteine, lysine, arginine, and methionine (with amino or thiol groups) were the primary binding sites. Spherical or rod‐like structures were observed in the scanning electron microscopy images of PPHs‐CGA, and its particle size was larger than that of the PPHs. After combining PPHs and CGA, the fluorescence intensity decreased, Fourier‐transform infrared spectrum showed a shift in the absorption peak, and electrophoresis band weakened, indicating that covalent binding was successful. PPHs with different degrees of hydrolysis have different levels of exposure to amino acids, and their antioxidant properties differ when combined with CGA. After combining, the surface hydrophobicity was reduced, and the secondary structure was unfolded and extended, thereby improving its emulsifying properties. In summary, PPHs‐CGA may be used in the food industry as a bi‐type food factor and proposes a new application for peanut protein in food.

Evaluation of protein and lipid ingredients through in vitro digestibility for tropical gar (Atractosteus tropicus) juveniles

Tropical gar (Atractosteus tropicus) is an ecologically, culturally, economically, and scientifically important species. Therefore, it is essential to know the available resources to design feeds for the culture of this species. This study evaluated the in vitro digestibility of meals and oils commonly used in aquaculture, using crude extract of the stomach and intestines of A. tropicus juveniles. We used the pH stat titration system to determine the degree of hydrolysis (DH%), the concentration of amino acids released (mg g of meal-1), and the speed of hydrolysis (SH%) of various animal and vegetable ingredients. The relative digestibility of each protein ingredient was calculated using bovine hemoglobin for acid digestion and Hammersten bovine casein for the alkaline phase as reference ingredients (100% digestibility). For lipid ingredients, fish oil was used as a reference. The highest relative digestibility for protein ingredients was observed with fish, crustaceans, beef, poultry meat and offal, and beef blood meals. The best lipid SH was observed with soybean lecithin and cod liver, soybean, olive, maize, and canola oils. It is possible to use a wide variety of terrestrial animal proteins and vegetable oils for manufacturing a diet for A. tropicus rearing.

Microscopic and Macroscopic Characterization of Hydrogels Based on Poly(vinyl-alcohol)-Glutaraldehyde Mixtures for Fricke Gel Dosimetry.

In recent decades, hydrogels have emerged as innovative soft materials with widespread applications in the medical and biomedical fields, including drug delivery, tissue engineering, and gel dosimetry. In this work, a comprehensive study of the macroscopic and microscopic properties of hydrogel matrices based on Poly(vinyl-alcohol) (PVA) chemically crosslinked with Glutaraldehyde (GTA) was reported. Five different kinds of PVAs differing in molecular weight and degree of hydrolysis were considered. The local microscopic organization of the hydrogels was studied through the use of the 1H nuclear magnetic resonance relaxometry technique. Various macroscopic properties (gel fraction, water loss, contact angle, swelling degree, viscosity, and Young's Modulus) were investigated with the aim of finding a correlation between them and the features of the hydrogel matrix. Additionally, an optical characterization was performed on all the hydrogels loaded with Fricke solution to assess their dosimetric behavior. The results obtained indicate that the degree of PVA hydrolysis is a crucial parameter influencing the structure of the hydrogel matrix. This factor should be considered for ensuring stability over time, a vital property in the context of potential biomedical applications where hydrogels act as radiological tissue-equivalent materials.

Characterization of the Effects of Low-Sodium Salt Substitution on Sensory Quality, Protein Oxidation, and Hydrolysis of Air-Dried Chicken Meat and Its Molecular Mechanisms Based on Tandem Mass Tagging-Labeled Quantitative Proteomics.

The effects of low-sodium salt mixture substitution on the sensory quality, protein oxidation, and hydrolysis of air-dried chicken and its molecular mechanisms were investigated based on tandem mass tagging (TMT) quantitative proteomics. The composite salt formulated with 1.6% KCl, 0.8% MgCl2, and 5.6% NaCl was found to improve the freshness and texture quality scores. Low-sodium salt mixture substitution significantly decreased the carbonyl content (1.52 nmol/mg), surface hydrophobicity (102.58 μg), and dimeric tyrosine content (2.69 A.U.), and significantly increased the sulfhydryl content (74.46 nmol/mg) and tryptophan fluorescence intensity, suggesting that protein oxidation was inhibited. Furthermore, low-sodium salt mixture substitution significantly increased the protein hydrolysis index (0.067), and cathepsin B and L activities (102.13 U/g and 349.25 U/g), suggesting that protein hydrolysis was facilitated. The correlation results showed that changes in the degree of protein hydrolysis and protein oxidation were closely related to sensory quality. TMT quantitative proteomics indicated that the degradation of myosin and titin as well as changes in the activities of the enzymes, CNDP2, DPP7, ABHD12B, FADH2A, and AASS, were responsible for the changes in the taste quality. In addition, CNDP2, ALDH1A1, and NMNAT1 are key enzymes that reduce protein oxidation. Overall, KCl and MgCl2 composite salt substitution is an effective method for producing low-sodium air-dried chicken.

Assessment of functional efficacy of sheep plasma protein hydrolysates and their utilization in mutton sausage

The present study examines the bioactive potential of sheep plasma protein hydrolysates (SPPH) produced by in-vitro gastrointestinal digestion as antioxidants, antimicrobials, anti-obesity agents, and inhibitors of lipid oxidation in sausage to address the oxidative stability and shelf-life issues of mutton. The antioxidant and antimicrobial activities, indicate a positive relationship between the degree of hydrolysis and digestion duration. The study finds that SPPH has a potent inhibitory effect on pancreatic lipase and cholesterol esterase. It has higher oil holding capacity than sheep plasma protein, observed at one hour of hydrolysis time. SPPH exhibit an improved behavior in foaming properties along alkaline pH and digestion time while display lower emulsifying activity and stability with hydrolysis advancement. The SPPH act as a natural preservative in developing functional mutton sausage by inhibiting lipid-oxidation. This study showed that the recovery of SPPH can be a cost-effective and sustainable strategy for generating available ingredients for enhanced shelf-life of meat products.

Use of olive and sunflower protein hydrolysates for the physical and oxidative stabilization of fish oil-in-water emulsions.

Olive and sunflower seeds are by-products generated in large amounts by the plant oil industry. The technological and biological properties of plant-based substrates, especially protein hydrolysates, have increased their use as functional ingredients for food matrices. The present study evaluates the physical and oxidative stabilities of 50 g kg-1 fish oil-in-water emulsions where protein hydrolysates from olive and sunflower seeds were incorporated at 20 g kg-1 protein as natural emulsifiers. The goal was to investigate the effect of protein source (i.e. olive and sunflower seeds), enzyme (i.e. subtilisin and trypsin) and degree of hydrolysis (5%, 8% and 11%) on the ability of the hydrolysate to stabilize the emulsion and retard lipid oxidation over a 7-day storage period. The plant protein hydrolysates displayed different emulsifying and antioxidant capacities when incorporated into the fish oil-in-water emulsions. The hydrolysates with degrees of hydrolysis (DH) of 5%, especially those from sunflower seed meal, provided higher physical stability, regardless of the enzymatic treatment. For example, the average D [2, 3] values for the emulsions containing sunflower subtilisin hydrolysates at DH 5% only slightly increased from 1.21 ± 0.02 μm (day 0) to 2.01 ± 0.04 μm (day 7). Moreover, the emulsions stabilized with sunflower or olive seed hydrolysates at DH 5% were stable against lipid oxidation throughout the storage experiment, with no significant variation in the oxidation indices between days 0 and 4. The results of the present study support the use of sunflower seed hydrolysates at DH 5% as natural emulsifiers for fish oil-in-water emulsions, providing both physical and chemical stability against lipid oxidation. © 2024 Society of Chemical Industry.

Influence of available oral collagen supplementation on the function of joints and skin

Introduction and purpose: In our work, we focused on presenting the role of collagen supplementation and its impact on skin and joint health. We considered studies discussing the differences in the effects of collagen from various plant and animal sources. We also examined the influence of the degree of hydrolysis on the absorbability of the supplement. The aim of the paper is to comprehensively summarize knowledge on collagen supplementation, differentiating between its sources and forms. Material and methods: In our paper, we endeavored to address the subject of oral collagen supplementation comprehensively. We explored studies about various forms and sources of collagen. We also delved into the collagen supplementation impact on skin and joints health and impact of additives in enhancing the positive effect of collagen . State of knowledge: Oral collagen supplementation can be beneficial for skin and joints health. It promote collagen synthesis in human body increasing strength of skin and joints. The higher the degree of collagen hydrolysis, the more easily it is absorbed by the body. Additives in the form of vitamin C and A can enhance collagen synthesis. Results and conclusions: This review article indicates a growing interest in oral collagen supplementation and its impact on health. Although there are encouraging findings regarding positive effects, further research is necessary to better understand and determine the optimal conditions for achieving maximum supplementation benefits. This information may be crucial for clinical decisions made by physicians regarding joint reinforcement and anti-aging effects on the skin.

Preparation and identification of antioxidant peptides from Quasipaa spinosa skin through two-step enzymatic hydrolysis and molecular simulation

Frog skin, a by-product of Quasipaa Spinosa farming, is rich in protein and potentially a valuable raw material for obtaining antioxidant peptides. This study used papain combined with acid protease to digest frog skin in a two-step enzymatic hydrolysis method. Based on a single factor and response surface experiments, experimental conditions were optimized, and the degree of hydrolysis was 30 %. A frog skin hydrolysate (QSPH-Ⅰ-3) was obtained following ultrafiltration and gel filtration chromatography. IC50 for DPPH, ABTS, and hydroxyl radical scavenging capacities were 1.68 ± 0.05, 1.20 ± 0.14 and 1.55 ± 0.11 mg/mL, respectively. Peptide sequences (17) were analyzed and, through molecular docking, peptides with low binding energies for KEAP1 were identified, which might affect the NRF2-KEAP1 pathway. These findings suggest protein hydrolysates and antioxidant peptide derivatives might be used in functional foods.

Antioxidant and Antimicrobial Potential of Peptide from Rabbit Meat Hydrolysate Prepared by Trypsin and Zingibain.

&lt;b&gt;Background and Objective:&lt;/b&gt; Rabbit meat is a livestock product potentially viable as a protein source to obtain peptides. Antioxidant and antimicrobial peptides are ingredients extracted from various foods through enzymatic hydrolysis, chemical hydrolysis and fermentation to produce health-promoting foods. This research aims to investigate the potential of rabbit meat as a source of antioxidant and antimicrobial peptides through hydrolysis using trypsin and zingibain enzymes. &lt;b&gt;Materials and Methods:&lt;/b&gt; This research conducted an explorative-descriptive approach, focusing on antioxidant and antimicrobial activity. Rabbit meat was extracted using trypsin, zingibain and a combination of trypsin and crude extract zingibain. The hydrolyzed rabbit meat extract was tested at intervals of 0, 2, 6, 16, 24, 40 and 48 hrs to determine the degree of hydrolysis and the profile of hydrolyzed proteins with electrophoresis SDS PAGE. The antioxidant activity was tested using the DPPH method and the antimicrobial activity using agar well diffusion method. &lt;b&gt;Results:&lt;/b&gt; The degree of hydrolysis increased with the hydrolysis time. The highest protein content of rabbit meat extract hydrolyzed with trypsin was 287.65 mg/mL, observed during 12 hrs hydrolysis. The optimum conditions for the hydrolysis of rabbit meat protein were obtained at 24 hrs, with an IC&lt;sub&gt;50&lt;/sub&gt; value of 52.45% hydrolyzed by trypsin. As per antimicrobial activities, &lt;i&gt;Escherichia coli&lt;/i&gt; and &lt;i&gt;Salmonella&lt;/i&gt; sp. were more effective in inhibiting rabbit meat hydrolysates compared to &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; and &lt;i&gt;Staphylococcus aureus&lt;/i&gt;. The inhibition of all pathogen increased until 12 hrs hydrolysis but decreased in 24 hrs hydrolysis. &lt;b&gt;Conclusion:&lt;/b&gt; The combination zingibain enzyme and trypsin is feasible for hydrolyzing rabbit meat and the optimum hydrolysis time was 24 hrs with IC&lt;sub&gt;50&lt;/sub&gt; 52.45 ppm, although accompanied by reduction in antibacterial activities.

Impact of Time and Enzyme Concentration on Sangyod Rice Bran Hydrolysate: Phytochemicals, Antioxidants, Amino Acids, and Cytotoxicity

This study investigated the production of Sangyod rice bran hydrolysate (SYRB) from Sangyod rice, focusing on incubation times (1, 3, and 5 h) and alcalase enzyme concentrations (0, 0.7, and 1% v/v). The results demonstrated a concentration-dependent relationship: higher alcalase concentrations increased hydrolysate yield. Prolonged incubation, especially with alcalase, enhanced substrate breakdown, further increasing hydrolysate production. The degree of hydrolysis, reflecting peptide bond cleavage, depended on both incubation time and enzyme concentration, emphasizing the role of enzyme activity in efficiency. Moreover, color analysis (L*, a*, b*) and color difference (∆E) revealed intricate changes from enzymatic hydrolysis. Proximate composition analysis showed higher protein and lipid content with increased enzyme concentration and longer incubation times, whereas ash content varied with both factors. Hydrolysate powders exhibited higher moisture content than raw rice bran, indicating the impact of the hydrolysis process. The study also explored SYRB's antioxidant properties and cytotoxicity, which were sensitive to incubation time and alcalase concentration. Longer incubation increased DPPH scavenging activity, with the highest efficacy at 3 h. Meanwhile, ABTS scavenging displayed a delicate balance with alcalase concentration. The cytotoxicity study of SYRB revealed that all concentrations of SYRB were non-toxic to C2C12 cells, with cell viability values exceeding 70%.