Protease Extract Research Articles

New Pipeline for Analysing Fruit Proteolytic Products Used as Digestive Health Nutraceuticals

Proteolytic products are extensively used in the nutraceutical sector to improve protein digestion and muscle quality in target populations (e.g., athletes or elderly). These products are processed using techniques that often lead to low purity but competitive pricing. Despite their widespread use and well-established production methods, the industry lacks standardized analytical methods for assessing these products and detecting potential fraud. This study proposes a comprehensive and harmonized pipeline for their analysis, which includes quantifying total soluble protein and proteolytic activity, as well as the determination of product stability and protein profile using SDS-PAGE and proteomic techniques. Despite the fact that protease extracts from pineapple had the highest protein content, most of the bromelain remained inactive, unlike in kiwi and papaya. SDS-PAGE revealed partial protein degradation of pineapple extracts, whereas kiwi extracts reflected a lower purification level but a higher protein integrity. The application of proteomic approaches strengthened the identification and origin tracing of the proteases. This study contributes to the development of a robust framework for analyzing proteolytic extracts, spanning from soluble protein quantification to protein profiling and activity determination. It may also ensure reliable supplier selection, high-quality manufacturing practices, and the implementation of optimal storage and formulation strategies in the nutraceutical industry.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Screening of Bacillus spp. bacterial endophytes for protease production, and application in feather degradation and bio-detergent additive

Research on proteases and secondary metabolites from endophytes is an area that requires attention from researchers. In this study, proteases from Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 endophytes were characterised, and their potential biotechnological applications were investigated. Optimum protease production was achieved when isolates were grown in media containing (g/L): glucose 10g, casein 5g, yeast extract 5g, KH2PO4 2g, Na2CO3 10g at pH 9. The crude protease extracts were active in alkaline environments, thus referred to as alkaline proteases with optimal pH of 10. Additionally, Bacillus sp. strain MHSD 16 and Bacillus sp. strain MHSD17 proteases were active at high temperatures, with optimum enzyme activity at 50 °C. Thermostability profiles of these proteases showed that the enzymes were highly stable between (40–60 °C), maintaining over 85 % stability after 120 min incubation at 60 °C. Furthermore, the enzymes were stable and compatible with various household and laundry detergents. In the presence of commercial laundry detergent, OMO® 68 % and 72 % activity was retained for Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17, respectively, while 67 % and 68 % activity were retained in the presence of Sunlight®. The potential application for use in detergents was investigated through the removal of blood stains with the crude alkaline extracts displaying efficient stain removal abilities. Feather degradation was also investigated and Bacillus sp. MHSD17 exhibited feather keratin degrading properties more effectively than Bacillus sp. MHSD16.

Partial purification and characterization of protease extracted from kinema

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

Partial Purification, Characterization of Protease Extract from Ginger and its Effect on lipid profile and Liver Function

A ginger rhizome from the local market, Zingiber officinale var. Bentong, was used to extract protease. The activity of the protease was estimate during the extraction, the effects of pH and stabilizer were investigated. The best conditions were found to be a buffer of sodium phosphate, pH 7, 10 mM cysteine, and 5 mM EDTA as stabilizer. During the extraction process, 1% of crude ginger protease (GP) yield with a specific activity of 198 U/mg protein was generated. The protease functioned best at a pH of 7.0 and 60 °C, respectively. The effect of partially purified ginger protease on cholesterol (lipid profile) and liver function in mice was investigated.

AKTIVITAS PROTEASE EKSTRASELULER BAKTERI YANG DIISOLASI DARI LIMBAH CAIR INDUSTRI TAHU

Tofu waste that is discharged into the aquatic environment has a negative impact on the quality of the water around the tofu liquid waste disposal site. Industrial enzyme production is currently dominated by proteases. Proteases in the pharmaceutical field support the absorption of proteins in the digestive tract, as active cosmetic ingredients. This study aims to isolate, identify protease-producing bacteria from tofu wastewater, determine the total protein content and enzyme activity index of 5 isolates of protease-producing bacteria. Tofu wastewater bacteria were isolated with Skim Milk Agar medium. 5 bacterial isolates were selected which produced the widest clear zones, different colony characters, identified by morphological tests, gram staining tests, and biochemical tests. Five isolates were made into a bacterial suspension isolated from crude protease extract and the protease level was determined using the Bicinchoninic Acid method, and the protease activity was tested. Five bacterial colonies showed that colony 1 and colony 2 included Gram positive bacteria, colonies 3, 4 and 5 included Gram negative bacteria. The value of total protein content in the tofu industrial wastewater from the five colonies respectively: 219.93; 833,267; 421,267; 81,267; and 38.6 µg/µL. The highest proteolytic activity test was in sample 2 of 2.13.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Peptidomic Fingerprints of Stored UHT Milk Inoculated with Protease Extracts from Different Pseudomonas Strains Relative to aprX Expression and Visible Spoilage

Lately, concern about the protease AprX produced by Pseudomonas has increased in the dairy industry due to its ability to survive UHT treatment and spoil UHT milk. Efficient prediction methods for UHT milk spoilage are currently lacking, mainly due to high diversity in proteolytic potential between Pseudomonas strains. The present study aimed to gain more insight into the variability between Pseudomonas strains regarding proteolytic potential by comparing their proteolytic capability with their aprX expression levels and differences in peptide formation. The variability in aprX expression levels in four Pseudomonas strains were related to physical stability, milk proteolysis and peptidomic cleavage patterns of milk proteins in a storage experiment of UHT milk inoculated with protease extracellular extracts and stored for 45 days at 20 °C. A positive relationship was observed between the relative expression of aprX and milk proteolysis during storage, with the strain Pseudomonas panacis DSM 18529 showing the highest level in both parameters. This strain was the only strain to show visual gelation, which occurred after 21 days. The peptide formation analysis showed a similar protein hydrolysis pattern between strains and high hydrolysis of αs1-caseins during long-term spoilage putatively due to the activity of AprX was observed.

Enzymatic Characterization, Antioxidant and Antimicrobial Activities of Protease from Chickpea (Cicer arietinum L.) Seed and Leaf Extracts

Chickpea (Cicer arietinum L.) proteins are appreciated due to their high biological values, well balanced amino acid content and low content of anti-nutritional factors. The present study was undertaken to investigate enzymatic characterization, antioxidant and antimicrobial activities of protease extract from chickpea (Cicer arietinum L.). The chickpea seed and leaf samples collected from farmer’s field were used for protease enzyme extraction. The protein concentration was determined by Lowry method. Protease activity was assayed by using casein as a substrate. Optimization of the enzyme activity was made based on pH and temperature. The antioxidant activity was tested based on hydrogen peroxide, and DPPH free radical scavenging activities. The antimicrobial experiment was conducted based on disc diffusion and broth dilution methods. The result of crude protein extracts and protease activity has demonstrated that significantly the highest protein concentration (74.53%) and protease activity (22. 20U/ml) in seed supernatant solution. The effect pH on protease activity demonstrated the optimum pH for the protease extract was > pH6. The effect of temperature on protease activity demonstrated that the optimum temperature for protease activity was around 40oC. Significantly the highest free radical scavenging activity of diphenyl-1-picrylhydrazyl DPPH (40.65%) and HPSA (57.95%) were recorded for seed supernatant enzyme extract. The the strongest antibacterial activity with maximum zone of inhibition (22.35mm), minimum inhibitory concentration MIC (1.5µg/ml) and corresponding minimum bactericidal concentration MBC (2.5µg/ml) against S. aureus were recorded for leaf supernatant enzyme extract against Staphylococcus aureus. The highest dose of the enzyme extract (30µg/ml) revealed maximum antifungal activity with the highest zone of inhibition (18.25mm), MIC (1.5µg/ml) and corresponding minimum fungicidal concentration MFC (2µg/ml) was recorded for leaf supernatant against Candida albicans. The finding of the present study demonstrated that the supernatant enzyme extract has got higher biological activities including antioxidant and antimicrobial potentials.

Effects of cold atmospheric plasma on endogenous enzyme activity and muscle protein oxidation in Trachinotus ovatus

In this study, we investigated the effects of cold atmospheric plasma (CAP) technology on endogenous enzyme characteristics and muscle protein properties of the golden pomfret (Trachinotus ovatus) under different treatment power and time conditions. Results showed that the enzymatic activity of cathepsin B, L, and calpain in crude protease extracts (CPE) decreased significantly as the treatment power and treatment time of CAP increased (p < 0.05). Oxidative degradation of the CPE after exposure to CAP resulted in significant changes in the structure, total sulfhydryl, and carbonyl content of the CPE (p < 0.05). CAP of an appropriate intensity resulted in significant improvements in the color parameters, hydration properties, and textural property parameters of muscle proteins (p < 0.05). These results suggest that CAP, as a non-thermophysical modification technique, can inhibit the activity of endogenous enzymes as well as alter the protein function in food.

Proteolysis Degree of Protein Corona Affect Ultrasound-Induced Sublethal Effects on Saccharomyces cerevisiae: Transcriptomics Analysis and Adaptive Regulation of Membrane Homeostasis.

Protein corona (PC) adsorbed on the surface of nanoparticles brings new research perspectives on the interaction between nanoparticles and fermentative microorganisms. Herein, the proteolysis of wheat PC adsorbed on a nano-Se surface using cell-free protease extract from S. cerevisiae was conducted. The proteolysis caused monotonic changes of ζ-potentials and surface hydrophobicity of PC. Notably, the innermost PC layer was difficult to be proteolyzed. Furthermore, when S. cerevisiae was stimulated by ultrasound + 0.1 mg/mL nano-Se@PC, the proportion of lethal and sublethal injured cells increased as a function of the proteolysis time of PC. The transcriptomics analysis revealed that 34 differentially expressed genes which varied monotonically were related to the plasma membrane, fatty acid metabolism, glycerolipid metabolism, etc. Significant declines in the membrane potential and proton motive force disruption of membrane were found with the prolonged proteolysis time; meanwhile, higher membrane permeability, membrane oxidative stress levels, membrane lipid fluidity, and micro-viscosity were triggered.

Commercial Extraction of Applicable Alkaline Protease and Chitinase by Bacillus thuringiensis dendrolimus IP 4A/4B

Objective: The study aims to produce alkaline protease and chitinase in the same fermentation process in the most economical way possible so as to reduce production costs and to investigate the application of alkaline protease in detergents compatibility. Methods: Inoculums were prepared in nutrient yeast-extract standard medium, grown, and extracted from the solid medium, while the enzyme activities were measured using Lowry protein assay and high-performance liquid chromatography. Results: The culture of Bacillus thuringiensis subspecies dendrolimus IP 4A/4B was performed under solid-state fermentation conditions. The efficiency and performance of extraction using tap water outperformed other solutions. The crude protease enzyme was compatible with some detergents available in the local market, such as Persil, Tide, and Ariel, where the crude enzyme retained its activity at rates that varied between 113%, 110% and 83%. No microbial growth and therefore no chitinase enzymatic activity was observed on the first and second days, while microbial growth and chitinase activity commenced on the third day and reached the peak of the curve on the fourth day and stabilized on the sixth day. The productivity of chitinase in solid-state was superior to submerged fermentation conditions, which were 2.050 and 0.693U (μmole/mL/min). Conclusion: Production of alkaline protease by Bacillus thuringiensis dendrolimus IP/4B is efficient using tap water for extraction. The alkaline protease is compatible with (Arial, Tide and Persil) detergents, and the same bacterial strain also produced chitinase.

Statistical improvement of protease production from a new isolate Bacillus thuringiensis strain-MA8 and its application in the production of enzyme-modified cheese

Enzyme-modified cheese (EMC) is a concentrated cheese flavor that is produced enzymatically from dairy substrates to provide an intense source of cheese flavor with broad applications. In this study, EMC was produced by enzymatic biotransformation from a new bacterial isolate described and molecularly identified as Bacillus thuringiensis strain-MA8. Optimization of protease production conditions using one-variable-at-a-time followed by multi-factorial (Plackett-Burman and Box-Behnken) designs increased production by 7-fold. Protease was used at different concentrations (300 and 900 U/100 g curd) as a cost-effective source of concentrated cheese flavor in the EMC preparation. Sensorial evaluation of EMC revealed that the overall acceptability, flavor, and texture were improved from the 2nd day compared to the control, and then decreased on the 4th day without any apparent bitterness. The chemical characteristics of EMC showed that the addition of protease extracts increased the total volatile fatty acids, water-soluble nitrogen, and acidity of EMC significantly (p≤0.05) compared to the control. The amino acids profile revealed that EMC1 which was treated with (300 U/100 g curd) protease had the highest essential amino acids (EAA) and EAA/total amino acids ratio. Nutritional parameters including protein efficiency ratio, biological value, and chemical score of EMC were higher than control based on Val, Met + Cys, Ile, Leu, and Phe + Tyr amino acids. Also, Scanning Electron Microscopy showed significant changes in EMC compared to the control. In conclusion, the addition of (300 U/100g curd) of protease revealed good EMC characteristics without any apparent defect.

PURIFICATION AND PARTIAL CHARACTERISATION OF A PROTEASE INHIBITOR FROM Mimosa diplotricha

Search for inhibitors to insect proteases is one of many strategies to control pests. Previous work has demonstrated successful purification of effective inhibitors from plant origin. Thus, the current study attempted to purify protease inhibitors from locally available medicinal plants. The study demonstrated that the ethanolic extracts of Mimosa diplotricha leaves caused a significant 80% reduction in bovine trypsin activity. The inhibitory property of the proteinaceous nature of the extract was reconfirmed through qualitative analysis using the detection of trypsin inhibitors on the SDS-PAGE technique. The ammonium precipitated trypsin inhibitor was purified using Hi-Trap G25 and resolved into a single band with a molecular weight of approximately 20.8 kDa. By using the Dixon plot the competitive inhibitor has a Ki value of 2.16 × 10-4 mM. The purified protein inhibited the protease extract of Chrysomya megacephala at IC50 of 28 μg/mL. The results highlighted the presence of trypsin inhibitor in Mimosa diplotricha and its potential as a pest control agent.

Amino acid sequence of two new milk-clotting proteases from the macroalga Gracilaria edulis

This study is aimed at identifying and characterising the proteases we previously extracted from the red seaweed Gracilaria edulis with the potential as milk-clotting enzymes. The protease extract was first analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Two protease bands with a molecular weight of 44 and 108 kDa were identified, and analysed using in-gel digestion and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS). Eight peptides from the LC-MS/MS analysis matched those in existing protein databases but they were not related to any protease of the genera Gracilaria and Hydropuntia. Further analysis revealed that more than 80% of the peptide sequence of the algal proteases matched with those from members of the bacteria kingdom, including Gallaecimonas and Alteromonas. Among these, twelve matching homolog proteases were identified as metalloprotease and serine protease. The results indicated that the algal proteases have a close relationship with both algae and bacteria, and suggest that the proteases might have resulted from past bacterial colonisation of the algae and subsequent horizontal gene transfer between bacteria and algae.

Production, extraction and characterization of a serine protease with fibrinolytic, fibrinogenolytic and thrombolytic activity obtained by Paenibacillus graminis

This study provides optimized parameters for producing and purifying a microbial serine protease by Paenibacillus graminis in submerged fermentation and the innovative use of endosperm of Gliricidia sepium as substrate. Biphasic aqueous systems were the method of choice for the partial purification of this protease. After extracting the enzyme, its biochemical characterization and fibrinolytic, fibrinogenolytic and thrombolytic activity were performed. The highest protease activity (16.25 U/mL) was obtained in the PEG-rich phase in the composing assay of MPEG (8000 g/mol), CPEG (12.5 g/mol), CPHO (10%) and pH 8.0 (optimum pH). Recoveries greater than 80% were obtained with a purification factor of 5.21, a partition coefficient of 1.13 and a yield of 145.52%. The fibrinolytic protease (50 kDa) purified achieved a 46.96% reduction of thrombus in vitro and managed to fully degrade the Bβ-chain of human fibrinogen and the γ-chain of bovine fibrinogen. The enzyme activity was enhanced by Cu2+, Zn2+, Na+, Ca2+ and K+. These results suggest that the protease purified by ATPS can potentially be used as a future agent thrombolytic.

Variations in active proteases of Blastocystis sp. obtained from water and animal isolates from the Philippines.

Blastocystis sp. is a commonly encountered gut protozoan with unclear pathogenicity. The presence of proteases in this organism may be related to its potential pathogenicity or other physiological differences. This study aimed to identify the various proteases that may be present in different subtypes (STs) of Blastocystis sp. using azocasein assay and gelatin zymography. In this study, cysteine, serine, aspartic protease, metalloproteases, and unknown proteases were identified in Blastocystis sp. cultures obtained from animal and water samples belonging to ST1-ST5 and ST7. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Cysteine protease was the most commonly encountered. Protease activity in general is not associated with ST or source (animal or water). However, the presence of cysteine protease alone was associated with animal samples, whereas the presence of more than two protease types was associated with water samples. Azocasein assay and gelatin zymography were conducted on different batches of protease extracts, which showed varying results. Protease activity and the types present may change over time. Blastocystis sp. shows high protease diversity, including possible novel types.

Extraction of protease from ora‐pro‐nobis ( Pereskia aculeata Miller) and partial purification in polyethylene glycol + sodium phosphate aqueous two‐phase system

This article studies the influence of pH and extraction solution, time and solid/liquid ratio on the extraction of protease from ora-pro-nobis. Aqueous two-phase systems (ATPS) formed by polyethylene glycol 1500 or 4000 and phosphate was investigated as a purification step for these proteases. The best results were with sodium phosphate pH 7.0, 60 min of extraction, and at the liquid/solid ratio 21 mL/g. ATPS formed by polyethylene glycol and phosphate were obtained. The increase in molar mass resulted in the expansion of the biphasic region, exclusion volume, and the free volume of the upper phase. The proteases were preferentially partitioned to the upper phase. The free energy indicates that the partition of proteases to the upper phase is a spontaneous process. High selectivity, activity recovery, and the purification factor indicate that the studied systems can be used as a good alternative for purification of this enzyme.

Optimization of spray-drying conditions for obtaining Bacillus sp. SMIA-2 protease powder

Microbial proteases, especially from Bacillus spp., have enormously been exploited for a broad variety of applications such as for physiological processes, food and feedstuff, detergents, as well as in the pharmaceutical and leather. In this work, proteases produced by the thermophilic Bacillus sp. SMIA-2 submerged cultures were spray-dried in an attempt to improve its stability for applications in industry. The enzymatic extract was dried using drying adjuvants, and optimal conditions for preserving enzymatic activity were studied following a statistical experimental design. The spray process factors studied were the drier air inlet temperature and the adjuvants concentrations. The responses analyzed were the enzymatic activity and mass recovered of the powder after spray drying. Additionally, the stability of the powder was assessed during 180 days at room temperature. The results revealed that satisfactory levels of enzymatic activity were obtained when 0.5% (w/v) carboxymethylcellulose and 1.0% (w/v) maltodextrin were incorporated to enzymes solutions and the spray drier inlet temperature was 110 ºC. Furthermore, this dried protease extracts showed potential for future commercial applications because of their stability at room temperature for 180 days.

Functional properties ofOryctes rhinoceros larvae protein hydrolysates affected by different enzyme concentration and hydrolysis time

Oryctes rhinoceros larva has the potential to be developed into protein hydrolysates. In this study,O. rhinoceros larva was hydrolysed with crude papain protease extract at different concentrations (1, 2, and 3% w/w) and hydrolysis time (60, 120, 180 min). The objective of this study was to investigate the functional properties of protein hydrolysates fromO. rhinoceros larva affected by different papain concentrations and hydrolysis time. Yield, protein concentration, and functional properties were measured and compared with whey protein. The result showed that the yield and protein concentration increased significantly (P&lt;0.05) with increasing papain concentration. The water holding capacity varied between 1.66-4.23 g/g and OHC varied in the range of 2.61-5.24 g/g, higher than control (P&lt;0.05).O. rhinoceros protein hydrolysate has good emulsifying activity and stability, ranging from 8.38 to 45.88 m2/g and 12.09 to 30.87 min, respectively, while foam capacity ranged from 21.11% to 59.78% and foaming stability up to 25.11%. Generally, the functional properties ofO. rhinoceros larva protein hydrolysate improved with the increase of the crude papain concentration and hydrolysis time. Several improved functional properties showed potential for developingO. rhinoceros larva protein hydrolysates as an alternative functional protein source in food ingredient formulation.