Protease Preparation Research Articles

The Effect of Various Domestically Produced Proteolytic Enzyme Preparations on the Organoleptic Properties of Pea Protein Isolates

The Effect of Various Domestically Produced Proteolytic Enzyme Preparations on the Organoleptic Properties of Pea Protein Isolates

Nanobiocatalyst based on covalent immobilization of proteases onto functionalized nanocellulose for efficient production of sunflower meal protein hydrolysates

This study aimed to engineer bio-based and environmentally friendly nanobiocatalyst systems using commercially available protease preparations, Alcalase® 2.4L (Alcalase) and Flavourzyme® (Flavourzyme), along with functionalized cellulose nanocrystals (CNC). The study highlights the advantageous properties of the obtained glutaraldehyde-activated amino-modified CNC support (GA-amino-CNC), characterized as nanorods with dimensions ranging between 100-800 nm in length and 40-60 nm in width, particularly for protease immobilization. Maximum specific activity of immobilized Alcalase (15 IU/mg of proteins) was achieved at an initial enzyme concentration of 52 mg/g of support after 3 hours, with 80% of covalently attached enzyme molecules. In the case of Flavourzyme, maximum exo-hydrolytic activity of 21 L-pNAU/g of support was observed after 20 min, while a higher yield of covalently bound enzyme molecules (53%) detected after 1 hour. The covalently bound proteases on glutaraldehyde-activated amino-modified cellulose nanocrystals were evaluated for their efficacy in producing high-quality sunflower-based protein hydrolysates suitable for food and feed applications. Sequential hydrolysis of sunflower meal protein isolate (SMPI) using the developed nanobiocatalysts yielded a maximum hydrolysis degree of 20%. In addition, more than 80% of the initial activity of nanobiocatalysts were retained after 3 reuses, suggesting that these protease nanobiocatalysts have potential application in production of SMP hydrolysates.

Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

Acute Microbial Protease Supplementation Increases Net Postprandial Plasma Amino Acid Concentrations After Pea Protein Ingestion in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Trial

BackgroundDigestibility is a primary factor in determining the quality of dietary protein. Microbial protease supplementation may be a strategy for improving protein digestion and subsequent postprandial plasma amino acid availability. ObjectivesTo assess the effect of co-ingesting a microbial protease mixture with pea protein on postprandial plasma amino acid concentrations. DesignA mixture of 3 microbial protease preparations (P3) was tested for proteolytic efficacy in an in vitro static simulation of gastrointestinal digestion. Subsequently, in a randomized, double-blind, placebo-controlled crossover trial, 24 healthy adults (27 ± 4 y; 12 females, 12 males) ingested 25 g pea protein isolate (20 g protein, 2.2 g fat) with either P3 or maltodextrin placebo (PLA). Blood samples were collected at baseline and throughout a 0‒5 h postprandial period and both the early (0–2 h) iAUC and total (0–5 h) iAUC were examined. ResultsPlasma glucose concentrations decreased in both conditions (P < 0.001), with higher concentrations after P3 ingestion compared with PLA (P < 0.001). Plasma insulin concentrations increased for both conditions (P < 0.001) with no difference between conditions (P = 0.331). Plasma total amino acid (TAA) concentrations increased over time (P < 0.001) with higher concentrations observed for P3 compared with PLA (P = 0.010) during the 0‒5 h period. There was a trend for elevated essential amino acid (EAA) concentrations for P3 compared with PLA (P = 0.099) during the 0‒5 h postprandial period but not for leucine (P = 0.282) or branched-chain amino acids (BCAA, P = 0.410). The early net exposure (0‒2 h iAUC) to amino acids (leucine, BCAA, EAA, and TAA) was higher for P3 compared with PLA (all, P < 0.05). ConclusionsMicrobial protease co-ingestion increases plasma TAA concentrations (0–5 h) and leucine, BCAA, EAA, and TAA availability in the early postprandial period (0‒2 h) compared with ingesting pea protein with placebo in healthy adults.

Obtaining a food coating with bioprotective properties

In this study, the food coating's bioprotective properties were investigated. Currently, in the food industry, special attention is paid to the creation of fundamentally new packaging materials – non–toxic, capable of providing effective protection of the product from microbial contamination, exposure to oxygen in the air, to prevent the drying of the product during storage - including those applied to the surface of the product and eaten with it. The use of protective coatings in the production of meat semi-finished products allows you to improve the quality and appearance, and also allow you to preserve the freshness of meat semi-finished products. The aim of the work is to obtain a food coating with bioprotective properties and extracts of biologically active substances of plant raw materials. As the objects the objects of the study were used secondary collagen-containing raw materials of the meat industry (veins, tendons, fasciae released during the venation of beef in sausage and canning industries), dispersed forms of collagen proteins isolated from animal tissues by directed biomodification under the action of proteolytic enzyme preparations, CO2 extracts of medicinal plants and spices, and composite bases with their individual and sharing, obtained in the laboratory of the Department of Technology of Animal Products of the Voronezh State University of Engineering Technologies and in the research institute of Food safety of the Almaty Technological University. The choice of CO2 extracts of plant raw materials as sources of natural antioxidants is due to their more pronounced antioxidant activity compared to extracts obtained on the basis of classical methods. The inclusion of antioxidants in the composition of food films increases the shelf life of the product, protecting it from oxidation and color change.

Optimisation of low temperature (8ᵒC) enzymatic hydrolysis of acid whey using design of experiments (DOE) for the generation of thermally stable whey protein hydrolysates

The impact of enzymatic hydrolysis at 8ᵒC of liquid whey protein concentrate (WPC35) derived from acid casein manufacture using Prolyve 1000®, a microbial protease preparation, under free-fall pH conditions on the physicochemical and the thermal stability properties of the resultant hydrolysates was investigated. The hydrolysis process was optimised using a design of experiments (DOE) approach. Eleven hydrolysates (H1–H11) were generated using 3 factors x 2 levels, i.e., enzyme:substrate (E:S) 0.25–1.00% (v/w), starting pH 7.5–9.0 and incubation time 10–96 h. Hydrolysate degree of hydrolysis (DH) values ranged from 0.75 (H10) to 4.74% (H6) which increased to 1.91 (H10) and 7.24% (H6) following subsequent thermal processing (mimicking the evaporation process) at 54ᵒC x 15 min and 64ᵒC x 10 min. The apparent viscosity (ηapp) of all hydrolysates was measured to assess their heat stability during heating at 85ᵒC for up to 20 min, this was shown to be E:S and pH-dependent. While the unhydrolysed samples formed a gel on heating at ∼74-80ᵒC. Following DOE analysis, it was found that E:S had a significant impact on the DH, the extent of intact protein degradation (%Deg) and the thermal stability. The %Deg following processing at 64ᵒC showed a strong correlation (R2 = 0.924; p < 0.001) with ηapp, and thus this parameter may be used to predict the thermal stability of the hydrolysate samples. The optimised hydrolysis conditions for the generation of heat stable WPC35 hydrolysates during 8°C incubation were achieved using an E:S = 0.625% at a starting pH = 8.65. Performance of protein hydrolysis reactions at low temperature has potential to enhance process sustainability.

Antifungal peptides from faba bean flour fermented by Levilactobacillus brevis AM7 improve the shelf-life of composite faba-wheat bread

Faba bean is an important crop from an ecological, nutritional, and economical perspective, hence its incorporation in plant-based products can play a key role in the sustainability of the whole food system. In this study, the antifungal properties of faba bean flour were investigated employing three bioprocessing treatments including hydrolysis with a commercial proteolytic enzyme preparation, and fermentation with selected or commercial mixed sourdough starters. Faba bean flour fermented by Levilactobacillus brevis AM7 showed the broadest inhibitory spectrum against the fungal species tested and antifungal activity ranging from 25 to 73 %. Bioactive proteins and peptides were found to be responsible for the antifungal activity. Polypeptides sequences corresponding to defensin-like and non-specific lipid-transfer proteins and seven antifungal peptides having 11–22 amino acid residues and mass from 1240.7 to 2624.2 Da, were identified via Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra (nano-LC-ESI-MS/MS). To assess the antifungal effect in food processing conditions, fermented faba bean was used as ingredient in breadmaking at a substitution level of 30 % of the dough weight. Wheat composite-bread containing faba bean sourdough, which could be claimed as “source of proteins”, according to the current EC Regulation, delayed the molds contamination, lasting up to 10 days before mold appearance, and behaving similarly to wheat bread containing calcium propionate.

Modeling the Biocatalytic Method of Lipid Extraction Using Artificial Neural Networks

The paper presents the data on lipid fraction extraction from the raw fat of hibernating hunting animals. The processing of valuable raw materials must be maximized. For this purpose, various methods of rendering are used. As a result of temperature exposure, the protein part of raw fat undergoes significant changes. The protein denatures under the influence of temperature, and the dross formed during the rendering process absorbs and retains up to 30% of the fat. The authors propose using proteolytic enzyme preparations for a more complete extraction of fats, as the enzymes will hydrolyze the protein into compounds of lower molecular weight both before and during the rendering process. The experiment proved that the biocatalytic method allows achieving a fat yield of more than 95%. The best result can be obtained if the rendering is carried out at optimal parameters, which can be defined using a mathematical model. Mathematical modeling was carried out using an artificial neural network. During the study, a fully connected neural network was designed; it had eight hidden layers with 64 neurons in each, and its accuracy was measured by mean relative error, which amounted to 5.16%. With the help of the network, the optimal values of applied concentration, temperature and duration of rendering, at which a fat yield of more than 98% is achieved, were determined for each enzyme preparation. After that, the obtained values were confirmed experimentally. Thus, the study showed the efficiency of using artificial neural networks for modeling the biocatalytic method of lipid extraction.

An eco-friendly approach of biodiesel production from tannery fleshing wastes by crude neutral protease enzyme

A new approach for extracting fat from tannery fleshing wastes (TFW) was established by hydrolyzing in water using a crude neutral protease enzyme. The bacterial strain for protease preparation was isolated from the waste dumping site of Institute of Leather Engineering and Technology of the University of Dhaka. The crude enzyme thus obtained was found highly active at 55 °C and pH 8.0, with a substrate concentration of 2%. Several reaction parameters, such as enzyme concentration, hydrolysis temperature and duration, were optimized using response surface methodology (RSM). The detailed analysis data by RSM showed 31.33% enzyme to dry weight of fleshing, 2.874 h of hydrolysis, and at 65.23 °C temperature to be the optimum for maximum theoretical fat yield of 38.46%. The laboratory experiments under optimum conditions resulted 34.9% fat yield with an error of 10.17%. The fleshing fat was transesterified in methanol into biodiesel using a recyclable magnetic nano catalyst with a 98% conversion and glycerol as a byproduct. The FT-IR spectroscopic results demonstrated an absorption of CO stretching at 1740 cm−1 for α, β-unsaturated esters in the biodiesel and its fatty acid ethyl ester. The 1H-NMR spectroscopy revealed a singlet peak of methoxy protons at 3.65 ppm and a triplet at 2.26–2.30 ppm for α-CH2 protons that confirmed the presence of unsaturated fatty acids with a carbonyl hydrogen double bond peak at 2.00–2.01 ppm. The GC-MS data of the biodiesel comprises of ethyl E−11-hexa-decanoate, ethyl myristate, palmitate, stearate, and decanoate. The results of various fuel parameters of the biodiesel were measured and compared with standard biodiesel EN14214 which revealed that it has comparable fuel properties to the standard. The higher calorific value (38.5 MJ kg−1) and flash point (152 °C) of the biodiesel than the standard, indicating its high potential and ease of storage stability than standard biodiesel.

Вплив параметрів ферментативної обробки на вихід пресової гарбузової олії та її властивості

The influence of parameters of pumpkin seeds pretreatment with enzyme preparations of proteolytic and cellulolytic activity on the yield of press oil, its composition and quality parameters were studied in this work. The enzyme preparation PENICILOPEPSIN (Enzym Biotech, Ukraine) with proteolytic activity and CELLULAD (Enzym Biotech, Ukraine) with cellulolytic, hemicellulase and xylanase activity were used for pretreatment. Enzymatic treatment of seeds was carried out with a mixture of these preparations at a ratio of 7:3 for 2 h at a pH 5.2 and 48—54°C. The following parameters of enzymatic pretreatment were studied: the amount of the enzyme mixture, which varied from 0.3% to 2.4% of the weight of the seeds and the amount of moisture added together with the enzymes — from 15% to 50%. It was established that the rational parameters of the enzymatic treatment of pumpkin seeds are the mass of the enzyme mixture of 0.6% and the amount of moisture added with enzymes — 35% of the seed mass. Under such technological parameters, the yield of press pumpkin oil was 65.6%, which is almost 6% higher than in the control sample. The acid and anisidine value of the control oil sample and the oil extracted after the enzymatic treatment of the seeds did not differ significantly. However, the content of peroxide compounds in the oil extracted after enzymatic treatment of seeds was lower. The total antioxidant activity of the oil, determined by the reaction of the 2,2-diphenyl-1- picrylhydrazyl radicals quenching within 30 min, was higher in the oil samples after seed pretreatment at the maximum tested moisture (50%). The obtained results indicate that the enzymatic pretreatment of pumpkin seeds with proteolytic and cellulolytic enzyme preparations is a promising method of oil yield increasing.

In silico prediction of the inhibitory effect of phytochemical components extracted from Knautia sarajevensis on the main protease of SARS-CoV-2 virus

Essential role in replication and transcription of coronavirus makes the main protease of SARS-CoV-2 a great traget for drug design. The aim of this study was to predict structural interactions of compounds isolated from the Bosnian-Herzegovinian endemic plant Knautia sarajevensis (G. Beck) Szabó against the 3CLpro of SARS-CoV-2 virus. The three-dimensional crystal structure of SARS-CoV-2 main protease was retrieved from the RCSB Protein Data Bank and the three-dimensional structures of isolated compounds were obtained from the PubChem database. Active site was predicted using PrankWeb, while the preparation of protease and compounds was performed using AutoDock Tools and OpenBabel. Molecular docking was carried out using AutoDock Vina. Structural interactions are visualised and analyzed using PyMOL, LigPlus and UCSF Chimera. Apigenin, kaempferol, myricetin and quercetin showed the highest binding affinity for SARS-CoV-2 main protease and formed significant hydrogen bonds with the given protein. Results obtained in this study are in accordance with previous studies and showed that these compounds could potentially have antiviral effects against SARS-CoV-2. These findings indicate that K. sarajevensis could be potentially utilized as an adjuvant in the treatment of coronavirus disease 2019, but further pharmacological studies are required in order to prove the potential medicinal use of the plant.

Use of fungal and bacterial protease preparations to enhance extraction of lipid from fish roe: Effect on lipidomic profile of extracted oil.

The present study investigated the hydrolysis of protein in hoki roe homogenate using a HT (bacterial), a FP-II (fungal) protease preparations and Alcalase (bacterial) to enhance lipid yield extraction. The degree of hydrolysis was determined at various pH, temperature and time using casein and hoki roe. Total lipid extraction and lipidomic analysis was carried out following proteolysis of hoki roe homogenate. The degree of hydrolysis and SDS-PAGE revealed that the hydrolytic capability of Alcalase was better than HT and FPII. The total extracted lipid yield was better following hydrolysis with Alcalase (19.29%), compared to HT (18.29%) and FPII (18.33%). However, the total phospholipid (PL) and n-3 fatty acid yields were better from HT hydrolysed hoki roe homogenate (PL=30.7μmol/g; n-3=10.5%), compared to Alcalase (PL=22μmol/g; n-3=5.95%). Overall, this study indicates that HT protease preparation hydrolysis of fish roe homogenate can both enhance lipid extraction and retain lipid quality.

Optimal concentration of proteolytic enzyme preparation for meat processing

AnnotationResearch work has been carried out on the effect of various concentrations of an enzyme preparation (EP) on some indicators of salted camel meat. The proteolytic activity, pH and temperature optimum of the enzyme protosubtilin G20x were studied. The effect of protosubtilin G20x on VVS, shear stress, consistency and yield after boiling of salted camel meat samples was studied. This research work indicates that the treatment of meat with an enzyme preparation causes moisture-binding capacity (WCC), with this there are significant changes in the consistency and mass of the samples after heat treatment.

Fluorescence dequenching assay for the activity of TEV protease

Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.

Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.

Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bβ, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague–Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.

Physical-mathematical model to predict the kinetic coagulation process by clotting activity of bacterial endopeptidases

A physical-mathematical model was used to evaluate the capability of an enzymatic pool of Bacillus sp. P7 (isolated from Piaractus mesopotamicus) to promote the bovine casein micelles coagulation. Experiments were designed to assess the effects of temperature, pH, and enzyme activity/mass of substrate ratio on the kinetic parameters of the coagulation process and the microstructure of the obtained clots. Descriptive and predictive equations indicate that the temperature and the pH modified these parameters significantly. In optimal conditions, the clot’s mean pore size was 3.6 times smaller using chymosin. On the other hand, rheological measurements evidence a moderate elasticity of clot, which indicates the usefulness of P7 protease preparation as a clotting agent in spreadable or soft cheese manufacture. Also, the hydrolysis products, which are in the whey after casein micelles coagulation, demonstrated antioxidant activities. Equations to model and predict the process kinetics were combined with rheological and microstructure analyses of the obtained clots, and whey bioactivities were evaluated. Nevertheless, the use of P7PP requires further investigation concerning the stability of the enzyme preparation during storage, its performance, and how these variables could be related to the proposed models.

Resource Utilization of Bovine Neck Ligament: Enzymatic Preparation of Elastin Peptide and Its Antioxidant Activity.

Elastin is considered an excellent resource for obtaining antioxidant peptides due to unique amino acid composition. However, it is hardly soluble in water or in dilute acid or alkali; most of the elastases have low yields for preparing elastin peptides, making it difficult to meet industrial applications. To address above problems, enzymes capable of hydrolyzing elastin into soluble peptides were preferred from typical commercial protease preparations. The optimal enzymatic hydrolysis process conditions for elastin peptides were obtained by response surface optimization design. The molecular weight, amino acid composition, and antioxidant activity of the enzymatic hydrolysis products were determined. The results show that the alkaline protease NUE has a strong hydrolysis effect. The optimized enzymatic hydrolysis conditions are as follows: substrate concentration is 5%, enzyme concentration is 650 U/mL, pH is 10.0, temperature is 60°C, time is 6h. The degree of hydrolysis of elastic protein peptides obtained through this method is 14.42%. The distribution of molecular weight is 200-6500Da, more than 85% of the component molecular amount is greater than 800Da; the amino acid content related to antioxidant activity has reached 68mg/100mg, so it has extremely high free radical clearance. Compared with acid and alkali methods, the anti-oxidation capacity of enzyme-based peptide is better, the reaction conditions are milder, the yield is higher, and by-products and pollutants are fewer. It provides an effective way to industrialized production of elastin peptides with high antioxidant activity and a basis for its widespread application in the food and pharmaceutical industries.

Whey proteins hydrolysis using Alcalase and Flavourzyme

One of the ways to solve the problem of whey utilization is to obtain enzymatic hydrolysates with bioactive properties and reduced antigenicity. A wide range of enzyme preparations are used for whey hydrolysis. The aim of the study was to select the conditions for enzymatic processing of whey protein concentrate to provide the most complete hydrolysis of all basic whey proteins using the most popular proteolytic enzyme preparations for the food industry – Alcalase and Flavourzyme. It was found that the completeness of whey proteins hydrolysis, determined by the yield of soluble and low molecular weight protein, as well as by the disappearance of protein bands on electrophoregrams of the obtained hydrolysates, largely depends on the enzyme preparations dosages and preliminary heat treatment of the substrate. Combined hydrolysis of the pretreated whey protein concentrate with 1% Alcalase and 2% Flavourzyme for 20.5 h at 50°C with stirring allows one to convert about 70% of whey proteins to a stable soluble state, not denatured by heating at 90°C, and 50% of the hydrolyzed protein has a molecular weight of less than 10 kDa. This treatment provides the elimination of the main antigenic whey proteins and obtaining the hydrolysates with a degree of hydrolysis of 36%, determined using the formol titration method.

Preparation and identification of dipeptidyl peptidase IV inhibitory peptides from quinoa protein

The objective of this study was to investigate the dipeptidyl peptidase IV (DPP-IV) inhibitory properties of quinoa protein-derived peptides. After germination, quinoa protein was extracted and then hydrolyzed by different enzymes such as papain, Alcalase, Neutrase, and Flavourzyme and pepsin-trypsin digestion. Results showed that the quinoa protein hydrolysates (QPH) by pepsin-trypsin digestion displayed the highest DPP-IV inhibitory activity. When ultrafiltrated, the fraction of quinoa protein hydrolysate with molecular weight less than 1 kDa (QPH1) exhibited a superior DPP-IV inhibitory activity with IC50 of 3.40 ± 0.20 mg/mL in vitro and 2.20 ± 0.29 mg/mL in Caco-2 based in situ. Furthermore, the peptide sequences of QPH1 were identified by UPLC-MS/MS. Twenty quinoa-derived peptides were determined with in vitro DPP-IV inhibitory activities with IC50 values less than 500 μM. The peptides IPI, IPV, VAYPL and IPIN showed the highest in vitro DPP-IV inhibitory activities with IC50 of 5.25 ± 0.16, 26.15 ± 0.58, 42.93 ± 1.15, and 56.58 ± 3.36 μM, respectively. The in situ DPP-IV activities of Caco-2 cells were also attenuated by these four peptides with IC50 of 10.75 ± 0.87, 29.11 ± 1.79, 61.9 ± 4.23, and 92.59 ± 12.89 µM, respectively. Moreover, these four peptides were identified as competitive inhibitors of DPP-IV. Molecular docking showed that quinoa peptides IPI and IPV were predicted to form multiple hydrogen bonds, attractive charge, and hydrophobic interactions with the residues of active site of DPP-IV. This study confirms that quinoa protein is a good source for DPP-IV inhibitory peptides and has potential as ingredients in functional foods for the prevention or management of type 2 diabetes.