Enzymatic Hydrolysis Of Whey Protein Research Articles

Ultrasonic monitoring of enzymatic hydrolysis of proteins. 2. Relaxation effects

The paper investigates the occurrence of relaxation processes in enzymatic hydrolysis of whey proteins in phosphate buffer using high-resolution ultrasonic spectroscopy. The hydrolysis produces a frequency-dependent evolution of ultrasonic velocity and attenuation originated by the proton transfer between the terminal group of protein hydrolysates and the phosphate ions. Such frequency dependence is absent in other systems, e.g., Tris buffer. Real-time profiles of ultrasonic velocity and attenuation measured during the hydrolysis of β–lactoglobulin, α–lactalbumin and bovine serum albumin by α–chymotrypsin were employed in quantitative analysis of the relaxation mechanisms, relaxation times, determination of the proton transfer rate constants and the reaction volume. The proton transfer is characterised by the rate constant 9.4 x 107 L mol-1 s-1 and the adiabatic reaction volume 24 x 10-6 m3 mol-1. The obtained reaction volume agrees well with thermodynamic data for ionisation volumes and enthalpies of the terminal amino group and phosphate. The described methodology for estimating the relaxation contribution to ultrasonic characteristics allows for precision real-time ultrasonic measurements of the concentration of peptide bonds cleaved during protein hydrolysis in a broad range of environmental conditions.

Enzymatic hydrolysis of whey proteins by the combination of Alcalase and Neutrase: Kinetic model and hydrolysis control

A combination of Alcalase and Neutrase was used to hydrolyse whey proteins and the hydrolysis kinetics mechanism was studied to achieve accurate control of the hydrolysis process. By measuring the degree of hydrolysis (DH) at different enzyme concentrations and different substrate concentrations, the hydrolysis kinetic model was established as: DH=6.536Ln[1+(4.687E0S0–0.0007)t]. The results showed that the theoretical and actual values of the model were in good agreement. Subsequently, the molecular mass distribution of the whey proteins hydrolysates at different DH was determined; it was found that the molecular mass components of 5–20 and >20 kDa gradually decreased with the increase of DH, while the percentage of <1 kDa gradually increased. Extensive hydrolysis of whey proteins was achieved when the DH reached 21–25%. The establishment of kinetic model has theoretical guidance for the accurate control of the whey proteins hydrolysis by the combination of Alcalase and Neutrase.

Transportation of whey protein-derived peptides using Caco-2 cell model and identification of novel cholesterol-lowering peptides.

The increasing morbidity and mortality of cardiovascular disease have become a major factor in human death. Serum cholesterol is considered to be an important risk factor for inducing coronary heart disease, atherosclerosis and other cardiovascular diseases. To screen intestinal absorbable functional small peptides with cholesterol-lowering activity by enzymatic hydrolysis of whey protein and develop cholesterol-based functional food that may become a substitute for chemically synthesized drugs, providing new ideas for diseases caused by high cholesterol. This study aimed to evaluate the cholesterol-lowering activity of intestinal absorbable whey protein-derived peptides hydrolyzed by alkaline protease, trypsin and chymotrypsin, respectively. The whey protein hydrolysates acquired by enzymatic hydrolysis under optimal conditions were purified by a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The fractions obtained by Sephadex G-10 gel filtration chromatography were transported through a Caco-2 cell monolayer. The transported peptides were detected in the basolateral aspect of Caco-2 cell monolayers using ultra- performance liquid chromatography-tandem mass spectrometry (UPLC-MS). His-Thr-Ser-Gly-Tyr (HTSGY), Ala-Val-Phe-Lys (AVFK) and Ala-Leu-Pro-Met (ALPM) were unreported peptides with cholesterol-lowering activity. The cholesterol-lowering activities of the three peptides did not change significantly during simulated gastrointestinal digestion. This study not only provides theoretical support for the development of bioactive peptides that can be directly absorbed by the human body, but also provides new treatment ideas for hypercholesterolemia.

Understanding the Factors Influencing the Ability of Calcium-Binding Peptides to Promote Calcium Absorption

The objective of this research was to determine the factors that influence the ability of calcium-binding peptides, obtained through in silico enzymatic hydrolysis of whey proteins, to promote calcium absorption. Isothermal titration calorimetry and density functional theory calculations revealed that all of the investigated peptides could spontaneously bind calcium through an entropy-driven endothermic binding reaction via their terminal and side-chain carboxyl groups at physiological pH. Among the investigated peptides, tripeptide EAC demonstrated the strongest ability to promote calcium absorption, with a calcium ion activity-based binding constant of 209 L/mol and a promotion factor of 2.57. The ability of calcium-binding peptides to promote calcium absorption was not correlated with their calcium-binding constants but rather with their chemical hardness and ionization potential. Machine learning modeling of all 8400 dipeptides and tripeptides indicated that those with higher log P values and aliphatic N/C-termini or aromatic C-termini were more likely to effectively promote calcium absorption. These findings provide valuable insights for the design of peptides to increase calcium bioavailability.

Peptidomic analysis of whey protein hydrolysates and prediction of their antioxidant peptides

Enzymatic hydrolysis of proteins is a breakdown process of peptide bond in proteins, releasing some peptides with potential biological functions. Previous studies on enzymatic hydrolysis of whey proteins have not identified the complete peptide profiles after hydrolysis. In this study, we reconstructed a profile of peptides from whey hydrolysates with two enzymes and different processing conditions. We also developed an ensemble machine learning predictor to classify peptides obtained from whey hydrolysis. A total of 2572 peptides were identified over three process conditions with two enzymes in duplicate. 499 peptides were classified and chosen as potential antioxidant peptides from whey proteins. The peptides classified as antioxidants in the hydrolysates had a proportion of 13.1%–24.5% regarding all peptides identified. These results facilitate the selection of promising peptides involved in the antioxidant properties during the enzymatic hydrolysis of whey proteins, aiding the discovery of novel antioxidant peptides.

The Influence of Enzymatic Hydrolysis of Whey Proteins on the Properties of Gelatin-Whey Composite Hydrogels.

Amino-acids, peptides, and protein hydrolysates, together with their coordinating compounds, have various applications as fertilizers, nutritional supplements, additives, fillers, or active principles to produce hydrogels with therapeutic properties. Hydrogel-based patches can be adapted for drug, protein, or peptide delivery, and tissue healing and regeneration. These materials have the advantage of copying the contour of the wound surface, ensuring oxygenation, hydration, and at the same time protecting the surface from bacterial invasion. The aim of this paper is to describe the production of a new type of hydrogel based on whey protein isolates (WPI), whey protein hydrolysates (WPH), and gelatin. The hydrogels were obtained by utilizing a microwave-assisted method using gelatin, glycerol, WPI or WPH, copper sulfate, and water. WPH was obtained by enzymatic hydrolysis of whey protein isolates in the presence of bromelain. The hydrogel films obtained have been characterized by FT-IR and UV-VIS spectroscopy. The swelling degree and swelling kinetics have also been determined.

Antioxidant and antihypertensive activity of bovine whey protein concentrate enzymatic hydrolysates

Whey is a highly polluting by-product of cheese processing. However, it has valuable nutritional properties since it is a rich and balanced source of proteins and amino acids. Therefore, it has a broad range of functional properties that can be exploited for diverse applications. Research has shown how the enzymatic hydrolysis of whey proteins releases bioactive peptides. In the present study, the hydrolysis of whey protein concentrate (WCP) was performed using purified Sporisorium reilianum aspartyl protease (Eap1), commercial enzymes chymotrypsin (C) and trypsin (T), as well as different enzymatic combinations in order to determine which enzyme or combination allowed for the release of peptides presenting the highest antioxidant and antihypertensive activity levels; our results indicated that hydrolysis with Eap1 releases the best-performing peptides in comparison with individual enzymes and their combinations.

Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin

Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate ≤3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2•− and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a low-cost dairy industry product, which could improve consumers’ health when used as functional ingredients.

Ферментативний гідроліз сироваткових білків молока

Ферментативний гідроліз сироваткових білків молока

Process optimization for enzymatic hydrolysis of whey protein concentrate

A study was under taken to optimize the enzymatic hydrolysis of whey protein concentrate for utilizing the resultant hydrolyzate in ragi based functional weaning food. Spray dried Whey protein concentrate (WPC) was reconstituted and heated to 80 o C for 5 min. The reconstituted medium was inoculated separately with Neutrase, papain and trypsin enzymes at various enzyme to substrate ratio (E: S) and incubated for a period of 180 h at their respective optimum pH and temperature. The effect of enzyme on degree of hydrolysis was monitored at a regular interval of 30 minutes by measuring change in pH. From among the three enzymes tried for enzymatic hydrolysis, it was observed that Neutrase enzyme is superior with respect to the extent of hydrolysis obtained followed by trypsin and papain. Neutrase enzyme could able to give hydrolysis of 6.08 per cent degree of hydrolysis within 150 minutes of duration. From the study, it was found that reconstituting the spray dried WPC at 15 per cent protein level and adding Neutrase enzyme at concentration of 1:50 and incubation for a period of 150 min is optimum for obtaining the maximum degree of hydrolysis. Spray dried WPC reconstituted to 15 per cent protein level and hydrolysed using Neutrase enzyme at 1:50 ratio for a period of 150 min was further used in the formulation of weaning food.

High hydrostatic pressure assisted enzymatic hydrolysis of whey proteins

Abstract Whey proteins, due to their high nutritional value, are generally hydrolyzed to reduce the allergenicity and used as ingredients in many special products, such as infant formulae, geriatric products, highly energetic supplements or dietetic foods or in foods produced to prevent nutrition related diseases, like food intolerances and allergies. The aim of this work was to assess the applicability of innovative technologies, such as high hydrostatic pressure (HHP) processes, to assist the enzymatic hydrolysis of target proteins, namely whey protein concentrate (WPC-80), in order to modify their antigenicity. Experiments were carried out to verify the effectiveness of HHP technology to accelerate whey protein hydrolysis reaction with selected enzymes (α-chymotrypsin, bromelain), and to affect the protein allergenic power. To this purpose, different HHP treatments were carried out at several pressure levels (100, 200, 300 and 400 MPa) and the untreated whey protein samples were used as control. A defined enzyme/substrate ratio of 1/10 w/w was used in the experiments, while the treatment time was changed from 0 to 30 min (0, 5, 15, or 30 min). The experimental data demonstrated that High Hydrostatic Pressure (HHP) induced WPC-80 unfolding at the highest value of the pressure applied (400 MPa) as indicated by the higher exposure of free sulfhydryl groups. When HHP was used in combination with enzymatic hydrolysis, the degree of hydrolysis increased not only with the pressure level applied but also with the processing time. These results suggested that, even if the exposure of hidden epitopes upon protein unfolding increased the antigenicity of whey proteins, further peptide bonds cleavage also took place after hydrolysis. This effect could modify whey proteins antigenic sequences, and thus their antigenic power.

PH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins

Enzymatic hydrolysis of a commercial whey protein isolate (WPI) using either trypsin or Protamex® was compared using controlled (pH-stat) and uncontrolled (free-fall) pH conditions. pH-stat control at the enzyme’s optimum value led to a more rapid rate of WPI hydrolysis by trypsin, while the opposite was the case when Protamex® was used. Furthermore, the choice of alkaline solution used to maintain constant pH during pH-stat experiments appeared to affect the reaction rate, being higher when KOH is added to the reaction mixture instead of NaOH. It would appear that potassium may play a role as co-factor or activator for the activity of this particular protease preparation. Although pH-stat techniques are usually considered to yield better hydrolysis kinetics, these findings suggest that the response of proteolytic enzyme preparations to static or free-fall pH control should be checked in advance, particularly when undertaking large scale production of WPI hydrolysates.

Enzymatic generation of whey protein hydrolysates under pH-controlled and non pH-controlled conditions: Impact on physicochemical and bioactive properties

Enzymatic hydrolysis of whey protein (WP) was carried out under pH-controlled and non pH-controlled conditions using papain and a microbial-derived alternative (papain-like activity). The impact of such conditions on physicochemical and bioactive properties was assessed. WP hydrolysates (WPH) generated with the same enzyme displayed similar degree of hydrolysis. However, their reverse-phase liquid chromatograph mass spectrometry peptide profiles differed. A significantly higher oxygen radical absorbance capacity (ORAC) value was obtained for WP hydrolysed with papain at constant pH of 7.0 compared to the associated WPH generated without pH regulation. In contrast, there was no significant effect of pH regulation on dipeptidyl peptidase IV (DPP-IV) properties. WP hydrolysed with papain-like activity under pH regulation at 7.0 displayed higher ORAC activity and DPP-IV inhibitory properties compared to the associated WPH generated without pH regulation. This study has demonstrated that pH conditions during WPH generation may impact on peptide release and therefore on WPH bioactive properties.

DETERMINATION OF PHYSICOCHEMICAL, IMMUNOCHEMICAL AND ANTIOXIDANT PROPERTIES, TOXICOLOGICAL AND HYGIENIC ASSESSMENT OF WHEY PROTEIN COMCENTRATE AND ITS HYDROLYSATE

Enzymatic hydrolysis of whey proteins is aimed to obtain products with low allergenic potential and high nutritional value. Whey peptides are protein fraction that possesses a variety of physicochemical, immunochemical and bioactive properties (antioxidant, antibacterial, immunomodulatory effects). Controlled parameters of enzymatic hydrolysates are the degree of hydrolysis of protein substrates, peptide composition, residual antigenicity, antioxidant capacity, etc. The purpose of this work is to characterize peptide profile, antigenicity and free radical scavenging properties of experimental hydrolysate sample, toxicological and hygienic assessment of its impact on a test object (infusoria Tetrahymena pyriformis). The research of properties of whey proteins and their enzymatic hydrolysates was conducted using classical and modern methodological approaches: SDS electrophoresis in polyacrylamide gel, HPLC, mass-spectrometry, competitive ELISA, TEAC (Trolox Equivalent Capacity Assay) technique. Peptide composition, antigenic and antioxidant properties of obtained enzymatic hydrolysate were determined, toxicity of raw and digested whey proteins were examined. We established that the investigated hydrolysate sample falls under the category of partial hydrolysates for functional products according to its physicochemical, immunochemical, free radical scavenging and organoleptic properties. According to the results of toxicological and hygienic assessment using T. pyriformis model, whey protein concentrate and its hydrolysates are non-toxic and do not possess cumulative properties. Thus, we obtained partial enzymatic hydrolysate of whey proteins from milk, which can be used as physiologically active component in the development of new specialized food.

Synthesis and functionality of proteinacious nutraceuticals from casein whey—A clean and safe route of valorization of dairy waste

Due to stricter environmental legislation, coagulation and precipitation technologies have been practiced for whey protein sedimentation from a long prior. Nowadays, in pharmaceutical and dairy industries, individual whey proteins have been isolated by size-exclusion membrane separation technology, ion-exchange membrane chromatography, gel-permeation chromatography, affinity-peptide ligand chromatography and electro-chromatography. Furthermore, whey protein-derived peptides have been synthesized by conventional batch or catalyst-immobilized packed-bed bioreactors or membrane-assigned bioreactor with high throughput and purity, considering raw whey or isolated individual protein as feedstock. Different types of enzymes, such as trypsin, chymotrypsin, alcalase, pepsin and chymosin, have been popularly used for enzymatic hydrolysis of whey proteins. Whey proteins, such as β-lactoglobulin, α-lactalbumin, bovine serum albumin, lactoperoxidase, lactoferrin and immunoglobulins, and enzymatic hydrolysis of whey proteins have their unique physiological, medicinal, functional and nutritional values. It could be expected that without going for mere treatment of whey (coagulation or sedimentation of whole protein), synthesis of proteinacious bioactives (separation of individual whey proteins and synthesis of their derivatives) will lead to zero effluent discharge within a domain of whey upgradation. The present review pretends to highlight the different process-related technological aspects for the synthesis of different proteinacious bioactives from whey, as well as their functionality.

Modification of peptide functionality during enzymatic hydrolysis of whey proteins

Enzymatic hydrolysis of whey proteins increased the reducing capacity and decreased reactive sulfhydryl (SH) content of the peptides. Hydrolysis yielded Maillard reaction products and the carbonyl compounds depleted SH by nucleophilic reaction.

Antioxidant effects of enzymatic hydrolysates of whey protein concentrate on cultured human endothelial cells

Enzymatic hydrolysis of whey protein concentrate (WPC) was performed using Alcalase, Neutrase, Corolase PP or Flavourzyme. The hydrolysates were filtered through membranes having different molecular mass cut-offs. The hydrolysate fractions had significantly (P < 0.001) greater antioxidant capacity than unhydrolysed WPC, as measured by the oxygen radical absorbance capacity assay (ORAC). Furthermore, incubation of human umbilical vein endothelial cells (HUVECs) with the hydrolysate fractions resulted in an increase in cellular glutathione and catalase activity. Microarray analysis showed beneficial regulation of various genes involved in the antioxidant/detoxification processes in HUVECs incubated with the 5 kDa permeates of WPC hydrolysed by Alcalase or Neutrase. In conclusion, peptides generated by the enzymatic hydrolysis of WPC using Alcalase, Neutrase, Corolase PP or Flavourzyme have a high antioxidant capacity and may have a role in the beneficial regulation of endothelial cell function.

Research on Enzymatic Hydrolysis of Whey Protein

Based on single factor analysis for the enzymatic hydrolysis of whey protein, papain was selected as the optimal enzyme and its enzymatic hydrolysis conditions were optimized by the quadratic regression orthogonal rotary test. The orthogonal regression model for degree of hydrolysis (DH) to three factors including temperature (X1), time (X2), enzyme dosage (X3) was established as follow: DH=10.40+0.22X1+0.30X2+1.31X3+0.019X1X2+0.011X1X3-0.039X2X3-0.39X12-0.16X22-0.40X32, Verification test showed a DH of 11.7% was obtained at the optimal hydrolysis condition of 56.6°C, 113.8 min and enzyme 8213.7 U /g protein, which basically consisted with the model theoretical value.

Insulinotropic properties of whey protein hydrolysates and impact of peptide fractionation on insulinotropic response

The influence of different substrates and the effect of pH regulation during enzymatic hydrolysis of whey protein on glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells were studied. No significant differences (P ≥ 0.05) were detected in terms of glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells exposed to whey protein hydrolysates (WPH1 and WPH2) obtained with two different whey protein substrates. The whey protein hydrolysate (WPH3) obtained without pH regulation during hydrolysis, had a significantly lower insulinotropic potential in BRIN-BD11 cells than the WPH1 hydrolysate that was manufactured with pH regulation. Fractionation of WPH1 was carried out to enrich bioactive peptides. Comparing the different fractionation techniques studied (solid-phase extraction and semi-preparative reverse phase-high performance liquid chromatography), the most potent insulinotropic fraction was enriched in free amino acids and contained relatively hydrophilic peptides. This indicates that amino acids and hydrophilic peptides may be involved in the insulinotropic effect of WPH1.

Enzymatic hydrolysis of whey protein concentrate: effect of enzyme type and enzyme:substrate ratio on peptide profile

Enzymatic hydrolysis of whey protein concentrate was used to prepare hydrolysates with appropriate peptide profiles for use in the development of dietary supplements. Effects of the type of enzyme (pancreatin and proteases from Bacillus licheniformis, Aspergillus oryzae and Aspergillus sojae) and enzyme:substrate ratio (0.5:100, 1:100, 2:100, 3:100, 4:100 and 8:100) were evaluated. Twenty-four hydrolysates were prepared, and their peptide profiles were characterized by chain length using size exclusion liquid chromatography fractionation, followed by a rapid Correct Fraction Area method for quantifying the peptide and amino acid contents. The use of a protease from Bacillus licheniformis (E:S 8:100) and a pancreatin (E:S 2:100, 3:100, 4:100 and 8:100) yielded preparations of hydrolysates with the highest sums of di- and tripeptide and free amino acid contents (9.99 and 11.28 % for the protease from Bacillus licheniformis and pancreatin, respectively) as well as the lowest amount of large peptides (44.61 and 44.13 %, respectively).