Effect Of Limited Enzymatic Hydrolysis Research Articles

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.

Innovative multistep modifications of β-Lactoglobulin for enhanced emulsifying and antioxidant activities

This aim of this study was to investigate the effects of limited enzymatic hydrolysis (H), glycation (G), and amyloid fibril (F) modification on the emulsifying and antioxidant activities of β-Lactoglobulin (β-Lg). Changes in the physicochemical properties of limited enzymatic hydrolysis-modified β-Lg (Hβ-Lg), glycation by maltodextrin (Mal) -modified Hβ-Lg (GHβ-Lg), and amyloid fibril-modified GHβ-Lg (FGHβ-Lg) were observed through free amino acid content, sodium dodecyl sulfate‒polyacrylamide gel electrophoresis profiles and ThT fluorescence intensity. The structural changes and microscopic morphology of the modified proteins were determined by circular dichroism, Fourier transform infrared spectroscopy and transmission electron microscopy. Circular dichroism analysis showed that the β-structure content of FHβ-Lg increased, while the secondary structure of FGHβ-Lg was preserved. Finally, the functional activities of the modified proteins were evaluated. Comparing β-Lg with FGHβ-Lg or GHβ-Lg, the emulsifying activity of FGHβ-Lg increased from 9.38 m2/g to 24.37 m2/g, while the emulsifying stability of GHβ-Lg increased from 48.93 min to 131.22 min. Finally, the antioxidant activities of the modified proteins were determined by reducing power (β-Lg: 0.17, FGHβ-Lg: 0.27), ABTS·+ scavenging activity (β-Lg: 75.91%, FGHβ-Lg: 86.33%), Fe2+ chelating activity (β-Lg: 54.30%, FGHβ-Lg: 75.49%), and DPPH scavenging ability (β-Lg: 67.43%, FGHβ-Lg: 77.13%). Overall, our findings suggest that FGHβ-Lg has great potential in the development of protein emulsifiers with high antioxidant activities.

Effects of limited enzymatic hydrolysis and polysaccharide addition on the physicochemical properties of emulsions stabilized with duck myofibrillar protein under low-salt conditions

This study aimed to investigate the role of hydrolysis and guar gum (GG) participation on the emulsification of the duck myofibrillar protein (MP) and the related stability of oil-in-water emulsion in low-salt condition. Emulsions were prepared using one of each or both treatments, and that prepared with trypsin hydrolysis and GG (T-GG) exhibited the highest stability. FTIR analysis confirmed the hydrogen bond interactions between the system components. T-GG treatment improved emulsion properties and decreased oil droplet size. Moreover, CLSM indicated that aggregation of T-GG oil droplets was prevented. Physical stability was assessed such as Turbiscan stability index, creaming index, and rheological properties. The adsorbed percentage for T-GG was the lowest. However, interfacial tension, droplet size, stability, and peroxide value analyses indicated that a denser interfacial membrane structure is formed with T-GG. Thus, T-GG treatment could be applied in the food industry, such as in nutrient delivery systems and fat mimetics.

Combined effects of limited enzymatic hydrolysis and high hydrostatic pressure on the structural and emulsifying properties of rice proteins

The combined effects of limited enzymatic hydrolysis and high hydrostatic pressure (HHP) treatment on the structural and emulsifying properties of rice proteins (RPs) were investigated. Alcalase exhibited higher hydrolysis efficiency than papain. Through alcalase-catalyzed hydrolysis and HHP treatment, emulsifying activity index (EAI) and emulsifying stability index (ESI) of RPs were improved from 14.58 m2/g and 22.54 min to 29.93 m2/g and 43.35 min, respectively. The content of random coil in RPs increased, while the content of α-helix decreased. HHP at 300 MPa led to the highest surface hydrophobicity (H0) and fluorescence intensity. The enzymatic hydrolysis-HHP (300 MPa) modified RPs could form an oil-in-water emulsion with a d4,3 of 0.347 nm, ζ-potential of −36.4 mV, and creaming index of 10.5% after 7 days storage. This work sheds light on employing limited enzymatic hydrolysis and HHP to modify RPs for better application in emulsion-based food products.

Effect of limited enzymatic hydrolysis on the structure and emulsifying properties of rice bran protein

The influence of the degree of hydrolysis (DH) on the structure, solubility and emulsifying properties of rice bran protein (RBP) was investigated. Sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE), intrinsic fluorescence, surface hydrophobicity and circular dichroism (CD) techniques were used for the analysis of the structural characteristics. Hydrolysis can change the molecular weight of the protein, increase its flexibility and fluidity, change its surface hydrophobicity, and increase its solubility. Oil-in-water emulsions were prepared from rice bran protein hydrolysates (RBPH) with different DH values (1%, 3%, and 6%). The stabilities of the RBPH-stabilized emulsions were investigated under the following environmental pressures: pH (3–9), ionic strength (0–300 mM NaCl) and thermal treatment (30–90 °C, 30 min). Particle size and surface charge (ζ-potential) were measured as an indication of the emulsion stability. Emulsions prepared with 3% DH RBP were much more stable than the emulsions prepared with RBPH with other DH values, and this RBP has potential as an emulsifier in the food industry.

Effect of limited enzymatic hydrolysis on structure and emulsifying properties of rice glutelin

The influence of the degree of hydrolysis (DH) on the structure, solubility, rheology, and emulsifying properties of rice glutelin was investigated. Structural properties were characterized using analysis of molecular weight distribution, surface hydrophobicity, intrinsic fluorescence, and Circular Dichroism (CD) spectra. Protein hydrolysis changed molecular weight, increased flexibility, altered surface hydrophobicity, and increased solubility. Oil-in-water emulsions were prepared from rice glutelin with different DH (0.5%, 2%, and 6%) and their stability to storage, pH, salt, and thermal processing was assessed. The storage, pH, salt, and temperature stability of the emulsions increased with decreasing hydrolysis. Emulsions prepared with 2% DH rice glutelin were stable over a range of environmental conditions: pH 7–9; NaCl <100 mM (pH 7); temperatures < 90 °C (pH 7, 0 mM NaCl) studied. These results will facilitate the formulation and production of natural emulsion-based products using rice glutelin as an emulsifier.

Effects of limited enzymatic hydrolysis, pH, ionic strength and temperature on physicochemical and functional properties of palm (Elaeis guineensis Jacq.) kernel expeller protein

Limited hydrolysates ranging from 6.82 to 16.74 % degree of hydrolysis (DH) were produced from palm kernel expeller protein (PKP) using Alcalase. The nutritional value, physicochemical and functional properties of PKP and the hydrolysates, especially effects of DH, pH, ionic strength and temperature on these properties were studied. Results showed that all the hydrolysates showed higher nutritive value, better thermal stability and solubility (70.68–99.13 g/100 g) than PKP. The surface hydrophobicity, emulsifying properties and foaming activity were improved by limited enzymatic hydrolysis (DH: 6.82 to 11.72 %), whereas the foam stability, viscosity, water and oil holding capacity were negatively correlated with DH. Increase in ionic strength (from 0 to 0.4 mol/L) or temperature (from 30 to 50 °C) enhanced the solubility, emulsifying properties and foaming activity. But high temperature remarkably weakened the foaming properties and viscosity. Solubility, surface hydrophobicity and molecular weight influenced the emulsifying and foaming properties. PKP and the hydrolysates were good protein source.

Influence of Degree of Hydrolysis on the Functional Properties of Cowpea Protein Hydrolysates

The effects of limited enzymatic hydrolysis with pepsin on the functional properties and molecular characteristics of cowpea protein concentrate (CPC) were investigated. Hydrolysates were prepared at degrees of hydrolysis (DH) 5–30%. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis demonstrated that globulin was the major protein component of CPC, and enzymatic breakdown of polypeptides was selective. As a result, significant changes in the functional properties of hydrolysates were noticed as compared to the protein concentrate. Then, cowpea protein hydrolysates (CPHs) showed good water solubility index at pH 7 (>95%) and pH 4.5 (>55%), at DH 20 and 30%, respectively. Oil-holding capacity, emulsifying activity and foaming capacity were also improved by the enzymatic hydrolysis, while proteolysis was detrimental to foam stability and did not improve emulsion stability at DH 10–30%. Therefore, CPHs showed good potential for utilization in food formulations. Practical Applications Cowpea is a good nutritional value and available protein source, which could be used for a large-scale production of protein hydrolysates. Enzymatic hydrolysis of food proteins is mostly used to produce ingredients or supplements in food and beverages. In this regard, functional properties of protein hydrolysates are largely dependent on the degree of hydrolysis (DH), which needs to be controlled to avoid excessive proteolysis that can impair functionality and cause unfavorable effects. Then, functional properties of cowpea protein concentrate hydrolyzed by pepsin at DH 5–30% were investigated. The results showed that solubility and oil-holding capacity were higher at DH 20–30%. Emulsifying activity and foaming capacity were also improved following hydrolysis, while de-decreasing molecular weight of protein was detrimental to foam stability of hydrolysates. In summary, cowpea protein hydrolysates showed adequate functional properties for utilization as food ingredient and the peptides produced could turn out to present some biological activities.

Effect of limited enzymatic hydrolysis on physico‐chemical properties of soybean protein isolate‐maltodextrin conjugates

SummaryThe effect of limited hydrolysis was investigated on the physico‐chemical properties of soy protein isolate–maltodextrin (SPI‐Md) conjugate. The hydrolysates at a degree of hydrolysis (DH) of 1.8% showed much higher surface hydrophobicity (H0; 71.39 ± 3.60) than that of the SPI control (42.09 ± 2.17) and SPI‐Md conjugates (53.46 ± 2.74). Intrinsic fluorescence analysis demonstrated the unfolding of protein molecule and exposure of hydrophobic groups of SPI‐Md conjugate hydrolysates. As evidenced by far‐UV circular dichroism (CD) spectroscopy, the limited hydrolysis increased the unordered secondary structures of SPI‐Md conjugates. The denaturation temperature (Td) of SPI‐Md conjugate was significantly increased by subsequent limited hydrolysis from 102.53 ± 0.60 °C to 108.11 ± 0.61 °C at DH 1.8%. In particular, the emulsifying activity index (EAI) was improved notably after limited hydrolysis of DH 1.8% (147.76 ± 4.39 m2 g−1) compared with that of native SPI (88.90 ± 1.44 m2 g−1) and SPI‐Md conjugate (108.97 ± 1.45 m2 g−1).

Effect of pH and Pepsin Limited Hydrolysis on the Structure and Functional Properties of Soybean Protein Hydrolysates

Effects of limited enzymatic hydrolysis with pepsin on the functional properties and structure characteristics of soybean proteins were investigated. Hydrolysates with different incubation time (10 to 900 min) were prepared. Results showed that SPI hydrolyzed for 60 min exhibited the best emulsibility and the ability of resisting freezing/thawing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis proved that pepsin can degrade glycinin but had little effect on the α' subunit of β-conglycinin. The structure unfolding reached the largest extent after incubation for 60 min and the soluble and flexible aggregates were formed. After 120 min, glycinin was degraded totally and β-conglycinin formed insoluble aggregates. Moreover, 2 methods were applied for the deactivation of pepsin to obtain final hydrolysates at pH 2.0 and 7.0, respectively. The structure analysis revealed that the unfolding extent and structure characteristic were different in these 2 conditions. When adjusting the pH value from 2.0 to 7.0, the unfolding protein molecular would reaggregate again at pH 7.0 due to the charge neutralization, and the hydrodynamic diameter and λmax absorbance decreased compared to pH 2.0. Moreover, some of the insoluble aggregates formed at pH 2.0 became soluble at pH 7.0, because of the salt-in phenomenon.

Effect of limited enzymatic hydrolysis on linoleic acid binding properties of β-lactoglobulin

β-Lactoglobulin (BLG) is a member of lipocalin family, proteins with ability to bind small hydrophobic ligands, such as retinol, vitamins and fatty acids. Moreover, BLG is susceptible to protease action producing a wide range of polypeptides depending on the hydrolysis degree (HD). In the present work, the effect of limited enzymatic hydrolysis on fatty acid binding properties of BLG was studied. Linoleic acid (LA) was used as a model fatty acid. Limited enzymatic hydrolysis was performed using α-chymotrypsin immobilised on agarose microparticles. BLG hydrolysates were produced at HD: 1%, 3% and 5%. In order to determine the influence of HD on BLG molecular weight SDS–PAGE was used. BLG structural modification and LA binding properties were monitored by means of fluorescence spectroscopic techniques. The increase in HD produced: (i) a BLG degradation and a molecular weight distribution of BLG hydrolysates and (ii) an increased exposition of buried hydrophobic residues, however it was observed a decrease in surface hydrophobicity possibly due to a deterioration of hydrophobic protein domains. It was observed that enzymatic hydrolysis treatment produced a decrease in BLG ability for binding LA. It was concluded that limited enzymatic hydrolysis could deteriorate the specific site on BLG structure necessary for binding LA.

The effects of limited enzymatic hydrolysis on the physicochemical and emulsifying properties of a lentil protein isolate

The physicochemical and emulsifying properties of lentil protein isolates (LPI) were investigated as a function of their degree of hydrolysis (DH of 4, 9 and 20%) following exposure to trypsin/heat. Specifically, interfacial tension, surface characteristics (charge and hydrophobicity) and intrinsic fluorescence were determined. These parameters were then related to changes in the emulsification activity (EAI) and stability indices (ESI) of unhydrolyzed (u-LPI) and hydrolyzed LPI (h-LPI) in a flaxseed oil–water emulsion. Interfacial tension was found to decrease from ~6.5 to ~6.1mN m−1 for u-LPI and h-LPI (DH 4–20%), respectively. A similar trend was observed for surface hydrophobicity, which declined from ~30 to ~24 for the u-LPI and h-LPI (DH 4–20%), respectively. In contrast, surface charge values were similar for all materials (~−37mV). Intrinsic fluorescence as a function of emission wavelengths (300–400nm) indicated a slight change in the tertiary conformation of LPI upon hydrolysis, where the magnitude of fluorescence intensity declined relative to that of u-LPI. Changes in physicochemical properties upon hydrolysis had a detrimental effect on EAI and ESI values, which declined from ~51 to ~47m2g−1 and ~12 to ~11min for u-LPI and h-LPI (DH 4–20%), respectively.

Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate

Effects of limited enzymatic hydrolysis with pepsin and/or high-pressure homogenization in acid condition on the functional properties and structure characteristic of soybean protein isolate (SPI) were investigated. Functional properties, including protein solubility, surface hydrophobicity, particle size distribution, the ability of resisting freezing/thawing, foaming properties and dynamic surface properties were evaluated. Result showed that, single acid treatment could improve functional properties of SPI, but was not as effective as single or combined pepsin hydrolysis and high-pressure homogenization in acid condition. Emulsibility, the ability of resisting freezing/thawing and foaming capacity of soybean proteins were remarkably improved by the combination treatment, but no improvement of foaming stability was detected. Changes of structures were detected by surface hydrophobicity, and hydrodynamic diameter. It was found that aggregates existed in all treated samples. Besides, more flexible and soluble aggregates were observed in samples treated by limited pepsin proteolysis, high-pressure homogenization and the combination treatments, which might contribute to their functional properties.

Enzymatic hydrolysis and their effects on conformational and functional properties of peanut protein isolate

The effects of limited enzymatic hydrolysis by Alcalase on the conformational and functional properties of peanut ( Arachis hypogaea L.) protein isolate (PPI) were investigated. Acid subunits of arachin were most susceptible to Alcalase hydrolysis, followed by conarachin and the basic subunits of arachin. Enzymatic hydrolysis increased the thermal stability of arachin and led to a sharp increase in the number of disulphide bonds with a decrease of the sulphydryl group in PPI hydrolysates in comparison with PPI. The analysis of intrinsic fluorescence spectra indicated a more moveable tertiary conformation of PPI hydrolysates than PPI. The limited emzymatic hydrolysis improved the functional properties of PPI, such as protein solubility and gel-forming ability, but impaired the emulsifying activity index.

Stability of oil: Water emulsions of amaranth proteins. Effect of hydrolysis and pH

Abstract In this contribution we have determined the effect of limited enzymatic hydrolysis on the emulsifying capacity of amaranth proteins. The action of enzyme (alcalase and trypsin) and the pH of the continuous phase of the oil/water emulsion (pH 2.0, 6.3 and 8.0) were the variables analyzed. The results obtained show that amaranth protein isolates, AI, contain proteins species capable of forming and stabilizing emulsions, mainly at acidic pH (2.0) and to a lesser extent at pH 8.0. While the emulsions obtained are sensitive to creaming and flocculation, they do not undergo destabilization by coalescence. The emulsions prepared from proteins subjected to low grade trypsin hydrolysis (TH2.2) are sensitive to creaming - flocculation, whereas alcalase-hydrolyzed proteins (AH1.7 and AH9.5) exhibited a significant destabilization by creaming, flocculation and coalescence, mainly at pH 6.3. The effect of the pH of the aqueous phase was determining on the emulsion stability beside the structural and physicochemical characteristics of protein species utilized as tensioactive. At acidic pH (pH 2.0) the unfolding and charge of polypeptides and the capacity of form a viscoelastic film at the interface were essential while at alkaline pH (pH 8.0) the balance among high and low molecular mass protein species and flexibility of the molecule fixed the emulsions properties.

Improving the functional properties of soy glycinin by enzymatic treatment. Adsorption and foaming characteristics

In this contribution we have determined the effect of limited enzymatic hydrolysis on the interfacial (dynamics of adsorption and surface dilatational properties) and foaming (foam formation and stabilization) characteristics of a soy globulin (glycinin, fraction 11S). The degree of hydrolysis (DH=0%, 2%, and 6%), the pH of the aqueous solution (pH=5 and 7), and the protein concentration in solution (at 0.1, 0.5 and 1 wt%) were the variables studied. The temperature and the ionic strength were maintained constant at 20 °C and 0.05 M, respectively. The rate of adsorption and surface dilatational properties (surface dilatational modulus, E, and loss angle) of glycinin at the air–water interface depend on the pH and DH. The adsorption decreased drastically at pH 5.0, close to the isoelectric point of glycinin, because of the existence of a lag period and a low rate of diffusion. The interfacial characteristics of glycinin are much improved by enzymatic treatment, especially in the case of acidic aqueous solutions. Hydrolysates with a low DH have improved functional properties (mainly foaming capacity and foam stability), especially at pH close to the isoelectric point (pI), because the native protein is more difficult to convert into a film at fluid interfaces at pH≈pI. The foam capacity depends on the rate of diffusion of protein to the interface and is much improved by the enzymatic treatment. Foam stability correlates with surface pressure and, to a minor extent, with surface dilatational modulus at long-term adsorption with few exceptions.

Effects of limited enzymatic hydrolysis with trypsin on the functional properties of hemp ( Cannabis sativa L.) protein isolate

Effects of limited enzymatic hydrolysis induced by trypsin on the physicochemical and functional properties of hemp ( Cannabis sativa L.) protein isolate (HPI) were investigated. The enzymatic hydrolysis was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). SEC and differential scanning calorimetry (DSC) analyses confirmed the presence of aggregates in the corresponding hydrolysates (with the degree of hydrolysis of 2.3–6.7%). Functional properties, including protein solubility (PS), thermal properties, emulsifying and foaming properties, and water holding and fat adsorption capacities (WHC and FAC) were evaluated. The PS was remarkably improved by the limited enzymatic hydrolysis at all tested pH values. However, the enzymatic hydrolysis led to the marked decreases in emulsifying activity index, foaming capacity and foam stability, WHC and FAC. These decreases were to a great extent related to the presence of aggregates in the hydrolysates.

Limited Enzymatic Hydrolysis Can Improve the Interfacial and Foaming Characteristics of β-Conglycinin

In this contribution, we have determined the effect of limited enzymatic hydrolysis on the interfacial (dynamics of adsorption and surface dilatational properties) and foaming (foam formation and stabilization) characteristics of a soy globulin (beta-conglycinin, fraction 7S). The degree of hydrolysis (DH = 0, 2, and 5%), the pH of the aqueous solution (pH = 5 and 7), and the protein concentration in solution (at 0.1, 0.5, and 1 wt %) were the variables studied. The temperature and the ionic strength were maintained constant at 20 degrees C and 0.05 M, respectively. The rate of adsorption and surface dilatational properties (surface dilatational modulus, E, and loss angle) of beta-conglycinin at the air-water interface depend on the pH and DH. The adsorption decreased drastically at pH 5.0, close to the isoelectric point of beta-conglycinin, because of the existence of a lag period and a low rate of diffusion. The interfacial characteristics of beta-conglycinin are much improved by enzymatic treatment, especially in the case of acidic aqueous solutions. Hydrolysates with a low DH have improved functional properties (mainly foaming capacity and foam stability), especially at pH values close to the isoelectric point (pI), because the protein is more difficult to convert into a film at fluid interfaces at pH approximately equal to pI.