Antioxidant, angiotensin-converting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides - A review.

Egg white contains many functionally important proteins: ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), ovoglobulin (G2 and G3, 8%), ovomucin (3.5%), and lysozyme (3.5%) are major proteins, while ovoinhibitors, ovomacroglobulin, ovoglycoprotein, ovoflavoprotein, thiamine-binding proteins, and avidin are minor proteins present in egg white. These proteins, as well as the peptides derived from the proteins, have been recognized for their functional importance as antioxidant, antimicrobial, metal-chelating, anti-viral, anti-tumour, and angiotensin-converting enzyme (ACE)-inhibitory activities. Among the functional properties of the peptides, antioxidant and antimicrobial activities are important characteristics for food processing while other properties such as ACE-inhibitory activity of the peptides can have important health-related functionalities. Bioactive peptides can be produced from egg white proteins by enzyme hydrolysis, chemical treatments, or thermal treatments at different pH conditions. The effective functional peptides produced from egg white proteins are usually smaller than 2 kDa in molecular size. However, these peptides are known for their beneficial activities in vitro only, and little work has been done to prove their beneficial effects in vivo. Therefore, further studies are needed to see if the bioactive peptides derived from egg white proteins are helpful for humans in the future.

Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates.

Ovomucin was hydrolyzed using enzymes or by heating under alkaline conditions (pH 12.0), and the functional, structural and compositional characteristics of the peptides in the hydrolysates were determined. Among the treatments, heating at 100 °C for 15 min under alkaline conditions (OM) produced peptides with the highest iron-binding and antioxidant capacities. Ovomucin hydrolyzed with papain (OMPa) or alcalase (OMAl) produced peptides with high ACE-inhibitory activity. The mass spectrometry analysis indicated that most of the peptides from OMPa were <2 kDa, but peptides from OMTr and OM were >2 kDa. OMAl hydrolyzed ovomucin almost completely and no peptides within 700-5000 Da were found in the hydrolasate. The results indicated that the number and size of peptides were closely related to the functionality of the hydrolysates. Considering the time, cost and activities of the hydrolysates, OM was the best treatment for hydrolyzing ovomucin to produce functional peptides.

Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates.

Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point.

Enzymatic hydrolysis of ovalbumin and the functional properties of the hydrolysates.

Ovalbumin is the predominant protein in egg white and is widely used in cell culture. However, it also can be used to produce peptides with various functional properties. The objectives of this study were to hydrolyze ovalbumin using various enzyme, incubation time, and temperature combinations, and to compare the functional properties of the hydrolysates. Ovalbumin (20 mg/mL) was hydrolyzed with 1% of pepsin, trypsin, α-chymotrypsin, papain, and alcalase, singly or in combination at 37°C, and then the enzymes were inactivated at 100°C for 15 min. Hydrolyzing ovalbumin with pepsin (OAPe), pepsin + papain (OAPePa), pepsin + alcalase (OAPeAl), alcalase + trypsin (OAAlTr), and α-chymotrypsin (OACh) was also effective in producing peptides from ovalbumin, and the peptides produced had strong iron- and copper-binding capacities and antioxidant capability. However, the best treatment of all was the OAAlTr treatment, which showed the highest iron-chelating and antioxidant activities among the enzyme treatments (P < 0.05). Electrospray-ionization mass spectrometry (MS/MS) analysis identified numerous peptides (<5 kDa) from the OAPe, OAPeAl, OACh, OAAlTr, and OAPePa hydrolysates of ovalbumin, but the number and size of peptides varied widely depending on the treatments. The enzymatic hydrolysis significantly increased the functionality of ovalbumin, and the improvement depended upon the composition of peptides produced rather than the number of the peptides produced.

Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents.

A study was conducted to develop a simple sequential separation protocol to separate phosvitin and IgY from egg yolk without using organic solvents. Egg yolk was diluted with 2 volumes of distilled water (DW), homogenized, and centrifuged. The precipitant was collected and homogenized with 4 volumes of 10% NaCl (wt/vol) in 0.05 N NaOH solution to extract phosvitin. The pH of the homogenate was adjusted to 4.0 and the precipitate was removed by centrifugation. The supernatant was collected and then heat-treated at 70°C for 30 min and centrifuged to remove impurities. The supernatant containing phosvitin was collected, had salts removed, and was concentrated and then freeze-dried. The supernatant from the centrifugation of diluted egg yolk was diluted again with 3 volumes of DW, and the precipitate was removed by centrifugation. The resulting supernatant was concentrated using ultrafiltration and then IgY was precipitated using 20% saturated (NH4)2SO4+ 15% NaCl (wt/vol). The precipitant was collected after centrifugation at 3,400 × g for 30 min at 4°C and dissolved with DW, had salts removed, and then was freeze-dried. The purity of separated phosvitin and IgY was checked using SDS-PAGE and the proteins were verified using Western blotting. The purity of phosvitin and IgY was 97.2 and 98.7%, and the yield was 98.7 and 80.9%, respectively. The ELISA results indicated that the activities of separated IgY and phosvitin were 96.3 and 98.3%, respectively. This study proved that both phosvitin and IgY can be separated in sequence from egg yolk without using an organic solvent. Also, the method is very simple and has a high potential for scale-up processing.

Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white.

Ovalbumin, ovotransferrin, ovomucin, and lysozyme are a few of the egg white proteins that can be used as functional components. The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using FPC3500 cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin first using 5.0% (wt/vol) (NH4)2SO4 and 2.5% (wt/vol) citric acid combination. After centrifugation, the supernatant (S1) was used for ovalbumin separation and the precipitant was dissolved in water, and reprecipitated using 2.0% ammonium sulfate (wt/vol) and 1.5% citric acid (wt/vol) combination. The precipitant was used as ovotransferrin fraction, and the supernatant (S2) was pooled with the first supernatant (S1), desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was >98% and that of ovotransferrin and lysozyme was >82% for both laboratory and scale-up preparations. The SDS-PAGE and western blotting of the separated proteins, except for ovomucin, showed >90% purity. The ELISA results indicated that the activities of separated ovalbumin, ovotransferrin, and lysozyme were >96%. The protocol separated 4 major proteins in sequence, and the method was simple and easily scaled up.

Separation of ovotransferrin and ovomucoid from chicken egg white.

Ovotransferrin and ovomucoid were separated using 2 methods after extracting the ovotransferrin- and ovomucoid-containing fraction from egg white. Diluted egg white (2×) was added to Fe(3+) and treated with 43% ethanol (final concentration). After centrifugation, the supernatant was collected and treated with either a high-level ethanol (61% final concentration) or an acidic salt combination (2.5% ammonium sulfate and 2.5% citric acid) to separate ovotransferrin and ovomucoid. For the high-level of ethanol method, ovotransferrin was precipitated using 61% ethanol. After centrifugation, the precipitant was dissolved in 9 vol. of distilled water and the residual ethanol in the solution was removed using ultrafiltration. The supernatant, mainly containing ovomucoid, was diluted with 4 vol. of water, had ethanol removed, and was then concentrated and used as the ovomucoid fraction. For the acidic salt precipitation method, the ethanol in the supernatant was removed first. The ethanol-free solution was then concentrated and treated with a 2.5% ammonium sulfate and 2.5% citric acid combination. After centrifugation, the precipitant was used as the ovotransferrin and the supernatant as the ovomucoid fraction. The ovomucoid fraction from both of the protocols was further purified by heating at 65°C for 20 min and the impurities were removed by centrifugation. The yields of ovomucoid and ovotransferrin were >96 and >92%, respectively. The purity of ovomucoid was >89% and that of the ovotransferrin was >88%. The ELISA results confirmed that the activity of the separated ovotransferrin was >95%. Both of the protocols separated ovotransferrin and ovomucoid effectively and the methods were simple, fast, and easy to scale up.

Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents--a review.

Egg white contains many functionally important proteins. Ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), ovomucin (3.5%), and lysozyme (3.5%) are among the major proteins that have high potentials for industrial applications if separated. The separation methods for these proteins from egg white have been developed since early 1900, but preparation methods of these proteins for commercial applications are still under development. Simplicity and scalability of the methods, use of nontoxic chemicals for the separation, and sequential separation for multiple proteins are very important criteria for the commercial production and application of these proteins. The separated proteins can be used in food and pharmaceutical industry as is or after modifications with enzymes. Ovotransferrin is used as a metal transporter, antimicrobial, or anticancer agent, whereas lysozyme is mainly used as a food preservative. Ovalbumin is widely used as a nutrient supplement and ovomucin as a tumor suppression agent. Ovomucoid is the major egg allergen but can inhibit the growth of tumors, and thus can be used as an anticancer agent. Hydrolyzed peptides from these proteins showed very good angiotensin I converting enzyme inhibitory, anticancer, metal binding, and antioxidant activities. Therefore, separation of egg white proteins and the productions of bioactive peptides from egg white proteins are emerging areas with many new applications.

Separation of ovotransferrin from chicken egg white without using organic solvents.

Ovotransferrin is one of the major egg white proteins that have antimicrobial activity as well as iron binding capability. The objective of this study was to develop a simple and easy method to separate ovotransferrin without using organic solvents. Egg white was separated from yolk, added in a 1:1 ratio to distilled water (DW), and then homogenized. The ovomucin in the diluted egg white was removed by centrifugation, adjusting the pH to 4.5 to 5.0. The resulting supernatant was added to different ratios of ammonium sulfate and citric acid, and then centrifuged after holding overnight at 4°C. The precipitant, which contains ovotransferrin, was dissolved in DW, and ovotransferrin was precipitated using different ratios of ammonium sulfate and citric acid. The precipitant collected after centrifugation was dissolved with DW and subjected to ultrafiltration to remove salts and concentrate the solution. The purity of the ovotransferrin was determined using SDS-PAGE, the protein identified using Western blot, and the estimated yield calculated by weighing the ovotransferrin after freeze drying. Over 85% purity and over 83% yield were obtained from the combinations of 5.0% (wt/vol) ammonium sulfate and 2.5% (wt/vol) citric acid followed by 2.0% (wt/vol) ammonium sulfate and 1.5% (wt/vol) citric acid. Activity of the ovotransferrin showed similar activity with previously separated ovotransferrin. However, this method is simpler and more cost effective than the previous method. The isolated ovotransferrin can be used as is or after modifications for various applications such as antimicrobial treatments, anticancer treatments, and iron-supplementing agents for humans.