Separation of ovotransferrin from chicken egg white without using organic solvents

Abeyrathne, E. D. N. S.,Lee, H. Y.,Ham, J. S.,Ahn, D. U. Poultry Science Association 2013 Poultry science Vol.92 No.4

<P>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.</P>

Utilization of ferroproteins by Candida albicans during candidastasis by apo-ovotransferrin

한용문 동덕여자대학교 종합약학연구소 2001 동덕약학연구지 Vol.5 No.-

Recent reports have shown that some pathogenic bacteria can obtain iron from ferroproteins, such as cytochrome C, ferritin, hemin, hemoglobin and myoglobin. These studies prompted to determine if the opportunistic pathogenic fungi, Candida albicans, can utilize ferroproteins to circumvent the iron-regulatory effect of ovotransferrin. Assays to measure growth stimulation in vitro by the ferroproteins were conducted as follows: C. albicans was cultured in iron-free (pretreated with apo-ovotransferrin for 24 h) culture medium. Once C. albicans yeast cell growth reached stasis from iron starvation, individual ferroproteins were added to the culture media. The results suggested that hemin, hemoglobin, and myoglobin were able to support a partial growth recovery. Additional studies with haptoglobin, a serum protein that interacts with the globin moiety of certain ferroproteins, established that C. albicans was able to obtain iron from the haptoglobin-ferroprotein complexes. This result suggests that the heme group may be the iron source from the ferroproteins.

Antioxidant, Antimicrobial, and Cytotoxic Activities of Ovotransferrin from Egg White

Sun Hee Moon,Jae Hoon Lee,Yong Ju Lee,Ji Yeon Paik,Dong Uk Ahn,Hyun Dong Paik 한국축산식품학회 2012 한국축산식품학회지 Vol.32 No.5

The antioxidant, antimicrobial, and cytotoxic activities of ovotransferrin were investigated in vitro. The antioxidant capacity of ovotransferrin was evaluated using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging method, antimicrobial effects using the agar well diffusion method, and cytotoxicity using the 3 (4,5-dimethylthizol-2-yl)-2,5-diphenylatetetrazolium bromide (MTT) assay. The DPPH radical-scavenging capacity of ovotransferrin at 1 mg/mL level reached approximately 60% after 48 h of reaction. The antimicrobial effects of ovotransferrin against common food-borne pathogens, Staphylococcus aureus KCCM 32395, Bacillus cereus KCCM 40935, Listeria monocytogenes ATCC 15313, Escherichia coli O157:H7 ATCC 43895, and Helicobacter pylori HpKCTC 26695 were dose dependant. Gram positive bacteria was more sensitive to ovotransferrin than gram-negative bacteria. Ovotransferrin showed stronger antimicrobial effect against L. monocytogenes than other gram-positive bacteria tested. The cytotoxicity of ovotransferrin was evaluated in human cancer cell lines, various tissue origins, including the larynx (Hep-2), stomach (AGS), lung (SK-MES 1), liver (HepG2), breast (MCF-7), cervix (HeLa), and colon (HT-29). Ovotransferrin displayed relatively high cytotoxicity (≤60% inhibition effects) at 40 mg/mL. At lower concentrations (≤10 mg/mL), however, ovotransferrin cytotoxic effects were not significant in all cancer cell lines tested. These results indicated that ovotransferrin has potential to be used as an antioxidant or antimicrobial agent in foods or a pharmaceutical agent against cancers.

Antioxidant, Antimicrobial, and Cytotoxic Activities of Ovotransferrin from Egg White

Moon, Sun-Hee,Lee, Jae-Hoon,Lee, Yong-Ju,Paik, Ji-Yeon,Ahn, Dong-Uk,Paik, Hyun-Dong Korean Society for Food Science of Animal Resource 2012 한국축산식품학회지 Vol.32 No.5

The antioxidant, antimicrobial, and cytotoxic activities of ovotransferrin were investigated in vitro. The antioxidant capacity of ovotransferrin was evaluated using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging method, antimicrobial effects using the agar well diffusion method, and cytotoxicity using the 3-(4,5-dimethylthizol-2-yl)-2,5-diphenylatetetrazolium bromide (MTT) assay. The DPPH radical-scavenging capacity of ovotransferrin at 1 mg/mL level reached approximately 60% after 48 h of reaction. The antimicrobial effects of ovotransferrin against common food-borne pathogens, Staphylococcus aureus KCCM 32395, Bacillus cereus KCCM 40935, Listeria monocytogenes ATCC 15313, Escherichia coli O157:H7 ATCC 43895, and Helicobacter pylori HpKCTC 26695 were dose dependant. Gram-positive bacteria was more sensitive to ovotransferrin than gram-negative bacteria. Ovotransferrin showed stronger antimicrobial effect against L. monocytogenes than other gram-positive bacteria tested. The cytotoxicity of ovotransferrin was evaluated in human cancer cell lines, various tissue origins, including the larynx (Hep-2), stomach (AGS), lung (SK-MES-1), liver (HepG2), breast (MCF-7), cervix (HeLa), and colon (HT-29). Ovotransferrin displayed relatively high cytotoxicity (${\leq}60%$ inhibition effects) at 40 mg/mL. At lower concentrations (${\leq}10mg/mL$), however, ovotransferrin cytotoxic effects were not significant in all cancer cell lines tested. These results indicated that ovotransferrin has potential to be used as an antioxidant or antimicrobial agent in foods or a pharmaceutical agent against cancers.

Antioxidant, Antimicrobial, and Cytotoxic Activities of Ovotransferrin from Egg White

문선희,이재훈,이용주,백지연,안동욱,백현동 한국축산식품학회 2012 한국축산식품학회지 Vol.32 No.5

The antioxidant, antimicrobial, and cytotoxic activities of ovotransferrin were investigated in vitro. The antioxidant capacity of ovotransferrin was evaluated using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging method, antimicrobial effects using the agar well diffusion method, and cytotoxicity using the 3-(4,5-dimethylthizol-2-yl)-2,5-diphenylatetetrazolium bromide (MTT) assay. The DPPH radical-scavenging capacity of ovotransferrin at 1 mg/mL level reached approximately 60% after 48 h of reaction. The antimicrobial effects of ovotransferrin against common food-borne pathogens, Staphylococcus aureus KCCM 32395, Bacillus cereus KCCM 40935, Listeria monocytogenes ATCC 15313, Escherichia coli O157:H7ATCC 43895, and Helicobacter pylori HpKCTC 26695 were dose dependant. Gram-positive bacteria was more sensitive to ovotransferrin than gram-negative bacteria. Ovotransferrin showed stronger antimicrobial effect against L. monocytogenes than other gram-positive bacteria tested. The cytotoxicity of ovotransferrin was evaluated in human cancer cell lines, various tissue origins, including the larynx (Hep-2), stomach (AGS), lung (SK-MES-1), liver (HepG2), breast (MCF-7), cervix (HeLa), and colon (HT-29). Ovotransferrin displayed relatively high cytotoxicity (≤60% inhibition effects) at 40 mg/mL. At lower concentrations (≤10 mg/mL), however, ovotransferrin cytotoxic effects were not significant in all cancer cell lines tested. These results indicated that ovotransferrin has potential to be used as an antioxidant or antimicrobial agent in foods or a pharmaceutical agent against cancers.

Enzymatic Hydrolysis of Ovotransferrin and the Functional Properties of Its Hydrolysates

Ethige Chathura Nishshanka Rathnapala,Dong Uk Ahn,Edirisingha Dewage Nalaka Sandun Abeyrathne 한국축산식품학회 2021 한국축산식품학회지 Vol.41 No.4

Bioactive peptides have great potentials as nutraceutical and pharmaceutical agents that can improve human health. The objectives of this research were to produce functional peptides from ovotransferrin, a major egg white protein, using single enzyme treatments, and to analyze the properties of the hydrolysates produced. Lyophilized ovotransferrin was dissolved in distilled water at 20 mg/mL, treated with protease, elastase, papain, trypsin, or α-chymotrypsin at 1% (w/v) level of substrate, and incubated for 0-24 h at the optimal temperature of each enzyme (protease 55℃, papain 37℃, elastase 25℃, trypsin 37℃, α-chymotrypsin 37℃). The hydrolysates were tested for antioxidant, metal-chelating, and antimicrobial activities. Protease, papain, trypsin, and α-chymotrypsin hydrolyzed ovotransferrin relatively well after 3 h of incubation, but it took 24 h with elastase to reach a similar degree of hydrolysis. The hydrolysates obtained after 3 h of incubation with protease, papain, trypsin, α-chymotrypsin, and after 24 h with elastase were selected as the best products to analyze their functional properties. None of the hydrolysates exhibited antioxidant properties in the oil emulsion nor antimicrobial property at 20 mg/mL concentration. However, ovotransferrin with α-chymotrypsin and with elastase had higher Fe3+-chelating activities (1.06±0.88%, 1.25±0.24%) than the native ovotransferrin (0.46±0.60%). Overall, the results indicated that the single-enzyme treatments of ovotransferrin were not effective to produce peptides with antioxidant, antimicrobial, or Fe3+-chelating activity. Further research on the effects of enzyme combinations may be needed.

In Vitro Immune-Enhancing Activity of Ovotransferrin from Egg White via MAPK Signaling Pathways in RAW 264.7 Macrophages

Jae Hoon Lee,Dong Uk Ahn,Hyun-Dong Paik 한국축산식품학회 2018 한국축산식품학회지 Vol.38 No.6

Ovotransferrin (OTF) is a well-known protein of the transferrin family with strong iron chelating activity, resulting in its antimicrobial activity. Furthermore, OTF is known to have antioxidant, anticancer, and antihypertensive activities. However, there have been few studies about the immune-enhancing activity of OTF. In current study, we investigated the immune-enhancing activity of OTF using the murine macrophage cells in vitro. The effect of OTF on production of pro-inflammatory mediators and cytokines were determined using Griess assay and quantitative real-time PCR. Using Neutral Red uptake assay, we confirmed the effect of OTF on phagocytic activity of macrophages. Ovotransferrin significantly increased the production of nitric oxide (NO) and secretion of inducible nitric oxide synthase (iNOS) mRNA with no cytotoxic activity. Ovotransferrin (2 mg/mL) stimulated NO production up to 31.9±3.5 μM. Ovotransferrin significantly increased the mRNA expression levels of pro-inflammatory cytokines which are tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), and IL-6: OTF (2 mg/mL) treatment increased the secretion of mRNA for TNF-α, IL-1β, and IL-6 by 22.20-, 37.91-, and 6.17-fold of the negative control, respectively. The phagocytic activity of macrophages was also increased by OTF treatment significantly compared with negative control. Also, OTF treatment increased phosphorylation level of MAPK signaling pathways. These results indicated that OTF has immune-enhancing activity by activating RAW 264.7 macrophages via MAPK pathways.

Functional properties of ovotransferrin from chicken egg white and its derived peptides: a review

Ethige Chathura Nishshanka Rathnapala,안동욱,Sandun Abeyrathne 한국식품과학회 2021 Food Science and Biotechnology Vol.30 No.5

With emerging trends in the food and pharmaceuticalindustries, potential applications of egg-derivedbioactive compounds were recognized. Ovotransferrin is amajor egg white functional protein responsible for multiplebioactivities. The objectives of this review are to providescientific evidence of the functional properties of chickenovotransferrin and its derived peptides and to identifyfuture research approaches and applications. Various easy,economical, and non-toxic methods have been reported toproduce ovotransferrin with high yield and purity, andchemical and enzymatic approaches have been employedto release bioactive peptides. The native ovotransferrin isknown to have antimicrobial, antioxidant, anticancer, andimmunomodulatory activities. The peptides produced fromovotransferrin also are reported to have antioxidant,antimicrobial, antihypertensive, and anticancer properties. However, little or no application of these compounds in thefood and pharmaceutical areas is available yet. Therefore,the practical application of OTF in nutraceutical andpharmaceutical areas are among the emerging areas ofresearch.

Improved immune-enhancing activity of egg white protein ovotransferrin after enzyme hydrolysis

( Jae Hoon Lee ),( Hyeon Joong Kim ),( Dong Uk Ahn ),( Hyun-dong Paik ) 한국축산학회 2021 한국축산학회지 Vol.63 No.5

Ovotransferrin (OTF), an egg protein known as transferrin family protein, possess strong antimicrobial and antioxidant activity. This is because OTF has two iron binding sites, so it has a strong metal chelating ability. The present study aimed to evaluate the improved immune-enhancing activities of OTF hydrolysates produced using bromelain, pancreatin, and papain. The effects of OTF hydrolysates on the production and secretion of pro-inflammatory mediators in RAW 264.7 macrophages were confirmed. The production of nitric oxide (NO) was evaluated using Griess reagent and the expression of inducible nitric oxide synthase (iNOS) were evaluated using quantitative real-time polymerase chain reaction (PCR). And the production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and the phagocytic activity of macrophages were evaluated using an ELISA assay and neutral red uptake assay, respectively. All OTF hydrolysates enhanced NO production by increasing iNOS mRNA expression. Treating RAW 264.7 macrophages with OTF hydrolysates increased the production of pro-inflammatory cytokines and the phagocytic activity. The production of NO and pro-inflammatory cytokines induced by OTF hydrolysates was inhibited by the addition of specific mitogen-activated protein kinase (MAPK) inhibitors. In conclusion, results indicated that all OTF hydrolysates activated RAW 264.7 macrophages by activating MAPK signaling pathway.

Improvement of Functional Properties of Ovotransferrin by Phosphorylation through Dry-heating in the Presence of Pyrophosphate

Hayashi, Yoko,Li, Can-Peng,Enomoto, Hirofumi,Ibrahim, Hisham R.,Sugimoto, Yasushi,Aoki, Takayoshi Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.4

Ovotransferrin (OTf) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4.0 and $85^{\circ}C$ for 1 and 5 d, and the functional properties of phosphorylated OTf (PP-OTf) were investigated. The phosphorus content of OTf increased to 0.91% as a result of phosphorylation and the electrophoretic mobility of PP-OTf also increased. Although the solubility of dry-heated OTf slightly decreased, the decrease was reduced by phosphorylation. The stability against heat-induced insolubilization of OTf was somewhat improved by phosphorylation, but more than 70% of PP-OTf was insolubilized when it was heated at $70^{\circ}C$ for 10 min at pH 7.0. However, heat-induced insolubilization of PP-OTf was reduced when it was heated in the presence of phosphorylated ovalbumin. This may explain the excellent stability of phosphorylated egg white protein against heat-induced insolubilization which was reported previously. The emulsifying property of OTf was also somewhat improved by phosphorylation. The calcium phosphate-solubilizing ability of PP-OTf was enhanced. Although the degree of phosphorylation of OTf by dry-heating in the presence of pyrophosphate was similar to that of ovalbumin, the improvement of properties of PP-OTf was considerably different from those of phosphorylated ovalbumin.