SHORT COMMUNCATION

Antioxidant and Immune-Modulating Activities of Egg Yolk Protein Extracts

Jae Hoon Lee1https://orcid.org/0000-0002-7440-6842, Yunjung Lee2https://orcid.org/0000-0003-4726-4671, Hyun-Dong Paik3https://orcid.org/0000-0001-9891-7703, Eunju Park2,*https://orcid.org/0000-0002-3462-6090
Author Information & Copyright
1Research Group of Food Processing, Korea Food Research Institute, Wanju 55365, Korea
2Department of Food and Nutrition, Kyungnam University, Changwon 51767, Korea
3Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Korea
*Corresponding author : Eunju Park, Department of Food and Nutrition, Kyungnam University, Changwon, 51767, Korea, Tel: +82-55-249-2218, Fax: +82-505-999-2104, E-mail: pej@kyungnam.ac.kr

© Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Nov 25, 2021 ; Revised: Jan 05, 2022 ; Accepted: Jan 07, 2022

Published Online: Mar 01, 2022

Abstract

Egg yolk is widely used to extract lecithin, which is utilized in the food and cosmetics industry. After lecithin is removed, the rest of egg yolk is generated as a by-product. Thus, it is necessary to properly utilize it. In this study, egg yolk protein extracts were produced using ethanol (EYE-E) and water (EYE-W). Their antioxidant and immunomodulatory effects were then evaluated. Antioxidant activities of EYE-E and EYE-W were determined using cellular antioxidant capacity (CAC) assay and comet assay. EYE-E and EYE-W showed significant (p<0.05) scavenging effects on intracellular reactive oxygen species (ROS) in a dose dependent manner. At a concentration of 50 μg/mL, EYE-W showed higher (p<0.05) antioxidant activity than EYE-E. EYE-E and EYE-W also exhibited protective effects against DNA damage caused by oxidative stress. After treatment with EYE-E and EYE-W, DNA damage level of 48.7% due to oxidative stress was decreased to 36.2% and 31.8% levels, respectively. In addition, EYE-E and EYE-W showed immunomodulatory effects by regulating Th1 cytokines (TNF-α and IL-2) and Th2 cytokines (IL-10 and IL-4) in Balb/c mouse splenocytes. These data suggest that EYE-E and EYE-W could be used as functional food ingredients with excellent antioxidant and immunomodulatory activities in the food industry.

Keywords: egg yolk protein; antioxidant activity; immunomodulatory activity; HepG2; splenocyte

Introduction

Reactive oxygen species (ROS), including hydrogen peroxide, hydroxyl radicals, and superoxide anions, are representative products of cellular oxygen metabolism in all organisms (Wu et al., 2017). ROS play an important role in cellular signaling and homeostasis for physiological functions (Zhao et al., 2014). These ROS are maintained at an appropriate level by the body’s antioxidant defense system, including enzymatic and non-enzymatic antioxidant systems. Catalase, glutathione peroxidase, and superoxide dismutase are enzymatic antioxidant system. Glutathione, ascorbic acid, and carotenes are non-enzymatic system (Zhao et al., 2017). However, when a problem occurs in these systems, ROS are overproduced, causing oxidative stress on various biological molecules (e.g., lipids, proteins, and DNA) in the body, resulting in damage (Qian et al., 2020). ROS are known to be highly associated with the development of various diseases such as cardiovascular diseases, coronary heart diseases, cancer, atherosclerosis, and diabetes (Thetsrimuang et al., 2011; Wang et al., 2016; Wu et al., 2017).

Antioxidants including natural and synthetic antioxidants can neutralize free radicals and inhibit oxidative diffusion to remove overproduced ROS (He et al., 2019). Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are inexpensive with excellent antioxidant activity. However, toxicity and side effects can occur when they are consumed for a long time (André et al., 2010). Therefore, studies are being actively conducted to find a substance that is economical with excellent antioxidant activity from natural sources (Lorenzo et al., 2018). Natural antioxidants include anthocyanin, isoflavone, and bioactive protein and peptides derived from soybean, whey, plant, and eggs (Lorenzo et al., 2018; Pisoschi et al., 2021; Wen et al., 2020). Meanwhile, some researchers have reported a relationship between antioxidant and immune-modulating activity (Ji et al., 2020; Kim et al., 2020a). ROS produced by oxidative stress can influences inflammation by promoting the production of inflammatory mediators such as pro-inflammatory cytokines, anti-inflammatory cytokines and chemokines. Inflammatory response then repeats the production of ROS and causes excessive inflammatory reactions (Saisavoey et al., 2016). Therefore, it is important to study both antioxidant activity and immune-modulating activity (Harikrishnan et al., 2020; Ji et al., 2020).

Hen eggs not only provide excellent nutrition, but also contain various functional components, making them one of the most popular ingredients in the food and pharmaceutical industries as a functional raw material (Moreno-Fernández et al., 2020). In particular, various proteins present in egg white and yolk (ovalbumin, ovotransferrin, ovomucin, IgY, phosvitin, etc.) have been reported to have various functional activities such as antioxidant, anti-inflammatory, immune-enhancing, and anti-biofilm activities (Abeyrathne et al., 2016; Kim et al., 2020b; Kim et al., 2020c; Lee et al., 2018). Egg yolk protein is produced as a by-product after extracting egg yolk lecithin with an organic solvent. Egg yolk lecithin is used in the food and cosmetic industries (Peñaranda-López et al., 2020). Egg yolk protein by-products after lecithin extraction are mostly denatured proteins with limited values and functionalities due to solvents used during the lecithin extraction process (Eckert et al., 2014). Therefore, ethanol and water extraction was performed to extract useful substances from the egg yolk protein by-products, which is a widely used method due to its advantages of low cost and safety as a food grade-solvent (Liau et al., 2017; Liu et al., 2021; Mani-López et al., 2021).

In this study, egg yolk protein extracts were produced using ethanol and water to utilize by-products after extracting egg yolk lecithin. Antioxidant activities of egg yolk protein extracts were investigated by cellular antioxidant capacity (CAC) assay and comet assay. Their immune-modulating activities were investigated by analyzing their effects on inflammatory cytokine production in Balb/c mouse splenocytes.

Materials and Methods

Chemicals

Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute Medium 1640 (RPMI-1640), fetal bovine serum (FBS), penicillin, streptomycin, phosphate-buffered saline (PBS), and Hank’s balanced salt solution (HBSS) were obtained from HyClone Laboratories (Logan, MI, USA). Thiazolyl blue tetrazolium bromide (MTT), lipopolysaccharide (LPS), concanavalin A (ConA), 2′,7′-dichlorofluorescin diacetate (DCF-DA), 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA kit for analyzing cytokines (IL-2, IL-4, IL-10, and TNF-α) were obtained from BD Biosciences (San Diego, CA, USA), and all other reagents and chemicals used were analytical grade.

Preparation of egg yolk protein extracts

The by-product egg yolk protein produced after extracting the yolk lecithin used in this study was obtained from the Join (Egg processing company, Yongin, Korea). The residue of the lecithin extraction from whole egg yolk extracted using 95% ethanol was provided by the company in a dry state as a by-product. Egg yolk protein powder was extracted with ethanol and water in laboratory. Briefly, the ethanol extracts were added to 100 mL of ethanol (70%) to 5 g of egg yolk protein powder, and extracted for 72 h at room temperature. The water extract was extracted with autoclave (DW-AC-131, Dong Won Scientific, Seoul, Korea) by adding 100 mL of distilled water to 5 g of egg yolk protein powder. After extraction, the ethanol and water extracts were vacuum filtered through Whatman No. 1 filter paper (Whatman, Buckinghamshire, UK). The ethanol extract was evaporated under reduced pressure using rotary evaporator (EYELA N-1000, Tokyo Rikakikai, Tokyo, Japan) at 37°C to remove solvent. Then solvent free extract was freeze dried for 3 days. The water extract was directly freeze dried for 3 days. Samples were classified as EYE-E (egg yolk protein extract-ethanol) and EYE-W (egg yolk protein extract-water).

Antioxidant activity assay
HepG2 cell lines and cell viability

HepG2 cells were purchased from American Type Culture Collection (Rockville, MD, USA) and were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO2. The effects of EYE-E and EYE-W on HepG2 cell viability were determined using MTT assay (Lee et al., 2017a). HepG2 cells (5×104 cells/well) were seeded on a 96 well-plate and incubated for 24 h. Various concentrations of samples (1, 5, 10, 50, and 100 μg/mL) were treated in each well and incubated for additional 24 h. After then, MTT (2.5 mg/mL in PBS) solution was added to each well for additional 4 h. The supernatant was then removed from each well and dimethyl sulfoxide was added to each well to dissolve the MTT formazan. The absorbance of each well was measured using OPTIMA microplate reader (BMG LABTECH, Ortenberg, Germany) at 570 nm.

Cellular antioxidant capacity (CAC assay)

HepG2 cells were seeded at a density of 5×104 cells/well on a 96 well-plate to test CAC of egg yolk protein extracts following the method of Song et al. (2018). After 24 h incubation, the medium was replaced with HBSS buffer and EYE-E and EYE-W were added in each well that the final concentration was 1, 5, 10, and 50 μg/mL and incubated for 30 min. After incubation, each well was washed with HBSS buffer and 80 mM AAPH was added to the sample except for the negative control. After an additional 30 min incubation, 40 mM DCFH-DA was added to each well and incubated for 30 min in the dark. The degree of fluorescence was measured at excitation wavelength 485 nm, and emission wavelength 535 nm using OPTIMA microplate reader.

Single cell gel electrophoresis (Comet assay)

HepG2 cells (2×104 cells/mL) were washed with 1 mL of PBS and used in comet assay. The cells incubated with EYE-E and EYE-W at a concentration of 50 μg/mL at 37°C for 30 min in the dark. To stimulate oxidation stress, 200 μM H2O2 were treated to cells and incubated 5 min at 4°C, then washed with PBS. After incubation, 0.7% low melting agarose were mixed with cells and distributed over slides pre-coated with 1% normal melting agarose. Slides were placed in cold alkali lysis buffer (100 mM Na2EDTA, 2.5 M NaCl, 10 mM Tris, 1% sodium lauryl sarcosine, 1% Triton X-100, and 10% DMSO) for 1 h. Slides were then placed in a gel electrophoresis tank containing fresh electrophoresis buffer (300 mM NaOH, 10 mM Na2EDTA, pH 13.0) for 20 min and subjected to electrophoresis (25 V, 300 mA) for 20 min. After electrophoresis, slides were washed and then immersed in cold ethanol for 5 min and dried. Slides were stained with 20 μg/mL ethidium bromide. Finally, DNA damage image was obtained using fluorescence microscopy (LEICA DMLB, Wetzlar, Germany) and CCD camera (Nikon, Tokyo, Japan), and the image was analyzed using comet image analyzing system (Komet version 5.0, Kinetic Imaging, Liverpool, UK). We quantified head and tail intensity of samples using 50 cells from two replicate slides.

Immuno-modulating activity assay
Isolation and culture of mouse splenocytes

The experimental protocol was approved by the Institutional Animal Care of Kyungnam University (KUIAC-18-02). Male Balb/c mouse (five weeks old) were purchased from Koatech (Pyeongtaek, Korea). The animals were placed in wire mesh bottomed individual cages and housed in climate controlled quarter (23±2°C at 50% relative humidity) with a 12-h light/dark cycle. All animals were acclimatized for seven days before the experiment and were sacrificed by cervical dislocation. Obtained spleens were placed in RPMI 1640 medium. Single cell suspensions were prepared by pressing the tissue through 40 μm cell strainer (Corning, Corning, NY, USA). Cells were centrifuged 450×g, 4°C for 5 min to obtain splenocytes. Splenocytes were maintained in RPMI 1640 medium, supplemented with 10% FBS, 1% penicillin/streptomycin.

Cytokine production assay

Splenocytes were cultured at 1×106 cells/well in 96 well-plate for cytokines measurement. LPS and ConA were treated to splenocytes to 5 μg/mL of final concentration. Samples were treated to final concentration of 200 μg/mL. After that, splenocytes were incubated 5% CO2 for 24 h at 37°C and cytokines (IL-2, IL-4, IL-10, and TNF-α) were quantitated in supernatants. Cytokines production were conducted using Mouse ELISA kit and were measured at 450 nm.

Statistical analysis

All analysis was performed in triplicate. Results are presented as mean values±SD. The results were compared by analysis of one-way ANOVA followed by Duncan's multiple-range test using SPSS for Window ver. 20 (IBM, Armonk, NY, USA). Statistical significance was determined at p<0.05.

Results and Discussion

Antioxidant activities of egg yolk protein extracts using cellular antioxidant capacity (CAC) assay and comet assay

Before determine antioxidant activities of EYE-E and EYE-W, their effects on viabilities of HepG2 cells were determined using MTT assay (data not shown). EYE-W showed no significant (p>0.05) effect on HepG2 cell viability at concentrations of 1 to 100 μg/mL. However, EYE-E at the highest concentration of 100 μg/mL showed significant (p<0.05) cytotoxicity, leading to cell viability of 88.8±4.6% compared to the control (viability of 100%). Therefore, EYE-E and EYE-W were used at concentrations of 1 to 50 μg/mL in subsequent experiments.

The CAC assay is an experimental method widely used to verify the antioxidant activity of a test sample. The DCFH-DA probe used in this study can be cleaved to DCFH in cells and oxidized to DCF by cellular oxidants such as ROS. When intracellular ROS content is reduced by antioxidants, the oxidation of DCFH to DCF is reduced, allowing the antioxidant activity to be expressed as a quantitative value (Wolfe and Liu, 2007).

Results of cellular antioxidant capacities of EYE-E and EYE-W are shown in Fig. 1. When HepG2 cells were treated with AAPH as an oxidative stress, DCF fluorescence intensity was significantly (p<0.05) increased by 183.7±2.5% compared to the negative control (NC; without treatment). However, after treatment with EYE-E and EYE-W, DCF fluorescence intensity increased by AAPH was decreased significantly (p<0.05) in a dose-dependent manner. After treatment with EYE-E or EYE-W at the highest concentration of 50 μg/mL (Fig. 1C), DCF fluorescence intensity was 120.7±0.6% for the EYE-E group and 112.7±3.5% for the EYE-W group (reduction of 34% and 38%, respectively). These results demonstrate that both EYE-E and EYE-W possess antioxidant activities by scavenging intracellular ROS induced by AAPH treatment, with EYE-W showing a significantly (p<0.05) higher antioxidant activity than EYE-E at a concentration of 50 μg/mL.

kosfa-42-2-321-g1
Fig. 1. Cellular antioxidant capacity (CAC) of (A) EYE-E, (B) EYE-W, and (C) 50 μg/mL of egg yolk protein extracts. Data are presented as means±SD of triplicate measurements. Various corresponding letters indicate significant differences (p<0.05) by Duncan’s multiple range test. NC (negative control), treated with HBSS buffer; PC (positive control), treated with 80 mM AAPH, EYE-E, egg yolk protein extract-ethanol; EYE-W, egg yolk protein extract-water; AAPH, 2,2′-Azobis (2-methylpropionamidine) dihydrochloride.
Download Original Figure

Excess ROS can attack biological molecules, resulting in cell damage (Qian et al., 2020). Therefore, many studies involving antioxidants have focused on scavenging or controlling ROS levels in cells (He et al., 2019; Yi et al., 2017). Previous studies have reported that pretreatment with antioxidant materials obtained from various sources such as hen egg protein (Yi et al., 2017) and duck egg protein (He et al., 2019) can reduce the production of ROS induced by H2O2 stimulation in cells. Results of the present study suggest that EYE-E and EYE-W can reduce oxidative stress by scavenging intracellular ROS induced by H2O2 and consequently protect HepG2 cells. Next, comet assay was performed to determine whether EYE-E and EYE-W with ability of decrease oxidative stress by scavenging ROS might be effective in protecting against DNA damage. The comet assay, also known as a single cell electrophoresis assay, is an assay that can analyze the level of DNA damage induced by oxidative stress in eukaryotic cells (Liao et al., 2009). Decrease of DNA damage in HepG2 cells due to antioxidant activity of egg yolk protein extract was measured. Results are shown in Fig. 2. Tail DNA intensity was 3.9±0.5% for the NC and 48.7±1.7% for the positive control (PC), meaning that DNA of HepG2 cells was damaged by oxidative stress due to H2O2 treatment. When egg yolk protein extract treated at a concentration 50 μg/mL, the tail intensity was significantly (p<0.05) decreased to 36.2±0.3% for the EYE-E group and 31.8±1.7% for the EYE-W group than that in the PC.

kosfa-42-2-321-g2
Fig. 2. Egg yolk protein extracts protect cells against DNA damage. Data are presented as means±SD of triplicate measurements. Various corresponding letters indicate significant differences (p<0.05) by Duncan’s multiple range test. NC (negative control), treated with PBS buffer; PC (positive control), treated with 200 μM H2O2; EYE-E, 50 μg/mL of egg yolk protein extract-ethanol; EYE-W, 50 μg/mL of egg yolk protein extract-water.
Download Original Figure

DNA damage induced by ROS can threaten genome stability and plays a role in the pathogenesis of many diseases such as cancer and coronary heart disease. It also affects ageing (Odongo et al., 2019). Many antioxidant substances have been reported to be effective in protecting against DNA damage by scavenging ROS (Lee et al., 2017b; Pukalskienė et al., 2018). Many egg proteins (such as ovotransferrin, ovalbumin, ovomucin) and egg yolk protein hydrolysates also have antioxidant activities by scavenging ROS (Lee and Paik, 2019; Liu et al., 2020). Among egg proteins, phosvitin can effectively protect human leukocytes stimulated by H2O2 against DNA damage due to its excellent antioxidant activity (Moon et al., 2014). Results of this study suggest that various egg yolk proteins with antioxidant activities present in EYE-E and EYE-W could effectively scavenge ROS and possess protective effect against cellular DNA damage. In addition, a significant difference in antioxidant activity between the EYE-E and EYE-W samples was confirmed (p<0.05; Fig. 2). This is considered to be due to the difference in the components according to the extraction solvent of the two extracts. The protein contents of EYE-E and EYE-W were 1,762.4±8.0 mg/100 g and 2,761.2±28.8 mg/100 g. And the total phenol contents of EYE-E and EYE-W were 55.9±1.2 mg GAE (gallic acid equivalent)/100 g and 79.9±0.9 mg GAE/100 g, and the total flavonoid contents of EYE-E and EYE-W were 6.8 mg±0.0 mg QE (quercetin equivalent)/100 g and 5.8±0.0 mg QE/100 g (data not shown). In previous studies, it was reported that the antioxidants flavonoids and polyphenols were present in egg yolk, and it was reported that there was a difference in the content depending on the feed used for poultry breeding (Iskender et al., 2017; Omri et al., 2019). This suggests that the difference in the extraction of active ingredients according to the extraction solvent contributed to the difference in the antioxidant activity of EYE-E and EYE-W.

Immuno-modulating activities of egg yolk protein extracts by regulating cytokine production

Cytokines play an important role in hormonal mediation in the host defense system. They are small proteins secreted by a wide range of immune cells (Liu and Lin, 2013). Cytokines can be classified to Th1 and Th2 type cytokines with different roles in the immune system (Ku and Lin, 2013). Th1 type cytokines are recognized as pro-inflammatory cytokines that trigger local inflammation, whereas Th2 type cytokines are recognized as anti-inflammatory cytokines that inhibit the synthesis of Th1 cytokines (Liu and Lin, 2013). Therefore, when determining immunomodulatory activities of test samples, it is important to know their effects on the secretion of both Th1 and Th2 types of cytokines.

Effects of EYE-E and EYE-W on secretion of Th1 cytokines are shown in Fig. 3A and 3B. LPS and ConA were used as mitogens to stimulate splenocytes. The production of TNF-α was significantly (p<0.05) increased to 322.0±11.0 pg/mL after stimulation with LPS compared to the control (197.4±28.8 pg/mL). TNF-α is a representative Th1 cytokine that can stimulate an innate immune response (Thieringer et al., 2000). However, EYE-E and EYE-W treatments significantly decreased TNF-α production levels to 177.4±21.5 and 217.4±28.8 pg/mL, respectively (p<0.05). Levels of IL-2 production were significantly increased to 158.5±40.8 μg/mL in the presence of ConA (p<0.05). EYE-W treatment significantly reduced the level of IL-2 production to 44.9±2.2 μg/mL, whereas EYE-E treatment did not significantly affect IL-2 production in splenocytes. IL-2 is a pro-inflammatory cytokine that can regulate immune cells (e.g., T-cells, B-cells, and macrophages; Ku and Lin, 2013).

kosfa-42-2-321-g3
Fig. 3. Effects of EYE-E and EYE-W on Th1 and Th2 type cytokines from splenocytes in Balb/c mouse treated with LPS or ConA. Data are presented as means±SD of triplicate measurements. Various corresponding letters indicate significant (p<0.05) differences by Duncan’s multiple range test. NC (negative control), cell only; LPS or ConA, treated with LPS (5 μg/mL) or ConA (5 μg/mL); EYE-E, 200 μg/mL of egg yolk protein extract-ethanol; EYE-W, 200 μg/mL of egg yolk protein extract-water; ConA, concanavalin A.
Download Original Figure

Effects of EYE-E and EYE-W on secretion of Th2 cytokines are shown in Fig. 3C and 3D. In the presence of LPS or ConA, IL-10 and IL-4 secretion levels were significantly increased to 1,788±0.0 ng/mL and 64±3.1 ng/mL (p<0.05), respectively. However, EYE-E and EYE-W treatment significantly (p<0.05) decreased IL-10 and IL-4 secretion levels. IL-10 is produced in the later stages of inflammation. It can reduce the release of ROS with an ability to present antigens of mononuclear phagocytes (Kim et al., 2017). IL-4 is also a representative anti-inflammatory cytokine that can suppress the production of inflammatory cytokines and chemokines (Woodward et al., 2012). Similar to our results, Ku and Lin (2013) have reported that terpenoid compounds can inhibit the production of both IL-10 and IL-2 cytokine simultaneously. It has been suggested that some terpenoid compounds have immunomodulatory effects by modulating Th1/Th2 cytokine production.

In conclusion, this study showed that EYE-E and EYE-W obtained by utilizing by-products of egg proteins after extracting lecithin from egg yolk had significant antioxidant activities. Both EYE-E and EYE-W could protect cells against DNA damage caused by oxidative stress. Furthermore, EYE-E and EYE-W could effectively regulate the secretion of pro- and anti-inflammatory cytokines. Therefore, EYE-E and EYE-W could be used as a functional food ingredient with excellent antioxidant and immunomodulatory activities in the food industry.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Author Contributions

Conceptualization: Paik HD, Park E. Data curation: Lee JH, Lee Y. Formal analysis: Lee JH, Lee Y. Methodology: Lee JH, Lee Y. Software: Lee JH, Lee Y. Validation: Lee JH, Park E. Investigation: Park E. Writing - original draft: Lee JH. Writing - review & editing: Lee JH, Lee Y, Paik HD, Park E.

Ethics Approval

All animal experimental procedures were approved and reviewed by the Institutional Animal Care of Kyungnam University (KUIAC-18-02).

References

1.

Abeyrathne EDNS, Lee HY, Jo C, Suh JW, Ahn DU. 2016; Enzymatic hydrolysis of ovomucin and the functional and structural characteristics of peptides in the hydrolysates. Food Chem. 192:107-113

2.

André C, Castanheira I, Cruz JM, Paseiro P, Sanches-Silva A. 2010; Analytical strategies to evaluate antioxidants in food: A review. Trends Food Sci Technol. 21:229-246

3.

Eckert E, Zambrowicz A, Pokora M, Setner B, Dąbrowska A, Szołtysik M, Szewczuk Z, Polanowski A, Trziszka T, Chrzanowska J. 2014; Egg-yolk protein by-product as a source of ACE-inhibitory peptides obtained with using unconventional proteinase from Asian pumpkin (Cucurbita ficifolia). J Proteomics. 110:107-116

4.

Harikrishnan R, Thamizharasan S, Devi G, Van Doan H, Kumar TTA, Hoseinifar SH, Balasundaram C. 2020; Dried lemon peel enriched diet improves antioxidant activity, immune response and modulates immuno-antioxidant genes in Labeo rohita against Aeromonas sorbia. Fish Shellfish Immunol. 106:675-684

5.

He Y, Bu L, Xie H, Liang G. 2019; Antioxidant activities and protective effects of duck embryo peptides against H2O2-induced oxidative damage in HepG2 cells. Poult Sci. 98:7118-7128

6.

Iskender H, Yenice G, Dokumacioglu E, Kaynar O, Hayirli A, Kaya A. 2017; Comparison of the effects of dietary supplementation of flavonoids on laying hen performance, egg quality and egg nutrient profile. Br Poult Sci. 58:550-556

7.

Ji Z, Mao J, Chen S, Mao J. 2020; Antioxidant and anti-inflammatory activity of peptides from foxtail millet (Setaria italica) prolamins in HaCaT cells and RAW264.7 murine macrophages. Food Biosci. 36:100636

8.

Kim CY, Yu QM, Kong HJ, Lee JY, Yang KM, Seo JS. 2020a; Antioxidant and anti-inflammatory activities of Agrimonia pilosa Ledeb. extract. Evid Based Complement Altern Med. 2020:8571207

9.

Kim HJ, Kim D, Lee M, Jang A. 2017; Anti-inflammatory effect of dietary pork extract on proliferation and cytokine secretion using mouse primary splenocytes. Food Res Int. 102:710-716

10.

Kim HJ, Lee JH, Ahn DU, Paik HD. 2020b; Anti-biofilm effect of egg yolk phosvitin by inhibition of biomass production and adherence activity against Streptococcus mutans. Food Sci Anim Resour. 40:1001-1013

11.

Kim HS, Lee JH, Moon SH, Ahn DU, Paik HD. 2020c; Ovalbumin hydrolysates inhibit nitric oxide production in LPS-induced RAW 264.7 macrophages. Food Sci Anim Resour. 40:274-285

12.

Ku CM, Lin JY. 2013; Anti-inflammatory effects of 27 selected terpenoid compounds tested through modulating Th1/Th2 cytokine secretion profiles using murine primary splenocytes. Food Chem. 141:1104-1113

13.

Lee JH, Ahn DU, Paik HD. 2018; In vitro immune-enhancing activity of ovotransferrin from egg white via MAPK signaling pathways in RAW 264.7 macrophages. Korean J Food Sci Anim Resour. 38:1226-1236

14.

Lee JH, Moon SH, Kim HS, Park E, Ahn DU, Paik HD. 2017a; Antioxidant and anticancer effects of functional peptides from ovotransferrin hydrolysates. J Sci Food Agric. 97:4857-4864

15.

Lee JH, Paik HD. 2019; Anticancer and immunomodulatory activity of egg proteins and peptides: A review. Poult Sci. 98:6505-6516

16.

Lee JH, Park E, Jin HJ, Lee Y, Choi SJ, Lee GW, Chang PS, Paik HD. 2017b; Anti-inflammatory and anti-genotoxic activity of branched chain amino acids (BCAA) in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. Food Sci Biotechnol. 26:1371-1377

17.

Liao W, McNutt MA, Zhu WG. 2009; The comet assay: A sensitive method for detecting DNA damage in individual cells. Methods. 48:46-53

18.

Liau BC, Ponnusamy VK, Lee MR, Jong TT, Chen JH. 2017; Development of pressurized hot water extraction for five flavonoid glycosides from defatted Camellia oleifera seeds (byproducts). Ind Crops Prod. 95:296-304

19.

Liu CJ, Lin JY. 2013; Anti-inflammatory effects of phenolic extracts from strawberry and mulberry fruits on cytokine secretion profiles using mouse primary splenocytes and peritoneal macrophages. Int Immunopharmacol. 16:165-170

20.

Liu Y, Benohoud M, Yamdeu JHG, Gong YY, Orfila C. 2021; Green extraction of polyphenols from citrus peel by-products and their antifungal activity against Aspergillus flavus. Food Chem X. 12:100144

21.

Liu Y, Sheng L, Ma M, Jin Y. 2020; Proteome-based identification of chicken egg yolk proteins associated with antioxidant activity on the Qinghai-Tibetan Plateau. Int J Biol Macromol. 150:1093-1103

22.

Lorenzo JM, Munekata PES, Gómez B, Barba FJ, Mora L, Pérez-Santaescolástica C, Toldrá F. 2018; Bioactive peptides as natural antioxidants in food products: A review. Trends Food Sci Technol. 79:136-147

23.

Mani-López E, Palou E, López-Malo A. 2021; Legume proteins, peptides, water extracts, and crude protein extracts as antifungals for food applications. Trends Food Sci Technol. 112:16-24

24.

Moon SH, Lee JH, Lee M, Park E, Ahn DU, Paik HD. 2014; Cytotoxic and antigenotoxic activities of phosvitin from egg yolk. Poult Sci. 93:2103-2107

25.

Moreno-Fernández S, Garcés-Rimón M, Miguel M. 2020; Egg-derived peptides and hydrolysates: A new bioactive treasure for cardiometabolic diseases. Trends Food Sci Technol. 104:208-218

26.

Odongo GA, Skatchkov I, Herz C, Lamy E. 2019; Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block. DNA Repair. 77:58-64

27.

Omri B, Alloui N, Durazzo A, Lucarini M, Aiello A, Romano R, Santini A, Abdouli H. 2019; Egg yolk antioxidants profiles: Effect of diet supplementation with linseeds and tomato-red pepper mixture before and after storage. Foods. 8:320

28.

Peñaranda-López AL, Brito-de la Fuente E, Torrestiana-Sánchez B. 2020; Fractionation of hydrolysates from concentrated lecithin free egg yolk protein dispersions by ultrafiltration. Food Bioprod Process. 123:209-216

29.

Pisoschi AM, Pop A, Iordache F, Stanca L, Predoi G, Serban AI. 2021; Oxidative stress mitigation by antioxidants: An overview on their chemistry and influences on health status. Eur J Med Chem. 209:112891

30.

Pukalskienė M, Slapšytė G, Dedonytė V, Lazutka JR, Mierauskienė J, Venskutonis PR. 2018; Genotoxicity and antioxidant activity of five Agrimonia and Filipendula species plant extracts evaluated by comet and micronucleus assays in human lymphocytes and Ames Salmonella/microsome test. Food Chem Toxicol. 113:303-313

31.

Qian L, Liu H, Li T, Liu Y, Zhang Z, Zhang Y. 2020; Purification, characterization and in vitro antioxidant activity of a polysaccharide AAP–3–1 from Auricularia auricula. Int J Biol Macromol. 162:1453-1464

32.

Saisavoey T, Sangtanoo P, Reamtong O, Karnchanatat A. 2016; Antioxidant and anti-inflammatory effects of defatted rice bran (Oryza sativa L.) protein hydrolysates on raw 264.7 macrophage cells. J Food Biochem. 40:731-740

33.

Song HY, Kim HM, Kim WS, Byun EH, Jang BS, Choi DS, Byun EB. 2018; Effect of gamma irradiation on the anti-oxidant and anti-melanogenic activity of black ginseng extract in B16F10 melanoma cells. Radiat Phys Chem. 149:33-40

34.

Thetsrimuang C, Khammuang S, Chiablaem K, Srisomsap C, Sarnthima R. 2011; Antioxidant properties and cytotoxicity of crude polysaccharides from Lentinus polychrous Lév. Food Chem. 128:634-639

35.

Thieringer R, Fenyk-Melody JE, Le Grand CB, Shelton BA, Detmers PA, Somers EP, Carbin L, Moller DE, Wright SD, Berger J. 2000; Activation of peroxisome proliferator-activated receptor γ does not inhibit IL-6 or TNF-α responses of macrophages to lipopolysaccharide in vitro or in vivo. J Immunol. 164:1046-1054

36.

Wang L, Ding L, Yu Z, Zhang T, Ma S, Liu J. 2016; Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells. Food Res Int. 90:33-41

37.

Wen C, Zhang J, Zhang H, Duan Y, Ma H. 2020; Plant protein-derived antioxidant peptides: Isolation, identification, mechanism of action and application in food systems: A review. Trends Food Sci Technol. 105:308-322

38.

Wolfe KL, Liu RH. 2007; Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agric Food Chem. 55:8896-8907

39.

Woodward EA, Kolesnik TB, Nicholson SE, Prêle CM, Hart PH. 2012; The anti-inflammatory actions of IL-4 in human monocytes are not mediated by IL-10, RP105 or the kinase activity of RIPK2. Cytokine. 58:415-423

40.

Wu J, Huo J, Huang M, Zhao M, Luo X, Sun B. 2017; Structural characterization of a tetrapeptide from sesame flavor-type Baijiu and its preventive effects against AAPH-induced oxidative stress in HepG2 cells. J Agric Food Chem. 65:10495-10504

41.

Yi J, Zhao J, Wu J. 2017; Egg ovotransferrin derived IRW exerts protective effect against H2O2-induced oxidative stress in Caco-2 cells. J Funct Foods. 39:160-167

42.

Zhao L, Su J, Li L, Chen J, Hu S, Zhang X, Chen T. 2014; Mechanistic elucidation of apoptosis and cell cycle arrest induced by 5-hydroxymethylfurfural, the important role of ROS-mediated signaling pathways. Food Res Int. 66:186-196

43.

Zhao M, Yang Q, Lin L, Sun B, Wang Y. 2017; Intracellular antioxidant activities of selected cereal phenolic extracts and mechanisms underlying the protective effects of adlay phenolic extracts on H2O2-induced oxidative stress in human erythrocytes. J Funct Foods. 31:160-171

Call for Special Section


We are pleased to invite you a paper for Food Science of Animal Resources for a special issue “New concept of probiotics for human and animal health”. Both research and review articles are welcome for possible publication in this issue. Both research and review articles are welcome for possible publication in this section. All manuscript will be peer-reviewed before their acceptance for publication.

Edited by Dr. Seok-Seong Kang
- Deadline for manuscript submissions: 30 June 2022
- We offer 50% reduction of the article processing charge and provide the fast review process (approximately 1 month).

The specific topics of interest for the special issue include:
 • New probiotics and alternative probiotics such as postbiotics
 • Probiotics/postbiotics for human and animal health
 • Probiotics/postbiotics and metabolic disorders
 • Probiotics/postbiotics and gut microbiome balance
 • The next generation of probiotics


I don't want to open this window for a day.