Antioxidant and anti-inflammatory properties of chicken egg vitelline membrane hydrolysates

Research Note Antioxidant and anti-inflammatory properties of chicken egg vitelline membrane hydrolysates Dillon Lee, Fatemeh Bamdad, Kevin Khey, and Hoon H. Sunwoo1 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 8613-114 St, Edmonton, Alberta, Canada T6G 2H7 ABSTRACT Vitelline membrane (VM) is a multilayered structure that surrounds the egg yolk serving to separate the yolk and the white. Due to its poor solubility in aqueous-based media, VM proteins and their biological properties have not been fully defined. In the current study, VM was hydrolyzed using different enzymes under the optimum hydrolysis conditions. Antioxidant and anti-inflammatory properties were evaluated in chemical and cellular models. Flavourzyme- and trypsin-treated samples showed the highest radical scavenging and ferric ion reducing effect (31% and 20 µM of Trolox equivalents/mg,

respectively). In cellular studies, all VM hydrolysates were cyto-compatible and inhibited nitric oxide production by RAW264.7 macrophage cells significantly. Lipopolysaccharide-stimulated upregulation of pro-inflammatory cytokines in RAW264.7 cells was suppressed by flavourzyme-treated VM. These results revealed that enzymatic hydrolysis of VM is a promising approach to produce peptides with several bioactivities (free radical scavenging, metal chelation, and anti-inflammatory) as valuable ingredients for cosmeceuticals and nutraceuticals.

Key words: Vitelline membrane, peptide, enzymatic hydrolysis, antioxidant, anti-inflammatory effect 2017 Poultry Science 96:3510–3516 http://dx.doi.org/10.3382/ps/pex125

INTRODUCTION In 2015, Canada produced about 700 million dozen eggs, with table eggs representing approximately 73% while the remaining 27% were processed as egg products (Agriculture and Agri-Food Canada, 2016). In addition, Canada exported 8.2 million kilograms of processed eggs. Processed egg production results in various types of by-products including eggshells, shell membranes, and yolk vitelline membranes (VMs), all of which are usually dumped into the environment as waste. Such disposal in large quantities can cause soil and ground water contamination and nitrogen release into the surrounding area (Glatz et al., 2011). Thus, proper waste management and value-added by-product utilization contribute to a sustainable poultry and egg industry by minimizing the environmental impact of waste and by increasing revenue through commercializing the value-added waste products. Eggshells are often used as biodegradable soil fertilizers and a calcium source in laying hen feed (Glatz et al., 2011), while the membranes separated from eggshells  C 2017 Poultry Science Association Inc. Received December 9, 2016. Accepted April 25, 2017. 1 Corresponding author: [email protected]

are included in cosmetics. About 10% of the shell membrane is collagen. The membrane proteins have been used to grow human skin fibroblasts for severe burn situations (Maeda and Sasaki, 1982). Many studies have also investigated the biological activities of eggshell components, specifically their antimicrobial properties (Rose-Martel, 2013). However, little is known about the biological activities and pharmaceutical potential of other processed egg by-products such as VM. Vitelline membrane is a multilayered structure that surrounds the egg yolk serving to separate the yolk and white (Back et al., 1982; Mann, 2008) to maintain osmotic balance as a diffusion barrier (Trziszka and Smolinska, 1982). VM is also the final barrier to bacterial infection (Mann, 2008). The membrane is composed of about 87% protein, 10% carbohydrate, and 3% lipid (Back et al., 1982). VM contains 3 layers, where a granular continuous membrane (lamina continua) is sandwiched between 2 fibrous layers: the inner and outer layers. The inner layer (lamina perivitellina) is in contact with the yolk and contains sodium dodecyl sulphate (SDS)-soluble glycoproteins (GP-I, GP-II, and GP-III) and zona pellucida proteins (ZPC/ZP3, ZP1, and ZPD) (Back et al., 1982; Mann, 2008). The outer layer (lamina extravitellina), that faces the egg albumen, is mainly composed of lysozyme, ovomucin, and VM outer proteins I and II (VMO-I and VMO-II) (Mann, 2008; Kido

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and Doi, 1988). Back et al. (1982) has suggested that the outer layer may serve as an antibacterial barrier because of its lysozyme content. Oxidative damage occurs when the body’s natural defense system fails to detoxify superfluous reactive oxygen species (ROS). Many research studies have been conducted to identify means to prevent, delay, or even repair oxidative damage in biological systems. Although ROS, such as the superoxide anion, hydrogen peroxide, and hydroxyl radical, are natural by-products of metabolism, the overproduction of these species in immune cells results in over-activation of inflammatory responses, which is detrimental to cells and organisms (Thomson et al., 1998). Egg-derived antioxidants currently receive worldwide attention because they are believed to be safe and effective in alleviating oxidative stress and subsequent damage. Antioxidant peptides, owing to their different amino acid constituents, inhibit oxidative damages to biological molecules through multiple pathways such as free radical scavenging, metal ion chelation and inactivation of ROS (Bamdad & Chen, 2013). Peptides produced from egg white and yolk proteins have shown antioxidant properties through various mechanisms (Manso et al., 2008). Although the VM structure and protein sequences have been identified previously, the biological significance of the VM proteins are presently unknown. In this study, we evaluated the antioxidant and antiinflammatory properties of VM and its hydrolysis products. This is the first report showing the potential of the VM and its enzymatic hydrolysates as antiinflammatory and antioxidant agents.

MATERIALS AND METHODS Materials Alcalase (Alc) and flavourzyme (Fla) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Trypsin (Try) was obtained from Thermo Fisher (Waltham, MA). Mini-PROTEAN Tris-Tricine precast gels and Precision Plus Dual Xtra protein standard (15-250 kDa) were obtained from Bio-Rad (Mississauga, ON, Canada). The mouse macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin/fungizone. Lipopolysaccharide (LPS) from E. coli 0111:B4 was purchased from SigmaAldrich. Cell culture reagents (i.e., DMEM, FBS, penicillin/streptomycin/fungizone, glutamine, phosphatebuffered saline, ultrapure distilled water, and TRIzol) were purchased from Invitrogen (Carlsbad, CA). AllIn-One RT MasterMix (5×) and EvaGreen (2×) qPCR MasterMix-Low ROX were from Applied Biological Materials (Richmond, BC, Canada). All primers were ordered from IDT (Coralville, IA).

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Isolation and Solubilisation of VM Eggs from White Leghorn laying hens were purchased from a local market in Edmonton, Canada. Eggs (approx. 100 dozen) were cracked, the yolk was carefully separated from albumen, and gently washed with distilled water. After chalaza was removed, the cleaned yolks were punctured and the yolk content was squeezed out carefully. The VM was rinsed with distilled water at room temperature to remove yolk material, lyophilized, and ground to obtain VM powder. Based on preliminary experiments, 1% SDS was selected as the appropriate solvent for VM powder. Five grams of VM powder were dissolved in 20 mL of 1% SDS and sonicated (42 Hz) in a water bath at 25◦ C for 5 min, followed by enzymatic digestion.

Enzymatic Hydrolysis of VM VM powder (10 mg/mL) was digested by Alc, Fla, and Try individually, for 24 h at a 1:100 enzyme to substrate ratio. All hydrolysis treatments were conducted in triplicate. The optimal pH and temperature condition for each enzyme, according to the manufacturer, was used. The same condition was used in the absence of enzyme to act as the control. At the end of hydrolysis, samples were heated at 100◦ C for 10 min to inactivate the enzyme and then centrifuged (4,000 × g, 20 min) to separate the insoluble material. The supernatants were lyophilized and kept at –20◦ C until analyzed.

Determination of the Degree of Hydrolysis The degree of hydrolysis (DH) was analyzed in triplicate by measuring free primary amines by the o-phthaldialdehyde (OPA) fluorometric assay with L-serine as the standard (Benkhelifa et al., 2005). The total content of amine groups, expressed as serine equivalents (mEq/L), was obtained from a standard curve prepared from different L-serine concentrations. The DH was calculated using the following equation: DH = (h/htot ) × 100

(1)

where, hydrolysis equivalents (h) is the amount of peptide bonds cleaved during hydrolysis, expressed as millimole serine equivalents per gram protein (mmoL/g protein); htot is the total amount of peptide bonds in the protein substrate determined from the protein sample totally hydrolyzed with 6 N HCl at 110◦ C for 24 h.

SDS Polyacrylamide Gel Electrophoresis Gel electrophoresis of VM proteins was performed according to the method of Schaegger (2006) using a Mini-PROTEAN electrophoresis kit with Tris-Tricine Precast 12% polyacrylamide gels. Precision Plus Dual Xtra protein standard was used as the molecular weight

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marker. Gels were stained with 0.05% Coomassie Blue R-250 and de-stained with 7% acetic acid. The gel was scanned with a Gel DocTM EZ Imager (Bio-Rad).

Antioxidant Assays 1,1-Diphenyl-2-Picryl Hydrazyl Radical Scavenging Assay The 1,1-diphenyl-2-picryl hydrazyl(DPPH-) free radical scavenging activity of VM hydrolysate (VMH) samples at 0.1 mg/mL was assessed in triplicate according to the method described previously (Sakanaka and Tachibana, 2006). Glutathione (GSH) was used as the positive control. Radical scavenging activity of the hydrolysates was calculated according to the following equation: % DPPH free radical scavenging = 1 − (As /Ac ) ×100 where, As and Ac represent the absorbance of the sample and control, respectively. Superoxide Radical Scavenging Assay In the superoxide radical scavenging assay, 80 μL of VMH samples (1 mg/mL) were mixed with 80 μL of 50 mM TrisHCl buffer (pH 8.3) in a 96-well microplate, followed by the addition of 40 μL of 1.5 mM pyrogallol in 10 mM HCl. The rate of superoxide-induced polymerization of pyrogallol (ΔAs /min) was measured as the increase in absorbance at 320 nm for 5 min at 23◦ C (Sakanaka and Tachibana, 2006). GSH was used as a positive control and Tris-HCl buffer was used instead of hydrolysates in control experiments (ΔAc /min). Superoxide scavenging activity of 3 independent replicates was calculated using the following equation: % superoxide scavenging activity = [(ΔAc /min − ΔAs /min)]/ (ΔAc /min) × 100 Iron Chelating Activity The chelation of ferrous ions by VMH (1 mg/mL) was estimated in triplicate according to the method described by Dinis et al. (1994). EDTA, a strong metal chelator, was used as a positive control. Ferrous ion chelating ability was calculated by the following equation: % iron chelating ability = (Bc −Bs )/Bc × 100 where, Bs and Bc represent the absorbance of the sample and control (everything except the protein hydrolysate), respectively. Ferric-Reducing Antioxidant Power Assay The antioxidant capacity of VMH samples (1 mg/mL) was estimated according to the ferric-reducing antioxidant power (FRAP) assay procedure described by Fogarasi et al. (2015). Trolox (1.0–200 μM) was used as the positive control. The results of triplicate assays

were corrected for dilution and expressed as μM of Trolox equivalents per mg sample. Cell Culture Studies The RAW264.7 murine macrophage cell line was used in all in vitro studies. Cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin/fungizone, and incubated at 37◦ C in 5% CO2 . All cell-based studies were conducted in at least 5 independent trials. Cell passages 7 to 15 were used in all experiments. Cytotoxicity of VMH Samples The cytotoxicity of VMH samples was measured through the (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay (Mosmann, 1983). RAW264.7 cells (1 × 104 cells/mL) were seeded into a 96-well culture plate and incubated for 24 h. The cells were then treated with VMH samples at 1 mg/mL for 20 h. To observe cell viability, 20 μL of a 500 μg/mL MTT solution was added to each well in the dark and the plate was incubated for another 4 h. The purple formazan precipitate that developed was dissolved in 100 μL of dimethyl sulfoxide, and the absorbance was recorded at 560 nm. Cytotoxicity for each sample was expressed as a percentage of absorbance of tested samples compared to the negative control (cells grown only in medium). Nitrite Production Assay The effect of VMH samples on the production of nitrite (as an index of nitric oxide [NO]) in RAW264.7 cell was evaluated through the Griess assay (Kim et al., 1999). RAW264.7 cells (1 × 105 cells/mL) were seeded into a 48-well culture plate and incubated for 24 h under 5% CO2 at 37◦ C. Cells were then treated with 300 μL of medium only (negative control), 1 μg/mL bacterial LPS (positive control), or 10 μg/mL of VMH samples along with 1 μg/mL LPS. Following the 24 h incubation, 100 μL of supernatant and 100 μL of Griess reagent were mixed in the 96-well plate and left for 10 min at room temperature. Absorbance was determined at 540 nm. After generating the standard curve using the stable conversion product of NO, nitrite, the concentration of nitrite was calculated and compared to the negative and positive controls. Quantitative Real-Time Polymerase Chain Reaction Analysis for Cytokine Gene Expression RAW264.7 cells were plated in 6-well plates at 5 × 104 cells/cm2 . After 24 h incubation at 37◦ C, cells were treated with LPS (1 μg/mL) and VM or FlaVMH (10 μg/mL). Cells incubated in media, or VMH samples (10 μg/mL), were taken as negative controls. Cells treated only with LPS (1 μg/mL) were taken as the positive control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. After 24 h, RNA was extracted from the cultures by the TRIzol-chloroform method. RNA purity was measured spectrophotometrically. First-strand cDNAs were synthesized from 1 μg of RNA samples (in 20 μL of reaction mixture) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a

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Quantstudio III (Applied Biosystems, Carlsbad, CA). The 10 μL qPCR reaction mixture consisted of 2.5 ng cDNA, 300 nM of each primer (tumor necrosis factorα (TNF-α), forward: 5 -TAC TGA ACT TCG GGG TGA TCG GTC C-3 , reverse: 5 -CAG CCT TGT CCC TTG AAG AGA ACC -3 ; interleukin 1β (IL1β ), forward: 5 -GGA GAA CCA AGC AAC GAC AAA ATA CC-3 , reverse: 5 -TGG GGA ACT CTG CAG ACT CAA AC-3 ; GAPDH, forward: 5 -ACT TTG TAC AGC TCA TTT CC-3 , reverse: 5 -TGC AGC GAA CTT TAT TGA TG-3 ) and 5 μL of EvaGreen 2 × qPCR MasterMix-Low Rox. Water was used as the control. mRNA expression was normalized against GAPDH mRNA (ΔΔCT -method). Data are presented as fold changes against the unstimulated control (Chanput et al., 2010).

Statistical Analysis All experiments were performed using one pooled sample of lyophilized VM powder in at least 3 independent trials. The results are reported as means ± SD. Results were subjected to analysis of variance using SAS software (SAS Institute, Cary, NC). Statistical significance of differences (P < 0.05) was evaluated by the least significant difference procedure.

RESULTS AND DISCUSSION SDS-PAGE Pattern of VM Proteins VM was solubilized by SDS to obtain proteins, including ovalbumin, lysozyme C, ovomucin, ovotransferrin, and the VM outer proteins (VMO-I and VMOII) (Mann, 2008). Figure 1-lane A presents the electrophoretic pattern of the VM proteins. Four major and 4 minor bands can be clearly distinguished. By comparing the mobilities of these bands with the standard molecular weight markers, the molecular masses of the bands were estimated as 250, 100, 75, 70, 44, 18, 14, and 9 kDa (major bands, with greater intensity and thickness, are shown in bold). The protein bands at approximate molecular weights of 250, 100, and 44 kDa are the glycoproteins GPIII, GPII, and GPI, respectively, that are present in the VM inner layer (Back et al., 1982; Mann, 2008). The VM outer layer contains VMO-I and VMO-II with molecular masses of 17.5 and 9 kDa, respectively (Kido et al., 1995). These proteins appeared as faint bands at 18 and 9 kDa. The mobility of VMO-II on SDS-PAGE is affected mainly by the redox state of the protein. In the presence of a reducing agent such as 2-mercaptoethanol, it gives a single band at 9 kDa, while under a non-reducing condition its apparent molecular weight is 15 kDa (Kido et al., 1992). One major band was a 14 kDa protein, which corresponded to the molecular weight of lysozyme. According to Schafer et al. (1998), these 3 proteins are

Figure 1. SDS-PAGE pattern of vitelline membrane proteins. M, marker; VM, vitelline membrane proteins.

present at the ratio of 80:15:5 (lysozyme:VMO-I:VMOII) (Schafer et al., 1998). Although ovomucin constitutes a large proportion of the total proteins in VM (43%), it has a very low solubility in the presence of SDS, remaining on the top of the gel.

Enzymatic Hydrolysis of VM Vitelline membrane was hydrolyzed by 3 commercial proteases (Alc, Fla, and Try), individually, and the DH was determined by the OPA fluorometric assay that measures the increase in free-amino groups after hydrolysis. After a 24 h digestion, VM hydrolyzed by Fla (Fla-VMH) had the highest DH (27.1 ± 1.4%; P < 0.05) compared to Alc-VMH and Try-VMH (20.8 ± 1.2 and 9.9 ± 1.4%, respectively). Alc is an endo-peptidase, Fla is a mixture of endo- and exo-peptidases, and Try cleaves the C-terminal side of arginine and lysine specifically, except when either is bound to proline. Therefore, Fla-VMH tends to contain more low-molecular weight compounds (amino acids and small-to-medium sized peptides), compared to the other 2 hydrolysates (Cumby et al., 2008).

Antioxidant Assays The antioxidant activity of peptides is based mainly on their free radical scavenging capacity, reducing power, and the ability to chelate transition metal ions.

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LEE ET AL. Table 1. Antioxidant activity of vitelline membrane hydrolysates. Antioxidant assay

VM ∗

DPPH scavenging activity (%) Superoxide radical scavenging activity (%)# FRAP value (TE/ mg)# Iron chelating activity (%)#

b

2.53 7.43c 0.61d 7.21c

± ± ± ±

Alc-VMH 1.2 1.9 0.06 1.3

a

12.29 17.2b 6.8b 19.1b

± ± ± ±

3.7 5.2 0.1 0.4

Fla-VMH a

15.62 31.1a 19.8a 21.1a,b

± ± ± ±

3.3 2.8 0.15 1.6

Try-VMH 18.73a 11.8b,c 4.7c 22.0a

± ± ± ±

6.0 3.8 0.4 1.4

#

Sample concentration was 1 mg/mL. Sample concentration in the DPPH scavenging assay was 0.1 mg/mL. a–d Values with different superscripts within a row indicate significant differences at P < 0.05. VM, vitelline membrane; Alc-VMH, alcalase hydrolyzed VM; Fla-VMH, flavourzyme hydrolyzed VM; Try-VMH, trypsin hydrolyzed VM. ∗

Free Radical Scavenging Activity and Reducing Power Free radical scavenging capacity of VM and VMH samples (Alc-, Fla- and Try-VMH) was evaluated using the DPPH and superoxide radical scavenging assays (Table 1). Enzymatic hydrolysis significantly improved the DPPH radical scavenging activity of VM (P < 0.05), indicating the possible generation of novel antioxidant peptides. However, no differences were found in the DPPH scavenging value among the 3 VMH samples at a constant concentration of 0.1 mg/mL. The positive control, GSH, showed a much higher activity (58.7%) at 0.1 mg/mL compared to the VMH samples. DPPH scavenging activity of egg white hydrolyzed by papain and trypsin was reported as 50.4 ± 2.1 and 37.2 ± 1.7% at 1 mg/mL, respectively (MemarpoorYazdi et al., 2012). Superoxide radical scavenging activity of VM was improved after enzymatic hydrolysis (Table 1). Fla-VMH showed the most potent activity (31.1 ± 2.8%), which was significantly higher than Alc-VMH and Try-VMH (P < 0.05). Radical scavenging activity of a protein hydrolysate depends on the size and amino acid sequence of its peptides (Shi et al., 2014), which is influenced by the degree of hydrolysis and the specificity of the enzyme (Liu et al., 2010). Similar scavenging activity (25% at a hydrolysate concentration of 0.125%) was reported by Sakanaka and Tachibana (2006) for egg yolk protein hydrolyzed sequentially by Orientase and proteinase from Bacillus sp. The reducing power of VMH was assessed using the FRAP assay, which is based on the ability to act as electron-donor to convert ferric to ferrous ions. Fla-VMH showed significantly stronger reducing power (19.8 ± 0.15) (P < 0.05), which was ∼2.9- and ∼4.2fold higher than Alc-VMH and Try-VMH, respectively (Table 1). Egg yolk protein hydrolysate was also reported to be effective in reducing ferric ions (Zambrowicz et al., 2015). Iron Chelating Activity The ability to chelate transition metals can prevent the metal-catalyzed generation of free radicals and oxidative chain reactions in food and biological systems. All 3 VMHs exhibited strong iron-chelating activity (Table 1), significantly higher than that of VM at 1 mg/mL (P < 0.05). Try-VMH showed the highest activity against ferrous ions (22 ± 1.4%). Peptides produced from egg shell membrane proteins showed lower iron-

chelating activity (IC50 of 9.79 ± 0.03 mg/mL) (Shi et al., 2014).

Cytotoxicity and Effects on Nitrite Production by VM and VMH In addition to various chemical assays to evaluate antioxidant activity, it is important to study the biological effects of the VM using a valid in vitro cell model. Macrophages are the major sources of cytokines involved in the immune response, inflammation, and other homeostatic processes. Thus, the RAW264.7 murine macrophage cell line was used for the rapid screening of the antioxidant and anti-inflammatory potentials of the VM and VMH samples. VM and VMH demonstrated no cytotoxicity to RAW264.7 cells in the concentration range used for this study (Figure 2a). VM and VMH were also evaluated for their effect on NO production in LPS-stimulated RAW264.7 cells (Figure 2b). All VM and VMH samples inhibited LPS-stimulated NO production, although VMH samples were significantly more effective than VM. Unhydrolyzed VM reduced the NO production by 78%, while VMH caused 92 to 96% decrease in NO content of the cell. There was no significant variation among the VMH samples (Figure 2b). Although the uptake mechanism of VM and VMH, and their effects on cell metabolism, require further investigation, the results of the Griess assay suggest that the VM samples have the potential to become effective antiinflammatory agents.

TNF-α and IL-1β mRNA Production by RAW264.7 Cells Of the VMHs studied, Fla-VMH yielded the highest DH and showed the most potent antioxidant properties. Therefore, the effect of Fla-VMH on the expression of pro-inflammatory cytokines (TNF-α and IL-1β ) was studied to determine its potential anti-inflammatory properties (Figure 3). The level of TNF-α in LPStreated RAW264.7 cells was 30 times higher than in the negative control cells (P < 0.01). TNF-α expression was inhibited significantly (P < 0.01) in RAW264.7 cells induced by LPS and treated with 10 μg/mL VM (47%) or Fla-VMH (89%). A similar trend was observed

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with the expression of IL-1β ; VM and Fla-VMH samples suppressed the expression of IL-1β by 54 and 83%, respectively, in cells induced by LPS. These results indicate that VM and Fla-VMH significantly suppressed the expression of TNF-α and IL-1β which may alleviate the inflammatory reaction.

CONCLUSIONS The present study provides novel information on the antioxidant and anti-inflammatory properties of egg VM. VM hydrolysates showed improved solubility in aqueous-based media and exhibited higher radical scavenging and ferric reducing activity than VM. VMH samples also inhibited inflammatory responses in macrophage cells. Therefore, enzyme-treated VM has the potential to be used as a functional ingredient with several bioactivities (free radical scavenging, metal chelation, and anti-inflammatory effects) in protein-rich supplements, cosmeceutical and nutraceutical products.

ACKNOWLEDGMENTS The authors are grateful to the Canadian Food Innovators (#CFI-009) and the Natural Sciences and Engineering Research Council of Canada (CRDPJ#485627-15) for financial supports.

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Figure 2. (a) The effects of vitelline membrane (VM) and its hydolysates (by alcalase (Alc-VMH), flavourzyme (Fla-VMH), and trypsin (Try-VMH)) on the viability of RAW264.7 macrophage cells. Viability was assessed after 24 h incubation of cells with different concentrations of VM and VMHs using the MTT assay. Data are expressed as means ± SD. (b) Nitrite content of RAW264.7 cell supernatants after treatment with Alc-VMH, Fla-VMH, or Try-VMH.

Figure 3. The effects of flavourzyme-hydrolyzed vitelline membrane (Fla-VMH) at a concentration of 10 μ g/mL on the mRNA levels of TNF-α and IL-1β , relative to GAPDH (ΔΔCt ), in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Data are expressed as means ± SD. Values with different lowercase letters differ significantly (P < 0.05).

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