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. 2023 Apr 5;102(7):102695. doi: 10.1016/j.psj.2023.102695

Isolation, identification, and mode of action of antibacterial peptides derived from egg yolk hydrolysate

Thippawan Pimchan *, Fu Tian *, Kanjana Thumanu †, Sureelak Rodtong ‡, Jirawat Yongsawatdigul *,1
PMCID: PMC10172704  PMID: 37120868

Abstract

Egg yolk is a coproduct of egg white processing. The protein hydrolysis of egg yolks to exhibit antimicrobial activity is a strategy for its valorization. The objective of this study is to fractionate antibacterial peptides from pepsin-hydrolyzed egg yolks using flash chromatography. In addition, the mode of actions of the fractionated peptides were elucidated and plausible antibacterial peptides were reported. The fraction 6 (F6) obtained from a C18-flash column exhibited antibacterial activity against Staphylococcus aureus ATCC 29213 and Salmonella typhimurium TISTR 292 at minimal inhibitory concentration (MIC) values of 0.5 to 1 mmol/L (Leucine equivalent). The fractionated peptides induced DNA leakage as monitored by 260 nm. Propidium iodide and SYTO9 staining observed under a confocal microscope suggested the disintegration of cell membranes. Synchrotron-based Fourier-transform infrared spectroscopy analysis revealed that the egg yolk peptides at 1 × MIC induced an alteration of phospholipids at cell membranes and modified conformation of intracellular proteins and nucleic acids. Scanning electron microscopy revealed obvious cell ruptures when S. aureus was treated at 1 × MIC for 4 h, whereas damage of cell membranes and leakage of intracellular components were also observed for the transmission electron microscopy. Egg yolk peptides showed no hemolytic activity in human erythrocytes at concentrations up to 4 mmol/L. Peptide identification by LC-MS/MS revealed 3 cationic and 10 anionic peptides with 100% sequence similarity to apolipoprotein-B of Gallus gallus with hydrophobicity ranging from 27 to 75%. The identified peptide KGGDLGLFEPTL exhibited the highest antibacterial activity toward S. aureus at MIC of 2 mmol/L. Peptides derived from egg yolk hydrolysate present significant potential as antistaphylococcal agents for food and/or pharmaceutical application.

Key words: egg yolk, protein hydrolysate, antibacterial peptide, flash chromatography, mechanism

INTRODUCTION

The control of food pathogenic bacteria can be achieved using antimicrobial agents, but their safety is a concern, in particular with synthetic chemical agents (Abdelhamid and El-Dougdoug, 2020). The adverse effects of synthetic preservatives include liver damage, asthma, and allergic reactions (Saeed et al., 2019). As a result, alternative antimicrobial agents should be sought. Antimicrobial peptides (AMPs) or host defense peptides are parts of the innate immune response that can be found naturally in various organisms, including mammals, poultry, plants, insects, and microbes. Currently, over 3,200 AMPs have been reported in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php) and the most common AMP commercially applied in food is nisin. AMPs are capable of inhibiting diverse microorganisms, including bacteria, fungi, viruses, and parasites (Haney et al., 2019; Huan et al., 2020; Lazzaro et al., 2020). From a health perspective, AMPs possess several advantages over antibiotics, including a broad spectrum of antimicrobial activity and selective cytotoxicity for hosts (Matsuzaki, 2009). As most AMPs target the cell membrane, it is more difficult for microorganisms to develop resistance in comparison to metabolic enzyme-targeting antibiotics (Hancock and Sahl, 2006; Sang and Blecha, 2008; Mahlapuu et al., 2016). Moreover, several AMPs are capable of triggering the cytotoxicity of human cancer cells, representing a potential source of effective cancer therapy agents (Tornesello et al., 2020). Thus, AMPs appear to be emerging antimicrobial agents for the food industry, functional food related to immune response, and therapeutic agents in oncology.

Protein hydrolysates from enzymatic digestion contain peptides exhibiting a variety of biological activities, such as antioxidant, antihypertensive, antithrombotic, antimicrobial, anti-inflammatory, and immunomodulatory properties (Nasri, 2017). The proteolytic digestion of different types of proteins could release distinct types of AMPs, paving a new efficient strategy to obtain new antimicrobial agents compared to natural AMPs extracted from host cells, which typically results in much lower yield. Milk lactoferrin demonstrates moderate antimicrobial activity against Escherichia coli and Staphylococcus aureus at minimum inhibitory concentration (MIC) of 130 mg/mL and 260 mg/mL, but proteolytic digestion by pepsin improved its activity to 65 mg/mL and 130 mg/mL, respectively (Wang et al., 2020).

Egg yolk is a good source of phospholipids, which is typically separated from yolk by nonpolar or weakly polar solvent extraction for food and pharmaceutical application as egg yolk lecithin. Solid residues remaining after phospholipids extraction are mainly proteins, which inevitably underwent denaturation by solvents, resulting in limited functionalities (Lesnierowski and Stangierski, 2018). Efficient utilization of these proteins would lead to valorization of egg yolk. Major egg yolk proteins include low-density lipoproteins, high-density lipoproteins, livetin, and phosvitin, accounting for 68, 16, 10, and 4% of total protein, respectively (Anton, 2007). The hydrolysis of phosvitin by trypsin yielded antioxidant peptides (Xu et al., 2007). Antioxidant, angiotensin converting enzyme inhibitory, and antidiabetic (α-glucosidase and dipeptidyl peptidase [DPP-IV] inhibitory) activities have also been reported in defatted egg yolk hydrolysate (Zambrowicz et al., 2015). However, the antibacterial potentials of egg yolk protein hydrolysates have not been widely explored. Therefore, the objectives of this study were to fractionate and identify potential antibacterial peptides from egg yolk hydrolysates, targeting food-borne pathogens. The antibacterial action of the fractionated peptides was also elucidated.

MATERIALS AND METHODS

Chemicals and Bacterial Strains

Fresh hen eggs were obtained from Betagro, Nakhon Ratchasima, Thailand. Bacterial strains, including S. aureus ATCC 29213, Salmonella typhimurium TISTR 292, E. coli TISTR 780, Bacillus cereus DMST 5040 and Listeria monocytogenes DMST 17303 were used. Chemicals and reagents were obtained from Sigma Aldrich (St. Louis, MO), unless otherwise stated.

Preparation of Egg Yolk Hydrolysate

Yolks were manually separated from the whole egg. Egg yolk lipids were removed by ethanol/hexane extraction (1:1, v/v). The residuals were collected and air dried at room temperature for 1 d. The defatted yolk residues were stored at -20°C until use.

The defatted egg yolk powder was dissolved in deionized water in a ratio of 1 to 15 and the pH was adjusted to 2 by 1 M HCl. Pepsin at a concentration of 1% protein was added and the mixture was incubated at 37°C for 4 h with constant shaking. The enzymatic reaction was terminated by heating at 95°C for 15 min and cooled immediately. The reaction mixtures were centrifuged at 10,000 × g for 15 min. Supernatants of egg yolk hydrolysates were collected and stored at -20°C until use. Peptide content in the hydrolysate was estimated by determining α-amino group content using 2,4,6-trinitrobenzenesulfonic acid method and L-leucine as a standard (Adler-Nissen, 1979).

Fractionation of Antibacterial Peptides

The egg yolk hydrolysate was fractioned using a Puriflash equipped with a C18 column (C18AQ-15μm, 30 × 250 mm, Interchim, Montluçon, France). Mobile phase A was 0.05% (v/v) trifluoroacetic acid, and mobile phase B was 0.05% trifluoroacetic acid in acetonitrile. The elution was as follows: 0 to 20 min, 2% B; 20 to 40 min, 10% B; 40 to 50 min, 20% B; 50 to 60 min, 20 to 100% B; 61 to70 min, 100 to 0% B; 70 to 75 min, 0% B. Eluted peptides were monitored at 218 nm. All fractions were collected and lyophilized (CHRIST Gamma 2-16 LSC, Germany) for antibacterial activity assay.

Determination of Antibacterial Activity

The antibacterial activity of the collected fractions against S. aureus ATCC 29213, S. Typhimurium TISTR 292, E. coli TISTR 780, B. cereus DMST 5040 and L. monocytogenes DMST 17303 was determined using the microdilution broth method in accordance with the Clinical Laboratory Standard Guideline with some modification. Pure cultures grown on tryptic soy agar for 18 h were adjusted to achieve 0.5 McFarland equivalent standard and diluted with 0.85% NaCl to a final concentration of approximately 5 × 105 CFU/mL. The diluted bacterial suspension was mixed with each fraction at various concentrations of 0.25, 0.5, 1, 2, 4, 8 mmol/L based on α-amino group content in a 96-well microplate and incubated at 37°C for 18 h. Bacterial inhibition was measured at 600 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Vantaa, Finland. The MIC was defined as the lowest concentration of peptide at which bacterial growth is completely inhibited. The minimum bactericidal concentration was also determined as the peptide concentration that showed no bacterial colonies on a plate count agar (Merck KGaA, Darmstadt, Germany) after incubation at 37°C for 18 h. Bacteria treated with 20 µg/mL kanamycin was used as a positive control.

Leakage of Nucleic Acids

Leakage of nucleic acids were performed to determine cell membrane integrity (Carson et al., 2002). Bacterial suspensions (100 μL) were added to the peptide fraction at concentrations of 0.5 × MIC and 1 × MIC, and subsequently incubated at 37˚C for 4 h. Samples were taken every 1 h, centrifuged at 10,000 × g for 5 min, and supernatants were filtered through a 0.22 µm syringe filter. Absorbance at 260 nm, reflecting DNA and RNA, were quantified by a NanoDrop™ 2000c spectrophotometers (Thermo Scientific, Waltham, MA).

Scanning Electron Microscopy (SEM)

SEM analysis was performed according to Xiang et al. (2019). with some modification. S. aureus ATCC 29213 in log phase were harvested, resuspended with phosphate buffered saline (PBS), and adjusted to an absorbance of 0.5 at 600 nm prior to adding the fractionated peptides at 0.5 × MIC and 1 × MIC. Cells were incubated at 37°C for 4 h with shaking at 120 rpm (Fisher Scientific, Hampton, NH). Then, samples were harvested by centrifugation at 3,000 × g for 10 min at 4°C and washed twice with PBS. Cell pellets were fixed with 2.5% (w/v) glutaraldehyde (Electron Microscope Sciences; EMS) in a 0.1 M phosphate buffer (pH 7.2) at 4°C for 12 h and then washed twice with a 0.1 M phosphate buffer and fixing with 1% osmium tetroxide (EMS) in a 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature. Samples were dehydrated for 15 min at each concentration of acetone of 0, 20, 40, 60, 80, and 100% in sequential order. Cells were placed on aluminum tape and then placed on a stub and coated with carbon and a thin layer of gold before visualizing under a field-emission scanning electron microscope (Zeiss AURIGA FESEM/FIB/EDX, Jena, Germany), with electron energy between 2-5 keV.

Transmission Electron Microscopy

Following the dehydration step as described in SEM preparation, samples were subsequently infiltrated with an epoxy resin in graded acetone (1:3, 1:1, 3:1) (Hartmann et al., 2010). Samples in 100% epoxy resin were transferred to embedding capsules and allowed to polymerize at 60°C for 24 h. Specimens were ultrathin sectioned by ultramicrotome equipped with a diamond knife to obtain a thickness of 70 to 90 nm. The sections were placed on copper grids and counterstained with 2% (w/v) uranyl acetate and 0.25% (w/v) lead citrate at room temperature for 15 min. The prepared samples were viewed with a Tecnai G2 electron microscope (FEI, Hillsboro, OR) at 120 kV.

Confocal Laser Scanning Microscopy

Membrane integrity was analyzed by confocal laser scanning microscopy as described by Rodrigues de Almeida et al. (2018). Bacteria were cultured to the mid-log phase in TSB and suspended to a final concentration of 107 CFU/mL. Bacterial suspension was then treated with fractionated peptides at 0.5 × MIC and 1 × MIC for 4 h. Bacterial cells were harvested by centrifugation at 3000 × g for 10 min, washed 2 times with PBS before staining with 5 μg/mL SYTO-9 and 10 μg/mL propidium iodide (PI). Samples were incubated at 4°C in the dark for 30 min. Samples were washed 3 times with PBS and dropped on a glass slide. Images were captured by a confocal laser scanning microscope (Nikon 90i A1R, Nikon, Tokyo, Japan). Cells stained by SYTO9 were observed at excitation and emission wavelengths of 488 and 530 nm, whereas those stained by PI were visualized at 538 and 617 nm, respectively.

Synchrotron-Based Fourier Transform Infrared Spectroscopy

The effect of the fractionated peptides on cellular components of S. aureus ATCC 29213 and S. Typhimurium TISTR 292 was elucidated using synchrotron-based Fourier transform infrared spectroscopy (SR-FTIR) spectroscopy. Bacterial cells in mid-log phase were harvested and adjusted with normal saline to attain 108 CFU/mL. Bacterial suspensions were treated with 0.5 × MIC and 1 × MIC of the fractionated peptides at 37°C for 4 h. Cells were collected by centrifugation at 3,000 × g for 10 min. Cell pellets were washed twice with normal saline, re-suspended in deionized water and placed on an IR-transparent 2-mm thick barium fluoride (BaF2) window. Samples were dried at room temperature for 30 min and kept in a desiccator before FT-IR analysis.

FT-IR spectra were recorded in transmission mode using a Bruker Vertex 70 spectrometer coupled with a Bruker Hyperion 2000 microscope (Bruker Optics Inc., Ettlin-Gen, Germany) connected to the Synchrotron Light Source at the beamline 4.1 (Synchrotron Light Research Institute, Nakhon Ratchasima, Thailand). FT-IR spectra were recorded at wavenumbers of 3000-800 cm−1 using an aperture size of 10 × 10 μm at a 4 cm−1 spectral resolution, with 64 scans. OPUS 7.5 software (Bruker Optics Ltd., Ettlingen, Germany) was used for spectral acquisition and instrument control. The signal intensity of second derivatives were calculated using Savitzky-Golay algorithms (17 smoothing points) and subjected to principal component analysis by the Unscramble X 10.4 software (CAMO Software AS, Oslo, Norway).

Hemolysis

The hemolytic activity of the fractionated peptides was assayed as described by Xu et al. (2020). Human red blood cells from healthy volunteers were collected in tubes containing heparin, then washed 3 times with 1 × PBS, and finally centrifuged at 1,000 × g for 5 min at 4 °C. Blood cells were resuspended in PBS to reach a final concentration of 1% (v/v) erythrocytes. Fifty microliters of red blood cell suspension were incubated with 50 µL of varying concentrations of peptide fractions for 1 h at 37°C. Red blood suspension incubated with PBS and 10% Triton-X-100 were used as negative and positive controls, respectively. Samples were centrifuged at 1,000 × g for 5 min at 4°C and supernatants were transferred to a 96-well microtiter plate. An absorbance at 570 nm was read using a Versamax EXT microplate reader (Molecular Devices, Sunnyvale, CA). The percent hemolysis was calculated using the following equation:

Hemolysis(%)=(OD570ofthetreatedsample−OD570ofthenegativecontrol)(OD570ofthepositivecontrol−OD570ofthenegativecontrol)×100

The experimental protocol was reviewed and approved by the Human Research Ethics Committee of Suranaree University of Technology (EC-64-32).

Peptide Identification

The peptide fraction exhibiting the highest antimicrobial activity was separated and identified by ultrahigh performance liquid chromatography (UHPLC) (Dionex Ultimate 3000 UHPLC, Thermo Scientific, Waltham, MA) equipped with electrospray ionization tandem mass spectrometer (micro-TOF-Q II) (Bruker, Rheinstetten, Germany). The peptide was loaded onto AdvanceBio Peptide Plus (4.6 mm × 150 mm, 3.5 μm, 100 A (Agilent Technologies, Santa Clara, CA) and eluted with 0.1% formic acid in DI as a solvent A and acetonitrile containing 0.1% formic acid as a solvent B. The elution was performed as follows; starting with 30% B and reaching 80% B in 30 min, and holding for additional 8 min, subsequently decreasing to 30% B in 2 min followed by holding at 30%B for 5 min. Electrospray ionization was performed at 1.6 kV using CaptiveSpray. Mass spectra (MS) and MS/MS spectra were obtained with a positive-ion mode in the range of 50 m/z to 1,500 m/z (Compass 1.9 software, Bruker Daltonics, Bremen, Germany).

Data were acquired and analyzed using the PEAKS Studio 10.0 software (Waterloo, ON, Canada). The pI values and net charge were calculated by the online ProtParam tool (https://web.expasy.org/protparam/). The hydrophobicity was analyzed in Peptide2.0 (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php). The secondary structure of the identified peptides was predicted using SOPMA (https://npsaprabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html).

Statistical Analysis

All experiments were carried out 3 times and data were expressed as mean ± SD. SPSS version 23.0 software (SPSS Inc., Chicago, IL,) was used for statistical analysis. Significant differences in each treated group were analyzed by analysis of variance (ANOVA). A P-value at < 0.05 with Tukey's HSD post-hoc test was considered as the statistically significant difference.

RESULTS AND DISCUSSION

Antibacterial Activity of Egg Yolk Hydrolysate

Egg yolk hydrolysate separated by Flash column chromatography yielded 7 fractions (F1-F7) as depicted in Figure 1. MIC results demonstrated that the F6 possessed the highest antibacterial activity against S. aureus ATCC 29213 and S. Typhimurium TISTR 292 (Table 1). The fractions F7 and F5 also showed inhibition, but to a lesser extent. The other 5 fractions did not show antibacterial activity. The MIC values of the F6 were 0.5 mmol/L and 1 mmol/L against S. aureus and S. Typhimurium, respectively (Table 1). Furthermore, the minimum bactericidal concentration of F6 was at 2 mmol/L. Antibacterial peptides typically contain positive net charges and hydrophobic amino acids. Positively charged peptides electrostatically interact with negatively charged bacterial cell membranes, resulting in a disruption of the cell membrane eventually leading to cell lysis (Kim et al., 2018; Lei et al., 2019). It has been reported that phosvitin hydrolysate prepared from pepsin digestion exhibited antibacterial activity against E. coli under thermal stress at 50°C (Choi et al., 2004). The combination of IgY hydrolysates (livetin fractions) and phosvitin also showed potential for the inhibition of enterotoxigenic E. coli (Gujral et al., 2017).

Figure 1.

Figure 1

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Representative chromatogram of egg yolk peptides fractionated by C18AQ flash chromatography.

Table 1.

Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of various egg yolk peptide fractions obtained by flash chromatography.

Bacteria strain Minimum inhibitory concentration (mmol/L)
 Minimum bactericidal concentration (mmol/L)
F5 F6 F7 F5 F6 F7
S. aureus ATCC 29213 1 0.5 1 8 2 2
B. cereus DMST 5040 >2 2 >2 >8 >8 >8
S. Typhimurium TISTR 292 >2 1 0.5 8 2 2
E. coli TISTR 780 >2 >2 >2 >8 >8 >8
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Membrane Disruption

An increase in OD260 indicates an increase in membrane permeability in a concentration-dependent manner (Figure 2). After 4 h of incubation, a drastic increase in optical density was noticed at 1 × MIC (P < 0.05).

Figure 2.

Figure 2

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Release of DNA/RNA from S. aureus ATCC 29213 treated with 0.5 × MIC and 1 × MIC of F6. All data are expressed as the mean values of triplicate ± standard deviation.

Both live and dead cell membranes are permeable to SYTO 9, which binds to DNA and RNA, and emits green fluorescence, whereas PI permeates damaged cell membranes and intercalates with DNA, emitting a red fluorescence (Benfield and Henriques, 2020). Only green fluorescence was observed in the control group (no peptide), indicating integrity of bacterial cell membranes (Figure 3A). Both PI and SYTO 9 signals were detected in S. aureus exposed at 1 × MIC, suggesting that the peptide fraction in the F6 induced membrane damage and a loss of membrane integrity (Figure 3B and 3C).

Figure 3.

Figure 3

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Confocal fluorescence microscopic images of S. aureus ATCC 29213 subjected to the egg yolk peptide F6 at 0.5 × MIC and at 1 × MIC and the control (no peptide). All samples were stained by SYTO-9 and PI. Scale bar is 10 μm.

It is well established that the primary target of AMPs is the bacterial membrane (Sato and Feix, 2006). The cationic AMP preferentially binds to negatively charged bacterial membranes, resulting in membrane disintegration and subsequently inducing cell lysis and death (Powers and Hancock, 2003). The peptide OVTp12 from egg white hydrolysates has also been reported to disrupt the cell membrane integrity of S. aureus and E. coli (Ma et al., 2020). This is the first report of the antimicrobial action of peptides fractionated from egg yolk hydrolysates.

Electron Microscopy

Morphological changes of S. aureus after exposing to F6 was observed in Figure 4. The control exhibited intact cocci shape and no damage on entire surface of bacterial cell envelop (Figure 4A). When cells were treated with the F6 at concentrations of 0.5 × MIC (0.25 mmol/L) and 1 × MIC (0.5 mmol/L), cell damage was evident with rough surface, and obvious cells lysis when exposing to the F6 for 4 h (Figure 4B and 4C). These results indicated that F6 caused morphological damages to S. aureus ATCC 29213.

Figure 4.

Figure 4

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Scanning and transmission electron microscopy images of S. aureus ATCC 29213, (A, D) Control sample without peptides, (B, E) cells treated with F6 at 0.5 × MIC, (C, F) cells treated with F6 at 1 × MIC. Exposure time was 4 h.

Transmission electron microscopy analysis of the control showed intact cocci-shaped with distinguishable cell wall and cell membrane (Figure 4D). S. aureus subjected to the egg yolk peptide at 0.5 × MIC (0.25 mmol/L) revealed abnormal morphology and disintegration of bacteria cell membrane (arrow, Figure 4E). S. aureus cells exposed at 1 × MIC (0.5 mmol/L) remarkably appeared to be distorted with cell membrane rupture and leakage of intracellular components (arrows, Figure 4F). S. aureus exposed to egg yolk peptides also showed lower density than the control, indicating lesser cytoplasmic membrane materials. Thinner cell envelopes were also evident in the egg yolk peptide treated cells.

SEM and transmission electron microscopy images clearly affirmed that treatment with egg yolk peptides induced destruction of cell membranes, resulting in changes of membrane permeability and leakage of cytoplasmic contents. The modified peptides, FRIRVRV-NH2, inhibited E. coli and S. aureus by forming pores and damage cell membranes, causing leakage of intracellular contents and cell death (Tan et al., 2017). Hartmann et al. (2010) reported that of PGLa or gramicidin caused damages to the cell envelope of E. coli DSM 1103 and S. aureus DSM 1104, including blisters, blebbing, membrane stacks, mesosomes, deep craters, and burst cells. Peptide cathelicidin from duck bone marrow also destroyed the cell membranes of E. coli, increasing membrane permeability and leakage of cytoplasmic contents (Feng et al., 2020).

SR-FTIR Microspectroscopy

FTIR microspectroscopy was used to evaluate changes of biomolecules of S. aureus after treatment with the egg yolk peptide F6 (Figure 5A). FTIR spectra illustrates 4 distinct regions: 3000 to 2800 cm−1 (fatty acids); 1700 to 1500 cm−1 (amides I and II of proteins); 1500 to 1200 cm−1 (the mixed regions of fatty acids, proteins and phosphate-containing molecules), and 1200 to 900 cm−1 (polysaccharides, nucleic acids, DNA and RNA) (Naumann, 2000). Second derivatives of spectra revealed explicit changes of protein bands at 1654, 1636, and 1546 cm−1 (Figure 5B). Wavenumbers 1654 and 1636 cm−1 correspond to the Amide I band, indicating stretching vibrations of the C=O, whereas 1546 cm−1 represents amide II from the C=N stretching and C-N-H bending vibrations (Al-Qadiri et al., 2008). Changes of Amide I signified disturbance of secondary structures of α-helix and β-pleated sheet. In addition, a higher intensity at 2963, 2925, and 2853 cm−1 corresponding to the symmetrical and asymmetrical vibrations of -CH groups of fatty acids on the membranes was observed in the cells treated with egg yolk peptides at 1 × MIC (Figure 5A). An increase at 1235 cm−1 indicated a change in the asymmetric stretching of the phosphate group P=O of the phosphodiester bond of nucleic acid, whereas P=O symmetric stretching at 1083 cm−1 implied changes of the phospholipids of cell membranes (Figure 5B). Results on the SR-FTIR suggested that egg yolk peptides inhibited the growth of S. aureus ATCC 29213 by altering membrane phospholipids and inducing structural changes of intracellular proteins and nucleic acids.

Figure 5.

Figure 5

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Average original SR-FTIR spectra and average the second derivative spectra of S. aureus without peptide treatment (control), cells treated 0.5 × MIC and 1 × MIC F6. (A) Average original FTIR spectra (3800-900 cm−1), (B) average the second derivative spectra of fatty acid regions (3000-2800 cm−1), and (C) average the second derivative spectra of protein, nucleic acid and other carbohydrate regions (1800-950 cm−1). Triplicate experiments were conducted and total 150 spectra were averaged.

Cells treated with egg yolk peptides displayed higher integral areas of wavenumbers assigned to cell membrane, protein and nucleic acid regions compared to the respective wavenumbers for the untreated cells (Table 2, P < 0.05). An increase in the integral area at 2963, 2925, 2825, and 1451 cm−1, corresponding to the symmetrical and asymmetrical vibrations of -CH groups of fatty acids (Naumann, 2000; Movasaghi et al., 2008), suggested a major disruption of bacterial cell membranes. In addition, changes at 1654, 1546 cm−1 indicated conformational changes of intracellular proteins induced by egg yolk peptides, whereas those at 1235 and 1121 cm−1 implied leakage of nucleic acids. All these intracellular changes could be the main cause of cell death.

Table 2.

Band assignments and the integral area of second derivative spectra (× 10−4 cm−1) of S. aureus treated with the F6 of egg yolk hydrolysate.

Wave number (cm−1) Assignments Integral area
Untreated 0.5 × MIC 1 × MIC
2963 Fatty acid 83.64 ± 0.62a 69.67 ± 0.09b 98.55 ± 1.03c
2925 Fatty acid 140.93 ± 5.30a 108.65 ± 0.63b 158.30 ± 2.14c
2853 Fatty acid 48.00 ± 0.79a 56.57 ± 0.21a 68.84 ± 3.60b
1748 Ester lipid 46.79 ± 0.22a 59.46 ± 0.28b 76.73 ± 1.00a
1654 Amide I 1035.94 ± 5.05a 1111.08 ± 2.24b 1035.87 ± 22.10a
1546 Amide II 460.08 ± 0.80a 465.24 ± 5.06a 538.54 ± 25.76b
1451 Cell membrane 69.82 ± 1.34a 73.66 ± 0.32a 108.59 ± 2.17b
1396 Cell membrane 252.12 ± 1.53a 199.63 ± 1.82b 267.28 ± 11.33a
1335 Cell membrane 24.72 ± 0.52a 30.64 ± 0.82b 38.35 ± 1.17c
1235 Phosphodiesters in nucleic acid 160.33 ± 4.23a 217.69 ± 2.27b 242.19 ± 9.82b
1121 Phosphodiesters in nucleic acid 24.23 ± 0.99a 14.53 ± 2.90a 49.10 ± 3.63b
1085 Nucleic acids and phospholipids 174.11 ± 3.62a 155.62 ± 0.57a 191.43 ±14.73a
965 Nucleic acid 49.22 ± 2.50a 35.15 ± 1.49b 35.72 ± 2.36b
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The values are the mean ± SD.

a,b,c

Different superscripts indicate significant difference (Tukey's HSD test, P < 0.05) in the row.

The 2-dimensional principal component analysis plot revealed that the control and the peptide-treated group at 1 × MIC was clearly separated along PC1 with a total variation of 69%, and the spectra of cells treated with 0.5 × MIC was differentiated by PC-2 (Figure 6A). The PC1 loading plots showed the highest negative spectrum variations at 2970, 2933, 1673 and 1632 cm−1 (Figure 6B), indicating that fatty acids and proteins were the main contributors to data variance between the control and the cells treated at 1 × MIC. In addition, the PC2 loading plot was discriminated by the loading spectra at 1664 and 1234 cm−1, corresponding to C=O stretching and P=O symmetric stretching, respectively. These results suggested that fatty acids at cell membranes, intracellular proteins, and the nucleic acids of S. aureus are greatly modified when exposed to egg yolk peptides.

Figure 6.

Figure 6

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PCA analysis of the SR-FTIR spectra of S. aureus without peptides (control) and cells subjected to 0.5 × MIC and 1 × MIC F6. (A) PCA of 2D score plot and (B) PCA loading plot. PCA, principal component analysis.

Identification of Antibacterial Peptides

Thirteen peptides were identified in the F6 including 3 cationic peptides and 10 anionic peptides (Table 3). All identified peptides showed 100% similarity to fragments of the Gallus gallus apolipoprotein B (Apo B) in egg yolk. Apo B is a major protein in low-density lipoproteins, containing hydrophobic region in β-sheet structure and amphipathic α-helices as lipid-binding domains (Olofsson et al., 1987; Bayly, 2014). After pepsin hydrolysis, peptides from yolk proteins were generated. Peptides derived from Apo B were expected to be predominant and to possess relatively high hydrophobicity, which were eluted at relatively high concentrations of acetonitrile (ACN) of 40 to 50% on the C18 column (Figure 1).

Table 3.

Characteristics of plausible antimicrobial peptides identified from the fraction F6 of egg yolk hydrolysate.

Amino acid sequence Length Mass (Da) de novo score Net charge1 Hydrophobicity2 (%) Secondary structure
KGGDLGLFEPTL 12 1245.66 84 −1 41 Random coil (100%)
FELPTGAGL 9 903.47 80 −1 55 Random coil (100%)
VTLKVPGVTL 10 1025.65 73 +1 60 Random coil (100%)
SLVPVPVGLGPA 12 1104.65 64 0 75 Random coil (100%)
PAASDTAGAGPSF 13 1147.51 63 −1 53 Random coil (100%)
CASALLGA 8 704.35 63 0 62 Random coil (100%)
HAAHTTGLGGPF 12 1164.57 58 0 41 Random coil (100%)
KVLPLPFGGRSPGGGA 16 1508.85 57 +2 50 Random coil (100%)
AEAPSPAAPLTGGAYEL 17 1613.79 55 −2 58 α- Helix (23.53%)
Random coil (76.47%)
LPLSSSSAADLSKGSKSDL 19 1861.96 55 0 36 α-Helix (38.84%)
β-Turn (5.26%)
Random coil (57.89%)
PSLSASGATPVWSCL 18 1474.71 54 0 53 Random coil (100%)
KGAGSGGTLHL 11 996.54 53 +1 27 Random coil (100%)
LPGVVLSLPVGPRLPASE 18 1800.05 51 0 66 α-Helix (33.33%)
Extended strand (22.22%)
Random coil (44.44%)
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1

Calculated based on negatively charged amino acids (E and D) and positively charged amino acids (K, R, and H) in the peptide sequence.

2

the percentage of hydrophobic residues (I, V, L, F, C, M, A, W) in the peptide sequence.

The molecular mass of these peptides ranged from 700 to 1,900 Da. Previous studies suggested that the net positive charge and hydrophobicity were critical characteristics for antibacterial activity (Kim et al., 2018). Most AMPs are cationic amphipathic peptides, containing one or no acidic residues and a high number of cationic amino acid residues including arginine (R), lysine (K) or histidine (H). Cationic AMPs inhibited the growth of bacteria by interacting with negatively charged bacterial cell membranes, leading to a change in the electrochemical potential of the bacterial cell membranes. This leads to cell membrane damage, increased membrane permeation, followed by leakage of cytoplasmic contents, and eventually cell lysis and death (Lei, et al., 2019). Hydrophobic amino acids such as tryptophan (W), valine (V), leucine (L) and isoleucine (I) are also important for secondary structure formation and interactions with negatively charged bacterial membranes (Dziuba and Dziuba, 2014). Typically, AMPs bind to the membrane surface with their hydrophobic side chains anchoring in the hydrophobic core of the lipid bilayer, causing membrane disintegration (Khandelia et al., 2008). In addition, fraction 6 also contained anionic peptides, which have been reported to exhibit antimicrobial activity. The mechanism underlying the antimicrobial action of anionic AMP appears to be its interaction with the membrane (Dennison et al., 2018). Some anionic AMPs can utilize metal ions, for instance Zn2+, to form cationic salt bridges with negatively charged bacterial membranes (Almarwani et al., 2020).

The mode of action of the F6, which targets cell membrane disruption and permeation, appeared to resemble that of cationic AMPs. Thus, cationic peptides contained in fraction 6 were presumed to play a vital role in the growth inhibition of S. aureus. All cationic peptides in the F6 displayed a random coil structure. Transformation of random coils (polymorphic behavior) in a water environment to either α-helix or β-strand of cell-penetrating peptides in the presence of phospholipid mono-/bi-layers has been reported. The α-helical structure occurring upon interactions with cell membranes is thought to be potent in its membrane-destabilizing activity (Eiríksdóttir et al., 2010). The structural transformation of egg yolk peptides from random coil to helical structure could have also occurred. Further studies on the structural transformation of peptides are still needed to verify this assumption.

Seven out of 13 peptides were synthesized and tested for their antimicrobial activity (Table 4). The peptide KGGDLGLFEPTL showed the highest antibacterial activity with MIC of 2 mmol/L against S. aureus ATCC 29213, B. cereus DMST 5040 and S. Typhimurium TISTR 292. In addition, FELPLGAG and CASALLGA exhibited antimicrobial activity with MIC values of 4 mmol/L. The rest showed weaker inhibition with MIC > 4 mmol/L. These peptides, particularly KGGDLGLFEPTL, likely contributed to antistaphylococcal activity of the F6 fraction. Hydrophobicity is one of prime factors contributing to antimicrobial activity of AMPs as it is required to bind with lipid components of bacterial cell membrane leading to membrane permeabilization and cell death (Kopiasz et al., 2021). Hydrophobicity value of 3 most active AMPs identified in this study, namely KGGDLGLFEPTL, FELPLGAG and CASALLGA, was estimated to be 41, 55 and 62%, respectively. This could partly contribute to antibacterial activity of these peptides and the fraction 6. The most potent AMP found in this study, KGGDLGLFEPTL, is categorized as anionic AMP containing aspartate (D) and glutamate (E) residues and net charge of -1 charge. It has been reported that anionic AMPs utilize metal ions and the negatively charged components of the microbial membrane to form salt bridges, leading to cell membrane interactions and penetration (Almarwani et al., 2020). Ovine pulmonary surfactant associated anion peptide containing 5 to 7 aspartate residues was the first discovered anionic AMP exhibited antimicrobial activity against the ovine pathogen Mannheimia haemolytica in the presence of Zn2+ (Miller et al., 2021). Mechanism underlying antimicrobial action of KGGDLGLFEPTL warrants further investigations.

Table 4.

Minimum inhibitory concentration (MIC) of identified peptides derived from egg yolk hydrolysate against various pathogenic bacteria.

Peptide MIC (mmol/L)
S. aureus B. cereus S. Typhimurium E. coli
ATCC 29213 DMST 5040 TISTR 292 TISTR 780
KGGDLGLFEPTL 2 2 2 >4
FELPLGAG 4 4 4 >4
CASALLGA 4 4 4 >4
KVLPLPFGGRSPGGGA >4 >4 >4 >4
VTLKVPGVTL >4 >4 >4 >4
HAAHTTGLGGPF >4 >4 >4 >4
KGAGSGGTLHT >4 >4 >4 >4
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Hemolysis

The hemolysis rate increased proportionally with peptide concentration (Figure 7). A hemolysis rate of more than 5% implies erythrocyte toxicity (Che et al., 2008). AMPs have ability to form pores on eukaryotic membranes. AMPs at higher concentrations could have greater interactions with hydrophobic core of the red blood cell membranes, resulting in greater extent of hemolysis. (Kondejewski et al., 2002; Chou et al., 2008). The F6 was considered safe at concentrations up to 4 mmol/L (leucine equivalent), and toxicity to erythrocytes was observed at 8 mmol/L (Figure 7). To be used as a therapeutic agent, the F6 should be applied below 4 mmol/L, which is about 4 times higher than its MIC value. Our results indicate that peptides from pepsin-hydrolyzed egg yolk are good candidates for an emerging antibacterial agent and bio-preservative in food and the pharmaceutical industry.

Figure 7.

Figure 7

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Hemolytic activity in human red blood cells of peptides fractionated from egg yolk hydrolysate prepared from pepsin. Data are the average of at least 4 independent experiments. Error bars represent the standard deviations.

CONCLUSIONS

The fraction 6 (F6) obtained from flash column chromatography of pepsin-hydrolyzed egg yolk exhibited antibacterial activity against S. aureus and S. Typhimurium. The fraction inhibited growth of S. aureus by disrupting and disintegrating cell membranes resulting in an increase in membrane permeability, cell lysis and death. SR-FTIR analysis indicated that F6 induced alteration of membrane phospholipids and various intracellular components, including proteins and nucleic acids of S. aureus. The F6 contained cationic and anionic peptides with hydrophobicity as high as 75%. Although the mode of action of F6 closely resembled that of cationic AMP, which mainly includes disrupting cell membranes and interacting with intracellular components, the role of anionic peptides should not be ruled out. Among identified peptides, KGGDLGLFEPTL was the most potent AMP against S. aureus with MIC value of 2 mM and likely contributed to antimicrobial activity of the F6. The F6 showed relatively low hemolytic activity suggesting its safety for use in the control of S. aureus in both food and pharmaceutical applications.

ACKNOWLEDGMENTS

A postdoctoral fellowship under the Industrial Post-doctorate Development for Agriculture, Food, Energy and Bio-materials for the Future from Khon Kaen University (KKU-PMU-B 63-012) was awarded to TP. Research funding was supported by 1) Suranaree University of Technology (SUT), 2) Thailand Science Research and Innovation (TSRI), and 3) the National Science, Research and Innovation Fund (NSRF) under the Fundamental Fund Project (FF3-305-66-12-35(B)). Additional funding from the National Research Council of Thailand (N42A650548) was also greatly appreciated.

DISCLOSURES

The authors declare no conflicts of interest.

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