1. Introduction
Natural antimicrobials from food sources are welcomed as food preservatives as they are not hazardous and have no side effects like those caused by synthetic ones [
1,
2,
3,
4,
5,
6]. These natural antimicrobials are quite promising for food bio-preservation to reduce the need for antibiotics, control of microbial spoilage process, and could kill the resistant variants of bacteria that can survive in foods and limit the occurrence of new food-borne disease outbreaks caused by pathogenic bacteria [
7,
8,
9,
10,
11,
12]. Natural extracts are compounds with a wide structural diversity, representing an important source of new chemical compounds with a possibly significant antibacterial action against food-borne pathogens with powerful action against multidrug-resistant bacteria [
13,
14,
15,
16,
17,
18,
19,
20,
21]. The possible negative impact of such chemicals on the environment and human health may preclude synthetic agents’ use [
19]. Therefore, novel antimicrobial agents from natural sources are highly required [
4,
15,
17,
20]. Many strategies have been suggested to improve proteins’ antimicrobial activities, including chemical modification, e.g., such as esterification [
22,
23,
24,
25,
26]. Food proteins have long been recognized as important source of bioactive peptides. In particular, peptides derived from protein-based foods have been shown to possess significant antimicrobial capacities. These peptides from either plant or animal sources can be generated through enzymatic hydrolysis, acid or alkali treatment, or fermentation [
26]. Bioactive peptides can be obtained from different protein sources, including milk [
27] and legume proteins [
28,
29]. Peptides from various protein resources have been reported to have multiple functions, including antioxidative ability, angiotensin I converting enzyme (ACE) inhibitor properties [
30], hepatoprotective activity [
31], and antibacterial activity [
6,
19,
20,
32,
33,
34,
35,
36].
Eggs contain various proteins exhibiting antimicrobial activities, including lysozyme and ovotransferrin, which are commonly used as natural food antimicrobial agents against a wide spectrum of bacteria [
37]. Natural antimicrobial peptides exist in different compartments (egg shell, albumen, and vitelline membranes) of eggs. Most of these peptides are cationic or amphipathic, but there are also hydrophobic α-helical peptides that possess antimicrobial activity [
38]. Although a wide variety of peptides with different chemical structures and peptide conformations have been reported to exhibit antimicrobial activity, these antimicrobial activities’ mechanism is less well understood in most cases. In fact, several observations suggest that natural peptides can alter cytoplasmic membrane permeability, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis and in turn, cause bacterial cell death [
39]. Therefore, antimicrobial peptides (AMPs) constitute a promising alternative as therapeutic agents against various pathogenic microbes [
40]. More than 60 peptide drugs have reached the market for patients’ benefit, and approximately 140 peptide therapeutics are currently being evaluated in clinical trials [
41]. In a previous study [
37], the methylated egg white protein inhibited many bacterial pathogens, simulating antibiotics’ inhibitory activity. In an endeavor to develop such work, the present study aimed to determine the antibacterial activity and mode of action of protein hydrolysates produced from egg albumin by enzymatic digestion using pepsin. The toxicity of both native egg albumin (NEA) and hydrolyzed egg albumin (HEA) was studied using Wistar albino rats.
3. Discussion
The occurrence and spread of antibiotic resistant bacteria are pressing public health problems worldwide [
13,
34,
42]. Many bacteria have become resistant against many antimicrobial agents. The resistance rates are higher in developing countries [
43]. The development of new prophylactic and therapeutic procedures is urgently required to meet the challenges imposed by the emergence of bacterial resistance [
44,
45,
46]. Cationic Antimicrobial Peptides (CAMPs) are promising new antibacterial agents due to their killing mechanism via interaction with bacterial cell walls and membranes [
20,
47].
Imparting cationic character on native proteins can be achieved through chemical modification, e.g., esterification which blocks free carboxyl groups, elevating the net positive charge on the modified proteins [
48,
49] or through the enzymatic hydrolysis [
50] which potentially affects the molecular size and hydrophobicity, as well as the polar and ionizable groups of protein hydrolysates. This modification produces potent bactericidal peptides that may be classified as effective antimicrobials [
51,
52].
In line with this trend, HEA was obtained by enzymatic hydrolysis of egg albumin using pepsin at its optimal conditions (pH 2 at 37 °C) to liberate bioactive peptides, a relatively short peptide residue length (e.g., 2–20 amino acids) possessing hydrophobic amino acid residues in addition to proline, lysine, or arginine groups and bioactive peptides. These peptides showed to be an effective antimicrobial [
52]. The large number of the peptides (44 fractions) released by peptic hydrolysis are apparently due to the broad specificity of pepsin and the high susceptibility of proteins to this enzyme [
53]. The majority of the peptides which had relatively high molecular weight (3–9 amino acids) agrees with the fact that pepsin attacks the protein molecules gradually from the C- and N-terminals [
53]. The obtained hydrolysate style, showing increasing and steady rate of hydrolysis with time during 4 h, reflects the nature of pepsin specificity which tends to attack protein molecules gradually from the C- and N-terminals [
54,
55], targeting preferentially linkages with aromatic or carboxylic L-amino acids [
56]. The higher relative quantity of the bigger sized peptides may indicate their higher participation in the biological activity of the hydrolysates; protein hydrolysis may give rise to basic peptides which can directly attack the cell walls and cell membranes of the pathogenic bacteria [
57,
58].
The higher relative quantity of the bigger sized peptides (3–9 amino acids) may indicate their higher participation in the biological activity of the hydrolysates, i.e., peptides of more than three amino acids may be essentially more responsible for the antibacterial activity since they represent more than 74% of the peptide mixture. However, the data available from the analysis cannot give information about the specific peptide that was most responsible for this activity.
The observed molecular mass range of NEA subunits between 50 and 180 kDa agrees with its ability to dissociate into more active subunits under different ionic strength and pH [
59,
60,
61,
62]. The electrophoretic patterns of HEA confirmed the transformation of NEA into lower molecular weight peptides. The faster migration from anode to cathode, in case of the hydrolyzed protein, than the native one refers to bigger positive charges. This may imply the liberation of some cationic peptides in the course of hydrolysis in accordance with previous studies [
63,
64]. HEA exhibited a distinctive inhibition against the pathogenic bacteria, while NEA showed only scant inhibition corroborating previous results [
65,
66,
67,
68].
The higher antimicrobial activity of HEA than NEA could be due to the more hydrophobicity and cationic nature of HEA [
26,
37,
69,
70,
71,
72]; initiating the electrostatic interaction between the positive charges of the antimicrobial agent and the negatives charges on the bacterial cell walls or membranes. The hydrophobic aggregation between the similar regions of the two reactants can further stabilize this complex. Oscillating random Brownian motion of these aggregations [
71] may produce large-sized pores channels and disintegrate the cell wall and cell membranes, engendering higher cell permeability, cell emptiness, and death. The mechanism of antimicrobial peptides is based on their amino acid composition, amphipathicity, and cationic charges allowing their attachment to cell membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’, or ‘toroidal-pore’ mechanisms [
37,
39].
Since HEA shows broad-spectrum antimicrobial activity, it can promote the activity of antibiotics against all tested bacteria. This was realized in the tested HEA-antibiotic combinations which showed greater antibacterial action than the single antibiotics, especially against
L. monocytogenes,
B. cereus, and
P. aeruginosa. This synergism between antibiotics and HEA may be due to accentuating the hydrogen bonding and hydrophobic–hydrophobic interactions with targeted microbes [
73] or through using different mechanisms of bacteria inhibition.
This Gram (−) bacteria (especially
P. aeruoginosa) is known for being notorious for its ability to survive in the environment, particularly in moist conditions. It may contaminate medicines, surgical equipment, clothing, and dressing with the ability to cause serious infections in immune compromised patients [
74]. Therefore, it was used in this study for TEM studies. Additionally,
S. aureus bacterium was selected from the Gram (+) group as it was more sensitive and because of its public health concerns.
S. aureus is a pathogen of skin abscesses, pharyngitis, sinusitis, meningitis, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, sepsis, and wound infections following surgery. Moreover, it responsible for food poisoning illness as it is capable of producing several virulence factors such as enterotoxins, adhesins, hemolysins, invasins, superantigens, and surface factors that inhibit its phagocytic engulfment [
73,
75,
76].
The signs of the irregular wrinkled outer surface, fragmentation, adhesion, and aggregation of damaged cells or cellular debris of HEA-treated bacterial cells, manifested by TEM examination confirm the antimicrobial action and follow previously published works [
23,
52,
77].
The potential toxicity of NEA and HEA was assessed by determining acute oral toxicity when administrated to Wistar Albino rats. Mortality absence after a single administration of up to 5000 mg/kg body weight/day of the two tested substances indicates their safety and their possible biological utilization. The absence of significant body weight changes or any abnormality signs during 14 days after the single high dose administration of HEA (5000 mg/kg body weight/day) indicates its harmlessness. Since no deaths emerged in response to the used range of doses, the LD
50 of HEA could not be calculated, but it may be assumedly more than 5000 mg/kg body weight/day. Therefore, HEA may be regarded nontoxic since its LD
50lies in the range 5000–15,000 mg/kg body weight/day according to [
78].
Extending the administration of two HEA doses (500 and 2500 mg/kg body weight/day) for 28 days did not either produce evident signs of toxicity or pathogenicity on the body or organ weights, indicating the absence of any specific organ toxicity. Changes in body or organ weights are taken as indicators of toxicity [
79] while normal body weight changes are indicators of food safety and lack of toxicity [
80]. The rats conserved normal healthy status in spite of repeated force-feeding with high doses. The obtained results revealed slight reductions in these two parameters and showed absence of any negative effects of the two studied substances on the renal function during repeated dose administration for 28 days. The rats conserved normal healthy status despite repeated force-feeding with high drug doses. The obtained results revealed the absence of any negative effects on the renal or hepatic function as well as the histological status during repeated-dose administration for 28 days in agreement with a previous study [
81]. Therefore, our data demonstrate the importance of hydrolyzed egg albumin in controlling resistant bacteria. Further work will be necessary to study pathogenic bacteria’s inhibition by HEA in processed foods based on egg albumin.