Lysozyme-Derived Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms

doi:10.21203/rs.3.rs-2994942/v1

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Lysozyme-Derived Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms

https://doi.org/10.21203/rs.3.rs-2994942/v1

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published 20 Feb, 2024

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Food-borne illnesses and microbial food contamination are crucial concerns and still issues of major worldwide concern. Lysozyme (Lz) is an active protein against Gram-positive bacteria through its muramidase lytic activity, however, several authors could identify some antimicrobial peptides derived from native Lz that have an exaggerated and broad-spectrum antibacterial activity. Therefore, a lysozyme peptides preparation (LzP) is developed in order to broaden the Lz spectrum. In this work, we investigated the potential efficacy of LzP as a novel Nutra-preservative (food origin) agent against some pathogenic and spoilage bacteria. Our results showed that LzP demonstrated only 11% of the lysozyme lytic activity. However, LzP exhibited strong antibacterial activity against Escherichia coli, Salmonella enteritidis, and Pseudomonas species, while Salmonella typhi and Aeromonas hydrophila exhibited slight resistance. Despite the lowest LzP concentration (0.1%) employed, it performs stronger antibacterial activity than weak organic acids (0.3%). Interestingly, the synergistic multi-component formulation (LzP, glycine, and citric acid) could inhibit 6 log₁₀ cfu/ml of E. coli survival growth. The effect of heat treatment on LzP showed a decrease in its antibacterial activity at 5 and 67% by boiling at 100ºC/30 min, and autoclaving at 121 ºC/15 min; respectively. On the other hand, LzP acquired stable antibacterial activity at different pH values (4–7). In conclusion, LzP would be an innovative, natural, and food-origin preservative to control the growth of food poisoning and spoilage bacteria in food.

Egg-white lysozyme

lysozyme-derived peptides

food poisoning

Food spoilage

Nutra-preservatives

natural food preservation

Food safety and quality are the most pressing issues and challenges facing the food sector for consumers and food technologists. One of the most important concerns for food manufacturers is microbial contamination, which includes the presence of spoilage microorganisms in general and pathogens in particular. Therefore, one of the main priorities of food manufacturers and regulatory organizations is to provide foodstuffs free of any microbial contaminants [1]. Interestingly, the frequency of foodborne illnesses in humans remains mostly unknown. Foodborne illnesses caused by pathogens present in various food considered a growing public health issue and encompass a wide spectrum of diseases [2]. In most nations, the prevalence of foodborne microbial illnesses has significantly grown during the previous ten years [3]. Despite, the recent food preservation technologies have advanced, foodborne bacterial disease outbreaks, food deterioration, and food waste still occur [4].

The widespread use of chemical food preservatives is a result of their affordable cost and straightforward manufacturing procedure. Food preservation agents are vital and play a critical role in the battle against food deterioration. Nevertheless, the improperly used or prolonged consumption of chemical preservatives like sodium benzoate, sodium nitrate, sodium nitrite, and benzoic acid has been related to major health problems [5]. Additionally, regarding the negative impacts of commercial chemical preservatives on public health, individuals are also growing more interested in the replacement of chemical preservatives with natural alternatives drawn from natural systems [6, 7].

As consumer demand for natural ingredients and clean labels grows, it is anticipated that the industry will abandon chemical techniques of food preservation and switch to more natural alternatives, more particularly, nutra-preservatives (food origin preservatives) such as food-derived antimicrobial peptides and hydrolyzed food proteins [7, 8].

Many pathogens are incriminated in food contamination and harm the consumers; such as; Salmonella spp., Shigella spp., Micrococcus spp., Enterococcus faecalis, Escherichia coli, Bacillus licheniformis, Staphylococcus aureus, Campylobacter jejuni, Listeria monocytogenes, Yersinia enterocolitica, Escherichia coli 0157:H7, Vibrio parahemolyticus, and Clostridium botulinum, and others. Numerous studies have established the efficiency of antimicrobial peptides originating from food proteins and demonstrated effectiveness against a range of food-borne pathogens. Hence these peptides can aid in food preservation in a natural, and safe way [9, 10].

Lysozyme is a protein with a molecular mass of about 14 kDa that is found in many mucosal secretions (including saliva, tears, and mucus), tissues of plants, and animals. It is crucial for innate immunity, providing protection against bacteria, viruses, and fungi. Due to its antibacterial qualities, it has long been the master important of numerous applications [11]. Historically, Lysozyme is traditionally associated with chicken eggs. Egg white includes 11% proteins, 3.5% of which is lysozyme. As a result, this enzyme is one of the primary proteins in egg white where it serves as a defensive protein and feeds the developing embryo [12]. Lysozyme-derived peptides (LzP) are a source of different biotechnological and health-promoting functions that can be used to create functional foods and nutraceuticals, besides, LzP showed a marvelous and broad-spectrum antibacterial activity [7, 13].

The current study aims to investigate the efficiency of LzP on a variety of spoilage and pathogenic bacteria which could contaminate foodstuffs as well as study the factors impacting their antibacterial activity. Moreover, assess the antibacterial properties of the multi-component food-grade antibacterial mixture compared to employing LzP alone. Aiming to find out an effective, safe, and more acceptable natural bio-preservative formulation as an alternative approach for controlling undesirable bacteria in food.

Microbial Strains and Culture Conditions

Hen egg white lysozyme (Lz) was received from Wako Chemicals (Osaka, Japan). Porcine pepsin A was supplied from Sigma-Aldrich (Meguro-ku, Tokyo, Japan). Test model indicator microorganisms for antibacterial assays, Escherichia coli K-12 (IFO 3301), Salmonella enteritidis (IFO 3313), and Salmonella typhimurium (ATCC 14028) strains were received from the Institute of Fermentation (Osaka, Japan). Pseudomonas aeruginosa (ATCC 27853), Pseudomonas fluorescence (ATCC 17386), and Aeromonas hydrophila (ATCC 7965) strains were obtained from the American Type Culture Collection (Rockville, MD, USA). Cultures were maintained in trypticase soy broth (TSB) with 15% glycerol at 20°C. Brain–heart infusion (BHI) broth, Trypticase soy broth (TSB), and nutrient agar were provided from Difco Laboratories (Detroit, MI, USA). The microbial substrate of Lz, Micrococcus lysodeikticus, and other chemical organic food additives including propionic acid, glycine, citric acid, potassium sorbate, and sodium sorbate were of food grade purchased from Sigma–Aldrich (St. Louis, MO, USA).

Lysozyme-derived Peptides

Lysozyme-derived peptides powder (LzP) was obtained from Pharma Foods International Co., Ltd (Kyoto, Japan). It was produced by partial enzymatic hydrolysis from egg white lysozyme using pepsin enzyme. The LzP powder is a mixture composed of 50% active peptides and 50% glycine. It was dissolved in 1.0% wt./vol in sterilized distilled water. The solution was gently stirred to avoid foaming, filtered, and kept refrigerated as a stock solution at 4°C [7].

Hydrolytic Activity

To examine the residual lytic activity of LzP in comparison to its native Lz. The turbidimetric approach depending on Micrococcus lysodeikticus was used according to the previously published method [14]. The turbidity of three mixtures, each containing either 1.9 ml of Micrococcus lysodeikticus mixed in 50 mM potassium phosphate buffer (pH 6.2) with 100 µl of solutions of LzP, Lz, and potassium phosphate buffer (50 mM, negative control), were tested for turbidity using a spectrophotometer (SmartSpec-3000, Bio-Rad, USA). The decrease in suspension absorbance at 450 nm at 25°C was used to quantify the lysis of Micrococcus lysodeikticus cells. The LZP enzymatic activity was expressed as a proportion of the activity of the untreated Lz.

Antimicrobial Activity

The liquid broth technique was used to assess the antibacterial activity, according to a previous study [15]. Aliquots of mid-logarithmic phase bacterial suspension grown in brain heart infusion (BHI) broth were harvested, washed, and resuspended trypticase soy broth (TSB), adjusted to a final concentration of 5 log₁₀ cfu/ml of cells, then were mixed with an equal volume of the medium containing test protein or peptides. The LzP-free controls were incubated (act as control). The suspensions were incubated at the given temperature for 4 h, serially diluted in physiological saline, and disseminated on nutrient agar. Colony-forming units were obtained after the plates were incubated at a certain temperature for a pre-determined time. The formula used to calculate the killing power was as follows: Killing power% = (log₁₀ Ctrl − log₁₀ T)/ log₁₀ Ctrl X100.

where Ctrl and T are CFU/ml of control and treated groups; respectively. All assays were performed in triplicates and the results, unless otherwise specified, are the mean of three independent experiments.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The protein profiles of the peptic digests were performed using SDS–PAGE Tris-Tricine ready gels (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted in Tris-Tricine sample buffer (BioRad, USA). Electrophoresis was carried out at 100 V, for 3 h, at room temperature, in Tris-Tricine SDS running buffer. To immediately see and stain the protein bands, silver nitrate was utilized as opposed to the following standards marker.

Characterization of LzP

Effect of Temperature

In a test tube, 4.5 ml of saline was combined with 0.5 ml of LzP. Then, each test tube was covered with paraffin oil to prevent evaporation; and then heated at different temperatures for various storage times (100°C/30 min; 121°C/15 lbs/15 min; 4°C /30 d; -20°C/ 7 d; and freeze dry). The preparations utilized in the previously described liquid broth assay to evaluate its antibacterial effectiveness against Escherichia coli [16].

Effect of pH

The effect of pH was investigated by adding 0.5 ml of LzP into 4.5 ml of nutritional broth at various pH levels (4 to 8), then the mixtures were incubated for 30 min at 37°C. Escherichia coli resistance testing was performed on each LzP sample that had been subjected to a variety of pH levels [16].

Comparing LzP Efficacy with Organic Weak Acids Against Escherichia coli

Different concentrations of frequently employed chemical organic food preservatives, such as glycine, citric acid, propionic acid, potassium sorbate, and sodium benzoate at a concentration of 0.3% were incubated with E. coli at 37°C / 4 h in comparison to LzP at concentration 0.1% in presence of negative control, which contained E. coli bacterial suspension only. After the plates had been incubated at 37°C for 18 h, colony-forming units were collected.

Formulation of Multi-component Antibacterial Mixture Against Escherichia coli

Different concentrations of LzP were used as one component of the antibacterial mixture. The multi-component of the antibacterial formulations was composed of LzP, glycine, and/or citric acid at different concentrations as shown in Fig. 4. Liquid broth assay, as mentioned previously, was used to test LzP and other formulations against E. coli.

Statistical Analysis

All data were presented as the means and standard deviations of triplicates. The significance of differences was determined via one-way ANOVA. Differences with p < 0.05 were considered statistically significant.

Antibacterial Activity of LzP Against Pathogenic and Spoilage Bacteria

Figure 1A showed the bacterial growth count expressed as log₁₀ cfu/ml against the most pathogenic food-borne tested bacteria in the presence of different concentrations (250, 500, 750, and 1000 µg/ml (w/v), of Lz and LzP after incubation for 4 h at 37ºC. It was clear that LzP performed higher antibacterial activity than Lz in a dose-dependent behavior against E. coli, Sal. enteritidis and Sal. typhi. The higher LzP concentration (1000 µg/ml) could result in complete bacterial inhibition with 100% killing power against the most pathogenic bacteria E. coli and Sal. enteritidis as illustrated in Fig. 1B. Although Sal. typhi showed slight resistance but, a 3.2 log₁₀ cfu/ml growth reduction could be achieved by LzP at a concentration of 1000 µg/ml with killing power at 67.27%.

Figure 2A demonstrated the bacterial survival rate expressed as log₁₀ cfu/ml against indicator spoilage bacteria in the presence of different concentrations (250, 500, 750, and 1000 µg/ml (w/v), of Lz and LzP for 4 h at 37ºC. There was a significant increase (p < 0.05) in the tested indicator bacterial growth, compared with Lz and LzP under similar conditions. It was worth noting that LzP performed higher antibacterial activity than Lz in a dose-dependent manner against Ps. Fluorescens, Ps. Aeruginosa, and A. hydrophila. Interestingly, LzP at concentrations 750, and 1000 µg/ml was effective against Ps. Aeruginosa, and Ps. fluorescence resulted in 100% killing power as illustrated in Fig. 2B. It was reported that Ps. aeruginosa was highly sensitive when treated with LzP, meanwhile, A. hydrophila showed slight resistance. At the time, Lz could reduce A. hydrophila growth rate 1.1 log₁₀ cfu/ml with a killing power of 18.03%, LzP could reduce 2.9 log₁₀ cfu/ml with a killing power of 47.54% at a concentration 1000 µg/ml.

Antibacterial Efficacy of LzP Compared with Organic Acids Against Escherichia coli

In the food industry, synthetic additives are used to improve the characteristics and properties of processed foods; and include antimicrobial preservatives (glycine, citric acid, propionic acid, potassium sorbate, and sodium benzoate). Figure 3 showed the antibacterial activity of LzP and many widely used chemical weak organic acids used as food preservatives against E. coli. Despite being utilized higher concentration of weak organic acids (0.3%) than LzP (0.1%) in the antibacterial assay, they performed less antibacterial impact on E. coli growth.

During the 4 h antibacterial assay incubation, the most powerful weak organic acids (citric acid and propionic acid both at a concentration of 0.3%) were able to reduce the E. coli survival rate to roughly 3.9 log₁₀ cfu/ml, which is nearly half of the control value 7.2 log₁₀ cfu/ml. On the other hand, LzP 0.1% showed the ability to reduce E. coli bacterial growth to a level of 1.1 log₁₀ cfu/ml.

Antibacterial Activity of LzP and its Formulations

The synergistic antibacterial effect of the formulations made of LzP and organic acids, namely, glycine and citric acid were studied using liquid broth assay. We added combinations of LzP and organic acids to the E. coli bacterial suspension and then incubated it at 37ºC/ 4 h after that the viable cell counts (cfu/ml) were enumerated as displayed in Fig. 4. The LzP could inhibit E. coli survival rate in a concentration-dependent manner as the following, the highest LzP concentration 0.1% could decrease E. coli growth to 1.1 log₁₀ cfu/ml followed by LzP concentrations 0.04%, and then 0.02% that could reduce the bacterial count to 3.2, and 5.7 log₁₀ cfu/ml; respectively, when compared to E. coli growth control which could reach 6.9 log₁₀ cfu/ml during the antibacterial assay.

Upon LzP 0.04% and glycine 0.1% being formulated, a potent powerful combination is created that could reduce the E. coli growth to a lower detectable level of 0.9 log₁₀ cfu/ml. It was observed that the addition of citric acid 0.04% to the formulation (LzP 0.02%, and glycine 0.1%) did not improve the formula's synergistic antibacterial power. On the other side, using glycine 0.1% or even formulated with citric acid 0.04% is less antibacterial effective as they could reduce the bacterial survival rate to 1.6 log₁₀ cfu/ml in comparison with E. coli growth control at 6.9 log₁₀ cfu/ml.

Stability of LzP Antibacterial Activity

Heat Stability

The LzP’s antibacterial activity was evaluated for its stability at various degrees of temperatures and storage time on a model strain, E. coli. Survival rate after incubation for 4 h then the results calculated as inhibitory percent are presented in Table 1. Strikingly, we found that temperature and storage time had greater impact on LzP antibacterial activity against E. coli. Treated LzP at 100ºC for 30 min resulted in maximum stability and the percentage of bacterial growth inhibition reached approximately 95%. Meanwhile, LzP subjected to autoclaving at 121ºC/ 15 lbs/ 15 min lost 67% of its antibacterial activity. On the contrary, the antibacterial activity of LzP was relatively resistant to different degrees of thermal storage conditions at 4ºC / 30 d, -20ºC / 7 d, and freeze drying as the antibacterial activity maintained 100% compared with the non-heat treated LzP.

Table 1

Effect of storage incubation time and thermal stability on the antibacterial activity of Lysozyme peptides (LzP) at different conditions against *E. coli.*
Treatment	Antibacterial activity%
No treatment 100°C/30 minutes 121 °C/15 lb in^-2/ 15 minutes 4°C/ 30 days -20°C/ 7 days Freeze-dried	100 95 33 100 100 100

pH Stability

Data in Fig. 5 showed the stability of LzP’s antibacterial activity at different pH values, ranging from 4 to 8. It was observed that the antibacterial activity is gradually decreased with increasing the pH values. The optimal antibacterial stability was observed at pH 4.0 to 7.0. LzP at a concentration of 1000 µg/ml showed about 6-fold E. coli bacterial growth reduction. Growth patterns of E. coli in the absence of LzP was varied according to the pH value; its optimum growth rates were at pH 4–7 reaching 6.9 log₁₀ cfu/ml. Whereas, by increasing the pH value to the alkaline side, E. coli showed a slight inhibitory decrease in the growth rate reached to 5.5 log₁₀ cfu/ml.

Lytic activity and SDS-PAGE

Table 2. revealed that LzP only had 11.05% (2409 u/mg) of the lytic activity of the untreated Lz (which is 100%: 21,799 u/mg). Although LzP lost about 89% of lytic activity, it demonstrated potent antibacterial action against E. coli compared to Lz.

Table 2

Muramidase activity of lysozyme and LzP.
	Lz	LzP
Muramidase activity (%)	100.00	11.05
Muramidase activity (µ/mg)	21.799	2.409

SDS–PAGE of the peptic Lz digests was screened using Tris-Tricine ready gels with 16.5% acrylamide stained by silver stain illustrated in Fig. 6. Clear band with molecular weight parallel to Lz at 4.4KDa. Other small molecular weight peptide fragments appear corresponding to 7.36, 5.43 and 1.04 KDa also produced.

Foodborne illness caused by microbes present in foods are considered an important emerging public health issue and encompasses a wide range of diseases [2]. Food proteins can be hydrolyzed by three approaches, enzymatically (using proteolytic enzymes from either plants or microorganisms), hydrolyzed with digestive enzymes (simulating gastrointestinal digestion), or fermented using starter cultures to yield bioactive peptides. In the approach enzymatic hydrolysis method, the target protein is subjected to enzymatic treatment at a certain pH and temperature within a specific time. The benefits of this approach include ease of scaling up and a generally faster reaction time than microbial fermentation [17]. Within the protein sequence, many bioactive peptides are encoded that serve as many functional active ingredients [18]. The peptides generated can be more precisely controlled when proteins are hydrolyzed by enzymes outside of the gastrointestinal tract. These can then be searched individually or in combination for biological functions and potential applications that diverge from those of the parent proteins [13].

Muramidase, a Lysozyme from hen egg whites that belongs to the family of glycosidic hydrolases, catalyzes the lysis of the β (1–4) link between N-acetylglucosamine and N-acetylmuramic acid in bacterial cell walls. Its primary structure is a single polypeptide chain with 129 amino acids, as illustrated in the protein data bank (PDB code 1HEW) displayed in Fig. 6C. Usually, it disintegrates into a compact, spherical secondary structure with a surface slit [19, 20]. Pepsin at pH 4.0, primarily breaks down peptide bonds that contain aromatic hydrophobic amino acids, with Phe, Trp, and Leu residues providing the best cleavage sites [21]. It is necessary to point out that a previous study [22] could identify and locate the peptide fragments using MALDI-TOF-MS analysis that supports our validated SDS-PAGE findings in Fig. 6C. It was noted that incomplete peptic hydrolysis of Lz produced more active, smaller peptides with molecular weights of 7.3, 5.4, and 1.04 KDa and left 60% of the Lz protein intact that referring to LzP. Although Lz's catalytic activity is more crucial for bacterial growth control, LzP has less lytic activity (11.05%). Even though LzP could perform more potent antibacterial activity which is attributed to the stronger generated peptides.

Our study was with particular emphasis on exploring the antibacterial activity of LzP on different pathogenic and spoilage bacteria as well as the variables influencing its efficacy on E. coli that serve as a model in the antibacterial assay. According to our findings, The LzP effectively combats the survival of E. coli and Sal. enteritidis but Sal. typhimurium exhibits a little amount of resistance which is frequently associated with food poisoning with a number of virulence characteristics and drug resistance, may be to blame for this resistance. The primary mechanisms of resistance are altered lactamase and penicillin-binding proteins, decreased permeability of the outer membrane, and activation and synthesis of efflux pumps [23, 24]. Both Ps. aeruginosa and Ps. fluorescence were more vulnerable to LzP while A. hydrophila showing slight resistance; where LzP was only 47% lethal at a concentration of 1000 µg/ml. A. hydrophila is a Gram-negative, anaerobic, oxidase-positive, facultative, opportunistic marine pathogen. It produces a variety of virulence factors as enterotoxins and lytic enzymes. It has been isolated from various food items as meat, milk, and vegetables. However, numerous research revealed that this opportunistic pathogen is resistant to commercial antimicrobials. Recognition of A. hydrophila as an anaerobe is required to make the antibacterial activity of LzP decrease due to the need for particular growing conditions during the antibacterial assay [25].

To prevent microbial growth, chemical preservatives like benzoate, propionate, sorbate, nitrate, and sulfites are frequently utilized [26]. Recently, it has been observed that synthetic preservatives have raised many health concerns and issues. As consumers are becoming more conscious of the relationship of health issues and their diet, consumer awareness become increasing about the synthetic-based antimicrobials in food formulations. Due to worries over these compounds' long-term use, which results in liver damage, asthma, numerous allergic reactions, and even cancer, therefore, most people are turning to natural antimicrobials [27]. Consequently, the use of synthetic preservatives has negative effects on human health, and food researchers and consumers discourage their usage. However, numerous studies have demonstrated a link between the overuse of synthetic food additives is related with gastrointestinal, respiratory, dermatological, and neurological adverse reactions [27]. Due to these public health risks caused by weak organic acids, it is imperative to find natural antimicrobials that can effectively combat these organic acid-based public health risks.

Using a liquid broth experiment, the pre-screening effects of food-grade weak organic acids on E. coli survival in comparison to LzP were studied. E. coli is less sensitive to the effect of organic acids despite the fact that 0.3% of organic acids were used as opposed to 0.1% of LzP, we observe E. coli had less sensitivity to the effect of organic acids as Gram-negative bacteria are typically less susceptible to weak acids action because the bacterial protective outer membrane, which serves as a protective barrier to organic acids action [28]. The LzP was tested for antibacterial activity against E. coli. Then formulation of LzP, glycine, and citric acids were tested in the current study. According to earlier studies, organic acids are frequently utilized as food preservatives due to their antibacterial qualities. Particularly, the undissociated form of the acid that can freely diffuse past the membrane of microbes and into their cell cytoplasm is what weak organic acids' antimicrobial activity depends on. The acid will dissociate, and anions will collect once inside the cell, where the pH is almost neutral, inhibiting cell enzymes (decarboxylases and catalases) and nutrition transport mechanisms [29]. Contrarily, antimicrobial LzP functions as membrane-disrupting antibacterial agents that engage with the bacterial membrane to create pores, which ultimately cause bacterial death [30]. Different interactions may arise when antibacterial agents are combined, leading to a variety of effects that could be additive, antagonistic, or synergistic [31]. Combining antibacterial agents produces stronger effects that boost antibacterial activity and enable the use of lower dosages of chemical organic antibacterial agents that are safe for use in food.

According to the findings of our experiments, LzP activity has stronger antibacterial activity than weak organic acids. However, in the time-kill assay, neither synergistic effects nor additional value between LzP and citric acid was observed. There was no discernible difference between glycine 0.1% (5.2 log₁₀ cfu/ml) or when coupled with citric acid 0.04% (5.2 log₁₀ cfu/ml). There were no appreciable differences observed between LzP 0.1% alone (1.1 log₁₀ cfu/ml) and when combined with glycine and/or citric acid. Otherwise, it would be beneficiary as we use LzP at low concentrations with glycine and/or citric acid, this may be of importance to reduce the preservation costs. The slight difference in bacterial inhibition may be attributed that the LzP mechanism creates pores or tunnels in the cell membrane making it easier for organic acids to pass inside the bacterial cells. Our results were in line with the previous study [31] which demonstrated that Lz and citric acid together had no added benefit.

Comparative studies of the relative impact of LzP, such as pH, temperature, and storage time on the growth survival of E. coli. To address this issue, we compared the antimicrobial action of treated LzP on E. coli under several circumstances. It is crucial to identify how thermal storage conditions and pH levels affect the antibacterial stability of LzP because many food-related factors can completely or partially affect the function of these compounds.

High-pressure treatment and autoclaving pressure affect the different forms of protein structure the secondary, tertiary, and quaternary resulting in reversible alterations with induction permanent denaturation [33]. Proteins undergo irreversible denaturation are supposedly to be due to the breakdown of the hydrogen bonds that stabilize and support the secondary structure [34]. This could account for why autoclaving LzP results in a significant decrease in its antibacterial effect. The highly inhibitory effect of LzP during boiling for 30 min or cooling storage gives it a great opportunity and a vital role in foods undergoing thermal processing.

In general, most pathogenic bacteria can typically grow in a pH range of 4.0 to 9.0, with the optimum pH range from 6.50 to 7.50 [35]. The influence of pH values on LzP antibacterial stability was considerable, with a weak acidic zone (pH 4.0–6.0), while less stable at higher pH over 6 (alkaline side).

Finally, the findings presented in this study add fresh knowledge about the ideal circumstances in which antibacterial peptides (LzP) execute their most effective antibacterial activity and offer an intriguing possibility for the prospective use of antibacterial peptides (LZP) as an effective, novel, food origin preservative (nutra-preservative), safe, and natural food preservative delegate is offered by the study's findings.

Due to adverse effects of industrial chemicals and their carcinogenicity and toxicity for humans, the debates have increased on using natural preservatives. It is therefore crucial to find alternatives to conventional food antimicrobials. In this work we present a viable alternative by simply using lysozyme derived peptides using pepsin (LzP). It performs potent antibacterial effect against most spoilage and food borne pathogens. LzP at low concentration acquired potent antibacterial activity than using weak organic acids (glycine and citric acid) which are commonly used in the food-processing industry. In addition, it was further found that the formulated mixture of food-grade weak acids to LzP have not add more synergistic antibacterial value. It was found, LzP effective at wide range of pH (4–8) and maintain good stability against thermal processing at 100 ºC/ 30 min in addition withstand the storage cooling conditions. We, therefore, suggest the prospective use of LZP as an eco-friendly approach to food preservation.

Acknowledgments

The authors thank Dr. Mujo Kim, CEO of Pharma Foods International Co., Ltd, Kyoto, Japan for his support of our work and for providing lysozyme peptides preparation samples for this study.

Funding Declaration

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Author Contribution

Adham M. Abdou: Conceptualization, Methodology, Resources, Validation, Supervision, Visualization, Data curation, Writing - original draft, Writing - review & editing. Dina A. Awad: Conceptualization, Methodology, Resources, Validation, Writing - original draft & Data software.

Conflict of Interest

The Authors declare that there is no conflict of interest.

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Lysozyme-Derived Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms

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