Biosynthesis and characterization of silver nanoparticles from Punica granatum (pomegranate) peel waste and its application to inhibit foodborne pathogens

Polyphenolics have been predicted to effectively develop antimicrobial agents for the food industry as food additives and promote human health. This study aims to synthesize pomegranate peel extract (PPE) with silver nanoparticles (AgNPs) against eight foodborne pathogens. Multispectroscopic analysis of UV–vis spectroscopy, Zeta potential, Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) analysis were used to characterize the interaction between PPE and AgNPs. Eight foodborne pathogenic strains (six bacterial and two fungal strains) Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8379, Klebsiella pneumoniae ATCC 00607, Salmonella typhi DSM 17058, Shigella sonnei DSM 5570, Aspergillus flavus ATCC 9643, and Rhizopus oryzae ATCC 96382 were used to test the inhibitory potential of PPW-AgNPs. The reaction colour of PPE-AgNPs from yellow to brown indicated that the nanoparticles were successfully formed. The UV absorption of PPE-AgNPs was detected at 440 nm of 0.9 SPR. SEM image of PPE-AgNPs exhibited spherical shapes with a zeta potential of − 20.1 mV. PPE-AgNPs showed high antimicrobial activity against all tested strains. The highest inhibition activity of PPE-AgNPs was recorded for the B. subtilis strain followed by K. pneumonia, while the highest resistance was noticed for R. oryzae. The components of pomegranate peel were analyzed using gas chromatography–mass spectrometry (GC–MS). The major constituents of pomegranate peel is phenol (51.1%), followed by Isocitronellol (19.41%) and 1-Propanol, 2-(2-hydroxypropyl)- (16.05%). PPE is key in the simple, eco-friendly green synthesis of extracellular stable AgNPs as an alternative source for harmful chemical disinfectants.


Standard inoculum
Standard inoculum for all tested pathogenic bacterial and fungal strains was prepared according to a previous method 18 .A loop of freshly prepared culture of bacterial inoculum was inoculated with 50 mL glucose broth and incubated at 37 °C for 24 h using the shake flask method at 150 rpm.The spore suspension was inoculated with a broth of malt media and incubated at 28 °C for 72 h at 150 rpm.

Preparation of pomegranate peel extract (PPE)
The pericarp was manually separated from pomegranate peels and used for extraction.According to a previous study 19 , the pomegranate peel aqueous extract was obtained.Twenty grams of fresh pericarp was soaked in 60.0 mL of distilled water and placed in the shaker at 150 rpm for 4 h.Samples were left in the dark at room temperature for 12 h and centrifuged at 10,000 rpm for 15 min.Supernatants were collected and used for subsequent studies.

Biosynthesis of pomegranate peel extract (PPE) loaded on silver nanoparticles (AgNps)
Fifty millilitres of AgNO 3 (1 mM) were dropwised into a glass beaker containing 2 mL of the previously prepared PPE solution (33.3% w/v in distilled water).Then, the reaction mixture was incubated overnight in the dark

Minimum lethal concentration (MLC) of PPE-AgNPs
Based on the results of MIC, Minimum lethal concentration (MLC) is expressed as the values of minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) is presented as the lowest AgNP concentrations inhibiting the growth of pathogenic bacterial and fungal strains.MBC value was performed by sub-culturing all negative MIC wells, then incubating at 37 °C and 28 °C for 24 h and 72 h, respectively.The least concentration that showed no growth on the growth medium was indicated as the MBC value 25 .

PPE-AgNPs mode of action
Based on MIC, MBC and MFC results.MBC/MIC and MFC /MIC ratios were calculated.PPE-AgNPs had a bactericidal and fungicidal effect when the ratio's value is greater than or equal to 4. On the other hand, it is considered a bacteriostatic or fungistatic agent when the value equals 2 or less 26 .

SEM preparation for the antimicrobial effect detection of PPE-AgNPs
Sterile distilled water (as control) was prepared in 50 mM PBS (pH 7.0).Subsequently, 100 µL of bacteria or fungi growth preparation (1 × 10 6 spores or bacteria/mL) were added to each tube and incubated at 150 rpm at 30 and 28 °C for 12 and 24 h for bacteria and fungi, respectively.Then, fungi spores and bacteria were collected thrice after centrifugation (10,000 rpm, 5 min), 4 °C, and washed with 50 mM PBS (pH 7.0).Then, samples were fixed with 3% glutaraldehyde 50 mM PBS (pH 7.0) at room temperature for 4 h without agitation and rinsed with PBS four times for 20 min each.After post-fixing in 1% osmic acid at room temperature for 2 h, the samples were washed with double distilled water for 15 min.Samples were dehydrated through a graded alcohol series of 30%, 50%, 70%, 80%, 90%, and 95%, then thrice at 100% for 15 min in each series.Following this, samples were incubated in isoamyl acetate overnight.Samples were then subjected to a critical point of dry carbon dioxide and coated with gold.Observation using a scanning electron microscope (S-3400N, SEM system, Hitachi, Tokyo, Japan) 27 .

Gas Chromatography (GC-MS) analysis for PPE
The pomegranate peel (PPE) powder was analyzed using Gas-Chromatography-Mass spectrometry (GC-MS, QQQ 7890B).10 μL of PPE ethanolic extract were diluted with hexane (≥ 99%, Sigma-Aldrich, Darmstadt, Germany) and injection volume at 2 μL was subjected to column chromatography (Agilent 19091S-433: 1 HP-5MS, www.nature.com/scientificreports/30 m × 250 μm × 0.25 μm).The separative chromatography included QQQ Collision Cell EPC (He Quench Gas with a flow rate of 2.25 mL/min, N 2 Collision Gas with a flow rate of 1.5 mL/min) was applied.After the sample was subjected, the oven temperature was set at 40-280 °C with a flow rate of 4 °C/min and a post-run of 2 min.Qualitative volatile PPE compounds were compared with their mass involved in library data 28 .

Statistical analysis
All samples and collected data were statistically analyzed and expressed as means using IBM® SPSS® Statistics software (2017).Tukey's test at a P-value of 0.05 was applied 29 .Each experiment was performed in triplicate, and the results are expressed as mean ± SD.

Results and discussion
Biosynthesis and characterization of PPE-AgNPs PPE-AgNP biosynthesis was detected immediately when the color changed from pale yellow to dark orange upon adding silver nitrate to the pomegranate waste extract under vigorous stirring conditions (Fig. 1).Similar color change observations have been reported in recent studies on green synthesis of silver nanoparticles using pomegranate peel extracts.In a similar study 30 , a color change was noticed from light yellow to brown during biosynthesis of silver nanoparticles from P. granatum peel extract after mixing with silver nitrate solution for 20 min.A study reported a color change from pale yellow to dark brown within 1 h of mixing pomegranate peel extract and silver nitrate, indicating silver ion reduction 5 .Similarly, comparable observations were noticed of a color change from light yellow to reddish brown in 5 min after adding silver nitrate to pomegranate peel extract 21,31 .The rapid color change from light to dark shades is attributed to the excitation of surface plasmon vibrations in the synthesized silver nanoparticles.These similar findings across recent literature validate the use of visual color change as a preliminary indicator of successful biosynthesis of silver nanoparticles using pomegranate peel extract.
UV-visible spectrophotometer UV spectrum range were recorded for control PPE and PPE-AgNPs at 200-700 nm.Three spectrum peaks were detected for PPE control at 256.6, and 383.12 nm, while for, PPE-AgNPs, only one peak was detected with a strong surface plasmon resonance (SPR) at 440, recording a score of 0.9 (Fig. 2).Many researchers revealed the detection of AgNPs at 432-442 nm after 5 min of mixing.On the other hand, some studies reported the detection of AgNPs within 10-3 min using different peel extracts 19,32 .
DLS and Zeta potential PPE-AgNPs had a diameter ranging from 75.5 to 4085.6 nm with a mean size of 624.4 nm, as shown in Fig. 3 and a zeta potential of − 20.1 mV, as illustrated in Fig. 4. Contrarily, 30 reported a much higher zeta potential of − 68.93 mV and less average particle size distribution between 57.7 and 42.4 nm for the AgNPs synthesized using pomegranate peel extract.Moreover, 33 found that the average particle size of AgNPs from onion plant leaf extract was 36 nm, and the zeta potential was − 24.1 mV.This wide variation in AgNP sizes is attributed to the reducing agent's structure and the chemical composition of plant leaf extract compounds 34 .The zeta potential   www.nature.com/scientificreports/value indicated moderate stability of biosynthesized PPE-AgNPs 0.3 mv, which could be attributed to the natural bioactive compounds responsible for capping, reduction of silver ions, and stability of nanoparticles 35 .

Fourier transform infrared (FTIR)
FTIR was carried out to identify the main biomolecules in plant extracts that help in Ag+ ions reduction to AgNPs.As shown in Fig. 5 and Table 1, the main phytochemicals compounds detected in pomegranate peel waste extract were flavonoids, alkaloids, glycosides, and terpenoids.A similar study by 20 detected phenolic compounds in pomegranate peel waste extract using FTIR with peaks at 3371/cm, 1635/cm, 1373/cm, and 2924/cm, which were assigned to the stretching of primary and secondary amines, the C-N stretching vibrations of aromatic groups 17, respectively which play a role as capping agents.
Recent studies have comprehensively characterized the diverse phytochemicals present in pomegranate peel waste extracts that contribute to the green synthesis of metallic nanoparticles.A study 36 detected flavonoids, alkaloids, terpenoids and polyphenols using phytochemical tests and proposed these biomolecules reduce silver ions and stabilize nanoparticles..In summary, these recent studies strongly indicate the diverse phytochemical composition of pomegranate peel that provides reducing, capping and stabilizing agents for green sustainable of nanoparticle synthesis.

Scanning electron microscope (SEM)
Scanning Electron Microscopy (SEM) determined the PPE-AgNP's shape and size from Punica granatum.SEM analysis showed semi-spherical coated AgNPs, as shown in Fig. 6.

Inhibitory activity of PPE-AgNPs from Punica granatum
Antibiotic susceptibility test of the biosynthesized PPE-AgNPs was performed using eight foodborne pathogens, including six bacterial and two fungal strains.Data in Table 2 showed that all tested pathogenic bacteria were highly sensitive to AgNPs with IZD ranging from 4.5 to 0.96 cm, while fungi were resistant with the lowest IZD of 0.96 cm when compared to the control antibiotic drug with IZD scored 4.1-0.96cm.The most sensitive bacterial strain was recorded for B. subtilis with an IZD of 4.5 cm and activity index (AI) of 1.10.However, the lowest IZD and activity index (AI) was observed for the fungal strains of A. flavus and R. oryzae ATCC 96382, reaching 1.30 and 0.96 with AI of 0.48 and 0.51, respectively.This inhibitory activity is attributed to the binding of silver to the phospholipid layer of the bacterial cell membrane, where the formation of pores occurs.This,  www.nature.com/scientificreports/ in turn, results in their destabilization and an increase in membrane permeability, causing leakage of cellular metabolites and cell death 30,38 .Also, 34 found that the AgNPs synthesized using pomegranate fruit peel extract as a reducing agent exhibited a fairly significant inhibition with 2.6 cm against S. aureus.The major mechanism of silver nanoparticles manifesting antibacterial properties was anchoring or penetrating the bacterial cell wall and modulating cellular signalling by dephosphorylating peptide substrate on tyrosine residues.

Minimum inhibition concentration (MIC) of PPE-AgNPs
MIC values of PPE-AgNPs against the tested pathogenic bacterial and fungal strains ranged from 1000 to 12.5 μg/ mL, as illustrated in Table 3.The MIC value was exhibited at 250 μg/mL for E. coli ATCC 8379 and E. faecalis ATCC 29212, while it was 125 μg/mL K. pneumoniae.On the other hand, it was 50 μg/mL against S. typhi and 25 μg/mL against B. subtilis.The results showed 100% of the antibacterial spectrum activity of PPE-AgNPs at concentrations ranging from 1000 to 250 μg/mL, whereas at concentrations of 125, and 75 μg/mL the activity was 66.7% and 50%, respectively.Whereas at a concentration of 50 μg/mL, the antibacterial spectrum showed 33.3%; at a 25 μg/mL concentration, it exhibited 16.6% activity.There was no antibacterial spectrum at 12.5 μg/mL.The PPE-AgNPs MIC antifungal activity investigation was at concentrations of 1000-12.5 μg/mL, both A. flavus and R. oryzae.The MIC was 250 μg/mL for R. oryzae and 75 μg/mL for A. flavus.At 1000-250 μg/mL concentrations, 50% of the spectrum of activity was attained for all fungi at 125-75 μg/mL.In addition, concentrations ranging from 25 to 12.5 μg/mL did not display any activity against the tested fungal strains.

Minimum lethal concentration (MLC) of PPE-AgNPs
The minimum lethal concentration MLC (MBC and MFC) for pomegranate peel silver nanoparticles (PPE-AgNPs) are presented in Table 4.The MBC value was 500 μg/mL for E. coli, E. faecalis and K. pneumoniae.On the other hand, it was 250 μg/mL for S. sonnei and 125 μg/mL for S. typhi and B. subtilis.The results showed 100% of the antibacterial spectrum activity of PPE-AgNPs at concentrations ranging from 1000 to 250 μg/mL, whereas at concentrations of 250-125 μg/mL, the activity was 33.3%.There was no antibacterial spectrum at a 75-12.5 μg/mL concentration.The MFC was 1000 μg/mL for R. oryzae ATCC 96382, while it was 500 μg/mL for A. flavus, as shown in Table 4.At the concentration 1000, the spectrum activity was 100%, while at 500 μg/ mL, only 50% of the activity was attained for all fungi at 250-12.5 μg/mL did not display any activity against the tested fungal strains.

Pomegranate peels silver nanoparticles (PPE-AgNPs) action mode
Finally, it could be observed that the mode of action of PPE-silver nanoparticles against pathogenic bacterial and fungal strains is shown in Table 5. Results indicated that the PPE-AgNPs have a bactericidal against E. coli and E. faecalis, while it was bacteriostatic with B. subtilis, K. pneumoniae, S. typhi, S. sonnei, and S. sonnei.Also, it revealed a fungistatic effect for both fungal strains.In Brief, PPE-AgNPs showed high antimicrobial activity against all tested strains.The highest inhibition activity of PPE-AgNPs was recorded for the B. subtilis strain followed by K. pneumonia, while the highest resistance was noticed for R. oryzae, Fig. 7. Previous research has elucidated the complex interactions between nanoparticles (NPs) and microorganisms including bacteria, fungi and viruses along with the resultant antimicrobial effects.Metal NPs like silver, gold, zinc oxide and copper have shown pronounced antibacterial activity against both Gram positive and Gram negative bacteria due to their positively charged surfaces interacting and damaging the negatively charged bacterial cell membrane leading to cell death 39,40 .The antimicrobial mechanisms involve generation of reactive oxygen species, inhibition of vital www.nature.com/scientificreports/microbial enzymatic and protein functions, and disruption of DNA replication and protein synthesis causing growth inhibition.Factors like NP size, shape, surface properties and concentration influence the antibacterial effects, with smaller NPs having greater impacts due to higher surface area to volume ratios 41 .However, some studies have reported microbial resistance development against NPs indicating dynamic interactions and adaptation.Overall, previous research has clearly demonstrated the broad-spectrum antimicrobial properties of diverse NPs and elucidated their complex interactions with microbial cells that induce cell damage and growth inhibition.But microbial adaptation mechanisms point to the need for further studies on these evolving NPmicrobe interactions.

SEM preparation for the antimicrobial effect detection of PPE-AgNPs
In our SEM findings, obvious signs of differences in cell morphology between control and treated cells with damage to the cell wall for the most influenced microorganisms of Bacillus subtilis, Enterococcus faecalis, and Aspergillus niger were found according to analysis with the aid of a scanning electron microscope (SEM).After 12 www.nature.com/scientificreports/and 24 h of incubation at 30 and 28 °C for 12 and 24 h for bacteria and fungi, respectively.The control microbes treated with distilled water displayed a normal and compact morphology (Fig. 8a, b, e), whereas the microbial cells treated with 1000 µg/mL PPE-AgNPs showed irregular modifications.As shown in Fig. 8c, d, cell surfaces appeared to suffer from PPE-AgNPs with irregular morphological shapes, disrupted cell walls and pores or invaginations on cells, and hypha broken down for fungi, as shown in Fig. 8f, g.In the same line, it was found that AgNPs, there was accumulation on S. aureus and P. aeruginosa cell surface, which results in pores or invaginations on cells that affect bacterial viability 27,42 .Cells treated with AgNPs showed destruction and distortion at cell poles due to an accumulation of nanoparticles that affected cell viability and caused cell death and pores or invaginations on cells.The antifungal SEM investigation 43 shows a great variation between a control with the normal shape of fungal mycelium, whereas the treated sample with AgNPs shows aggregations of small micro nanoparticles at an earlier stage of formation on the hyphae 44 .

Gas Chromatography (GC-MS) analysis for PPE
The phytochemical composition of pomegranate peel (PPE) was analyzed using GC-MS and presented in Table 6 and Fig. 9.The major constituents of pomegranate peel were phenol (51.1%), followed by Isocitronellol (19.41%) and 1-Propanol, 2-(2-hydroxypropyl)-(16.05%).Additionally, other terpenoids such as geraniol (4.89%), styrene (4.47%), and isopolygol (4.07%) have been identified in the ethanolic extract of pomegranate peel (Fig. 9).Geraniol is a monoterpenic alcohol with a pleasant rose-like aroma, is found in many essential oils and has antibacterial and antifungal potential 45,46 .Phenolic compounds have gained attention, especially in the food research sector, as microbial growth inhibitors for foodborne pathogenic and spoilage bacteria.All phenols also have promising anti-quorum sensing potential and inhibit the biofilm formation and toxin production of food-related pathogens 47 .

Conclusion
The present study demonstrates a simple, eco-friendly biosynthesis of PPE-AgNPs using pomegranate peel extract as a reducing agent.The phytochemicals detected by the FTIR, such as flavonoids, alkaloids, glycosides, and terpenoids, acted as better reducing and capping agents.Besides the physicochemical advantages of PPE-AgNPs, these have shown improved antimicrobial activity, which can contribute to solving the global problem of multi-drug resistant (MDR) microorganisms soon.

Figure 1 .Figure 2 .
Figure 1.Colour change of PPE solution from Punica granatum during reduction of silver nitrate to form PPE-AgNPs. Left: pomegranate peel waste extract.Right: biosynthesis of PPE-AgNPs revealing change in colour from yellow to brown.

Figure 8 .
Figure 8. SEM images of (a) Bacillus subtilis, (b) Enterococcus faecalis, and (e) Aspergillus niger cells control treated with distilled water, while (c) Bacillus subtilis, (d) Enterococcus faecalis, and (e, f) Aspergillus niger cells treated with 1000 µg/mL PPE-AgNPs.Arrows refer to the morphologic alterations in cells and contact of PPE-AgNPs cell membrane and collapsed cell walls.

. Intensity Peak position Functional group Compound
Detected peaks at 3396, 3278, and 3169/cm that were attributed to various (OH) and (NH) groups as well as H-bonding water-OH, alcohols, and amides that possibly are a part of NPs-stabilizing compounds, and the capping agents.Hence, it is reported that the amide group, amino, carbonyl group, and polyphenolic compounds in the PPE are a part of the redox reaction, dispersion, capping, and stabilizers involved in the production of nanoparticles during the process of synthesis Vol.:(0123456789) Scientific Reports | (2023) 13:19469 | https://doi.org/10.1038/s41598-023-46355-xwww.nature.com/scientificreports/

Table 6 .
Chemical composition (%) of PPE analyzed by GC-MS.a Retention time.b Compound percentage.