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Article

Investigating the Potential of Green-Fabricated Zinc Oxide Nanoparticles to Inhibit the Foodborne Pathogenic Bacteria Isolated from Spoiled Fruits

by
Amr Fouda
1,2,*,
Mohammed Ali Abdel-Rahman
1,
Ahmed M. Eid
1,
Samy Selim
3,*,
Hasan Ejaz
3,
Muharib Alruwaili
3,
Emad Manni
3,
Mohammed S. Almuhayawi
4,
Soad K. Al Jaouni
5 and
Saad El-Din Hassan
1
1
Botany and Microbiology Department, Faculty of Science, AL-Azhar University, Nasr City, Cairo 11884, Egypt
2
School of Nuclear Science and Technology, University of South China, Hengyang 421001, China
3
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka 72388, Saudi Arabia
4
Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia
5
Department of Hematology/Oncology, Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Catalysts 2024, 14(7), 427; https://doi.org/10.3390/catal14070427
Submission received: 5 June 2024 / Revised: 22 June 2024 / Accepted: 1 July 2024 / Published: 4 July 2024
(This article belongs to the Section Biocatalysis)
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Versions Notes

Abstract

In the current investigation, the antibacterial activity of zinc oxide nanoparticles (ZnO-NPs) formed by an aqueous extract of Psidium guajava leaves against foodborne pathogenic bacterial strains was investigated. To achieve this goal, 33 bacterial isolates were obtained from spoiled fruits. Among these isolates, 79% showed cellulase activity, 82% showed amylase activity, 81% exhibited xylanase potential, and 65% exhibited lipase activity. Moreover, 12 isolates showed complete hemolysis (β-hemolysis). The identification of these isolates was done using sequencing and amplification of 16s rRNA as Staphylococcus aureus (two strains), Pseudomonas syringae (one strain), E. coli (two strains), Salmonella typhimurium (two strains), Listeria monocytogenes (one isolate), Bacillus cereus (two isolates), and Bacillus subtilis (two isolates). The formed ZnO-NPs by aqueous Psidium guajava leaf extract were characterized using UV, FT-IR, TEM, EDX, XRD, DLS, and Zeta potential. The data revealed the successful formation of a spherical shape, crystallographic structure, and well-arranged ZnO-NPs. FT-IR showed the effect of different functional groups in the plant extract in the formation of ZnO-NPs through reducing, capping, and stabilizing of end products. Moreover, EDX analysis showed that the Zn ion occupied the main component of the produced NPs. Interestingly, the obtained bacterial strains showed varied sensitivity toward green-synthesized ZnO-NPs. The growth inhibition of foodborne pathogenic strains by ZnO-NPs was concentration dependent, forming a zone of inhibition in the range of 20–23 mm at a concentration of 200 µg mL−1, which decreased to 15–18 mm at 100 µg mL−1. Moreover, the values of MIC were 25 and 50 µg mL−1 based on the bacterial strain. Overall, the green-synthesized ZnO-NPs can be a useful approach for inhibiting the growth of spoilage bacterial strains that destroy fruits and hence reduce the harmful effects of traditional treatment methods on the environment and human health.

1. Introduction

Fruits are important for a healthy lifestyle and should be incorporated into the daily diet due to being a rich source of minerals, vitamins, carbohydrates, antioxidant compounds, proteins, dietary fiber, and phytochemical substances. However, they are considered a rich source of microbial contaminants, which cause deterioration and spoilage [1]. Microorganisms that contaminate fruits can be obtained from the soil, air, water, human handling, equipment during harvesting, transportation or processing, dust, seeds, and post-harvest treatment [2]. Different bacterial species are recognized as spoilage strains and have negative impacts on human health (foodborne pathogens). Among these strains are Bacillus spp., such as B. cereus, B. anthracis, B. pseudomycoides, B. subtilis, B. mycoides, B. toyonensis, B. thuringiensis, and B. cytotoxicus [3]. Also, Listeria monocytogenes, Salmonella spp., E. coli, Staphylococcus spp., and Pseudomonas spp. are known as foodborne pathogenic bacteria [4].
Hence, the main challenges for researchers is discovering new active substances that have multivaried bioactivities, such as antibacterial, antifungal, antioxidant, and food preservation, for maintaining fruit safety, quality, and freshness [5]. Storage and handling methods are important to avoid microbial contamination and physical spoilage. Preservation is achieved through gentle handling to avoid scratches and optimize the storage conditions at optimum temperature and suitable humidity levels [6]. Optimizing these factors is a critical step for preventing the growth of microbes and hence reducing the enzymatic reactions that cause fruit spoilage. Also, washing and sanitization of packaging materials such as bags, moisture-resistant films, and preservation containers are considered important steps in the preservation of fruits via reducing the microbial load, leading to a decrease in spoilage and foodborne pathogens [7]. Post-harvest treatment using chemical substances such as 1-methylpropene (inhibit the ethylene action in the fruits), calcium chloride (delay the ripening and senescence), and antimicrobial agents such as fungicides and bactericides is a crucial step for fruit preservation [8]. Although these chemical compounds may have strong actions for protecting fruits, they have harmful effects on the health of human and reduce the nutritional value of fruits; therefore, they should be approved by food organizations to ensure their safety.
Recently, nanotechnology has been considered a promising approach for post-harvest disease management and is considered one of the best strategies to increase the shelf-life of edible and storage fruits [9]. Nanotechnology is a multidisciplinary science concerned with the formation of new active substances on the nanoscale (1–100 nm) [10]. These nanoscale structures have promising features that do not exist in bulk materials and hence increase their applications and incorporation into various sectors [11]. Among these sectors, the food industry and fruit preservation can use nanoparticles as antimicrobial agents, to monitor fruit and food quality, produce nanosystems to mitigate spoilage factors, form coating and barrier films, establish nanosystems for safe packaging, and act as scavengers for ethylene [12]. Due to the antimicrobial activity of nanoparticles, they can be integrated as coating agents on the surface of fruits or in packaging materials, leading to an increase in fruit shelf-life. Also, they can be used for the production of nanosensors to monitor various parameters that cause fruit spoilage, such as pH, humidity, and temperature. Ethylene is a plant hormone used for the acceleration of ripening and senescence of fruits. Recently, some nanomaterials have been integrated into packaging materials to adsorb or scavenge this plant hormone, leading to an increase in fruit shelf-life [13]. Interestingly, the use of these substances has some advantages compared to chemicals, such as target selectivity, lower toxicity, decreased toxic effects, and increased preservation time [14]. Because of various advantages of green synthesis, such as low cost, biological compatibility, safety for the environment, and scalability, it has recently emerged as the superior approach for producing nanoparticles when compared to physical and chemical synthesis techniques. Different bacterial, fungal, actinomycetes, plants, and algal strains are used to create stable nanoparticles [15,16]. In this sense, nano zinc oxide (ZnO-NPs) is unique among nanomaterials due to its special optical and chemical characteristics besides its extensive antibacterial action. Moreover, it has been effectively fabricated by bacteria, plants, and marine macroalgae [17,18]. The Food and Drug Administration defines ZnO-NPs as safe substances; hence, they can be used in the preservation of foods due to their activity to inhibit the growth of foodborne pathogens [19]. Moreover, the treatment of guava and its juice with CuO-NPs and ZnO-NPs reduced water loss, reduced the growth of microorganisms through inhibition of the respiration rate during storage, as well as delayed fruit ripening for a period of 15 days [20].
Guava (Psidium guajava) is one of the tropical fruits whose leaves contain a treasure trove of phytochemicals, such as caffeic acid, chlorogenic acid, gallic acid, epigallocatechin gallate, guaijaverin, hyperin, avicularin, kaempferol, myricetin, quercetin, apigenin, catechin, and avicularin. Guava leaf extract has been used as an alternative medicine for oral ulcers, diarrhea, gum swelling wounds, and cough. In addition, it has been investigated for its antimicrobial, antioxidant, anticancer, antidiarrheal, antidiabetic, hepatoprotective, and lipid-lowering properties [21]. The mixing of chemically synthesized ZnO-NPs with P. guajava leaf aqueous extract increased the inhibition of enteric E. coli compared to ZnO-NPs only due to synergistic effects [22]. In recent studies, the highest bacterial reduction of Bacillus subtilis, Staphylococcus aureus, and Candid albicans was attained by treatment with biogenic ZnO-NPs produced by either water or ethanol guava leaf extract compared to the reduction caused by nanocomposites, ZnO-NPs/chitosan, ZnO-NPs/alginate, and ZnO-NPs/CMC [23,24]. Although the using of aqueous extract of P. guajava leaves to form nano-ZnO is not novel, the current study has several unique contributions, such as the isolation of different bacterial species from spoiled fruits, detection of human pathogenic strains (which cause complete hemolysis), and identification by gene sequence analysis; validation of the green ZnO-NPs formed with a plant extract and a focus on scalability and reproducibility; and a comprehensive assessment of the efficacy of biogenic ZnO-NPs in the inhibition of foodborne pathogenic bacterial strains. For this reason, in this study, we aimed to synthesize zinc oxide nanoparticles using guava leaf extract and examine their activity against a group of fruit-borne bacterial pathogens. The following approaches have been employed to describe the obtained ZnO-NPs; UV–Vis spectroscopy, FT-IR, TEM, EDX, XRD, DLS, and Zeta potential.

2. Results and Discussion

2.1. Bacterial Isolation and Enzymatic Activities

Fruits are characterized by their high content of vitamins, minerals, carbohydrates, and other active substances. Due to their high-water content, fruits are subjected to high microbial attack, leading to spoilage. In this investigation, three types of fruits, namely strawberries, apples, and bananas, suffered from spoilage and were used as sources for bacterial isolation. Thirty-three bacterial isolates were obtained, represented by percentages of 36% (12 isolates) obtained from strawberries, 30% (10 isolates) obtained from apples, and 33% (11 isolates) obtained from bananas. Similarly, 92 bacterial isolates were obtained from the waste of food processing, including compost, vegetables, and rice grounds [25]. Also, Hasan and Zulkahar reported that four bacterial strains were the most common species isolated from spoiled pineapples, bananas, and papayas [26].
The Gram reaction of the obtained bacterial isolates revealed that 18 isolates were Gram-positive strains with bacilli and cocci shapes, while 15 isolates were Gram-negative with rods and bacilli shapes (Table 1, Figure 1). On the other hand, the bacterial isolates showed varied hydrolytic enzyme activities. Among 33 bacterial isolates, 26 isolates (79%) produced cellulase enzymes with different clear zones. The diameter of these clear zones was in the ranges of 1–10 mm for 3 isolates, 10–15 mm for 14 isolates, and more than 15 mm for 9 isolates (Table 1, Figure 1). Moreover, 82% (27 isolates) were amylase producers. Among these isolates, 7 were selected as the highest amylase producers (clear zone > 15 mm), 15 isolates were identified as moderate (10–15 mm clear zone), and 5 isolates showed low amylase activity (1–10 mm clear zone) (Table 1, Figure 1). For xylanase and lipase activities, 81% and 65% were enzyme producers, respectively, with high, moderate, and low activities (Table 1, Figure 1). In a similar study, among 92 bacterial isolates obtained from food processing waste, 48, 23, 60, and 11 bacterial isolates had the potential to protease, cellulase, ligninolytic, and lipase enzymes production, respectively [25].
The potential of the obtained bacterial isolates to secrete different hydrolytic enzymes, such as cellulase, amylase, xylanase, and lipase, opens the way to understanding the role of these strains in food spoilage. The degradation of cellulose, a main plant cell wall component, is achieved by the cellulase enzyme, leading to the softening and deterioration of the fruit tissue, ultimately shortening storage life [27]. As a result of cellulase action, the bacteria can penetrate the fruit cell wall to access their nutrients, leading to spoilage acceleration. Similarly, the starch in fruits is reserved as a carbohydrate during the ripening process. The bacterial strains that have amylase activity can degrade these components to simple sugars to be used as an energy source. The high content of simple sugars promotes the proliferation of bacteria and enhances fruit spoilage [28]. Xylan is a complex plant cell wall polysaccharide that can be degraded by the xylanase enzyme into xylose and hence destroy the plant cell wall, enhancing the entrance of bacteria to fruit tissues and causing their spoilage [29]. Lipids and fats are deposited in plant cell membranes as phospholipid bilayers. The lipase enzyme catalyzes the hydrolysis of these structures, compromising their integrity and accelerating the degradation of fruit tissue [30].
The assessment of hemolytic activity between bacterial strains isolated from spoilage foods is an important indicator for several reasons. For instance, the presence of hemolytic strains, especially those causing complete hemolysis of red blood cells, among foodborne bacteria indicates the pathogenicity of these strains and could pose a health risk to humans and animals. Also, hemolytic bacterial strains can accelerate the breakdown of cell membranes, producing enzymes and toxins that facilitate food spoilage. Moreover, the presence of hemolytic bacteria among those obtained from spoiled foods is monitored by regulations and standards concerning food safety and helps to ensure compliance with food safety protocols [31]. In the current study, the bacterial strains obtained from spoiled fruits showed varied hemolytic activity. Among 33 bacterial isolates, 12 isolates (36%) were characterized as having β-hemolytic activity (complete hemolysis), 13 isolates (39%) were identified as having α-hemolytic activity (partial hemolysis), and 8 isolates (24%) lacked hemolytic activity (γ-hemolysis) (Table 1 and Figure 1). In a similar investigation, 4 isolates (16.7%) from 24 Cronobacter spp. obtained from commercial-ready foods, including nuts, sprouts, fruits, and leaf vegetables, had the potential to completely break down sheep blood cells (β-hemolysis) [32].

2.2. Molecular Identification of β-Hemolysis Bacterial Isolates

The pathogenic bacterial strains with β-hemolytic activity (12 strains) underwent identification by molecular method based on 16sr RNA gene sequencing. Sequence analysis revealed that bacterial strains S3 and S10 were similar to Staphylococcus aureus (accession number, NR115606), with percentages of 99.33% and 98.83%, respectively (Table 2). Strains S5 and B31 were identified with percentages of 99.21% and 98.66%, respectively, as Salmonella enterica (accession number, NR074910). On the other hand, bacterial strain S8 obtained from spoiled strawberries and strain B27 isolated from damaged bananas were identical to Escherichia coli (accession number, NR024570), with percentages of 99.08% and 98.23%, respectively. The bacterial strain coded A16 obtained from spoiled apples was similar to Pseudomonas syringae (accession number, NR117820) with a percentage of 98.93%. Amongst Gram-positive bacteria, the strain coded A18, which was obtained from spoiled apples, was similar to Listeria monocytogenes (accession number, NR044823), with a percentage of 98.56%. Bacillus spp. were found among the obtained strains as B. subtilis (S11 and B24) and B. cereus (A14 and B25), which were identical to those loaded in GenBank with accession numbers of NR027552 and NR115526, with similarity percentages in the range of 98.25–98.88% (Table 2). Therefore, the obtained food spoilage bacterial strains were identified as S. aureus (S3 and S10), S. enterica (S5 and B31), E. coli (S8 and B27), B. subtilis (S11 and B24), B. cereus (A14 and B25), P. syringae (A16), and L. monocytogenes (A18) (Figure 2). The sequences retrieved from gene analysis were loaded in GenBank with accession numbers PP683392−PP683403.
As shown, among 12 bacterial strains that were identified using 16sr RNA, Gram-positive strains were represented by 58.3% whereas Gram-negative strains represented 41.7%. Among Gram-positive bacteria, Bacillus species represented 57%. The most identified bacterial strains were reported in published investigations as foodborne pathogenic strains and had the potential to spoil fruits. For instance, the pathogenic strains of Pseudomonas spp., Salmonella spp., Staphylococcus aureus, Klebsiella spp., and E. coli, with percentages of 23.7, 21.9, 22.8, 8.9, and 22.3%, respectively, were isolated from spoiled samples including fruits, dairy products, vegetables, bakery products, spoiled rice, and poultry products and identified using conventional methods followed by genotypic methods [33]. Among 17 bacterial strains obtained from spoiled fermented vegetables, 16 strains were identified as Bacillus spp. and one strain was Staphylococcus saprophyticus based on amplification and sequencing of the 16sr RNA gene [34].
In addition to the spoilage features of the obtained bacterial strains in the current investigation, most of them are harmful to humans and animals. For example, L. monocytogenes is characterized as a foodborne pathogenic strain causing listeriosis and affecting newborns, pregnant women, and immune-suppressed individuals. The symptoms of infection range from mild, such as muscle aches and fever, to severe symptoms such as meningitis, miscarriage in pregnant women, and septicemia [35]. Also, S. aureus is a common foodborne pathogenic bacteria related to spoiled dairy, salads, meat, and fruit products. It is able to produce toxins that cause gastrointestinal illnesses such as diarrhea, vomiting, nausea, and abdominal cramps [36]. Usually, the harmful effects due to Gram-negative bacteria are more serious. This finding could be related to the presence of endotoxins in the Gram-negative cell wall, which induce the response of the immune system. Also, Gram-negative bacteria produce potent endotoxins that increase damage to infected tissues [37].

2.3. Psidium Guajava-Mediated ZnO-NPs Synthesis

Nanoparticles are widely integrated into different fields due to their unique and superior features shown in their nanoscale structures. Among these sectors, in the food industry, NPs are used either during manufacturing as supplementary agents or after finishing and during packaging as preservation tools to increase and prolong the shelf-life period [38]. To improve their activity and increase biocompatibility, green synthesis is preferable to chemical and physical methods. Therefore, the water extract of Psidium guajava leaves was utilized as a reducing agent of metal precursor (Zn(CH3COO)2.2H2O) to form a nanoscale structure (ZnO-NPs) followed by capping and stabilizing for the final product. These activities could be attributed to the metabolites present in the water extract. The leaves of P. guajava are enriched with different bioactive compounds. For instance, acids such as guavacoumaric, triterpenoids guavanoic, jacoumaric, asiatic acid, isoneriucoumaric, and hydroxyursolic are obtained from guava leaves [39]. Also, glucosides such as guavin A and B, pedunculagin, ellagic acid 4-gentiobioside, and strictinin amritoside, as well as flavonoids such as quercetin, kaempferol, and leucocyanidin were collected from guava leaves [40]. Due to these high metabolic contents, the water extract of guava leaves has been used for producing different metal and metal oxide NPs, such as Ag-, Au-, TiO2-, CuO-, and FeO-NPs [41]. Recently, manganese dioxide nanoparticles (MnO2-NPs) were fabricated with aqueous extract of guava leaves and showed high photocatalytic activity [42]. Interestingly, an aqueous extract of guava leaves reduced Zn(CH3COO)2·2H2O and formed ZnO-NPs, which were utilized to inhibit the Staphylococcus aureus and E. coli growth as well as investigate their efficacy in the breakdown of methylene blue dye (photocatalysis) [24].

2.4. ZnO-NPs Characterization

2.4.1. Optical Characteristics

The optical features of synthesized NPs are an important parameter that is used as a first indicator of size, shape, and interaction of light with the NP surface to detect surface plasmon resonance (SPR). Also, the optical features of NPs detected by UV–Vis analysis are easy, rapid, and simple for exploring their electronic transition stability before being used in various applications [43]. The appearance of a white precipitate after reaction of the water guava leaf extract with the precursor indicated ZnO-NP formation. This transition in color is related to the efficacy of metabolites in the aqueous extract of guava leaves, such as flavonoids and terpenoids, to act as reducing, capping, and stabilizing agents [41]. The complete reduction of Zn(CH3COO)2·2H2O to form ZnO-NPs was monitored as the color change stopping, which was observed after 24 h of mixing the plant extract with metal oxide [44]. The SPR of produced ZnO-NPs was detected by measuring the absorbance of the white color at wavelengths in the range of 200–800 nm. As shown, the maximum SPR was observed at 370 nm (Figure 3), which is typical for the ZnO absorption peak [45]. In a similar investigation, the SPR for nano-ZnO formed by water guava leaf extract was localized at a wavelength of 376 nm [24]. Hameed et al. [46] reported that the UV absorption of ZnO-NPs formed using an aqueous extract of Spirogyra hyaline varied based on the metal precursors used. The authors reported that the maximum SPRs of ZnO-NPs were observed at 358 nm and 363 nm for metal oxide precursors of zinc acetate and zin nitrate, respectively. Mani and coauthors reported that the absorption peak of ZnO-NPs at wavelengths of 320 to 380 nm indicated the successful of monodispersed nanostructures production that have spherical shapes and smaller sizes [47].

2.4.2. Morphological Analysis

Transmission electron microscopy (TEM) is a rapid analysis tool for the detection of shapes and sizes of plant-based ZnO-NPs. As shown, the water extract of guava leaves produced spherical shapes without aggregation and had sizes in the range of 5–30 nm with an average of 14.97 ± 7.2 nm (Figure 4A,B). In a similar study, TEM analysis was used to investigate the shape and size of ZnO-NPs fabricated by four aqueous plant extracts of Buddleja globosa, Aristotelia chilensis, Galega officinalis, and Eucalyptus globulus [48]. The authors reported that the aqueous extracts showed high potential to form spherical shapes with average sizes of 31.2, 13.6, 31.9, and 15.7 nm for B. globosa, A. chilensis, G. officinalis, and E. globulus, respectively. The integration of nanomaterials in various applications is dependent of several parameters, the most important of which are shape and size. For instance, different shapes (spherical, polyhedron, and plate-like) of ZnO-NPs with different sizes (38.8, 23.3, and 161.8 nm) were successfully produced using sol-gel, polyol, and hydrothermal methods, respectively [49]. The authors reported that the highest toxicity (82.9%) of human osteosarcoma MG-63 cell lines was attained by treatment of ZnO-NPs formed using a hydrothermal method followed by those produced by the sol-gel method (66.2%) and polyol method (62.5%). The authors attributed these activities to the variation in size and shape of the synthesized ZnO-NPs. Interestingly, some authors reported that the NP production process is affected by some parameters, particularly temperature and pH, to produce smaller sizes [50]. They showed that the high temperature of the process and alkaline conditions are preferred to produce smaller sizes. Therefore, the ZnO-NP production conditions were achieved at 70 °C and a pH of 8.0, which reflected this hypothesis in the production of small-sized particles.

2.4.3. Elemental Composition Analysis

Energy-dispersive X-ray (EDX) is a common technology for the detection of elemental components of NPs. The chemical and physical behaviors of biosynthesized NPs in various sectors are correlated with their purity based on their metal constituents. The EDX chart (Figure 5A) confirmed the formation of ZnO because of the presence of emission peaks of the Zn and O ions at bending energies of (1.0, 8.7, and 9.8 KeV) and (0.5 KeV) respectively [18,51]. As shown, the Zn and O ions occupied the highest weight percentages (69.84 and 17.15%) and atomic percentages (53.56 and 25.76%) (Figure 5A). The deviation in the O ratio from the theoretical 1:1 ratio in our EDX analysis could be attributed to various factors, such as the presence of plant residues as capping agents, which introduced additional O-containing groups. Also, the adsorption of atmospheric O on the surface of nanostructure led to an increase in the O percentage in the EDX analysis. Moreover, incomplete reduction of the Zn ion to ZnO-NPs or production of intermediate compounds such as Zn(OH)2 led to distortion of the percentages of elemental composition.
Similarly, the Zn ion and O ion occupied the major contents of nano-ZnO formed by water Artemisia absinthium leaves extract with weight and atomic percentages of (71.28 and 18.12%) and (47.88 and 49,78%), respectively [52]. The EDX profile showed that the presence of emission peaks at 0.3 and 2.7 KeV corresponded to the C and Cl ions, respectively [46]. Interestingly, these ions were present with weight and atomic percentages of 9.58 and 3.43% and 14.53 and 6.15%, respectively (Figure 5A). The presence of these ions could be related to the emission of capping agents from the plant extract [52]. The EDX analysis for ZnO-NPs formed using an aqueous guava leaf extract obtained by Saha et al. [24] was incompatible with the current analysis. The authors reported that the synthesized ZnO-NPs were highly pure due to the absence of any peaks in the EDX analysis other than the Zn and O ions.

2.4.4. Structural Features

The structural properties of biogenic ZnO-NPs, including their crystalline or amorphous nature, were detected using X-ray diffraction (XRD). As shown in Figure 5B, there were nine diffraction peaks located at two theta values (indexed to Bragg’s plane) of 31.5° (100), 34.6° (002), 36.4° (101), 47.3° (102), 56.6° (110), 62.3° (103), 67.5° (112), 69.4° (201), and 76.8° (202). Similarly, the XRD chart of ZnO-NPs produced using leaf extracts of Cassia fistula and Melia azedarach showed nine peaks with two theta values corresponding to 31.8° (100), 34.5° (002), 36.3° (101), 47.6° (102), 56.6° (110), 66.4° (103), 67.9° (112), 69.1° (201), and 76.9° (202) [53]. In the current investigation, the biogenic ZnO-NPs were crystalline in nature according to the standard file JCPDS No. 36-1451 [53,54].
The Debye–Scherrer formula was utilized to calculate the crystalline size of biosynthesized ZnO-NPs based on the half-maximum of Bragg’s diffraction peak (101) at the two-theta degree of 36.4° [55]. In this research, the crystalline size of biogenic ZnO-NPs was ~16 nm. Similarly, the average crystallite sizes of nano-ZnO produced by water extracts of B. globosa, A. chilensis, and E. globulus were in the range of 2.2–17.8 nm, as calculated by the Debye–Scherrer formula [48]. The authors attributed the small crystalline sizes by XRD to plant metabolites, such as polyphenolic substances that acted as capping agents for producing NPs followed by increasing their stabilization.

2.4.5. DLS Analysis

The average size of guava leaf extract-mediated ZnO-NPs in colloidal solution as well as the size distribution were detected using DLS. As shown, the average particle size of biogenic ZnO-NPs was 69.15 nm (Figure 6), indicating the ultrafine structure was less than 100 nm in diameter. The size analysis based on TEM, XRD, and DLS revealed that the obtained size by TEM and XRD was lower than the size by DLS. This finding has different reasons, for instance, DLS measures the hydrodynamic residue due to the detection of the size in colloidal fluid, compared to TEM, which detects the size as dry powder. Besides that, the detection of size in solution (DLS analysis) is affected by various factors, such as coating and stabilizing agents that may increase the particle size, the purity of the solvent used during sample preparation, and the colloidal solution itself, which is affected by the homogenous or non-homogenous distribution of particles [56,57]. The obtained results were compatible with published investigations. For instance, the aqueous leaf extract of Pelargonium odoratissimum was utilized for ZnO-NP formation with average sizes of 34.1 nm detected using TEM, 21.6 nm detected by SEM, 14 nm using XRD, and 76 nm as detected by DLS [58]. Also, the average sizes of nano-ZnO particle formed using peel aqueous extract of Punica granatum were 25.1, 43.0, and 62.3 nm as detected by TEM, XRD, and DLS analysis, respectively [59].
The polydispersity index (PDI) is a critical factor that provides useful data regarding the hetero- or homogeneity of nanoparticles in the colloidal fluid, which gives background about NP properties and their prospective applications. The PDI is a numerical indicator, which between 0–0.4 indicates uniformity and narrow size distribution, whereas above 0.5 indicates that the particles are heterogeneous (broad size distribution) [60]. Herein, the PDI value of guava leaf extract-mediated ZnO-NPs was 0.24, which indicated the homogeneity of biogenic NPs in the colloidal solution. In a similar investigation, the PDI values of ZnO-NPs formed using aqueous extracts of B. globosa, E. globulus, A. chilensis, and G. officinalis were 0.3, 0.2, 0.2, and 0.5, respectively, indicating the particle homogeneity of the synthesized ZnO-NPs except those formed using the plant extract of G. officinalis [48].
The electrokinetic potential or Zeta (ζ) potential describes the surface charge or electric potential of NPs in colloidal solution. It is the key parameter for assessing the stability, surface modification, and interaction between particles in solution. These advantages make the ζ-value an important factor in specific applications, such as environmental engineering, pharmaceuticals, food processing and preservation, and material sciences [61]. The particles in a solution are defined as unstable when 1 > ζ-value < −10, whereas they are characterized as relatively stable when +10 > ζ-value < −20, stable at values of +20 > ζ < −30, and highly stable at +30 > ζ-value < −30 [62]. Here, the ζ-value of the biosynthesized nanostructure was 29.8 mV (Figure 6), which confirmed their high stability in colloidal fluids. Also, the positive charge as only charge on the nanostructure surface raises the stability through the repulsion of particles from each other, leading to prevention of aggregation [63]. Recently, the ζ-value of Punica granatum-based nano-ZnO was 35.7 mV, which confirmed the high NP stability [59]. On the other hand, the ZnO-NPs formed using Moringa oleifera seed extract bore a negative charge with a ζ-value of −38 mV, indicating the stability of NPs in fluids [64].

2.5. Inhibition of the Growth of Foodnorne Pathogenic Bacteria Using Green-Synthesized ZnO-NPs

Herein, the potential of guava leaf extract-based synthesis of nano-ZnO to prevent the growth of foodborne pathogenic bacteria and hence prevent food spoilage was investigated using agar well diffusion methods at different nano concentrations (200 - 6.25 µg mL−1). The antibacterial activity was performed toward 12 β-hemolytic strains. As shown, the ZnO-NPs formed using guava leaf extract had superior activity for inhibiting bacterial growth in a concentration-dependent manner. The obtained data were compatible with those that recorded the antimicrobial activity of green-synthesized ZnO-NPs against different pathogenic microbes [53,65]. The Food and Drug Administration defined ZnO-NPs as a safe material that can be used during food processing, food packaging, or food and fruit preservation against microbial attack [66]. This phenomenon could be attributed to their antimicrobial activity. Jin and coauthors reported the high antibacterial activity of ZnO-NPs in different forms (powder, coating agent, and film) against Salmonella enteritidis and Listeria monocytogenes [67]. Also, compared to bulk materials, ZnO in the nanoform showed promising bioactivity against food pathogenic strains including E. coli, B. subtilis, S. enteritidis, and S. aureus [68].
In this investigation, plant-based ZnO-NPs showed maximum antibacterial activity against selected pathogenic strains at 200 µg mL−1 of nano-ZnO, with varied inhibition zones. The highest zone of inhibition (22.5 ± 0.7 mm) was recorded for bacterial strain P. syringae A16 followed by strains E. coli S8 and B27 (21.7 ± 0.6 mm), S. typhimurium B31 (20.7 ± 0.5 mm), and S. typhimurium S5 (20.2 ± 0.5 mm) (Table 3, Figure 7). The less susceptible bacterial strain to ZnO-NPs was S. aureus, which recorded the lowest inhibition zone (15.3 ± 0.6 mm) at the highest concentration. These inhibition zones were decreased at low doses. For example, the inhibition zones were decreased to 17.0 ± 1.0 mm for strain A16, and 17.7 ± 0.6, 18.0 ± 1.0, 16.3 ± 0.5, and 13.7 ± 0.6 mm for bacterial strains S8, B27, B31, and S5, respectively, at a concentration of 50 µg mL−1 (Table 3, Figure 7). Similarly, biosynthesized ZnO-NPs were used to protect strawberries from spoilage caused by E. coli and S. aureus due to their inhibitory effects [69]. Also, the inhibition of the growth of food pathogenic Gram-positive and Gram-negative bacterial strains by biogenic ZnO-NPs was achieved in a concentration-dependent manner [17].
The MIC value, the lowest ZnO-NP concentration to inhibit bacterial growth, was investigated for each strain. Analysis of variance revealed that the MIC value of biogenic ZnO-NPs against S. aureus (S3 and S10) was 50 µg mL−1, whereas it was 25 µg mL−1 for B. cereus A14 and 12.5 µg mL−1 for the other bacterial strains (Table 3, Figure 7). In a similar investigation, the MIC values for plant-synthesized ZnO-NPs against E. coli and S. aureus were 40 and 20 mg/L, respectively [69]. Also, the MIC value of ZnO-NPs formed using extract of plantain peel against foodborne pathogenic strains K. pneumoniae, S. enterica, B. subtilis, and S. aureus was 100 µg mL−1 [70].
As shown, the inhibitory activity of ZnO-NPs toward Gram-positive strains was lower than those toward Gram-negative ones. This finding may be attributed to the change in cell wall structure. Gram-negative strains contain thin peptidoglycan layers compared to Gram-positive, which have thick layers that reduce or stop NP penetration and hence decrease their activity [71]. Also, the lipopolysaccharide (negative charge) present in Gram-negative cell walls will be attracted to positive NP charge and increase the susceptibility of Gram-negative to ZnO-NPs [11]. Data analysis revealed that S. aureus was the least sensitive bacterial strain to ZnO-NPs. This phenomenon could be related to the presence of carotenoid pigment, which decreases the effect of oxidative stress formed by ZnO-NPs [72]. Data obtained by Al-Odayni and coauthors were incompatible with the obtained results. The authors reported that the inhibition of Gram-positive bacteria (S. aureus) due to treatment with biogenic ZnO-NPs formed by aqueous extract of guava leaves was higher than that of negative strains (E. coli and K. pneumoniae) [73].
The antibacterial activity of ZnO-NPs can be attributed to different mechanisms, such as the production of reactive oxygen species (ROS), the accumulation of toxic ions (Zn2+) within the cells, or the direct interaction of nanostructure with bacterial cells. The transition of electrons from the valence to conduction band leads to the production of electron (e−) and hole (h+) pairs, which react with H2O and O to form ROS ions such as OH• (hydroxyl radicals), O2− (superoxide radicals), and HOO• (perhydroxyl radicals) [74,75]. These ROS ions are very toxic to bacterial cells due to inducing oxidative stress. Moreover, H2O2, which is highly toxic to bacterial cells, is formed during oxidative stress [76]. Also, the evaluated concentration of ROS can disrupt the cellular components to enhance the lipid peroxidation process [77]. Ramya et al. [78] reported that the antibacterial activity of guava leaf extract-mediated biosynthesis of ZnO-NPs was higher toward Gram-positive bacteria compared to Gram-negative bacteria due to the high secretion of ROS. The second antibacterial mechanism is the liberation of toxic ions (Zn2+) upon entrance into bacterial cells. These toxic ions cause dysfunction of cytoplasmic membrane integrity, block the respiratory and transport systems, and disrupt cell components such as nucleic acids, proteins, and amino acids [68]. The media used, pH, shape of NPs, their size and concentration, and bacterial type are factors that affect the dissolution and liberation of Zn2+. Pasquet and coauthors showed that using Mueller Hinton media at pH 8.0 increased the liberation of toxic ions. Also, the production of these ions was faster for the smallest size compared to larger ones [79]. The interaction of plant-based ZnO-NPs with bacterial cells interferes with enzymatic action, inhibits the replication or forming of mutations in DNA, and disrupts bacterial biofilm formation, leading to improved antibacterial activity [80].

3. Materials and Methods

3.1. Sample Collection

Three different spoiled fruits, designated as strawberries, apples, and bananas, were collected in sterilized plastic bags from a market in Cairo, Egypt. The most common signs of spoilage were softening, alcoholic odor due to bacterial fermentation, and flavor, texture, and appearance changes. The collected spoiled fruits were transferred directly to the lab and kept in the refrigerator for future studies.

3.2. Bacterial Isolation

The serial dilution method was used for bacterial isolation from the collected spoiled fruits. In this method, 1.0 g of each collected fruit was suspended separately in 10 mL of distilled H2O containing antifungal agent, mixed well, and allowed to settle. After that, the suspension was serially diluted to obtain dilutions in the range of 10−1 to 10−5. Approximately 1 mL of each dilution was added over the nutrient agar plate surface (containing, g/L: peptone, 5.0; beef extract, 3.0; NaCL, 5.0; agar 15.0) using a sterilized glass rod before being incubated at 37 °C for 24 h. Afterward, the bacterial colonies were re-streaked on a new nutrient agar plate for complete purification. The purified bacterial isolates were kept in a refrigerator on a slant for subsequent experiments [34].

3.3. Hydrolytic Enzyme Production

The potential of the obtained bacterial strains to produce different hydrolytic enzymes was investigated using the agar plate method. The enzymes assayed were cellulase, amylase, xylanase, and lipase. In this method, each isolate was separately inoculated on an agar plate supplemented with appropriate substrate (1%), which was carboxymethyl cellulose (CMC) [81], soluble starch [82], xylan [83], and olive oil [84] for cellulase, amylase, xylanase, and lipase enzymes, respectively. The inoculated plates were incubated overnight at 35 ± 2 °C before being flooded with a specific reagent (Logule’s iodine solution for cellulase, amylase, and xylanase). The results were recorded as the diameter of the clear zone (mm) around bacterial growth and represented as follows: + for clear zones in the range of 1–10 mm, ++ for clear zones of 10–15 mm, and +++ for clear zones > 15 mm.

3.4. Hemolysis Test

The potential of the obtained isolates to break down blood cells was investigated using an agar plate supplemented with 5% (w/v) sheep blood. Each bacterial strain was inoculated over the surface of the blood agar plate and incubated for 48 h at 35 ± 2 °C. The hemolytic activity of the bacterial cells was designated as follows: α-hemolysis for strains that formed greenish zones around their growth, β-hemolysis for those that formed clear zones, and γ-hemolysis for strains that lacked the potential to degrade the blood cells (no zones around bacterial growth) [85].

3.5. Bacterial Identification

The bacterial isolates designated as having β-hemolytic activity were subjected to identification based on 16S rRNA. The primers 27f (5′-AGAGTTTGA TCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) were utilized for this purpose [86]. Separate bacterial colonies were picked up and resuspended in sterilized deionized H2O (50 µL), heated for 10 min at 97 °C in a water bath, followed by centrifugation (20,000 for 10 min) to collect the upper layer containing DNA. The PCR tube contained 0.5 mM MgCl2, 1× PCR buffer, Taq DNA polymerase enzyme (2.5 U, QIAGEN, Germantown, MD, USA), deoxynucleoside triphosphate (0.25 mM), primer (0.5 µM), and bacterial DNA (5 ng). The following conditions were conducted for PCR cycling: 3 min at 94 °C, 0.5 min at 94 °C for 30 cycles, 0.5 min at 55 °C, 1 min at 72 °C, and finally 10 min at 72 °C. The sequence of the obtained PCR products was analyzed with primers using the Applied Biosystems 3730 × l DNA Analyzer at Sigma Company, Cairo, Egypt. The obtained sequences were compared with those deposited in GenBank using the BLAST method.
The phylogenetic tree was constructed using the neighbor-joining method with MEGA V6.1 software. The confidence level of each branch (1000 repeats) was tested by bootstrap analysis.

3.6. Green Synthesis of ZnO-NPs by Psidium Guajava

To form zinc oxide nanoparticles (ZnO-NPs), the leaves of Psidium guajava L. (common name is guava) were collected from market. The gathered leaves were rinsed three times with tap water to eliminate any contaminants, then dried in an oven set at 40 °C until they could be ground into a powder. The water extract was prepared by heating 5.0 g of the powdered substance to 40 °C for one hour while stirring at 150 rpm in distilled water (dH2O, 100 mL). To acquire 2 mM concentration of ZnO-NPs, the precursor, zinc acetate (Zn(CH3COO)2·2H2O), was dissolved in 80 mL of dH2O and diluted to 100 mL by adding 20 mL of the produced water extract. Next, 1N NaOH was added to the solution to optimize the pH at 8, mixed thoroughly, and let sit at 70 °C under stirring conditions (150 rpm) for 60 min. before being left overnight at room temperature to ensure that the plant extract completely reduced the metal oxide precursor. This was recognized by a change in color from colorless to white, which were known as zinc oxide nanoparticles (ZnO-NPs) [53]. Centrifugation at 15,000 rpm was used to collect the white precipitate from the solution. It was then washed thrice with dH2O and calcinated at 200 °C for four hours.

3.7. ZnO Nanoparticles Characterization

3.7.1. UV–Vis Analysis

UV–Vis spectroscopy (JENWAY-6305, Staffordshire, UK) was used to identify surface plasmon resonance (SPR) and quantify the absorbance of the newly formed color of ZnO-NPs. In this investigation, a quartz cuvette was filled with 2 mL of the nanostructure solution, and measuring their absorbance at wavelengths ranging from 200 to 800 nm.

3.7.2. Morphological and Elemental Component Detection

Using transmission electron microscopy (JEOL 1010, Tokyo, Japan), the shapes and sizes of the ZnO-NPs, in addition to their aggregation, were evaluated. During this investigation, ZnO-NPs were dissolved in 2 mL of pure ethanol at a concentration of 60 µg mL−1 and thoroughly mixed by sonicating for 20 min. Following that, the transmission electron microscopy carbon-coated copper grid was loaded with a few drops of ethanol suspension and then allowed to air dry [87]. No additional sputtering was applied to the TEM grid to obtain optimal imaging. The TEM photos were recorded under high vacuum mode with pressure of 10−5 Toor. Through the utilization of EDX (JEOL, JSM6360LA, Tokyo, Japan), the elemental composition of biosynthesized ZnO-NPs was detected.

3.7.3. X-ray Diffraction Analysis

This analysis was conducted to examine the structure (crystalline or amorphous nature) of the plant-synthesized ZnO-NPs using X-ray diffraction (XRD, XC Pert Pro, Philips, Eindhoven, the Netherlands). The theta values ranged from 10° to 80°, and the conditions of analysis were 40 Kv (voltage), 30 mA (current), and a λ degree of 1.54 Å for the Cu, which served as the source of X-rays being used. Utilizing the Debye–Scherrer equation, the following formula was utilized in order to determine the average crystallite size of the NPs that were formed [87]:
A = 0.9 × 1.54/β × cosθ
where A is the average crystallite size; 0.9 and 1.54 are the Debye–Scherrer constant and λ degree of the X-ray source, respectively; and β is the half-maximum intensity; and θ is the Bragg’s angle.

3.7.4. Dynamic Light Scattering (DLS)

Through the use of DLS, the distribution of ZnO-NPs and the sizes of the particles in colloidal solution were investigated. Following resuspension of the sample in dH2O at a concentration of 60 µg mL−1, it underwent DLS measurements from Malvern, United Kingdom. The measurements were performed in triplicate at a temperature of 24.6 °C and pH of 6.5. The measurement was performed after one minute for each run. The instrument utilized a 633 nm He-Ne laser, and the scattering angle was fixed at 173° [88]. The stability of nanostructure was further tested using Zeta potential measurement instruments under the same conditions.

3.8. Antibacterial Activity

The inhibitory activity of synthesized ZnO-NPs against β-hemolytic bacterial strains was evaluated at different concentrations (200, 100, 50, 25, 12.5, and 6.25 µg mL−1) using the agar well diffusion method [89]. For this purpose, the selected bacterial strains were refreshed on the surface of nutrient agar plates overnight at 35 ± 2 °C incubation conditions. After that, separate colonies for each selected strain were picked up and reinoculated separately on the surface Mueller Hinton agar plates (Oxid, ready prepared) before making wells (0.6 mm in diameter). Each well received 100 µL of each ZnO-NP concentration and was kept in the refrigerator for one hour to complete diffusion, followed by incubation at 35 ± 2 °C for 24 h. In each plate, one well received 100 µL of DMSO (solvent system) as a negative control, which did not show any antibacterial activity. The inhibition zones formed around each well were measured in mm and recorded as positive results. The lowest concentration of ZnO-NPs that formed an inhibition zone was recorded as the minimum inhibitory concentration (MIC) [90]. The experiment was conducted in triplicate.

3.9. Statistical Analysis

The SPSS (version 18, SPSS Inc., Chicago, IL, USA) was utilized for data statistical analysis. ANOVA was applied to compare samples before conducting Tukey’s multiple comparison test.

4. Conclusions

In this study, 33 foodborne bacterial isolates were obtained from spoiled fruits and tested for enzymatic activities including cellulase, amylase, xylanase, and lipase. Those isolates exhibited varied enzymatic activities responsible for fruit spoilage. Surprisingly, out of those isolates, 12 isolates showed β-hemolytic activity toward blood cells. Those isolates were identified using 16s rRNA sequencing as Staphylococcus aureus S3, Salmonella enterica S5, Escherichia coli S8, Staphylococcus aureus S10, Bacillus subtilis S11, Bacillus cereus A14, Pseudomonas syringae A16, Listeria monocytogenes A18, Bacillus subtilis B24, Bacillus cereus B25, Escherichia coli B27, and Salmonella enterica B31. ZnO-NPs were green-synthesized using an aqueous leaf extract of Psidium guajava. Those NPs were characterized using UV, FT-IR, TEM, EDX, XRD, DLS, and Zeta potential, which indicated the production of spherical shape, crystallographic structure, and well-arranged ZnO-NPs. Interestingly, those NPs exhibited strong antibacterial activities against the identified foodborne pathogenic strains in a concentration-dependent, with a value of MIC ranging from 25 to 50 µg mL−1 based on the foodborne strain. This indicates the potential of the green-synthesized ZnO-NPs as a possible approach for preserving fruits and controlling the growth of pathogenic foodborne strains.

Author Contributions

Conceptualization, A.F., M.A.A.-R., A.M.E. and S.E.-D.H.; methodology, A.F., M.A.A.-R., A.M.E. and S.E.-D.H.; software, A.F., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; validation, A.F., M.A.A.-R., A.M.E., S.S. and S.E.-D.H.; formal analysis, A.F., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; investigation, A.F., M.A.A.-R., A.M.E., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; resources, A.F., S.S., H.E., M.A., E.M., M.S.A. and S.K.A.J., data curation, A.F., M.A.A.-R., A.M.E., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; writing—original draft preparation, A.F., M.A.A.-R., A.M.E., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; writing—review and editing, A.F. and M.A.A.-R. visualization, A.F., M.A.A.-R., A.M.E., S.S., H.E., M.A., E.M., M.S.A., S.K.A.J. and S.E.-D.H.; supervision, A.F., M.A.A.-R., A.M.E. and S.E.-D.H.; project administration, A.F., M.A.A.-R., A.M.E. and S.E.-D.H.; funding acquisition, S.S., H.E., M.A., E.M., M.S.A. and S.K.A.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available upon request from the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Percentages of different activities of the bacterial strains obtained from spoiled fruits.
Figure 1. Percentages of different activities of the bacterial strains obtained from spoiled fruits.
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Figure 2. Molecular identification of β-hemolytic strains obtained from collected spoiled fruits. ◆ indicates 16S rRNA fragments of the bacterial strain. The analysis was done by MEGA 6.1 using the neighbor-joining method.
Figure 2. Molecular identification of β-hemolytic strains obtained from collected spoiled fruits. ◆ indicates 16S rRNA fragments of the bacterial strain. The analysis was done by MEGA 6.1 using the neighbor-joining method.
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Figure 3. UV–Vis spectrum of formed ZnO-NPs using an aqueous extract of guava leaves showing maximum SPR at a wavelength of 370 nm.
Figure 3. UV–Vis spectrum of formed ZnO-NPs using an aqueous extract of guava leaves showing maximum SPR at a wavelength of 370 nm.
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Figure 4. (A) TEM analysis showing a spherical shape and well-arranged ZnO-NPs, and (B) size distribution according to TEM analysis.
Figure 4. (A) TEM analysis showing a spherical shape and well-arranged ZnO-NPs, and (B) size distribution according to TEM analysis.
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Figure 5. Characterization of guava leaf extract-mediated biosynthesis of ZnO-NPs. (A) EDX analysis showing the components of the sample; and (B) XRD analysis showing the crystalline nature.
Figure 5. Characterization of guava leaf extract-mediated biosynthesis of ZnO-NPs. (A) EDX analysis showing the components of the sample; and (B) XRD analysis showing the crystalline nature.
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Figure 6. Detection of particle size in colloidal solution by dynamic light scattering and surface charge by ζ-potential analysis.
Figure 6. Detection of particle size in colloidal solution by dynamic light scattering and surface charge by ζ-potential analysis.
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Figure 7. Antibacterial activity of biogenic ZnO-NPs against selected hemolytic bacterial strains isolated from spoiled fruits. Different letters on the bars at the same NPs concentration designate the data were significant (p ≤ 0.05) (n = 3).
Figure 7. Antibacterial activity of biogenic ZnO-NPs against selected hemolytic bacterial strains isolated from spoiled fruits. Different letters on the bars at the same NPs concentration designate the data were significant (p ≤ 0.05) (n = 3).
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Table 1. Morphological characteristics, enzymatic activities, and hemolysis of the bacterial strains obtained from spoiled fruit samples.
Table 1. Morphological characteristics, enzymatic activities, and hemolysis of the bacterial strains obtained from spoiled fruit samples.
Isolation SourceCodeGram ReactionCell ShapeEnzymatic Activity:Hemolysis
CellulaseAmylaseXylanaseLipase
StrawberriesS1+Bacilli−+−−γ-hemolysis
S2+Cocci+++++−α-hemolysis
S3+Cocci, clusters+++++++++++β-hemolysis
S4−Rods−−−+α-hemolysis
S5−Rods++++++++++β-hemolysis
S6+Bacilli++++++++α-hemolysis
S7−Rods+−−+γ-hemolysis
S8−Rods++++++++++β-hemolysis
S9−Rods++++++−α-hemolysis
S10+Cocci, clusters++++++++++++β-hemolysis
S11+Bacilli++++++++++++β-hemolysis
S12+Bacilli+++++++−γ-hemolysis
ApplesA13−Rods−++−−α-hemolysis
A14+Bacilli++++++++++β-hemolysis
A15−Rods+++++++++α-hemolysis
A16−Rods++++++++++β-hemolysis
A17+Cocci+++++++α-hemolysis
A18+Rods+++++++++β-hemolysis
A19+Cocci++++++++α-hemolysis
A20−Bacilli−−−−γ-hemolysis
A21−Rods++++−γ-hemolysis
A22+Cocci++-++−γ-hemolysis
BananasB23−Rods−++++α-hemolysis
B24+Bacilli+++++++++++β-hemolysis
B25+Bacilli++++++++++++β-hemolysis
B26−Bacilli−−−+γ-hemolysis
B27−Rods+++++++++β-hemolysis
B28+Cocci+++++−α-hemolysis
B29+Cocci+−+−α-hemolysis
B30+Bacilli+++++++++++γ-hemolysis
B31−Rods++++++++++β-hemolysis
B32+Bacilli+++++++++−α-hemolysis
B33−Rods−++++−α-hemolysis
− is meaning no activity; +, for clear zones in the range of 1–10 mm; ++, for clear zones of 10–15 mm; and +++, for clear zones > 15 mm.
Table 2. The identification of β-hemolytic bacterial strains isolated from spoiled fruits based on 16S rRNA sequence analysis.
Table 2. The identification of β-hemolytic bacterial strains isolated from spoiled fruits based on 16S rRNA sequence analysis.
Bacterial StrainHomolog SequenceIdentity
Percentage (%)		NCBI Accession NumberAccession Number of the Obtained Strain
S3Staphylococcus aureus99.33%NR115606 PP683392
S5Salmonella enterica99.21% NR074910PP683393
S8Escherichia coli99.08%NR024570PP683394
S10Staphylococcus aureus98.83%NR115606PP683395
S11Bacillus subtilis98.76%NR027552PP683396
A14Bacillus cereus98.72%NR115526PP683397
A16Pseudomonas syringae98.93%NR117820PP683398
A18Listeria monocytogenes98.56%NR044823PP683399
B24Bacillus subtilis98.25%NR027552PP683400
B25Bacillus cereus98.88%NR115526PP683401
B27Escherichia coli98.23%NR024570PP683402
B31Salmonella enterica98.66%NR074910PP683403
Table 3. The inhibitory action of guava leaf extract-mediated ZnO-NP synthesis at different concentrations. The data are represented as the mean diameter of three replicates (n = 3) ± SD. The value of MIC is recognized as the lowest ZnO-NP concentration that has antibacterial activity (forming an inhibition zone).
Table 3. The inhibitory action of guava leaf extract-mediated ZnO-NP synthesis at different concentrations. The data are represented as the mean diameter of three replicates (n = 3) ± SD. The value of MIC is recognized as the lowest ZnO-NP concentration that has antibacterial activity (forming an inhibition zone).
Bacterial StrainZnO-NPs Concentration (µg mL−1)/Inhibition Zone (mm)
200 100 50 25 12.5 6.25
Staphylococcus aureus S315.7 ± 0.613.3 ± 0.611.3 ± 0.60.0 ± 0.00.0 ± 0.00.0 ± 0.0
Salmonella enterica S520.3 ± 0.618.2 ± 0.313.7 ± 0.611.7 ± 0.612.7 ± 0.50.0 ± 0.0
Escherichia coli S821.7 ± 0.619.3 ± 0.617.7 ± 0.615.3 ± 0.613.3 ± 0.60.0 ± 0.0
Staphylococcus aureus S1015.3 ± 0.613.0 ± 1.011.7 ± 0.60.0 ± 0.00.0 ± 0.00.0 ± 0.0
Bacillus subtilis S1118.3 ± 0.614.7± 0.613.0 ± 1.011.3 ± 0.69.0 ± 1.00.0 ± 0.0
Bacillus cereus A1417.0 ± 1.015.0 ± 1.014.0 ± 1.012.0 ± 1.00.0 ± 0.00.0 ± 0.0
Pseudomonas syringae A1622.5± 1.021.0 ± 1.018.0 ± 1.015.0 ± 1.112.5 ± 1.00.0 ± 0.0
Listeria monocytogenes A1818.0 ± 1.015.3 ± 0.613.7 ± 0.612.3 ± 0.69.7 ± 0.60.0 ± 0.0
Bacillus subtilis B2418.0 ± 1.014.3 ± 0.612.7 ± 0.611.3 ± 0.68.7 ± 1.20.0 ± 0.0
Bacillus cereus B2517.0 ± 1.014.0 ± 1.011.7 ± 1.210.7 ± 0.68.3 ± 0.60.0 ± 0.0
Escherichia coli B2721.7 ± 0.620.0 ± 1.018.0 ± 1.014.7 ± 1.212.3 ± 0.60.0 ± 0.0
Salmonella enterica B3120.7 ± 0.618.3 ± 0.616.3 ± 0.613.7 ± 0.611.3 ± 1.20.0 ± 0.0
0.0, means no inhibition zone.
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Fouda, A.; Abdel-Rahman, M.A.; Eid, A.M.; Selim, S.; Ejaz, H.; Alruwaili, M.; Manni, E.; Almuhayawi, M.S.; Al Jaouni, S.K.; Hassan, S.E.-D. Investigating the Potential of Green-Fabricated Zinc Oxide Nanoparticles to Inhibit the Foodborne Pathogenic Bacteria Isolated from Spoiled Fruits. Catalysts 2024, 14, 427. https://doi.org/10.3390/catal14070427

AMA Style

Fouda A, Abdel-Rahman MA, Eid AM, Selim S, Ejaz H, Alruwaili M, Manni E, Almuhayawi MS, Al Jaouni SK, Hassan SE-D. Investigating the Potential of Green-Fabricated Zinc Oxide Nanoparticles to Inhibit the Foodborne Pathogenic Bacteria Isolated from Spoiled Fruits. Catalysts. 2024; 14(7):427. https://doi.org/10.3390/catal14070427

Chicago/Turabian Style

Fouda, Amr, Mohammed Ali Abdel-Rahman, Ahmed M. Eid, Samy Selim, Hasan Ejaz, Muharib Alruwaili, Emad Manni, Mohammed S. Almuhayawi, Soad K. Al Jaouni, and Saad El-Din Hassan. 2024. "Investigating the Potential of Green-Fabricated Zinc Oxide Nanoparticles to Inhibit the Foodborne Pathogenic Bacteria Isolated from Spoiled Fruits" Catalysts 14, no. 7: 427. https://doi.org/10.3390/catal14070427

APA Style

Fouda, A., Abdel-Rahman, M. A., Eid, A. M., Selim, S., Ejaz, H., Alruwaili, M., Manni, E., Almuhayawi, M. S., Al Jaouni, S. K., & Hassan, S. E.-D. (2024). Investigating the Potential of Green-Fabricated Zinc Oxide Nanoparticles to Inhibit the Foodborne Pathogenic Bacteria Isolated from Spoiled Fruits. Catalysts, 14(7), 427. https://doi.org/10.3390/catal14070427

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