Antimicrobial potential of floral extract-decorated nanoparticles against food-borne pathogens

Abstract

Green nanoparticles are economically beneficial and do not harm the environment as they are eco-friendly when compared with chemically synthesized silver nanoparticles. Contamination of food and food products with micro-organisms can cause food spoilage and food-borne diseases. This research mainly focuses on United Nations Sustainable Development Goals (SDGs 2, 3, 6, 9, 12), particularly in the areas of health, food safety, and sustainable innovation. The aim of the study was to synthesize Moringa oleifera flower mediated silver nanoparticles to control the growth and biofilm formation in isolated food - borne pathogens. The fresh extract obtained from the flowers of Moringa oleifera has been utilized for the synthesis of silver nanoparticles (Mo-AgNPs). The Mo-AgNPs were characterized by using various analytical techniques. In silico analysis has been carried out to know the binding potential of phytocompounds of Moringa oleifera with the virulent proteins of bacterial strains. The toxicity effect of Mo-AgNPs was evaluated by using seed germination studies with the seeds of Vigna radiata and evaluated the toxicity effect in Artemia nauplii based on its mortality rate. The novelty of the work is to evaluate the antibacterial efficacy of the synthesized Mo-AgNPs, antimicrobial assays including agar well diffusion, Minimum Inhibition Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Biofilm formation assay were performed in the bacterial strains isolated from spoiled food. Mo-AgNPs confirmed its nanosize by depicting the particle size as 12.73 nm with 0.115 mV. Mo-AgNPs showed potential benefit for plant growth and exhibited toxicity to Artemia nauplii at higher concentration. The maximum concentrations of Mo-AgNPs that inhibit and kill the isolated food - borne pathogens were 3.125 and 50 µg/ml respectively. Mo-AgNPs effectively reduced the biofilm formation in all the tested strains. Molecular docking studies confirmed that the Ellagic acid has the least value of − 8.6 and − 8.9 kcal/mol with beta lactamase of Enterobacter cloacae and beta lactamase OXY1 of Klebsiella oxytoca respectively. Quercetin, Apigenin, Riboflavin and kaempferol have lower values of − 7.7, − 7.6, − 7.8 and − 7 kcal/mol (Enterobacter cloacae) and − 8.3, − 7.8, − 7.9 and − 7.7 kcal/mol (Klebsiella oxytoca), respectively. Through this study it was proven that the synthesized Mo-AgNPs could have the potential to fight against the bacterial pathogens that are responsible for food - borne diseases and food spoilage. In the future, Mo-AgNPs can be utilized to develop food packaging biomaterials that can increase the shelf life and prevent food from spoilage.

Introduction

The contamination of food may occur at any stage from production to packaging till consumption. Bacteria existing in the surroundings can be both beneficial and harmful. Bacteria play a crucial role in food industries including fermentation of food products namely cheese, yoghurt, pickles, vitamins and vinegar production and many more. Whereas on other hand, some bacteria are pathogenic in nature and are often contaminating food and food products. These pathogenic bacteria affect the quality of food and cause spoilage. Consumption of contaminated foods can have negative impact on health [13] and sometimes it may lead to death of the consumer.

With the rise in antibiotics resistance, the need for alternative to existing antimicrobials can solve the problem of antimicrobial resistance. A thorough analysis of mechanisms of bacterial resistance have been studied from ancient times to the present, as well as more recent developments in nanotechnology and their resistance-fighting methods, has been published in Vinothini et al. [53]. The pathogens found in food cause many diseases that have serious consequences for human health and the economy [9]. Members of the Enterobacteriaceae family have been identified as a major cause of food poisoning [34]. Enterobacteriaceae are Gram-negative bacteria that includes Salmonella sp, Klebsiella sp, Enterobacter sp, Citrobacter sp, Escherichia coli, Shigella sp, and other species. They can cause diarrhea, intestinal infections, urinary tract infections (UTIs), food poisoning and sometimes can lead to life threatening complications. Antibiotic treatment against Enterobacteriaceae is ineffective because of the development of antibiotic resistance [35]. Though the antibiotics are cost effective solution, multidrug resistant bacteria are ineffective to the antibiotics treatment due to inactivation of antibiotics by using its enzymatic degradation mechanisms namely β-lactamases. The alteration of bacterial cell wall and development of efflux pumps offer resistance to certain antibiotics. To tackle the current scenario of emerging drug resistance, nanoparticles are a viable substitute for antibiotics in the fight against harmful bacteria causing food spoilage and food poisoning. Consequently, synthesizing novel nanoparticles with improved bactericidal action has attracted a great deal of attention.

The recent surge towards the interest in nanotechnology in the scientific and technological community has enabled the synthesis and fabrication of various sized nanoparticles [23]. Metal-based nanoparticles are tiny particles with the size ranges from 1 to 100 nm at least in one dimension. Because of their smaller size, shape, and composition, nanoparticles have special qualities that make them useful in a variety of industries including mechanical, civil, electronics, biomedical, catalysis and many more [21, 22, 44]. Among many noble metal-based nanoparticles, silver nanoparticles (AgNPs), have garnered special interest because of their unique characteristic nature including electrical conductivity, chemical stability, catalytic activity, and antibacterial activity [18]. Distinct physico-chemical properties including their high surface area, mass ratio, high reactivity, and nanometer-sized particles are responsible for their multifunctional properties. Unique properties of AgNPs gained significant edge in the development of substitute that combat multidrug-resistant microbes.

Various kinds of nanoparticles are synthesized by using physical, chemical, biological, and hybrid techniques. Although physical and chemical techniques are frequently used, their application is restricted due to the possibility of hazardous substances as byproducts. Utilising plants (bark, roots, leaves, fruit, flowers, rhizoids, seeds, and latex), bacteria, fungi, algae in the green synthesis of nanoparticles, exhibits synergistic properties, which are more effective than those synthesized by employing chemical and physical processes. Green synthesis offers ecofriendly products by overcoming the usage of hazardous chemicals and reaction conditions during the synthesis of nanoparticles which are compatible for biomedicaland other biological applications. The smaller size and the greater surface area of the green silver nanoparticles, contributes to their effective antibacterial activity against the pathogens [25, 33, 38–40]. Green nanoparticles directly disrupt the bacterial cell wall and stop the development of resistance strains by means of synergism. The green synthesized silver nanoparticles are economically beneficial and do not harm the environment as they are eco-friendly when compared with chemically synthesized silver nanoparticles [43]. In depth analysis of various types of nanoparticles made us focus on synthesizing green based silver nanoparticles to combat food—borne pathogens.

Medicinal plants play a key role in controlling and preventing the spread of food-borne pathogens. Moringa oleifera a family of Moringaceae (order Brassicales) is native to many tropical areas and is abundantly found in many Asian countries [12]. Since eighteenth century, Moringa oleifera has been utilized in Indian medicine system as traditional healers. Every part of this Moringa plant is used as traditional medicine for treating various diseases [26]. Numerous medical applications have long been acknowledged as part of the Ayurvedic and Unani medical systems. The indigenous medical system has utilised nearly every part of the plant, including the leaves, bark, flowers, fruits, pods, gum, root, seeds and its oil to treat a variety of illnesses, including anaemia, asthma, bronchitis, conjunctivitis, diabetes, diarrhoea, digestive disorders, earache, fevers, goitre, gout, haemorrhoids, headache, heart problems, joint pain, measles, rheumatism, skin infections, smallpox, swelling, wounds, and many more [30]. Moringa plant possesses antibacterial, antifungal, antiviral and antiparasitic activities [51]. The flowers of Moringa reported to have anti-arthritic, hypocholesterolemic properties and it can treat colds and urinary tract infections. The bioactive compounds present in Moringa flower exhibit antibacterial activity which has the potential to fight against a wide range of bacterial strains. Moringa flowers are reported to contain [30] higher amount of monounsaturated fatty acids than polyunsaturated fatty acids which are known for their nutritional benefit in supporting heart’s health. Moringa flowers are reported to contain 74 essential oils which are packed with essential nutrients, vitamins A, E, and C, and other fatty acids which support skin and hair health. Moringa flowers are reported to contain glucosinolates which is the precursor to isothiocyanates. Isothiocyanates are well known compounds which prevent and manage chronic diseases by exhibiting anti-inflammatory, antioxidant and anticancer properties. Moringa flowers contain ascorbic acids, flavonoids, and polyphenols. The flowers Moringa oleifera contain quercetin, which is well known for its hepatoprotective function. Quercetin, a type of flavonoid, demonstrates antibacterial activity by disrupting the cell wall of bacteria and its biofilm formation, inhibits nucleic acid synthesis.

Green techniques, which use plant resources for synthesis, are comparatively safe and environmentally beneficial ways to overcome the toxicity issue in nanoparticles synthesis. Additionally, the potentially active compounds used in plant-mediated nanoparticles synthesis are biocompatible for a variety of biological uses. Considering the aforementioned information, this study focused on synthesizing and characterizing Mo-AgNPs by using the aqueous extract of Moringa flowers. Floral extracts are less often utilized in nanoparticles synthesis compared to other parts of Moringa. In addition, unique secondary metabolites of floral extracts work synergistically to contribute to its role as capping/stabilizing of nanoparticles as well as enhance the antimicrobial activity of Mo-AgNPs. One of the novel features of the work was to perform in silico analysis to determine the binding potential of phytocompounds of Moringa flower against the antibiotic-resistant proteins of food—borne pathogens. The other novelty of the study can be highlighted through exploring the unique aspects of Mo-AgNPs in controlling the growth and biofilm of foodborne pathogens namely Enterobacter kobei, Enterobacter cloacae, Enterobacter bugandensis, Enterobacter chuandaensis, Enterobacter huaxiensis and Klebsiella oxytoca in invitro condition. The efficacy of the Mo-AgNPs could pave the way for developing products for practical applications in food safety and preservation.

Materials and methods

Chemicals and reagents

All chemical reagents utilized for this research work were of high purity standards (AR grade). Antibiotics discs, LB broth and LB agar medium were procured from Hi Media Laboratories Pvt. Ltd. Silver nitrate, crystal violet, glacial acetic acid, ethanol and all other chemicals and solvents of reagent grade were procured from Sisco Research Laboratories Pvt. Ltd. (SRL).

Synthesis of Mo-AgNPs by using floral extract of Moringa oleifera

The fresh Moringa oleifera flowers were collected from B. S. Abdur Rahman Crescent Institute of Science and Technology, Chennai, India. Moringa oleifera were identified and authenticated by Dr. D. Narasimhan, Botanist, Chennai. A specimen of the leaves was deposited at School of Life Sciences, BSACIST, Chennai (Accession number: SLS-BSAU-22010). The collected flowers were washed, shade dried completely for 2 days (without moisture content) and powdered by using a blender. Ten grams of powdered Moringa flower was added to 100 ml of distilled water and macerated overnight and was boiled for 15 min. To obtain the clear extract, the content was centrifuged for 5 min at 2500 revolutions per minute (RPM) and filtered using Whatman filter paper. The resulting filtrate was separately collected and stored in a refrigerator for experiments. The silver nanoparticles were synthesized from Moringa flower extract by mixing 1 part of 1 mM silver nitrate solution with 5 parts of flower extract [8, 11, 14]. The solution was incubated in dark for overnight. The colour change was monitored for confirming the synthesis of nanoparticles.

Characterization of Mo-AgNPs

The optical property of Mo-AgNPs was measured by using Ultraviolet–visible spectroscopy. The formation of strong plasmon resonances confirms the synthesis of Mo-AgNPs [37]. The spectrum was recorded for both the Moringa oleifera flower extract and for the Mo-AgNPs. The synthesized nanoparticles were characterized to evaluate their size, shape, surface area, chemical composition, topography, etc., [42]. High-resolution transmission electron microscopy (HR-TEM) was a useful and important method of classifying nanomaterials, determining the particle size, distribution and its formation. Field Emission Scanning Electron Microscopy (FESEM) was performed to analyze the shape and surface of Mo-AgNPs. The energy dispersive X-ray analysis (EDAX) analysis was conducted to ascertain the elemental composition of the synthesized silver nanoparticles. Mo-AgNPs was subjected to XRD analysis to study the crystalline nature of the nanoparticles. Fourier Transform Infrared Spectroscopy (FTIR) technique was employed to detect the chemical composition, by identifying the presence of several functional groups in Mo-AgNPs. Dynamic Light Scattering (DLS) analysis was used to determine the size distribution of Mo-AgNPs by measuring the shift in the intensity of light scattering caused by the Brownian particle movement [32].

Ecotoxicity studies using seeds of Vigna radiata and Artemia nauplii

The toxicity effect of Mo-AgNPs on plant growth and viability was investigated on the seedlings of Vigna radiata. The seeds of Vigna radiata were soaked overnight in clean water and the best sprouted seeds were selected for toxicity studies. The selected seeds were sowed in seed tray and the nanoparticles were added in the concentration of 1, 5, and 10 μg/ml. The control was also grown separately without any treatment (water alone). The growth was monitored for 14 days and the observation on their root length, shoot length and plant mass were recorded [41]. The eggs of Artemia were obtained from Central Institute of Brackishwater Aquaculture (CIBA, ICAR), Chennai. The toxic effect of Mo-AgNPs in Artemia nauplii was observed. Approximately 1 gm of pre-filtered cysts were placed in 1 L of sea water in a circular plastic beaker with supported aeration. After 24 h, the cysts hatched. The alive Artemia nauplii were collected and placed in 12 well plates. Different concentrations of nanoparticles 1, 5, and 10 μg/ml were added, and it was fed with activated yeast solution to avoid starvation. After 24 h, the mortality rate was observed [50].

In vitro analysis

Isolation, identification and antibiogram of organisms isolated from spoiled food

The isolation of bacteria from spoiled fruits and vegetables, including bell pepper, banana and pumpkin, was achieved by spreading a diluted fruit suspension (1:10) on Luria–Bertani (LB) agar plates. Plates were incubated at 37 °C for 24 h to allow bacterial growth. Distinct colonies were subcultured three times on fresh LB agar plates to obtain pure cultures. Gram staining and colony morphology confirmed the purity of isolated colonies.

Selected individual colonies were inoculated in LB broth. Tubes were incubated at 37 °C to reach 0.5 McFarland standard. Cultured organisms were used for DNA isolation by adopting heat lysis method. Isolated DNA was amplified by using 16SrRNA primers namely forward (5’-AGA GTT TGA TCC TGG CTC AG-3’) and reverse (5’- GGT TAC CTT GTT ACG ACT T-3’) with initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, elongation at 72 °C for 1.5 min for 35 cycles and final elongation at 72 °C for 5 min. The amplified products of respective bacterial strains were subjected to sequencing. The obtained sequence was subjected to BLAST and the top 10 evolutionarily related organisms were selected for constructing phylogenetic trees by using MEGA X. Organisms were identified and deposited in NCBI GenBank with respective accession numbers [17].

Antibiogram was performed for the isolated strains by using Ampicillin 2 µg, Penicillin 10 µg, Streptomycin 10 µg, Gentamycin10 µg, Ceftriaxone 30 µg, Cefoperozone 75 µg, and Enroflaxacin, 5 µg, Ciprofloxacin 5 µg, Cefoperazone/ Sulbactam 50 µg, Tetracycline 10 µg, Norfloxacin 10 µg (Himedia Pvt Ltd.).

Antibacterial and biofilm formation assays

All the test organisms were inoculated in LB broth for an overnight period and then diluted to achieve a colony-forming unit (CFU) of 10⁶/ml. Agar well diffusion is used to test the antibacterial activity of Mo-AgNPs. Wells were punctured and filled with 1 mM silver nitrate solution, 25, 50 and 75 μg/ml of Mo-AgNPs, distilled water, and Ampicillin 25 μg/ml. The plates were incubated for the period of 12 to 24 h and the zone of inhibition was measured at 24 h [36].

The minimum inhibitory concentration of Mo-AgNPs was determined by using 96-well plate method [11]. Each well was filled with 100 μl of LB broth. 100 μl of 1 mg/ml Mo-AgNPs was added serially diluted till the last well. 2 μl of food—borne pathogen strains of selected bacterial isolates were added in triplicates in their respective wells. Plates were maintained with positive control and standard antibiotic ampicillin treatment (25 ug/ml). These plates were then incubated at 37 °C for 24 h. The inhibitory effect of the Mo-AgNPs was observed by viewing the visual turbidity at each concentration and noted for minimum inhibitory concentration (MIC). The growth rate of organism after 24th hour was determined by measuring the absorbance at 600 nm (Perkin Elmer Multimode reader).

Minimum bactericidal concentration (MBC) is the lowest concentration at which Mo-AgNPs completely kills tested micro-organisms. From MIC plate, 2 μl of culture for MIC plates were inoculated into LB agar plates and plates were incubated at 37 °C for 24 h. After 24 h of incubation, the concentration at which the complete inhibition of bacterial growth was considered as the minimum bactericidal concentration of Mo-AgNPs for the respective bacterial strains.

The biofilm assay was performed in the same method as that of MIC in microtiter plate method. However, after 48 h of incubation, the formation of biofilm was measured after staining with 0.1% crystal violet and destaining with 30% glacial acetic acid. The destained solution was measured at 580 nm to measure the quantity of biofilms formed in the microtiter plate [24, 45].

All the experimental analysis were performed three times in triplicates and statistical significance was carried out by using student t-test in MS Excel. Where * Denotes T-test (*P < 0.05, **P < 0.01, ***P < 0.001).

In silico analysis

The in silico analysis was performed to understand the protein binding capacity of target proteins of microorganisms with phytocompounds of Moringa oleifera (flower). The proteins beta lactamase (4XUX from Enterobacter cloacae) and beta lactamase OXY 1 (3BYD from Klebsiella oxytoca) were retrieved from Protein Data Bank (PDB). In order to select the best, soluble, lead-like, and non-violating ligands, the phytocompounds were subjected to absorption, distribution, metabolism, and excretion (ADME) analysis by using Swiss ADME. 14 Compounds that were screened by using Swiss-ADME software (Table 1). Molecular docking of proteins and ligands were carried out by using PyRx software by following the steps given in the bioinformatics magazine and their interactions were visualized [3, 4, 28, 50].

Table 1.

Screening of ligands by ADMET property analysis by using SwissADME

Compound name	Molecular weight (g/mol)	TPSA (Ų)	No. of heavy atoms	Log P	Log S	No. of H donors	No. of H acceptors	No. of rotatable bonds	GI Absorption	Drug likeness (Lipinski rule)	BBB
Quercetin	302.24	131.36	22	1.23	Soluble	5	7	1	High	Yes, 0 violation	No
Kaempferol	286.24	111.13	21	1.58	Soluble	4	6	1	High	Yes,0 violation	No
Apigenin	270.24	90.9	20	2.11	Soluble	3	5	1	High	Yes,0 violation	No
Gallic acid	170.12	97.99	12	0.21	Soluble	4	5	1	High	Yes,0 violation	No
Ellagic acid	302.9	141.34	22	1	Soluble	4	8	0	High	Yes,0 violation	No
Ferulic acid	194.18	66.76	14	1.36	Soluble	2	4	3	High	Yes,0 violation	Yes
Caffeic acid	180.16	77.76	13	0.93	Soluble	3	4	2	High	Yes,0 violation	No
O- coumaric acid	164.16	57.53	12	1.4	Soluble	2	3	2	High	Yes,0 violation	Yes
Vitamin A	286.45	20.23	21	5.04	Soluble	1	1	5	High	Yes,1 violation	Yes
Thiamine	265.35	104.15	18	0.53	Soluble	2	3	4	High	Yes,0 violation	No
Riboflavin	376.36	161.56	27	-0.32	Soluble	5	8	5	Low	Yes,0 violation	No
Niacin	123.11	50.19	9	0.32	Soluble	1	3	1	High	Yes,0 violation	Yes
Pyridoxine	169.18	73.58	12	-0.02	Soluble	3	4	2	High	Yes,0 violation	Yes
Palmitoleic acid	254.41	37.3	18	4.92	Moderately soluble	1	2	13	High	Yes,0 violation	Yes

Results and discussion

Synthesis and characterization of Mo-AgNPs

The UV spectroscopy was used to confirm the formation of surface plasmon resonance (SPR) peaks in the instance of Mo-AgNPs synthesis. Light is absorbed and scattered by nanoparticles because of the collective oscillation of conduction electrons [27]. After colour change (Fig. 1a, b) UV spectra exhibited the SPRs peak between 350 and 450 nm. The prominent peak was obtained at 409 nm (Fig. 1c), which confirming the synthesis of Mo-AgNPs. CgAgNPs synthesized by using C. gloeosporioides showed a similar SPR peak around 400 nm [27]. A concentrated electron beam is utilized in the HRTEM technique to provide high-resolution images of the shape and structure of nanoparticles. HRTEM shows that Mo-AgNPs are polydisperse, spherical in nature and the particle size ranges between 5 and 50 nm (Fig. 1d). HRTEM results showing spherical, polydisperse Mo-AgNPs ranging from 5 to 50 nm are consistent with previous literature on plant-mediated AgNP synthesis. The SAED pattern of Mo-AgNPs confirms their crystalline nature with distinct diffraction rings. Polycrystalline nature of Mo-AgNP is confirmed by the SAED pattern, which shows clear concentric diffraction rings with particle sizes ranging from 5 to 50 nm (Fig. 1e). These rings, which match the distinctive planes of face-centered cubic (fcc) silver, show that the crystalline silver nanoparticles made with Moringa oleifera floral extract were confirmed. FESEM works on the premise of scanning a sample with a focused electron beam and gathering the signals produced by the electrons interactions with the samples surface. In the study, FESEM imaging at the nanoscale showed a flower-like pattern (Fig. 1f) that might be attributed to particle aggregation during sample preparation. Energy dispersive x-ray spectroscopy (EDS) works based on the principle when high-energy electron beam interacts with the sample, it emits distinctive X-rays. X-rays have distinct energies that are particular to each element. EDS can determine and measure the elemental composition of the nanoparticles [16]. The elemental makeup of the sample may be discovered by detecting and examining these released X-rays. In the investigation, the discovery of a peak at 3 keV (Fig. 1g) in the EDS spectra represents the presence of roughly 52.75% silver content, was used to corroborate the presence of silver (Ag) in the Mo-AgNPs [15]. (Fig. 1h). The sample is exposed to infrared light, and the absorption or transmission of particular wavelength gives information of functional groups. FTIR results exhibited the different stretches of bands at different peaks (Fig. 2a, b) which were listed (Table 2). Different lengths of bonds at various peaks were observed by comparing the FTIR spectra of the Moringa flower extract and Mo-AgNPs, showing the existence of certain chemical groups involved in the production of Mo-AgNPs. DLS is based on the Brownian motion of particles and an approach to determine the size distribution of the particles. The Mo-AgNPs in the study had a Z-average size (mean size) of 12.73 nm, and the polydispersity index (PdI) value of 0.605 (Fig. 2c) indicating polydispersity of the particles. The results from DLS were in agreement with those obtained with HRTEM. Zeta potential is a measurement of the electric potential difference between the surface of particles and the surrounding liquid media. The zeta potential measurement sheds light on the stability and interaction potential of nanoparticles. The Mo-AgNPs and Gram-negative bacteria in the study interacted strongly, improving the structural stability of the nanoparticles and avoiding their agglomeration, as shown by the positive zeta potential value of 0.115 mV (Fig. 2d) [54]. The XRD diffractogram provided the crystallite structure and size of the synthesized nanoparticles. The diffraction peaks were obtained at 38, 44, 64, 77 and 82 and were assigned to the miller indices (111), (200), (220), (311), (222) respectively which corresponds to JCPDS file No. 04–0783. The size of the nanoparticles was calculated using Full Width at Half Maximum values and from Scherrer’s equation was found as 6 nm.

Fig. 1.

Synthesis and characterization of Mo-AgNPs: (a) MOF extract (b) Synthesis of Mo-AgNPs which is indicated by the change of color to dark brown (c) UV-vis spectrum of Mo-AgNPs (d) HRTEM image pattern of Mo-AgNPs which shows the spherical shaped nanoparticles in nanometer in range (e) SAED analysis of Mo-AgNPs (f) FESEM image of Mo-AgNPs exhibiting flower like pattern (g) EDAX analysis indicating the presence of silver at 3KeV (h) XRD diffractogram of Mo-AgNPs

Fig. 2.

Physicochemical characterization of Mo-AgNPs (a) FTIR analysis of Mo-AgNPs (b) FTIR analysis of Moringa flower extract (c) Peak showing the diameter of Mo-AgNPs by measuring the Brownian movement of Mo-AgNPs (d) Analysis of surface charge of Mo-AgNPs by measuring the zeta potential

Table 2.

FTIR footprints ofMoringa flower extract and Mo-AgNPs (https://instanano.com/all/characterization/ftir/ftir-functional-group-search/)

Peak wavelength (cmˉ¹)	Group	Compound class
Moringa flower extract
3882	O–H stretching	Alcohol
3761.47	O–H stretching	Alcohol
3739.3	O–H stretching	Alcohol
3286.11	O–H stretching	Alcohol
2981.41	C–H stretching	Alkane
2920.66	C–H stretching	Alkane
2853.17	C–H stretching	Alkane
2318.98	O= C =O stretching	Carbon dioxide
2191.7	CΞC stretching	Alkyne
2054.78	N = C = S stretching	Isothiocyanate
1987.29	N = C = S stretching	Isothiocyanate
1779.01	C = O stretching	Acid halide
1628.59	C = C stretching	Alkene
1257.36	C–O stretching	Aromatic ester
1022.09	C–N stretching	Amine
Mo-AgNPs
3815.47	O–H stretching	Alcohol
3771.12	O–H stretching	Alcohol
3726.76	O–H stretching	Alcohol
3696.87	O–H stretching	Alcohol
3234.04	O–H stretching	Alcohol
2911.99	C–H stretching	Alkane
2842.46	C–H stretching	Alkane
2365.26	O = C = O stretching	Carbon dioxide
2108.78	CΞC stretching	Alkyne
1991.14	N = C = S stretching	Isothiocyanate
1799.26	C = O stretching	Acid halide
1594.84	N–H bending	Amine
1405.85	S = O stretching	Sulfate
1312.32	O–H bending	Phenol
1192.76	C–O stretching	Ester
1119.48	C–O stretching	Secondary alcohol
1077.05	C–O stretching	primary alcohol
1000.87	C = C bending	Alkene
942.056	C = C bending	Alkene
703.89	C = C bending	Alkene
661.464	C = C bending	Alkene
592.039	C–Br stretching	Halo compound
556.363	C–Br stretching	Halo compound

Toxicity studies of Mo-AgNPs in Vigna radiata and Artemia nauplii

Nanoparticles have raised concerns about their potential toxicity to living organisms and the environment. Mo-AgNPs have gained attention due to their unique properties. This study aimed to assess the effects of Mo-AgNPs on plant growth and Artemia nauplii. The contrasting effects observed between plant growth and Artemia nauplii highlighted the complexity of nanoparticles interactions with different organisms [48]. The treatment of plants with different concentrations of Mo-AgNPs resulted in significant effects on growth parameters (Fig. 3a, b). The shoot length increased by 17.36% (1 μg/ml), 43.17% (5 μg/ml), and 55.38% (10 μg/ml) of Mo-AgNPs. Similarly, the root length increased by 33.33%, 39.21%, and 49.01% for the respective concentrations. The plant mass increased by 12.3%, 33.84%, and 51.53%, and the growth rate increased by 5.88%, 35.29%, and 41.17% with increasing concentrations. Mo-AgNPs showed potential as a beneficial nanoparticles for plant growth, as evidenced by the positive effects on shoot length, root length, plant mass, and growth rate [50]. The toxicity of Mo-AgNPs on Artemia nauplii depicted in Fig. 3c. Mo-AgNPs exhibited toxicity to Artemia nauplii at its higher concentration. After 24 h of exposure, the mortality rate increased with higher concentrations of Mo-AgNPs. At 1 μg/ml, the mortality rate increased by 20%, while at 5 μg/ml and 10 μg/ml, it increased by 60% and 80%, respectively. Toxicity was reported at higher concentrations due to the accumulation of silver nanoparticles in the gut region of Artemia nauplii during food uptake which led to their mortality. However, their toxicity to Artemia nauplii underscores the importance of considering environmental implications and further studies will be carried out in higher animal models. Arulvasu et al. [5] demonstrated the toxic effect of silver nanoparticles in brine shrimps. The study showed that the percentage of Artemia cysts that hatched decreased with increase in the concentration of AgNPs. Further it was observed increase in the mortality rate, aggregation of nanoparticles in the gut area, apoptosis, and DNA damage in treated nauplii. Love et al. [20] studied the toxic effect of silver nanoparticles synthesized by using endophytic bacteria Cronobacter sakazakii in Artemia salina and in Vigna radiata seeds. Upon treatment there was good hatching of eggs with the LC₅₀ value of 688.41 µg/mL. The nanoparticles also enhanced plant growth in Vigna radiata seeds treated with 2.5 ppm of nanoparticles. This study supports that the toxicity of nanoparticles varies from model system as well as the physico chemical nature of the nanoparticles and its concentration dependent.

Fig. 3.

Toxicity studies of Mo-AgNPs: (a) Qualitative analysis of the growth of Vigna radiata seedlings at 1 µg/ml, 5 µg/ml and 10 µg/ml of Mo-AgNPs (b) Growth rate of Vigna radiata (c) Mortality rate Artemia nauplii at 1 µg/ml, 5 µg/ml and 10 µg/ml of Mo-AgNPs

In vitro antibacterial analysis of Mo-AgNPs in food—borne pathogens

Isolation, identification, and antibiotic susceptibility of organisms isolated from spoiled food

The bacterial strains isolated from spoiled food were identified by employing 16SrRNA sequencing. Based on the construction of evolutionary relationships by using phylogenetic tree analysis, the organisms were identified and deposited in NCBI GenBank with their respective accession numbers are followed Enterobacter kobei—MZ245730, Enterobacter cloacae—MZ246448, Klebsiella oxytoca—MZ246548, Enterobacter bugandensis- MZ246580, Enterobacter chuandaensis—MZ246629, Enterobacter huaxiensis- MZ246628 (Fig. 4a-f). Antibiogram was performed and it was observed that Enterobacter kobei, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, Enterobacter huaxiensis, were resistant to Ampicillin 2 µg, and Penicillin 10 µg. Antimicrobial resistance (AMR) in Enterobacteriaceae isolated from spoilt food is an increasing global concern. The existence of opportunistic and clinically relevant pathogens in the spoiled food was highlighted by the identification of Enterobacter sp. and Klebsiella oxytoca. Recent research revealing intrinsic and acquired β-lactam resistance in Enterobacter and Klebsiella species is in line with the antibiotic resistance profiles found, especially resistance to Ampicillin (2 µg) and Penicillin (10 µg). The rising frequency of Enterobacter bugandensis and Klebsiella oxytoca in food sources has been brought to light by studies by Singh et al. [46]. These bacteria frequently carry extended-spectrum β-lactamase (ESBL) genes, which contribute to their resistance profiles. Once thought to be ambient or opportunistic, these species are now commonly found in research on food contamination, indicating that improperly handled or spoilt food may serve as a breeding ground for resistant bacteria.

Fig. 4.

Phylogenetic relationships of organisms isolated from spoiled fruits and vegetables: (a) Enterobacter kobei - MZ245730, (b) Enterobacter cloacae - MZ246448, (c) Klebsiella oxytoca - MZ246548, (d) Enterobacter bugandensis- MZ246580, (e) Enterobacter chuandaensis - MZ246629, (f) Enterobacter huaxiensis- MZ246628

Antibacterial activity of Mo-AgNPs in bacterial strains

Antibacterial activity of Mo-AgNPs was evaluated in isolated Gram negative bacterial strains namely Enterobacter kobei, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, Enterobacter huaxiensis. Antibacterial activity can function by interfering with cell wall integrity, blocking DNA replication or transcription, or inhibiting essential enzyme activities. Antibacterial activity's mode of action may entail rupturing the bacterial cell membrane, meddling with vital cellular functions, preventing protein synthesis, or focusing on particular metabolic pathways [46]. The overall process of Mo-AgNP synthesis using Moringa oleifera flower extract and the antibacterial mechanisms involved including ROS generation, membrane disruption, protein leakage, and interference with metabolic pathways are diagrammatically represented in Fig. 5. This figure provides a comprehensive visual summary of the synthesis steps and modes of bacterial inhibition by Mo-AgNPs.

Fig. 5.

Synthesis of Mo-AgNPs using Moringa oleifera flower extract and the proposed antibacterial mechanisms, including membrane disruption, and interference with cellular functions

In agar well diffusion assay, the antibacterial activity of Mo-AgNPs was confirmed by the formation of zone of inhibition (Fig. 6a). The zone of inhibition (ZOI) was measured with varied concentration of Mo-AgNPs: 25, 50 and 75 μg/ml. At the concentration of 25 μg/ml, the ZOI was 19, 20, 20, 19, 21, and 23 mm for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis respectively. At the concentration of 50 μg/ml, the ZOI was 20, 21, 22, 27, 20 and 25 mm for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis respectively. At the concentration of 75 μg/ml, the ZOI was 20, 22, 23, 28, 21 and 26 mm for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis respectively (Table 3). This was due to the penetration of Mo-AgNPs into the cell walls of Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis and disturbs the regular functioning of the cell [29]. Notably, Table 3 reveals that ampicillin failed to show any inhibition zones ("–") against Enterobacter kobei, Enterobacter chuandaensis, and Enterobacter huaxiensis, while Mo-AgNPs demonstrated clear and measurable zones of inhibition against all these strains, even at the lowest concentration. This lack of response to ampicillin is likely due to antibiotic resistance mechanisms, such as β-lactamase enzyme production, commonly found in Enterobacter species, which degrade β-lactam antibiotics like ampicillin and render them ineffective. In contrast, Mo-AgNPs bypass these resistance mechanisms by acting through non-specific modes such as cell wall disruption, oxidative stress (ROS generation), and interference with intracellular biomolecules. This explains their effectiveness even against strains that are resistant to conventional antibiotics.

Fig. 6.

Antibacterial efficacy of Mo-AgNPs against isolated strains: (a) Mo-AgNPs showing zone of inhibition. The wells are loaded with: A– AgNO₃ (1:10, 25 µg/mL), B– Mo-AgNPs (25 µg/mL), C– Mo-AgNPs (50 µg/mL), D– Mo-AgNPs (75 µg/mL), E– Distilled Water (25 µg/mL, negative control), F– Ampicillin (25 µg/mL, positive control). (b) Growth of bacteria at 24 h of treatment (c) Biofilm formation at 48 h of treatment

Table 3.

Assessment of antibacterial activity of Mo-AgNPs in food - borne pathogens by using Agar-well diffusion assay

SI.no	Strains	Zone of inhibition in mm
		Mo-AgNPs 25 µg/ml	Mo-AgNPs 50 µg/ml	Mo-AgNPs 75 µg/ml	Ampicillin 25 µg/ml
1	Enterobacter kobei	19 ± 1.2	20 ± 1.5	20 ± 1.2	–
2	Enterobacter cloacae	20 ± 1.6	21 ± 1.8	22 ± 1.0	18 ± 1.4
3	Klebsiella oxytoca	20 ± 2.1	22 ± 1.3	23 ± 1.5	19 ± 1.0
4	Enterobacter bugandensis	19 ± 1.5	27 ± 1.2	28 ± 1.2	15 ± 1.2
5	Enterobacter chuandaensis	21 ± 1.9	20 ± 1.4	21 ± 1.4	–
6	Enterobacter huaxiensis	23 ± 1.5	25 ± 1.9	26 ± 1.1	–

Moreover, while the size of the inhibition zones for Mo-AgNPs was significantly larger than that of ampicillin, the difference in inhibition zones between Mo-AgNPs and AgNPs alone was modest, though still notable in certain strains like E. bugandensis and E. huaxiensis, suggesting that molybdenum functionalization enhanced antibacterial potency in a strain-specific manner. Silver nanoparticles exhibit broad-spectrum antibacterial activity in both Gram-negative and Gram-positive strains. Gram-negative bacteria responds to the silver nanoparticles treatment than the Gram positive strains due to their thinner peptidoglycan layer and higher membrane permeability. Agar well diffusion assay confirms the antibacterial potential of Moringa flower-mediated silver nanoparticles as antimicrobial agent in comparison with standard antibiotic ampicillin. Antibacterial mechanism was exhibited because of the phytocompounds which are capped in Mo-AgNPs. Moringa flowers are reported to contain various phytocompounds namely flavonoids, phenolic acids, gallic acid, alkaloids, tannins, saponins, terpenoids, steroids. The activity of Mo-AgNPs is due to various phytocompounds of Moringa flower and physico chemical nature of the nanoparticles. The nanometer sized and positive charge of Mo-AgNPs made nanoparticles to easily interact with negatively charged cell wall of Gram negative bacteria. The smaller size and electrostatic interaction between the bacterial cell and nanoparticles rupture the bacterial cell and prevent the multiplication of bacteria which makes the clear zone around the wells which were filled with Mo-AgNPs. Similar results were observed in the studies by Malaikozhundan et al. [25] showing the antibacterial activity of Withania somnifera leaf extract mediated zinc oxide nanoparticles in E. faecalis, S. aureus, E. coli and P. aeruginosa. Liaqat et al. [19] tested antimicrobial activity of E. camaldulensis, T. arjuna, and its combinations mediated nanoparticles in Bacillus subtilis, Staphylococcus aureus, Pasteurella multocida, and Escherichia coli. Singh et al. [47] synthesized nanoparticles using neem and turmeric extract which have exhibited significant antimicrobial activity in Staphylococcus aureus, Streptococcus mutans, and Lactobacillus species. Researchers have investigated the antibacterial properties of AgNPs against bacteria that are resistant to many drugs, and the results have demonstrated the effectiveness of AgNPs in drug resistant pathogens. Inhibition zones obtained by testing the efficacy of silver nanoparticles from various research have demonstrated a wide range of variation. However, from this result Mo-AgNPs showed dose dependent activity against the tested strains as the concentration increases the zone of inhibition increases. This is due to the amount/ quantity of nanoparticles interaction with bacterial strains increases hence showing larger zone of inhibitions at the higher concentration of 75 μg/ml.

The MIC assay establishes the lowest dose of Mo-AgNPs that prevents visible microbial growth. The results of MIC showed the Mo-AgNPs concentration, which inhibits the bacterial growth at 24 h were 0.39 μg/ml for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, and Enterobacter bugandensis, 3.12 μg/ml for Enterobacter chuandaensis and 1.56 μg/ml for Enterobacter huaxiensis. This was due to the entry of Mo-AgNPs in the bacterial cell wall that induces high stress [55]. On comparison with control, the growth percentage of bacterial strains of was inhibited up to 79.59%, 63.94%, 64.31%, 73.51%, 96.77% and 97.15% respectively in Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, Enterobacter huaxiensis (Fig. 6b). Mo-AgNPs successfully controlled the growth of Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis in comparison to the commonly used antibiotic ampicillin (Table 4). Ahmed et al. [2] studied the antibacterial effect of ethanolic and aqueous leaf extracts and compared with green Ag-NPs in foodborne pathogenic bacterial strains Staphylococcus aureus ATCC 13565, Staphylococcus sciuri 2–6, and Bacillus cereus EMCC 1080, Salmonella enterica SA19992307, Salmonella typhi ATCC 25566, Escherichia coli 0157 H7 ATCC 51659, and Pseudomonas aeruginosa NRRL B-272 and the MIC values of the Moringa nanoparticles was observed in the range of 0.05 mg/mL to 0.13 mg/mL. The similarity in inhibitory concentration (MIC) for the majority of tested bacteria may be due to the broad-spectrum and non-specific mechanism of Mo-AgNPs, which uniformly target essential cellular structures like membranes, proteins, and DNA across different bacterial species. This leads to consistent susceptibility among various pathogens, especially those with similar membrane compositions and metabolic pathways. However, the moringa flower nanoparticles exhibited higher antibacterial potential even at lower concentrations. Further studies help to optimize the concentration of Mo-AgNPs against wide range of pathogenic bacteria.

Table 4.

MIC and MBC of Mo-AgNPs in isolated food - borne pathogens

SI.NO	Name of the organism	Inhibitory concentration of Mo-AgNPs (µg/ml)	Bactericidal concentration of Mo-AgNPs (µg/ml)
1	Enterobacter kobei	0.39 µg/ml	3.12 µg/ml
2	Enterobacter cloacae	0.39 µg/ml	6.25 µg/ml
3	Klebsiella oxytoca	0.39 µg/ml	50 µg/ml
4	Enterobacter bugandensis	0.39 µg/ml	3.12 µg/ml
5	Enterobacter chuandaensis	3.12 µg/ml	50 µg/ml
6	Enterobacter huaxiensis	1.56 µg/ml	50 µg/ml

MBC is compliant with MIC; while MIC test shows a lower concentration of Mo-AgNPs that inhibits bacterial growth whereas MBC shows a lower concentration of Mo-AgNPs leading to bacterial death [36]. MBC is the concentration of Mo-AgNPs that completely kills the bacterial strains. The MBC results showed the Mo-AgNPs concentration that kills the Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis were 3.12 μg/ml, 6.25 μg/ml, 50 μg/ml, 3.12 μg/ml, 50 μg/ml and 50 μg/ml respectively (Table 4). The result of MBC supports that the Mo-AgNPs can kill bacterial pathogens by attaching itself to a bacterial cell, penetrate into the bacterial cell and lead to cell death [10]. Asif et al. [6] studied the antibacterial activity of Moringa leaf extract nanoparticles in E. coli and the MIC was observed at 5 mM. Varthini et al. [52] studied the antibacterial activity of Moringa seed extract nanoparticles in E. coli, Klebsiella, Pseudomonous, Staphylococcus bacteria and MIC was observed at 200 µL. The antibacterial activity of Mo-AgNPs was associated with several mechanisms (i) production of Reactive Oxygen Species (ROS), such as hydroxyl radicals (OH^·) and super oxide anions (O2 −), (ii) Ag + ions in Mo-AgNPs interact with sulfhydryl groups which results in denaturation of proteins in the bacteria which are vital for its survival and multiplication and (iii) Mo-AgNPs penetrate into the bacterial cell wall and leads to bacterial cell death due to leaking of sugars and proteins. Moreover, nanosized Mo-AgNPs enter the cell and cause major disturbance in various metabolic pathways which are important for the energy production and cell cycle.

Moringa flowers contain several phytocompounds responsible for their antibacterial activity. These bioactive constituents contribute to disrupting bacterial cell membranes, inhibiting protein synthesis, and inducing oxidative stress. Flavonoids including quercetin and kaempferol act as antioxidants and can disrupt bacterial cell wall. Phenolic compounds damage bacterial cell wall and disrupt membrane integrity. Alkaloids are reported to interfere with synthesis of DNA, RNA. Tannins are known to denature the proteins and enzymes. Saponins increases the permeability of cell membrane and leads to cell lysis. Terpenoids impairs the membrane integrity and cell signalling pathways.

Biofilm formation is the process by which microorganisms connect to surfaces, develop, and create polymers that help in matrix growth and adhesion. Biofilm assay is a useful way to test bacterial attachments by measuring the staining of adhesive biomass. The biofilm assay was used to test the Mo-AgNPs anti-biofilm ability. The formation of biofilm decreases with the increased dose of Mo-AgNPs. From the biofilm formation assay, it was clear that Mo-AgNPs treatment reduced the biofilm formation than ampicillin treatment. Decrease in biofilm formation proofs the that the bacterial strains are more susceptible to Mo-AgNPs treatment than ampicillin. The concentration which inhibits the biofilm formation were 0.39 μg/ml for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, and Enterobacter bugandensis, 3.12 μg/ml for Enterobacter chuandaensis and 1.56 μg/ml for Enterobacter huaxiensis. The Mo-AgNPs reduces the biofilm formation by 76.32%, 77,93%, 81%, 73.48%, 70.81% and 82.75% (Fig. 6c) for Enterobacter kobei, Enterobacter cloacae, Klebsiella oxytoca, Enterobacter bugandensis, Enterobacter chuandaensis, and Enterobacter huaxiensis respectively. Quorum sensing promotes the production of biofilms, which are essential for the survival and growth of microorganisms. Quorum sensing is a signaling system which coordinates with the genes which are responsible for biofilm formation. Silver nanoparticles may effectively enter the biofilm matrix utilizing proximity sensing and exhibit antibiofilm actions without directly harming the bacteria. The phytocompounds and silver ions in Mo-AgNPs prevent the formation of biofilms (Ranjani and Hemalatha 2024a). Disturbance in the cell signaling mechanism results in reduced bacterial adhesion, and production of extracellular polymeric substance and overall attenuation of virulence in food—borne pathogens.

Mo-AgNPs inhibit biofilm formation through multiple mechanisms by applying the synergistic mechanism of their phytochemical constituents and silver ions. Biofilm formation is a multi-step process where bacteria adhere to surfaces, proliferate, and produce extracellular polymeric substances (EPS). Mo-AgNPs inhibits the biofilm formation at several stages in biofilm formation namely inhibition of bacterial cell adhesion, quorum sensors, matrix formation by stimulating oxidative stress and acting as quorum quenchers. Flavonoids and tannins are reported to interfere with bacterial adhesion by interacting with responsible proteins under a low hydrophobic environment. Flavonoids and terpenoids inhibit QS signalling molecules namely acyl-homoserine lactones (AHLs) which reduces the expression of virulent genes which are responsible for EPS production. Phenolic compounds, saponins can degrade EPS or inhibit its biosynthesis, weakening the biofilm matrix and making bacteria more susceptible to nanoparticles. Nanoparticles enter the biofilm layer and induces oxidative stress which results in intracellular damage and disturbs the metabolic pathways which are crucial for maintaining the existing biofilms.

The results of Agar well diffusion, MIC, MBC, and biofilm formation show the efficacy of Mo-AgNPs for halting and eliminating bacterial growth and eliminating the bacteria and can be utilized as potential bacteriostatic, bactericidal and antibiofilm agent. This is accomplished by Mo-AgNPs adhering to the outer layer of bacterial cells and expanding the membrane's porosity, which ultimately causes cell death. Antibacterial activity is further increased by the synergistic interaction of silver ions with numerous phytochemicals present in Moringa oleifera flowers, which are released by the silver nanoparticles inside the cells. Phenolic chemicals and terpenoids, among other phytocompounds, are found in Moringa oleifera flowers and help the plant's antibacterial properties. Mo-AgNPs effective penetrating capabilities along with their charge and size plays a major role in its bactericidal activity. The formation of pits on the bacterial cell wall is caused by Mo-AgNPs' ability to electrostatically attach to bacterial membranes quickly due to their tiny size. The cell membrane's integrity becomes risk prone, and oxygen radicals are produced as a result of the invasion. Bacterial cells are rendered inactive by the interaction of Ag ions with their thiol groups. Further, the architecture of the bacterial cell membrane changes into tightly packed granules of electrons, and nanoparticles make bacterial strains lose their ability to replicate [31]. Ag + ions can denature and inactivate the membrane proteins and triggers the cellular leakage. This study observed effective antibacterial activity of Ag nanoparticles against drug-resistant bacteria. These findings show that the investigated organism’s growth has been consistently inhibited by Mo-AgNPs.

In silico analysis of phytocompounds of Moringa flower against antibiotic resistant enzymes

Based on molecular docking studies, Ellagic acid has the least value of -8.6 and -8.9 kcal/mol with beta lactamase of Enterobacter cloacae and beta lactamase OXY1 of Klebsiella oxytoca respectively. Quercetin, Apigenin, Riboflavin and kaempferol have lower values of -7.7, -7.6, -7.8 and -7 kcal/mol (Enterobacter cloacae) (Fig. 7a- f) and -8.3, -7.8, -7.9 and -7.7 kcal/mol (Klebsiella oxytoca) respectively (Fig. 8a- f). A lower binding energy, which indicates a stronger interaction between the protein and ligand, suggests the possibility of antibacterial activity. Ellagic acid showed the highest binding affinity with both beta lactamases among the investigated phytocompounds. This study raises the possibility that ellagic acid has a significant ability to suppress the beta lactamase enzyme activity in Klebsiella oxytoca and Enterobacter cloacae. Other phytocompounds such as Quercetin, Apigenin, Riboflavin and Kaempferol have also shown potential for inhibiting beta-lactamases [28]. The identification of natural compounds with antibacterial properties in Moringa flowers, particularly ellagic acid, has the potential to combat antibiotic resistance and develop new treatments for foodborne infections. Similar to our study, Bhat et al. [7] reported that the ellagic acid as a potent inhibitor of CTX-M152. Talbot et al., [49] reported the ability of ellagic acid to inhibit the CTX-M-15 by its negative charge and acylation of serine at the active site in in vitro studies. Antibiotics inactivate enzymes in food—borne pathogens able to inactivate the antibiotics by means of degradation and develop antibiotic resistance. The phytocompounds of Moringa flowers showed strong binding affinities with beta-lactamases, suggesting that these phytocompounds target synergistically and enhance their ability to combat foodborne pathogens. Abdallah et al. [1] reported the antibacterial effect of Moringa leaf extract in food borne pathogens namely E. coli O157:H7, Salmonella enterica serovar typhimurium, and Staphylococcus aureus in ground beef. Based on the in-silico findings, phytocompounds offer a promising avenue for combating antibiotic resistance and offer an alternative strategy for the treatment of multi drug resistant bacterial infections [32]. Strong antibacterial properties against foodborne pathogens are demonstrated by floral extract-decorated nanoparticles, providing a natural alternative for food preservation. These findings confers safer, more environmentally friendly methods of ensuring food safety. To increase shelf life and lessen the use of artificial preservatives, future studies should focus on mechanism of action, toxicity evaluation, scalability, and incorporation into food packaging systems.

Fig. 7.

Interaction of phytochemicals present in Moringa flower with beta lactamase of Enterobacter cloacae: (a) Beta lactamase of Enterobacter cloacae (b) Ellagic acid (c) Quercetin (d) Apigenin (e) Riboflavin (f) Kaempferol

Fig. 8.

Interaction of phytochemicals present in Moringa flower with beta lactamase OXY1 of Klebsiella oxytoca: (a) Beta lactamase OXY1 of Klebsiella oxytoca (b) Ellagic acid (c) Quercetin (d) Apigenin (e) Riboflavin (f) Kaempferol

Conclusion

This study concludes the effective binding nature of the target protein with the phytocompounds of Moringa flower during in silico analysis. Mo-AgNPs were synthesized and also characterized for its nature. Through in-vitro analysis, the antibacterial activity of Mo-AgNPs was indicated by zone of inhibition, MIC and MBC results showed minimum concentration of Mo-AgNPs which inhibit and kills the bacterial growth. Biofilm formation assay showed that the Mo-AgNPs are potential antibiofilm agents. Finally, it was proved that the Mo-AgNPs can be used as an alternative to treat food—borne pathogens that cause food—borne diseases and food spoilage. Future studies on Mo-AgNPs can examine the mechanisms of action, broad-spectrum antibacterial activity, and optimisation for scaling up. Drug-resistant food-borne infections can be efficiently and sustainably combated by focussing on food safety, toxicity, regulatory requirements, synergistic effects with antibiotics, and applications in food preservation. The limitations and challenges include large-scale production, regulatory permission, environmental impact assessment, and maintaining food system stability. Long-term safety assessments and thorough in vivo research are essential to confirm their usage in food preservation. The cost of synthesis can be optimized based on scale-up challenges faced during the collection of Moringa flowers and green synthesis. Regulatory compliance, cost-effectiveness, and consistency are some of the issues that scalability faces. Commercial viability depends on industrial-friendly techniques and synthesis process optimisation. By optimizing various strategies, the benefits and the advantages of Mo-AgNPs can be utilized in future to develop nano or bio-based product food packaging material which will maintain the integrity and shelf life of food after proper testing and validation.

This research work mainly focus on United Nations Sustainable Development Goals (SDGs), particularly in the areas of health, food safety, and sustainable innovation. SDG 2- Mo-AgNPs contributes food safety and prevent food- borne diseases, SDG 3- Good Health and Well-Being by addressing AMR, SDG 9- Industry, Innovation, and Infrastructure- nanobiotechnology offers innovative solutions of developing novel food packaging materials, SDG 12—Responsible Consumption and Production- green nanoparticles encourages eco-friendly practices, SDG 6– Clean Water and Sanitation (indirectly) Mo-AgNPs controls the outbreak of MDR’s in water system hence offering hygiene and waste management practices.

Author contributions

“SH conceived and designed research. NFMYS, SR conducted experiments. SH analyzed data. All authors wrote the manuscript. All authors read and approved the manuscript.”

Funding

Nil.

Availability of data and materials

“Data will be available on request from the corresponding author”.

Declarations

Ethics approval and Consent to participate

Not Applicable.

Consent to publish

All authors read and approved the manuscript for publication.

Competing interests

The authors declare no competing interests.