Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents.

In this study, a facile, ecological and economical green method is described for the fabrication of iron (Fe), copper (Cu) and silver (Ag) nanoparticles (NPs) from the extract of Syzygium cumini leaves. The obtained metal NPs were categorized using UV/Vis, SEM, TEM, FTIR and EDX-ray spectroscopy techniques. The Fe-, Cu- and Ag-NPs were crystalline, spherical and size ranged from 40-52, 28-35 and 11-19 nm, respectively. The Ag-NPs showed excellent antimicrobial activities against methicillin- and vancomycin-resistance Staphylococcus aureus bacterial strains and Aspergillus flavus and A. parasiticus fungal species. Furthermore, the aflatoxins (AFs) production was also significantly inhibited when compared with the Fe- and Cu-NPs. In contrast, the adsorption results of NPs with aflatoxin B1 (AFB1) were observed as following order Fe->Cu->Ag-NPs. The Langmuir isotherm model well described the equilibrium data by the sorption capacity of Fe-NPs (105.3 ng mg-1), Cu-NPs (88.5 ng mg-1) and Ag-NPs (81.7 ng mg-1). The adsorption was found feasible, endothermic and follow the pseudo-second order kinetic model as revealed by the thermodynamic and kinetic studies. The present findings suggests that the green synthesis of metal NPs is a simple, sustainable, non-toxic, economical and energy-effective as compared to the others conventional approaches. In addition, synthesized metal NPs might be a promising AFs adsorbent for the detoxification of AFB1 in human and animal food/feed.

Introduction

Apart from recent advancement, pathogenic moulds contamination associated in food and feed products are still major threat world-wide. The gram-positive bacteria Staphylococcus aureus is considered a historically and most frequently associated human pathogen and able to produce various diseases in human [1]. Furthermore, aflatoxins (AFs) contamination are considered one of the most hazardous toxins and commonly found in food and feed crops [2, 3]. AFs are derivative of difurocoumarin and produced by toxigenic species of Aspergillus flavus and A. parasiticus. To date, at least 20 different types of AFs such as aflatoxin B1, B2, G1 and G2 have been identified. However, aflatoxin B1 (AFB1) is considered one of the utmost potent carcinogens, hepatotoxic, mutagenic, immunosuppressive, neoplastic and teratogenic [4]. Various environmental elements such as agricultural practices, moisture, temperature, unseasonal rains, geographical location, storage and transportation are also favored the growth and production of Aspergillus and AFB1 contamination.

The massive economic effects on the agriculture segment can also be considered because this contamination is able to decrease nutritional significance of food and feedstuff, reduction in meat production, reduced kidney and liver function, immune system suppression and finally created harmfulness to the consumers of dairy/ foodstuffs. So, numerous strategies have been recommended for the AFs detoxification from contaminated food and feedstuff [5-7]. Though, most of the reported methods are having certain drawbacks.

The one of the most effective method to adsorb AFB1 is the addition of non-nutritive adsorbent in the contaminated consumption that decreased the bioavailability of toxin in the gastrointestinal tract. However, the non-specificity and the higher cost are the major issues of these adsorbents. In addition, due to non-degradable property, cause deposited in environment when excreted in manure is another issue that restricted to use [8].

Currently, nanotechnology is emerging field with its applications in science and technology such as bioengineering and nanomedicines through targeted drug delivery for cancer therapy, biomedical applications, pharmaceutical industry, contrast agents in magnetic resonance imaging (MRI), biosensing and antibacterial and photocatalytic agent [9-16]. Nanoparticles (NPs) are using in some cosmetics, sunscreens, toothpaste, pharmaceutical and even in food products [17]. Metal NPs directly encounter with the human/ animal body, therefore, there is necessary to develop environmentally pleasant approaches for the synthesis of NPs without using of toxic or dangerous chemicals.

A variety of techniques have been reported for the NPs synthesis, which are too expensive, low compound conversions, high energy requirement or involved environmentally hazardous chemicals [18, 19]. Biosynthesis of metal NPs using different plants leaves extracts [14], roots [20], microorganisms like fungi [21] and bacteria [22] have been developed as a new method to improve or remove the above-mentioned physical and chemical approaches challenges. Amongst the numerous well-known approaches, plant-mediated green synthesis of NPs is considered a widely accepted technology recently. Green nanotechnology has become most popular over the chemical techniques as a facile, economical, environment-pleasant, readily scaled up, without high temperature, pressure and energy, biocompatible, without using toxic and redundant chemicals and safe for human therapeutic uses. Various plants leaves extracts such as Gongronema Latifolium, Moringa oleifera, bitter leave (Veronica amygdalina), Psidium guajava have been utilized for the biosynthesis of metal NPs [23-26]. The flavonoids, phenolics, polysaccharides and terpenoids are the secondary metabolites present in the plant that are accountable primarily for the metal ions reduction into bulk metallic NPs preparation in the redox reaction to obtain nano-sized NPs that are non-toxic by transforming M+ to M0 and also act as a capping agent of the obtained NPs [25].

So, in this study, iron, copper and silver NPs were prepared using Syzygium cumini leaves extract and characterized using different techniques. Furthermore, antibacterial, antifungal and adsorption of AFB1 properties of each NPs were assessed under various conditions. Additionally, the adsorption mechanism was also judged by the assessment of adsorption isotherm models, kinetic and thermodynamic parameters.

Materials and methods

Synthesis of metal nanoparticles

Ten grams chopped leaves were accurately weighed and boiled in the presence of 100 mL DI-H2O for 20 min at 80°C, cooled at room environment and filtered using Whatman filter paper no. 1. The solutions of FeCl3, CuSO4 and AgNO3 (0.010 mol/L) was separately added to the plant extract in the ratio of 1:1, 4:1 and 9:1 by volume, respectively. All mixtures were stirred and NPs were obtained through centrifugation for 10 min at 10,000 rpm. The solid masses were then separately rinsed with DI-H2O in triplicate to eliminate boundless biological particles followed by drying at 80°C for 3 hours in a vacuum oven.

Characterization of metal nanoparticles

The confirmation of the NPs formation was assessed by the changes in color of reaction suspension. However, pH of the originator/ reducing agents before and after mixing was also used as another indicator. UV–Vis absorption spectrum of individual NPs were recorded using double beam spectrophotometer (Model no. UV-1700, Shimadzu, Japan) between 200–800 nm. The size distribution and surface morphology of each NPs were analyzed using SEM (Model # JEOL- JSM 6380A, Japan) and TEM measurements (Model # JEOL JEM-2100F, Japan) operating at 160 kV with a point-to-point resolution of 1.9 Å. EDX spectroscopy of the synthesized NPs was assessed for the confirmation of elemental composition using the SEM with EDX detector (Model # EX-54175jMU, Jeol Japan). FTIR spectra for the evaluation of functional groups were obtained in the range of 500−4000 cm-1 using Perkin-Elmer 100 spectrometer (Perkin-Elmer Inc, Waltham, MA) by the potassium bromide pellet (FTIR grade).

Antibacterial assay

The antibacterial property of each NPs was assessed against gram-positive methicillin- (MRSA) and vancomycin-resistance (VRSA) Staphylococcus aureus infectious strains using modified Oxford cup diffusion inhibition assays [27]. In brief, the new culture was prepared in the concentration of 0.2 OD600 containing 106 colony-forming units (CFU) per mL. The diluted culture was spread on petri plates containing 1.5% w v-1 nutrient agar using a disinfected glass rod spreader. Then, sterilized Oxford cups (6 mm) were placed on the upper of nutrient agar, dispensed 50 μL of 1 mg mL-1 (50 μg) of each NPs suspension into each Oxford cup and kept at 37°C for 24 h. The activity was recorded by the formation of inhibition zone (mm) round the Oxford cup by calliper. Antibiotics, Vancomycin and Cefoxitin (30 μg disc-1) (Oxoid, UK) were considered as a control. Each study was obtained in triplicate and results are stated as average ± SEM.

Minimum inhibitory concentration (MIC)

Dilution broth method was utilized to evaluate the MIC for each metal NPs. In brief, each NPs in a concentration between 1‒512 μg mL-1 was prepared in 10 mL 0.8% (w v-1) nutrient broth medium separately and sterilized for 15 min at 121°C using Rexall autoclave (Kaohsiung, Taiwan). An aliquot of 25 μL comprising bacterial strains (106 CFU mL-1) was dispensed in each concentration and incubated overnight under rotation at 120 rpm and 37 ± 2°C. After incubation time, an aliquot of 200 μL was transported into sterilized ELISA plates (96-well) and optical densities (ODs) were noted using an ELISA reader (Infinite 200; Tecan Systems, USA) at 600 nm. However, positive control with only NPs and negative control with only inoculums were too executed. Each trial was done in triplicate and described as average ± SD.

Antifungal assay

The antifungal property of NPs was carried out against toxigenic strains of Aspergillus flavus and A. parasiticus and the production of aflatoxin (AFs) contamination [28]. Briefly, separately metal NPs in the amount of 10, 25, 50, 100 μg mL-1 were added into the separate flask containing 100 mL of Czapek Dox Liquid medium. All flasks were autoclaved for 15 min at 121°C and then let to cool at room temperature. An aliquot 1 mL (about 106 spores mL-1) of pathogenic Aspergillus was introduced into each flask. The same protocols were applied for each kind of toxigenic strain (A. flavus and A. parasiticus). The positive control with alone spore suspension whereas negative control with only NPs were also tested with the mean time. Then, mixtures were retained for 15 days at 25 ± 2°C. The mixture was blended for 2 min at 5000 rpm and purified using Whatman filter paper. The filtrates were utilized to determine the level of AFs by HPLC as described underneath.

Determination of AFs using HPLC

The AFs levels were determined using our previously described technique [2]. In short, AFs was extracted in triplicate with 50 mL of chloroform from culture filtrates. The chloroform extract was collected and evaporated in a rotary flask evaporator. The residue of each chloroform extract was re-suspended in 100 mL of methanol:water: (80:20; v v-1) and purified using Whatman filter paper. Two mL filtrate diluted with PBS (pH 7.4) was passed through AflaStarTM immunoaffinity column (IACs). The column was washed two times with 20 mL of DI-H2O. The elution of AFs was done by passing 1.5 mL of HPLC grade methanol and DI-H2O in an amber vial. AFs were examined using HPLC attached with fluorescence sensor and post-column (Kobra Cell) derivatization. The mobile phase preparation was H2O:ACN:MeOH (65:17.5:17.5; v v-1) with KBr (119 mg L-1) and HNO3 (154 ml L-1) and delivered at 1 mL min-1. A volume of 99 μL of samples was introduced and separated using a LiChroCART® 100Å RP-18 column (40°C).

Adsorption of aflatoxin B1

The adsorption of aflatoxin B1 (AFB1) with each NPs was studied according to the previously defined method with some modifications [29]. NPs with the amount of 0.1−5 mg mL-1 were introduced separately in Erlenmeyer containers comprising AFB1 solution (100 ng mL-1) to examine the optimal NPs amount on which greater adsorption occurred. All flasks were placed in thermo shaker incubator for 45 min at 120 rpm at 37 ± 2°C. After a specific time period, centrifugation was performed for 2 min at 10,000 rpm. The supernatant was attained and free AFB1 was determined by HPLC as described above. The adsorption amount of AFB1 was obtained by the comparison of initial and equilibrium AFB1 concentrations in solution ().

Where, Ci / Ce (ng mL-1) are the initial / equilibrium AFB1 concentrations in solution respectively. The entire procedure was validated using the positive and negative controls.

The adsorption experiments were performed with each NPs equivalent to 1 mg mL-1 and different initial concentrations of AFB1 solutions (20–200 ng mL-1). To reveal the release efficiency directly and adsorption isotherm, the adsorption experiments were performed with the constant amount of each NPs (1 mg mL-1) and different initial concentrations of AFB1 solutions (20–200 ng mL-1). The solutions were agitated in thermo shaker at 120 rpm and 37°C for 45 min. The AFB1 amount was determined by HPLC at equilibrium and time t = 0. Entirely trials were executed in triplicate and the AFB1 amount at equilibrium Qe (ng mg-1) was obtained by mass balance equation (): where

M (mg mL-1) and V (mL) are the amount and volume of NPs, respectively. The influence of pH was performed at initial pH of 1, 3, 5, 7 and 9. However, further factors were kept constant (37 ± 2°C, 45 min, NPs amount 1 mg and 100 ng mL-1 of AFB1 concentration). The kinetic study was evaluated as same with the equilibrium execution to investigate the effect of contact time. The solutions were composed by centrifugation after 15 min interval vigorous mixing. On regular time intervals, the AFB1concentrations in the supernatant was assessed at time t = 0 and t by HPLC. The temperature effect and thermodynamic parameters were estimated by incubation the samples at 10, 25, 37 and 45°C. The supernatant was utilized for the analysis of un-attached AFB1 by HPLC procedure. Entirely trials were carried out in three times.

Results and discussion

Formation and characterization of metal nanoparticles

The color variations from pale yellow to brownish black were the first indication for the formation of NPs. The further confirmation was done by the variation in the solutions pH. The mixture pH was tested pre- and post-reduction as mentioned in . Before and after reduction, the pH was observed as acidic. Though, the pH of the solution further decreased in the reduction route and moved to the more acidic range.

The SEM micrographs displayed that prepared NPs were agglomerated and spherical in nature ( The diameter of Fe-, Cu- and Ag-NPs ranged (average) from 55−65 (58)-, 42−52 (45)- and 22−38 (32) nm, respectively (. The difference in size was due to the different volume of leaves extract was used for the metal ions reduction. Consequently, the volume of leaves extract directly effects on the morphology, size and production of NPs. The presence of higher amount of polyphenols in the Syzgium cumini was the main reason for the larger size particles formation as reported by Kumar et al., (2010), as observed in the larger-sized Fe-NPs, where a lot of polyphenols present [30].

SEM images of metal nanoparticles (a) iron (b) copper and (c) silver nanoparticles.

The TEM images of synthesized metal NPs ( showed that a large number of NPs were in spherical form. The images revealed that the most of the NPs were agglomerated, due to hydroxyl arrangement of extract. Fe-, Cu- and Ag-NPs was observed to be around 40-, 30- and 10 nm in size, respectively (, which is comparable with the NPs size as obtained from SEM results.

TEM images of metals nanoparticles (a) iron (b) copper and (c) silver nanoparticles.

The FTIR spectrum ( showed the incidence of various biomolecules such as flavonoids and polyphenols in Syzgium cumini leaves extract and able to produce NPs. The peak seemed at 1065 cm-1, which linked to C-N aliphatic amines stretching vibration or alcohols/ phenols. The sorption peak appears at 3425 cm-1 specifies polyphenolic OH molecule. The peak at about 3000 cm-1 corresponded with aromatic C-H broadening. At 3258 cm-1 peak can be attributed to primary aliphatic amines. The peaks appeared at 1620 and 1065 cm-1 shows the existence of carbonyl groups (C = O) and C-O single bonds, respectively. The aromatic C-H out of plane deformation bands found at 614 cm-1 [31]. displays the FTIR spectrum of Fe-NPs. The peaks detected at 3442 and 2924 cm-1 specifies to O−H broadening vibrations of the C−OH or H2O and CH2 stretching vibrations, respectively. However, the peaks at 1649 and 1091 cm-1 seems to be C = C and C-O particular bonds, respectively. The C−O−C of carboxylic acid exist at the boundaries of Fe-NPs appeared at 790 cm-1 [32]. At 569 cm-1 peak was appeared owing to Fe–O strecting [33]. presented the Cu-NPs FTIR spectrum which indicated the broad peaks at 3425 cm-1 and 1622 cm-1 associated to O–H groups and un-reacted ketone group indicating the existence of flavonones which adsorbed on the Cu-NPs surface, respectively. The peaks at 1471 and 1394 cm-1 indicated the polyphenolic O–H bend, verify the existence of aromatic cluster. The peak at 1111 cm-1 was allotted to C–O–C group in Cu-NPs [34]. The characteristic spectrum of synthesized Ag-NPs presented three bands among 823 and 1384 cm-1 (. A broad band also appears at about 3412 cm-1 may be attributed to O−H and representing the promising participation in the NPs synthesis. However, two less intense and narrower bands appear between 1710 and 2385 cm-1. The peak at 1641 cm-1 is labelled to the C = O broadening vibration of acid derivatives. The peaks at 1381 cm-1 corresponding to C = C of amide groups and aromatic rings. The peak at 1070 cm-1 agrees to aliphatic amines C−N stretching vibration confirms the incidence of polyphenols [30].

FTIR spectra of Syzygium cumini leaves extract and metal nanoparticles (a) Syzygium cumini leaves extract (b) iron (c) copper and (d) silver nanoparticles.

presented the EDX spectrum of Fe-NPs contains peaks of Fe, O and C in addition Na and Cl and C confirming the formation of Fe. The Cl signals must be resulting from FeCl3 precursors used in NPs formation. The EDX spectrum of Cu-NPs is presented in , indicates the C, O, S and Cu peaks. The peak of S obtained from CuSO4 precursors used in the synthesis protocol. The EDX profile of Ag-NPs showed in , a strong Ag signal in conjunction with Cl, C and O peaks, which may have created from the biomolecules present on the Ag-NPs surface.

EDX spectra of metal nanoparticles (a) iron (b) copper and (c) silver nanoparticles.

The optical property of synthesized Fe-, Cu- and Ag-NPs was assessed by the evaluation of UV-Vis spectra. Before the mixing of FeCl3, CuSO4 or AgNO3 solutions, the mustard or yellow colour of each plant extract don’t produces spectral features in UV/Vis range due to unavailability of surface plasmon resonance (SPR). However increasing in the absorbance after addition of precursor solutions indicated the onset of particles formation. UV-Vis spectrum of Fe-NPs revealed characteristic SPR absorption peak at 262 nm ( and agreement with the previous study [35]. UV/Vis spectrum of Cu-NPs presented maximum SPR absorption peak at 572 nm (). The strong SPR absorption band might be due to the SPR band of Cu colloids and the formation of NPs [36]. UV/Vis spectrum of Ag-NPs showed characteristic SPR absorption peak at 426 nm (), as compare with the previous study [23]. The increases in SPR absorption peak with respect to time indicating polydispersity nature of the Ag-NPs [37]. Only one sharp peak appeared in all spectrums, can be attributed to the NPs absorption and validates the NPs formation only. This phenomenon was occurred due to the SPR absorption and confirms that the protein of plant leaves act as a template and stabilizing agent during the synthesis of NPs. SPR is an important optical biosensing tool that occurs due to the combined vibration of electrons of metal NPs in resonance with the light wave.

UV-Visible spectra of metal nanoparticles (a) iron (b) copper and (c) silver nanoparticles.

Antibacterial study of metal nanoparticles

The zone of inhibition (ZOI) produced by NPs against MRSA and VRSA is presented in . However, the values of ZOI and MIC are presented in . The order of antibacterial property was found to be Ag- > Cu- > Fe-NPs, which shows that the NPs size is the important factor in the effect of antibacterial activity. It was clear from SEM and TEM studies that the size of Ag-NPs was smaller as compared with Cu- and Fe-NPs. The antibacterial effect of each NPs was considerably dissimilar with each other at p ≤ 0.05.

Zone of inhibition of produced by iron, copper and silver nanoparticles against Staphylococcus aureus strains (a) MRSA and (b) VRSA.

The greater surface area is provided by the smaller size of Ag-NPs, which delivers better contact to the microorganisms and showed superior antibacterial activity. Furthermore, Ag-NPs with small size comprises easier uptake, greater external area and as a results provides the efficiency in preventing the bacterial growth [38]. The MIC rate of Ag-NPs existed 4 and 8 times greater as related with Cu- and Fe-NPs, respectively. Whereas, the Cu-NPs shown 4 times additional bactericidal effect as Fe-NPs against MRSA / VRSA (.

Various mechanisms have been proposed to understand the antibacterial performance of NPs. Oxidative stress caused by reactive oxygen species (ROS) is considered the key mechanism by which these particles showed antibacterial activity. NPs can generate ROS that is responsible for damaging the proteins and DNA of bacteria’s cells and finally death. This is the main mechanism by which antibacterial drugs and antibiotics work [38]. Furthermore, the NPs connect to the cell membrane of microbes and penetrated in to the cell. NPs interconnect with the phosphorus-comprising compounds such as DNA and sulfur-holding proteins, resulting in the distortion of helical arrangements and interferes essential biochemical reactions. A small molecular load area in the bacteria center is produced by NPs, consequently, protecting the DNA with the metal ions. NPs may hit the respiratory circle, blocked the cell partition finally leading to cell decease [39].

Antifungal study of metal nanoparticles

The antifungal action of NPs was in the increasing order Fe- < Cu- < Ag-NPs and associated with the dose of particles (. Ag-NPs presented a strong antifungal effect in contradiction of Aspergillus species and inhibit the production of aflatoxins (AFs). For instance, no AFs (100% inhibition) was produced at 100 μg mL-1 of Ag-NPs in both strains. However, about 43−49% and 76−80% reduction was achieved using Fe- and Cu-NPs, respectively. The antifungal effect of individually NPs was considerably dissimilar with each other at p ≤ 0.05. As mentioned above, the smaller size NPs have significantly antibacterial/ antifungal activity as compare to the larger particles. The greater antimicrobial effect of Ag-NPs can be correlated with their size and shape which provides greater surface area [38]. Venkatesan et al., (2016) also described the superior antifungal effect of Ag-NPs amongst other NPs and commercially accessible antifungal agents, as agree with this study [40]. The inhibition rate was increased by the increased amount of NPs.

Different reports are available in which the antifungal mechanism of NPs was described. The best mechanism in which NPs is joined with the cell wall and move inside the cell. NPs interrelate with protein and DNA and generate mutation process. As a result, DNA not capable to reproduce and stop the cell partition finally in cell decease [41]. Furthermore, NPs are able to create a spot of low molecular area at the midpoint of fungi cell. The particles then disrupt the respiratory arrangement and eventually cell splitting is entirely stopped subsequent in cell death [42]. The damage and rupture cell membrane can also be seen in the SEM micrograph after the treated with NPs and lastly cell death [28].

In the present study, the adsorption ability of NPs with AFB1 was optimized by altering the amount of NPs, AFB1 concentration, pH, incubation temperature and time.

Effect of nanoparticles amount

provides the outcome of NPs quantity on the adsorption route, which indicates that the rate of AFB1 adsorption improved with increasing amount of NPs. When the amount of adsorbent rises, the sorption places on the surface of adsorbent was also increases, consequently increasing the AFB1 adsorption rate [43]. The adsorption ability of individually NPs was also likened; it was noticed that Fe-NPs having greater adsorption ability with AFB1 than Cu- and Ag-NPs.

Effect of different parameters on the adsorption of aflatoxin B1 using iron, copper and silver nanoparticles (a) Effect of nanoparticles amount (b) Effect of initial concentration of aflatoxin B1 (c) Effect of pH (d) Effect of incubation time and (e) Effect of incubation temperature. Blue Iron nanoparticles red copper nanoparticles green silver nanoparticles.

Effect of aflatoxin B1 concentration

The influence of AFB1 concentration on the sorption method is presented in . The adsorption of AFB1 declined with increasing the primary concentration of AFB1. At the higher AFB1 concentration, the adsorption positions of NPs completely saturated. The adsorption sites were completely engaged and vacancies for adsorption were not available. Consequently, a gradual decline was documented in the sorption amount.

Effect of pH

The adsorption ability of NPs with respect to pH is shown in , that adsorption of AFB1 continuously increases with arise in solution pH. At lower pH, more positive charges (H+) exist on the NPs surface, which provide large electrostatic repulsion strength between AFB1 and NPs. As pH increases, H+ ions can be certainly dissociated and the static repulsion forces drops and the AFB1 adsorption proliferations [44]. Furthermore, the charges and chemical interface among NPs and AFB1 are also contributed to this phenomenon. The pKa (constant of acid dissociation) is accountable for the AFB1charge [45].

Effect of contact time

The AFB1adsorption with NPs was observed time-related and increases with increase in contact time (). The plot of AFB1 adsorption versus time presented that the rate of AFB1 adsorption was fast at the early periods and then extend progressively rises as the system achieved equilibrium. This suggests that the electrostatic attraction is the base of adsorption process [46]. The higher interval might be promoting the contact of ions to the adsorbent active sites; consequently, the maximum adsorption amount was attained. Moreover, the Fe-NPs provide higher adsorption degree than that of Cu- and Ag-NPs, that can be documented to its more interlamellar positioning that is promising to the distribution of AFB1 molecules [27].

Effect of incubation temperature

The adsorption ability of NPs was increased with rising the temperature (). This behavior indicates that the process was attained in endothermic nature. This may be due to increase AFB1 motility and creates or increases the number of active sites for the sorption procedure at greater temperature [47].

Adsorption isotherm

The sorption mechanism was assessed by the calculation of three isotherm models such as Langmuir, Freundlich and Dubinin–Radushkevich (D-R) [48-50]. The fitness of isotherm model was done by the comparison of experimental and theoretical adsorption capacities (Qe) and regression correlation coefficient (R2) values. The isotherm parameters and R2 values are described in . The sorption data were well fixed by the Langmuir model. The experimental and theoretical Qe values are adjacent to each other with high R2 (0.970−0.979) values in contrast to Freundlich and D-R models. The Langmuir model describes the monolayer coverage on the NPs surface with AFB1. The phenomena occur due to the uniform spreading of active positions on the NPs edges.

The maximum Qe values of Fe-, Cu- and Ag-NPs with AFB1 were 105.3-, 88.5- and 81.7 ng mg-1, respectively. Furthermore, the RL values of Langmuir constant were found > 0 but < 1, showing that the process was quite favorable and other contents is not affected the process. In addition, the values of Es were estimated by D-R model were between 0.0407−0.0439 kJ mol-1, indicating inside the physical adsorption energy range equivalent to 8 kJ mol-1 [51].

Kinetic study

In order to examine the rate of sorption process of NPs with AFB1, pseudo-first and pseudo-second order kinetics models were applied. The selection of model existed on the values of experimental and theoretical Qe and R2. The kinetic parameters and R2 values are accessible in . The sorption data were well verifying the condition by the pseudo-second order kinetics model. The experimental and theoretical values of Qe were agreed to each with the linear plot (R2 close to 1). The data suggested that the environment of the process was a chemical monitoring procedure. Furthermore, the electrostatic interface among NPs and AFB1 could also be deliberated in the adsorption manner. The chemisorption is recognized as the rate limiting step and attributed the very fast kinetics to adsorbate being adsorbed onto the adsorbent surface [52]. The initial adsorption rate (h) values were found higher due to more mass transfer action from higher initial concentrations. However, the adsorption rate (k2) values designates that the higher AFB1concentration should have caused in more driving forces and quicker rates for AFB1. As a result, the structural arrangement of active sites or functional groups available on the NPs surface was more promising for the binding of AFB1 [27].

Thermodynamic parameters

The thermodynamic parameters such as Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of the AFB1 adsorption with NPs were determined using Van’t Hoff equation at different temperatures [53]. The obtained parameters are showing in . The ΔG° values of process were calculated negative for each tested temperatures, specified that the process was feasible, spontaneous and endothermic. Additionally, as the temperature rises the ΔG° values improved, proposing that the sorption may be more feasible at greater temperature. A similar pattern for the adsorption of AFB1 using grape pomace, banana peel and magnetic carbon nanocomposites obtained from bagasse have been studied and indicate that the adsorption process occurs spontaneously at a higher temperature [47, 54, 55]. The positive values of ΔH° showing the endothermic nature of adsorption process and improved the randomness of the solid-solution. The amount of the ΔH° values also offers indication of the environment of the adsorption, values < 20 kj mol-1 reflected indicative of physiosorption rather than chemisorptions. The ΔH° values determined for AFB1 on NPs ranged from 5.66 to 7.42 kj mol-1 suggesting physiosorption process and confirms the endothermic process. Whereas, the ΔS° values were also positive and higher for each NPs, which surmise that the adsorption process was favorable in terms of entropy. The ΔS° values also designated the increased randomness at the adsorbent/solution interface during the adsorption process [56].

Conclusion

In this study, Fe-, Cu- and Ag-NPs were effectively produced using Syzgium cumini leaves extract. Ag-NPs display the more significant antimicrobial activity and reduced the production of AFs contamination as compare to Cu- and Fe-NPs. In addition, Fe-NPs showed the higher adsorption capacity with AFB1 in contrast to Cu- and Ag-NPs. In conclusion, metal NPs synthesized by Syzgium cumini leaves may be applied in biomedical uses, remedial in industrial purposes, the alternative of Cefoxitin and Vancomycin antibiotic and preparation of antimicrobial drugs. Metal NPs can also be apply as an active fungicide in the cultivated process, preparation of medicine and pesticide and controlling phytopathogenic microorganisms and AFs contamination. In addition, NPs can be used as an alternative of commercially available AFs adsorbent for the detoxification of AFB1 in human and animal food/ feed.

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18 May 2020

PONE-D-20-11741

Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Attached file

Minor –

1. Line number 47 and 55 – they should add “and”.

2. Line number 61 – “Although about 20 AFs have been discovered”; it looks incomplete.

3. Line number 79 – reference format

4. The editor could ask for better resolution or contrast of images. If possible they could add statistical particle distribution of metal – nanoparticles.

5. In FTIR study – they should provide reference for Fe – O stretching.

6. UV – Visible study should be discussed in details and should provide appropriate reference for absorption peak as it is a signature peak for metal nanoparticles.

7. Please provide supporting reference for section 3.4.4 (Effect of contact time) and 3.4.5 (Effect of incubation temperature).

8. Reference for “he calculation of three isotherm models such as Langmuir, Freundlich and Dubinin–Radushkevich (D-R)”.

9. More analysis and supporting reference needed for section 3.7 kinetic study.

10. Kinetic study and thermodynamic parameters needs more explanation.

11. For supporting document the editor can ask for procedure to estimate parameters in Table – 4 , Table – 5 and Table – 6.

12. Figure captions need to be modified

a. For example Figure I - SEM images of metal nanoparticles (a) iron (b) copper (c) and silver nanoparticles

- (c) should be after ‘and’ (do in all captions)

13. Please provide better quality of TEM images (with proper brightness and contrast ration)

-

Reviewer #2: The authors presented

Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents.

The work is okay with minor correction as stated below. The work can be accepted after the minor corrections

1. Section 2.1, line 1

About 10 g of chopped leaves were accurately weighed and boiled in the presence of 100 mL of

Section 2.3.1, line 2

….a concentration between of 1‒512 µg mL-1 was prepared in 10 mL of 0.8% (w v-1) nutrient broth 133

2. Section 2.5, line 1&2

The adsorption of aflatoxin B1 (AFB1) with NPs with studied as a were studied following the previously defined method with some modifications (15).

3. After equation one

Where, where

4. Section 3.1Line 9&10

The volume of the leaf extracts directly influences on the size of the NPs

5. Section 3.1 Paragraph 7 line 3-5

It was occur caused by surface plasmon resonance (SPR) and verifying that the available leaves protein plays a significant role as a template as well as preservative during synthesis route. (This sentence is ambiguous kindly re-cast)

6. Conclusion, line 2&3

Ag-NPs the promising potential to reveal the excellent antibacterial and antifungal effect

and inhibit the AFs production. (This sentence is ambiguous kindly re-cast)

7. Conclusion, line 3

In addition, Fe-NPs showed the higher highest adsorption capacity with

8. Acknowledgement

The authors thankfully are thankful to the Feed and Feed Safety Laboratory, FMRRC, PCSIR Laboratories

9. The introduction lack current citation. The suggested articles below should be added to the introduction

1. Materials Chemistry and Physics 237:121859 https://doi.org/10.1016/j.matchemphys.2019.121859

2. Polym Res (2019) 26: 225. https://doi.org/10.1007/s10965-019-1897-z

3. Journal of Photochemistry and Photobiology B: Biology Volume 199, October 2019, 111601 https://doi.org/10.1016/j.jphotobiol.2019.111601

4. Appl Nanosci (2019). https://doi.org/10.1007/s13204-019-01099-x

5. Surfaces and Interfaces Volume 17, December 2019, 100359

https://doi.org/10.1016/j.surfin.2019.100359

6. European Polymer Journal 2020, 122:109371. https://doi.org/10.1016/j.eurpolymj.2019.109371

7. Applied Physics A 2020, 126:72. https://doi.org/10.1007/s00339-019-3249-6

8. Surfaces and Interfaces, 18, 100419. https://doi.org/10.1016/j.surfin.2019.100419

9. General English and grammar correction should be done, few has been suggested above

**********

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Reviewer #1: Yes: Amol Nande

Reviewer #2: No

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Submitted filename: comments.docx

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25 May 2020

THE EDITORIAL OFFICE Dated 23-05-2020

PLOS ONE

Corrections in the manuscript entitled “Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents (Ms. Ref. No.: PONE-D-20-11741)”

Dear Editor,

First of all, authors are thankful to you for your useful comments and suggestions about our manuscript. The authors have modified the manuscript accordingly and the detailed corrections are listed below point by point. All the changes in manuscript are highlighted using Track Changes.

Response to Editor comments:

Manuscript has been formatted according to the attached guidelines.

Response to Reviewer comments:

Reviewer # 1:

Minor

1. Line number 47 and 55 – they should add “and”.

Response to Reviewer comment no. 1: The word has been added “and” in both lines as mentioned below.

Abstract

The present findings suggests that the green synthesis of metal NPs is a simple, sustainable, non-toxic, economical and energy-effective as compared to the others conventional approaches.

1. Introduction

Apart from recent advancement, pathogenic mould contamination associated in food and feed products are still major threat world-wide.

2. Line number 61 – “Although about 20 AFs have been discovered”; it looks incomplete.

Response to Reviewer comment no. 2: The sentence has been rephrased as mentioned below.

1. Introduction

To date, at least 20 different types of AFs such as aflatoxin B1, B2, G1 and G2 have been identified.

3. Line number 79 – reference format

Response to Reviewer comment no. 3: Correction has been made as per reviewer’s recommendation in text of manuscript as mentioned below.

1. Introduction

Currently, nanotechnology is emerging field with its applications in science and technology. Nanoparticles (NPs) are using in some cosmetics, sunscreens, toothpaste, pharmaceutical and even food products (9).

4. The editor could ask for better resolution or contrast of images. If possible they could add statistical particle distribution of metal – nanoparticles.

Response to Reviewer comment no. 4: The resolution or contrast of each image has been improved. However, in the present study, the SEM and TEM analysis was used to evaluate the particle size distribution. TEM is a direct counting method, which measures a size value for each particle selected for the analysis, thus providing a number-based particle size distribution. The statistical particles distribution in terms of range and mean has been incorporated in Table 1.

5. In FTIR study – they should provide reference for Fe – O stretching.

Response to Reviewer comment no. 5: The missing reference has been added as mentioned below.

5. References

Sun X, Zheng C, Zhang F, Yang Y, Wu G, Yu A, Guan N. Size-controlled synthesis of magnetite (Fe3O4) nanoparticles coated with glucose and gluconic acid from a single Fe (III) precursor by a sucrose bifunctional hydrothermal method. The Journal of Physical Chemistry C. 2009 Sep 10;113(36):16002-8.

6. UV – Visible study should be discussed in details and should provide appropriate reference for absorption peak as it is a signature peak for metal nanoparticles.

Response to Reviewer comment no. 6: The UV-Visible study has been discussed in detail with appropriate references as mentioned below.

The optical property of synthesized Fe-, Cu- and Ag-NPs was assessed by the evaluation of UV-Vis spectra. Before the mixing of FeCl3, CuSO4 or AgNO3 solutions, the mustard or yellow colour of each plant extract don’t produces spectral features in UV/Vis range due to unavailability of surface plasmon resonance (SPR). However, on the addition of precursor solutions an increase in the absorbance arisen that maybe indicated the onset of particle formation. UV-Vis spectrum of Fe-NPs revealed characteristic SPR absorption peak at 262 nm (Fig. 5a) and agreement with the previous study (21). UV/Vis spectrum of Cu-NPs presented maximum SPR absorption peak at 572 nm (Fig. 5b). The strong SPR absorption band might be due to the SPR band of Cu colloids and the formation of NPs (22). UV/Vis spectrum of Ag-NPs showed characteristic SPR absorption peak at 426 nm (Fig. 5c), as compare with the previous study (23). The increases in SPR absorption peak with respect to time indicating polydispersity nature of the Ag-NPs (24). Only one sharp peak appeared in all spectrums, can be attributed to the NPs absorption and validates the NPs formation only. This phenomena was occurred due to the SPR absorption and confirms that the protein of plant leaves act as a template and stabilizing agent during the synthesis of NPs. SPR is an important optical biosensing tool that occurs due to the combined vibration of electrons of metal NPs in resonance with the light wave.

7. Please provide supporting reference for section 3.4.4 (Effect of contact time) and 3.4.5 (Effect of incubation temperature).

Response to Reviewer comment no. 7: References have been provided for both sections as mentioned below.

5. References

Shar ZH, Fletcher MT, Sumbal GA, Sherazi STH, Giles C, Bhanger MI, et al. Banana peel: an effective biosorbent for aflatoxins Part A Chemistry, analysis, control, exposure & risk assessment. 2016.

Asghar MA, Zahir E, Shahid SM, Khan MN, Asghar MA, Iqbal J, et al. Iron, copper and silver nanoparticles: Green synthesis using green and black tea leaves extracts and evaluation of antibacterial, antifungal and aflatoxin B1 adsorption activity. LWT. 2018;90:98-107.

Zahoor M, Khan FA. Adsorption of aflatoxin B1 on magnetic carbon nanocomposites prepared from bagasse. Arabian Journal of Chemistry. 2018;11(5):729-38.

8. Reference for “the calculation of three isotherm models such as Langmuir, Freundlich and Dubinin–Radushkevich (D-R)”.

Response to Reviewer comment no. 8: References for the calculation of three isotherm models such as Langmuir, Freundlich and Dubinin–Radushkevich (D-R) have been provided as mentioned below.

5. References

Langmuir I. The constitution and fundamental properties of solids and liquids. Part I. Solids. Journal of the American chemical society. 1916;38(11):2221-95.

Freundlich H. Over the adsorption in solution. J Phys Chem. 1906;57(385471):1100-7.

Dubinin M. The potential theory of adsorption of gases and vapors for adsorbents with energetically nonuniform surfaces. Chemical Reviews. 1960;60(2):235-41.

9. More analysis and supporting reference needed for section 3.7 kinetic study.

Response to Reviewer comment no. 9: The detail for the kinetic study has been provided with appropriate references.

3.7. Kinetic study

Furthermore, the electrostatic interface among NPs and AFB1 could also be deliberated in the adsorption manner. The chemisorption is recognized as the rate limiting step and attributed the very fast kinetics to adsorbate being adsorbed onto the adsorbent surface (38). The initial adsorption rate (h) values were found higher due to more mass transfer action from higher initial concentrations. However, the adsorption rate (k2) values designates that the higher AFB1concentration should have caused in more driving forces and quicker rates for AFB1. As a result, the structural arrangement of active sites or functional groups available on the NPs surface was more promising for the binding of AFB1 (13).

10. Kinetic study and thermodynamic parameters needs more explanation.

Response to Reviewer comment no. 10: The more explanation for the kinetic study and thermodynamic parameters has been provided as per reviewer recommendation as mentioned below.

3.7. Kinetic study

In order to examine the rate of sorption process of NPs with AFB1, pseudo-first and pseudo-second order kinetics models were applied. The selection of model existed on the values of experimental and theoretical Qe and R2. The kinetic parameters and R2 values are accessible in Table 5. The sorption data were well verifying the condition by the pseudo-second order kinetics model. The experimental and theoretical values of Qe were agreed to each with the linear plot (R2 close to 1). The data suggested that the environment of the process was a chemical monitoring procedure. Furthermore, the electrostatic interface among NPs and AFB1 could also be deliberated in the adsorption manner. The chemisorption is recognized as the rate limiting step and attributed the very fast kinetics to adsorbate being adsorbed onto the adsorbent surface (38). The initial adsorption rate (h) values were found higher due to more mass transfer action from higher initial concentrations. However, the adsorption rate (k2) values designates that the higher AFB1concentration should have caused in more driving forces and quicker rates for AFB1. As a result, the structural arrangement of active sites or functional groups available on the NPs surface was more promising for the binding of AFB1 (13).

3.8. Thermodynamic parameters

The thermodynamic parameters such as Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of the AFB1 adsorption with NPs were determined using Van’t Hoff equation at different temperatures (39). The obtained parameters are showing in Table 6. The ΔG° values of process were calculated negative for each tested temperatures, specified that the process was feasible, spontaneous and endothermic. Additionally, as the temperature rises the ΔG° values improved, proposing that the sorption may be more feasible at greater temperature. A similar pattern for the adsorption of AFB1 using grape pomace, banana peel and magnetic carbon nanocomposites obtained from bagasse have been studied and indicates that the adsorption process occurs spontaneously at a higher temperature (33, 40, 41). The positive values of ΔH° showing the endothermic nature of adsorption process and improved the randomness of the solid-solution. The amount of the ΔH° values also offers indication of the environment of the adsorption, values < 20 kj mol-1 reflected indicative of physiosorption rather than chemisorptions. The ΔH° values determined for AFB1 on NPs ranged from 5.66 to 7.42 kj mol-1 suggesting physiosorption process and confirms the endothermic process. Whereas, the ΔS° values were also positive and higher for each NPs, which surmise that the adsorption process was favourable in terms of entropy. The ΔS° values also designated the increased randomness at the adsorbent/solution interface during the adsorption process (42).

11. For supporting document the editor can ask for procedure to estimate parameters in Table – 4 , Table – 5 and Table – 6.

Response to Reviewer comment no. 11: The parameters in Table 4, 5 and 6 shows the adsorption, isotherm, kinetic and thermodynamic study, respectively. All parameters of each study were calculated according to the related equations. All the calculations are based on the previous reported procedures. The references have been provided for procedure to estimate parameters in each table. Furthermore, the authors can provide the calculations for each parameter on the request of reader.

12. Figure captions need to be modified

a. For example Figure I - SEM images of metal nanoparticles (a) iron (b) copper (c) and silver nanoparticles

- (c) should be after ‘and’ (do in all captions)

Response to Reviewer comment no. 12: All figures captions have been modified as per reviewer’s recommendation.

13. Please provide better quality of TEM images (with proper brightness and contrast ration)

Response to Reviewer comment no. 13: The quality of TEM images have been improved as per reviewer’s recommendation.

Reviewer # 2:

The authors presented Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents.

The work is okay with minor correction as stated below. The work can be accepted after the minor corrections.

The authors grateful thanks to the reviewer’s for the above mentioned comments. However, minor comments as per reviewer recommendation das been incorporated in the manuscript.

1. Section 2.1, line 1

About 10 g of chopped leaves were accurately weighed and boiled in the presence of 100 mL of

Section 2.3.1, line 2

….a concentration between of 1‒512 µg mL-1 was prepared in 10 mL of 0.8% (w v-1) nutrient broth 133

Response to Reviewer comment no. 1: Corrections have been made in both sentences as mentioned below.

2. Experimental

2.1. Synthesis of metal nanoparticles

Ten grams chopped leaves were accurately weighed and boiled in the presence of 100 mL DI-H2O for 20 min at 80oC, cooled at room environment and filtered using Whatman filter paper no. 1.

2.3.1. Minimum inhibitory concentration (MIC)

Dilution broth method was utilized to evaluate the MIC for each metal NPs. In brief, each NPs in a concentration between 1‒512 µg mL-1 was prepared in 10 mL 0.8% (w v-1) nutri¬ent broth medium separately and sterilized for 15 min at 121oC using Rexall autoclave (Kaohsiung, Taiwan).

2. Section 2.5, line 1&2

The adsorption of aflatoxin B1 (AFB1) with NPs with studied as a were studied following the previously defined method with some modifications (15).

Response to Reviewer comment no. 2: Corrections have been made in the sentence as mentioned below.

2.5. Adsorption of aflatoxin B1

The adsorption of aflatoxin B1 (AFB1) with each NPs was studied according to the previously defined method with some modifications (15).

3. After equation one

Where, where

Response to Reviewer comment no. 3: Correction has been done in both equations 1 and 2.

4. Section 3.1 Line 9&10

The volume of the leaf extracts directly influences on the size of the NPs

Response to Reviewer comment no. 4: Correction has been made as mentioned below.

3.1. Formation and characterization of metal nanoparticles

Consequently, the volume of leaves extract directly effects on the morphology, size and production of NPs.

5. Section 3.1 Paragraph 7 line 3-5

It was occur caused by surface plasmon resonance (SPR) and verifying that the available leaves protein plays a significant role as a template as well as preservative during synthesis route. (This sentence is ambiguous kindly re-cast)

Response to Reviewer comment no. 5: The sentence has been revised as mentioned below.

3.1. Formation and characterization of metal nanoparticles

This phenomena was occurred due to the SPR absorption and confirms that the protein of plant leaves act as a template and stabilizing agent during the synthesis of NPs.

6. Conclusion, line 2&3

Ag-NPs the promising potential to reveal the excellent antibacterial and antifungal effect

and inhibit the AFs production. (This sentence is ambiguous kindly re-cast)

Response to Reviewer comment no. 6: The sentence has been revised in conclusion of manuscript as mentioned below.

4. Conclusion

Ag-NPs displays the more significant antimicrobial activity and reduced the production of AFs contamination as compare to Cu- and Fe-NPs.

7. Conclusion, line 3

In addition, Fe-NPs showed the higher highest adsorption capacity with

Response to Reviewer comment no. 7: Correction has been made as mentioned below.

4. Conclusion

In addition, Fe-NPs showed the higher adsorption capacity with AFB1 in contrast to Cu- and Ag-NPs.

8. Acknowledgement

The authors thankfully are thankful to the Feed and Feed Safety Laboratory, FMRRC, PCSIR Laboratories

Response to Reviewer comment no. 8: Correction has been made as mentioned below.

Acknowledgement

The authors gratefully thanks to the Feed and Feed Safety Laboratory, FMRRC, PCSIR Laboratories Complex, Karachi-Pakistan for the supporting of current research work.

9. The introduction lack current citation. The suggested articles below should be added to the introduction

1. Materials Chemistry and Physics 237:121859 https://doi.org/10.1016/j.matchemphys.2019.121859

2. Polym Res (2019) 26: 225. https://doi.org/10.1007/s10965-019-1897-z

3. Journal of Photochemistry and Photobiology B: Biology Volume 199, October 2019, 111601 https://doi.org/10.1016/j.jphotobiol.2019.111601

4. Appl Nanosci (2019). https://doi.org/10.1007/s13204-019-01099-x

5. Surfaces and Interfaces Volume 17, December 2019, 100359

https://doi.org/10.1016/j.surfin.2019.100359

6. European Polymer Journal 2020, 122:109371. https://doi.org/10.1016/j.eurpolymj.2019.109371

7. Applied Physics A 2020, 126:72. https://doi.org/10.1007/s00339-019-3249-6

8. Surfaces and Interfaces, 18, 100419. https://doi.org/10.1016/j.surfin.2019.100419

9. General English and grammar correction should be done, few has been suggested above

Response to Reviewer comment no. 9: All the above mentioned articles have been cited as per reviewer’s recommendation. Furthermore, the general English and grammar corrections have been also done.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

8 Jun 2020

Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents

PONE-D-20-11741R1

Dear Dr. Asghar,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Mohammad A. Al-Ghouti

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please adjust the minor edits raised by the reviewer 2.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It sound OK to me.

All comments are addressed properly. They added the references and performed the required changes also.

The TEM diagram changed to good pixel.

Thank you for consideration

Reviewer #2: The authors should see attached file for my comments. Little corrections are needed. try and effect them corretly

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Amol Nande

Reviewer #2: No

9 Jun 2020

PONE-D-20-11741R1

Facile, one-pot biosynthesis and characterization of iron, copper and silver nanoparticles using Syzygium cumini leaf extract: As an effective antimicrobial and aflatoxin B1 adsorption agents

Dear Dr. Asghar:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Mohammad A. Al-Ghouti

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PLOS ONE

Table 1

Morphology and particles size distribution of metal NPs produced by Syzygium cumini (Jamun) leaves extract.

Nanoparticles IDs	Change in color		change in pH		Final yield (mg)	Morphology	Size (nm) Range (Mean)
	Plant extract	After reduction	Plant extract	After reduction			SEM	TEM
Fe-NPs	Pale Yellow	Brownish Black	5.86	3.25	52.2	Spherical	40−52 (46)	30−42 (40)
Cu-NPs		Brown		3.36	36.9	Spherical	28−35 (31)	20−35 (30)
Ag-NPs		Brown		4.21	18.5	Spherical	11−19 (15)	7−14 (10)

NPs = nanoparticles, SEM = scanning electron microscope, TEM = Transmission Electron Microscopy

Table 2

The values of inhibition zone and minimum inhibitory concentration (MIC) of Fe-, Cu- and Ag-NPs against methicillin-resistance and vancomycin-resistance Staphylococcus Aureus.

NPs IDs	MRSA		VRSA
	Zone of inhibition (mm ± SEM)	MIC ± SD (μg mL-1)	Zone of inhibition (mm ± SEM)	MIC ± SD (μg mL-1)
Fe-NPs	11 ± 0.45	128 ± 1.8	13 ± 0.28	128 ± 2.1
Cu-NPs	14 ± 0.33	32 ± 1.1	16 ± 0.31	32 ± 0.9
Ag-NPs	18 ± 0.32	8 ± 0.40	20 ± 0.40	8 ± 0.27

NPS = nanoparticles, SEM = standard error mean, SD = standard deviation.

The antibacterial activity of each NPs was considerably dissimilar from each other at p ≤ 0.05.

Table 3

Influence of various amounts of Fe-, Cu- and Ag-NPs on aflatoxins contamination produced by Aspergillus flavus and A. parasiticus.

Amount of nanoparticles (μg mL-1)	Reduction (%) of aflatoxins at different amount of Fe-, Cu- and Ag-NPs
	Aspergillus flavus			Aspergillus parasiticus
	Fe-NPs	Cu-NPs	Ag-NPs	Fe-NPs	Cu-NPs	Ag-NPs
10	3.4 ± 0.4	9.9 ± 1.2	35.8 ± 2.2	4.1 ± 0.4	15.2 ± 1.9	38.6 ± 2.7
25	9.1 ± 1.9	25.8 ± 1.8	48.1 ± 3.2	11.5 ± 1.4	32.5 ± 1.6	53.6 ± 3.0
50	25.6 ± 1.3	47.8 ± 2.2	66.4 ± 2.8	30.3 ± 1.5	55.8 ± 3.2	69.7 ± 3.1
100	43.9 ± 1.4	75.7 ± 3.2	100 ± 0.0	49.0 ± 1.8	80.0 ± 2.1	100 ± 0.0

The antifungal activity of each NPs was considerably dissimilar from each other at p ≤ 0.05.

Table 4

The correlation and equilibrium isotherm constants for the sorption of aflatoxin B1 with Fe-, Cu- and Ag-NPs.

Nanoparticles IDs	Qe (exp)	Langmuir				Freundlich			D-R
		Qmax	b	RL	R2	KF	n	R2	Qmax	Es	R2
Fe-NPs	102.6	105.3	0.158	0.863	0.970	0.351	3.300	0.944	38.0	0.0263	0.923
CU-NPs	87.4	88.5	0.138	0.879	0.979	0.375	4.329	0.968	43.7	0.0229	0.907
Ag-NPs	80.0	81.7	0.115	0.897	0.979	0.324	4.054	0.971	39.1	0.0256	0.942

Qe = equilibrium concentration, Qmax = capacity of monolayer, b = Langmuir constant, RL = separation constant factor, R2 = determination coefficients, KF = sorption capacity, n = sorption intensity, Es = mean free energy of adsorption, DR = Dubinin–Radushkevich adsorption isotherm.

The Qmax values of each NPs was considerably dissimilar each other at p ≤ 0.05.

Table 5

Kinetic parameters for aflatoxins B1 adsorption on Fe-, Cu- and Ag-NPs.

Nanoparticles IDs	Qe (exp)	First Order			Second Order
		R2	K1 (min-1)	Qe (Calc)	R2	K2 (min-1)	Qe (Calc)	h
GT-Fe-NPs	102.6	0.8390	0.1059	1.79	0.9920	0.0004	106.42	3.97
BT-Fe-NPs	87.4	0.7780	0.1105	1.82	0.9790	0.0005	90.91	3.86
GT-Cu-NPs	80.0	0.8730	0.0484	2.31	0.9860	0.0004	84.75	2.75

Qe = equilibrium concentration, R2 = determination coefficients, K1 = Lagergren (pseudo-first order) adsorption rate constant, K2 = pseudo-second order adsorption rate constant, h = initial sorption rate.

The Qe values of each NPs was considerably dissimilar each other at p ≤ 0.05.

Table 6

Thermodynamic considerations of the adsorption aflatoxins B1 with Fe-, Cu- and Ag-NPs.

Nanoparticles IDs	ΔH° (kJ mol-1)	ΔS° (J mol.K-1)	ΔG° (kJ mol-1) at different temperature (K)
			298	303	308	313
Fe-NPs	7.42	34.17	-2.23	-2.86	-3.04	-3.52
Cu-NPs	6.70	29.67	-1.70	-2.14	-2.48	-2.75
Ag-NPs	5.66	23.77	-1.07	-1.42	-1.69	-1.91

K = Kelvin; ΔG° = Gibbs free energy; ΔH° = enthalpy; ΔS° = entropy

The ΔG°, ΔH° and ΔS° values of each NPs were considerably dissimilar at p ≤ 0.05.