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Effect of size and shape controlled biogenic synthesis of gold nanoparticles and their mode of interactions against food borne bacterial pathogens
⁎Corresponding author. Tel.: +82 063 270 2566, mobile: +82 010 315 94466; fax: +82 063 270 2572. siyun@jbnu.ac.kr (Soon-Il Yun)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In this study, size and shape controlled biogenic synthesis of gold nanoparticles and their antibacterial activity against food borne bacterial pathogens were investigated. Synthesis of gold nanoparticles was carried out using two medicinally important plants Cucurbita pepo and Malva crispa and the size and shape of the nanoparticles were controlled by altering various parameters in the reaction medium. Results obtained from UV–Vis, FE-SEM, EDS and HR-TEM analyses supported the nanoparticles formation. FT-IR analysis confirmed the presence of biomolecules in the plant leaves extracts responsible for reducing and capping agents. Interestingly, the plant extract synthesized gold nanoparticles showed effective inhibition zone against Gram-positive and Gram-negative pathogens. The minimum inhibitory concentration (MIC) of synthesized gold nanoparticles at 400 μg/ml concentration showed effective inhibitory activity against Escherichia coli and Listeria monocytogenes. Conductivity of the medium continuously increased during the nanoparticles treatment with food borne bacterial pathogens resulting in indirect indication of the disruption of bacterial cell membranes. In addition, mode of interactions of gold nanoparticles against food borne bacterial pathogens was demonstrated using Bio-TEM analysis which is clear evident for the disruption of bacterial cell membranes.
Keywords
Cucurbita pepo
Malva crispa
Gold nanoparticles
Antibacterial activity
Conductivity
Bio-TEM
1 Introduction
Today, synthesis of metallic nanoparticles is carried out mainly on biological systems such as bacteria (Rajashree and Suman, 2012), fungi (Mishraa et al., 2012), yeast (Sathish Kumar et al., 2011), algae (Rajeshkumar et al., 2013) and plant extracts (Krishnaraj et al., 2010). So the synthesized nanoparticles have versatile applications in medicine (Rana et al., 2012) hence it is necessary to avoid the toxic chemicals for nanoparticles preparation. In particular, bacteria, fungi, yeast and algae took more time for nanoparticles synthesis and avoiding the cross contamination is also one of the most important tasks. Keeping this in mind, synthesis of nanoparticles using plant extracts is always preferable, in which no need to focus more on cross contaminations as like microbes and the rate of nanoparticles formation is also very fast. There are a number of solvent systems used for extracting the plant components for nanoparticles synthesis like methanol (Rajan et al., 2012), ethanol (Rang Im et al., 2012), diethyl ether (Et2O), chloroform (CHCl3), ethyl acetate (EtOAc) (Singh and Kundu, 2013) and water (Poinern et al., 2013; Khalil et al., 2012; Majumdar et al., 2013). As the research is focused on eco-friendly synthesis of nanoparticles, it is always preferable to use aqueous solvent (water) for extraction procedures, thereby avoiding the use of toxic chemicals. Moreover, water is a powerful high polar solvent and it is very simple to extract the high polar phytochemical compounds from plant leaves into medium. Very recently, one research group have reported that the water soluble organics present in the plant leaves extracts were found to be responsible for nanoparticles synthesis (Arunachalam and Annamalai, 2013). There are many research groups successfully synthesized gold nanoparticles from aqueous plant leaves extracts at different time intervals and studied the stability of nanoparticles under optimized conditions (Yasmin et al., 2014; Ghosh et al., 2011). In other view, a growing number of food borne bacterial infections is giving clear instruction to researchers for finding a new way to control. Hence, this research work is mainly focused on gold nanoparticles synthesis from aqueous leaves extracts of Cucurbita pepo (Pumpkin-Cucurbitaceae) and Malva crispa (Curled Mallow-Malvaceae). There are several parameters optimized for achieving the uniform size and shape of the nanoparticles and also proved their potential as antibacterial agents against food borne pathogens.
2 Materials and methods
The fresh and healthy leaves of C. pepo were collected from the campus of Chonbuk National University and M. crispa was purchased from local market, Jeonju, South Korea. HAuCl4 was purchased from Sigma Aldrich (254169), Whatman filter paper (1820–110) was purchased from GE Health care UK Ltd, Muller Hinton Agar (MHA) was purchased from Difco (Lot.8339486), MTT (Methyl thiozolyl diphenyl-tetrazolium bromide) was purchased from Sigma Aldrich (M-2128), food borne bacterial pathogens of Bacillus cereus (ATCC 14579), Staphylococcus aureus (KCTC 1927), Listeria monocytogenes (KCCM 40307), Escherichia coli (KCCM 11234), Salmonella typhi and Salmonella enterica (KCCM 11806) were obtained from Korean Collection for Type Cultures (KCTC).
2.1 Synthesis of gold nanoparticles
Gold nanoparticles used in this study were synthesized by standard published procedures with little modifications. Three different quantities (5, 10, 15 g) of fresh and healthy leaves of C. pepo and M. crispa were surface cleaned with tap water, followed by D.H2O at several times to remove any external particles adhered on the surface of leaves. Then, the leaves were separately boiled with 100 ml of Dist.H2O in microwave oven (3–6 min) and the resulting extracts were filtered through Whatman filter papers. Six, 10 and 15 ml of filtered extracts were mixed with 44, 40 and 35 ml of 1, 2, 3 mM HAuCl4 solutions with the final reaction volume of 50 ml in each flasks and this setup was kept in room temperature under static conditions. A control setup was also maintained throughout the experiment with leaves extracts alone.
2.2 UV–Vis spectroscopy study
Synthesized gold nanoparticles were confirmed by sampling the reaction mixture at regular time intervals and the absorption maxima was scanned by UV–Vis spectra, at the wavelength of 200–800 nm in Shimadzu UV-1800 spectrophotometer.
2.3 Study for the influence of pH, time, concentration of HAuCl4 solution, concentration ratio of leaves extract and HAuCl4 solution and stability of gold nanoparticles synthesis
Synthesized gold nanoparticles were optimized using different pH (2–12), time (10–210 min.), concentration of HAuCl4 (1–3 mM), concentration ratio of leaves extract (6 ml extract + 44 ml D.H2O; 12 ml extract + 38 ml D.H2O; 18 ml extract + 32 ml D.H2O). Then the absorbance maxima of the reaction solution were measured using UV–Vis spectrophotometer at 200–800 nm. Also, the stability of synthesized nanoparticles was further confirmed by treating with acid, alkali, UV irradiation and monitored the performance.
2.4 Characterization of gold nanoparticles
Further, the reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min and collected the supernatant. An aliquot of this filtrate containing gold nanoparticles was used for HR-TEM and EDS analyses. For electron microscopic studies, 25 μl of reaction solution was coated on TEM grid and the images of nanoparticles were studied using HR-TEM (JEM-2010) and osmium coated dried samples were observed in FE-SEM (S-4800, Hitachi, Japan). Presence of elements in the sample was confirmed by EDS analysis. Then the reaction samples were freeze dried using freeze drier with concentrator from Ilshin biobase (Model No: MCF D8512) for getting the solutions in a powder form. For FT-IR analysis dried powder was pellet out in KBr pelletizers using Perkin Elmer model spectrum GX operated at a wavelength of 350–4500 cm−1 at a resolution of 0.4 cm−1 with the wavelength accuracy of 0.1 cm at 1600 cm−1.
2.5 Preliminary investigation for the presence of phytochemicals
Aqueous leaves extracts of C. pepo and M. crispa were investigated for the presence of phytochemicals viz. tannin, saponin, flavonoids, phenol, glycosides, reducing sugar and alkaloids by following standard biochemical methods (Kardong et al., 2013).
2.6 Antibacterial activity of gold nanoparticles against food borne pathogens
Gold nanoparticles synthesized from aqueous leaves extract of C. pepo and M. crispa were tested for their potent antibacterial activity against few food borne Gram-positive and Gram-negative pathogens.
2.7 Efficacy of gold nanoparticles against food borne bacterial pathogens
2.7.1 Antibacterial activity
In this study, Gram-positive bacteria such as B. cereus, S. aureus, L. monocytogenes and Gram negative bacteria such as E. coli, S. typhi, S. enterica were used as test organisms and initially cultured in nutrient broth. Well diffusion assay was performed to determine the antibacterial activity of gold nanoparticles synthesized from plant extracts. First 3 wells were acted as controls in which 1st well was added with D.H2O and 2nd well was loaded with plant extract while 3rd well was treated with 1 mM HAuCl4 solution. The 4th well was added with 40 μl of 800 μg/ml concentration of gold nanoparticles and the plates were incubated at 37 ± 2° C for 12–24 h and then the zone of inhibition was measured in terms of centimeters.
2.8 Minimal inhibitory concentration of gold nanoparticles
Minimal inhibitory concentration (MIC) of gold nanoparticles was determined by MTT assay using 96-well microtitre plates (SPL life sciences, Korea). The mean of live cells of both Gram-positive and Gram-negative bacteria was recorded using ELISA reader (Epoch, Biotech instruments). The MIC was determined based on different concentrations, where there was no increase in the OD595 and was zero.
2.9 Conductivity study
Interactions of biologically synthesized gold nanoparticles against Gram-positive and Gram-negative bacteria were studied using conductivity meter. For this study, viable bacterial cultures grown in nutrient broth were treated with gold nanoparticles. Ten milliliters of log phase cultures centrifuged at 6000 rpm for 10 min and the pellet was suspended in sterile D.H2O. Five milliliters of this suspension was exposed to 1 ml of 800 μg/ml concentration of gold nanoparticles and the conductivity was recorded using conductivity meter (Horiba ES-14) after the incubation time (1, 8, 16 and 24 h).
2.9.1 Mode of interactions of gold nanoparticles into bacterial cell membranes
Mode of interactions of gold nanoparticles on Gram-positive and Gram-negative bacterial cell membranes was studied using Bio-TEM (H-7650, Hitachi, Japan). For this study, 10 ml of log phase cultures was centrifuged at 6000 rpm for 10 min and the pellet was suspended in sterile D.H2O. Five milliliters of this suspension was exposed to 1 ml of 800 μg/ml concentration of gold nanoparticles for 8 h, stained with negative staining and observed under Bio-TEM.
3 Results and Discussion
There are number of methods employed for synthesis of gold nanoparticles in which chemical (Li et al., 2012) and biological (Correa-Lianten et al., 2013) methods are most popular. Synthesis of gold nanoparticles using chemical method is simple process and controlling the size and shape of the nanoparticles is also possible. But in the case of biological systems mainly in bacteria (Sharma et al., 2012), fungi (Das et al., 2012) and plants (Adavallan and Krishna kumar, 2014), controlling the size and shape of nanoparticles is a very tedious process. Previously, Yasmin et al. (2014) have synthesized gold nanoparticles from herbal plant extract Hibiscus rosa-sinensis, optimized various parameters in the reaction solution and achieved the in vitro stability of nanoparticles. Pumpkin, one of the most important vegetables coming under the family Cucurbitaceae, contains several nutritional facts like essential oils, vitamins, calcium, iron (Iwu, 1983) and phytochemicals such as saponins, alkaloids, tannins, phenolic (Oyewole and Abalaka, 2012) and is being used in many traditional medicines (Gbile, 1986). Similarly, curled mallow coming under the family Malvaceae has been used in folk medicine of Brazil and other countries for various treatments (Esteves et al., 2009). In South Korea, these plant leaves extracts were commonly used as a soup and as ingredients in baby food.
The present study demonstrated the simple synthesis, characterization and optimization of gold nanoparticles from C. pepo and M. crispa. Based on the various optimization processes, almost uniform size and shape of the nanoparticles was achieved. Further, the bacterial cell membrane damage mechanism was indirectly demonstrated using conductivity meter. For supporting this study, mode of interactions of gold nanoparticles on Gram-positive and Gram-negative bacterial cell membranes was demonstrated using Bio-TEM (Fig. 1).
Schematic flow diagram of biogenic synthesis of gold nanoparticles from plant leaves extracts and its mode of interactions with bacteria.
3.1 Synthesis of gold nanoparticles
Ten grams of extract boiled in 100 ml of D.H2O for 5 min in microwave oven showed a positive response for gold nanoparticles synthesis by turning the pale yellow color into pinkish violet. The characteristic absorption band observed at 540 nm further confirmed the synthesis. These results were in good agreement with the previously published research work done by Arunachalam and Annamalai (2013), who have obtained a similar kind of absorbance band at 540 nm. Further, different concentrations of leaves extract were also optimized to get uniform sized nanoparticles; 6 ml of extract in 44 ml D.H2O was found to be suitable for nanoparticles synthesis. Curled mallow took 120 min for the synthesis while it was 150 min for pumpkin. Intensity of pinkish violet color was continuously increased in the reaction solution during the incubation period and this may be due to the reduction of HAuCl4 and excitation of surface plasmon resonance (SPR) bands (Mulvaney, 1996). The control leaves extract alone did not turn to color formation. Similarly, different concentrations of HAuCl4 were optimized for the maximum synthesis of gold nanoparticles. Interestingly, in both the cases, 1 mM HAuCl4 supported the formation of gold nanoparticles whereas peak got shifted at 2 mM and no particles formation was observed at 3 mM concentration of HAuCl4 (Figs. 2 and 2a). Similar to our study, other research group also optimized nanoparticles formation using different concentrations of HAuCl4 and 1 mM concentration was found to be suitable for the synthesis (Ghosh et al., 2011).
Optimization of biogenic synthesis of gold nanoparticles from pumpkin leaves extract at different leaves extracts concentration (top left); time (top right); pH (bottom left); different molar concentration of HAuCl4 (bottom right).
Optimization of biogenic synthesis of gold nanoparticles from curled mallow leaves extract at different leaves extracts concentration (top left); time (top right); pH (bottom left); different molar concentration of HAuCl4 (bottom right).
pH is one of the important factors for nanoparticles synthesis. There is no pinkish violet color formation and no characteristic absorption band observed at acidic pH (2) but in pH 3, color formation was observed; however, the characteristic absorption band was absent. In both the cases, pinkish violet color formation and absorption bands were noticed from pH 4 to 6, whereas nanoparticles formation was absent as it can be seen from HR-TEM analysis. Dark pinkish violet color formation was very rapid in alkaline pH (9–12) but the characteristic absorption band was not observed from pH 9–11 instead peak got shifted. But in the case of pH 12, rapid pinkish violet color was formed within 10 min after adding HAuCl4 into pumpkin leaves extract and the characteristic band was prominent at 540 nm. Similarly, in curled mallow extract, pH 10 supported the dark pinkish violet color formation and the characteristic band was noticed at 540 nm but very less nanoparticles observed in HR-TEM analysis. At neutral pH (7, actual pH of the extract) and pH 8, the reaction was started as soon as HAuCl4 was added into the reaction medium and the formation was observed within 150 min. for pumpkin and 120 min. for curled mallow during the incubation at room temperature under static condition (Figs. 3 and 3a).
Nanoparticles formation at different pH in pumpkin leaves extract. Absence of particle formations observed at pH 4 (left); immature particle formations observed at pH 10 (right).
Nanoparticles formation at different pH in curled mallow leaves extract. Absence of particle formations observed at pH 4 (left); Very less particle formations observed at pH 12 (right).
To access the stability of gold nanoparticles formed in the reaction solution at 1 mM HAuCl4 in 6 ml leaves extract and 44 ml D.H2O at pH 8, UV–Vis analysis was carried out. Synthesized nanoparticles were treated with acidic and alkaline solutions, continuous UV irradiation and checked the nanoparticles stability. Based on the results obtained from UV–Vis spectrum, there was no alteration in the characteristic absorption peak at 540 nm even after a month of incubation period, indicating strong stability of biosynthesized gold nanoparticles. Hence, before taking up the nanoparticles into various biological applications it is much important to do research on various optimization processes for nanoparticles stability as well as formation. Based on the optimization process, it is very much possible to control the uniform size and shape of the nanoparticles synthesis using plant extracts.
3.2 Electron microscopic study
On the basis of HR-TEM analysis, poly dispersed nanoparticles were observed in unoptimized condition with the size ranging from 1 to 100 nm. But, in the optimized conditions, almost uniform size and shape of the nanoparticles was achieved in both pumpkin and curled mallow leaves extract (Figs. 4 and 4a). Further, FE-SEM analysis confirmed the formation of gold nanoparticles. Presence of strong signal identical to gold was observed in EDS analysis of both leaves extracts (Fig. 5).
Formation of gold nanoparticles from pumpkin leaves extract in HR-TEM. Poly dispersed biogenic gold nanoparticles (top left). Size and shape controlled biogenic gold nanoparticles (top right) and their particle size distribution histogram; Phytochemicals capped gold nanoparticles (bottom).
Formation of gold nanoparticles from curled mallow leaves extract in HR-TEM. Poly dispersed biogenic gold nanoparticles (top left); Shape controlled biogenic gold nanoparticles (top right); Phytochemicals capped gold nanoparticles (bottom).
Characterization of gold nanoparticles. FE-SEM analysis of gold nanoparticles synthesized from pumpkin leaves extract (top left); EDS analysis of gold nanoparticles synthesized from pumpkin leaves extract (top right); FE-SEM analysis of gold nanoparticles synthesized from curled mallow leaves (bottom left); EDS analysis of gold nanoparticles synthesized from curled mallow leaves extract (bottom right).
3.3 FT-IR spectral study
The alcoholic hydroxyl groups (—OH) of C. pepo showed the quite intense and smooth curved peak at 3375 cm−1. The peak at 2800–3300 cm−1 was due to C—H stretching vibrations and the absorption observed at the right of sp3 CH stretch at 2700–2900 cm−1 confirmed the reaction changes in the leaves extract after adding HAuCl4. In continues, variations in the carbonyl C⚌O peak at 1606 cm−1 was altered because of metal carbonyl complex, NH2 scissoring, N—H bending and these variations were due to the presence of aromatic cyclic ether compound from the leaves extract. The out-of-phase stretch at 2927 cm−1 was responsible for the presence of linear alkane alcohols, ethers, primary and secondary amines in the reaction solutions. Control and synthesized nanoparticles showed the peaks between 700 and 580 cm−1 inferred the presence of aromatics, which did not played a major role in the nanoparticles synthesis. Further, the percentage of transmittance at 1070–1080 cm−1 was increased due to the presence of substitution reactions in the aromatic ring. A doublet near 1600 cm−1 and 1390 cm−1 region was assuming the presence of alkyl-substituted naphthalene. Further, the characteristic band observed at 1390 cm−1 and 1370 cm−1 followed by 1080 cm−1 and 1050 cm−1 inferred the formation of gold nanoparticles in the leaves extract of C. pepo. Hence, based on the overall results, it is here concluded that phenolic substances present in the plant leaves extract of C. pepo played a major role in the gold nanoparticles synthesis. These results were in good agreement with the previously published research work done by Ratul Kumar Das et al. (2010), and they also found the phenolic compounds responsible for capping and efficient stabilization of gold nanoparticles from leaves extract.
Similarly, the alcoholic hydroxyl groups (-OH) in M. crispa plant leaves extract showed the quite intense and smooth curved peak at 3400 cm−1 and confirmed the reaction changes in the leaves extract after adding HAuCl4. The peak at 1634 cm−1 is the characteristic of amide or ester carbonyl stretch and altered because of the metal carbonyl complex, NH2 scissoring and N—H bends which lead to aromatic cyclic amide or ether compounds from the leaves extract. Control and synthesized nanoparticles showed peaks between 650 and 590 cm−1 which inferred the presence of aromatic sulfur substances and an insignificant change was observed but the characteristic band at 1080 and 1050 cm−1 was due to the presence of sulfur containing substances. Further, the sharp peak of doublet at 1634 cm−1 and 1385 cm−1 assumed the presence of esters of alkyl-substituted naphthalene. Hence, based on the overall results, presence of polyphenol substances in M. crispa plant leaves extract played a major role in the gold nanoparticles synthesis (Fig. 5a).
FT-IR analysis of pumpkin leaves extract alone (top left); synthesized gold nanoparticles (top right) from pumpkin leaves extract; curled mallow leaves extract alone (bottom left); synthesized gold nanoparticles (bottom right) from curled mallow leaves extract. Insert picture shows the preliminary phytochemical analysis of phenolic compounds responsible for capping and reducing agents (i) leaves extract alone (ii) presence of dark green color indicates phenolic compounds.
3.4 Phytochemical screening study
The results of the qualitative screening of phytochemical components in the aqueous leaves extracts of C. pepo and M. crispa were shown in Table 1. The aqueous leaves extract of C. pepo revealed the presence of tannin, saponin, flavonoids, phenol, glycosides, reducing sugar and alkaloids. But, in the case of M. crispa leaves extract, presence of all the above phytochemicals except tannin was observed. (+) indicates presence of constituents (−) indicates absence of constituents.
Phytochemical constituents
Cucurbita pepo leaves extract
Malva crispa leaves extract
Tannin
+
−
Saponin
+
+
Flavonoids
+
+
Phenol
+
+
Glycosides
+
+
Reducing sugar
+
+
Alkaloids
+
+
3.5 Antibacterial activity
The antibacterial activity of biosynthesized gold nanoparticles from both the plant leaves extracts was performed against three different Gram-positive bacteria such as B. cereus, S. aureus, L. monocytogenes and three different Gram-negative bacteria such as E. coli, S. typhi and S. enterica. The maximum zone of inhibition was observed against Gram-negative bacteria than the Gram-positive ones due to the thick peptidoglycan layer in the Gram-positive cell wall (Figs. 6 and 6a) (Table 2 and 2a). (–) indicates no zone formation. (–) indicates no zone formation.
Antibacterial activity of synthesized nanoparticles from pumpkin leaves extract by well diffusion assay (1) Bacillus cereus (2) Staphylococcus aureus (3) Listeria monocytogenes (4) Escherichia coli (5) Salmonella typhi (6) Salmonella enterica. (i) D.H2O (ii) Aqueous leaves extract alone (iii) 1 mM HAuCl4 (iv) Gold Nanoparticles synthesized from plant leaves extract.
Antibacterial activity of synthesized nanoparticles from curled mallow leaves extract by well diffusion assay (1) Bacillus cereus (2) Staphylococcus aureus (3) Listeria monocytogenes (4) Escherichia coli (5) Salmonella typhi (6) Salmonella enterica. (i) D.H2O (ii) Aqueous leaves extract alone (iii) 1 mM HAuCl4 (iv) Gold Nanoparticles synthesized from plant leaves extract.
Food borne bacterial pathogens
Zone of inhibition (mm in diameter)
D.H2O (Control)
Plant extract (800 μg/ml
1mM HAuCl4
Biogenic gold nanoparticles from plant leaves extract (800 μg/ml)
Gram positive
Bacillus cereus
–
–
11
11
Staphylococcus aureus
–
–
12
11
Listeria monocytogenes
–
–
–
–
Gram negative
Escherichia coli
–
–
12
12
Salmonella typhi
–
–
12
12
Salmonella enterica
–
–
11
12
Food borne bacterial pathogens
Zone of inhibition (mm in diameter)
D.H2O (Control)
Plant extract (800 μg/ml
1 mM HAuCl4
Biogenic gold nanoparticles from plant leaves extract (800 μg/ml)
Gram positive
Bacillus cereus
–
–
11
11
Staphylococcus aureus
–
–
12
12
Listeria monocytogenes
–
–
10
12
Gram negative
Escherichia coli
–
–
12
11
Salmonella typhi
–
–
11
12
Salmonella enterica
–
–
11
12
3.6 Minimal inhibitory concentration of gold nanoparticles
Among different concentrations of gold nanoparticles tested, 400 μg/ml was proved to be MIC for E. coli and Listeria monocytogens while 800 μg/ml was found to be MIC for S. typhi, S. enterica, B. cereus and S. aureus.
3.7 Conductivity study
Interactions of biologically synthesized gold nanoparticles on Gram-positive and Gram-negative pathogens were studied using conductivity meter. Ten ml of log phase cultures of both Gram-positive and Gram-negative bacteria exposed to 1 ml of 800 μg/ml concentration of gold nanoparticles showed higher conductivity levels in all the tests. The increase of conductivity was due to rupture of bacterial cell membranes which oozed out all the inner contents into outside the medium (Oyewole and Abalaka, 2012) (Fig. 7).
Mode of interactions of gold nanoparticles treated with bacteria using conductivity study. Values were represented as mean of average ± SD of three independent experiments.
3.8 Bacterial cell membrane damage study
Mode of interactions of gold nanoparticles on both Gram-positive and Gram-negative bacterial pathogens was studied using Bio-TEM. Bacterial cultures were exposed to 1 ml of 800 μg/ml concentrations of gold nanoparticles for 8 h and stained with negative staining. The Bio-TEM showed the clear destruction of bacterial cell membranes (Figs. 8 and 8a).
Food borne Gram positive bacterial cell wall membrane damage study using Bio-TEM. Mode of interactions of phytochemicals capped gold nanoparticles against Bacillus cereus (top) Staphylococcus aureus (middle) Listeria monocytogenes (bottom).
Food borne Gram negative bacterial cell wall membrane damage study using Bio-TEM. Mode of interactions of phytochemicals capped gold nanoparticles against Escherichia coli (top). Salmonella typhi (middle). Salmonella enterica (bottom).
In this study, bacterial cultures treated with gold nanoparticles synthesized from plant leaves extracts increased the conductivity. This could be well attributed to the dissolution of the cellular contents in the culture broth, by the disruption of the cell membrane structures with the loss of membrane permeability or the inability to sustain with the ATP production, necessary for maintaining the membrane dynamics. Presence of phytochemicals in the plant leaves extracts was believed to be responsible for the nanoparticles formation. Based on the concentrations, availability and chemical structures, any of the following phytochemicals such as alkaloids, glycosides, flavonoids, phenolic compounds, tannins, saponins, catechins, steroids and terpenoids were playing a major role in reducing HAuCl4 and act as reducing and stabilizing agents for nanoparticles formation. Although, much research is focused on the rapid synthesis of gold nanoparticles from plant leaves extract, the rate of formation is purely depended upon the available sources of phytochemicals in the reaction solution (Smitha et al., 2009; Nune et al., 2009; Begum et al., 2009; Wang et al., 2009). In addition, gold nanoparticles synthesized from aqueous plant leaves extract carrying the phytochemical properties which are helpful in antimicrobial as well as other biological applications.
4 Conclusions
The biogenic synthesis of gold nanoparticles and the various characterization techniques confirmed the formation of nanoparticles from two medicinally important aqueous plant leaves extracts C. pepo and M. crispa. Moreover, a variety of parameters optimized for achieving the size and shape controlled nanoparticles. Interestingly, the plant extracts synthesized gold nanoparticles proved to be potent antibacterial agent against food spoilage pathogens. Today, there are a number of methods employed for conjugating the gold nanoparticles with various biomolecules such as antibiotics, and polyvinyl pyrollidone (PVP), for improving the efficiency of gold nanoparticles against microbes. But if the study is focused on phytochemicals conjugated gold nanoparticles and its efficiency for improving the antimicrobial and other biological applications will give good mileage on research.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2007953) and also funds from Chonbuk National University in 2011, Republic of Korea.
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