Received date: 04/11/2015 Accepted date: 19/11/2015 Published date: 30/11/2015
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Traditionally, antimicrobial activities of silver have been known for human, but recently effects of silver nanoparticles and their antimicrobial effects have evaluated by researchers. In this study silver nanoparticles were produced by chemical reduction and then antimicrobial effects of these particles for two of the most threatening bacteria in food industries, Staphylococcus aureus and Escherichia coli as gram-positive and gramnegative bacteria examined by concentrations of 5, 10, 25, 50 mg L-1. The Results indicated that the chemical reduction method is a good and suitable procedure for production of silver nanoparticles, but it needs high accuracy. The minimum inhibitory effect of silver nanoparticles for Escherichia coli and Staphylococcus aureus were 5 and l0 mg L-1 respectively, which Escherichia coli was more resistance. So, these particles could be considered as a suitable alternative to conventional antimicrobial agents.
Silver Nanoparticles, Chemical Reduction, Antimicrobial Activity, Process Variable
Reduction in size of particles is one of the effective and useful tools to improve particles’ effectiveness; in fact nano technology assists to reduce the size and to eliminate restriction of sizes that had existed from the past [1,2]. Microorganisms like bacteria, yeast and molds exist in environment frequently infected people . Increasing rate of infectious diseases which caused by pathogenic bacteria also increasing their resistance against antibiotics, pushed Physicians to use these new antimicrobial agents [4-6].
The antimicrobial activities of silver have been known from ancient time . It is supposed that due to small size of silver nanoparticles, specific surface of atom and considerable amount of it has antibacterial activities in comparison with mass silver [3,8-15]. Various mechanisms are suggested for the impacts of silver’s germicide but silver is different from other germicide materials and contrary to them, it seems that microorganisms have no resistance against silver in process of time and this apparently is because silver has more than one method to reveal the its antimicrobial impact on microorganisms .
In recent years to synthesis of metal nanoparticles a variety of chemical and physical methods have been developed, including chemical reduction, electrochemical, photochemical, sonochemical, thermal reduction, physical synthesis of steam, condensation neutral gases, chemical synthesis of steam, compression of steam atomic metal and etc. [17-23]. Physical methods are used rarely due to their need to high temperatures more than 1000°C, expensive equipment and complexity of how to control synthesis circumstances. Since facility of controlling conditions of reaction, inexpensive process and simple equipments and raw material, chemical methods are so common instead and among these methods, chemical reduction utilize more than others because of simple equipment’s and operations . As yet different components have been used as reducer and stabilizer which are mostly toxic compounds.
Purposes of this research are synthesis of stable silver nanoparticles by using chemical reduction without using toxic solvents and evaluation of antibacterial effects of silver nanoparticles on S. aureus and E. coli as two harmful and problematic bacteria in food.
Silver nitrate, Ethanol, medium of Eosin-Methylene blue, Nutrient Broth, Muller-Hinton agar, Mannitol-Salt-agar also peptone water provided from Germany Merck Company. Polyvinylpyrrolidone with 55000 molecular mass was purchased from Sigma- Aldrich Germany. Moreover, S. aureus PTCC No. 1112 (ATCC No: 6537) and E. coli PTCC No. 1330 (ATCC No: 8739) supplied from Iranian Research Organization for Science and Technology.
Synthesis of silver nanoparticles
Chemical reduction was utilized to synthesis silver nanoparticles in this study. Some of the advantages of this method in comparison to other methods for production of silver nanoparticles are simplicity, inexpensive, ability to control the conditions of reaction .Silver salt, reducer, stabilizer and solvent, silver nitrate, ethanol, polyvinylpyrrolidone 55000 molecular mass and deionized water were required to synthesis of silver nanoparticles by this method. 0.1 g polyvinylpyrrolidone and 20 mL de-ionized water were added into a twin-neck flask. Then, exposed to nitrogen at 90°C for 10 min and stirred it strongly in order to solve completely beside de-oxygenation. Next, 0.01 g of silver nitrate with 1 mL ethanol added to the twin-neck flask, frequently color of solution bring to change. Which, after 20 min, the color changed from colorless into tawny which indicated synthesis of silver nanoparticles and end of the reaction. Examining UV-Vis (Ultraviolet-visible) spectrum of colloid solutions is a more accurate method to ensure of completing synthesis of silver nanoparticles. Appearance of a peak on 420 nm indicated the synthesis of silver nanoparticles was spheroid. Then, the volume of 1 mL of sample was reached to 10 mL with de-ionized water and read the UV-Vis spectrum of it with spectrophotometer. This procedure should be repeated every 5 min. At the beginning, advent of peak on 300 nm indicated existence of silver ions in the colorless solution. By time passed, peak moved to higher wavelengths which indicated reduction of silver ions to silver nanoparticles which in this step colorless solution changed into yellow. In the final step, appearance of peak on 420 nm and tawny color of solution presented production of silver nanoparticles and end of the reaction.
After the reaction ends, an image of silver nanoparticles was taken by Transmission electron microscopy (TEM); this is to ensure of synthesis of silver nanoparticles, moreover, determine mean of the particles’ size.
After obtaining images by TEM, size and scattering of particles were calculated by Manual Microstructure Distance Measurement software which was a production of Nahamin Pardazane Asia Company. Biggest and smallest sizes of silver nanoparticles were 34 and 3 nm which most of them distributed in range of 8 to 15 nm. Then, colloidal solution of silver nanoparticles by concentrations of 5, 10, 25, 50 mg L-1 were prepared and applied to evaluate antimicrobial effects of those particles on S. aureus and E. coli.
First, McFarland Standard (0.5) for control treatment was prepared, then put 0.1 mL of S. aureus and E. coli in each test tubes containing Nutrient Broth medium (one type of bacteria per tube) and put tubes in incubator for 24 h on 37°C. After 24 h, the tubes removed from incubator which showed turbidity same to McFarland (0.5) turbidity with Light Absorption Coefficient (LAC) of 1. In accordance with McFarland (0.5) Standard it was accounted for 105 ×108 CFU mL-1 of S. aureus in the solution.
Subsequently different dilutions of 103-107 of both mentioned bacteria were prepared and then all dilutions cultured with both bacteria in separate specific medium as Eosin-Methylene blue for S. aureus, Mannitol-Salt-agar for E. coli and Muller-Hinton agar applied to evaluate antimicrobial effects of both bacteria. Next, put each culture plate into incubator for 24 h on 37°C in order to those bacteria grow on foresaid culture medium. All plates which were cultured by this method consider as control plates.
After that, when the time passed for each one as 1, 12, 24 and 48 h, put 1 mL of all silver nanoparticles concentrations into each prepared bacteria concentrations on specific medium for each bacteria and also on Muller-Hinton agar for both bacteria; then placed in incubator for 24 h on 37°C to determine rate of inhibition effects silver nanoparticles on bacteria. All plates which were cultured by this method considered as test plates. Incubated control and test plates compared in order to understand silver nanoparticles impacts on percentage reduction of bacterial count.
All plates including control and test plates have been evaluated after 24 h. Initial evaluation of those plates indicated that growth inhibition in 50 mg L-1 treatment of silver nanoparticles for all given times. Figure 1 demonstrated inhibited growth of S. aureus and E. coli (107 CFU mL-1) exposed to silver nanoparticles 50 mg L-1 after 1 h contact.
After observing the plates, the results must be compared statistically to evaluate independent and interaction effects between factors on percentage reduction count of S. aureus and E. coli. So, it would be cleared whether they are significant or not. Table 1 indicated the results obtained from analysis of variance (ANOVA) by full-factorial model.
|Source||SOS**||Degree of freedom||mean squares||F value||P-value|
B-Type of Bacteria
C-Concentration of nano
Table 1: Analysis of variance for variables impacts on percentage reduction count of bacteria by full- factorial model.
The results demonstrated that effects of independent factors like time, type of bacteria, silver nanoparticles concentration and also interaction effects between time and silver nanoparticles concentration, type of bacteria and silver nanoparticles concentration and time, type of bacteria, silver nanoparticles concentration were significant in level of 1% and effect of time and type of bacteria were significant in level of 5%. The outputs presented a linear relationship among three given factors on percentage reduction count of bacteria. Desired fitting of data could be evaluated by information such as R2, R2 adj, C.V or coefficient of variation that those amounts were equal to R-Squared= 0.9974, Adj R-Squared= 0.9969 and C.V= 2.11%.
Figure 2 indicated the comparisons effects of different time growth on bacterial reduction count in presence of silver nanoparticles with concentration of 5 mg L-1 and at the temperature of 25°C. Also, The interaction of time and type of bacteria on reduction of bacterial count in presence of silver nanoparticles at the temperature of 25°C has been shown in Figure 3 which indicated that the effect of time on reduction of E. coli was not significant but by increasing time from 1 to 48 h, the number of survived S. aureus bacterial cell was reduced.
According to Figure 2, which showed the interaction effects of concentration of silver nanoparticles and type of bacteria on reduction of bacterial cells at the temperature of 25°C, the concentration of 5 mg L1 silver nanoparticles reduced the number of S. aureus bacterial cells but this concentration hadn’t any effects on E. coli bacterial cells. The inhibitory effect of silver nanoparticles on E. coli was obscured in concentration of 5 mg L1. So, the concentrations of silver nanoparticles which can inhibit the growth of S. aureus (Gram-positive) and E. coli (Gram-negative) were respectively 5 and 10 mg L-1. Moreover, concentration of 50 mg L-1 silver nanoparticles inhibited the growth of both bacteria completely. Structure of membrane Gram-positive and Gram-negative bacteria is different from each other and also peptidoglycan layer may be in different thicknesses, which were reasons that E. coli had more resistance than S. aureus. Gram-positive bacteria as S. aureus have thick and multi-layer peptidoglycan. However, Gram-negative bacteria as E. coli have thinner layer and its outer membrane containing lipopolysaccharide which protected the bacteria from antibiotic and antimicrobial factors.
According to Figure 3 which demonstrated interaction effects of concentration of silver nanoparticles and type of bacteria on percentage reduction count of bacterial cells at the temperature of 25°C, the concentration of 5 mg L-1 silver nanoparticles hadn’t any effect on reduction of number of bacterial cells in any time. But, the concentration of 10 mg L-1 silver nanoparticles by increasing the time can reduce more number of bacterial cells. Moreover, the concentration of 25 and 50 mg L-1 silver nanoparticles were important. The results obviously showed that after 1 h exposure of bacteria to these concentrations of nanoparticles causes high antibacterial effects on E. coli and S. aureus.
Table 2 demonstrated comparison of mean between effects of silver nanoparticles in different concentration on percentage reduction count of bacteria. As they cleared, there was a significant difference between silver nanoparticles by concentration of 5, 10, 25 or 5, 10, 50 mg L-1; but there was no significant difference among silver nanoparticles by concentration of 25 and 50 mg L-1. Silver nanoparticles with concentrations of 25 and 50 mg L-1 had the most also 5 and 10 mg L-1 had the least effectiveness on bacteria. The effects of type of bacteria on bacterial reduction count in presence of silver nanoparticles with concentration of 5 mg L-1 and temperature 25°C were compared in Table 3. Obviously there was a significant difference between types of bacteria which the most reduction occurred in S. aureus. These results were like to those had been obtained by Cho et al., who also found that inhibitory concentration were at least respectively 5 and 10 mg L-1 for S. aureus and E. coli. However, they described 50 mg L-1 could be reasonable concentration for S. aureus and subsequently for E. coli would be 100 mg L-1. Ruparelia also reported same outputs that E. coli is more resistance than S. aureus. They evaluated effects of silver nanoparticles which were produced through reduction on S. aureus and E. coli and determined E. coli is more resistance than S. aureus 
|Concentration of anoparticles (mg L-1)||Decrease of Bacterial Count (%)||Duncan Grouping|
|Data reported are average of 6 replication, Different Latin letters shows significant difference between data reported in each row (P<0.05) *|
Table 2: Effects of different concentration of silver nanoparticles on reduction count of bacteria at temperature 25°C .
|Type of Bacteria||Decrease of Bacterial Count (%)||Duncan Grouping|
|Data reported are average of 6 replication, Different Latin letters shows significant difference between data reported in each row (P<0.05) *|
Table 3. Effects of type of bacteria on bacterial count in presence of silver nanoparticles with concentration of 5 mg L-1 and temperature 25°C.
Silver nanoparticles are caused destruction of bacteria by impact on bacteria cell-wall [26-28]. There are several reports about mechanism of silver nanoparticles’ impacts on bacteria. Aggregation of silver nanoparticles on bacteria cell-wall and penetration to cell has been explained as bacteria destruction . Also similar studies have been demonstrated that size and shape of silver nanoparticles effect on germicide activities; as smaller silver nanoparticles with spherical shape have the most germicide effects. Cho  reported that surface of E. coli cell-wall strongly damage in contact with silver nanoparticles. Lack of S. aureus and E. coli growth on test plates consisting silver nanoparticles with concentration of 50 mg L-1 presented high inhibitory effectiveness which is a result of destructing cell-wall from both bacteria. Our results support the theory that production of silver nanoparticles could provide a simple and inexpensive method in order to inhibit bacterial growth.
Generally, the results of study demonstrated that chemical reduction method is a good and suitable procedure for production of silver nanoparticles, but it needs high accuracy and the most important step is preventing the aggregation of particles by use of appropriate stabilizer.
Moreover, according to the results, silver nanoparticles can destroy S. aureus and E. coli in low concentrations which are both harmful bacteria in food industries. This can considerably assist to food industry in all over the world; including food packing and antiseptic of assembly line by silver nanoparticles.
The minimum inhibitory concentration of silver nanoparticles for S. aureus and E. coli were 5 and 10 mg L-1 respectively. Moreover, both bacteria were killed in concentration of 50 mgL-1 of silver nanoparticles. Also, the results showed that there was no resistance to silver nanoparticles in bacteria by passing the time. So, these particles can be considered as a suitable alternative to conventional antimicrobial agents.
This study was partially supported by National Nutrition and Food Technology Research Institute (NNFTRI) Master of Science project, Iran. Hereby, this support is appreciated.