Zohreh Didar
Abstract
Introduction: Metal oxide nanoparticles have unique physical and chemical properties. These components have shown antimicrobial effects against a wide range of microorganisms. In order to improve the physical properties of metal oxide nanoparticles, doping other elements with metal oxide nanoparticles ...
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Introduction: Metal oxide nanoparticles have unique physical and chemical properties. These components have shown antimicrobial effects against a wide range of microorganisms. In order to improve the physical properties of metal oxide nanoparticles, doping other elements with metal oxide nanoparticles is an effective way. Bacillus cereus is a gram-positive bacteria causing food-borne diseases. In this study, the antimicrobial effects of doped zinc oxide nanoparticles with manganese or iron on Bacillus cereus have been studied. To investigate the synergistic effects of the combined nanoparticles with two common biocides, including hydrogen peroxide and sodium hypochlorite, have been used.
Materials and methods: Co- precipitation method was used to prepare nanoparticles of manganese-zinc oxide and iron-zinc oxide. In this method, zinc sulfate and manganese sulfate were used to prepare manganese-zinc oxide and iron sulfate and zinc sulfate are used for Zn- Fe doped nanoparticles. After preparing the sulfate solutions, the sulfate solutions were mixed and placed in an ultrasonic apparatus at a frequency of 57 kHz for 2 hours at 50ºC. Then, it was stirred at 80°C. A solution of NaOH was added until the pH of the solution reached 12. In these conditions, the mixing was done for 30 minutes. The solution was placed at ambient temperature for 18 hours. Then the centrifuge was performed to separate the sediment. Purification was done through washing with distilled water and ethanol. The precipitates were dried in the vacuum oven. In this way, the doped nanoparticles of manganese-zinc oxide and iron-zinc oxide were obtained. The Fourier transform infrared spectrum (FTIR) was carried out by the Perkin-Elmer apparatus of the Spectroma2 model, using a dry potassium bromide tablet at a frequency range of 4500-4000 cm-1. The X-ray diffraction was tested using the Phillips PW1820 from 2º to 80º. Structure of produced nanoparticles was assessed by the HITACHI electron microscope, the H-7500 model, by placing a drop of nanoparticles dissolved in methanol on a special lining with carbon coating and air drying, and performing microscopic images using an electron microscope in 100kv. The bacteria used in this study included Bacillus cereus (PTCC 1665) was purchased from the Iranian Scientific and Industrial Research Center and was transferred to the BHI medium in sterile condition and incubated for 32 hours at a temperature of 32°C. Microbial cells were isolated by centrifugation at 4000 rpm. McFarland's method was used for determining the bacterial population. Dilution was carried out to reach a population of about 106 CFU/ml. Agar disc diffusion method was used for assessing the antimicrobial effect of the doped nanoparticles alone or in combination with tested biocides (hydrogen peroxide, sodium hypochlorite). At first, 106 CFU/ml of Bacillus cereus were inoculated on the surface of Blood Agar. Then, 5, 10, 20, 30, 50, 100 and 200 mg/L of each of the nanoparticles were placed on the surface of the culture medium and then the plates was incubated at 37°C for 24 hours. Inhibition zone was considered as antibacterial activity. In order to investigate synergistic effects, inhibitory fraction index test was calculated. All experiments were performed in three replications. Statistical analyzes were performed using STATISTICA software.
Results and discussion: Results obtained from X-ray and FTIR analysis of doped nanoparticles confirmed that co- precipitation is a suitable method for producing doped nanoparticles of zinc oxide. TEM analysis of produced nanoparticles also affirm formation of doped nanoparticles of zinc oxide with manganese and iron. The results of antimicrobial tests showed that Mn-Zn oxide nanoparticles have more antimicrobial effects on Bacillus cereus than zinc oxide (32mm inhibition zone) whereas Fe- Zn oxide nanoparticles cause inhibition zone about 12 mm. In addition, both doped nanoparticles have more antimicrobial effects than zinc oxide nanoparticles alone, resulted in doping process improves antimicrobial properties of zinc oxide. The synergistic effects of synthetic nanoparticles in the combination of two common antimicrobial agents, including hydrogen peroxide and sodium hypochlorite, have been identified. Both nanoparticles show synergistic effects in combination with two tested biocides (especially in high concentrations). A mixture of two biocides with nanoparticles increases their antimicrobial properties. Manganese-zinc oxide nanoparticles with hydrogen peroxide and sodium hypochlorite showed a partial synergistic effect at low concentrations (5 + 20) and a complete synergistic effect at higher concentrations. In the case of iron-zinc oxide, combination of this nanoparticle with hydrogen peroxide and sodium hypochlorite, has complete synergistic effects at high concentration (100 + 200) and at other conditions, shows partial synergistic effects.
Mahmoud Yolmeh; Mohammad Bagher Habibi Najafi; Mahmoud Najafzadeh
Abstract
Introduction: One of the most important aspects of food preservation is controlling the growth of microorganisms, which if overlooked it leads to uncontrolled growth of microorganisms associated with food spoilage and food poisoning. Microbial contamination of foods is important because of pathogens ...
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Introduction: One of the most important aspects of food preservation is controlling the growth of microorganisms, which if overlooked it leads to uncontrolled growth of microorganisms associated with food spoilage and food poisoning. Microbial contamination of foods is important because of pathogens are capable to transfer to foods during the processing, distribution, and storage. Escherichia coli and Bacillus cereus can cause spoilage in food; intake food contains plenty of bacteria and toxic. Therefore it is important to eliminate or control these bacteria safely. Ultraviolet (UV) radiation is considered as non-ionizing radiation and the first time in 1940 was used as a method for infection elimination in air. This approach nowadays is widely used for controlling microbial growth and as disinfection in food industry. Wavelength range of ultraviolet radiation is approximately 328-210 nm. The beam is naturally present in the sunlight. The bactericidal effect of UV irradiation depends on the type of bacteria, the distance, and dose of radiation. The most cytotoxic effect of UV irradiation is obtained at the wavelength of 260 nm, which corresponds to the intense absorption of energy by organic bases in the nucleic acid. UV irradiation causes radicals generation, which subsequently attack the nucleic acid and develop mutations in their genomes and gene transcription and translation processes. In this study, the antibacterial effect of different exposure times of ultraviolet radiation on the growth of E. coli and B. cereus was evaluated.Materials and methods: All media used in this study was procured from Merck Company. Ultraviolet device (Camag, USA) was used at wavelength of 254 nm, Nr= 29000, Amp= 0.25. B. cereus isolation: 1 mL of different dilutions of rice (0.1, 0.01, and 0.001) was transferred to Brain-heart infusion (BHI) and it was incubated at 32 °C for 24 h. A loop containing the bacteria was then transferred to Mannitol Egg Yolk Polymyxin (MYP) agar and it was incubated at 35 °C for 24 h. B. cereus produce big and round colonies, with a halo around the colonies. Starch test was carried out as confirming test for B. cereus colonies. Briefly, some colonies of B. cereus were added to test tube with sterile distilled water containing starch and a few drops of lugol. Development of blue color indicates the presence of B. cereus due to starch hydrolysis.E. coli isolation: E. coli was isolated from raw milk following the method described by Kargar et al. (2005). Briefly, raw milk was first homogenized; 0.1 ml of each dilution of homogenized raw milk was inoculated on Escherichia coli broth medium containing 20 mg novobiocin. E. coli was then isolated after transferring the former media on EMB specific culture and incubation at 36 °C for 24 h. After confirming colonies by Durham tube and complementary tests, pure cultures were obtained from them by streak-plate method.UV irradiation: A loop of E. coli colonies was transferred to nutrient broth and it was treated with UV beam (254 nm) at three times (40, 60, and 80 s). After preparing dilution of 0.0001 for each of the treatments and incubating for 24 h, survival curve was plotted. These operations were also carried out on B. ceruse colonies. A control sample also was considered for each examined bacterium.Results and Discussion: Rate of Bacillus cereus growth was reduced under UV radiation. As it is shown, Death curve of E. coli, E. coli count was decreased by increasing the time of UV radiation, so that count of this bacteria reached to about zero after UV radiation for 80 s. However, reduction of B. cereus count was less than E. coli count at same wavelength (254 nm) and time of irradiation. This revealed that B. cereus have more resistance to UV radiation compared to E. coli. These results were consistent to observation of Sharp (1940) who evaluate the effects of UV light on bacteria suspended in air and reported that required energy for air sterilization containing B. cereus is more than twice the energy is needed to eliminate E. coli. UV light more penetrates to cell wall of Gram-negative bacteria compared to Gram-positive bacteria due to having a small amount of peptidoglycan in the cell wall and caused mutations in regulating genes of transcription and translation. Conclusion: The efficiency of the two main processes of cell is reduced in the presence of UV irradiation and leads to growth reduction and death. The more resistance of B. cereus can be for several reasons, such as having a thicker cell wall compared to E. coli, and the capability to produce spore, and the capability to proofing mutations.
