Increasing the Shelf Life of Milk by Metal Oxide Nanoparticles and Mild Heat
 
Mahbooubeh Mirhosseini; PhD^*1 & Roghayeh Dehestani; MSc²

 
¹ Department of Biology, Payame Noor University, Yazd, Iran.
² Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran.
 

 
Introduction

Considering the recent emergence of pathogenic bacteria, which are resistant to a broad spectrum of antibiotics, and the adverse effects of some antibiotics on human health, effective antimicrobial compounds with minimum side effects should be found urgently to eliminate infectious agents from the food and environment (Kimiaee Sadr et al., 2016, Li et al., 2008, Magiorakos et al., 2012). Numerous efforts
have been made to eradicate multiple- antibiotic-resistant pathogenic bacteria using antimicrobial nanomaterials. The aim was to reduce the food-borne bacteria and life-threatening infections with low toxicity (Li et al., 2008). One of the significant properties of antimicrobial nanoparticles (NPs) is their ability to inhibit the growth of gram-negative bacteria and food-borne pathogen (Akbar and Anal, 2014). Applicability of NPs in different fields of science such as biology, medicine, and pharmacology, especially in medications has attracted much attention. The researchers found that NPs could be employed in food preservation and food packing, leading to increased food safety in industrial food processes (Akbar and Anal, 2014). Metal oxide NPs, a new class of compounds with unique physical and chemical properties, have antibacterial activities against various types of harmful microorganisms. Among NPs, metal oxide NPs, such as ZnO, Fe₂O_3, and MgO are the most considered research subjects  (Xie et al., 2011). Application of NPs is not exclusively confined to medicines, rather they are widely used in industrial productions such as cosmetics, plastics, paints, ceramics, and textiles (Rizzo et al., 2013). Metal oxides NPs were reported to have antibacterial activity against microorganism pathogens, such
as Staphylococcus aureus, Escherichia coli, Aspergillus niger, Saccharomyces spp, and Mushroom species  (Hewitt et al., 2001). However, gram-positive bacteria are more prone to be affected by metal oxide NPs in comparison with gram-negative bacteria (Li et al., 2008). Due to the broad spectrum antibacterial properties of metal oxide NPs, these nanomaterials are considered as potential candidates to be applied in food industry (Stoimenov et al., 2002). The size of NPs was inversely associated with the inhibitory effects of these materials on bacterial growth (Akbar and Anal, 2014). Prophylactic strategies, rapid diagnosis, and advanced antibiotic therapy have significantly improved the life of human beings in recent years. However, antibiotic resistance is still the main enigma in public health that urges scientists to seek more effective compounds against multi-drug resistant bacteria with low toxicity in order to overcome the bacterial resistance (Rizzo et al., 2013).
The spread of infectious diseases caused by microorganisms in the food and increased bacterial resistance to antibiotics have arisen a major problem for the public health (Makhluf et al., 2005). Hence, new compounds should be explored and designed to fight against such bacteria. Metal oxide NPs have evolved as new antimicrobial agents, which can be potentially used against
food-borne pathogens (Akbar and Anal, 2014, Dastjerdi and Montazer, 2010). Considering
the heat resistance, antibacterial activity,
targeting abilities, high durability, and thermal conductivity of metal oxide NPs, they are very promising in the discovery and delivery of bioactive molecules for food-borne bacteria, spores, and viruses. Therefore, the present study aimed to monitor the antibacterial activity of some metal oxide NPs including ZnO, Fe₂O₃, and MgO against food-borne bacteria in the culture
medium and milk.
Materials and Methods
Bacterial strains, medium culture, and materials: The strains of bacteria used in this study were Escherichia coli (E. coli) PTCC1394 and Staphylococcus aureus (S. aureus) PTCC1431, which were both obtained from the culture collection of Iran. Stock cultures were maintained
at −70 °C until use. Bacteria were active on tryptic agar at 37 °C and maintained at 0–2 °C before the experiments. Furthermore, MgO NPs (with the particle size of 20-30 nm) were prepared from US Research Nanomaterials Inc (Houston, TX); ZnO NPs were prepared from Teconan (Navarra, Spain) with the particle size of 20–25 nm; and Fe₂O₃ NPs (>50 nm particle size) were obtained from Sigma Chemical Company (Darmstadt, Germany). The soluble form of all NPs was prepared in double-distilled water and then sonicated in an ultrasonic bath (S30H Elmasonic, Germany) for 20 min. As a result, the homogenous colloidal suspension was achieved for each nanoparticle (Mirhosseini and Firouzabadi, 2013).
Antimicrobial effect of NPs: The antibacterial activity of all NPs, namely, ZnO, Fe₂O₃, and MgO was evaluated against E. coli and S. aureus. For this purpose, the spot-on-lawn assay was employed to measure the potency of NP types in the suppression of bacterial growth. Briefly, 20 µL of the colloidal suspensions of NPs at concentrations of 0, 0.65, 1.25, 2.5, and 5 mg/ml were spotted onto tryptone soya agar (TSA)soft-agar lawns (0.6%). Later, they were inoculated by 10⁷ bacteria/mL E. coli or S. aureus according to 0.5 McFarland standards. Each plate of bacteria was incubated at 37 °C for 24 h. Afterwards, the inhibition zone around each spot was calculated to measure the inhibitory effect of each metal oxide NP (Mirhosseini and Firouzabadi, 2013).
Minimum inhibitory concentration (MIC) determination of NPs: The minimal concentration of a given antimicrobial agent that inhibits the growth of microorganisms is known as the MIC (Stoimenov et al., 2002). To measure the MIC of ZnO, Fe₂O_3, and MgO NPs colloidal suspension from each NP was prepared in which the concentration of each NP was 100 mg/ml. At first, 1 ml of TSB media was added to the tubes and then 1 ml of each NP was poured into the tube. In the next step, a serial two-fold dilution method (100- 50- 25-12.5-6.25,….mg/ml) was applied to achieve the lowest concentration of each NP to inhibit the growth of bacteria, starting from tube 1 to tube 7. The 8^th tube was considered as the control tube. Later, 100 µL of the 10⁷ bacteria/mL corresponding to 0.5 McFarland standards of either S. aureus or E. coli was added to all tubes. Subsequently, all tubes were incubated at 37 °C overnight. The rate of bacterial growth was determined by the measurement of optical density (OD) at 620 nm performed by the spectrophotometry (Optizen 2120 UV Plus, Korea) analysis. The obtained value for each NP was designated as the MIC (Stoimenov et al., 2002, Xie et al., 2011).
Minimum bactericidal concentration (MBC) determination of NPs: The MBC is characterized by determination of the lowest concentration of an antibacterial agent that can reduce viability of the initial bacterial inoculum by ≥ 99.9%. The value of MBC for each test tube, without visible growth, was obtained by subculturing 50 µl on tryptone soya agar (TSA). After incubation at 37 °C for 24 h, the MBC was defined as the lowest concentration leading to a negative subculture or giving the presence of only one colony after the incubation process (Stoimenov et al., 2002).
Determination of the effect of NPs and heat on bacterial load of milk: Traditional cow's milk was used for the present study. Metal oxide NPs namely, ZnO, Fe₂O₃, and MgO were dissolved in milk to reach the concentration of 1.5 mg/ml. Milk samples were then inoculated with 100 µl of the mixed bacteria for each strain (equivalent to 10⁷ CFU mL^-1). The heating process was carried out in a water bath at 100 °C in which the temperature was controlled by the insertion of a thermometer in milk containing NPs. During the thermal treatment, milk samples were homogeneously agitated to obtain uniform distribution of the inoculums. Once the temperature of milk samples reached the target treatment temperature of 50-60 °C, samples were removed from water bath and immediately transferred into the ice box (Premanathan et al., 2011).
One milliliter of the treated samples was dissolved in 9 ml of 0.2% (w/v) peptone water and then serially diluted (10^-1–10^-5) in 0.1% peptone water. Mannitol salt agar (MSA, Merck, Darmstadt, Germany) and eosin methylene blue (EMB, Merck, Darmstadt, Germany) agar were employed for the separating and counting S. aureus and E. coli, respectively. Microscopic analysis of bacteria was accomplished by the Gram stain smear. The biochemical and sugar fermentation tests in phenol read broth were also performed for the identification and confirmation of the bacterial isolates contained in milk samples (Brayner et al., 2006, Phillips, 1993).
Analysis of cell morphology by SEM: Cultivated bacteria namely, S. aureus and E. coli, at the mid-log phase of growth corresponding to 0.5 McFarland standards, were treated with 1.5 mg mL^-1 MgO NPs for 18 h. One milliliter of the treated and untreated bacterial suspensions were centrifuged at 10,000 rpm for 5 min and the bacterial sediments were dissolved in 100 µL tryptic soy broth. Afterwards, 20 µL of the bacterial suspensions were put on glass coverslips. The glass coverslips were air-dried for 45 min and then fixed with a solution containing 2.5% glutaraldehyde and 0.1 M imidazole buffer solution (pH 7.2) for 2 h. Later, the fixative solution was replaced by 0.1 M imidazole buffer, followed by dehydration with an ascending ethanol series (50, 80 and 100%). The coverslips were placed on SEM stubs by means of carbon adhesive disks, which were then sputter-coated with a thin layer of gold using Sputter Coater device (DSR1, Nano-Structured Coatings Company, Iran). Finally, digital images of the treated and untreated S. aureus and E. coli were captured using the SEM (Phenom ProX, Netherland) that was operated at an accelerating voltage of 10 kV and instrumental magnifications of 35,000 (Jin and He, 2011).
Data analysis: The values obtained in experiments were presented as mean ± standard deviation (SD). One-way analysis of variance (one-way ANOVA) was applied followed by Duncan's multiple range test (Duncan 1955) in order to compare the significance of values between different groups. The analysis of the data was conducted by the SPSS software (version 16, SPSS, Chicago, USA). The level of significance was set at P-value < 0.05.
Results
Antimicrobial effect of NPs: The results showed that the suspension solutions of ZnO, Fe₂O_3, and MgO NPs did not affect the growth rate of E. coli and S. aureus in a solid medium.
MIC determination of NPs: The results indicated that the MIC value for MgO NPs against E. coli was 2.5 mg/ml, which showed a potent inhibitory effect on bacterial growth. The MIC value for ZnO NPs against E. coli was 4.5 mg/ml, indicating a moderate inhibitory effect on bacterial growth. The MIC value for Fe₂O₃ NPs against E. coli was 80 mg/ml, exhibiting a weak inhibitory effect on the growth of the bacteria. Our findings revealed that ZnO and MgO NPs had acceptable impacts on inhibiting the growth of S. aureus. The MIC values for MgO and ZnO NPs against S. aureus were 2 mg/ml and 2.2 mg/ml, respectively. It can be concluded that both MgO and ZnO NPs have the same potency against the bacterial growth when applied for the infection caused by S. aureus. The MIC value for Fe₂O₃ NPs against S. aureus was 45 mg/ml, showing a weak potency to inhibit the growth of S. aureus.
MBC determination of NPs: The result showed that the MBC values of MgO, ZnO, and Fe₂O₃ NPs against E. coli were 3 mg/ml, 25 mg/ml, and 90 mg/ml, respectively. Furthermore, the MBC values for MgO, ZnO, and Fe₂O₃ NPs against S. aureus were 2.5 mg/ml, 5 mg/ml, and 50 mg/ml, respectively. According to the results of the MBC, it seems that S. aureus is more susceptible to be influenced by NPs.
The effect of NPs and heating process on milk: Figure 1 illustrates the survival of E. coli exposed to MgO, ZnO, and Fe₂O₃ NPs, as well as the heat or combination of NPs and heat. The treatment of milk with MgO NPs significantly decreased the growth rate of E. coli (P < 0.05); however, application of ZnO and Fe₂O₃ NPs did not considerably reduce the growth of E. coli as compared with the control sample for 24 h (P > 0.05). Additionally, co-treatment of milk with the heat and MgO, ZnO, and Fe₂O₃ NPs had synergistic effects on inhibition of the bacterial growth when compared with the individual treatment (P < 0.05).
Figure 2 depicts the survival of S. aureus treated with MgO, ZnO, and Fe₂O₃ NPs, as well as the heat or in the combination of heat and NPs. Similar to E. coli, the efficacy of the anti-staphylococci activity of NPs was dependent on the type of NPs and temperature. Our findings showed that combinatory treatment of milk with heat and NPs enhanced the antibacterial activity of MgO, ZnO, and Fe₂O₃ NPs against S. aureus significantly (P < 0.05). Furthermore, S. aureus was more prone to be affected by treatment with MgO, ZnO, and Fe₂O₃ NPs when compared with E. coli. This effect would be more tangible when the same amount of NPs or temperatures (during 8-24 h) was exposed to both types of bacteria, leading to a further decrease in the growth rate of S. aureus.
Analysis of cell morphology by scanning electron microscopy (SEM): Considering the results obtained from the SEM analyses, the morphological features of S. aureus exposed to MgO NPs were significantly distorted since some irregularities and heterogeneities were evident in this bacterium (Figure 3b). Conversely, S. aureus exposed to the control colloidal solution exhibited a regular shape as well as a smooth surface with spherical grape-like clusters (Figure 3a). Most of S. aureus treated with MgO NPs were completely ruptured as depicted by the atypical and disintegrated cell wall. However, some bacteria treated with MgO NPs only showed some abnormalities in bacterial shape.
Figure 4 displays the SEM analyses of E. coli that received no treatment (Figure 4a) and bacteria treated with MgO NPs (Figure 3b) for 18 h in TSB. Regarding the control bacteria, the untreated bacteria showed rod-like forms in regular sizes with an intact cell wall (Figure 4a). Treatment of bacteria with MgO NPs impaired E. coli and caused some deformities in cell surface and homogeneity of the cell wall significantly (Figure 4b).