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Effect of Ti[O.sub.2] nanoparticle on the Listeria and Pseudomonas bacteria segregated from raw cow milk.

INTRODUCTION

As the increasingly developing of food production, distribution and processing, increasing food safety has been also critically important. Nanotechnology is one of the new methods to enhance food safety. The nanotechnology-based products not only are healthier and safer, but also inexpensive with higher quality [1]. Nanotechnology can be used in food processing and perseverance, coverage (using nanoparticles), disinfecting food manufactures' equipments (by antimicrobial nanoemulsion) as well as in making biosensors to diagnose pathogenic impurities (pathogens) [2]. Nanotechnology, particularly in food packaging, can be widely applied in controlled microbial growth and smart packaging [3].

Applying nanoparticles in packaging is considered as one prevention way of food spoilage which initially requires assessing its effectiveness [4]. Using nanoparticles with various features is among the applications of nanotechnology in different sciences and industries including food industry [5]. Regarding high potential of food industry in using nano knowledge, nanoparticles metal oxides can be utilized in antiseptic and germicide systems. Ti[O.sub.2] nano-particles are one of the most common semiconductor nanoparticles with special characteristics of hydrophilic and photo-catalytic, UV absorbent and antibacterial which can be widely used in making polymeric nano-composites focusing on food packaging [6]. Ti[O.sub.2] nano-particles as a photo-catalyst effect on most environmental pollutants not only eliminate them, but also convert them into environmental compatible products. This compound is cheap, high-yielded and is extremely used in industrial technology to remove pollutants since its recycle option. So, Ti[O.sub.2] can be used as a proper substance to remove environmental pollutants such as non-renewable and toxic organic materials, different heavy metals from sewage, drinking water filtration, and bacteria, fungus and viruses demolition [7]. Of these protecting food against microorganisms' spoilages is one of the new goals in applying the nano scale of these particles.

Milk and its products by having valuable sources of Zerotein, vitamin, salts and fat, in addition to other rich food elements is a perfect place for growing many microbes [8]. Recently, it has been reported a strong relation between presence of some bacteria especially Listeria and Pseudomonas in infection resulted from using dairy products in comparison to other food products [9]. Little studies have been done on antimicrobial and inhibitive features of Dioxide Titanium photo-catalyst. Jung et al, studying Titanium nanoparticles' antimicrobial individually and doped with silver behavior, reported that as nanoparticles' concentration increases in the medium, higher inhibition can be seen in the growth of microorganisms. The study revealed that more contact area, collision possibility and more penetration of nanoparticles at higher concentrations critically influences on creating antimicrobial property [4]. Saadatmand et al also studied Chitosan- Ti[O.sub.2] antimicrobial characteristic and its application on the hospital sterile gauze and reported that at the given concentrations almost 100% of the bacteria's growth will be suspended as the presence of this titanium-contained composite [10]. Using various forms of Ti[O.sub.2] nano-particles can also be seen in the studies of some researchers on teeth microbial flora and /or dentistry equipments. For instance, Elsaka et al experiments can be mentioned that tested the antibacterial feature of photo optical glass ionomer containing various amounts of Ti[O.sub.2] through studying its effect on the growth of Streptococcus mutans. The results indicated that there is only a small difference between employed nanoparticle treatments with control medium in terms of inhibiting microbial growth [11].

The potential effects of chemical factors and toxic compounds can be determined through using different creatures. Alga, nematodes as well as crustaceans determined Ti[O.sub.2] nanoparticles toxicity. However, as more sensitive animals can provide higher confidence in toxicity determination tests; so, studying toxicity using bacteria can, indeed, help in determining nanoparticles' toxicity. On the other hand, as the ultimate goal of toxicity determination is to be extended to human beings; hence, studying toxicity using different animals can enhance the confidence coefficient of the previous experiments. Listeria and Pseudomonas among bacteria are considered as the critical species in forming some transferring pathogens through food due to high-resistant characteristics outbreak in milk, unfavorable spoilage and tolerating maintenance and cooling conditions as the two groups of positive and negative warm bacteria. Thus, the present research tried to study the inhibitive effect of Ti[O.sub.2] nanoparticles onListeria and Pseudomonas pathogens.

MATERIALS AND METHODS

2.1. Food substances:

This research used raw (uncooked) cow milk produced by several ranches and yoghurt centers in Gonbad-eKavous, Golestan County, within June to September in 2014.

2.2. Microorganisms:

As psychrophilic microorganisms are regarded as the major causes of milk spoilage and some even survive in pasteurization temperature by producing heat resistant enzymes [12]; therefore, this research tested the isolation and identification of two groups of these microorganisms, through routine experimental tests and mediums, including Listeria and Pseudomonas that cause undesired milk color and taste and reduced temperature stability as well as digestive and other acute diseases to study the effect of dioxide titanium nanoparticles [13-14]. All the mediums used in this research were made by Merck Company.

2.3. Nanoparticles:

Ti[O.sub.2] nanoparticles with the approximate 100 nm diameter were purchased from Sigma Aldrich Company.

[FIGURE 1 OMITTED]

10 g/lit stock solution of this nanoparticle was exposed to UV beam (260 nm) for 30 min to be activated; and then, was applied in the desired concentrations.

3. Methodology:

The sensitivity of the microorganisms under study to nanoparticles was determined through two standard methods of determining minimum growth inhibition concentration (MIC) and minimum bacteria static concentration (MBC). So, a microbial suspension of a 24-hour incubation of each bacterium was initially provided in the sterile physiology serum such that the turbidity of the standard Mac Farland 0.5 tube (equal to 1.5 x [10.sup.8] bacteria/ milt) is obtained. Next, by preparing serial attenuation of Ti[O.sub.2] particles in the liquid medium, the tubes containing medium and nanoparticle at 10000, 5000, 2500, 1250, 625, 156.25, 78.12, 39.06 and 0 ppm (control group) concentrations were provided to investigate the effect of Ti[O.sub.2] on the growth and surviving strength of the target microorganisms. MIC was determined by a spectrophotometer method through reading each tube absorbance in 600 nm wavelength. Hence, after injecting 0.1 ml bacterium suspension into the tubes and the incubation at 37[degrees]C on the shaker at 200 rpm, each tube density level (OD) was registered within a 2hour period for 24 hours and the microbial growth curve was plotted [15]. In the next step, following the minimum bacteria static concentration (MBC) was determined through measuring turbidity; the homogenous incubation was conducted on the plate agar to obtain MBC. 0.1 ml of the inhibitive tubes was injected on the agar Hinton Muller plate surface and uniformly distributed. Presence or absence of colonies and their numbers were observed and assessed followed by 24 hours incubation [16].

RESULTS AND DISCUSSION

Following the two materials of Listeria and Pseudomonas were identified and isolated as the critical bacteria in milk contamination as a food and employing several doses of Ti[O.sub.2] nanoparticles, the obtained data were measured and registered. Regarding the fixed bacterium number rate over time after suspension injection for 24 hours at different Ti[O.sub.2] concentrations, the results showed that 5000 ppm concentration equal to 1.2 tube turbidity was the minimum inhibitive concentration of Pseudomonas growth; in addition, this bacterium was unable to raise population at this and higher concentrations which the 1 tube turbidity was equal to 10000 ppm (Fig. 2).

[FIGURE 2 OMITTED]

A slight increase in the Pseudomonas growing was recorded at 1250 and 2500 ppm concentrations which approximately showed a similar performance. Increased population of this microorganism was significant from 625 ppm down to zero concentration within 24 hours which was less in comparison to control group. Ti[O.sub.2] nanoparticles presented more inhibitive effects on Listeria rather than Pseudomonas within a 24-hour period. This evaluation was seen as the minimum inhibitive concentration of this bacterium growth through recording 625 ppm concentration of Ti[O.sub.2] equal to 1.16 (Fig. 3).

Thus, Ti[O.sub.2] particles were much effective in the Listeria growth inhibitive effects' outbreak, occurred by employing 625 ppm concentration, as compared to Pseudomonas with the Ti[O.sub.2] MIC of 5000 ppm.

Bacteria injection was conducted in agar medium, in the next phase, to study Ti[O.sub.2] nanoparticles microbial strength on the food pathogens of this test and to determine MBC through 6 elementary tubes containing the highest nanoparticle concentration in the liquid medium. The results indicated that none of Ti[O.sub.2] concentrations being applied for antibacterial property has the ability to demolish the raw milk segregated bacteria; moreover, it can merely restrain their growth at certain concentrations. Hence, the injected bacteria raised in all agar incubation mediums used for MBC determination and demonstrated their survival potential at the presence of Ti[O.sub.2] nanoparticles.

[FIGURE 3 OMITTED]

Comparing the performances of two Listeria and Pseudomonas bacteria, being segregated from raw milk, revealed that Listeria inhibitive range of growing was larger than Pseudomonas at the presence of Ti[O.sub.2] nanoparticles. The required Ti[O.sub.2] concentration to influence on Listeria and Pseudomonas bacteria was assessed 625 ppm and 5000 ppm, respectively. Pseudomonas is a negative gram organism covered by a two-layered Lipopolysaccharide; whereas, Listeria is a positive gram bacterium. There are seen many reports of higher resistance of Pseudomonas species comparing to other studied bacteria groups to antimicrobial compounds and factors [17-19]. Furthermore, Listeria has been known as food pathogens, in recent years, particularly in milk and dairy products [20].

It is critically important to determine the milk microbial load whether it is raw or pasteurized. The large amount of milk is processed turning into yoghurt and cheese and the milk initial microbial load may also influence on these products. Moreover, less milk microbial load after pasteurization can lead to longer maintenance even up to 6 months [9]. Of these Ti[O.sub.2] is a nanotechnology product which one of its important application is removing food spoilage microorganisms and hygiene especially in drinking water. Ti[O.sub.2], as an especial organic additive, possess considerable characteristics such as chemical stability, biologic compatibility as well as non-toxicity [21-22]. Producing the substances with active oxygen such as hydrogen peroxide is regarded a probable mechanism to justify Ti[O.sub.2] nanoparticles antibacterial property which cause preventing microbes' growth. The performance reaction of Ti[O.sub.2] nanoparticles versus bacteria results from hydrogen peroxide and other active oxygen [23].

Peroxide or the produced active oxygen react to bacterium coverage components (such as proteins) and or bacteria's free ions producing hydroxyl free radicals which prevent bacteria growth interfering with their survival. This study, also, verified preventing bacteria growth at the presence of Ti[O.sub.2] nanoparticles. Hence, data consistency can be justified in titanium nanoparticle antibacterial outbreak. As it was previously mentioned, the Pseudomonas bacterium segregated from milk samples represented a higher inherent resistance to Ti[O.sub.2] nanoparticles comparing to Listeria. Pseudomonas was also enable to grow in nanoparticle environment up to 5000 ppm concentration; while, Listeria growth was inhibited at 625 ppm concentration of nanoparticle in incubation. Therefore, it can be stated that nanoparticles' higher MIC in Pseudomonas comparing to Listeria is probably due to this bacterium low penetration into external substances.

The structural difference of positive and negative gram bacteria can be considered as a factor of sensitivity to antimicrobial factors. Negative gram bacteria have a two-layered wall with a Lipopolysaccharide film providing the possibility of more strength for organism. Some also reported positive gram bacteria more resistant to antimicrobial causes as their thick peptide glycol wall. Selvam et al, in this regard, studied the effect of nanoparticles on two positive and negative gram bacteria and reported a higher relative sensitivity of positive gram bacteria to nanoparticles' presence [24].

Other scholars, studying nanoparticles antibacterial effects, also presented reports in contrast to the high resistance of negative gram bacteria against positive grams [25]. However, generally, Pseudomonas is known as the most resistant and less-needed bacteria up to now. It is able to grow in hard and stressed conditions; beside, it is believed that it can grow in the distilled water, too [26]. Thus, its high potential in resisting Ti[O.sub.2] nanoparticles can be justified in this study. It is necessary to mention that the applied concentration of Ti[O.sub.2] nanoparticle to suspend Pseudomonas growth and activity in this study was slightly higher than the same concentration for Listeria; whereas, in particular, it can be easily understood that the applied value of Ti[O.sub.2] to inhibit Pseudomonas, regarding its high resistance, is completely logical. However, studying nanoparticles' microbial power in the solid condition after 24 hours revealed that both bacteria have the growth potential in the agar medium making many colonies on the agar plate. So, it is obviously understood that Titanium nanoparticles are only able to inhibit growing milk spoilage organisms lacking the required potential to kill these organisms at the used concentrations. Interpreting the results leads us toward this point that Ti[O.sub.2] nanoparticles presence, even at small concentrations, can be followed by bacteria's higher retardation phase and longer generation time which is justified by nanoparticles especial mechanisms in preventing microbial growth. Saadatmand et al. [27] studied Ti[O.sub.2]- Chitosan nanocomposite antimicrobial feature and its application on the hospital sterile gauze and reported that almost 100% of bacteria growing, at the given concentrations, are suspended at the presence of this titanium composite [10]. Using various forms of Ti[O.sub.2] nanoparticles can be observed in the studies of some scholars on teeth (oral) microbial flora and or dentistry equipments. Among these, Elsaka et al (2011) experiments can be mentioned that tested the antibacterial feature of optical glass ionomer containing various amounts of Ti[O.sub.2] through studying its effect on the growth of Streptococcus mutants. The results revealed that there is only a slight difference between the employed nanoparticle treatment and control group in terms of inhibiting microbial growth. Perhaps it depends on the diffusion disc methodology being applied in their study.

The present research methodology was dilution broth focused on determining growth MIC. This method was more precise than other methods such as disc or sink and higher sensitive since not only uniformly distributes antimicrobial materials in the medium, but also intensifies the possibility of microorganisms' encounter and more effective inhibition. Further, Nonami and Funakoshi assessed the effect of nano Ti[O.sub.2] coverage on self-cleaning and antibacterial property of dental mirror surfaces and reported positive results [28].

5. Conclusion:

According to this research goals including studying the possibility of applying Ti[O.sub.2] nanoparticles in milk maintenance, pasteurization and packaging, it can be expressed that the aforementioned nanoparticles showed positive significant effects on the two segregated milk-bacteria which are regarded as the most possible spoilage causes in prolonged maintenance in cold places. This inhibition was observed more in Listeria bacterium which is highly important according to its higher frequency in milk samples. Therefore, further researches can test its application in pilot test through combining nanoparticles or other antimicrobial elements in order to expand Ti[O.sub.2] nanoparticles efficacy achieve the highest level and to consider the economic and commercial advantages of this process by reducing the nanoparticles and other used compounds doses.

ARTICLE INFO

Article history:

Received 26 September 2014

Received in revised form 20 November 2014

Accepted 25 December 2014

Available online 10 January 2015

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(1) Zahra Samei, (2) Mohammad T. Baei, (3) Hadi Koohsari

(1) Department of Chemical Engineering and Food Technology, Azadshahr Branch, Islamic Azad University, Azadshahr, Golestan, Iran

(2) Department of Chemistry, Azadshahr Branch, Islamic Azad University, Azadshahr, Golestan, Iran

(3) Department of Microbiology, Azadshahr Branch, Islamic Azad University, Azadshahr, Golestan, Iran

Corresponding Author: Mohammad T. Baei, Department of Chemistry, Azadshahr Branch, Islamic Azad University, Azadshahr, Golestan, Iran.

Ph: (+98) 9111751399. E-mail: Baei52@yahoo.com
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Author:Samei, Zahra; Baei, Mohammad T.; Koohsari, Hadi
Publication:Advances in Environmental Biology
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Date:Nov 1, 2014
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