ЗВІТ З НДР 29-81 ЗА 2007 – 2009 Р 306 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XV, Issue 4 – 2016, pag. 306 - 311 NAN OSIZED TITAN IUM D IO XIDE AS AN A NTIBA CTER IAL ADMIXTUR E FOR FOO D PACK AG IN G MATER IA LS *Igor KOBASA1, Mariia VOROBETS1, Larysa ARSENIEVA2 1Yu. Fedkovych National University of Chernivtsy, Ukraine, * imk-11@hotmail.com 2National University of Food Technologies, Kyiv, Ukraine * Corresponding author Received 15th November 2016, accepted 29th December 2016 Abstract: The article deals with research on antibacterial activity of the composite materials based on titanium dioxide. The influence of synthesis condition on phase composition of titania as well as its photocatalytic activity and antibacterial effect against the pathogenic microbes such as Staphy- lococcus aureus and Escherichia coli have been investigated. It was proved that the structure of nanosized titania samples synthesized by the high temperature hydrolysis of titanium tetrachlo- ride in the air-hydrogen atmosphere at 700-1100 0C is non-stoichiometric. These samples consist of both anatase and rutile crystalline modifications and exhibit comparatively high antibacterial activity. Keywords: TiO2, photocatalytic activity, antimicrobial activity, Staphylococcus aureus, Escherichia coli. 1. Introduction Contagious diseases set acute challenge for the health care field all over the world de- spite rapid progress achieved in pharmacy and new drugs research and development [1]. That is why special attention is paid to the search of new technological solutions related to antibacterial protection. In this context, it seems topical to design and de- velop new non-toxic packaging materials providing protection of food from patho- genic microbes and other adverse effects during the production and storage stages. Main efforts are being spent to develop- ment of the antibacterial composite materi- als to be used in the bactericide coverings for medical, pharmaceutical, sanitary- technical and household needs. It has been reported that some antibacterial glass/enamel coverings were synthesized on the base of copper and silver com- pounds [2], titanium dioxide [3] and calci- um hydroxylapatite [4, 5]. Other authors reported new antibacterial composite mate- rials containing the ions of some noble or heavy metals Ag+, Au3+, Hg2+, Cd2+, Cu2+ [6] or nanosized particles of silver and gold [7, 8]. TiO2 nanoparticles have been recently pro- posed as a source material for synthesis of the composites with high antibacterial ac- tivity [9]. Such composites can be used as components of antimicrobial ceramics, lacquer/dye coverings or microbe-proof packaging. It is known [10-12] that antibacterial and photocatalytic activity (PA) of TiO2 can vary depending on the conditions of its synthesis, temperature of preliminary treatments, nature and concentrations of the admixtures added to the source synthe- http://www.fia.usv.ro/fiajournal mailto:imk-11@hotmail.com Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XV , Issue 4 – 2016 Igor KOBASA, Mariia VOROBETS, Larysa ARSENIEVA, Nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, Food and Environment Safety, Volume XV, Issue 4 – 2016, pag. 306 – 311 307 sis composition, specific surface area, presence or absence of the crystalline in- clusions, TiO2 structure defects, their na- ture and concentration. Therefore, it is pos- sible to govern photocatalytic and antibac- terial activity of TiO2 through changes in these parameters and obtain either highly or moderately active products. It is known that anatase is the most photo- catalytically active modification of titania (rutile and brookite are two other modifica- tions) [13]. Different crystal lattices, vari- ous degree of the lattice imperfectness, presence of some admixtures can be men- tioned as the factors causing different pho- tocatalytic activity of various titania modi- fications. TiO2 is one of the semiconduct- ing photocatalysts used most widely be- cause of stability of its aqueous mixtures, cheapness, non-toxicity and high activity of the material that is based on strong oxi- dation efficiency of the photogenerated holes. Besides, TiO2 is registered as a food additive E171. This interest towards TiO2 has initiated numerous investigations aimed onto development of the TiO2-based materials with predefined photocatalytic and antibacterial characteristics, phase composition and texture. Our efforts also followed this direction and were targeted onto synthesis of the TiO2 material with some required properties. This paper reports results of investigation of the activity of the anatase and rutile modifications of titania and the non- stoichiometric product containing a mix- ture of anatase and rutile that has been car- ried out in order to develop the antibacteri- al packaging materials. 2. Experimental Three kinds of the TiO2 samples were used in this work: (1) nanosized titania synthe- sized by the high temperature hydrolysis of titanium tetrachloride vapors in the air- hydrogen flame at 700-1100 0C according to [13], specific surface area (SBET) = 50 m2/g; the anatase (2) and rutile (3) modifi- cations of TiO2 with SBET of 20 and 10 m2/g respectively, which have been syn- thesized by the low temperature hydrolysis of titanium chloride and sulfate [10]. The phase composition and particles size of all the samples were checked prior other in- vestigations. The powder surface was analyzed with X- ray Photoelectron Spectroscopy (XPS) also referred to as Electron Spectroscopy for Chemical Analysis (ESCA) using ESCA- LA™ XI+ X-ray Photoelectron Spectrome- ter Microprobe. XPS is an elemental anal- ysis technique which is capable of detect- ing all elements except for H and He and it has a nominal detection limit of ~0.1 at- om%. Spectral interferences may prohibit the detection of some elements in relative- ly low concentrations. Samples were measured at a 90° Take-Off-Angle yield- ing a sampling depth of ~10 nm. The anal- ysis area was ~500 µm in diameter. Anal- yses were performed with a monochro- matic Al kα x-ray source. The powder par- ticle size analysis was performed with a Field Emission Scanning Electron Micros- copy (FESEM) using Hitachi SU70 Elec- tron microscope. FESEM images depict topographic features of the sample surface. FESEM imaging was performed at 2 keV. One hundred particles of each powder were measured to provide an average par- ticle size. Both powders were coated with ~100 Å of gold to facilitate analysis. A reaction of methylene blue reduction has been used to measure photocatalytic activi- ty of the samples [14]. Antibacterial activi- ty of the samples has been investigated us- ing the diffusion method (also known as the disks method) according to the stand- ard ISO 27447:2009(E) and methods [4]. The bacterial colonies were cultivated from the ATCC standard strains Staphylo- coccus aureus and Escherichia coli while a paper disk impregnated with antibiotic no- vobiocin was used as a control experiment. Antimicrobial activity has been determined Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XV , Issue 4 – 2016 Igor KOBASA, Mariia VOROBETS, Larysa ARSENIEVA, Nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, Food and Environment Safety, Volume XV, Issue 4 – 2016, pag. 306 – 311 308 by the diffusion (“holes”) method accord- ing to the standard ISO 27447:2009(E) as described in details below. The inoculation material was prepared as follows: fresh bacterial colonies were tak- en after the 18 hours of growing in the sterile physiological medium (0.85 % NaCl) in such a way to ensure formation of the suspension with 0.5 McFarland’s den- sity degrees, which is approximately equal to 1-2·108 CFU/ml. Then the material was ten times diluted forming the 0.05 degrees system and then ten times again to form the 0.005 degrees system. Density of the systems has been determined nephelomet- rically. The latter (0.005 degrees) system was used for the next investigations. A cot- ton tampon with the inoculation material was immersed in the sterile solution, ex- tracted and squeezed to remove the exces- sive solution. Then the inoculation material was seeded on the substrate by triple appli- cation of the tampon with rotation 600 after each application. The substrate plates were dried and then 50 μl of various nanodis- persed TiO2 suspensions were added to investigate their antimicrobial activity. Each sample consisted of 0.1; 1.0; 5.0 and 10.0 % suspensions of the nanosized tita- nium dioxide. All the suspensions were prepared on the basis of physiological so- lution. The probes were placed into the in- cubator and stayed in the oxygen contain- ing medium under 370 for 16-18 hours. Then the diameter of the microbes deceler- ated growth area was measured as the source parameter to evaluate the antimi- crobial activity. The standard disk impreg- nated by novobiocin (5 μg) was used as the reference sample of the antimicrobial ac- tivity. 3. Results and Discussion It was found that phase composition and photocatalytic activity of the TiO2 samples depend on the method used to synthesize each sample (see Table 1). The pyrogenic TiO2 obtained by the high temperature synthesis from TiCl4 vapors is a highly defective material that contains both rutile and anatase crystalline modifi- cations. There are many paramagnetic cen- ters in such material, which serve as active photocatalytic centers [15]. The size of these particles ranged between 45 to 174 nm (see Fig. 1). A mechanism of photocatalytic and anti- bacterial activity of nanosized TiO2 de- pends on the process or origination of the photoexcitated electrons initiated by the light quanta. These electrons can be trans- ferred from the allocated valence band of a semiconductor into its unallocated molecu- lar orbital in the conductivity band. A pair electron-hole (е-–h+) is formed as a result of these processes and then further trans- formations can run in different directions [16]: - photoexcitation: TiO2 + hν → e- + h+; - absorption of electron by oxygen:   22 OeO ; - ionization of water: H2O → OH- + H+; - peroxide formation by protonation of the ion-radical:   HOOHO2 . The hydroperoxide radical formed in the latter process can take part in the following processes: HOO• + e- → HO2- or HOO- + H+ → H2O2. Therefore, the processes of oxidation and reduction can run simultane- ously on the surface of photoexcitated semiconducting photocatalyst. An elec- tron-hole recombination can take place if oxygen is capable to accept electrons and form peroxide ( 2O ), which participates in the follow-up protonation and form hy- dro peroxide radical ( HOO ) and then hydrogen peroxide (Н2О2). Particles sizes for the anatase (2) and rutile (3) samples of TiO2 synthesized by the liq- uid phase hydrolysis are 0.2 and 0.5 m simultaneously, these samples are low- defective and exhibit low PA. As it will be explained below, phase composition and structure of TiO2 also provides a signifi- Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XV , Issue 4 – 2016 Igor KOBASA, Mariia VOROBETS, Larysa ARSENIEVA, Nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, Food and Environment Safety, Volume XV, Issue 4 – 2016, pag. 306 – 311 309 cant influence on antibacterial activity of the titania-based composites. For instance, no antibacterial activity has been found for the TiO2 samples synthesized by the liquid phase hydrolysis of titanium chloride and sulfate. Table 1 Photocatalytic activity and phase composition of the TiO2 samples synthesized by various methods Synthesis method Phase composi- tion PA*, mg/(ml×min×m2) Liquid phase hydrolysis of TiCl4 anatase 1.2×10-2 ± 0.05 Liquid phase hydrolysis of Ti2(SO4)3 rutile 1.7×10-3± 0.05 High tem- perature hydrolysis of TiCl4 vapour anatase 50 % + rutile 50 % 1.2 ± 0.09 * - all the data are obtained in the 5-repetitions series and the mean values with standard errors are shown in the column As seen from the antibacterial activity in- vestigation results (see Fig. 2), the colony extension inhibition area depends on TiO2 concentration in suspension. Figure 1. SEM image of the TiO2 sample. Antibacterial activity of TiO2 suspensions containing 1.0, 5.0 and 10.0 wt % of TiO2 as against the gram-negative microbes Escherichia coli is higher than that of no- vobiocin (see Fig. 2, C and D); the novobi- ocin deceleration area diameter was of 13 mm, while the nanosized TiO2 suspensions areas were 7, 15, 16 and 20 mm for TiO2 concentrations 0.1; 1.0; 5.0 and 10 % re- spectively. Therefore, three later composi- tions exhibit higher antibacterial activity than antibiotic novobiocin. Antibacterial activity of TiO2 suspensions containing 5.0 and 10.0 wt % of TiO2 against the gram-positive microbes Staphylococcus aureus is higher than that of novobiocin (see Fig. 2, C and D): the novobiocin deceleration area diameter was 12 mm while the nanosized TiO2 suspen- sions areas were 1, 7, 13 and 15 mm for TiO2 concentrations 0.1; 1.0; 5.0 and 10 % correspondingly. Therefore, two later compositions exhibit higher antibacterial activity than antibiotic novobiocin. А B C D E Figure 2. Antibacterial activity of various com- positions against Escherichia coli: А – 0.1, В – 1.0, С – 5.0 and D – 10 % of TiO2; E – novobiocin. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XV , Issue 4 – 2016 Igor KOBASA, Mariia VOROBETS, Larysa ARSENIEVA, Nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, Food and Environment Safety, Volume XV, Issue 4 – 2016, pag. 306 – 311 310 The activity rises with increase in the con- centration so that antibacterial activity of the 1.0 % TiO2 suspension is approximate- ly similar to that of the standard sample test performed with antibiotic novobiocin (5 μg) (Fig. 2 E). Moreover, antibacterial activity of the 5.0 and 10.0 % TiO2 suspen- sions in relation to Escherichia coli (Fig. 2 C, D) is higher than that of novobiocin. Antibacterial activity of the nanosized tita- nia-based compositions against gram- positive Staphylococcus aureus cultivated in the beef-extract agar is less noticeable than the activity against Escherichia coli. Only after the concentration of TiO2 in the suspensions becomes equal to 5.0 %, the activity reaches same value as for the ref- erence novobiocin sample (Fig. 3). A B C D E Figure 3. Sensitivity of Staphylococcus aureus to the nanodispersed suspensions of TiO2 with con- centrations: А – 0.1, В – 1.0, С – 5.0 і D – 10 %; E – novobiocin. As seen from analysis of experimental re- sults of the TiO2 samples antibacterial ac- tivity investigation, only nanosized titania reveals some activity against pathogenic organisms. That is why such material can be used in further antibacterial research as a component of special bactericide cover- ings. In this context, the antibacterial activity of TiO2 samples was tested by adding 15 wt % of this source material to acryl-based lacquer/dye compositions. An aqueous suspension of the composition was applied by spraying over the metal surface in such a way to ensure formation of a thin homo- geneous layer that covers the entire sur- face. Then the covering was dried in the open air at the room temperature during 24 hours and used to carry out the antibacteri- al activity test according to ISO/WD 17094: “Fine ceramics (advanced ceramics , advanced technical ceramics) – Test method for antibacterial activity of semi- conducting photocatalytic materials under indoor lighting environment” [17]. All samples were preliminary disinfected by 70 % ethanol and then kept under the fol- lowing incubation conditions: 4 or 8 hours at 25 0C, humidity 65 %. Illumination in- tensity was 1000 lx. All the coverings re- ceived by this method ensured compara- tively high antibacterial activity against pathogenic microbes Escherichia coli and Staphylococcus aureus. It was found that 99.4 and 93.7 % of the above colonies re- spectively were extinguished after 4 hours long contact with the surfaces containing the nanodispersed TiO2 while 8 hours long contact caused total extinguishing of the colonies of both bacterial species. No anti- bacterial activity has been revealed in the control experiments with the same cover- ings containing no TiO2. That is why it can be assumed that the non- toxic bactericide packaging materials can be obtained by adding some TiO2 as a source component. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XV , Issue 4 – 2016 Igor KOBASA, Mariia VOROBETS, Larysa ARSENIEVA, Nanosized titanium dioxide as an antibacterial admixture for the food packaging materials, Food and Environment Safety, Volume XV, Issue 4 – 2016, pag. 306 – 311 311 4. Conclusion The TiO2 samples synthesized by high temperature hydrolysis of TiCl4 vapors in the air/hydrogen mixture have shown high antibacterial activity against the standard strains of Staphylococcus aureus and Escherichia coli (total extinguishing of both microbial colonies has been achieved after the 8 hours long application of the TiO2 samples). A comparatively high antibacterial activity has also been registered by the acryl-based lacquer-dye compositions containing some nanosized TiO2. These materials can po- tentially find wide applicability in the area of food packaging materials production. 5. References [1]. K.E. JONES, N.G. 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VINGOPAL. Photochemistry on surfaces: photodegradation of 1,3- diphenylisobenzofuran over metal oxide particles. J. Phys. Chem. 96, (12), 5053-5059 (1992). [17]. ISO 17094:2014: Fine ceramics (advanced ceramics , advanced technical ceramics) – Test method for antibacterial activity of semiconducting photocatalytic materials under indoor lighting environment http://www.iso.org/iso/iso_catalogue/catalogu e_tc/catalogue_detail.htm?csnumber=58094 http://link.springer.com/journal/10789 http://link.springer.com/journal/10789 http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=58094 http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=58094