**2.1 Mechanistic action of TiO2-NPs antimicrobial activity**

TiO2-NPs are the photocatalysts used to destroy unwanted organic compounds in the air, water, soil, and, more recently, in food [21].

Photocatalysis can be defined as the catalyst-driven acceleration of a lightinduced reaction [49–52]. Homogeneous and heterogeneous photocatalytic processes utilize metal complexes (transition metal complexes like iron, copper, chromium, etc.) and semiconducting materials such as TiO2, ZnO, SnO2, and CeO2 as catalysts. In the presence of light and heat, metal complexes become excited and form metal ion complexes, in contrast, semiconducting materials become excited due to the combination of electronic structures which is characterized by a filled valence band, empty conduction band, and light absorption properties, resulting in the generation of reactive oxygen species (ROS) or hydroxyl radicals. These hydroxyl radicals inflict damage to microbial cells [49–51, 53–55]. The subsequent hole in the valence band could further react with H2O in the grains or hydroxide ions adsorbed on the surface of TiO2-NPs to generate hydroxyl radicals (OH•), with electron in the conduction band reduce O2 to superoxide ions (O2 <sup>−</sup>) [21]. Gogniat and Dukan [56] demonstrated that DNA was denatured by hydroxyl radicals generated via the Fenton reaction resulting in cell death.

Electron paramagnetic resonance (EPR) spectroscopy study confirmed the photoproduction of hydroxyl radicals (OH•) from different TiO2. The efficiency of hydroxyl radical generation depends on the source/origin of TiO2 [57].

Cells are negatively charged [58] under optimum physiological condition due to heparan sulfate proteoglycans [59]. However, disease could trigger the cells to synthesize certain compounds which cause cell surface to become positively charge. Microbial cell could act as a hole for electron transfer between organism and its components [60]. The iron cluster on cell surface, in the periplasmic space, or inside the cell (proteins (such as ferritin)), could act as a precursor for iron-catalyzed Haber-Weiss reaction, which generates additional hydroxyl radicals in the presence of H2O2 and the superoxide ion [61].

Different treatments (photocatalysis, water, TIO2, UV-A) applied to elucidate the effects of lipid peroxidation on *S*. *cerevisiae* revealed high malondialdehyde (MDA) in TiO2 -treated subjects with 2 hours. The results demostrated that TiO2 was sufficient to damage membrane, thus interfered with permeability of the cell which led to the leakage of vital intracellular molecules (**Figure 1**) [48]. Similarly, Draper and Hadley [62] found photocatalysis-induced cell wall damage on S. cerevisiae [48]. This may decrease intracellular enzymatic activity as well as leaking of amino acids and NH4 + , suggesting a drastic impact on proteins [63].

Cellular respiratory enzymes lost their activity after been exposed to irradiated TiO2 (0.5 mg/mL), and the kinetics correlated with the losses of cell viability. Furthermore, when glucose was used instead of succinate as the electron donor, similar effects were observed. From this outcome, Li et al. [78] proposed that ROS generated from an irradiated TiO2 surface, interacted with the polyunsaturated phospholipids in *E*. *coli*. Moreover, cell membrane structure was perforated due to lipid peroxidation creating a hole for more TiO2-NPs to pass into interior of the cell, thus rendering respiratory proteinsinactive and subsequent cell death.

A progressive decrease in esterase activity was observed after exposing *S*. *cerevisiae* to irradiated TiO2 [63]. Other researchers documented overexpression and inhibition (expressed at lower levels, including those encoding six cbb3-type cytochrome C oxidase subunits, an electron transfer flavoprotein, and

#### **Figure 1.**

*Schematic illustration of the solar photocatalytic process for microbial cell inactivation in the presence of an aqueous suspension of TiO2. Modified with permission from ref 4498160008350 [72]. Contact between the cells and TiO2-NPs affects membrane permeability; however, this is reversible. The availability of more NPs could enhance the damage to cell wall, thus allowing leakage of small molecules such as ions. Damage at this stage may be irreversible, and this accompanies cell death. Higher molecular weight components such as proteins could further be leaked followed by protrusion of the cytoplasmic membrane into the surrounding medium through degraded areas of the peptidoglycan and lysis of the cell. Intracellular components are then degraded progressively especially from the point of contact with photocatalyst, followed by complete mineralization.*

**139**

*The Potential Application of Nanoparticles on Grains during Storage: Part 2 – An Overview…*

two oxidoreductases) of genes associated with energy production and conversion processes. TiO2-NPs exerted a stimulating effect on the respiratory chain and the

Likewise, Matsunaga et al. [13] observed that incubating TiO2/Pt NPs under

Kubacka et al. [65] examined genome/proteome-wide expression profiles of *P*. *aeruginosa* PAO1 cells treated with TiO2-based nanocomposite films. An increase and decrease in the levels of 165 and 151 transcripts were respectively reported in cells with TiO2-coated Ethylene vinyl alcohol (EVOH) particles. Few proteins were detected at a statistically significant level (p ≤ 0.1) in cells treated with TiO2-coated EVOH particles compared to the control. TiO2-UV treatment significantly suppressed (from 5.4- to 15.1-fold) the expression levels of genes essential for cell wall. However, 14 genes encoding for lipid metabolism essential for cell membrane were over-expressed (from 5.6- to 23.0-fold), unexpectedly, 2 were expressed at a lower

In vivo and in vitro studies confirm that hydroxyl radicals inflict damage (breakage) on DNA strands. The extent of damage was minimized when dimethyl sulfoxide, catalase, or mannitol were incorporated in the reaction mixture [66]. However, the findings [66] contradicts previous studies [21, 67]. Exposing either purine or pyrimidine bases to TiO2 and light from a 100-W Hg lamp resulted in the detec-

subject to the same conditions, unknown peroxide species, along with phosphate and carbon dioxide, were detected, suggesting the breakage and mineralization of

Kikuchi et al. [67] demonstrated the role of ROS on photocatalytic bactericidal activity. They utilized a porous polytetrafluoroethylene (PTFE) membrane in their system to physically separate the *E*. *coli* suspension from the TiO2 thin. The results showed an impressive photokilling capability of the system with and without (control) PTFE - which was attributed to the generated H2O2. A group [69] demonstrated the stimulating effect of TiO2-NPs on lipolytic activity in *A*. *niger*. The results showed that TiO2-NPs significantly increased lipase biosynthesis (more than 1.5 times) compared to the control experiment. Treatment with TiO2-NPs (size: 40 nm, concentration: 10 mg/L) in all culture media, enhanced lipolytic activity by 78.57% and 57.49% on the 4th and 5th day of cultivation, respectively. This finding reaffirms that smaller NPs can penetrate the cell membrane easily than bigger NPs,

sugar-phosphate backbone of DNA and RNA molecules, respectively [68].

thus easily interact with molecular proteins, resulting in stimulating effects.

and bulk TiO2 against *Enchytraeus crypticus* with and without UV radiation.

nisms, suggesting that size of the TiO2-NPs contributes to biocidal activity.

Gomes et al. [70] assessed the effects TiO2-NMs (NM103, NM104, and NM105)

Microarray analysis revealed 10431 differentially expressed genes (DEGs) (*p* < 0.01) triggered as a result of exposure to TiO2-NMs under no-UV. All samples under UV exposure registered an up-regulation of several transcripts, including caspase apoptosis-related cysteine peptidases, a signature of apoptosis activation, whereas under darkness the apoptotic signaling pathway was inhibited, suggesting that the oxi-radicals generated during the photoactivation of TiO2 might substantially contribute to the apoptotic response and damage to the cell membrane. DNA damage was triggered after exposing samples to bulk/nano TiO2 [71]. However, the findings of Gomes et al. [70] contradicted the [71] as reported that TiO2-NMs\_under no-UV impaired DNA repair, while bulk\_TiO2 under no-UV activated DNA repair mecha-

ion. However, when native DNA and RNA molecules were

metal halide lamp irradiation with *E. coli, Ch. vulgaris, L. acidophilus*, and *S. cerevisiae* inhibited cell respiration mechanisms and subsequent cell death. However, the results were not consistent as *Ch*. *vulgaris* had a thick cell wall mainly composed of polysaccharides and pectin hence, had comparative advantages

*DOI: http://dx.doi.org/10.5772/intechopen.93213*

(protection) over the other microbes.

level (from 5.5- to 7.4-fold).

<sup>−</sup> and NH4

+

tion of NO3

electron transfer mechanism of the microorganism [64, 65].

#### *The Potential Application of Nanoparticles on Grains during Storage: Part 2 – An Overview… DOI: http://dx.doi.org/10.5772/intechopen.93213*

two oxidoreductases) of genes associated with energy production and conversion processes. TiO2-NPs exerted a stimulating effect on the respiratory chain and the electron transfer mechanism of the microorganism [64, 65].

Likewise, Matsunaga et al. [13] observed that incubating TiO2/Pt NPs under metal halide lamp irradiation with *E. coli, Ch. vulgaris, L. acidophilus*, and *S. cerevisiae* inhibited cell respiration mechanisms and subsequent cell death. However, the results were not consistent as *Ch*. *vulgaris* had a thick cell wall mainly composed of polysaccharides and pectin hence, had comparative advantages (protection) over the other microbes.

Kubacka et al. [65] examined genome/proteome-wide expression profiles of *P*. *aeruginosa* PAO1 cells treated with TiO2-based nanocomposite films. An increase and decrease in the levels of 165 and 151 transcripts were respectively reported in cells with TiO2-coated Ethylene vinyl alcohol (EVOH) particles. Few proteins were detected at a statistically significant level (p ≤ 0.1) in cells treated with TiO2-coated EVOH particles compared to the control. TiO2-UV treatment significantly suppressed (from 5.4- to 15.1-fold) the expression levels of genes essential for cell wall. However, 14 genes encoding for lipid metabolism essential for cell membrane were over-expressed (from 5.6- to 23.0-fold), unexpectedly, 2 were expressed at a lower level (from 5.5- to 7.4-fold).

In vivo and in vitro studies confirm that hydroxyl radicals inflict damage (breakage) on DNA strands. The extent of damage was minimized when dimethyl sulfoxide, catalase, or mannitol were incorporated in the reaction mixture [66]. However, the findings [66] contradicts previous studies [21, 67]. Exposing either purine or pyrimidine bases to TiO2 and light from a 100-W Hg lamp resulted in the detection of NO3 <sup>−</sup> and NH4 + ion. However, when native DNA and RNA molecules were subject to the same conditions, unknown peroxide species, along with phosphate and carbon dioxide, were detected, suggesting the breakage and mineralization of sugar-phosphate backbone of DNA and RNA molecules, respectively [68].

Kikuchi et al. [67] demonstrated the role of ROS on photocatalytic bactericidal activity. They utilized a porous polytetrafluoroethylene (PTFE) membrane in their system to physically separate the *E*. *coli* suspension from the TiO2 thin. The results showed an impressive photokilling capability of the system with and without (control) PTFE - which was attributed to the generated H2O2. A group [69] demonstrated the stimulating effect of TiO2-NPs on lipolytic activity in *A*. *niger*. The results showed that TiO2-NPs significantly increased lipase biosynthesis (more than 1.5 times) compared to the control experiment. Treatment with TiO2-NPs (size: 40 nm, concentration: 10 mg/L) in all culture media, enhanced lipolytic activity by 78.57% and 57.49% on the 4th and 5th day of cultivation, respectively. This finding reaffirms that smaller NPs can penetrate the cell membrane easily than bigger NPs, thus easily interact with molecular proteins, resulting in stimulating effects.

Gomes et al. [70] assessed the effects TiO2-NMs (NM103, NM104, and NM105) and bulk TiO2 against *Enchytraeus crypticus* with and without UV radiation. Microarray analysis revealed 10431 differentially expressed genes (DEGs) (*p* < 0.01) triggered as a result of exposure to TiO2-NMs under no-UV. All samples under UV exposure registered an up-regulation of several transcripts, including caspase apoptosis-related cysteine peptidases, a signature of apoptosis activation, whereas under darkness the apoptotic signaling pathway was inhibited, suggesting that the oxi-radicals generated during the photoactivation of TiO2 might substantially contribute to the apoptotic response and damage to the cell membrane. DNA damage was triggered after exposing samples to bulk/nano TiO2 [71]. However, the findings of Gomes et al. [70] contradicted the [71] as reported that TiO2-NMs\_under no-UV impaired DNA repair, while bulk\_TiO2 under no-UV activated DNA repair mechanisms, suggesting that size of the TiO2-NPs contributes to biocidal activity.

*Mycotoxins and Food Safety*

of H2O2 and the superoxide ion [61].

+

of amino acids and NH4

Electron paramagnetic resonance (EPR) spectroscopy study confirmed the photoproduction of hydroxyl radicals (OH•) from different TiO2. The efficiency of

Cells are negatively charged [58] under optimum physiological condition due to heparan sulfate proteoglycans [59]. However, disease could trigger the cells to synthesize certain compounds which cause cell surface to become positively charge. Microbial cell could act as a hole for electron transfer between organism and its components [60]. The iron cluster on cell surface, in the periplasmic space, or inside the cell (proteins (such as ferritin)), could act as a precursor for iron-catalyzed Haber-Weiss reaction, which generates additional hydroxyl radicals in the presence

Different treatments (photocatalysis, water, TIO2, UV-A) applied to elucidate the effects of lipid peroxidation on *S*. *cerevisiae* revealed high malondialdehyde (MDA) in TiO2 -treated subjects with 2 hours. The results demostrated that TiO2 was sufficient to damage membrane, thus interfered with permeability of the cell which led to the leakage of vital intracellular molecules (**Figure 1**) [48]. Similarly, Draper and Hadley [62] found photocatalysis-induced cell wall damage on S. cerevisiae [48]. This may decrease intracellular enzymatic activity as well as leaking

Cellular respiratory enzymes lost their activity after been exposed to irradiated TiO2 (0.5 mg/mL), and the kinetics correlated with the losses of cell viability. Furthermore, when glucose was used instead of succinate as the electron donor, similar effects were observed. From this outcome, Li et al. [78] proposed that ROS generated from an irradiated TiO2 surface, interacted with the polyunsaturated phospholipids in *E*. *coli*. Moreover, cell membrane structure was perforated due to lipid peroxidation creating a hole for more TiO2-NPs to pass into interior of the cell,

thus rendering respiratory proteinsinactive and subsequent cell death.

A progressive decrease in esterase activity was observed after exposing *S*. *cerevisiae* to irradiated TiO2 [63]. Other researchers documented overexpression and inhibition (expressed at lower levels, including those encoding six cbb3-type cytochrome C oxidase subunits, an electron transfer flavoprotein, and

*Schematic illustration of the solar photocatalytic process for microbial cell inactivation in the presence of an aqueous suspension of TiO2. Modified with permission from ref 4498160008350 [72]. Contact between the cells and TiO2-NPs affects membrane permeability; however, this is reversible. The availability of more NPs could enhance the damage to cell wall, thus allowing leakage of small molecules such as ions. Damage at this stage may be irreversible, and this accompanies cell death. Higher molecular weight components such as proteins could further be leaked followed by protrusion of the cytoplasmic membrane into the surrounding medium through degraded areas of the peptidoglycan and lysis of the cell. Intracellular components are then degraded progressively especially from the point of contact with photocatalyst, followed by complete mineralization.*

, suggesting a drastic impact on proteins [63].

hydroxyl radical generation depends on the source/origin of TiO2 [57].

**138**

**Figure 1.**
