Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.
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Method Article
Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate
In This Article
Summary
Abstract
Riboflavin-5'-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (
) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.
Introduction
Riboflavin-5′-phosphate (FMN) is formed by phosphorylation at the riboflavin 5′-position of the ribityl side-chain and is required by all flavoproteins for numerous cellular processes to generate energy. It also plays the role of vitamin for some functions in the human body1. FMN is approximately 200 times more soluble in water than riboflavin2.
The antibacterial photodynamic inactivation (aPDI) of bacteria is an efficient way to control resistance to bacteria3,4 because it does not depend on the mode of bacterial resistance. Clinicall....
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Protocol
1. Photolysis system setup
- Mount six light-emitting diodes (LED) (DC 12 V) on the inside of an opaque plastic cup (8 cm x 7 cm) as shown in Figure 1 to establish a photolysis system31.
- Add reactants (see below) into the glass test tubes (13 mm in diameter and 100 mm in height) and secure the tubes at the top of the cup as shown in Figure 1. Place the experimental setup in a room with a steady temperature of 25 ± 3 °C.
- Monitor and record the temperature of test units throughout the photolytic reactions by an infrared thermometer.
NOT....
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Results
Effect of light wavelength on FMN
The absorbance spectra of 0.1 mM FMN that is irradiated using blue, green, red, and violet LEDs are shown in Figure 3. There are two peaks for FMN (372 nm and 444 nm) for the dark control. Green and red light have no effect because changes in the spectra are insignificant. On the other hand, the respective absorbance of FMN at 444 nm is reduced by about 19% and 34%, respectively, after blue and violet light irradiation at 10 W/m2
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Discussion
A photosensitizer increases the photochemical reaction of chemical compounds to generate ROS. Pathogenic microorganisms can be inactivated by light irradiation in the presence of photosensitizers. This study determines the aPDI of S. aureus due to ROS generated by violet light irradiation of an exogenous photosensitizer, FMN.
As shown in Figure 3, for FMN, the absorbance at 444 nm is reduced significantly after 5 min of irradiation using violet or blue li.......
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Disclosures
The authors declare no conflicts of interest.
Acknowledgements
The authors are grateful to Dr. Tak-Wah Wong and Mr. Zong-Jhe Hsieh for their support with experiments.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Blue, green and red LED lights | Vita LED Technologies Co., Tainan, Taiwan | DC 12 V 5050 | |
| Dimethyl Sulfoxide | Sigma-Aldrich, St. Louis, MO | 190186 | |
| Infrared thermometer | Raytek Co. Santa Cruz, CA | MT4 | |
| LB broth | Difco Co., NJ | ||
| L-Methionine | Sigma-Aldrich, St. Louis, MO | 1.05707 | |
| NBT | Bio Basic, Inc. Markham, Ontario, Canada | ||
| Power supply | China tech Co., New Taipei City, Taiwan | YP30-3-2 | |
| Riboflavin 5′-phosphate | Sigma-Aldrich, St. Louis, MO | R7774 | |
| RNase | New England BioLabs, Inc. Ipswich, MA | ||
| Solar power meter | Tenmars Electronics Co., Taipei, Taiwan | TM-207 | |
| Staphylococcus aureus subsp. aureus | Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan | 10451 | |
| UV-Vis optical spectrometer | Ocean Optics, Dunedin, FL | USB4000 | |
| UV-Vis spectrophotometer | Hitachi High-Tech Science Corporation,Tokyo, Japan | U-2900 | |
| Violet LED | Long-hui Electronic Co., LTD, Dongguan, China |
References
- Jian, H. L., Cheng, C. W., Chen, L. Y., Liang, J. Y. The photochemistry of riboflavin. MC-Transaction on Biotechnology. 3, 1-11 (2011).
- Lin, Y., Eitenmiller, R. R., Landen, W. O. Riboflavin. Vitamin analysis for the health and food sciences. , CRC Press. 329-360 (2008).
- Xie, L. J., Wang, R. L., Wang, D., Liu, ....
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