Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate

Summary

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.

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 bacteria^3,4 because it does not depend on the mode of bacterial resistanc....

Protocol

1. Photolysis system setup

1. 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.
2. 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.
3. Monitor and record the temperature of test units throughout the photolytic reactions by an infrared thermometer....

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/m

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.......

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