inactivation-pathogens-via-visible-light-photolysis-riboflavin-5

Inactivation of Pathogens <em>via</em> Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Ming Chuan University

A vascular mimetic membrane system was used to investigate the effect of cholesterol content in lipid bilayer on the dynamics of the melittin-membrane penetration reaction with real-time monitoring by a piezoelectric sensor and the assessment morphology using atomic force microscopy (AFM). In the presence of 30% cholesterol in a noncharged phosphatidylcholine (PC) phospholipid membrane, KA1 (binding affinity constant) and KA2 (insertion affinity constant) derived from a two-step model decreased significantly. This result suggests that the high dose of cholesterol in phospholipid membrane inhibits both the binding and the insertion of melittin. Next, dynamic laser scattering and AFM were used to verify the structural changes of lipid bilayers in solutions and interfaces, respectively. The superstructures in both 0 and 10% cholesterol lipid bilayers were disrupted with penetration of melittin according to these verifications. However, kinetic analysis reveals that the different mechanisms are dependent on cholesterol, particularly for the insertion step.

Exploring the effect of cholesterol in lipid bilayer membrane on the melittin penetration mechanism.

A piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect Schistosoma japonicum circulating antigens (SjCAg). Analytical strategy utilizes the polyclonal antibodies with broad-spectrum recognition to a complex target with high specificity. The immobilized antibodies were purified from immunized rabbit's sera (im-S) and infected rabbit's sera (inf-S) by S. japonicum. The detection capacities of antibodies were compared between the sera of different phenotypes and purified fractions. The sample dilution ratios were also evaluated and optimized. Additionally, the sera with a variety of infection degrees for validation could be discriminated quantitatively. The linear dose-response relationship indicates that the systematic sensitivity of this method is below 150 Hz and the lowest limit of detectable range is above 500 cercariae of S. japonicum infection for 2 weeks. The novel immunosensor technique is well potential to determine the SjCAg in serum samples for clinical diagnosis of parasitosis in early stage.

Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement.

Aspergillus ficuum grown on plates and in liquid cultures were illuminated by a white fluorescent light and four different colour LED lights (white, blue, green and red) to evaluate the regulation of LED lights on fungal growth. Biomass conversion, pellet size and phytase activity were examined. In liquid culture, luminous intensity was highly correlated with the rate of biomass conversion but did not affect pellet size. The white fluorescent light contained several different wavelengths, and therefore, its effect on A. ficuum represents the cooperative effect of these wavelengths. Strong luminance of a white fluorescent light inhibited growth of A. ficuum mycelia on plates, whereas white LED light enhanced growth. The development of mycelia was also inhibited by blue LED light and enhanced by red LED light illumination. Investigating the effect of LED lights on the growth of A. ficuum could provide evidence on the luminous intensity that is sufficient for regulating fermentation by light.

Effect of colour LEDs on mycelia growth of Aspergillus ficuum and phytase production in photo-fermentations.

The micronutrients in many cellular processes, riboflavin (vitamin B(2)), FMN, and FAD are photo-sensitive to UV and visible light to generate reactive oxygen species (ROS). The riboflavin photochemical treatment with UV light has been applied for the inactivation of microorganisms to serve as an effective and safe technology. Ultra-violet or high-intensity radiation is, however, considered as a highly risky practice. This study was working on the application of visible LED lights to riboflavin photochemical reactions to development an effective antimicrobial treatment. The photosensitization of bacterial genome with riboflavin was investigated in vitro and in vivo by light quality and irradiation dosage. The riboflavin photochemical treatment with blue LED light was proved to be able to inactivate E. coli by damaging nucleic acids with ROS generated. Riboflavin is capable of intercalating between the bases of bacterial DNA or RNA and absorbs lights in the visible regions. LED light illumination could be a more accessible and safe practice for riboflavin photochemical treatments to achieve hygienic requirements in vitro.

Blue light induced free radicals from riboflavin on E. coli DNA damage.

The Folin-Ciocalteu method is widely applied for the determination of the total phenolic contents in natural products. This method is significantly affected by the addition of sodium carbonate. The currently applied Folin-Ciocalteu methods may have been modified without any validation in the quantitative standards and the order of processes. In this study, serial experiments were performed to investigate the effect of phenolic calibrations based on the classic Folin-Ciocalteu method. Esterification condensations were observed in the assays with prior basification for gallic acid and catechin used as quantitative standards. The phenolic contents obtained in the samples differed depending on when basification occurred compared with the gallic acid calibration. The bias of the classic Folin-Ciocalteu method derived from cross-linkage of molecules was first defined in this study. The performance of the Folin-Ciocalteu method is optimised and validated again. 

Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols.

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing. 

Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli.

Determination of the superoxide dismutase activity is an important issue in the fields of biochemistry and the medical sciences. In the riboflavin/nitro blue tetrazolium (B2/NBT) method, the light sources used for generating superoxide anion radicals from light-excited riboflavin are normally fluorescent lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the light source on the light-excitation of riboflavin. The effectiveness of the photolysis was controlled by the wavelength of the light source. The spectra of fluorescent lamps are composed of multiple colour lights, and the emission spectra of fluorescent lamps made by different manufacturers may vary. Blue light was determined to be the most efficient for the photochemical reaction of riboflavin in visible region. The quality of the blue light in fluorescent lamps is critical to the photo-decomposition of riboflavin. A blue light is better than a fluorescent lamp for the photo-decomposition of riboflavin. The performance of the B2/NBT method is thereby optimized. 

Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity.

The light sensitive compound riboflavin-5'-phosphate (or flavin mononucleotide, FMN) generates reactive oxygen species (ROS) upon photo-irradiation. FMN is required by all flavoproteins because it is a cofactor of biological blue-light receptors. The photochemical effects of FMN after irradiation by blue or violet light on the inactivation of Staphylococcus aureus strains, including a methicillin-resistant strain (MRSA), were investigated in this study. Upon blue- or violet-light photo-treatment, FMN was shown to inactivate S. aureus due to the generated ROS. Effective bacterial inactivation can be achieved by FMN photolysis without an exogenous electron provider. Inactivation rates of 94.9 and 95.2% in S. aureus and MRSA, respectively, can be reached by blue light irradiation (2.0mW/cm) with 120μM FMN for 120min. A lower FMN concentration and a shorter time are required to reach similar effects by violet light irradiation. Inactivation rates of 96.3 and 97.0% in S. aureus and MRSA, respectively, can be reached by violet light irradiation (1.0mW/cm) with 30μM FMN for 30min. The sensitivity of the inherent photosensitizers is lower under blue-light irradiation. A long exposure photolytic treatment of FMN by blue light is required to inactivate S. aureus. Violet light was found to be more efficient in S. aureus inactivation at the same radiant intensity. FMN photolysis with blue or violet light irradiation enhanced the inactivation rates of S. aureus and MRSA. FMN photochemical treatment could be a supplemental technique in hygienic decontamination processes.

Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5'-phosphate photolysis.

The adaptability of bacterial resistance to antibiotics contributes to its high efficiency during evolution. Tetracycline (TC) is a broad-spectrum antimicrobial agent. Chromatographic analyses and mass spectrometry were used to study the effects of the light illumination of a 462 nm light-emitting diode (LED) on the conformational changes of TC in a phosphate buffer solution (PBS, pH 7.8). Especially, the inactivation of superoxide anion radicals (O₂•) and  (), including that of a multidrug-resistant  (), were investigated during the photolysis of TC. A photolysis product of TC (PPT) was generated in an alkaline solution after the illumination of a blue light. The mass spectra of PPT had characteristic ion signals in m/z 459, 445, and 249.1 Da. The PPT has the molecular formula of CHN₂O₉, and the exact mass is 458.44 g/mol. The inactivation of  is not significant with TC treatment. The drug-resistant ability of  has a less significant effect on PPT, and the changed conformation of TC retained the inactivation ability of  upon blue light photoreaction. With TC, illuminated by a blue light in a pH 7.8 PBS, O₂• was generated from TC photolysis, which enhanced the inactivation of  and . A 96.6% inactivation rate of  was reached with TC under 2.0 mW/cm² blue light illumination at 25 ± 3 °C for 120 min, and the effects of the TC-treated photoreaction on  viability repressed the growth of  by 4 to 5 logs. The present study of the blue light photoreaction of TC offers a new approach to the inactivation of 

Effects of 462 nm Light-Emitting Diode on the Inactivation of  and a Multidrug-Resistant by Tetracycline Photoreaction.

Tetracycline (TC) is a broad-spectrum antibiotic compound. Wastewater with TC may have an adverse effect on ecosystems. Riboflavin-5'-phosphate (FMN or flavin mononucleotide) is a non-toxic product of the phosphorylation of vitamin B and is required for the proper functioning of the humans. FMN is sensitized to ultraviolet (UV) and blue light radiation, as evidenced by the generation of reactive oxygen species (ROS). This study inspects feasible applications of blue light on FMN so as to develop a valid way of degrading TC by FMN photolysis. We used the increased rate of bacterial survival as a practical indicator of antibiotic degradation. TC in the presence of FMN solution decomposed completely after 20 W/m of blue light irradiation (TCF treatment), and the degradation of TC (D-TCF) occurred after the photolytic process. After TCF treatment, colony-forming units (CFUs) of  ( were determined for the D-TCF solution. The CFU of  preservation was 93.2% of the D-TCF solution (50 μg/mL of TC in the presence of 114 μg/mL of FMN solution treated with 20 W/m of blue light irradiation at 25 °C for 1 h) cultivation. The mass spectrum of D-TCF showed diagnostic ion signals at m/z 431.0 and 414.0 Da. The molecular formula of D-TCF was CHNO, and the exact mass was 430.44 g/mol. TC degradation by FMN photolysis can significantly decrease the antimicrobial ability of TC. The results expressed here regarding the influence of FMN photolysis on TC degradation offer an environmentally sound wastewater treatment method.

The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5'-Phosphate Photolysis on Microbial Viability.

Tea is rich in catechins and aluminum. In this study, the process of catechin photolysis was applied as a model for examining the effects of aluminum chloride (AlCl) on the structural changes of catechin and the alteration of aluminum complexes under blue light irradiation (BLI) at pH 8 using liquid chromatography and mass spectrometry techniques. Additionally, the effects of anions on catechin upon the addition of AlCl and treatment with BLI were also studied. In this study, when 1 mM catechin was treated with BLI, a superoxide anion radical (O2•-) was generated in an air-saturated aqueous solution, in addition to forming a dimeric catechin (proanthocyanidin) via a photon-induced redox reaction. The relative percentage of catechin was found to be 59.0 and 95.7 for catechin treated with BLI and catechin upon the addition of 1 mM AlCl treated with BLI, respectively. It suggested that catechin treated with BLI could be suppressed by AlCl, while AlCl did not form a complex with catechin in the photolytic system. However, under the same conditions, it was also found that the addition of AlCl inhibited the photolytic formation of O2•-, and reduced the generation of proanthocyanidin, suggesting that the disconnection of proanthocyanidin was achieved by AlCl acting as a catalyst under treatment with BLI. The influence of 1 mM fluoride (F-) and 1 mM oxalate (C2O42-) ions on the photolysis of 1 mM catechin upon the addition of 1 mM AlCl and treatment with BLI was found to be insignificant, implying that, during the photolysis of catechin, the Al species were either neutral or negatively charged and the aluminum species did not form a complex with anions in the photolytic system. Therefore, aluminum, which is an amphoteric species, has an inherent potential to stabilize the photolysis of catechin in an alkaline conditions, while suppressing the O2•- and proanthocyanidin generation via aluminum ion catalysis in the catechin/Al system under treatment with BLI.

Catechin Photolysis Suppression by Aluminum Chloride under Alkaline Conditions and Assessment with Liquid Chromatography-Mass Spectrometry.

Doxycycline hyclate (DCH) and minocycline hydrochloride (MH) are tetracycline antibiotics and broad-spectrum antimicrobial agents. The changes in DCH and MH under blue light (λ = 462 nm) irradiation in alkaline conditions (BLIA) were investigated. Deactivation caused by superoxide anion radical (O) and deactivation from DCH and MH during photolysis on Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), were studied. DCH is relatively unstable compared to MH under BLIA. The level of O generated from the MH-treated photoreaction is lower than that from DCH photolysis, and the DCH-treated photoreaction is more efficient at inactivating S. aureus and MRSA at the same radiant intensity. DCH subjected to BLIA decreased the viability of S. aureus and MRSA by 3.84 and 5.15 log, respectively. Two photolytic products of DCH (PPDs) were generated under BLIA. The mass spectra of the PPDs featured molecular ions at m/z 460.8 and 458.8. The molecular formulas of the PPDs were CHNO and CHNO, and their exact masses were 462.44 and 460.44 g/mol, respectively. These results bolster the photolytic oxidation that leads to DCH-enhanced deactivation of S. aureus and MRSA. Photochemical treatment of DCH could be applied as a supplement in hygienic processes.

Effects of free radicals from doxycycline hyclate and minocycline hydrochloride under blue light irradiation on the deactivation of Staphylococcus aureus, including a methicillin-resistant strain.

Article	Year
Exploring the effect of cholesterol in lipid bilayer membrane on the melittin penetration mechanism. Analytical biochemistry\| PubMed ID: 17570332	2007
Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement. Biosensors & bioelectronics\| PubMed ID: 18490150	2008
Effect of colour LEDs on mycelia growth of Aspergillus ficuum and phytase production in photo-fermentations. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 22082775	2012
Blue light induced free radicals from riboflavin on E. coli DNA damage. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 23347966	2013
Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols. Food chemistry\| PubMed ID: 25306311	2015
Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 25617617	2015
Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 25985146	2015
Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5'-phosphate photolysis. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 28715781	2017
Effects of 462 nm Light-Emitting Diode on the Inactivation of and a Multidrug-Resistant by Tetracycline Photoreaction. Journal of clinical medicine\| PubMed ID: 30213146	2018
The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5'-Phosphate Photolysis on Microbial Viability. Microorganisms\| PubMed ID: 31661888	2019
Catechin Photolysis Suppression by Aluminum Chloride under Alkaline Conditions and Assessment with Liquid Chromatography-Mass Spectrometry. Molecules (Basel, Switzerland)\| PubMed ID: 33348758	2020
Effects of free radicals from doxycycline hyclate and minocycline hydrochloride under blue light irradiation on the deactivation of Staphylococcus aureus, including a methicillin-resistant s... Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 34864528	2022

Chien-Wei Cheng

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