CN113068791A - Method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water - Google Patents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
- A23L5/276—Treatment with inorganic compounds
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Apparatus For Disinfection Or Sterilisation (AREA)
Abstract
The invention discloses a method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water, which comprises the steps of mixing a photosensitizer dispersion liquid with a sample to be treated, wherein the photosensitizer dispersion liquid comprises a photosensitizer and the alkaline electrolyzed water; incubating the mixed sample; irradiating the incubated sample with a light source. The method creatively combines the photodynamic technology with the water electrolysis technology, and develops a brand-new channel for improving the PDT sterilization efficiency; the technology can be applied to the food industry by matching alkaline electrolyzed water with a food-grade photosensitizer, so that the risk caused by the pollution of harmful microorganisms is reduced; the operation is extremely simple, the applicability is good, the cost is low, the safety is good, the environment is protected, no pollution is caused, the sterilization efficiency (for vibrio parahaemolyticus and Shewanella putrefaciens) can be obviously improved, and the application prospect is very good.
Description
Technical Field
The invention belongs to the field of food safety and biotechnology, and relates to a method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water.
Background
Photodynamic technology (PDT), also known as photodynamic inactivation (PDI), is a widely used new method in clinical medicine for the inactivation of tumor cells and pathogenic microorganisms, including bacteria, fungi and viruses. Three requirements for PDT sterilization are: oxygen, light and photosensitizers. The combination of the three can generate Reactive Oxygen Species (ROS) including singlet oxygen, hydrogen peroxide, hydroxyl radicals and superoxide anions. ROS have a strong oxidizing effect, causing damage or death of cells. The technology has the advantages of low cost, greenness, safety, no toxicity, no pollution and the like, and is applied to the field of food safety in recent years. However, PDT also has some significant drawbacks: the efficiency of killing some harmful microorganisms and biofilm thereof is not high or the selected photosensitizer has good antibacterial effect but has toxicity and is difficult to apply to the food industry.
At present, the most common method for improving the PDT sterilization efficiency is a photosensitizer modification method. In particular, photosensitizers are combined with nanoparticles or some compounds and their derivatives. For example, some nanoparticles (silver nanoparticles or gold nanoparticles and the like) can be used as carriers for conveying the photosensitizer to a target with a targeting effect, and the nanoparticles have good stability, strong targeting property and large specific surface area, can carry more photosensitizer molecules to approach or enter cells, and further improve the sterilization efficiency. The mechanism of improving the sterilization efficiency by combining some compounds with the photosensitizer is to utilize the special properties of the compounds themselves to directly or indirectly change the molecular structure of the photosensitizer and improve the solubility, the binding rate with bacteria, the self-stability and the like of the photosensitizer. As indicated in the literature I (Misba Lama, Kulshresta Shatavari, Khan Asad U.Antibiotilm action of a cellulose blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy [ J ] biofoulding, 2016,32(3):313-328.) toluidine blue-mediated photodynamic technology in combination with nanoparticles enhances the biofilm-resistant effect on Streptococcus mutans. Although the method improves the sterilization efficiency to a certain extent, the cost of the nano particles or compounds is high, the synthesis process is complex, and the nano particles or compounds are fatally toxic, so that the application range of the nano particles or compounds in the field of food safety is greatly reduced.
Yet another common approach is to combine PDT with other non-thermal sterilization techniques (ultrasound, ultraviolet, high voltage pulsed electric fields, etc.). PDT has poor biofilm removal due to the fact that the biofilm is covered by a thick extracellular polymer which can block photosensitizer molecules from entering the interior of bacteria. When the PDT and the ultrasonic wave are combined to treat the biofilm, the ultrasonic action can destroy extracellular polymers of the biofilm, and the combination rate of the photosensitizer and a target object is improved, so that the sterilization efficiency is enhanced. As shown in the second publication (Buchovic Irina, Lukseviute Viktorija, Kokstai Rita, et al. Inactiva of Gram (-) bacteria Salmonella enterica by chlorophyllin-based photosensistion: Mechanism of action and new strategies to enhance the activity efficiency [ J ]. Journal of Photochemistry and Photobiology B-Biology,2017,172,1-10.) it is noted that the efficiency of inactivation of Salmonella is significantly improved by the combination of the mediated photodynamic and pulsed high power UV technology. Although such methods improve the sterilization efficiency to some extent, the cost is too high and the process is complex, and each technique has a certain application range, so the more the techniques are used in combination, the smaller the application range is.
Therefore, the development of a method for improving PDT sterilization efficiency with low cost and good applicability and food safety level is of practical significance.
Disclosure of Invention
The invention aims to overcome the defects that the conventional method for improving PDT (photodynamic therapy) sterilization efficiency is high in cost, complex in process, small in application range and incapable of efficiently removing mixed species biofilm with stronger resistance in the food industry and the like, and provides a method for improving PDT sterilization efficiency with low cost and good applicability, which is in food safety level. The invention creatively combines the photodynamic technology with the water electrolysis technology, develops a brand-new channel for improving PDT sterilization efficiency, and has great application potential in the field of future food safety prevention and control.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for improving sterilization efficiency by combining a photodynamic technology with alkaline electrolyzed water comprises the following steps:
(1) mixing a photosensitizer dispersion liquid with a sample to be treated, wherein the photosensitizer dispersion liquid comprises a photosensitizer and alkaline electrolyzed water;
(2) incubating the mixed sample;
(3) irradiating the incubated sample with a light source.
The invention improves the existing PDT sterilization technology by using alkaline electrolytic water to improve the sterilization efficiency, wherein the alkaline electrolytic water is water with special properties, and is electrolytic water which is generated at the cathode of an electrolytic cell by an electrolytic device through an electrolyte solution and has reducibility; meanwhile, due to the electrolytic action, the solution not only has an alkaline environment, but also contains a large amount of free charge ions and functional groups, has good rust resistance, good oxidation resistance and high permeability. The source of the photosensitizer is green, safe, nontoxic and harmless, the sterilization efficiency can be obviously improved by matching the photosensitizer, the operation of the photosensitizer is extremely simple, the applicability is good, and the food-grade photosensitizer is selected. The technology is expected to be applied to the food industry, thereby reducing the risk caused by the pollution of harmful microorganisms and having great application prospect.
As a preferred technical scheme:
the invention is applicable to all water-soluble photosensitizers, such as sodium copper chlorophyllin, riboflavin and the like or a mixture of the photosensitizers (the mixed photosensitizers are adopted on the premise that the mixed photosensitizers do not react with each other and the maximum absorption wavelength is close), and other water-insoluble photosensitizers such as curcumin and the like can also be applicable, and certainly, the effect of the mixed photosensitizers is slightly lower than that of the water-soluble photosensitizers due to poor solubility. The sodium copper chlorophyllin selected here is water-soluble, its source is wide, the price is low, non-toxic and pollution-free and is regarded as a food additive in many countries, namely food safety grade, use this photosensitizer can apply the technology of the invention to the sterilization of food, in order to reduce the risk brought by pollution of the harmful microorganism.
The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water comprises the following steps:
preparing alkaline electrolyzed water by using an electrolyzed water preparation instrument, wherein a water inlet is common tap water, a water outlet is target electrolyzed water, and the parameters of the electrolyzed water preparation instrument are as follows: the pH is 7-10, and the oxidation-reduction potential (ORP) is 0-400 mV (preferably, pH is 9-10, ORP is-300-400 mV, more preferably, pH is 9.80, ORP is-388 mV). Wherein, the pH value is inversely proportional to the ORP value, the higher the pH value is, the better the sterilization efficiency is, but the pH value is not too high, because the harm can be generated to the food quality and even the human health. The alkaline electrolyzed water adopted by the invention is novel environment-friendly water which is green, safe, nontoxic and harmless.
The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water has the advantages that the concentration of the photosensitizer in a sample mixing system to be treated is 0-1000 mu M, preferably the concentration of the photosensitizer in the sample mixing system to be treated is 80-1000 mu M, more preferably the concentration of the photosensitizer in the sample mixing system to be treated is 150-300 mu M. Of course, the scope of the present invention is not limited thereto, and those skilled in the art can adjust the amount of the photosensitizer to be added according to actual needs in practical applications, and only one possible technical solution is given here.
In the method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water, in the step (2), the incubation is performed under a dark condition for 0-60 min, preferably for 10-60 min, and more preferably for 20 min. In addition, the incubation temperature has no influence on the method, the method can work normally at 0-40 ℃, the normal use of the photosensitizer and the illumination device can be influenced when the temperature is too low or too high, and the method is carried out at room temperature, namely 25 ℃.
The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water comprises the step (3), wherein the light source is a blue light source, and the wavelength of the blue light source is 455-460 nm. Only one possible technical solution is given here, and those skilled in the art can adjust the relevant conditions such as the light source according to the actual needs in the practical application.
The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water comprises the step (3), wherein the luminous power density irradiated by the light source is 0-500 mW/cm2Preferably, the optical power density irradiated by the light source is 2-50 mW/cm2More preferably, 3.8mW/cm is selected for use in the present invention2The optical power density of the (C) is extremely low in energy consumption and remarkable in sterilization effect. Although the scope of the present invention is not limited thereto, those skilled in the art can adjust the optical power density according to actual requirements in practical applications, and only one possible technical solution is given here, and theoretically, the higher the power is, the better the sterilization effect is. In addition, the invention is not limited to blue light sources, and any light source which can lead the photosensitizer to excite to generate ROS to generate photodynamic reaction and the optical power density thereof can improve the sterilization efficiency of the light source by combining alkaline electrolyzed water with the light source. The duration of light source irradiation is 0-120 min. Preferably, the irradiation time of the light source is 10-120 min, more preferably, the irradiation time is 20-120 min, experiments show that the sterilization efficiency is gradually improved along with the extension of the irradiation time, the maximum sterilization effect is achieved when the irradiation time is extended to 20min, and the sterilization efficiency is not obviously improved when the irradiation time is continuously extended, so that the optimal irradiation time is 20 min.
The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water is characterized in that the sterilization efficiency of vibrio parahaemolyticus (pathogenic bacteria) and shewanella putrefaciens (putrefactive bacteria) is improved. The scope of the present invention is not limited thereto, and only a part of the strains specifically tested herein are shown, and the method of the present invention can also be applied to kill other common pathogenic and putrefying bacteria.
In addition, the invention also provides the application of the method in sterilization.
The present invention also provides a photosensitizer composition (corresponding to the above photosensitizer dispersion) comprising a photosensitizer and alkaline electrolytic water.
Has the advantages that:
(1) the method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water creatively combines the photodynamic technology with the electrolyzed water technology, and develops a brand new channel for improving the PDT sterilization efficiency;
(2) the photodynamic technology is combined with the method for improving the sterilization efficiency by alkaline electrolyzed water, and the alkaline electrolyzed water is matched with the food-grade photosensitizer, so that the technology can be applied to the food industry, and the risk caused by the pollution of harmful microorganisms is reduced;
(3) the method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water has the advantages of extremely simple operation, good applicability, low cost, good safety, greenness, no pollution, capability of obviously removing the biofilm, capability of obviously improving the sterilization efficiency (for vibrio parahaemolyticus and Shewanella putrefaciens), and extremely wide application prospect.
Drawings
FIG. 1 is a schematic diagram of an apparatus structure of an 455-460 nm LED illumination system used in an embodiment;
FIG. 2 is a graph showing a comparison of ROS and singlet oxygen expression levels in PDT in combination with alkaline electrolyzed water under different concentrations of sodium copper chlorophyllin (FIG. 2-A, FIG. 2-B) and illumination time (FIG. 2-C, FIG. 2-D) treatment conditions;
FIG. 3 is a graph showing the effect of PDT in combination with alkaline electrolyzed water on the total biofilm biomass of mixed species under different concentrations of sodium copper chlorophyllin (FIG. 3-A) and illumination time (FIG. 3-B) treatment conditions;
FIG. 4 is a graph of the effect of PDT in combination with alkaline electrolyzed water treatment on mixed species biofilm cell viability (FIG. 4-A) and total colony count (FIG. 4-B);
FIG. 5 shows the effect of PDT in combination with alkaline electrolysis water treatment on the total biofilm biomass and cell viability of mixed species on the surface of stainless steel (FIG. 5-A, FIG. 5-C) and fish scales (FIG. 5-B, FIG. 5-D);
the LED shooting lamp comprises a 1-LED shooting lamp box, a 2-lifting table, a 3-24 pore plate and a 4-LED blue light source.
Detailed Description
The present invention will be described in more detail with reference to the accompanying drawings, in which embodiments of the invention are shown and described, and it is to be understood that the embodiments described are merely illustrative of some, but not all embodiments of the invention.
The strains used in the examples are as follows:
vibrio Parahaemolyticus (VP) ATCC 17802 was obtained from institute of microorganisms of Chinese academy of sciences; shewanella putrescentiae (Shewanella putrefeaciens, SP) strain E05 was isolated from prawns by this laboratory.
The reagents and materials used in the examples are as follows:
sodium copper chlorophyllin (food grade, purity > 95%) manufactured by Shanghai Biotechnology, Inc.; preparing alkaline electrolyzed water (pH is 9.80, ORP is-388 mV) by an electrolyzed water preparation instrument; thiosulfate citrate bile salt sucrose agar medium (TCBS), tryptone soy broth medium (TSB), iron agar medium, Beijing Luqiao technology GmbH; ROS active oxygen fluorescent probe Beijing Solaibao Biotech limited; 9,10 Anthranediyl-bis (methylene) dicarboxylic acid (AMDA,10mM) Sigma, USA; 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT reagent, 5mg/mL), PBS phosphate buffer solution, Crystal Violet solution, Shanghai Biotech engineering Co., Ltd.; other chemical analysis reagents are purchased from chemical reagents of the national drug group, ltd. 304 stainless steel discs (d 14mm) and fresh round fish scales (d 14mm) are commercially available on the market.
The instruments used in the examples are as follows:
455-460 nm LED lighting system is shown in FIG. 1, and comprises an LED shooting lamp box 1, a lifting platform 2, a 24-hole plate 3 and an LED blue light source 4, wherein the LED lighting system is surrounded by the LED shooting lamp box and can prevent the outside from being coveredLight enters, the LED blue light source is arranged in the LED photographing lamp box top inner side, the 24-hole plate is arranged on the lifting platform, the distance between samples in the LED blue light source and the 24-hole plate is adjusted to be 5.0cm by the lifting platform, the LED blue light source (the wavelength is 455-460 nm, the length of the lamp tube is 30cm, and the irradiation dose per second is 3.80mJ/cm2) Purchased from Shanghai Philips Lighting electronics, Inc.;
full automatic bench centrifuge 5417R, available from Eppendorf, Germany;
a multifunctional microplate reader SynergyHTX, available from beten instruments ltd, usa;
an electrolytic water producing apparatus FW-200, available from AMANO, Japan.
Culture method of Mixed biofilm (i.e., preparation method of sample to be treated)
Streaking Vibrio parahaemolyticus (-80 deg.C in 25% glycerol tube) on TCBS plate, culturing at 37 deg.C for 12 hr, picking green single bacterium, and culturing in TSB (containing 3% NaCl) broth overnight (200rpm) at constant temperature (37 deg.C) in incubator; shewanella putrefaciens (-80 ℃ in 25% glycerol tubes) was streaked onto iron agar plates, incubated at 25 ℃ for 24h, and black single colonies were picked up into TSB broth and placed in a thermostatted (30 ℃) incubator overnight (200 rpm). The concentrations of the two bacteria are respectively adjusted to 8log CFU/mL by 0.85% NaCl solution, and then the bacterial liquids of the two bacteria are mixed in equal volume for later use.
mu.L of the mixed bacterial liquid and 990. mu.L of sterile fresh TSB culture medium are added into a 24-well polystyrene plate, and then the plate is placed into an incubator at 25 ℃ for static culture for 18h to form a mature mixed species biofilm.
Preparation of photosensitizer dispersion
0.0361g of sodium copper chlorophyllin powder is added into 100mL of alkaline electrolytic aqueous solution (100 mL of 0.85% NaCl solution is used for replacing a control group), and stirred and mixed uniformly to obtain 500 mu M of sodium copper chlorophyllin stock solution for later use.
Determination of ROS and singlet oxygen:
after mixed species biofilms were incubated for the indicated time, planktonic bacteria were removed and washed 3 times with PBS. 1mL of sodium copper chlorophyllin solutions (0, 20, 50, 100, 150, 200. mu.M) at different concentrations were added to the well plate, respectively, with 0. mu.M being replaced with an equal volume of 0.85% NaCl solution or an aqueous electrolyte solution. Then 10. mu.L of ROS reactive oxygen species fluorescent probe (for monitoring the production of ROS) or 100. mu.L of AMDA reagent (for monitoring the production of singlet oxygen) is added, and after standing and incubating for 10min, illumination is carried out for different time periods (0, 5, 10, 15, 20 and 30 min). The absorbance at wavelengths of 504nm and 399nm was measured with a multifunctional microplate reader, respectively.
Effect of different concentrations of photosensitizer and illumination time on biofilm removal efficiency of mixed species
After mixed species biofilms were incubated in 24-well polystyrene plates (plastic) for 18h, planktonic bacteria were removed and washed with PBS solution.
(1) Four sets of experiments were set to explore the effect of photosensitizers of different concentrations in combination with alkaline electrolyzed water on biofilm removal by mixed species. First group (negative control group): adding 1mL of 0.85% NaCl solution; second group (control group): adding 1mL of sodium copper chlorophyllin prepared by dissolving 200 mu M NaCl solution with concentration of 0.85%; third group (control group): adding 1mL of alkaline electrolyzed water; fourth group (experimental group): 1mL of sodium copper chlorophyllin prepared by dissolving in alkaline aqueous electrolyte solution at different concentrations (20, 50, 100, 150, 200. mu.M) was added. Standing and incubating the second, third and fourth groups for 10min, and irradiating for 20 min.
(2) Four groups of experiments are also set to explore the effect of the different illumination time combined with the alkaline electrolyzed water on removing the biofilm of the mixed species. First group (negative control group): adding 1mL of 0.85% NaCl solution; second group (control group): adding 1mL of sodium copper chlorophyllin prepared by dissolving 150 mu M NaCl solution with concentration in 0.85%; third group (control group): adding 1mL of alkaline electrolyzed water; fourth group (experimental group): 1mL of sodium copper chlorophyllin prepared by dissolving in an alkaline aqueous electrolyte solution at a concentration of 150. mu.M was added. The second and third groups are incubated for 10min and then irradiated for 30min, and the fourth group is incubated for 10min and then irradiated for 5, 10, 15, 20 and 30 min.
The residual biofilms were quantified by crystal violet assay in all treatment groups as follows: after the treatment, the plate was washed with PBS to remove floating bacteria, and then dried in an oven at 55 ℃ and then 1mL of 0.1% (v/v) crystal violet solution was added to each well and allowed to stand at room temperature for 30 min. Washing with PBS solution for 3 times to remove excessive crystal violet, adding 1mL 95% ethanol into each well, standing at room temperature for 30min, and measuring absorbance at 600nm with multifunctional microplate reader.
MTT method and plate counting method for representing removal effect on mixed species biofilm
Likewise, four sets of experiments were set up. A first group: adding 0.85% NaCl solution for treatment; second group: adding sodium copper chlorophyllin prepared by dissolving 0.85% NaCl solution with concentration of 150 μ M; third group: adding alkaline electrolyzed water for treatment; and a fourth group: adding sodium copper chlorophyllin prepared by dissolving in alkaline electrolytic aqueous solution at concentration of 150 μ M. All groups were incubated and then illuminated for 20min, planktonic bacteria were removed and washed with PBS. 1mL of fresh TSB medium and 100. mu.L of MTT reagent were added to each well, and the mixture was incubated at 25 ℃ in an incubator for 2 hours or more. After washing planktonic bacteria with PBS solution, 1mL of DMSO solution was added to each well and allowed to stand at room temperature for 30min, and then the absorbance at a wavelength of 570nm was measured.
And detecting the number of live bacteria in the biofilm of the mixed species by using a plate counting method. The treatment of the biofilm was completely the same as that in the MTT method. After the treatment, 1mL of 0.85% NaCl solution is added into each hole, the gun head is used for repeatedly blowing and beating to suspend the residual biomembrane cells at the bottom of the hole plate, the mixture is transferred to a centrifuge tube and then is subjected to gradient dilution and is coated on a TCBS plate (12h/37 ℃) and an iron agar plate (24h/25 ℃). After incubation for the indicated time, the counts were counted.
The crystal violet method and the MTT method characterize the removal effect of biofilm of mixed species formed on stainless steel and fish scales
Placing the sterilized stainless steel disc and the circular fish scale in the bottom of a 24-pore plate in advance, adding 10 mu L of mixed bacterial liquid and 990 mu L of sterile fresh TSB culture medium, and then placing the pore plate in an incubator at 25 ℃ for standing culture for 18h to form a mixed species biofilm on the surface. And then, the mixed biofilm is totally consistent with the characteristics of the mixed biofilm removing effect by an MTT method and a plate counting method, the mixed biofilm is divided into four groups, and after the treatment is finished, the biofilm removing effect is verified by a crystal violet method and an MTT method respectively.
Data analysis
Each set of samples was set to 3 replicates and averaged after 3 independent replicates. Data processing was performed by Microsoft Excel 2010; single factor anova by SPSS 21.0, significance difference (P < 0.05); mapping analysis was performed by Graphpad Prism 8.4.3 and Origin 8.0.
A, b, c, d, e, f in FIGS. 3-5 are automatically generated by SPSS software when analyzing data, and are used to express significant differences.
Example 1
Effect of PDT in combination with alkaline electrolyzed water on ROS and singlet oxygen
The effect of PDT in combination with alkaline electrolyzed water on ROS and singlet oxygen production levels is shown in FIG. 2. when no photosensitizer is added (0.85% NaCl solution or alkaline electrolyzed water is added), weak ROS levels are detected in the system, since microorganisms produce endogenous ROS during normal metabolic processes (FIGS. 2-A, 2-C). The illumination time was maintained for 20min, and when the photosensitizer concentration was 20 μ M, ROS levels were detected by PDT alone at 0.101 and by PDT in combination with alkaline electrolyzed water treatment at 0.202. With increasing photosensitizer concentration, ROS levels in PDT treatment alone increased slowly, with ROS levels of only 0.288 when photosensitizer concentrations reached 200 μ M. However, ROS levels increased dramatically in the PDT in combination with alkaline electrolyzed water treatment group as the photosensitizer concentration increased, reaching 0.646 for photosensitizer concentrations of 200 μ M. The growth rate was significantly higher than that of PDT-only treatment group. There is a similar trend in the effect of light exposure time on ROS levels. Briefly, there was no significant difference in ROS levels in the PDT treated group alone versus the PDT combined alkaline electrolyzed water treated group when not illuminated and the photosensitizer concentration was maintained at 150 μ M. With the increase of the illumination time, the ROS level of the two gradually increases. When the illumination time is 30min, the ROS level of PDT single treatment group is only 0.269, while the ROS level of PDT combined alkaline electrolyzed water group is obviously higher than that of PDT single treatment group, reaching 0.552 and more than 2 times of that of PDT single treatment group.
The AMDA reagent has a maximum absorbance at 399nm, and singlet oxygen can react with it and consume AMDA. The level of singlet oxygen production is therefore directly proportional to the decrease in absorbance at 399nm of AMDA. As shown in FIGS. 2-B and 2-D, the absorbance of AMDA at 399nm was 1.262 without the addition of photosensitizer or without light. The illumination time of both groups was kept for 20min, and when the concentration of sodium copper chlorophyllin was 20 μ M, the absorbance value of the PDT-only treated group was reduced to 1.176 and the absorbance value of the PDT-combined alkaline electrolyzed water group was reduced to 1.058. The absorbance at 399nm decreased continuously for both groups with increasing photosensitizer concentration. But the drop rate of PDT in combination with alkaline electrolysis water was significantly higher than that of PDT alone treatment group. When the chlorophyll concentration increased to 200 μ M, the absorbance of the PDT-only treatment group decreased to 0.750 and the PDT-combined alkaline electrolyzed water group decreased to 0.183. The trend of the influence of the illumination time on PDT alone treatment and PDT combined with alkaline electrolysis water treatment is similar. The above results indicate that, compared with PDT alone, after PDT combined with alkaline electrolyzed water, the production level of ROS and singlet oxygen is greatly increased, which indicates that there is a coupled interaction between PDT and electrolyzed water, thereby possibly improving the sterilization efficiency thereof.
Example 2
PDT combined with alkaline electrolytic water under different conditions for removing biofilm of mixed species
To investigate whether PDT combined with alkaline electrolyzed water would enhance biofilm removal in mixed species, we characterized the removal in 24-well plates (plastic) using crystal violet. As shown in FIG. 3-A, the total biofilm biomass of the negative control group was 1.963 (OD)600nm) When PDT alone was treated (photosensitizer concentration 200 μ M, illumination time 20min), the total biomass decreased by 19.8%; when alkaline electrolyzed water was treated alone, the total biomass decreased by 42.2%. However, when PDT was combined with alkaline electrolysis of water, the photosensitizer concentration was only 20. mu.M, and the total biomass was reduced by 46.6%. Further increasing the concentration of the photosensitizer and obviously reducing the total biomass of the biofilm. When the photosensitizer concentration was increased to 150. mu.M, the total biomass had decreased by 72.6% compared to the control group. The light irradiation time has similar action trend to the effect of PDT combined with alkaline electrolytic water on removing the biofilm. Similarly, PDT alone reduced the total biofilm biomass by 20.6% and alkaline electrolyzed water alone reduced the total biofilm biomass by a fixed photosensitizer concentration of 150 μ M for a 30min exposure time43.2 percent. However, PDT combined with alkaline electrolyzed water illumination is only 5min, and the total biomass is reduced by more than half. After prolonged light exposure, the total biomass was further reduced. When the illumination time reaches 20min, the total biomass is reduced by more than 70%. The above experimental results strongly demonstrate that the removal efficiency of biofilm of mixed species after PDT in combination with alkaline electrolyzed water is significantly higher than that of PDT alone or alkaline electrolyzed water alone. We have also found that the removal efficiency of PDT in combination with alkaline electrolysis water for biofilm is greater than the sum of the removal efficiency of PDT treatment alone and alkaline electrolysis water treatment alone. This further verifies the coupling interaction between PDT and alkaline electrolysis water.
Cell viability and total colony counts before and after treatment were determined by MTT and plate count, respectively. As shown in FIG. 4, in the negative control group, the initial cell viability of the mixed species biofilm was 0.844 (OD)570nm). When the alkaline electrolyzed water is treated alone, the cell viability is reduced to 0.326. When the photosensitizer concentration is 150 μ M and the illumination time is 20min, the cell viability decreases to 0.557. However, when PDT was combined with alkaline electrolyzed water, cell viability decreased to 0.124 under equivalent conditions, which was 85.3% less than the control group. Similarly, the plate count results showed that the total number of colonies in the biofilm of the initial mixed species was 8.28Log CFU/mL (negative control). After the alkaline electrolytic water and PDT are respectively and independently treated, 6.12 Log CFU/mL and 7.36Log CFU/mL are remained in the total colony number. After PDT and alkaline electrolytic water are combined for treatment, the total colony number is reduced to 4.01Log CFU/mL, and the sterilization efficiency is up to more than 99.99%. The results of cell viability and total number of colonies further verify that the experimental results of the crystal violet method support the experimental results, and meanwhile prove the high efficiency of PDT combined with alkaline electrolyzed water.
Example 3
Cleaning effect of PDT (photodynamic therapy) combined with alkaline electrolytic water on biofilm of mixed species on different contact surfaces
Stainless steel and fish scales are common adhesion carriers in the actual life of biofilms. Therefore, the effect of PDT combined with alkaline electrolyzed water on removing the biofilm of mixed species adhered to the surfaces of stainless steel and fish scales is further explored. As shown in FIG. 5-A and FIG. 5-BThe total biomass of the stainless steel and the fish scale surface biofilm in the negative control group are 1.79 and 3.94 (OD)600nm). When PDT was treated alone (photosensitizer concentration: 150. mu.M; light time: 20min), there was no significant difference in total biomass from the control group. When the single treatment is carried out by using the alkaline electrolyzed water, the total biomass of the biofilm adhered on the stainless steel is reduced by 37 percent, and the total biomass of the biofilm adhered on the fish scales still does not obviously reduce. However, when PDT was combined with alkaline electrolysis of water, the total biofilm biomass adhered to stainless steel and fish scales was reduced by 52.1% and 13.6%, respectively. We have found that characterization of PDT in combination with alkaline electrolyzed water by the crystal violet method is not efficient in removing biofilm from fish scale surfaces, because crystal violet not only binds to biofilm but also stains fish scales, ultimately leading to inaccurate results. Therefore, we further explored the change in the total number of biofilm colonies by plate counting. As shown in FIGS. 5-C and 5-D, the total number of stainless steel and fish scale surface biofilm colonies in the negative control group were 8.11 Log CFU/mL and 8.51Log CFU/mL, respectively. When treated with PDT alone, there was no significant decrease in both; when the single treatment is carried out by using alkaline electrolyzed water, the total number of the bacterial colonies of the biofilm on the surfaces of the stainless steel and the fish scales is 6.83 and 8.29Log CFU/mL respectively. However, when PDT is combined with alkaline electrolytic water treatment, the total number of biofilm colonies on two contact surfaces is remarkably reduced, and the sterilization rate reaches 99.93 percent and 99.22 percent respectively. These results show that PDT combined with alkaline electrolyzed water also has good effect of removing biofilm on the surfaces of stainless steel and fish scales, which further reflects the practical application value of PDT combined with alkaline electrolyzed water in the food industry. Therefore, the technology is expected to become a new means for controlling harmful microorganism pollution in the food industry in the future.
Proved by verification, the method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water creatively combines the photodynamic technology with the electrolyzed water technology, and develops a brand new channel for improving the PDT sterilization efficiency; the technology can be applied to the food industry by matching alkaline electrolyzed water with a food-grade photosensitizer, so that the risk caused by the pollution of harmful microorganisms is reduced; the operation is extremely simple, the applicability is good, the cost is low, the safety is good, the environment is protected, no pollution is caused, the sterilization efficiency (for vibrio parahaemolyticus and Shewanella putrefaciens) can be obviously improved, and the application prospect is very good.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and various changes or modifications may be made without departing from the principles and spirit of the invention.
Claims (10)
1. A method for improving sterilization efficiency by combining a photodynamic technology with alkaline electrolyzed water is characterized by comprising the following steps:
(1) mixing a photosensitizer dispersion liquid with a sample to be treated, wherein the photosensitizer dispersion liquid comprises a photosensitizer and alkaline electrolyzed water;
(2) incubating the mixed sample;
(3) irradiating the incubated sample with a light source.
2. The method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water according to claim 1, wherein the photosensitizer is selected from more than one of sodium copper chlorophyllin and riboflavin.
3. The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water as claimed in claim 2, wherein the preparation method of the alkaline electrolyzed water is as follows:
preparing alkaline electrolyzed water by using an electrolyzed water preparation instrument, wherein a water inlet is common tap water, a water outlet is target electrolyzed water, and the parameters of the electrolyzed water preparation instrument are as follows: the pH value is 7-10, and the oxidation-reduction potential is 0-400 mV.
4. The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolyzed water as claimed in claim 1, wherein the concentration of the photosensitizer in the mixed system of the sample to be treated is 0-1000 μ M.
5. The method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water as claimed in claim 1, wherein in the step (2), the incubation is performed in dark for 0-60 min.
6. The method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolyzed water as claimed in claim 1, wherein in the step (3), the light source is a blue light source, and the wavelength of the blue light source is 455-460 nm.
7. The method for improving sterilization efficiency by combining photodynamic technology with alkaline electrolysis water as claimed in claim 6, wherein in the step (3), the light power density irradiated by the light source is 0-500 mW/cm2(ii) a The duration of light source irradiation is 0-120 min.
8. The method for improving the sterilization efficiency by combining the photodynamic technology with the alkaline electrolysis water as claimed in claim 3, wherein the improvement of the sterilization efficiency is the improvement of the sterilization efficiency on Vibrio parahaemolyticus and Shewanella putrefaciens.
9. Use of a method according to any one of claims 1 to 8 for sterilization.
10. A photosensitizer composition comprising a photosensitizer and alkaline electrolyzed water.
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