A vitamin-mediated photodynamic bactericide, its usage and application

Abstract

This invention discloses a vitamin-mediated photodynamic sterilization reagent, its usage method, and its application, belonging to the field of food sterilization technology. The sterilizing agent provided by this invention can rapidly generate high concentrations of active substances with sterilization functions, such as hydrogen peroxide, after being irradiated with LED blue light, achieving thorough sterilization with a sterilization rate exceeding 99.999%. The inactivated components pose no safety risks to consumers. It can effectively delay the proliferation of total bacterial count and the production of volatile basic nitrogen in fresh beef at 4°C, thereby extending its shelf life. The generated ROS has a long half-life, overcoming the serious defects of poor penetration and migration effects in traditional PDI sterilization and significantly extending the sterilization duration. The required light source is an LED blue light lamp, which has high luminous efficiency, stable wavelength, does not produce significant thermal effects, and is durable and has low sterilization costs. It does not require real-time illumination, is easy to use, and can effectively avoid the adverse effects of prolonged light exposure on food quality during PDI sterilization.

Description

Technical Field

[0001] This invention relates to the field of food sterilization technology, and in particular to a vitamin-mediated photodynamic sterilization reagent and its method of use and application. Background Technology

[0002] Microbial contamination can occur at any stage of food raw material handling, processing, packaging, and storage. Microorganisms utilize nutrients in food to maintain their own metabolism, producing metabolic waste that reduces food freshness, shortens shelf life, and ultimately leads to spoilage. Statistics show that approximately one-third of the world's food production is wasted annually. Furthermore, food waste disposal accelerates greenhouse gas emissions, causing significant economic losses. More seriously, the metabolic waste produced by microorganisms often contains toxins harmful to humans, easily triggering food safety issues.

[0003] Microbial sterilization is an effective method to minimize food loss and waste caused by microbial contamination. Various technologies are currently used to eradicate harmful microorganisms and ensure food quality and safety, such as heat sterilization and chemical methods. However, these strategies often alter the nutritional and sensory characteristics of food and lead to microbial resistance. In recent years, photodynamic inactivation (PDI) has attracted increasing attention from researchers due to its advantages of being green and safe, not inducing microbial resistance, low cost, and high efficiency. The principle of PDI is to generate reactive oxygen species (ROS) through the combination of photosensitizers, oxygen, and light, including singlet oxygen, hydroxyl radicals, superoxide anions, and hydrogen peroxide, to achieve sterilization. Specifically, after absorbing photons of specific wavelengths and energies, the photosensitizer transitions from the ground state to the singlet state, and then is excited to the triplet state through intersystem crossing (ISC). On its return to the ground state, it collides with neighboring molecules, causing energy transfer and subsequently generating ROS. ROS ultimately lead to bacterial death by oxidizing macromolecules such as proteins, lipids, and nucleic acids within microbial cells.

[0004] However, PDI also has significant drawbacks: most ROS have extremely short half-lives, making it difficult for them to diffuse and migrate within solutions or food matrices. Therefore, PDI sterilization relies on real-time and continuous light exposure; once the light source is removed, most of the generated ROS essentially quench and become ineffective, unable to exert any further sterilization effect. Furthermore, when applied to solid foods, PDI can only perform surface sterilization because the extremely short-lived ROS cannot penetrate or migrate into the food matrix for sterilization. Even if photosensitizers are pre-added to the food, light cannot penetrate, thus preventing the generation of ROS for sterilization within the food. Summary of the Invention

[0005] The purpose of this invention is to provide a vitamin-mediated photodynamic sterilization reagent and its usage and application, in order to solve the defects of the PDI method currently used for food sterilization, such as the extremely short half-life of reactive oxygen species, difficulty in diffusion and transfer in solution or matrix, the inability to act on the food surface, the need for real-time light irradiation, low sterilization efficiency, incomplete bacterial death, narrow application scenarios, and the serious heat generated by some photodynamic light sources (such as xenon lamps) which is detrimental to the quality of the food itself.

[0006] To achieve the above objectives, the present invention provides a vitamin-mediated photodynamic bactericide, comprising the following components: vitamin B2, vitamin C, and a solvent.

[0007] Preferably, the solvent is one of deionized water, physiological saline, and sterilized drinking water.

[0008] Preferably, the molar ratio of vitamin B2 to vitamin C is 2.5:1.

[0009] Preferably, the working concentration of vitamin B2 is not less than 5 μM.

[0010] Preferably, the working concentration of vitamin C is greater than 50 μM.

[0011] A method for using the vitamin-mediated photodynamic sterilization reagent as described above involves dissolving vitamin B2 and vitamin C separately, mixing them evenly in a certain proportion, irradiating them with light for a certain period of time, and then soaking food in the irradiated solution or placing it together with the food in a sealed space for sterilization.

[0012] Preferably, the illumination is provided by LED blue light for 1-20 minutes.

[0013] Preferably, soaking food refers to immersing the food to be sterilized in the solution after the light exposure has ended and then removing it.

[0014] The application of a vitamin-mediated photodynamic bactericide as described above in food sterilization and in killing bacteria.

[0015] Vitamin B2 is a water-soluble vitamin with extremely important physiological functions. It mainly participates in energy production in the cellular respiratory chain, and the synthesis and metabolism of proteins and some hormones, making it an indispensable trace element for the human body. Furthermore, vitamin B2 has excellent photosensitizing properties. This natural photosensitizer has high photosensitivity, reliable sources, and low toxicity, making it one of the most promising photosensitizers in the food industry. Moreover, because water-soluble vitamins are often excreted from the body through urine, the amount that can be used as a food additive is relatively limited, making it a green and safe food-grade photosensitizer.

[0016] Vitamin C is a well-known vitamin with important physiological functions. It is easily soluble in water, has strong reducing properties, participates in complex metabolic processes in the human body, promotes growth and disease resistance, and is often used as a nutritional supplement and antioxidant. It is an essential trace element for the human body. At the same time, vitamin C is also a common food additive; its antioxidant properties help extend the shelf life of food and inhibit oxidative spoilage.

[0017] PDI sterilization involves continuously irradiating a photosensitizer solution / carrier with a light source, thereby generating ROS (reactive oxygen species) in the system. When the photosensitizer molecule—vitamin B2—is irradiated with blue light, it transitions from its ground state to an excited singlet state, and then undergoes ISC (isolated spin-orbit coupling) to reach a triplet state. ISC is a photophysical process mediated by electron spin-orbit coupling, which enables the triplet photosensitizer molecule to transfer electrons or energy with surrounding molecules, thereby generating ROS (mainly short-lived singlet oxygen, superoxide anions, etc.). In this invention, we innovatively introduce vitamin C into the system, which can reduce the triplet photosensitizer molecule, and then react with oxygen in the air to return vitamin B2 to its ground state and generate a large amount of hydrogen peroxide molecules with a longer half-life and bactericidal effect. In addition, the dual-vitamin-mediated photodynamic sterilization system provided by this invention can also generate ascorbic acid radicals, hydroxyl radicals, and other oxidizing substances to play an auxiliary bactericidal role. The synergistic effect of multiple active substances achieves maximum sterilization efficiency.

[0018] The present invention provides a vitamin-mediated photodynamic bactericide, its method of use, and its application. The specific technical effects are as follows:

[0019] (1) The bactericide provided by the present invention can rapidly generate high concentrations of hydrogen peroxide, ascorbic acid free radicals and hydroxyl free radicals and other substances with bactericidal function after being irradiated by LED blue light. The yield of bactericidal substances is high. Only 2 minutes of irradiation of the bactericide with LED blue light is required to kill more than 99.999% of common and highly viable spoilage bacteria, Pseudomonas berries. It can effectively delay the proliferation of total colony count and the production of volatile basic nitrogen in fresh beef at 4°C, thereby delaying the spoilage of beef and extending its shelf life. The half-life of hydrogen peroxide is relatively long, ranging from several hours to several days, which can significantly extend the duration of PDI sterilization.

[0020] (2) The bactericide provided by the present invention can both soak food in a solution containing a high concentration of hydrogen peroxide and use the gaseous hydrogen peroxide volatilized in the solution to fumigate and sterilize the food, thereby achieving sterilization of the food inside; thus completely making up for the serious defects of poor penetration and migration effect in the traditional PDI sterilization process.

[0021] (3) The bactericide provided by the present invention can cause bacteria to break, fracture and dent, thereby completely inactivating the bacteria; the bactericidal components are all vitamins contained in food that are essential for the human body, and will not change the quality of food, and pose no safety hazard to consumers.

[0022] (4) The bactericide provided by the present invention is a substance that has bactericidal function after being exposed to light, and then sterilizes the target object. It does not require real-time light exposure, is easy to use, and can also effectively avoid the adverse effects of long-term light exposure on food quality during PDI sterilization, such as the heat generated by xenon lamp light source, which can lead to food moisture loss and affect color and texture parameters.

[0023] (5) The sterilization method provided by the present invention requires a light-emitting diode (LED) as the light source. LEDs have a compact structure, high luminous efficiency, stable wavelength, no obvious thermal effect, good durability, long service life, and low energy consumption, which effectively reduces the cost of sterilization. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is the standard curve between hydrogen peroxide concentration and absorbance plotted in Embodiment 1 of the present invention;

[0026] Figure 2 This describes the effect of different concentrations of vitamin B2 on hydrogen peroxide production (A) and bactericidal effect (B) in the PDI system in Example 1 of the present invention.

[0027] Figure 3 This describes the effect of different concentrations of vitamin C on hydrogen peroxide production (A) and bactericidal effect (B) in the PDI system in Example 1 of the present invention.

[0028] Figure 4 This describes the effect of different light exposure times on hydrogen peroxide production (A) and bactericidal effect (B) in the PDI system in Embodiment 1 of the present invention.

[0029] Figure 5 The images show (A) of the antibacterial effects of each component against *Pseudomonas berries* in Example 2 of this invention, and (B) the statistical results of the number of viable bacteria remaining on the plates.

[0030] Figure 6 These are SEM images of *Pseudomonas berries* in the control and treatment groups in Example 3 of this invention;

[0031] Figure 7 The results of the membrane potential (A) and hydrophobicity (B) of Pseudomonas berries in the control group and treatment group in Example 4 of this invention are as follows:

[0032] Figure 8 The images (A) and (B) are flow cytometry images of *Pseudomonas berries* in the control and treatment groups in Example 4 of this invention.

[0033] Figure 9 The results of the investigation of tricarboxylic acid cycle activity (A) and respiratory chain dehydrogenase activity (B) of Pseudomonas berries in the control group and treatment group in Example 5 of the present invention;

[0034] Figure 10 This is the actual application effect of sterilization and preservation of fresh beef in Embodiment Six of the present invention; where A is the result of the investigation on the effect on the appearance of beef; B is the result of the investigation on the effect on the total number of colonies in beef; and C is the result of the investigation on the effect on the volatile basic nitrogen in beef. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] The reagents, materials, and instruments used in the examples were all commercially available, as detailed below:

[0038] Vitamin B2 (food grade, purity >99%) and Vitamin C (food grade, purity >99%) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the LED blue light source (wavelength ~460nm, power 5W) was manufactured by Shanghai Philips Lighting Co., Ltd.; Pseudomonas CFC selective medium (powder), Pseudomonas CFC selective medium additive (lyophilized), nutrient broth (NB), and plate counting agar (PCA) were purchased from Qingdao Haibo Biotechnology Co., Ltd.; 3,3′,5,5′-tetramethylbenzidine (TMB, 98%) Horseradish peroxidase (HRP) and 2.5% glutaraldehyde electron microscopy fixative were purchased from Beijing Solarbio Science & Technology Co., Ltd.; dimethyl sulfoxide (DMSO), hexadecane, and anhydrous ethanol were from Sinopharm Chemical Reagent Co., Ltd.; 3% hydrogen peroxide standard solution, catalase, 0.01M hydrochloric acid standard solution, boric acid, methyl red, bromocresol green, rhodamine 123 fluorescent probe, iodonitrobenzolium chloride tetrazolium blue, and 2,3,5-triphenyltetrazolium chloride were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; SYTO 9 and PI dye were purchased from Thermo Fisher Scientific China Co., Ltd.

[0039] Velocity 17R Pro fully automatic high-speed benchtop centrifuge, Tianmei Instruments Laboratory Equipment (Shanghai) Co., Ltd.; SynergyHTX multi-functional microplate reader, Porton Instruments, Inc. (USA); Biochemical incubator, Shanghai Boxun Medical Bio-Instrument Co., Ltd.; Scanning electron microscope (SEM, EM30+), Kusem Corporation (Korea); Guava EasyCyte flow cytometer, Luminex Corporation (USA); Kjeldahl nitrogen analyzer K9860, Shandong Haineng Future Technology Group Co., Ltd.

[0040] Pseudomonas fragi ATCC 4973, a Gram-negative bacterium, was purchased from Beina Biotechnology.

[0041] Example 1

[0042] The conditions for a vitamin-mediated photodynamic sterilization system were optimized. The production of hydrogen peroxide and the sterilization effect in the system were used as the detection indicators for condition optimization.

[0043] The amount of hydrogen peroxide produced in the system is quantified by a TMB oxidation colorimetric reaction: the TMB substrate is oxidized to a cationic blue product under the catalysis of horseradish peroxidase (HRP). This product then reacts with hydrogen peroxide to form a soluble final product, which is dark blue. The hydrogen peroxide content can be quantified by measuring the absorbance of the blue product using a microplate reader and combining it with a standard curve.

[0044] The specific steps for condition optimization are as follows:

[0045] S11. Prepare the required solution.

[0046] Accurately weigh 0.072 g of TMB powder and add it to 10 mL of DMSO solution. After the TMB is completely dissolved, stir and mix well to obtain a 30 mM stock solution I.

[0047] Accurately weigh 0.01g of horseradish peroxidase (HRP) and add it to 1mL of sterile deionized water. Shake well to dissolve. Take 50μL of this solution and mix it with 11.3mL of sterile deionized water to obtain 1μM stock solution II.

[0048] Accurately weigh 0.38g of VB2 and add it to 100mL of deionized water to obtain a VB2 stock solution with a concentration of 10mM.

[0049] Accurately weigh 1.76g of vitamin C and add it to 100mL of deionized water to obtain a 100mM vitamin C stock solution.

[0050] Pseudomonas CFC Selective Medium Plates (for the culture of Pseudomonas berries): Weigh 49.4g of medium powder and add 10g of glycerol. Heat and dissolve in 1000mL of distilled water. Dispense into 200mL bottles. Autoclave at 121℃ for 15min. When cooled to about 50℃, add one vial of Pseudomonas CFC Selective Medium Additive to each bottle, mix well, pour into plates and solidify for later use.

[0051] To prepare a 5 log CFU / mL *Pseudomonas berryense* culture: *Pseudomonas berryense* (stored at -80°C, containing 25% glycerol by volume) was streaked onto a prepared *Pseudomonas* CFC selective medium plate and incubated at 25°C for 48 h. Typical single colonies were picked from the plate and transferred to 10 mL of NB broth, and incubated overnight on a shaker at 25°C (150 rpm / min). The culture was then centrifuged at 4000 g to remove the medium and resuspended in PBS buffer for later use.

[0052] S12. Prepare a standard curve of hydrogen peroxide absorbance values.

[0053] Hydrogen peroxide standard solutions of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 μM were prepared sequentially. 20 μL of stock solution I and 20 μL of stock solution II prepared in step S11 were added to each solution, and their absorbance at 652 nm was measured using a multi-mode microplate reader. A standard curve was obtained by plotting hydrogen peroxide concentration versus absorbance. Figure 1 As shown.

[0054] S13. Investigate the effects of different concentrations of vitamin B2 on hydrogen peroxide production and bactericidal effect. The specific steps are as follows:

[0055] Take 7 portions of deionized water, 0.5 mL for each portion, and add 2 μL of the VC stock solution prepared in step S11 (final VC concentration of 200 μM) to each portion. Then add 0, 0.1, 0.5, 1, 3, 5, and 8 μL of the VB2 stock solution prepared in step S11 (final VB2 concentrations of 0, 1, 5, 10, 30, 50, and 80 μM, respectively), and make up the total volume of the system to 1 mL with deionized water.

[0056] After thoroughly mixing the components, irradiate under LED blue light for 10 min. Each treatment was performed in triplicate. After irradiation, 1 mL of the solution was added sequentially to 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm was measured using a multi-mode microplate reader. Substituting this into the standard curve of hydrogen peroxide concentration versus absorbance prepared in step S12, the amount of hydrogen peroxide produced at different concentrations of VB2 was obtained.

[0057] The results are as follows Figure 2 As shown in Figure A, when the VC concentration is 200 μM and the illumination time is 10 min, the yield of hydrogen peroxide increases with the increase of VB2 concentration, and reaches the highest level near 50 μM. The synergistic effect of further increasing the concentration is not obvious.

[0058] Add 1 mL of the light-exposed solution to a 5 log CFU / mL (5 orders of magnitude) solution of *Pseudomonas berries*, mix and incubate, then take 100 μL and spread it evenly on the surface of a PCA plate. Incubate at 25°C for 48 h and count the number of viable bacteria. Each group has 3 replicates.

[0059] Statistical results are as follows Figure 2 As shown in Figure B, the bactericidal efficiency increases significantly with the increase of VB2 concentration. When the VB2 concentration reaches 30 μM, 5 log CFU / mL of Pseudomonas berries can be completely killed.

[0060] S14. Investigate the effects of different concentrations of vitamin C (VC) on hydrogen peroxide production and bactericidal effect. The specific steps are as follows:

[0061] Take 7 portions of deionized water, 0.5 mL for each portion, and add 5 μL of the VB2 stock solution prepared in step S11 (final VB2 concentration of 50 μM) to each portion. Then add 0, 0.5, 1, 2, 5, 7, and 10 μL of the VC stock solution prepared in step S11 (final VC concentrations of 0, 50, 100, 200, 500, 700, and 1000 μM, respectively), and make up the total volume of the system to 1 mL with deionized water.

[0062] After thoroughly mixing the components, irradiate under LED blue light for 10 min. Each treatment was performed in triplicate. After irradiation, 1 mL of the solution was added sequentially to 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm was measured using a multi-mode microplate reader. Substituting this into the standard curve of hydrogen peroxide concentration versus absorbance prepared in step S12, the amount of hydrogen peroxide produced at different concentrations of VB2 was obtained.

[0063] The results are as follows Figure 3 As shown in Figure A, when the VB2 concentration is 50 μM and the illumination time is 10 min, the production of hydrogen peroxide increases with the increase of VC concentration, reaching a maximum value near 200 μM. Further increasing the VC concentration reduces the production of hydrogen peroxide. When the VC concentration reaches 1000 μM, the production of hydrogen peroxide is close to 0. This may be because excess VC can neutralize the produced hydrogen peroxide.

[0064] Add 1 mL of the light-exposed solution to a 5 log CFU / mL *Pseudomonas berryans* bacterial suspension, mix well, incubate, and then spread 100 μL evenly onto a PCA plate. Incubate at 25°C for 48 h, and count the viable cells. Each group has three replicates. Statistical results are shown below. Figure 3 As shown in Figure B, the system exhibits the highest hydrogen peroxide production at VC concentrations of 200 and 500 μM, effectively inactivating all *Pseudomonas berryans*. However, with further increases in VC concentration, especially at 1000 μM, the hydrogen peroxide concentration approaches zero, yet it still demonstrates good bactericidal activity against *Pseudomonas berryans* (inactivation rate >99% from 5 log CFU / mL to 3 log CFU / mL). Therefore, it is speculated that the bactericidal system provided by this invention generates other substances with antibacterial activity in addition to hydrogen peroxide.

[0065] S15. Investigate the effect of different LED illumination times on hydrogen peroxide production and sterilization effect. The specific steps are as follows:

[0066] Take 7 portions of deionized water, each 0.5 mL, add 5 μL of the VB2 stock solution (final VB2 concentration of 50 μM) prepared in step S11 and 2 μL of the VC stock solution (final VC concentration of 200 μM) prepared in step S11 to each portion, and then add deionized water to make up the total volume of the system to 1 mL.

[0067] After thoroughly mixing the components, irradiate them under LED blue light for 0, 1, 2, 5, 10, 15, and 20 min, respectively. Each treatment was performed in triplicate. After irradiation, 1 mL of the solution was added sequentially to 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm was measured using a multi-mode microplate reader. Substituting this into the standard curve of hydrogen peroxide concentration versus absorbance prepared in step S12, the amount of hydrogen peroxide produced at different concentrations of VB2 was obtained.

[0068] The results are as follows Figure 4 As shown in Figure A, when the light exposure lasts for only 1 minute, the hydrogen peroxide content in the system rises rapidly and reaches its peak at around 10 minutes. Continuing to extend the irradiation time will not increase the hydrogen peroxide concentration.

[0069] Add 1 mL of the light-exposed solution to a 5 log CFU / mL *Pseudomonas berryans* bacterial suspension, mix well, incubate, and then spread 100 μL evenly onto a PCA plate. Incubate at 25°C for 48 h, and count the viable cells. Each group has three replicates. Statistical results are shown below. Figure 4 As shown in B, light exposure for only 2 minutes is sufficient to completely inactivate Pseudomonas berries in the system.

[0070] In summary, the optimal conditions for the obtained vitamin-mediated photodynamic sterilization system are: VB2 concentration of 50 μM; VC concentration of 200 μM; and light exposure time of 10 min.

[0071] Example 2

[0072] The effects of each component in the vitamin-mediated photodynamic bactericidal system provided by this invention on the bactericidal effect were investigated, and the specific steps are as follows:

[0073] A *Pseudomonas berryis* bacterial suspension with a concentration of 5 log CFU / mL was prepared using the method described in Example 1. Nine aliquots of the bacterial suspension, each with a total volume of 1 mL, were taken and treated with different reagents. The specific treatments are as follows: No reagents were added (control group); 5 μL of the VB2 stock solution prepared in Example 1 (final VB2 concentration of 50 μM) was added (vitamin B2 group); 5 μL of the VB2 stock solution prepared in Example 1 (final VB2 concentration of 50 μM) was added and irradiated with an LED blue light for 10 min (vitamin B2 / light group); 2 μL of the VC stock solution prepared in Example 1 (final VC concentration of 200 μM) was added and irradiated with an LED blue light for 10 min (vitamin C / light group); 5 μL of the VB2 stock solution prepared in Example 1 (final VC concentration of 200 μM) was added (vitamin C group); 5 μL of the VB2 stock solution prepared in Example 1 (final VC concentration of 200 μM) was added and irradiated with an LED blue light for 10 min (vitamin C / light group); The following groups were treated with VB2 (final concentration 50 μM) and 2 μL of LVC (final concentration 200 μM), designated as the Vitamin B2 / Vitamin C group. The following groups were treated with LED blue light for 10 min, and the following groups were treated with VB2 (final concentration 50 μM) and 2 μL of LVC (final concentration 200 μM) prepared in Example 1, designated as the Vitamin B2 / Vitamin C / Light group. This group represents the optimal treatment condition in Example 1. The following groups were treated with 1 μL of catalase at a concentration of 1000 U / mL, designated as the catalase group. The following groups were treated with VB2 (final concentration 50 μM), 2 μL of LVC (final concentration 200 μM), and 1 μL of catalase at a concentration of 1000 U / mL prepared in Example 1, designated as the Vitamin B2 / Vitamin C / Light + Catalase group.

[0074] The results are as follows Figure 5 As shown: Treatment of *Pseudomonas berryae* with only VB2, VC, VB2 / VC, or VC / light showed no difference compared to the blank control group, indicating no sterilization effect. The VB2 / light group could inactivate 1 log CFU / mL (one order of magnitude, equivalent to 90%) of bacteria. This is because VB2 is a common photosensitizer in PDI, which can produce a small amount of ROS with a long half-life under light, thus having a certain bactericidal effect. In this invention, when VC is added to the VB2-mediated PDI system and light is applied, the bactericidal efficiency of the original PDI against *Pseudomonas berryae* is greatly improved, with the inactivation rate increasing from 1 log CFU / mL to 5 log CFU / mL (inactivation rate increasing from 90% to over 99.999%).

[0075] In addition, we added catalase to the system to verify whether any other substances besides hydrogen peroxide had a bactericidal effect. Catalase itself has no bactericidal effect; however, when added to the VB2 / VC / light system, it neutralized all the hydrogen peroxide produced in the system. The results showed that after adding catalase, the system still had an inactivation efficiency of approximately 1 log CFU / mL (one order of magnitude, equivalent to 90%). This proves that other active substances in the system also play a bactericidal role, and also demonstrates that hydrogen peroxide plays a dominant bactericidal role in the system. It has been reported that VC and hydrogen peroxide undergo a redox reaction to further produce ascorbic acid free radicals, hydroxyl free radicals, and other active substances, thus playing an additional auxiliary bactericidal role.

[0076] Example 3

[0077] The effect of the optimal bactericidal system obtained in Example 1 on the surface morphology of *Pseudomonas berries* was investigated. The specific steps are as follows:

[0078] *Pseudomonas berries* were obtained in the treatment group (vitamin B2 / vitamin C / light group) and the control group (no reagent added) using the same method as in Example 2. 1 mL of bacterial suspension from each treatment / control group was centrifuged at 6000 rpm for 5 min and the supernatant was discarded. The bacterial pellet was then resuspended in 1 mL of 2.5% glutaraldehyde electron microscopy fixative and incubated overnight at 4°C to fix the bacteria. Excess fixative was discarded, and the pellet was washed with physiological saline. The bacterial sample was then dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol (twice), with 10 min intervals between each dehydration. 10 μL of the dehydrated bacterial suspension was added to a sterile cell slide, freeze-dried, and then sputter-coated with gold. Three different fields of view were randomly selected for observation and photography under SEM (2000x and 10000x magnification).

[0079] The results are as follows Figure 6 As shown, compared with the control group, the bacteria in the treatment group showed signs of breakage, fracture, and indentation, which proves that the bacteria have been completely inactivated and there is no risk of revival and regeneration.

[0080] Example 4

[0081] The effect of the optimal bactericidal system obtained in Example 1 on the cell membrane of *Pseudomonas berries* was investigated. The specific steps are as follows:

[0082] The treated and control groups of *Pseudomonas berries* were obtained using the same method as in Example 3. The effects of the vitamin-mediated photodynamic bactericidal system provided by this invention on bacterial cell membranes were investigated by detecting the membrane potential, hydrophobicity, and permeability of the cell membranes of the treated and control groups.

[0083] The membrane potential was detected as follows: Rhodamine 123 fluorescent probe was dissolved in sterile water to prepare a stock solution with a concentration of 1 mg / mL. 2 μL of Rhodamine 123 was added to 1 mL of the obtained treatment / control group bacterial solution to bring the final concentration to 2 μg / mL. After incubation in the dark for 30 min, the fluorescence intensity was detected using a fluorescence spectrophotometer with an excitation wavelength of 480 nm, an emission wavelength of 530 nm, a scanning range of 500-600 nm, and a gain factor of 10.

[0084] Metabolically active bacteria possess a normal membrane potential, which is crucial for their physiological metabolism. When bacteria are stimulated by external factors, their membrane potential typically changes. Rhodamine 123 is a cationic dye with strong fluorescence properties; its intensity is correlated with the cell membrane potential. When the membrane potential is negative, its fluorescence intensity increases, indicating that the bacterial cell membrane has undergone hyperpolarization due to adverse influences. Figure 7 Part A shows that the fluorescence intensity of the treated group increased significantly at 530 nm, proving that the bacteria changed from a normal physiological state to a hyperpolarized imbalance state after treatment.

[0085] The hydrophobicity was determined by adding 1 mL of hexadecane to the obtained bacterial suspensions of the treatment and control groups (1 mL) and mixing thoroughly for 1 min. After incubation for 30 min, the OD of the aqueous phase before and after incubation was measured using a multi-functional microplate reader. 600nm The value of is then used to calculate the hydrophobicity using the following formula.

[0086] Hydrophobicity (%) = (AB) / A × 100%, where A is the OD before incubation. 600nm The value of B is the OD value after incubation. 600nm The value of .

[0087] The cell membranes of Gram-negative bacteria are composed of abundant proteins and polysaccharides, which determine the hydrophobic properties of *Pseudomonas fruticosa*. When the bacterial cell membrane is disrupted, its hydrophobicity decreases. The result is as follows... Figure 7 As shown in Part B, the hydrophobicity of bacteria in the treatment group was significantly lower than that in the control group, which also proves that the bacterial cell membrane was affected.

[0088] The membrane permeability was measured as follows: 1 mL of sample was placed in each centrifuge tube, and 200 μL was used for testing. 300 μL of bacterial resuspension was transferred from each tube to a new centrifuge tube, vortexed, and divided into two equal portions. One portion was left untreated, and the other was boiled for 5 minutes. The two treated suspensions were remixed and divided into three equal portions, labeled as Blank, SYTO 9, and SYTO 9+PI for staining. Blank tubes were left unstained; SYTO 9 tubes: 2 μL of SYTO 9 staining solution diluted 10-fold was added; SYTO 9+PI tubes: 2 μL of SYTO 9 staining solution diluted 10-fold and 2 μL of PI staining solution diluted 10-fold were added; all other tubes: 2 μL of SYTO 9 staining solution diluted 10-fold and 2 μL of PI staining solution diluted 10-fold were added. Changes in the apoptosis rate of bacterial cells in the treatment and control groups were detected using the Green-B and Red-B channels of a flow cytometer.

[0089] By observing the penetration of SYTO9 and PI dyes into the cell membrane, it can be seen that the bacterial state in the treatment group changed from live apoptotic cells to necrotic cells. Figure 8 ).

[0090] In summary, after treatment with the novel PDI system, the cell membrane of *Pseudomonas berries*, which maintains essential physiological functions, was severely affected and damaged (membrane potential increased, hydrophobicity decreased, and membrane permeability increased), further demonstrating the thoroughness and high efficiency of this invention in inactivating bacteria.

[0091] Example 5

[0092] The effects of the optimal bactericidal system obtained in Example 1 on the respiratory metabolism of *Pseudomonas berries* were investigated to examine the thoroughness of the vitamin-mediated photodynamic bactericidal system provided by this invention. The specific steps are as follows:

[0093] The *Pseudomonas berries* in the treatment and control groups were obtained using the same method as in Example 3. Iodonitrobenzolium chloride (1 mM) was added to 1 mL of the bacterial culture in both groups and incubated at 37°C for 30 min. The absorbance at 630 nm was measured using a microplate reader to assess the effect of treatment on the intracellular tricarboxylic acid cycle activity of the bacteria. The results are as follows: Figure 9 As shown in A.

[0094] 0.1M glucose and 1 mg / mL triphenyltetrazolium chloride were added to 1 mL of bacterial culture in both the treatment and control groups, and the mixture was incubated at 37°C for 30 min. Two drops of concentrated sulfuric acid were then added to terminate the reaction, followed by the addition of 1 mL of n-butanol and shaking. The absorbance was measured at 490 nm using a microplate reader to assess the effect of treatment on the activity of intracellular respiratory chain dehydrogenases. The results are as follows: Figure 9 As shown in B.

[0095] The intracellular metabolism of bacteria in the treatment group was assessed by evaluating the activities of the tricarboxylic acid cycle and respiratory chain dehydrogenases. The tricarboxylic acid cycle and respiratory chain dehydrogenases play crucial roles in bacterial life activities, responsible for important physiological functions such as energy production, metabolic regulation, and electron transport. Figure 9 It can be seen that, compared with the control group, the activity of tricarboxylic acid cycle and respiratory chain dehydrogenase in the treated group of *Pseudomonas berries* was significantly reduced, which further proves that the bacteria were completely killed and unable to carry out normal physiological activities.

[0096] Example 6

[0097] The effects of the optimal sterilization system obtained in Example 1 on the respiratory metabolism of *Pseudomonas berrieseri* in beef were investigated to examine the effectiveness of the vitamin-mediated photodynamic sterilization system provided by this invention in practical applications. The specific steps are as follows:

[0098] Fresh beef from the same batch was purchased from a supermarket. After removing the fascia, it was cut into small pieces of approximately 3cm*3cm*1cm (10g) in a sterile laminar flow hood to ensure that each piece of beef had the same appearance and quality. The beef was then irradiated with ultraviolet light for 1 hour to maximize the elimination of background microorganisms. Subsequently, each piece of beef was inoculated with *Pseudomonas berryis* solution to a concentration of 5 log CFU / g and air-dried in the laminar flow hood for half an hour to ensure that the bacteria were adhered to and absorbed by the beef. The beef was then placed in a sterile homogenizing bag and immersed in a novel PDI solution containing the optimal sterilization system obtained in Example 1 for sterilization. The control group was immersed in an equal volume of sterile deionized water. After treatment, the beef was transferred to sterile petri dishes and stored at 4°C for 0, 1, 3, 5, and 7 days. Changes in appearance (photographed from above in a studio), total colony count, and volatile basic nitrogen were recorded at specified time points. Each group was repeated three times with three parallel operations.

[0099] The method for detecting the total bacterial count in beef is as follows: After storing fresh beef for a specified time, remove it and place it in a sterile homogenization bag. Then add 90 mL of sterile physiological saline and homogenize on a homogenizer for 5 min to suspend the bacteria in the physiological saline. Take 100 μL of the supernatant and spread it evenly on the surface of a PCA plate. Incubate in a 25℃ biochemical incubator for 48 h, and count the viable bacteria. Each group is set up with 3 replicates.

[0100] The method for detecting volatile basic nitrogen in beef is as follows: (1) Reagent preparation: Weigh 20g of boric acid and dissolve it in 1L of water to prepare a boric acid solution with a concentration of 20g / L; weigh 0.1g of methyl red and dissolve it in 100mL of 95% ethanol to prepare a methyl red solution with a concentration of 1g / L; weigh 0.1g of bromocresol green and dissolve it in 100mL of 95% ethanol to prepare a bromocresol green solution with a concentration of 1g / L; weigh 1g of magnesium oxide and dissolve it in 100mL of water to prepare a 1% magnesium oxide suspension. Mix 1.37mL of methyl red solution and 8.63mL of bromocresol green solution and add them to 1L of boric acid solution to prepare a boric acid absorption solution and add it to the Kjeldahl nitrogen analyzer. (2) Sample preparation: Take 3g of beef and 27mL of deionized water, homogenize the beef in a homogenizing bag and filter it through filter paper to obtain a filtrate containing volatile basic nitrogen. (3) Determination of volatile basic nitrogen: The fully automatic mode was turned on on the Kjeldahl nitrogen analyzer, and the following settings were set: 25 mL of boric acid was added, the distillation time was 5 min, the rinsing water volume was 30 mL, and the hydrochloric acid concentration used for calibration was 0.01 M. Finally, the content of volatile basic nitrogen in beef stored for different days was determined according to the calculation method of national standard GB 5009.228-2016.

[0101] The results are as follows Figure 10 As shown: After treatment with the novel PDI sterilization system developed in this invention, the color change of the contaminated fresh beef was significantly delayed, while the beef in the control group turned black and severely rotten by day 7. Figure 10 Part A of the document.

[0102] The total bacterial count in the beef was further tested. Figure 10 Part B) and volatile basic nitrogen ( Figure 10 (Part C) The results showed that treatment with the novel PDI sterilization system provided by this invention significantly inhibited the proliferation of putrefactive bacteria in beef. On the third day, the total colony count in the control group beef increased rapidly, indicating severe spoilage, while the treated group beef maintained a low level of microorganisms. On the other hand, the content of volatile basic nitrogen represents the level of protein decomposition in meat products; the more severe the spoilage, the higher the content of volatile basic nitrogen. According to the National Food Safety Standard for the Determination of Volatile Basic Nitrogen in Food (GB 5009.228-2016), when the content of volatile basic nitrogen in meat products exceeds 15 mg / 100g, it indicates food spoilage. Figure 10 As can be seen from C, the beef in the treatment group only began to spoil on the 5th day, while the beef in the control group had already reached the national standard limit on the 3rd day.

[0103] Therefore, the vitamin-mediated photodynamic bactericide provided by this invention not only exhibits a highly efficient and thorough bactericidal effect in vitro, but also significantly delays the spoilage of fresh beef in real life, thus showing good and efficient application prospects.

[0104] In summary, the bactericide provided by this invention can rapidly generate high concentrations of bactericidal substances such as hydrogen peroxide, ascorbic acid free radicals, and hydroxyl free radicals after being irradiated with LED blue light, achieving a sterilization rate of over 99.999%. Its long half-life significantly extends the duration of PDI sterilization. It can sterilize the interior of food, thus completely overcoming the serious defects of poor penetration and migration in traditional PDI sterilization. It can completely inactivate bacteria. The sterilizing components pose no safety risks to consumers. It requires no real-time illumination, making it convenient to use and effectively avoiding the adverse effects of prolonged light exposure on food quality during PDI sterilization. The required light source is a light-emitting diode (LED), which has a compact structure, high luminous efficiency, stable wavelength, does not produce significant thermal effects, and is durable, has a long service life, and low energy consumption, effectively reducing sterilization costs. It demonstrates good and efficient practical application potential and can significantly delay the spoilage of fresh beef.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A vitamin-mediated photodynamic bactericide, characterized in that, It consists of the following components: vitamin B2, vitamin C, and solvent; the photodynamic therapy is performed by irradiation with an LED blue light lamp; When the final concentration of vitamin C is 200 μM, the concentration of vitamin B2 is 50-80 μM; when the final concentration of vitamin B2 is 50 μM, the concentration of vitamin C is 100-1000 μM.

2. The vitamin-mediated photodynamic bactericide according to claim 1, characterized in that: The solvent is one of deionized water, sterilized drinking water, or physiological saline.

3. A method for sterilization using the vitamin-mediated photodynamic bactericidal reagent according to claim 1 or 2, characterized in that: After dissolving vitamin B2 and vitamin C separately, they are mixed evenly in a certain proportion and then exposed to light for a certain period of time. Food is then soaked in the solution after being exposed to light to sterilize it. Irradiate with LED blue light for 1-20 minutes; Soaking food refers to immersing food to be sterilized in a solution after the light exposure has ended, and then taking it out directly.

4. The application of a vitamin-mediated photodynamic bactericide as described in claim 1 or 2 in food sterilization, characterized in that: Applications in killing bacteria.

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