Vitamin-mediated photodynamic bactericidal reagent as well as use method and application thereof

Abstract

The invention discloses a vitamin-mediated photodynamic sterilization reagent as well as a use method and application thereof, and belongs to the technical field of food sterilization. The bactericide provided by the invention can quickly generate active substances with sterilization functions, such as high-concentration hydrogen peroxide, after being irradiated by LED blue light, and can thoroughly sterilize, and the sterilization rate can reach 99.999% or above; the inactivated components have no potential safety hazard to consumers; the proliferation of the total colony count of the fresh beef at 4 DEG C and the generation of volatile basic nitrogen can be effectively delayed, so that the shelf life of the beef is prolonged; the generated ROS has a long half-life period, the serious defect of poor permeation and migration effects in the traditional PDI sterilization process can be overcome, and the sterilization duration can be greatly prolonged; the needed light source is an LED blue light lamp, the light emitting efficiency is high, the wavelength is stable, no obvious heat effect is generated, the durability is good, and the sterilization cost is low; real-time illumination is not needed, the use is convenient, and the adverse effect on the food quality caused by long-time illumination in the PDI sterilization process can be effectively avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of food sterilization, and in particular to a vitamin-mediated photodynamic sterilization agent and a use method and application thereof. Background Art

[0002] Microbial contamination may occur in every process of food raw material handling, processing, packaging and storage. Microorganisms use nutrients in food to maintain their own metabolism and produce metabolic waste, which reduces the freshness of food and shortens its shelf life, leading to food spoilage. According to statistics, about one-third of the food produced in the world is wasted each year. In addition, the treatment of food waste also accelerates greenhouse gas emissions, causing significant economic losses. What's more serious is that the metabolic waste produced by microorganisms is mostly toxins that are harmful to the human body and can easily cause food safety problems.

[0003] Microbial sterilization is an effective method to minimize food loss and waste caused by microbial contamination. Various technologies have been applied to eradicate harmful microorganisms and ensure food quality and safety, such as thermal sterilization and chemical methods. However, these strategies often change the nutritional and sensory properties of food and lead to microbial resistance. In recent years, photodynamic inactivation (PDI) has begun to attract more and more researchers' attention due to its advantages of being green and safe, not producing microbial resistance, low cost, and high efficiency. The principle of PDI is to achieve the purpose of sterilization by combining photosensitizers, oxygen and light to produce reactive oxygen species (ROS), including singlet oxygen, hydroxyl radicals, superoxide anions, and hydrogen peroxide. Specifically, after absorbing photons of specific wavelengths and energies, the photosensitizer will transform from the ground state to the singlet state, and then be excited to the triplet state through intersystem crossing (ISC). On the way back to the ground state, it collides with neighboring molecules, resulting in energy transfer, and then produces ROS. ROS eventually kill bacteria by oxidizing macromolecules such as proteins, lipids, and nucleic acids in microbial cells.

[0004] However, PDI also has obvious disadvantages: the half-life of most ROS is extremely short, which makes it difficult for them to diffuse and migrate in solutions or food matrices. For this reason, PDI sterilization relies on real-time and continuous illumination. Once the light source is removed, most of the ROS generated are basically quenched instantly and fail to play a further bactericidal role. In addition, when PDI is applied to solid foods, it can only sterilize the surface, because ROS with extremely short lifespans cannot penetrate or migrate into the food matrix for killing. Even if the photosensitizer is added to the food in advance, the light cannot penetrate, so ROS sterilization cannot be generated inside. Summary of the invention

[0005] The purpose of the present invention is to provide a vitamin-mediated photodynamic sterilization reagent and a method for using and application thereof, so as to solve the defects of the PDI method currently used for food sterilization, such as the active oxygen produced has an extremely short half-life, is difficult to diffuse and transfer in a solution or matrix, can only act on the surface of food, cannot sterilize continuously, requires real-time illumination, has low sterilization efficiency, does not completely kill bacteria, has narrow application scenarios, and some photodynamic light sources (such as xenon lamps) generate severe heat that is not conducive to the quality of the food itself.

[0006] To achieve the above object, the present invention provides a vitamin-mediated photodynamic sterilization agent, 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: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 is to dissolve vitamin B2 and vitamin C separately, mix them evenly in proportion, and then expose them to light for a certain period of time, and then use the solution after exposure to light to soak food or place it in a closed space with food for sterilization.

[0012] Preferably, the illumination is performed using LED blue light for 1-20 min.

[0013] Preferably, soaking the food means immersing the food to be sterilized into the solution and then taking it out after the light irradiation is completed.

[0014] An application of the vitamin-mediated photodynamic sterilization agent as described above in food sterilization and in killing bacteria.

[0015] Vitamin B2 is a water-soluble vitamin with extremely important physiological functions. It is mainly involved in the energy production of the cell respiratory chain, the anabolism of proteins and some hormones, etc. It is an indispensable trace element for the human body. In addition, vitamin B2 has good photosensitivity. This natural photosensitizer has high light efficiency, reliable source, and low toxicity and side effects. It has become one of the most promising photosensitizers in the food field. In addition, since water-soluble vitamins are often excreted from the body through urine, the amount of them that can be used as food additives is relatively limited. It is a green and safe food-grade photosensitizer.

[0016] Vitamin C is a well-known vitamin that also has important physiological functions. This vitamin is easily soluble in water and has strong reducing properties. It participates in the complex metabolic process of 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 are beneficial to prolonging the shelf life of food and inhibiting food oxidation and deterioration.

[0017] PDI sterilization is to continuously irradiate the photosensitizer solution / carrier through a light source, thereby generating ROS sterilization in the system. When the photosensitizer molecule-vitamin B2 is irradiated with blue light, it will change from its ground state to an excited singlet state, and then reach a triplet state through ISC. ISC is a photophysical process mediated by electron spin-orbit coupling, which enables triplet photosensitizer molecules to transfer electrons or energy with surrounding molecules, thereby generating ROS (mainly singlet oxygen with extremely short half-life, superoxide anions, etc.). In the present invention, when we innovatively introduce vitamin C into the system, it can reduce triplet photosensitizer molecules, and then further react with oxygen in the air to return vitamin B2 to the ground state again and produce 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 the present invention can also produce ascorbic acid free radicals, hydroxyl free radicals and other oxidizing substances to play an auxiliary bactericidal role, and multiple active substances act synergistically to achieve the maximum bactericidal efficiency.

[0018] The present invention provides a vitamin-mediated photodynamic sterilization agent and a method and application thereof, and the specific technical effects thereof are as follows:

[0019] (1) The bactericide provided by the present invention can rapidly generate high-concentration hydrogen peroxide, ascorbic acid free radicals, hydroxyl free radicals and other bactericidal substances after being irradiated with LED blue light. The yield of bactericidal substances is high. It only takes 2 minutes of LED blue light irradiation to kill more than 99.999% of the common and highly viable spoilage bacteria, Pseudomonas fragariae. It can effectively delay the proliferation of the total colony count and the generation 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 greatly extend the duration of PDI sterilization.

[0020] (2) The bactericide provided by the present invention can be used to soak food in a solution containing high concentration of hydrogen peroxide, or to fumigate and sterilize food using the gaseous hydrogen peroxide volatilized from the solution, thereby achieving sterilization inside the food; thereby completely making up for the serious defects of poor penetration and migration effects 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 killing components are all vitamins contained in food and essential to the human body, which will not change the quality of food and pose no safety risks to consumers;

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

[0023] (5) The sterilization method provided by the present invention requires a light emitting diode (LED) as the light source. The LED has a compact structure, high luminous efficiency, and stable wavelength. It will not produce obvious thermal effects. It also has good durability, long service life, and low energy consumption, which effectively reduces the sterilization cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

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

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

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

[0028] Figure 4 The effect of different illumination times on the production of hydrogen peroxide (A) and the bactericidal effect (B) in the PDI system in Example 1 of the present invention;

[0029] Figure 5 The antibacterial effect of each component in Example 2 of the present invention on Pseudomonas fragariae is shown in a plate photograph (A) and the statistical result of the number of viable bacteria remaining on the plate (B);

[0030] Figure 6 is the SEM image of Pseudomonas fragariae in the control group and the treatment group in Example 3 of the present invention;

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

[0032] Figure 8 Flow cytometry images (A) and statistical results of the cell ratios of each component (B) of Pseudomonas fragariae in the control group and the treatment group in Example 4 of the present invention;

[0033] Fig. 9 These are the results of investigating the tricarboxylic acid cycle activity (A) and respiratory chain dehydrogenase activity (B) of Pseudomonas fragariae in the control group and the treatment group in Example 5 of the present invention;

[0034] Fig.10 It is the actual application effect of Example 6 of the present invention on the sterilization and preservation of fresh beef; wherein A is the result of the investigation on the impact on the appearance of beef; B is the result of the investigation on the impact on the total number of colonies in beef; and C is the result of the investigation on the impact on volatile basic nitrogen in beef. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0036] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0037] The reagents, materials, and instruments used in the examples were all obtained through commercial channels, and the specific information is as follows:

[0038] Vitamin B2 (VB2, food grade, purity> 99%), Vitamin C (VC, food grade, purity> 99%), were purchased from Shanghai Biotech Co., Ltd.; LED blue light source (wavelength ~ 460nm, power 5W), produced by Shanghai Philips Lighting Co., Ltd.; Pseudomonas CFC selective medium (powder), Pseudomonas CFC selective medium additive (lyophilized), nutrient broth (NB), plate count 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 Solebow Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO), hexadecane, and anhydrous ethanol were from Sinopharm Chemical Reagent Co., Ltd.; 3% hydrogen peroxide standard solution, catalase, 0.01 M hydrochloric acid standard solution, boric acid, methyl red, bromocresol green, rhodamine 123 fluorescent probe, iodonitrovinyl tetrazolium chloride blue, and 2,3,5-triphenyltetrazolium chloride were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; SYTO 9 and PI dyes were purchased from Thermo Fisher Scientific China Co., Ltd.

[0039] Fully automatic high-speed desktop centrifuge Velocity 17R Pro, Technic Instruments Laboratory Equipment (Shanghai) Co., Ltd.; multifunctional microplate reader SynergyHTX, American Boten Instrument Co., Ltd.; biochemical incubator, Shanghai Boxun Medical Biological Instrument Co., Ltd.; scanning electron microscope (SEM, EM30+), Korean Kusem Co., Ltd.; flow cytometer Guava EasyCyte, American Luminex Co., Ltd.; Kjeldahl nitrogen analyzer K9860, Shandong Haineng Future Technology Group Co., Ltd.

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

[0041] Embodiment 1

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

[0043] The amount of hydrogen peroxide produced in the system is quantified by TMB oxidation color reaction: TMB substrate is oxidized to a cationic blue product under the catalysis of horseradish peroxidase HRP, and then the product reacts with hydrogen peroxide to form a soluble final product, showing a dark blue color. The absorbance of the blue product is measured by an ELISA instrument and combined with a standard curve to quantify the content of hydrogen peroxide.

[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 TMB is completely dissolved, stir and mix to obtain 30 mM stock solution I.

[0047] Accurately weigh 0.01 g of horseradish peroxidase HRP, add 1 mL of sterile deionized water and shake thoroughly to dissolve. Take 50 μL of the dissolved solution and mix it with 11.3 mL of sterile deionized water to obtain a 1 μM stock solution II.

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

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

[0050] Pseudomonas CFC selective medium plate (for the culture of Pseudomonas fragariae): weigh 49.4g of medium powder and add 10g of glycerol, heat and dissolve in 1000mL of distilled water, divide into 200mL bottles, sterilize at 121℃ for 15min, cool to about 50℃, add one tube of Pseudomonas CFC selective medium additive to each bottle, mix well, pour into a plate to solidify for later use.

[0051] Prepare 5 log CFU / mL of Pseudomonas fragariae bacterial solution: Streak Pseudomonas fragariae (stored at -80°C, containing 25% volume of glycerol) on the prepared Pseudomonas CFC selective medium plate and culture at 25°C for 48 hours. Pick a typical single colony from the plate into NB broth (10 mL) and culture overnight on a shaker at 25°C (speed of 150 rpm / min). Centrifuge the bacterial solution at 4000g to remove the culture medium and add PBS buffer to resuspend for later use.

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

[0053] Prepare 0, 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 μM hydrogen peroxide standard solutions in sequence, add 20 μL of the stock solution I and 20 μL of the stock solution II prepared in step S11, respectively, detect the absorbance at 652 nm by a multifunctional microplate reader, and plot the hydrogen peroxide concentration and the absorbance value to obtain a standard curve between the hydrogen peroxide concentration and the absorbance, as shown in FIG. Figure 1 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 each, and add 2 μL of the VC stock solution prepared in step S11 (the final concentration of VC is 200 μM), then add 0, 0.1, 0.5, 1, 3, 5, and 8 μL of the VB2 stock solution prepared in step S11 (the final concentration of VB2 is 0, 1, 5, 10, 30, 50, and 80 μM, respectively), and add deionized water to the total volume of the system to 1 mL.

[0056] After the components are fully mixed, they are placed under LED blue light for 10 minutes. Three replicates are set for each treatment. After the illumination is over, 1 mL is taken and added to the system in sequence with 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm is detected by a multifunctional microplate reader. Substitute the standard curve between hydrogen peroxide concentration and absorbance prepared in step S12 to obtain the amount of hydrogen peroxide produced under different concentrations of VB2.

[0057] The results are as follows Figure 2 As shown in 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 benefits brought by further increasing the concentration are not obvious.

[0058] 1 mL of the illuminated solution was added to a 5 log CFU / mL (5 orders of magnitude) bacterial solution of Pseudomonas fragariae. After mixing and incubation, 100 μL was evenly spread on the surface of the PCA plate. The plate was placed in a 25°C biochemical incubator for 48 hours and the number of live bacteria was counted. Three replicates were set for each group.

[0059] The statistical results are as follows Figure 2 As shown in Figure B, the bactericidal efficiency increased significantly with the increase of VB2 concentration. When the VB2 concentration reached 30 μM, 5 log CFU / mL of Pseudomonas fragrans could 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 each, add 5 μL of the VB2 stock solution prepared in step S11 (the final concentration of VB2 is 50 μM), then add 0, 0.5, 1, 2, 5, 7, and 10 μL of the VC stock solution prepared in step S11 (the final concentration of VC is 0, 50, 100, 200, 500, 700, and 1000 μM, respectively), and add deionized water to the total volume of the system to 1 mL.

[0062] After the components are fully mixed, they are placed under LED blue light for 10 minutes. Three replicates are set for each treatment. After the illumination is over, 1 mL is taken and added to the system in sequence with 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm is detected by a multifunctional microplate reader. Substitute the standard curve between hydrogen peroxide concentration and absorbance prepared in step S12 to obtain the amount of hydrogen peroxide produced under different concentrations of VB2.

[0063] The results are as follows Figure 3 As shown in 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 and reaches a maximum value near 200 μM. As the VC concentration continues to increase, the production of hydrogen peroxide decreases. 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] 1 mL of the solution after illumination was added to the Pseudomonas fragariae liquid with a concentration of 5 log CFU / mL. After mixing and incubation, 100 μL was evenly spread on the surface of the PCA plate, and then placed in a 25°C biochemical incubator for 48 hours and the number of viable bacteria was counted. Three replicates were set for each group. The statistical results are shown in Figure 1. Figure 3 As shown in Figure B, when the VC concentration is 200 and 500 μM, the production of hydrogen peroxide in the system is the highest, and all Pseudomonas fragariae can be inactivated. However, when the VC concentration continues to increase, especially when the VC concentration reaches 1000 μM, the concentration of hydrogen peroxide in the system is close to 0, but it still has a good bactericidal effect on Pseudomonas fragariae (reduced from 5log CFU / mL to 3log CFU / mL, inactivation rate>99%). Therefore, it is speculated that the sterilization system provided by the present invention produces other substances with antibacterial activity in addition to hydrogen peroxide.

[0065] S15. Investigate the effects 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, 0.5 mL each, add 5 μL of the VB2 stock solution prepared in step S11 (the final concentration of VB2 is 50 μM) and 2 μL of the VC stock solution prepared in step S11 (the final concentration of VC is 200 μM), and add deionized water to the total volume of the system to 1 mL.

[0067] After the components are fully mixed, they are placed under LED blue light for 0, 1, 2, 5, 10, 15, and 20 minutes. Three replicates are set for each treatment. After the illumination is completed, 1 mL is taken and added to the system in sequence with 20 μL of stock solution I and 20 μL of stock solution II, and the absorbance at 652 nm is detected by a multifunctional microplate reader. Substitute the standard curve between the hydrogen peroxide concentration and the absorbance prepared in step S12 to obtain the amount of hydrogen peroxide produced under different concentrations of VB2.

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

[0069] 1 mL of the solution after illumination was added to the Pseudomonas fragariae liquid with a concentration of 5 log CFU / mL. After mixing and incubation, 100 μL was evenly spread on the surface of the PCA plate, and then placed in a 25°C biochemical incubator for 48 hours and the number of viable bacteria was counted. Three replicates were set for each group. The statistical results are shown in Figure 1. Figure 4 As shown in B, only 2 minutes of illumination is enough to completely inactivate the Pseudomonas fragariae in the system.

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

[0071] Embodiment 2

[0072] The effects of various components in the vitamin-mediated photodynamic sterilization system provided by the present invention on the sterilization effect are investigated, and the specific steps are as follows:

[0073] The method in Example 1 was used to prepare a bacterial solution of Pseudomonas fragariae with a concentration of 5log CFU / mL. Nine portions of bacterial solution were taken, each with a total volume of 1 mL. Different reagents were added and different treatments were performed. The specific treatments were as follows: no reagent was added and recorded as the control group; 5 μL of the VB2 stock solution prepared in Example 1 was added (the final concentration of VB2 was 50 μM), which was recorded as the vitamin B2 group; 5 μL of the VB2 stock solution prepared in Example 1 (the final concentration of VB2 was 50 μM) was added and irradiated with an LED blue light for 10 min, which was recorded as the vitamin B2 / light group; 2 μL of the VC stock solution prepared in Example 1 was added (the final concentration of VC was 200 μM), which was recorded as the vitamin C group; 2 μL of the VC stock solution prepared in Example 1 (the final concentration of VC was 200 μM) was added, and irradiated with an LED blue light for 10 min, which was recorded as the vitamin C / light group; 5 μL of the VB2 stock solution prepared in Example 1 ( VB2 final concentration is 50 μM), 2 μL VC stock solution (VC final concentration is 200 μM), recorded as VB2 / vitamin C group; 5 μL VB2 stock solution prepared in Example 1 (VB2 final concentration is 50 μM), 2 μL VC stock solution (VC final concentration is 200 μM) were added, and irradiated with LED blue light for 10 min, recorded as VB2 / vitamin C / light group, which is the treatment group with the optimal conditions in Example 1; 1 μL of catalase with a concentration of 1000 U / mL was added, recorded as catalase group; 5 μL VB2 stock solution prepared in Example 1 (VB2 final concentration is 50 μM), 2 μL VC stock solution (VC final concentration is 200 μM), 1 μL catalase with a concentration of 1000 U / mL were added, and irradiated with LED blue light for 10 min, recorded as VB2 / vitamin C / light+catalase group.

[0074] The results are as follows Figure 5 As shown: there is no difference between the blank control group and the VB2 or VC or VB2 / VC or VC / light treatment of Pseudomonas fragariae only, that is, there is no sterilization effect. The VB2 / light group can inactivate 1log CFU / mL (1 order of magnitude, equivalent to 90%) of bacteria. This is because VB2 is a common photosensitizer in PDI and can produce a small amount of ROS with a long half-life when illuminated, so it has a certain bactericidal effect. In the present invention, when VC is added to the VB2-mediated PDI system and light is applied, the bactericidal efficiency of the original PDI against Pseudomonas fragariae can be greatly improved, and the inactivation rate is increased from 1log CFU / mL to 5log CFU / mL (the inactivation rate is increased from 90% to more than 99.999%).

[0075] In addition, we added catalase to the system to verify whether there are other substances besides hydrogen peroxide that have a bactericidal effect. Catalase itself does not have any bactericidal effect. When catalase is added to the VB2 / VC / light system, it will neutralize all the hydrogen peroxide produced in the system. The results showed that after adding catalase to the system, the system still had an inactivation efficiency of about 1log CFU / mL (1 order of magnitude, equivalent to 90%). This proves that there are other active substances in the system that play a bactericidal role, and it also proves that hydrogen peroxide plays a leading role in the bactericidal effect in the system. It is reported that VC and hydrogen peroxide will undergo a redox reaction to further produce active substances such as ascorbic acid free radicals and hydroxyl free radicals, thereby playing an additional auxiliary bactericidal role.

[0076] Embodiment 3

[0077] The effect of the optimal sterilization system treatment obtained in Example 1 on the surface morphology of Pseudomonas fragariae was investigated, and the specific steps were as follows:

[0078] The same method as in Example 2 was used to obtain Pseudomonas fragariae in the treatment group (vitamin B2 / vitamin C / light group) and the control group (group without any reagent added). 1 mL of the bacterial solution of the treatment / control group was drawn, centrifuged at 6000 rpm for 5 min and the supernatant was discarded. Subsequently, 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to resuspend the bacterial precipitate, and then allowed to stand overnight at 4°C to fix the bacteria. Excess fixative was discarded and the precipitate was washed with physiological saline. The bacterial samples were dehydrated continuously with 30%, 50%, 70%, 90%, and 100% (twice) of ethanol, each time with an interval of 10 min. 10 μL of the dehydrated bacterial solution was drawn and added dropwise to a sterile cell slide, freeze-dried and then sprayed with gold, and three different fields of view were randomly selected for observation and photography under SEM (2000 times and 10000 times).

[0079] The results are as follows Figure 6 As shown, compared with the control group, the bacteria in the treatment group appeared broken, fractured and concave, which proved that the bacteria had been completely inactivated and there was no danger of resuscitation and regeneration.

[0080] Embodiment 4

[0081] The effect of the optimal sterilization system obtained in Example 1 on the cell membrane of Pseudomonas fragariae was investigated, and the specific steps were as follows:

[0082] The same method as in Example 3 was used to obtain Pseudomonas fragariae in the treatment group and the control group. The membrane potential, hydrophobicity and permeability of the cell membrane of Pseudomonas fragariae in the treatment group and the control group were detected to investigate the effect of the vitamin-mediated photodynamic sterilization system provided by the present invention on the bacterial cell membrane.

[0083] The membrane potential detection method is as follows: dissolve the rhodamine 123 fluorescent probe in sterile water to prepare a stock solution with a concentration of 1 mg / mL. Add 2 μL of rhodamine 123 to the obtained 1 mL of the treatment group / control group bacterial solution to make the final concentration 2 μg / mL. After incubation for 30 minutes in the dark, set the excitation wavelength to 480 nm, the emission wavelength to 530 nm, the scanning range to 500-600 nm, and the gain factor to 10 in the fluorescence spectrophotometer to detect its fluorescence intensity.

[0084] Metabolically active bacteria have normal membrane potential, which is essential for the physiological metabolism of bacteria. When bacteria are stimulated by external stimuli, their membrane potential usually changes. Rhodamine 123 is a cationic dye with strong fluorescence properties, and its intensity is related to the membrane potential of the cell membrane. When the membrane potential is negative, its fluorescence intensity increases, proving that the bacterial cell membrane is adversely affected and hyperpolarization occurs. Figure 7 Part A shows that the fluorescence intensity at 530 nm of the treated group increased significantly, proving that the bacteria were transformed from a normal physiological state to a hyperpolarized imbalance state after treatment.

[0085] The method for detecting the hydrophobicity is as follows: 1 mL of hexadecane was added to the bacterial solution (1 mL) of the treatment group and the control group and mixed and shaken for 1 min. After incubation for 30 min, the OD of the aqueous phase before and after incubation was measured by a multifunctional microplate reader. 600nm The hydrophobicity is then calculated using the following formula.

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

[0087] The cell membrane of Gram-negative bacteria is composed of a large amount of proteins and polysaccharides, which determine the hydrophobicity of Pseudomonas fragrans. When the cell membrane of bacteria is destroyed, its hydrophobicity will decrease. Figure 7 As shown in Part B, the bacterial hydrophobicity rate in the treated group was significantly lower than that in the control group, which also proved that the bacterial cell membrane was affected.

[0088] The method for detecting membrane permeability is as follows: take 1 mL of each sample in a centrifuge tube, and take 200 μL for detection. Take 300 μL of bacterial resuspension from each tube into a new centrifuge tube, vortex and mix, and divide into 2 parts, one of which is not treated, and the other is boiled for 5 minutes; remix the two differently treated suspensions and divide them into 3 parts, marked as Blank, SYTO 9, and SYTO 9+PI for staining. The Blank tube is not stained; SYTO 9 tube: add 2 μL of 10-fold diluted SYTO 9 staining solution; SYTO 9+PI tube: add 2 μL of 10-fold diluted SYTO 9 staining solution and 2 μL of 10-fold diluted PI staining solution; other tubes: add 2 μL of 10-fold diluted SYTO 9 staining solution and 2 μL of 10-fold diluted PI staining solution. The changes in the proportion of bacterial cell apoptosis in the treatment group and the control group were detected by flow cytometry Green-B channel and Red-B channel.

[0089] Through the penetration of SYTO9 and PI dyes into the cell membrane, we can see that the bacterial state in the treatment group changes from live apoptotic cells to necrotic cells ( Figure 8 ).

[0090] In summary, after Pseudomonas fragariae was treated with the new PDI system, the cell membrane that maintained the necessary physiological activity functions was seriously affected and damaged (the membrane potential increased, the hydrophobicity decreased, and the membrane permeability increased), which further proved the thoroughness and high efficiency of the present invention in inactivating bacteria.

[0091] Embodiment 5

[0092] The effect of the optimal sterilization system obtained in Example 1 on the respiratory metabolism of Pseudomonas fragariae was investigated to investigate the thoroughness of the vitamin-mediated photodynamic sterilization system provided by the present invention. The specific steps are as follows:

[0093] The same method as in Example 3 was used to obtain the treated group and the control group of Pseudomonas fragariae. Iononitrochlorotetrazolyl blue chloride with a final concentration of 1 mM was added to the bacterial solution (1 mL) of the treated group and the control group and incubated at 37°C for 30 min. The absorbance was measured at 630 nm by an ELISA reader to evaluate the effect of the treatment on the intracellular tricarboxylic acid cycle activity of the bacteria. The results are shown in FIG. Fig. 9 As shown in A.

[0094] 0.1M glucose and 1mg / mL 2,3,5-triphenyltetrazolium chloride were added to the bacterial solution (1mL) of the treatment group and the control group, and then incubated at 37°C for 30min. Then two drops of concentrated sulfuric acid were added to the system to terminate the reaction, and 1mL of n-butanol was added to mix and shake. The absorbance was measured at 490nm by an ELISA reader to evaluate the effect of the treatment on the activity of intracellular respiratory chain dehydrogenases. The results are shown in Figure 2. Fig. 9 As shown in B.

[0095] The intracellular metabolism of the treated bacteria was evaluated by the activity of tricarboxylic acid cycle and respiratory chain dehydrogenases. The tricarboxylic acid cycle and respiratory chain dehydrogenases play an important role in the life activities of bacteria and are responsible for important physiological functions such as energy production, metabolic regulation and electron transfer. Fig. 9 It can be seen that compared with the control group, the activities of tricarboxylic acid cycle and respiratory chain dehydrogenases in the treated group's Pseudomonas fragrans were significantly reduced, which further proves that the bacteria were completely killed and could not carry out normal physiological activities.

[0096] Embodiment 6

[0097] The effect of the optimal sterilization system obtained in Example 1 on the respiratory metabolism of Pseudomonas fragariae in beef was investigated to investigate the effect of the vitamin-mediated photodynamic sterilization system provided by the present invention in actual application scenarios. The specific steps are as follows:

[0098] Fresh beef of the same batch was purchased from a supermarket, and the fascia was removed and cut into small pieces of about 3cm*3cm*1cm (10g) on ​​a sterile clean bench. The appearance and quality of each piece of beef were ensured to be the same as much as possible and irradiated with ultraviolet light for 1h to kill the background microorganisms in the beef to the maximum extent. Subsequently, each piece of beef was inoculated with a bacterial solution of Pseudomonas fragariae to a concentration of 5log CFU / g, and air-dried in a clean bench for half an hour to ensure that the bacteria were adhered and absorbed by the beef. The beef was then placed in a sterile homogenizing bag, and the new PDI solution of the optimal sterilization system obtained in Example 1 was added and soaked for sterilization, and the control group was soaked in an equal amount of sterile deionized water. After the treatment, the beef was transferred to a sterile culture dish and placed in a 4°C refrigerator for storage for 0, 1, 3, 5, and 7 days, respectively, and the appearance changes of the beef (photographed at a top-down angle in a studio), the dynamic changes of the total colony count and volatile basic nitrogen were recorded at the specified time points. Each group was repeated three times and three parallels were set.

[0099] The method for detecting the total colony count in beef is as follows: After the fresh beef is stored for a specified time, it is taken out and placed in a sterile homogenizing bag, followed by adding 90 mL of sterile saline and homogenizing on a homogenizer for 5 minutes to suspend the bacteria in the saline. Take 100 μL of the supernatant and spread it evenly on the surface of the PCA plate, place it in a 25°C biochemical incubator for 48 hours, and then count the number of live bacteria. Set up 3 replicates for each group.

[0100] The method for detecting volatile basic nitrogen in beef is as follows: (1) Preparation of reagents: weigh 20g of boric acid, add 1L of water to dissolve and 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 1g / L methyl red solution; weigh 0.1g of bromocresol green and dissolve it in 100mL of 95% ethanol to prepare a 1g / L bromocresol green solution; weigh 1g of magnesium oxide and dissolve it in 100mL of water to prepare a 1% magnesium oxide suspension. Take 1.37mL of methyl red solution and 8.63mL of bromocresol green solution, mix them, add them to 1L of boric acid solution to prepare a boric acid absorption solution and add it to the Kjeldahl nitrogen analyzer. (2) Preparation of samples: 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 addition of 25 mL of boric acid, the distillation time was 5 min, the elution water volume was 30 mL, and the hydrochloric acid concentration used for calibration was 0.01 M. Finally, the volatile basic nitrogen content in beef stored for different days was determined according to the calculation method of the national standard GB 5009.228-2016.

[0101] The results are as follows Fig.10 As shown in the figure, after the contaminated fresh beef was treated with the new PDI sterilization system developed by the present invention, the change of its appearance color was significantly delayed, while the beef in the control group turned black and seriously corrupted on the 7th day ( Fig.10 Part A of the .

[0102] The total colony count in beef was further tested ( Fig.10 Part B) and volatile basic nitrogen ( Fig.10 Part C), the results showed that the new PDI sterilization system provided by the present invention can significantly inhibit the proliferation of spoilage bacteria in beef. On the third day, the total colony count in the beef of the control group increased rapidly, which means that the beef was seriously corrupted, while the beef of the treatment group still maintained a low microbial level. On the other hand, the content of volatile basic nitrogen represents the decomposition level of meat product protein. The more serious the corruption of the meat product, the higher the content of volatile basic nitrogen it produces. 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 15mg / 100g, it means that the food is corrupt and deteriorated. Fig.10 It can be seen from C that the beef in the treatment group began to spoil on the 5th day, while the beef in the control group reached the limit of the national standard on the 3rd day.

[0103] Therefore, the vitamin-mediated photodynamic sterilization reagent provided by the present invention not only exhibits an ultra-efficient and thorough sterilization effect in vitro, but also can significantly delay the corruption of fresh beef in real life, thereby showing a good and efficient application prospect.

[0104] In summary, the bactericide provided by the present invention can rapidly generate high-concentration hydrogen peroxide, ascorbic acid free radicals, hydroxyl free radicals and other substances with bactericidal functions after being irradiated by LED blue light, and the bactericidal rate can reach more than 99.999%; the half-life is relatively long, which can greatly extend the duration of PDI sterilization; the sterilization of the inside of the food can be achieved; and thus the serious defects of poor penetration and migration effects in the traditional PDI sterilization process are completely compensated; the bacteria can be completely inactivated; the killing components have no safety hazards to consumers; no real-time illumination is required, it is easy to use, and the adverse effects of long-term illumination on food quality during the PDI sterilization process can be effectively avoided; the required light source is a light emitting diode (LED), which has a compact structure, high luminous efficiency, stable wavelength, no obvious thermal effect, good durability, long service life, low energy consumption, and effectively reduces the sterilization cost; it shows 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 solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A vitamin-mediated photodynamic bactericidal agent, characterized in that: It comprises the following components: vitamin B2, vitamin C and a solvent.

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

3. A vitamin-mediated photodynamic bactericidal agent according to claim 1, characterized in that: The molar ratio of vitamin B2:vitamin C is 2.5:

1.

4. The vitamin-mediated photodynamic bactericidal agent according to claim 1, characterized in that: The working concentration of vitamin B2 is not less than 5 μM.

5. The vitamin-mediated photodynamic bactericidal agent according to claim 1, characterized in that: The working concentration of vitamin C is greater than 50 μM.

6. A method for using the vitamin-mediated photodynamic bactericidal agent according to any one of claims 1 to 5, characterized in that: Dissolve vitamin B2 and vitamin C separately, mix them evenly in proportion and expose them to light for a certain period of time. Use the solution after exposure to light to soak food or place it in a closed space with food for sterilization.

7. The method for using a vitamin-mediated photodynamic sterilization reagent according to claim 6, characterized in that: The illumination was carried out using LED blue light for 1-20 minutes.

8. The method for using a vitamin-mediated photodynamic bactericidal agent according to claim 6, characterized in that: Soaking food means immersing the food to be sterilized into the solution and taking it out directly after the light exposure ends.

9. Use of the vitamin-mediated photodynamic sterilization reagent according to any one of claims 1 to 5 in food sterilization, characterized in that: Application in killing bacteria.

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