Application of Curcumin-Mediated Photodynamic Degradation of Staphylococcal Enterotoxin

Abstract

The present invention discloses an application of curcumin-mediated photodynamic degradation of Staphylococcus aureus enterotoxin. The present invention effectively proves through experiments that water-soluble curcumin-mediated photodynamic can effectively reduce the enterotoxin content and its immunological activity. The present invention also provides a method for effectively inactivating Staphylococcus aureus and its enterotoxin. This method has high safety, low equipment requirements, simple operation, causes non-specific damage to the bacteria, is not prone to generating resistance, is non-toxic, has low cost, and can effectively control the risk of food poisoning caused by Staphylococcus aureus and its enterotoxin. The water-soluble curcumin-mediated photodynamic technology provided by the present invention has the potential for application in food processing.

Description

Technical Field

[0001] The present invention belongs to the technical field of photodynamic sterilization, and particularly relates to the application of curcumin-mediated photodynamic degradation of Staphylococcus aureus enterotoxin. Background Art

[0002] Staphylococcus aureus is one of the most concerned foodborne pathogenic bacteria, belonging to Gram-positive bacteria, and often causes diarrhea, vomiting, headache, low fever and even death. However, all the reported incidents of diseases caused by Staphylococcus aureus are related to the Staphylococcus enterotoxins (SEs) produced by it. SEs are extracellular proteins with low molecular weight (25-34 kDa), which can be produced at all stages of the growth period of Staphylococcus aureus, mainly in the middle and late stages, and have high pathogenicity. They are resistant to proteolytic enzymes such as pepsin and trypsin, and have thermal stability and are not easily destroyed by conventional food processing methods (such as heat treatment technology).

[0003] SEA and SEB are the most common Staphylococcus aureus enterotoxins causing food poisoning and have strong emetic activity. The amount required for the diseases caused by them is less than 1 μg. SEB has high toxicity and strong stability, and causes the most serious food poisoning. 0.004 μg / kg of SEB can induce disease symptoms, and a dose of 0.02 μg / kg may be lethal. At the same time, as superantigens, SEA and SEB do not need to be transmitted by antigen-presenting cells, but bind to T cell receptors through major histocompatibility complex class II molecules (MHC-II) on the surface of antigen-presenting cells, stimulate the activation and proliferation of T cells, secrete cytokines, and cause cell death. Therefore, the bacterial food poisoning caused by Staphylococcus aureus and its enterotoxins has brought great impacts on human life health and property safety, and also brought new challenges to the control and prevention of foodborne diseases.

[0004] At present, the sterilization technologies in the food industry include thermal sterilization and non-thermal sterilization. Thermal sterilization is a traditional sterilization technology. By using expensive high-pressure heat-resistant equipment, the food is heated to 121°C - 140°C. After treatment, it can effectively kill bacteria and heat-resistant spores, and has a good effect of extending the shelf life. However, this method usually affects the sensory and rheological properties of the food and causes changes in food components, which is not conducive to the sterilization treatment of fresh food. Ultraviolet-visible radiation is a non-thermal sterilization technology. After treatment, it can maintain the sensory and flavor of the food. The treatment mechanism is to produce pyrimidine dimers in the microbial genome, which has strong biological activity and destroys the DNA replication process. However, the ultraviolet light spectrum has a low frequency, and ultraviolet radiation is harmful to the human body.

[0005] Photodynamic non-thermal sterilization technology is a new type of sterilization technology and a research hotspot in the food industry. This method irradiates microorganisms with visible light of a specific wavelength. This process can stimulate the endogenous photosensitizing porphyrin molecules in microorganisms to generate a large amount of reactive oxygen species, causing damage to the bacterial cells and further leading to the death of the bacteria. Different tolerance effects of different microorganisms to photodynamic treatment are caused by different contents of endogenous porphyrin molecules in different microorganisms. Therefore, to effectively sterilize, exogenous photosensitizers can be added before illumination. After dark incubation, the small molecule photosensitizers enter the microorganisms, and then after illumination treatment, the ROS content in the microorganisms can be significantly increased. ROS can oxidatively damage cell membranes, proteins, and DNA, thereby effectively sterilizing. This method does not require the use of large equipment or expensive instruments. Effective sterilization can be achieved by irradiating with a portable LED light source. It has low cost, is safe and non-toxic, and is easy to operate. At the same time, non-thermal sterilization can maximize the preservation of food nutritional components, structural tissues, product color and other characteristics. Therefore, photodynamic non-thermal sterilization technology can meet the dual requirements of consumers for food safety and quality.

[0006] Curcumin (C 21 H 20 O 6 , CAS registration number 458-37-7) is a natural extract that can be extracted from plants of the genus Curcuma. A potential limitation of curcumin is its low solubility in water. Purified curcumin is usually dissolved in ethanol with or without a solubility enhancer (such as dimethyl sulfoxide), and then diluted with distilled water. However, as the ethanol volatilizes and the concentration decreases, curcumin is prone to precipitation. Summary of the Invention

[0007] To solve related problems, the primary object of the present invention is to provide the application of curcumin-mediated photodynamic degradation of Staphylococcus aureus enterotoxin.

[0008] Another object of the present invention is to provide the application of curcumin-mediated photodynamic degradation of Staphylococcus aureus enterotoxin and inactivation of Staphylococcus aureus.

[0009] To achieve the above-mentioned invention objects, the present invention adopts the following technical solutions:

[0010] The application of curcumin-mediated photodynamics, and the application is any one of the following:

[0011] I. The application of curcumin-mediated photodynamic degradation of Staphylococcus aureus enterotoxin: Mix the curcumin aqueous solution and Staphylococcus aureus enterotoxin or a sample containing it, incubate in the dark, and irradiate under a light source to complete the degradation of Staphylococcus aureus enterotoxin;

[0012] II. Application of curcumin-mediated photodynamic degradation of staphylococcal enterotoxin and inactivation of Staphylococcus aureus: Mix an aqueous solution of curcumin with staphylococcal enterotoxin, Staphylococcus aureus or a sample containing both, incubate in the dark, and irradiate under a light source to complete the degradation of staphylococcal enterotoxin and the inactivation of Staphylococcus aureus.

[0013] Incubate under dark conditions so that water-soluble curcumin can better bind to Staphylococcus aureus or enterotoxin.

[0014] Preferably, the staphylococcal enterotoxin includes staphylococcal enterotoxin SEA and staphylococcal enterotoxin SEB.

[0015] Preferably, the curcumin is water-soluble curcumin, food-grade, and can be completely dissolved in water; more preferably, the curcumin is UC3 CLEAR, purchased from Sabinsa Corporation, USA.

[0016] Preferably, the concentration of curcumin in the mixed system is 0 - 300 μM (not 0); more preferably, the final concentration of curcumin in the mixed system is 0 - 150 μM (not 0); most preferably, the final concentration of curcumin in the mixed system is 100 μM.

[0017] Preferably, the light source is an LED light source; more preferably, the light source is a blue visible light source with a wavelength of 420 ± 5 nm.

[0018] Preferably, the light intensity of the irradiation is 69.97 mW / cm 2 , the time is 0 - 40 min (not 0), and the corresponding light dose is 0 - 167.93 J / cm 2 ; more preferably, the time is 20 min.

[0019] Light dose (J / cm 2 ) = light intensity (W / cm 2 ) × irradiation time (s).

[0020] Preferably, the sample described in I or II is a food sample.

[0021] This invention effectively proves through experiments that water-soluble curcumin-mediated photodynamic can effectively reduce the content of enterotoxin. The content of supernatant enterotoxin can be reduced by up to 10%, and the content of enterotoxin in the supernatant after cell wall disruption can be reduced by up to 34%; it can reduce its immunological activity, manifested as significantly reducing the proliferative activity of lymphocytes (by 19%); at the same time, it can inactivate Staphylococcus aureus (the inactivation rate can reach 99.99%); this method has a certain control effect on foodborne poisoning caused by Staphylococcus aureus and its enterotoxin.

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] (1) Water-soluble curcumin-mediated photodynamic therapy can effectively reduce the content of Staphylococcus aureus enterotoxin and reduce the immunological activity of enterotoxin standard. Therefore, this method can have a certain control effect on foodborne poisoning caused by Staphylococcus aureus enterotoxin;

[0024] (2) The combination of water-soluble curcumin and LED blue light source has obvious inactivating effect on Staphylococcus aureus and its enterotoxin. A large amount of ROS is generated during the photo-inactivation process, resulting in non-specific damage to the bacteria and enterotoxin. Therefore, resistance is not easily generated during repeated use.

[0025] (3) Curcumin has good photosensitizing activity and no obvious chemical toxicity. Moreover, the color of curcumin becomes lighter after light treatment, which has little impact on the sensory properties of food. It can maximize the preservation of food nutritional components, structural tissues and product color and other characteristics, meeting the dual requirements of food safety and quality.

[0026] (4) Water-soluble curcumin has good solubility and a small molecular weight. When incubated with Staphylococcus aureus, it can better penetrate into the bacterial cells. Photodynamic therapy mediated by it can effectively inactivate Staphylococcus aureus, and the inactivation rate reaches 99.99%, with remarkable effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a research result diagram of the effect of water-soluble curcumin-mediated photodynamic inactivation of Staphylococcus aureus at different concentrations.

[0028] Figure 2 It is a research result diagram of the effect of water-soluble curcumin-mediated photodynamic therapy on reducing the content of Staphylococcus aureus enterotoxin at different concentrations.

[0029] Figure 3 It is a research result diagram of the effect of illumination time on water-soluble curcumin-mediated photodynamic inactivation of Staphylococcus aureus.

[0030] Figure 4 It is a research result diagram of the effect of illumination time on water-soluble curcumin-mediated photodynamic therapy on reducing the content of Staphylococcus aureus enterotoxin.

[0031] Figure 5 It is a research result diagram of the effect of water-soluble curcumin-mediated photodynamic therapy on the ROS level in Staphylococcus aureus cells under different conditions.

[0032] Figure 6 It is a research result diagram of the effect of water-soluble curcumin-mediated photodynamic therapy on the immunological activity of Staphylococcus aureus enterotoxin standard. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0034] The water-soluble curcumin used in the present invention is UC3 CLEAR (0337, Sabinsa Corporation, USA).

[0035] The Staphylococcus aureus used in the present invention is Staphylococcus aureus ATCC29213.

[0036] The Staphylococcus aureus enterotoxin sample used in the embodiment of the present invention:

[0037] First step: Centrifuge to collect the Staphylococcus aureus cells and supernatant after overnight pre-enrichment, and prepare the Staphylococcus aureus enterotoxin sample, where:

[0038] Preparation of supernatant enterotoxin: Collect 15 mL of supernatant and perform sterile filtration, concentrate it by centrifugation using an ultrafiltration tube, and collect 1 mL of the concentrated solution, which is regarded as the Staphylococcus aureus enterotoxin sample (supernatant);

[0039] Preparation of cell wall-broken supernatant enterotoxin: The cells are washed and resuspended with PBS to make the total number of colonies 10 8 CFU / mL; The cells are broken by ultrasonic disruption on an ice bath, the ultrasonic power is 61%, working for 10 s and pausing for 10 s, for a total of 4 min. After disruption, centrifuge to take the supernatant and perform sterile filtration, which is regarded as the Staphylococcus aureus enterotoxin sample (cell wall-broken supernatant).

[0040] Data analysis used in the embodiment of the present invention:

[0041] Excel, SPSS 19.0, and Graphpad Prism 7.0 are used to process and analyze the experimental data. The significant differences in the data are analyzed by one-way ANOVA (p < 0.05). Different letters abcdef in the figure indicate significant differences.

[0042] Example 1: Effects of water-soluble curcumin at different concentrations on photodynamic inactivation of Staphylococcus aureus

[0043] Prepare water-soluble curcumin at different concentrations, mix the water-soluble curcumin with the bacterial suspension at a ratio of 1:1, incubate in the dark for 10 min, and irradiate with LED blue light for 10 min.

[0044] Carry out experiments according to the above method for photodynamic treatment of Staphylococcus aureus, where: the final concentration of water-soluble curcumin in the system is 20, 50, 100, 150 μM, incubate in the dark for 10 min, and irradiate with LED blue light for 10 min (light dose is 41.98 J / cm 2) Meanwhile, a non-irradiation group (l-) and a group without photosensitizer (p-) were set up for comparison. Figure 1 To show the inactivation effect of photodynamic therapy mediated by different concentrations of water-soluble curcumin on Staphylococcus aureus.

[0045] As shown in the figure, when the concentration of the photosensitizer was 20 μM, the total number of colonies of Staphylococcus aureus decreased by 3 logs; with the increase in the concentration of water-soluble curcumin, the sterilization effect was significantly enhanced. When the concentration of the photosensitizer reached 50 μM, the total number of colonies decreased by 5 logs; when the concentration continued to increase, the effect change was not significant. The inactivation rate of Staphylococcus aureus was 99.99% at 100 μM. After photodynamic treatment, the colors of 50 μM and 100 μM water-soluble curcumin became significantly lighter, that is, it did not affect the sensory quality of food. Therefore, to enhance the sterilization effect, a curcumin concentration of 100 μM was selected for subsequent bactericidal experiments.

[0046] Example 2: Effect of photodynamic degradation of Staphylococcus aureus enterotoxin mediated by different concentrations of water-soluble curcumin

[0047] Prepare water-soluble curcumin with different concentrations, mix the water-soluble curcumin with the enterotoxin sample at a ratio of 1:1, incubate in the dark for 10 min, and irradiate with LED blue light for 10 min.

[0048] Experiments were carried out according to the above method of photodynamically treating enterotoxin samples (supernatant, cell wall-broken supernatant). Among them: the final concentration of water-soluble curcumin in the system was 20, 50, 100, 150 μM, incubated in the dark for 10 min, irradiated with LED blue light for 10 min, and the light intensity was 69.97 mW / cm 2 (The light dose was 41.98 J / cm 2 ). Meanwhile, a non-irradiation group (l-) and a group without photosensitizer (p-) were set up for comparison. The content of enterotoxin was detected using an ELISA (R4105) kit. The change in the content of enterotoxin (%) = 1 + (S 1 - S 0 ) / S 0 (Note: S 1 is the experimental group, the light control group, and the photosensitizer control group; S 0 is the blank group). Figure 2 To show the degradation effect of photodynamic therapy mediated by different concentrations of water-soluble curcumin on enterotoxin.

[0049] As shown in the figure, the inactivating effects of water-soluble curcumin at different concentrations on enterotoxins at different concentrations are different. For the concentrated supernatant enterotoxin solution (with a relatively high enterotoxin concentration), when the concentration of water-soluble curcumin increases to 150 μM, the enterotoxin content in the supernatant is 93.37%, showing no significant difference (p > 0.05) compared with the value (94.54%) at a water-soluble curcumin concentration of 20 μM. The change in the enterotoxin content of the broken-wall supernatant enterotoxin (with a relatively low enterotoxin concentration) shows a dose-dependent relationship at different water-soluble curcumin concentrations. As the treatment concentration of water-soluble curcumin increases, the enterotoxin content decreases significantly. When the water-soluble curcumin concentration is 100 μM, the enterotoxin content in the broken-wall supernatant is 69.53% (a 30% reduction), showing no significant difference (p > 0.05) compared with that at a concentration of 150 μM. In summary, the concentration of the photosensitizer for inactivating enterotoxin mediated by water-soluble curcumin in photodynamic therapy is 100 μM.

[0050] Example 3: Effect of illumination time on the inactivation of Staphylococcus aureus by water-soluble curcumin-mediated photodynamic therapy

[0051] Prepare 200 μM of water-soluble curcumin, mix the water-soluble curcumin with the bacterial suspension in a ratio of 1:1, incubate in the dark for 10 min, and irradiate with LED blue light for 10 - 40 min.

[0052] Conduct experiments according to the above method for treating Staphylococcus aureus by photodynamic therapy, where: the final concentration of water-soluble curcumin in the system is 100 μM, incubate in the dark for 10 min, and irradiate with LED blue light for 10, 20, 30, 40 min (the light doses are 41.98, 83.96, 125.94, 167.92 J / cm 2 ), and at the same time, set up a non-irradiation group (l-) and a group without photosensitizer (p-) for comparison. Figure 3 Show the inactivating effects of different illumination times on Staphylococcus aureus treated by water-soluble curcumin-mediated photodynamic therapy.

[0053] As shown in the figure, the bactericidal effects of different illumination times on water-soluble curcumin-mediated photodynamic therapy are different. In a relatively short illumination time, the bactericidal effect shows a dose-dependent relationship. When the illumination time is 10 min, the reduction can reach 3 log; when the illumination time increases to 20 min, the total number of Staphylococcus aureus can be reduced by 6 log (the inactivation rate reaches 99.99%). However, with the extension of the illumination time, the change in the number of Staphylococcus aureus colonies is not obvious. Therefore, in this study, an illumination time of 20 min for water-soluble curcumin-mediated photodynamic therapy has a better bactericidal effect and is used for subsequent experiments.

[0054] Example 4: Effect of illumination time on the degradation of Staphylococcus aureus enterotoxin by water-soluble curcumin-mediated photodynamic therapy

[0055] Prepare 200 μM of water-soluble curcumin, mix the water-soluble curcumin with the enterotoxin sample at a ratio of 1:1, incubate in the dark for 10 min, and irradiate with LED blue light for 10 - 40 min.

[0056] Conduct experiments according to the above method of photodynamic treatment of enterotoxin samples (supernatant, broken cell supernatant). Among them: the final concentration of water-soluble curcumin in the system is 100 μM, incubate in the dark for 10 min, irradiate with LED blue light for 10, 20, 30, 40 min (the light doses are 41.98, 83.96, 125.94, 167.92 J / cm 2 ), at the same time, set up a non-irradiation group (l-) and a non-photosensitizer group (p-) for control. The content of enterotoxin is detected using an ELISA (R4105) kit. The change in the content of enterotoxin (%) = 1 + (S 1 -S 0 ) / S 0 (Note: S 1 is the experimental group, the light irradiation control group and the photosensitizer control group; S 0 is the blank group). Figure 4 Show the degradation effect of water-soluble curcumin-mediated photodynamic on enterotoxin under different irradiation times.

[0057] As shown in the figure, the water-soluble curcumin-mediated photodynamic based on different irradiation times also has an impact on the degradation effect of Staphylococcus aureus enterotoxin. For the supernatant enterotoxin concentrate, the content of enterotoxin after photodynamic treatment is 92.49% (irradiation time is 20 min). Continuing to increase the irradiation time, there is no significant difference in the change of the supernatant enterotoxin content (p > 0.05). For the broken cell supernatant, with the increase of the irradiation time, the change in the reduction of enterotoxin is obvious. When the irradiation time is 10 min, the content of enterotoxin is 84.31% (the reduction can reach 15%). Continuing to increase the irradiation time to 20 min, the content of enterotoxin is 75.01%, showing a good treatment effect. Continuing to extend the irradiation time, there is no significant change (p > 0.05), and the maximum reduction of enterotoxin can reach 35%.

[0058] Therefore, when the photodynamic mediated by 100 μM water-soluble curcumin is irradiated for 20 min, the inactivation effect on enterotoxin is the best, and this condition is used for subsequent experiments.

[0059] In addition, there is no significant change in the number of colonies in the light irradiation control group and the photosensitizer control group of the water-soluble curcumin-mediated photodynamic in Examples 1 and 3 compared with the blank group. That is, the non-thermal sterilization technology of photodynamic inactivating Staphylococcus aureus requires the synergistic treatment of photosensitizer and irradiation;

[0060] The enterotoxin content in the light control group and photosensitizer control group of water-soluble curcumin-mediated photodynamic therapy in Examples 2 and 4 changed significantly compared with the blank group, but the change value of the control group was smaller than that of the blank group and the experimental group.

[0061] Example 5: Changes in the content of ROS in Staphylococcus aureus cells during the treatment of Staphylococcus aureus by water-soluble curcumin-mediated photodynamic therapy under different conditions

[0062] The Staphylococcus aureus culture solution was centrifuged and washed twice, and resuspended in ECFH-DA (10 μM). The concentration of Staphylococcus aureus was 10 6 ~10 7 CFU / mL. Incubate in a 37 °C constant temperature incubator for 20 min, and invert and mix every 3-5 min to ensure full contact between the bacteria and the probe. After loading the probe, wash three times with sterile PBS to fully remove the DCFH-DA that did not enter the bacterial cells. Mix 1 mL of the bacterial solution bound to the probe with 1 mL of water-soluble curcumin (100 μM), incubate in the dark at 37 °C for 10 min, and irradiate with 420 nm LED blue light for 20 min (l+, light dose: 83.96 J / cm 2 ); At the same time, set up a non-irradiated group (l-) and a group without photosensitizer (p-) for control. After photodynamic treatment, place the experimental group and the control group under a microplate reader for detection, and set up a positive control group for reactive oxygen species; The excitation wavelength for detection is 488 nm, and the emission wavelength is 525 nm. The relative content of ROS (%) = (R 1 -R 0 ) / R 0 (Note: R 1 is the experimental group, light control group and photosensitizer control group; R 0 is the blank group). Figure 5 Shows the changes in the content of ROS in Staphylococcus aureus cells during the treatment of Staphylococcus aureus by water-soluble curcumin-mediated photodynamic therapy under different conditions.

[0063] As shown in the figure, there is a significant difference in the ROS content between the experimental group and the control group of water-soluble curcumin-mediated photodynamic therapy (p < 0.05). The change in the ROS content of the photodynamic control group is ±30, while the change in the ROS content of the experimental group is as high as 120, indicating that during the process of water-soluble curcumin-mediated photodynamic bactericidal, it mainly causes an increase in the intracellular reactive oxygen content, leading to cell apoptosis and a decrease in the total number of colonies.

[0064] Example 6: Effect of water-soluble curcumin-mediated photodynamic therapy on the immunological activity of Staphylococcus aureus enterotoxin standard

[0065] The enterotoxin standard SEB was diluted to solutions with mass concentrations of 10, 1, and 0.1 μg / mL in equal volumes. One part was used for photodynamic treatment as the enterotoxin sample in the experimental group; the other part was untreated as the enterotoxin sample in the control group. The photodynamic treatment method was as follows: Prepare 200 μM of water-soluble curcumin, mix the water-soluble curcumin with the bacterial suspension and the enterotoxin sample at a ratio of 1:1, incubate in the dark for 10 min, and irradiate with LED blue light for 20 min. At the same time, set up a non-irradiation group (l-) and a non-photosensitizer group (p-) for comparison.

[0066] Mice aged 6 - 8 weeks were sacrificed by cervical dislocation. Their fresh spleens were carefully removed with scissors and forceps in a biosafety cabinet and placed in sterile RPMI-1640 medium. The mouse spleens were transferred to the laminar flow hood in the cell room. Using sterile forceps, the spleens were placed on a 40-μm cell sieve, and the spleen was gently ground into a single-cell suspension with a syringe without a needle inside the cell sieve. Add a certain volume of RPMI-1640 medium to wash the single cells into a 15-mL centrifuge tube and centrifuge with a high-speed refrigerated centrifuge (1400 rpm, 7 min), then discard the supernatant. The cell pellet was first resuspended with 1 mL of erythrocyte lysate, then another 1 mL of erythrocyte lysate was added, and it was left standing at room temperature for 5 minutes to fully lyse the erythrocytes. Add 10 mL of medium to terminate the erythrocyte lysis, invert and mix well, and then filter through a 40-μm cell sieve into a new 15-mL centrifuge tube. After centrifuging at 1400 rpm for 7 min, discard the supernatant, and resuspend the pellet with 1 mL of medium and mix well. Place it in a 4°C refrigerator or on ice for standby to maintain cell viability. Use a cell counting chamber to count. Usually, about 5×10 7 cells can be obtained from each spleen, and the cell survival rate is more than 80%;

[0067] 100 μL of the mouse spleen lymphocyte suspension with a concentration of 1×10 7 cells / mL was successively pipetted into a 96-well plate. After filtering the enterotoxin samples in the experimental group and the control group through a 0.22-μm filter membrane, 100 μL was pipetted into the corresponding wells. The negative control group, positive control group, and blank control were 10 μg / mL BSA, Con A (concanavalin A) with a final concentration of 5 μg / mL, and RPMI-1640 medium containing 10% FBS respectively, and 5 replicates were set for each sample. After plating, it was continuously cultured in an incubator at 37°C and 5% CO 2 for 72 h. After the culture was completed, 20 μL of MTT (5 mg / mL) solution was successively added to the wells to be tested in the 96-well plate, mixed well, and then incubated at 37°C and 5% CO 2Continue to culture in the incubator for 4 h; after the culture is completed, centrifuge at 2000 rpm for 10 min, carefully aspirate the supernatant, add 120 μL of DMSO, place the plate in the microplate reader and shake for 20 s, and then measure the absorbance of each well on the microplate reader (the measurement wavelength is 570 nm and the reference wavelength is 630 nm); Proliferation Index (PI) = absorbance of the experimental well / absorbance of the negative control well. Figure 6 Show the treatment effect of water-soluble curcumin-mediated photodynamic on the immunological activity of staphylococcal enterotoxin standard.

[0068] As shown in the figure, ConA is used as the positive control to stimulate lymphocyte proliferation, BSA is used as the negative control, and the proliferation indices of the experimental group and the control group of the enterotoxin standard SEB are significantly higher than those of the negative group, indicating that SEB has immunological activity and can stimulate lymphocyte proliferation. The proliferative effect of the enterotoxin standard on lymphocytes is dose-related. The proliferation index of lymphocytes can reach up to 40% with 10 μg / mL enterotoxin and 30% with 0.1 μg / mL enterotoxin. The proliferative effects of different concentrations of enterotoxin standard treated by photodynamic on lymphocytes are all significantly reduced, with a maximum reduction of 19%. The results show that water-soluble curcumin-mediated photodynamic can reduce the immunological activity of enterotoxin and has a certain control effect on foodborne poisoning caused by enterotoxin.

[0069] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of curcumin-mediated photodynamic therapy, Characterized in that: The application is any one of the following: I. Application of curcumin in the preparation of a product for mediating photodynamic degradation of staphylococcal enterotoxin: Mix an aqueous solution of curcumin and staphylococcal enterotoxin or a sample containing the same, incubate in the dark, irradiate under a light source, and complete the degradation of staphylococcal enterotoxin; II. Application of curcumin in the preparation of a product for mediating photodynamic degradation of staphylococcal enterotoxin and inactivating Staphylococcus aureus: Mix an aqueous solution of curcumin with staphylococcal enterotoxin, Staphylococcus aureus, or a sample containing both, incubate in the dark, irradiate under a light source, and complete the degradation of staphylococcal enterotoxin and the inactivation of Staphylococcus aureus; The curcumin is water-soluble curcumin UC3 CLEAR from Sabinsa Corporation; The light source is a blue light visible light source with a wavelength of 420 ± 5 nm; The sample is a food sample.

2. The application of curcumin-mediated photodynamic therapy according to claim 1, Characterized in that: The staphylococcal enterotoxin includes staphylococcal enterotoxin SEA and staphylococcal enterotoxin SEB.

3. The application of curcumin-mediated photodynamic therapy according to any one of claims 1-2, Characterized in that: The concentration of curcumin in the mixed system is 0-300 μM and not 0.

4. The application of curcumin-mediated photodynamic therapy according to any one of claims 1-2, Characterized in that: The concentration of curcumin in the mixed system is 0-150 μM and not 0.

5. The application of curcumin-mediated photodynamic therapy according to any one of claims 1-2, Characterized in that: The final concentration of curcumin in the mixed system is 100 μM.

6. The application of curcumin-mediated photodynamic therapy according to any one of claims 1-2, Characterized in that: The light density of the irradiation is 69.97 mW / cm 2 , the time is 0 to 40 min and not 0.

7. The application of curcumin-mediated photodynamic therapy according to claim 6, Characterized in that: The irradiation time is 20 min.

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