PHOTODYNAMIC INACTIVATION METHOD OF SALMONELLA

Abstract

The present invention discloses a photodynamic inactivation method of Salmonella. The method using riboflavin as a photosensitizer adopts a blue light source to photodynamically inactivating Salmonella, which belongs to the technical field of sterilization for inactivating food-borne pathogenic Salmonella. The photosensitizer used in the present invention is one of essential vitamins of the human body. The riboflavin (a food-grade photosensitizer) is safe and non-toxic, and has a significant effect for inactivation of Salmonella. Moreover, the present invention is capable of controlling the risk of salmonellosis, short in treatment time, simple in operation, and capable of thoroughly inactivating Salmonella, and has certain control and prevention effects. The present invention provides a method for effective inactivation of Salmonella in food, which is low in cost, simple in operation and wide in application and can better promote the development of the food sterilization technology.

Claims

1. A photodynamic inactivation method of Salmonella, comprising the following steps: (1) mixing riboflavin serving as a photosensitizer and a to-be-treated sample; (2) incubating the mixed sample under a dark condition; and (3) illuminating the sample after the dark incubation with a blue light source.

2. The photodynamic inactivation method of Salmonella according to claim 1, wherein a wavelength of the blue light source is 455 to 460 nm.

3. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (1), a concentration of the riboflavin in a to-be-treated sample mixed system is 100 to 300 μmol/L.

4. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (1), the concentration of the riboflavin in the to-be-treated sample mixed system is 150 to 200 μmol/L.

5. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (3), when the blue light source is used for illumination, only the blue light source is used, and the illumination of other light sources is prevented.

6. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (3), the illumination light energy density of the blue light source is 6 to 16 J/cm.sup.2.

7. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (3), the illumination light energy density of the blue light source is 9.36 to 15.6 J/cm.sup.2.

8. The photodynamic inactivation method of Salmonella according to claim 1, wherein in the step (3), the illumination light energy density of the blue light source is 12.48 to 15.6 J/cm.sup.2.

9. The photodynamic inactivation method of Salmonella according to claim 1, wherein, in the step (2), the dark incubation time is 35 to 50 min.

10. An application of the riboflavin serving as the photosensitizer in inactivation of Salmonella.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 Structural schematic diagram of a device of a 455-460 nm LED illumination system.

[0037] In the figure: 1—LED photographing light box; 2—lifting platform; 3—24-well plate; 4—LED blue light source.

[0038] FIG. 2 Effects of different riboflavin concentrations on photodynamic inactivation of Salmonella.

[0039] FIG. 3 Effects of different blue light source illumination time on photodynamic inactivation of Salmonella.

[0040] FIG. 4 Effects of different dark incubation time on photodynamic inactivation of Salmonella.

[0041] FIG. 5 Experimental effect of a photodynamic inactivation method of Salmonella in treating fresh eggs.

[0042] FIG. 6A-6F Effect diagram of riboflavin-mediated photodynamic inactivation on an outer membrane of Salmonella.

[0043] In the figure: 6A—negative reference group; 6B—single illumination group; 6C—single photosensitizer group; 6D—photodynamic experiment group I; 6E—photodynamic experiment group II; 6F—photodynamic experiment group III. Magnifications: A1-F1: 10000 times; A2-F2: 20000 times.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Preparation of Salmonella Bacterial Solution

[0044] Salmonella species used in embodiments are Salmonella typhimurium CICC 21484 and Salmonella enteritidis CMCC 50041 respectively. The Salmonella typhimurium CICC 21484 was bought from China Center of Industrial Culture Collection (CICC), and Salmonella enteritidis CMCC 50041 was bought from China Medical Culture Collection (CMCC).

[0045] A preparation method of Salmonella bacterial solution: standard strains Salmonella typhimurium CICC 21484 and Salmonella enteritidis CMCC 50041 stored in glycerin tubes at −80° C. were scribed and inoculated to bismuth sulfite agar plates and culture for 24 to 48 h at 37° C. Single colonies were picked into 9 mL TSB test tubes respectively and cultured for 13 h in a table concentrator with a rotation speed of 180 r/min at 37° C. to obtain a stable initial bacteria culture solution. Equal amounts of two Salmonella culture solutions were mixed in a centrifugal tube and centrifuged for 5 min (4° C., 4000 g). 0.85% sterile saline solution was used to re-suspend the bacteria. The concentration of the bacteria was adjusted to about 1×10.sup.7 CFU/mL.

Preparation of the Riboflavin Solution

[0046] Riboflavin in embodiments was bought from a Sangong Biotechnological company, and it was under a USP grade.

[0047] A preparation method of the riboflavin solution: riboflavin was mixed with 0.85% sterile saline solution to prepare the riboflavin solution. The riboflavin solution was prepared for immediate use and stored in darkness at room temperature.

Blue Light Source

[0048] The blue light source in embodiments was an LED blue light source (455-460 nm, 30 cm, 5 W) and was bought from Shenzhen in China (Shenzhen Kerun Optoelectronics Inc., China). A structure of a blue light source illumination device is shown in FIG. 1. An LED system includes an LED photographing light box, a lifting platform and an LED blue light source. The LED system was surrounded by the LED photographing light box, so that the entrance of external light can be prevented. The LED blue light source is disposed at the top inner side of the LED photographing light box. The 24-well plate is disposed on the lifting platform. A distance between the LED blue light source and a sample solution in the 24-well plate (a diameter of 14 mm) is adjusted to 5.0 cm by the lifting platform. The illumination intensity of the LED blue light source is 5.2 mW/cm.sup.2. An energy meter console (PM100D) with a photoelectric diode power sensor (S130C) (American Newton) was used for measurement. The following formula was used to calculate the dose of each obtained sample:

E=Pt

where E=Dose (energy density) in J/cm.sup.2, P=Irradiance (power density) in W/cm.sup.2, and t=time in sec.

[0049] As shown in FIG. 1, the lifting platform 2 is arranged inside the LED photographing light box 1. No dotted line is used to indicate the lifting platform 2, which does not affect those skilled in the art to understand a specific structure of a blue light illumination device.

[0050] Photodynamic Method for Treating Salmonella:

[0051] The riboflavin solution and the bacterial solution were mixed in a 5 mL centrifugal tube. In the mixed system, the concentration of the bacterial solution is about 1×10.sup.6 CFU/mL, and the riboflavin concentration is 0 μmol/L, 50 μmol/L, 100 μmol/L, 150 μmol/L, 200 μmol/L, 250 μmol/L and 300 μmol/L respectively. The mixed system was subjected to the dark incubation for a given time at the rotation speed of 2 r/min in a PTR-60 multifunctional vertical rotating mixer (the temperature is the room temperature: 22-25° C.). 500 μL mixed bacterial solution was sucked into the 24-well plate and illuminated by the LED blue light source with a wavelength of 455-460 nm for a given time. Thereafter, 0.85% sterile saline solution was used for dilution, and appropriate dilution degrees were selected. 100 μL diluent was coated on a plate, and then the plate was cultured for 24-48 h at 37° C. The number of bacterial colonies was calculated. Three parallel samples were prepared for each treatment. Each dilution degree was repeated for three times.

[0052] The PTR-60 multifunctional vertical rotating mixer was Grant-bio. A 9272 waterproof thermostatic incubator was from Shanghai Yiheng Sci-Tech Co., Ltd.

[0053] Data Analysis:

[0054] The experimental data were expressed as the mean±standard deviation. One-way analysis of variance was used to compare the value differences (P<0.05) using SPSS 17.0 (SPSS Inc., Chicago, Ill.).

[0055] Embodiments are used to describe the implementation of the present invention in detail below. An implementation process of the present invention for using the technical means to solve the technical problems and to achieve the technical effect is fully explained and implemented on this basis.

Embodiment 1

Effect Experiment of Different Riboflavin Concentrations on Photodynamic Inactivation of Salmonella

[0056] The experiment was carried out according to a photodynamic treatment method of Salmonella. The concentrations of the riboflavin solutions in the to-be-treated sample mixed system were 0 μmol/L, 50 μmol/L, 100 μmol/L, 150 μmol/L, 200 μmol/L, 250 μmol/L and 300 μmol/L respectively. The dark incubation time was 40 min. The illumination time of the LED blue light source was 30 min. FIG. 2 shows an inactivation status of Salmonella after being treated with different concentrations of riboflavin.

[0057] An initial inoculation amount of Salmonella in the system was about 4.5×10.sup.6 CFU/mL. When the riboflavin concentrations were 100 μmol/L, 150 μmol/L and 200 μmol/L, the amount of Salmonella could be reduced by 1.12 Log CFU/mL, 6.14 Log CFU/mL and 6.21 Log CFU/mL respectively with the incubation time of 40 min, the illumination time of 30 min. When the riboflavin concentrations were 150 μmol/L and 200 μmol/L respectively, the lethality of Salmonella can reach 99.99993% and 99.99995% respectively. When the riboflavin concentration was relatively low, with the increase of the concentration, the lethality of the photodynamic inactivation of Salmonella was improved. When the concentration reached 200 μmol/L, the lethality was maximal. When the riboflavin concentration is continuously increased, the lethality of Salmonella is apparently decreased. The possible reason is that the surplus photosensitizer in the solution absorbs a majority of light, so that the effective light of the photosensitizer on the surface of the bacteria is reduced, and the lethality of the bacteria is decreased. It is indicated that by selecting the appropriate riboflavin concentration, the inactivation effect on Salmonella can be improved.

[0058] According to the above photodynamic method for treating Salmonella, two groups of reference experiments were set.

[0059] The illumination time of the LED blue light source in one group was 0 min, the riboflavin concentration in the mixed system was 0 μmol/L, and the dark incubation time was 40 min. The illumination time of the LED blue light source in the other group was 0 min, the riboflavin concentration in the mixed system was 150 μmol/L, and the dark incubation time was 40 min. As shown in FIG. 2, when there is no riboflavin, no blue light, but only the dark incubation for treating the sample, or when there is only riboflavin and dark incubation but no blue light for treating the sample, the lethality of Salmonella was extremely low, and the sterilization effect was not significant.

Embodiment 2

Effect Experiment of Different Incubation Time on Photodynamic Inactivation of Salmonella

[0060] The experiment was carried out according to a photodynamic method for treating Salmonella. The riboflavin concentration in the mixed system was 150 μmol/L. The incubation time was 0 min, 20 min, 40 min and 60 min respectively. The illumination time of the LED blue light source was 30 min. As shown in FIG. 3, with the increase of the incubation time, the inactivation effect for Salmonella was also increased. When the incubation time was 40 min, the fatality rate of Salmonella was 99.99993%. However, the excessive long incubation time may also affect the sterilization effect. Therefore, selecting the appropriate incubation time can improve the inactivation effect for Salmonella.

[0061] According to the above photodynamic method for treating Salmonella, two groups of reference experiments were set. The illumination time of the LED blue light source in one group was 0 min, the riboflavin concentration in the mixed system was 0 μmol/L, and the dark incubation time was 40 min. The illumination time of the LED blue light source in the other group was 0 min, the riboflavin concentration in the mixed system was 150 μmol/L, and the dark incubation time was 40 min. As shown in FIG. 3, when there is no riboflavin, no blue light, but only dark incubation for treating the sample, or when there is riboflavin and only dark incubation but no blue light for treating the sample, the fatality rate of Salmonella was extremely low, and the sterilization effect was not significant.

Embodiment 3

Effect Experiment of Different Illumination Time on Photodynamic Inactivation of Salmonella

[0062] The experiment was carried out according to a photodynamic method for treating Salmonella. The riboflavin concentration in the to-be-treated sample mixed system was 150 μmol/L. The dark incubation time was 40 min. The illumination time of the LED blue light source was 0 min, 10 min, 20 min, 30 min, 40 min and 50 min. FIG. 4 shows an inactivation status of Salmonella after being treated with different illumination time. With the increase of the illumination time, the inactivation effect for Salmonella is also increased significantly. The initial inoculation amount of Salmonella in the system was about 6.75 Log CFU/mL. With the riboflavin concentration of 150 μmol/L, the incubation time of 40 min, and the illumination time of 20 min, 30 min, 40 min and 50 min, the amount of Salmonella can be reduced by 2.23 Log CFU/mL, 6.21 Log CFU/mL, 6.75 Log CFU/mL and 6.75 Log CFU/mL respectively. When the illumination time was 40 min, the fatality rate of Salmonella reached up to 99.99995%.

[0063] According to the above photodynamic method for treating Salmonella, two groups of reference experiments were set. The illumination time of the LED blue light source in one group was 0 min, the riboflavin concentration in the mixed system was 0 μmol/L, and the dark incubation time was 40 min. The illumination time of the LED blue light source in the other group was 0 min, the riboflavin concentration in the mixed system was 150 μmol/L, and the dark incubation time was 40 min. As shown in FIG. 4, when there is no riboflavin, no blue light, but only dark incubation for treating the sample, or when there is riboflavin and dark incubation but only no blue light for treating the sample, the fatality rate of Salmonella was extremely low, and the sterilization effect was not significant.

Embodiment 4

Salmonella Inactivation Experiment for Fresh Egg Shells

[0064] Sterile egg shells were mixed with a Salmonella bacterial solution to be contaminated and divided into three groups as follows: 1, a single photosensitizer group with riboflavin concentration of 150 μmol/L, was subjected to the dark incubation for 40 min without the illumination of a blue light source; 2, a photodynamic experiment group with riboflavin concentration of 150 μmol/L, was subjected to the dark incubation for 40 min under the illumination of the blue light source for 40 min; and 3, a blank reference group with a saline solution having a volume equal to that of the riboflavin solution. Each group was provided with two parallel groups.

[0065] Bacteria culture: under a sterile condition, each group of egg shells was diluted with the saline solution for two times and then homogenized by a sterile homogenizing device and diluted. 100 μL diluent with appropriate dilution degrees was inoculated into the TSA solid culture medium and cultured for 24 to 48 h in the thermostatic incubator at 37° C. The number of bacterial colonies in each group was counted.

[0066] As shown in FIG. 5, the result shows that the number of Salmonella bacterial colonies in the blank reference group of egg shells is 7.4×10.sup.5 CFU/g, the number of the bacterial colonies in the egg shells after the PDI treatment is 8.4×10.sup.2 CFU/g, and the number of the bacterial colonies in the egg shells of the single photosensitizer group is 7.0×10.sup.5 CFU/g. Through multiple experiments, it is discovered that the sterilization rate of the photodynamic inactivation method of Salmonella using the riboflavin as the photosensitizer can reach 99.88%, and the sterilization effect is apparent.

Embodiment 5

Effect Diagram of a Riboflavin-Mediated PDI Method on an Outer Membrane of Salmonella

[0067] The bacterial solution was treated under different conditions. As shown in FIG. 6, FIG. 6A is a negative reference group: this group was a pure bacterial solution without any treatment. FIG. 6B is a single illumination group: with no riboflavin solution, the bacterial solution was not subjected to the dark incubation but only illuminated with the blue light source with the illumination energy density of 15.6 J/cm.sup.2. FIG. 6C is a single photosensitizer group: with 150 μmol/L riboflavin solution, the bacterial solution was subjected to the dark incubation for 40 min without the illumination of the blue light source. FIG. 6D is a photodynamic experiment group I: with 50 μmol/L riboflavin solution, the bacterial solution was subjected to the dark incubation for 20 min and illuminated with the blue light source with the illumination energy density of 3.12 J/cm.sup.2. FIG. 6E is a photodynamic experiment group II: with 150 μmol/L riboflavin solution, the bacterial solution was subjected to the dark incubation for 40 min and illuminated with the blue light source with the illumination energy density of 9.63 J/cm.sup.2. FIG. 6F is a photodynamic experiment group III: with 150 μmol/L riboflavin solution, the bacterial solution was subjected to the dark incubation for 40 min and illuminated with the blue light source with the illumination energy density of 15.6 J/cm.sup.2.

[0068] Bacterial suspension (500 μL) was centrifuged for 5 min at 10,000 g. Supernatant was removed, and precipitates were mixed with 500 μL glutaraldehyde (2.5%) and formaldehyde (4%) in 0.1M Cacodylate buffer for overnight at 4° C. Then the sample was gradually dehydrated level by level through a consecutive 30%-100% ethanol solution. The sample was placed on a support by using double-sided transparent adhesive and coated with gold. A high-resolution desktop SEM (SNE-4500M, JEOL, Japan) was used for scanning, and results are shown in FIG. 6.

[0069] A scanning electron microscope (SEM) was used to represent morphologic change of bacteria cells. Compared with the negative reference group (FIG. 6A), the morphology of Salmonella cells in the single illumination group (FIG. 6B) and the single photosensitizer group (FIG. 6C) is not apparently changed, as shown in FIGS. 6B-C, and the cells are in a full rhabditiform. As shown in FIG. 6D, the surfaces of the cells in the photodynamic experiment group I had slight morphological deformation and grooves. As shown in FIG. 6E, the apparent deformed morphology of the cells and the fold cells can be observed in the photodynamic experiment group II. As shown in FIG. 6F, in the photodynamic experiment group III, the morphologic deformation of Salmonella cells is significantly changed, and a great number of cells are broken. Therefore, it can be concluded that the riboflavin-mediated PDI may inactivate Salmonella by attacking cell walls and cell membranes.

[0070] The above only describes specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or replacements made without contributing creative effort shall fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subjected to the protection scope defined by the claims.