Mild thermal enhanced photodynamic spore inactivation method

Abstract

The invention relates to a mild thermal enhanced photodynamic spore inactivation method, which combines a photodynamic technology with 65 DEG C mild thermal treatment, and is used for killing bacillus altitudinis spores and bacillus safensis spores in a germination stage. The method disclosed by the invention has an excellent killing effect on spores of bacillus altitudinis and spores of bacillus safensis, can realize efficient inactivation of the spores in meat products, solves the defects of the existing photodynamic technology for inactivating the spores, adopts a sterilization temperature far lower than a commercial sterilization temperature, can better maintain the flavor and quality of foods, and is suitable for industrial production. And the method has universality in food industry application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing, and particularly relates to a mild heat-enhanced photodynamic inactivation method of spores. BACKGROUND

[0002] Bacterial spores formed by Bacillus and Clostridium have dormancy characteristics, which enable them to survive in adverse environmental conditions. Dormant spores exhibit high resistance to many extreme conditions, which is attributed to the unique multilayer structure and characteristics of spores. Generally, spores are composed of a coat, an outer membrane, a cortex, a cell wall, an inner membrane, and a core. However, this unique survival strategy of spores is conducive to their survival under various food processing conditions, and they are easy to recover and grow into vegetative cells during food storage, which then leads to food spoilage and foodborne diseases.

[0003] In the food industry, the "direct sterilization" strategy is widely used, and heat treatment is a common means of spore inactivation, but the required temperature is extremely high. Studies have shown that exposure to a temperature of 120 ℃ for 1 h can effectively inactivate Bacillus cereus spores in rice. However, excessively high temperatures can severely damage the nutritional value and sensory quality of food. Emerging technologies, including microwaves, ultrasound, and photodynamic therapy, combined with heat treatment, can reduce the temperature required for spore inactivation, thereby minimizing potential harm to food.

[0004] Photodynamic inactivation (PDI) has become a promising sterilization technology in the food industry due to its unique non-specific therapeutic effect on foodborne pathogens. The working principle of PDI is that photosensitizers (PSs) produce reactive oxygen species (ROS) under specific wavelength light, which causes oxidative damage to microbial cells. PSs of plant and natural origin are favored in food and food processing environments due to their reliable source, low toxicity, and fast in vivo clearance rate. Studies have shown that exposure of riboflavin to blue LED light can significantly inactivate Escherichia coli and Salmonella typhimurium in apple juice. However, PDI still faces significant bottlenecks in spore inactivation. The metabolic activity of dormant spores is extremely low, and the multilayer structure of spores poses a strong physical barrier to the diffusion of ROS with ultra-short lifespan (≈ 3 μs), resulting in the killing effect of traditional PDI on spores being far inferior to that on vegetative cells. In addition, most photosensitizers require expensive lasers or specific wavelength light sources to be activated, limiting their universality in food industrial production and application. When spores begin to germinate, the resistance of spores is weakened and they are easily killed. This provides more possibilities for the application of some non-thermal technologies in spore inactivation.

[0005] Therefore, how to overcome the application limitations of PDI technology in spore inactivation and reduce the damage of traditional sterilization methods to food quality is a problem that needs to be solved urgently. SUMMARY

[0006] The present application aims to provide a mild heat-enhanced photodynamic inactivation of spores method.

[0007] To achieve the above object and other related objects, the technical solution provided by the present application is: a mild heat-enhanced photodynamic inactivation of spores method, characterized in that: a photosensitizer, a germinant and a to-be-treated substance containing spores are uniformly mixed to obtain a to-be-treated spore suspension; the to-be-treated spore suspension is subjected to an induction germination treatment; and finally, the spore suspension is subjected to a mild heat photodynamic treatment.

[0008] The preferred technical solution is: the photosensitizer contains curcumin and thymol, wherein the final concentration of curcumin is greater than or equal to 50 μM, and the final concentration of thymol is greater than or equal to 0.0625 mg / mL.

[0009] The preferred technical solution is: the germinant is L-aspartic acid, and the final concentration is greater than or equal to 5 mM.

[0010] The preferred technical solution is: the killed spores include spores of Bacillus altitudinis and spores of Bacillus safensis; the concentration of spores in the to-be-treated spore suspension is 10 6 ~10 7 CFU / mL.

[0011] The preferred technical solution is: the induction germination treatment includes: first, heat activation at 70-80℃ for 10-20 minutes, then ice bath cooling for 3-8 minutes, and then incubation at 30-40℃ for 30-60 minutes.

[0012] The preferred technical solution is: during the mild heat photodynamic treatment, the wavelength of the LED white light used is 448 nm, the output power is 0.8 mW / cm 2 , the illumination distance is 20-25 cm, the heat environment temperature is 65℃, and the treatment time is 1-2.5 h.

[0013] Due to the use of the above technical solution, the present application has the following advantages compared with the prior art:

[0014] The application provides a mild heat-enhanced photodynamic inactivation spore method, which combines photodynamic technology with 65 DEG C mild heat treatment (TL) to kill spores in the germination stage. That is, the spores are first induced to germinate, and then natural curcumin and thymol are used as high-efficiency photosensitizer systems, and the penetration of the multi-layer structure of the spores is enhanced by using 65 DEG C heat treatment to enhance the photodynamic technology, the damage of the photodynamic to the core of the spore is enhanced, and the inactivation efficiency of the spore is significantly improved. The method of the application has excellent killing effect on Bacillus altitudinis spores and Bacillus safensis spores, and the inactivation effect is significantly enhanced with the increase of the treatment time, the shortcomings of the existing photodynamic technology for inactivating spores are solved, and the sterilization temperature used is much lower than the commercial sterilization temperature, so that the flavor and quality of food can be better maintained, and the method has universality in food industry applications. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Table 1 is the killing effect of each group on Bacillus altitudinis spores under different treatment times in the experimental example 1 of the application.

[0016] Figure 2 Table 2 is the killing effect of each group on Bacillus safensis spores under different treatment times in the experimental example 2 of the application.

[0017] Figure 3 Table 3 is the influence of each group on the DPA release rate of Bacillus altitudinis spores in the experimental example 3 of the application.

[0018] Figure 4 Table 4 is the influence of each group on the DPA release rate of Bacillus safensis spores in the experimental example 4 of the application. DETAILED DESCRIPTION

[0019] The embodiments of the application are described below by specific examples, and those skilled in the art can easily understand other advantages and effects of the application from the content disclosed in the examples.

[0020] Please refer to Figures 1-4 It should be understood that the structures, proportions, sizes and the like shown in the drawings attached to the present specification are only used to understand and read the content disclosed in the present specification by those skilled in the art, and do not have technical significance to limit the conditions that can be implemented by the application, so any modification of the structure, change of the proportion relationship or adjustment of the size. The following examples are provided to better understand the application, but not to limit the application. The experimental materials used in the following examples are commercially available from conventional consumables and biochemical reagent stores unless otherwise specified.

[0021] Example 1: A mild heat-enhanced photodynamic inactivation spore method

[0022] A preparation method of a high-sterilization-performance edible photosensitizer, comprising the following steps:

[0023] Step 1. Tween 80 (1 % w / v) was dissolved in deionized water as solvent;

[0024] Step 2. 125 mg of curcumin (Cur) and 368 mg of thymol (Thy) were dissolved in 100 mL of 1 % (w / v) Tween 80 aqueous solution;

[0025] Step 3. The mixture was homogenized by ultrasonic treatment for 10 min using an ultrasonic cleaner (100 W) to obtain CTNPs. The prepared CTNPs solution was stored in a 4 ℃ refrigerator and used after 10-fold dilution.

[0026] Example 2: A method for mild heat-enhanced photodynamic inactivation of spores

[0027] A method for preparing a spore suspension, comprising the following steps:

[0028] Step 1. The activated Bacillus spores were inoculated into a manganese salt nutrient agar medium and cultured at 37 ℃ for 5-7 days. The formation rate of spores was observed by phase contrast microscopy and was more than 95 %.

[0029] Step 2. The spores were collected with sterile water and the residual nutrient cells were eliminated by water bath at 80 ℃ for 10 min to obtain a crude spore suspension.

[0030] Step 3. The crude spore suspension was centrifuged (8000 rpm, 15 min, 4 ℃) and washed three times. The supernatant and the top layer of the precipitate containing cell debris were discarded.

[0031] Step 4. The bottom layer was resuspended in sterile distilled water to a final concentration of 1.5×10 8 CFU / mL to obtain a spore suspension. The spore suspension was stored in a -40 ℃ refrigerator and the spores needed to be washed before each experiment.

[0032] Example 3: A method for mild heat-enhanced photodynamic inactivation of spores

[0033] A method for preparing a germinant, comprising the following steps:

[0034] L-aspartic acid was dissolved in distilled water to a final concentration of 5 mM.

[0035] Example 4: A method for mild heat-enhanced photodynamic inactivation of spores

[0036] A method for mild heat-enhanced photodynamic inactivation of spores, comprising the following steps:

[0037] Step 1. A food-grade photosensitizer, a germinant, and a spore suspension were mixed uniformly at a ratio of 1:1:1 to obtain a spore suspension to be treated.

[0038] Step 2, the spore suspension to be treated was induced to germinate by first heat-activating at 75 °C for 15 minutes, then immediately cooling in an ice bath for 5 minutes, and then incubating at 37 °C for 40 minutes;

[0039] Step 3, the spore suspension to be treated was placed under LED white light irradiation, the LED white light wavelength was 448 nm, the output power was 0.8 mW / cm 2 , the light irradiation distance was 20-25 cm, the thermal environment temperature was 65 °C, and the treatment time was 1-2.5 h.

[0040] Step 4, the treated spore suspension was gradiently diluted by dropping onto PCA medium, and then incubated at 37 °C for 15 hours to determine the count (CFU / mL).

[0041] Example 5: A method for mild heat-enhanced photodynamic inactivation of spores

[0042] A method for measuring the DPA release rate of spores, comprising the following steps:

[0043] The spore suspension after 2 h of treatment was centrifuged (8000 rpm, 10 min, 4 °C), and the supernatant was filtered through a 0.45 μm microporous filter. 100 μL of the supernatant was added to a 96-well plate, and an equal amount of 20 μmol / L TbCl3 solution was added. The fluorescence intensity at 545 nm emission wavelength and 270 nm excitation wavelength was detected using a microplate reader. Untreated spores and autoclaved spores (121 °C, 20 minutes) were used as negative and positive controls, respectively. The DPA release rate of spores was calculated by the following formula:

[0044] DPA (%) =

[0045] Where F0, F1 and F2 represent the fluorescence intensity of untreated, treated and 121 °C, 20 minutes treated samples, respectively.

[0046] Comparative Example 1:

[0047] The difference between this comparative example and Example 4 is that in Step 3, the spore suspension to be treated is not subjected to LED white light irradiation and 65 °C mild heat treatment.

[0048] Comparative Example 2:

[0049] The difference between this comparative example and Example 4 is that in Step 3, the spore suspension to be treated is subjected to LED white light irradiation at room temperature.

[0050] Comparative Example 3:

[0051] The difference between the present comparative example and experimental example 4 is that the spore suspension to be treated in step 3 is not subjected to LED white light irradiation, but only subjected to mild heat treatment at 65 ℃.

[0052] Experimental example 1:

[0053] First, the photosensitizer and the germinant are prepared according to example 1 and example 3, respectively, and the B. altitudinis spores are prepared according to example 2; the B. altitudinis spores are inactivated according to example 4. The blank control is sterile water mixed with the spore suspension at a ratio of 2:1. Then, comparative example 1, comparative example 2 and comparative example 3 are used for comparative experiments, and the experimental results are shown in Table 1. Figure 1

[0054] Experimental example 2:

[0055] First, the photosensitizer and the germinant are prepared according to example 1 and example 3, respectively, and the B. altitudinis spores are prepared according to example 2; the B. altitudinis spores are inactivated according to example 4. The blank control is sterile water mixed with the spore suspension at a ratio of 2:1. Then, comparative example 1, comparative example 2 and comparative example 3 are used for comparative experiments, and the experimental results are shown in Table 1. Figure 2

[0056] The mild heat enhanced photodynamic effect on the killing of spores in the germination stage will be described in detail below with reference to the accompanying drawings.

[0057] Referring to Figure 1 and Figure 2 Compared with the number of B. altitudinis and B. safensis spores in the blank control group, comparative example 1 and comparative example 2 have no significant inactivation effect on the spores. This is because the complex structure of the spores makes them more resistant to light than vegetative cells. The spores have a small acid-soluble protein associated with DNA, as well as a physical barrier caused by the thick protein coating of the spores. The inactivation effect of comparative example 3 on the spores is enhanced with the increase of the treatment time, and the inactivation logarithm of B. altitudinis and B. safensis spores is 2.17 log and 3.98 log, respectively, when the treatment time is 150 min. Example 1 has a stronger and more excellent inactivation effect on the spores, and 99.98% of B. altitudinis spores and 99.99% of B. safensis spores can be killed when the treatment time is 120 min, and the inactivation logarithm is 3.75 log and 4.05 log, respectively. The spores can be completely killed when the treatment time is 150 min. This is because the heat treatment enhances the penetration of photodynamic into the spores, and the two have a synergistic effect. This effect may be due to the weakened resistance of the spores in the germination stage, so that the low temperature of 65 ℃ can enhance the permeability of the spore inner membrane, destroy the structure of the spores, and promote the penetration of reactive oxygen species (ROS) generated by photodynamic into the core of the spores. Photodynamic and mild heat treatment work synergistically to cause irreversible inactivation of the spores. ​​

[0058] DPA is abundant in the core of spores, and it plays an important role in the resistance of spores to various sterilization techniques. When the membrane of spores is damaged and the permeability is increased, a large amount of DPA is released. As shown in Figure 3 and Figure 4 The DPA release amount of Comparative Example 1 and Comparative Example 2 has no significant difference, less than 5%. The DPA release amount of Bacillus gaertneri and Bacillus safensis of Comparative Example 3 is 57.03% and 67.49% respectively. This is because the temperature treatment promotes the germination of spores, and the hydration of the core of spores promotes the release of DPA. The DPA release amount of Bacillus gaertneri and Bacillus safensis of Example 1 of the present application is as high as 81.18% and 92.77%, which is significantly higher than that of other comparative examples. This synergistic effect is due to the combination of 65℃ temperature and germinant, which can maximize the germination of spores, increase the permeability, and induce core hydration. At the same time, the heat treatment promotes the penetration of the internal structure of the spores by the photodynamic effect, enhances the high sensitivity of the spores to oxidative damage during the germination stage, and improves the inactivation of the spores.

[0059] In general, the present application makes up for the shortcomings of traditional photodynamic technology, and has multiple advantages: the treatment system is safe, non-toxic, and non-polluting; the equipment is simple, easy to obtain, and the treatment adjustment is easy to meet, the cost is low, and it is easy to directly apply to the food industry, and has certain control and prevention effect, providing strong technical support for maintaining public health.

[0060] The purpose of the present application is to provide a mild heat enhanced photodynamic inactivation method of spores, which combines photodynamic technology with 65℃ mild heat treatment (TL) for killing spores in the germination stage. The method of the present application has excellent killing effect on spores, can realize efficient inactivation of spores in meat products, solves the shortcomings of existing photodynamic technology for inactivating spores, and uses sterilization temperature much lower than commercial sterilization temperature, which can better maintain the flavor and quality of food, and has universality in food industry applications.

[0061] The above is only to explain the preferred embodiments of the present application, and is not intended to limit the present application in any form. Any modification or change related to the present application made under the same inventive spirit shall still be included in the scope intended to be protected by the present application.

Claims

1. A method for mild heat enhanced photodynamic inactivation of spores, characterized by: The photosensitizer, the germinant and the to-be-treated substance containing spores are uniformly mixed to obtain a spore suspension to be treated; the spore suspension to be treated is subjected to induction germination treatment; and finally, the spore suspension is subjected to mild heat-photodynamic treatment.

2. The method of mild heat enhanced photodynamic inactivation of spores according to claim 1, characterized in that: The photosensitizer comprises curcumin and thymol, wherein the final concentration of curcumin is greater than or equal to 50 μM, and the final concentration of thymol is greater than or equal to 0.0625 mg / mL.

3. The method of mild heat enhanced photodynamic inactivation of spores according to claim 1, wherein: The germinant is L-aspartic acid, and the final concentration is greater than or equal to 5 mM.

4. The method of mild heat enhanced photodynamic inactivation of spores according to claim 1, wherein: The killed spores include spores of Bacillus altitudinis and spores of Bacillus safensis; the concentration of spores in the spore suspension to be treated is 10 6 CFU / mL. 7 CFU / mL.

5. The method of mild heat enhanced photodynamic inactivation of spores according to claim 1, wherein: The induction germination treatment comprises: first, heat activation at 70-80 ℃ for 10-20 minutes, then ice bath cooling for 3-8 minutes, and subsequent incubation at 30-40 ℃ for 30-60 minutes.

6. The method of mild heat enhanced photodynamic spore inactivation of claim 1, wherein: In the mild heat-photodynamic treatment process, the wavelength of the LED white light used is 448 nm, the output power is 0.8 mW / cm 2 , the illumination distance is 20~25 cm, the thermal environment temperature is 65 ℃, and the treatment time is 1~2.5 h.

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