Repair medium for sub-damaged lactic acid Escherichia coli and its preparation and repair method
The use of the repair medium of E. coli in lactate sub-damage, including the preparation of double-displacement water culture medium with components such as spirulina protease degradation products and high-pressure steam sterilization, solved the problem of low repair efficiency of existing culture medium and achieved E. coli Efficient repair and accurate detection of sub-damage bacteria.
Patent Information
- Application Number
- CN202211260755.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-14
- Publication Date
- 2025-01-14
- Estimated Expiration
- 2042-10-14
AI Technical Summary
The existing culture medium for repairing sub-damage E. coli is not repaired at high efficiency, resulting in low or high detection results, affecting the accurate quantification of E. coli in food.
Using lactate sub-damage E. coli repair medium, including spirulina protease degradation products, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose and Tween 80, was sterilized by high-pressure steam sterilization and bacterial filters, and prepared double-displacement water culture and repaired in a constant temperature shaking incubator.
It is efficiently repaired about 100% of E. coli sub-damaged bacteria within 20 minutes, and there is no obvious amplification of live bacteria during the repair time, meeting the E. coli detection needs in precise quantitative foods.
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Figure CN115838660B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial culture medium and food microbial testing technology, in particular to a repair culture medium for sub-damaged lactic acid Escherichia coli and a preparation and repair method thereof. Background Art
[0002] Foodborne pathogenic microorganisms cause foodborne diseases including food poisoning, endangering human or animal health. Food spoilage microorganisms cause food spoilage including corruption, rancidity and fermentation, which on the one hand causes sensory changes in food color, aroma, taste and shape, and on the other hand destroys food ingredients, reduces or loses edible value, causing significant economic losses. In addition, spoiled food is prone to breed bacteria, and even breeds pathogens and toxin-producing molds, leading to infectious diseases or food poisoning. Therefore, it is necessary to effectively preserve and handle food to control microorganisms.
[0003] Food preservation and processing methods, such as heating, drying, freezing, irradiation, acid treatment and alkali treatment, lead to the formation of the following three types of bacteria in food: 1) dead bacteria; 2) sub-damaged bacteria; 3) live bacteria. Among them, sub-damaged bacteria can be restored to health or spoil food under good conditions. Therefore, sub-damaged bacteria and live bacteria should be taken seriously and need to be effectively inspected and monitored.
[0004] In food microbiological analysis, sub-lesional bacteria cannot grow on selective culture media and thus escape detection, resulting in a low number of detected food microorganisms. This can lead to people mistakenly believing that unsafe food is safe, which can easily lead to food safety incidents. Therefore, on the one hand, it is necessary to effectively repair sub-lesional bacteria, and on the other hand, the repair culture medium must be extremely efficient to ensure that live bacteria have not yet reproduced within the repair time, otherwise it is easy to cause a high number of detected food microorganisms. For this reason, it is extremely important to develop an efficient repair culture medium.
[0005] Escherichia coli is considered to be the best microorganism to indicate fecal contamination of food, and will effectively characterize the contamination of food microorganisms. In addition, some strains of Escherichia coli can cause intestinal infections, such as enterotoxigenic Escherichia coli, pathogenic Escherichia coli, hemorrhagic Escherichia coli, invasive Escherichia coli and adherent Escherichia coli. Therefore, the detection of Escherichia coli in food is of great significance.
[0006] Lactic acid exists naturally in some foods, or is added to food in the form of an antibacterial agent. Thus, lactic acid causes sub-injury of Escherichia coli. At present, trypticase soy broth (TSB) is considered to be the culture medium with the best repair effect on sub-injury of lactic acid Escherichia coli, for example, the literature: (Shi, H., Chen, Z., Chen, D., and Kan, J. (2017). Sublethal injury and recovery of Escherichia coli O157: H7 and K-12 after exposure to lactic acid. Food Control 82, 190-195.). However, the repair efficiency of this culture medium is not high, and it takes 120 minutes to complete about 100% repair. In a nutrient-rich culture medium such as TSB, the time required for Escherichia coli to divide for one generation is about 20 minutes. Therefore, during the time when TSB is used for about 100% repair, the live bacteria are amplified in large quantities, which will make the test results higher, which is not conducive to the accurate quantification of Escherichia coli in food. Summary of the invention
[0007] The technical problem to be solved by the present invention is that the existing culture medium for repairing sub-damaged Escherichia coli has low repair efficiency. On the one hand, if the sub-damaged bacteria are not completely repaired without repair or the repair time is too short, the test result will be low; on the other hand, if the repair time is extended to make about 100% of the sub-damaged bacteria completely repaired, the live bacteria will be amplified in large quantities during the repair time, which will make the test result high. Therefore, it is not conducive to the accurate quantification of Escherichia coli in food detection.
[0008] The technical solution of the present invention to solve the above technical problems is as follows:
[0009] The invention provides a lactic acid sub-damaged Escherichia coli repair culture medium, comprising: a spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, Tween 80 and a solvent.
[0010] Preferably, the solvent is double distilled water, and each liter of double distilled water contains: 100-180 g of spirulina protease degradation product; 5-15 g of glucose, 3.74-8.51 g of dipotassium hydrogen phosphate, 0.1-0.7 g of potassium dihydrogen phosphate, and 1-10 ml of Tween 80.
[0011] Furthermore, the preparation method of the above-mentioned lactic acid sub-damaged Escherichia coli repair culture medium includes the following steps: adding 100-180g of spirulina protease degradation product per liter of double distilled water, adjusting the pH value to 6.0-8.0 with sodium hydroxide, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 5-15g of glucose, 3.74-8.51g of dipotassium hydrogen phosphate and 0.1-0.7g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 1-10ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
[0012] Preferably, each liter of double distilled water contains: each liter of double distilled water contains: 110-170 g of spirulina protease degradation product; 7-13 g of glucose, 4.24-8.01 g of dipotassium hydrogen phosphate, 0.2-0.6 g of potassium dihydrogen phosphate, and 2.5-8.5 ml of Tween 80.
[0013] Furthermore, the preparation method of the above-mentioned lactic acid sub-damaged Escherichia coli repair culture medium includes the following steps: adding 110-170g of spirulina protease degradation product per liter of double distilled water, adjusting the pH value to 6.3-7.7 with sodium hydroxide, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 7-13g of glucose, 4.24-8.01g of dipotassium hydrogen phosphate and 0.2-0.6g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 2.5-8.5ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
[0014] Preferably, each liter of double distilled water contains: 120-160 g of spirulina protease degradation product; 9-11 g of glucose, 4.74-7.51 g of dipotassium hydrogen phosphate, 0.3-0.5 g of potassium dihydrogen phosphate, and 4-7 ml of Tween 80.
[0015] Furthermore, the preparation method of the above-mentioned lactic acid sub-damaged Escherichia coli repair culture medium includes the following steps: adding 120-160g of spirulina protease degradation product per liter of double distilled water, adjusting the pH value to 6.6-7.4 with sodium hydroxide, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 9-11g of glucose, 4.74-7.51g of dipotassium hydrogen phosphate and 0.3-0.5g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 4-7ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
[0016] The present invention also provides a method for repairing sub-damaged lactic acid Escherichia coli, which uses the above-mentioned sub-damaged lactic acid Escherichia coli repair culture medium and comprises the following steps:
[0017] Step 1, transferring the sub-damaged lactic acid Escherichia coli solution into a centrifuge tube, centrifuging at 8000 g for 5 minutes, removing the supernatant and adding it into the sub-damaged lactic acid Escherichia coli repair culture medium;
[0018] Step 2: Repair in a constant temperature shaking incubator at 150 rpm and 37°C for 20 minutes, immediately centrifuge at 8000g for 5 minutes to remove the supernatant to terminate the repair and obtain the E. coli repair solution.
[0019] Preferably, the initial bacterial count of the sub-damaged lactic acid Escherichia coli solution in step 1 is OD600=0.1. OD600=0.1 is equivalent to 10 8 CFU / ml of bacteria.
[0020] Preferably, the lactic acid sub-damaged Escherichia coli solution in step 1 comprises Escherichia coli sub-damaged by L-lactic acid solution.
[0021] The beneficial effects of the present invention are:
[0022] The best existing TSB repair culture solution can only repair about 37.68% of sub-damaged Escherichia coli within 20 minutes, while the repair culture medium provided by the present invention can efficiently repair about 100% of sub-damaged Escherichia coli within 20 minutes, and during the repair time, there is no obvious amplification of live Escherichia coli, which meets the needs of accurate quantitative detection of Escherichia coli in food. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Schematic diagram of the sub-damage rate of lactic acid-damaged Escherichia coli.
[0024] Figure 2 This is a comparison chart of the colony counts after repair by the present invention and the reference repair medium TSB.
[0025] Figure 3 It is a comparison chart of the repair rate of the present invention and the reference repair medium TSB.
[0026] Figure 4 This is a comparison chart of the colonies of the repaired bacterial solution of the present invention and the unrepaired bacterial solution on TSA.
[0027] Figure 5 This is a colony statistics chart of the repaired bacterial solution of the present invention and the unrepaired bacterial solution on TSA. DETAILED DESCRIPTION
[0028] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0029] The invention provides a lactic acid sub-damaged Escherichia coli repair culture medium, comprising: a spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, Tween 80 and a solvent.
[0030] Preferably, the solvent is double distilled water, and the preparation method of the lactic acid sub-damaged Escherichia coli repair culture medium is: put 100-180g of spirulina protease degradation product per liter of double distilled water, adjust the pH value to 6.0-8.0 with sodium hydroxide, sterilize with high-pressure steam at least 121°C for at least 15 minutes, add 5-15g of glucose, 3.74-8.51g of dipotassium hydrogen phosphate and 0.1-0.7g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally add 1-10 ml of Tween 80, shake and mix, and filter and sterilize with a 0.22-micron bacterial filter.
[0031] Preferably, 110-170 g of spirulina protease degradation product is added per liter of double distilled water, the pH value is adjusted to 6.3-7.7 with sodium hydroxide, and sterilized with high-pressure steam at least 121° C. for at least 15 minutes. After cooling, 7-13 g of glucose, 4.24-8.01 g of dipotassium hydrogen phosphate and 0.2-0.6 g of potassium dihydrogen phosphate are added in a sterile environment, and finally 2.5-8.5 ml of Tween 80 is added. After oscillation and mixing, the mixture is filtered and sterilized with a 0.22-micron bacterial filter.
[0032] Preferably, 120-160 g of spirulina protease degradation product is added per liter of double distilled water, the pH value is adjusted to 6.6-7.4 with sodium hydroxide, and sterilized with high-pressure steam at least 121° C. for at least 15 minutes. After cooling, 9-11 g of glucose, 4.74-7.51 g of dipotassium hydrogen phosphate and 0.3-0.5 g of potassium dihydrogen phosphate are added in a sterile environment, and finally 4-7 ml of Tween 80 is added. After oscillation and mixing, the mixture is filtered and sterilized with a 0.22-micron bacterial filter.
[0033] The present invention also provides a method for repairing sub-damaged lactic acid Escherichia coli, which uses the above-mentioned sub-damaged lactic acid Escherichia coli repair culture medium and comprises the following steps:
[0034] Step 1, transfer 1 ml of the lactic acid sub-damaged Escherichia coli solution to a 1.5 ml centrifuge tube, centrifuge at 8000 g for 5 minutes, remove the supernatant and add it to the lactic acid sub-damaged Escherichia coli repair culture medium;
[0035] Step 2: Repair in a constant temperature shaking incubator at 150 rpm and 37°C for 20 minutes, immediately centrifuge at 8000g for 5 minutes to remove the supernatant to terminate the repair and obtain the E. coli repair solution.
[0036] Note: For 8000g centrifugation, the conversion formula between centrifugal force G and speed RPM is as follows: G = 1.11 × 10 -5 ×R×(RPM) 2Where G is the centrifugal force, which is usually expressed in multiples of g (gravitational acceleration) (such as 8000g in this article). R is the radius, in centimeters. For example, if the centrifugal radius is 10 cm and the speed is 8000 rpm, the centrifugal force is: G = 1.11*10 -5 *10*(8000) 2 =7104, that is, the centrifugal force is 7104g. When the centrifugal force is 8000g, the speed should be: 8489rpm, which is about 8500rpm. Different centrifuges have different radii, G is universal, and this paragraph is just a calculation example.
[0037] Results verification: According to the method of using the culture medium of the present invention, the lactic acid sub-damaged E. coli repair culture medium described in step 1 is replaced with TSB repair culture medium, and the E. coli control repair solution obtained in step 2 is used as the control group. Take the E. coli repair solution and the E. coli control repair solution obtained in step 2, add 1 ml of sterile water, shake evenly, dilute and spread on trypticase soy agar (TSA) plates and selective culture medium TSA-SC plates, and culture in a constant temperature incubator at 37°C for 16 hours to count the number of colonies and calculate the repair rate.
[0038] Note: The formula of the TSB repair culture medium is (per liter): 17 g tryptic peptone, 3 g soy peptone, 2.5 g D-glucose, 2.5 g sodium chloride, and 2.5 g potassium dihydrogen phosphate.
[0039] Note: The formula of TSA plate is (per liter): 15 g trypticase, 5 g soy papain hydrolysate, 5 g sodium chloride and 15 g agar. This medium supports the growth of sub-lesional bacteria and live bacteria of Escherichia coli.
[0040] Note: The formula of the selective culture medium TSA-SC plate is (per liter): 15 grams of trypticase, 5 grams of soybean papain hydrolysate, 20 grams of sodium chloride and 15 grams of agar. This culture medium only supports the growth of live Escherichia coli and does not support the growth of sub-lesional bacteria.
[0041] Note: The dilution and coating is carried out by using sterile water to make a 10-fold dilution so that the number of colonies grown on the plate is between 20 and 300; when coating, 0.02 ml of the diluted bacterial solution is used to coat the plate using a glass coater.
[0042] Note: The repair experiment for the above results was repeated 3 times and the average value was taken to calculate the repair rate.
[0043] Note: The calculation formula of the repair rate is as follows:
[0044]
[0045] Among them, %recovery is the repair rate; CFU TSA-SC,time CFU is the number of colonies on the selective medium TSA-SC after repair (average of 3 experiments); TSA-SC,start CFU is the number of colonies (average of 3 experiments) of the same bacterial solution without any repair applied on the selective medium TSA-SC. TSA,start The number of colonies obtained when the same bacterial solution without any repair was spread on TSA medium (the average value of 3 experiments).
[0046] According to the calculation results, the best existing TSB repair culture solution can only repair about 37.68% of sub-damaged Escherichia coli bacteria within 20 minutes, while the repair culture medium provided by the present invention can efficiently repair about 100% of sub-damaged Escherichia coli bacteria within 20 minutes, and during the repair time, there is no obvious amplification of live Escherichia coli bacteria, which meets the needs of accurate quantitative detection of Escherichia coli in food.
[0047] Preferably, the initial bacterial count of the sub-damaged lactic acid Escherichia coli solution in step 1 is OD600=0.1. OD600=0.1 is equivalent to 10 8 CFU / ml of bacteria.
[0048] Preferably, the 1 ml of the sub-damaged lactic acid Escherichia coli solution in step 1 contains 0.02 mmol of L-lactic acid solution, and the pH value of the L-lactic acid solution is 5.82.
[0049] Preferably, the preparation process of the L-lactic acid solution is: adding L-lactic acid into double distilled water, vortexing for 1 minute and filtering with a 0.22 micron bacterial filter for sterilization.
[0050] Note: The damage process of the lactic acid sub-damaged Escherichia coli liquid is as follows: add 0.5 ml of fresh Escherichia coli culture liquid to 5 ml of L-lactic acid solution, incubate in a constant temperature incubator at 37°C and 150 rpm to damage the bacteria for 60 minutes, immediately pipette 1 ml of the lactic acid sub-damaged Escherichia coli liquid into a 1.5 ml centrifuge tube, centrifuge at 8000 g for 5 minutes, and remove the supernatant to terminate the lactic acid sub-damage process.
[0051] Example 1
[0052] 1. Preparation of culture medium: Take 100 ml of 15% spirulina protease degradation product and adjust the pH value to 7 with sodium hydroxide, and sterilize with high pressure steam at 121°C for 15 minutes. After cooling, add 1 g of glucose, 0.6513 g of dipotassium hydrogen phosphate and 0.0355 of potassium dihydrogen phosphate in a sterile environment, and finally add 0.5 ml of Tween 80, shake and mix, and filter and sterilize with a 0.22 micron bacterial filter to obtain the final culture medium.
[0053] 2. Sub-injury and calculation of sub-injury rate: Take a 10ml glass test tube, add 5ml of 0.02mmol L-lactic acid solution, and then add 0.5ml of Escherichia coli solution with OD600 value of 0.1. Incubate the damaged bacteria in a constant temperature incubator at 37℃ and 150 rpm for 60 minutes. Immediately pipette 1ml of lactic acid sub-injured Escherichia coli solution into a 1.5ml centrifuge tube, centrifuge at 8000g for 5 minutes, and remove the supernatant to terminate the lactic acid sub-injury process to obtain sub-injured Escherichia coli. Add 1ml of sterile water and shake evenly, then dilute to 10 of the original -5 Spread on trypticase soy agar (TSA) plates and dilute to 10% of the original -4 Spread the selective medium TSA-SC plate. Incubate at 37°C for 16 hours in a constant temperature incubator. The sub-injury rate of lactic acid-damaged Escherichia coli can be calculated by the number of colonies (the results are shown in Figure 1 ). The results showed that the sub-damage rate of lactic acid-damaged Escherichia coli was 94.35%.
[0054] 3. Comparative evaluation of repair effect: The culture medium described in this article and the reference repair culture medium TSB were added to the centrifuge tube containing the sub-damaged E. coli precipitate, and repaired for 20 minutes at 150 rpm and 37°C in a constant temperature shaking incubator. Immediately centrifuge at 8000g for 5 minutes to remove the supernatant to terminate the repair, add 1 ml of sterile water, shake evenly, and dilute to 10% of the original -5 Spread on the selective medium TSA-SC plate, incubate at 37℃ for 16 hours and count the number of colonies (the results are shown in Figure 2 ) and calculate the repair rate (the result is as follows Figure 3 The results showed that the repair rate at 37°C for 20 minutes was 102.53% (note that the repair rate was slightly higher than 100%, mainly because the plate coating had certain operational contingencies), while the repair rate of the TSB repair medium was only 37.68%. The repair rate of the repair medium described in this article was much higher than that of the TSB repair medium.
[0055] 4. No proliferation of live bacteria occurred during the repair period: Take 1 ml of undamaged E. coli and dilute it directly to 10 of the original -6 The mixture was spread on a trypticase soy agar (TSA) plate; 1 ml of undamaged Escherichia coli was taken, centrifuged at 8000 g for 5 minutes to remove the supernatant, and then the culture medium of the present invention was added, and the mixture was incubated at 37° C. at 150 rpm in a constant temperature shaking incubator for 20 minutes, and the supernatant was removed immediately after centrifugation at 8000 g for 5 minutes to terminate the incubation, and 1 ml of sterile water was added to shake evenly and then diluted to 10% of the original -6, and spread on trypticase soy agar (TSA) plates. After incubation at 37°C for 16 hours, the number of colonies was counted (the results are shown in Figure 4 )Observe and calculate and compare (the results are as follows Figure 5 ). The results showed that the number of colonies before and after incubation was basically the same, indicating that no significant increase in live E. coli was observed during the 20-minute repair time.
[0056] Results: The repair culture medium provided by the present invention is far superior to the best TSB repair culture medium, and can effectively repair about 100% of sub-damaged Escherichia coli bacteria within 20 minutes, and no obvious amplification of live Escherichia coli bacteria was observed during the repair time.
[0057] Comparative Example 1
[0058] Take a 10ml glass test tube, add 5ml of 0.02mmol L-lactic acid solution, and then add 0.5ml of Escherichia coli liquid with OD600 value of 0.1. Incubate the damaged bacteria in a constant temperature incubator at 37℃ and 150 rpm for 60min, and immediately pipette 1ml of lactic acid sub-damaged Escherichia coli liquid into a 1.5ml centrifuge tube, centrifuge at 8000g for 5 minutes, and remove the supernatant to terminate the lactic acid sub-damage process, and obtain sub-damaged Escherichia coli.
[0059] TSB repair culture solution and the repair culture medium provided by the present invention were added to centrifuge tubes of different sub-damaged Escherichia coli, respectively, and repaired for 20 minutes at 150 rpm and 37°C in a constant temperature shaking incubator. The repair was immediately terminated by centrifugation at 8000 g for 5 minutes, and the supernatant was removed. 1 ml of sterile water was added and diluted to 10 after being shaken evenly. -5 The selective medium TSA-SC plates were cultured in a constant temperature incubator at 37°C for 16 hours and then counted (the results are shown in Figure 2 ) and calculate the repair rate (the result is as follows Figure 3 ).
[0060] from Figure 3 It can be concluded that under the same conditions, the repair rate of the repair medium provided by the present invention is 102.53%, while the traditional optimal TSB repair medium is only 37.68%, indicating that the repair efficiency of the repair medium provided by the present invention is far superior to that of the TSB repair medium.
[0061] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art, within the scope of information available through public channels and in combination with the technical inspiration provided by the present application documents, may make changes, modifications, substitutions and variations to the above embodiments, which still fall within the scope of the present invention.
Claims
1. A repair medium for sub-damaged lactic acid Escherichia coli, characterized in that: It is composed of the following ingredients: spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, Tween 80 and double distilled water; Per liter of double distilled water, 100-180 g of spirulina protease degradation product, 5-15 g of glucose, 3.74-8.51 g of dipotassium hydrogen phosphate, 0.1-0.7 g of potassium dihydrogen phosphate, and 1-10 ml of Tween 80 are added.
2. The repair medium for sub-damaged lactic acid Escherichia coli according to claim 1, characterized in that: The double distilled water is added with: 110-170 g of spirulina protease degradation product, 7-13 g of glucose, 4.24-8.01 g of dipotassium hydrogen phosphate, 0.2-0.6 g of potassium dihydrogen phosphate, and 2.5-8.5 ml of Tween 80 per liter.
3. The repair medium for sub-damaged lactic acid Escherichia coli according to claim 2, characterized in that: The double distilled water contains: 120-160 g of spirulina protease degradation product, 9-11 g of glucose, 4.74-7.51 g of dipotassium hydrogen phosphate, 0.3-0.5 g of potassium dihydrogen phosphate, and 4-7 ml of Tween 80 per liter.
4. A method for preparing a repair medium for sub-damaged lactic acid Escherichia coli according to claim 1, characterized in that: The method comprises the following steps: firstly, adding 100-180g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.0-8.0, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 5-15g of glucose, 3.74-8.51g of dipotassium hydrogen phosphate and 0.1-0.7g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 1-10ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
5. The method for preparing the repair medium for sub-damaged lactic acid Escherichia coli according to claim 4, characterized in that: The method comprises the following steps: adding 110-170g of spirulina protease degradation products per liter of double distilled water, adjusting the pH value to 6.3-7.7, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 7-13g of glucose, 4.24-8.01g of dipotassium hydrogen phosphate and 0.2-0.6g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 2.5-8.5ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
6. The method for preparing the repair medium for sub-damaged lactic acid Escherichia coli according to claim 5, characterized in that: The method comprises the following steps: adding 120-160g of spirulina protease degradation products per liter of double distilled water, adjusting the pH value to 6.6-7.4, sterilizing with high-pressure steam at least 121°C for at least 15 minutes, adding 9-11g of glucose, 4.74-7.51g of dipotassium hydrogen phosphate and 0.3-0.5g of potassium dihydrogen phosphate in a sterile environment after cooling, and finally adding 4-7ml of Tween 80, shaking and mixing, and filtering and sterilizing with a 0.22-micron bacterial filter.
7. A method for repairing sub-damaged lactic acid Escherichia coli, characterized in that: The steps include: Step 1, transferring the sub-damaged lactic acid Escherichia coli solution to a centrifuge tube, centrifuging at 8000 g for 5 minutes, removing the supernatant and adding it to the sub-damaged lactic acid Escherichia coli repair medium according to any one of claims 1 to 3; Step 2: Repair in a constant temperature shaking incubator at 150 rpm and 37°C for 20 minutes, immediately centrifuge at 8000g for 5 minutes to remove the supernatant to terminate the repair and obtain the E. coli repair solution.
8. The method for repairing sub-damaged lactic acid Escherichia coli according to claim 7, characterized in that: The initial bacterial count of the sub-damaged lactic acid Escherichia coli solution in step 1 is OD600=0.
1.
9. The method for repairing sub-damaged lactic acid Escherichia coli according to claim 7, characterized in that: The lactic acid sub-damaged Escherichia coli solution described in step 1 contains sub-damaged Escherichia coli caused by L-lactic acid solution.