CN115838660B - Lactobacillus helveticus repair culture medium for lactic acid sub-injury and preparation and repair method thereof - Google Patents
Lactobacillus helveticus repair culture medium for lactic acid sub-injury and preparation and repair method thereof Download PDFInfo
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Abstract
本发明提供了乳酸亚损伤大肠杆菌修复培养基及其配制和修复方法，该培养基包括：螺旋藻蛋白酶降解产物、磷酸氢二钾、磷酸二氢钾、葡萄糖、吐温80和溶剂。该修复方法包括：将1毫升乳酸亚损伤大肠杆菌液立即转移到1.5毫升离心管中，8000g离心5分钟，去上清液后加入到所述乳酸亚损伤大肠杆菌修复培养基中；在恒温振荡培养箱中以150转/分，37℃修复20分钟，立即8000g离心5分钟去掉上清液终止修复，获得大肠杆菌修复液。本发明提供的修复培养基在20分钟内能高效修复约100％的大肠杆菌亚损伤菌，且在修复时间内，大肠杆菌活菌未见明显扩增，满足精准定量食品中的大肠杆菌检测的需求。
The present invention provides a lactic acid sub-damaged Escherichia coli repair medium and its preparation and repair method, the medium comprising: spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, Tween 80 and solvent. The repair method comprises: immediately transferring 1 ml of lactic acid sub-damaged Escherichia coli liquid to a 1.5 ml centrifuge tube, centrifuging at 8000g for 5 minutes, removing the supernatant and adding it to the lactic acid sub-damaged Escherichia coli repair medium; repairing at 150 rpm and 37°C in a constant temperature shaking incubator for 20 minutes, immediately centrifuging at 8000g for 5 minutes to remove the supernatant to terminate the repair, and obtain the Escherichia coli repair liquid. The repair medium provided by the present invention can efficiently repair about 100% of the sub-damaged Escherichia coli bacteria within 20 minutes, and during the repair time, the live Escherichia coli bacteria are not significantly amplified, meeting the needs of accurate quantitative detection of Escherichia coli in food.
Description
Technical Field
The invention relates to the technical field of microbial culture media and the technical field of food microbial inspection, in particular to a lactic acid sub-injury escherichia coli repair culture medium and a preparation and repair method thereof.
Background
Food-borne pathogenic microorganisms cause food-borne diseases including food poisoning, which are harmful to human or animal health. Food spoilage microorganisms cause spoilage, rancidity, fermentation and the like of foods, on the one hand, cause sensory changes in color, aroma, taste, shape and the like of foods, and on the other hand, destroy food ingredients, reduce or lose eating value, and cause great economic loss. In addition, spoilage of foods is liable to grow bacteria, and even pathogenic bacteria and toxigenic mold, resulting in infectious diseases or poisoning of things. Therefore, there is a need for efficient preservation and treatment of food products to control microorganisms.
Food preservation and treatment methods, such as heating, drying, freezing, irradiation, acid treatment, and alkali treatment, result in the formation of bacteria in the food in three types, 1) dead bacteria, 2) sub-damaging bacteria, and 3) living bacteria. Wherein, under the condition of better conditions, the sub-injury bacteria can restore vitality to endanger health or spoil food. Thus, sub-damaging bacteria and living bacteria are also important and need to be effectively tested and monitored.
In food microbiological analysis, sub-damaging bacteria escape detection due to inability to grow on selective media, resulting in a lower number of detected food microorganisms, mistakenly considering unsafe food as safe, and easily causing food safety events to occur. Therefore, on one hand, effective repair of sub-damaged bacteria is necessary, and on the other hand, the repair culture medium is required to have extremely high efficiency, so that the living bacteria are not propagated yet in the repair time, otherwise, the quantity of the detected food microorganisms is easy to be higher. For this reason, it is extremely important to develop a high-efficiency repair medium.
Coli is considered to be the microorganism that indicates the best contamination of food faeces and will strongly characterize the contamination of food microorganisms. In addition, some strains of E.coli can cause intestinal infections such as enterotoxigenic E.coli, pathogenic E.coli, hemorrhagic E.coli, invasive E.coli, adhesive E.coli, and the like. Therefore, the detection of E.coli in food is of great importance.
Lactic acid occurs naturally in certain foods or is added to foods in the form of a bacteriostatic agent. Thus, milk causes coliform sub-injury. Currently, tryptone soy broth (TRYPTICASE SOY BROTH, TSB) is considered to be the best medium for repair of lactic acid sub-injured escherichia coli, e.g. document ：(Shi,H.,Chen,Z.,Chen,D.,and Kan,J.(2017).Sublethal injury and recovery of Escherichia coli O157:H7 and K-12after exposure to lactic acid.Food Control 82,190-195.). but this medium is not efficient in repair, requiring 120 minutes to complete about 100% of the repair. Coli requires about 20 minutes for its cell division into one generation in nutrient rich media such as TSB. Therefore, when TSB is adopted for 100% of repair time, a large amount of live bacteria are amplified, so that the detection result is high, and the accurate quantification of escherichia coli in food is not facilitated.
Disclosure of Invention
The invention aims to solve the technical problems that the existing culture medium for repairing the sub-injured escherichia coli is low in repairing efficiency, on one hand, if the sub-injured bacteria are not repaired or are not completely repaired under the condition of short repairing time, the detection result is low, and on the other hand, if the repairing time is prolonged, about 100% of the sub-injured bacteria are completely repaired, a large amount of live bacteria are amplified in the repairing time, and the detection result is high. Therefore, the method is not beneficial to the accurate quantification of the escherichia coli in food detection.
The technical scheme for solving the technical problems is as follows:
The invention provides a lactic acid sub-injury escherichia coli repair culture medium which comprises a spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, tween 80 and a solvent.
Preferably, the solvent is double distilled water, and each liter of double distilled water comprises 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.
Further, the preparation method of the lactic acid sub-damage escherichia coli repair culture medium comprises the steps of adding 100-180 g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.0-8.0 by sodium hydroxide, sterilizing for at least 15 minutes by high-pressure steam with the temperature of at least 121 ℃, cooling, adding 5-15 g of glucose, 3.74-8.51 g of dipotassium hydrogen phosphate and 0.1-0.7 g of potassium dihydrogen phosphate into a sterile environment, finally adding 1-10 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
Preferably, each liter of double distilled water comprises 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.
Further, the preparation method of the lactic acid sub-injury escherichia coli repair culture medium comprises the steps of adding 110-170 g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.3-7.7 by sodium hydroxide, sterilizing for at least 15 minutes by high-pressure steam with the temperature of at least 121 ℃, cooling, adding 7-13 g of glucose, 4.24-8.01 g of dipotassium hydrogen phosphate and 0.2-0.6 g of potassium dihydrogen phosphate into a sterile environment, finally adding 2.5-8.5 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
Preferably, each liter of double distilled water comprises 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.
Further, the preparation method of the lactic acid sub-damage escherichia coli repair culture medium comprises the steps of adding 120-160 g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.6-7.4 by sodium hydroxide, sterilizing for at least 15 minutes by high-pressure steam with the temperature of at least 121 ℃, cooling, adding 9-11 g of glucose, 4.74-7.51 g of dipotassium hydrogen phosphate and 0.3-0.5 g of potassium dihydrogen phosphate into a sterile environment, finally adding 4-7 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
The invention also provides a method for repairing the lactic acid sub-injury escherichia coli, which is applied to the lactic acid sub-injury escherichia coli repairing culture medium and comprises the following steps:
transferring the lactic acid sub-injury escherichia coli liquid into a centrifuge tube, centrifuging 8000g for 5 minutes, removing supernatant, and adding the supernatant into the lactic acid sub-injury escherichia coli repairing culture medium;
And step two, repairing for 20 minutes at a temperature of 37 ℃ in a constant-temperature shaking incubator at 150 revolutions per minute, immediately centrifuging for 5 minutes at 8000g, and removing the supernatant to stop repairing to obtain the escherichia coli repairing liquid.
Preferably, the lactic acid sub-damage escherichia coli solution in the step one has an initial bacterial load of od600=0.1. Od600=0.1, corresponding to 10 8 CFU/ml of bacteria.
Preferably, the lactic acid sub-damage escherichia coli solution in the step one comprises escherichia coli which is sub-damaged by the L-type lactic acid solution.
The beneficial effects of the invention are as follows:
The existing best TSB repair culture solution can only repair about 37.68% of escherichia coli sub-damaged bacteria within 20 minutes, but the repair culture medium provided by the invention can efficiently repair about 100% of escherichia coli sub-damaged bacteria within 20 minutes, and the escherichia coli viable bacteria are not obviously amplified within repair time, so that the requirements of escherichia coli detection in accurate quantitative foods are met.
Drawings
FIG. 1 is a graph showing the sub-damage rate of lactic acid-damaged E.coli.
FIG. 2 is a graph comparing colony counts after TSB repair in accordance with the present invention with a reference repair medium.
FIG. 3 is a graph comparing the repair rates of TSB according to the present invention with that of a reference repair medium.
FIG. 4 is a graph comparing colonies on TSA of the repaired and unrepaired bacterial solutions of the present invention.
FIG. 5 is a graph showing colony statistics of the repaired and unrepaired bacterial solutions of the present invention on TSA.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
The invention provides a lactic acid sub-injury escherichia coli repair culture medium which comprises a spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, tween 80 and a solvent.
Preferably, the solvent is double distilled water, 100-180 g of spirulina protease degradation product is added into each liter of double distilled water, the pH value is regulated to 6.0-8.0 by sodium hydroxide, high-pressure steam with the temperature of at least 121 ℃ is used for sterilization for at least 15 minutes, 5-15 g of glucose, 3.74-8.51 g of dipotassium hydrogen phosphate and 0.1-0.7 g of potassium dihydrogen phosphate are added into the sterile environment after cooling, finally 1-10 ml of tween 80 is added, and after shaking and mixing, bacteria filter filtration sterilization is carried out by a bacterial filter with the particle size of 0.22 microns.
Preferably, 110-170 g of spirulina protease degradation product is placed in each liter of double distilled water, the pH value is regulated to 6.3-7.7 by sodium hydroxide, high-pressure steam at the temperature of at least 121 ℃ is used for sterilization for at least 15 minutes, 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 into a sterile environment after cooling, finally 2.5-8.5 ml of Tween 80 is added, and after shaking and mixing, bacteria are filtered and sterilized by a bacterial filter of 0.22 microns.
Preferably, 120-160 g of spirulina protease degradation product is placed in each liter of double distilled water, the pH value is regulated to 6.6-7.4 by sodium hydroxide, high-pressure steam sterilization is carried out for at least 15 minutes at a temperature of at least 121 ℃, 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 into a sterile environment after cooling, finally 4-7 ml of Tween 80 is added, and after shaking and mixing, bacteria filtration and sterilization are carried out by a bacterial filter of 0.22 microns.
The invention also provides a method for repairing the lactic acid sub-injury escherichia coli, which is applied to the lactic acid sub-injury escherichia coli repairing culture medium and comprises the following steps:
Transferring 1 ml of lactic acid sub-injury escherichia coli liquid into a 1.5 ml centrifuge tube, centrifuging 8000g for 5 minutes, removing supernatant, and adding the supernatant into the lactic acid sub-injury escherichia coli repairing culture medium;
And step two, repairing for 20 minutes at a temperature of 37 ℃ in a constant-temperature shaking incubator at 150 revolutions per minute, immediately centrifuging for 5 minutes at 8000g, and removing the supernatant to stop repairing to obtain the escherichia coli repairing liquid.
Note that the conversion between centrifugal force G and rotational speed RPM is given by the conversion formula g=1.11×10 -5×R×(RPM)2, where G is the centrifugal force, typically expressed as a multiple of G (gravitational acceleration) (e.g. 8000G herein). R is the radius in cm. For example, the centrifugal radius is 10 cm, the rotational speed is 8000rpm, and the centrifugal force is G=1.11× -5*10*(8000)2 =7104, namely, the centrifugal force is 7104G. And when the centrifugal force is 8000g, the rotating speed is 8489rpm, namely about 8500rpm. Different centrifuge radii are different, G is common, and this section is merely an example of calculation.
The result proves that according to the using method of the culture medium, the lactic acid sub-injury escherichia coli repair culture medium in the step one is replaced by TSB repair culture solution, and escherichia coli control repair solution is obtained in the step two and is used as a control group. And (3) taking the escherichia coli repair liquid and the escherichia coli control repair liquid obtained in the step two, respectively adding 1 milliliter of sterile water and uniformly shaking, diluting and coating the mixture on a tryptone soybean agar (TRYPTICASE SOY AGAR, TSA) plate and a selective culture medium TSA-SC plate, culturing the mixture in a constant temperature incubator at 37 ℃ for 16 hours, counting a plurality of colonies, and calculating the repair rate.
Note that the formulation of the TSB repair broth was (per liter) tryptone 17 g, soytone 3 g, D-glucose 2.5g, sodium chloride 2.5g, dipotassium hydrogen phosphate 2.5 g.
Note that the TSA plates were formulated (per liter) of tryptone 15 g, soy papain hydrolysate 5g, sodium chloride 5g and agar 15 g, and the medium supported the growth of E.coli sub-damaging and live bacteria.
Note that the formulation of the TSA-SC plate for the selective medium was (per liter) tryptone 15g, soy papain hydrolysate 5g, sodium chloride 20 g and agar 15g, which medium only supported the growth of E.coli viable bacteria and not the growth of sub-damaging bacteria.
The dilution coating is performed by adopting 10 times of dilution with sterile water to ensure that the number of colonies growing on a flat plate is between 20 and 300, and the flat plate coating is performed by adopting a glass coater by adopting 0.02 milliliter of diluted bacterial liquid during the coating.
Note that the above results were verified by repeating the repair experiment 3 times, and the repair rate was calculated by taking the average value.
Note that the repair rate is calculated as follows:
Wherein,% recovery is repair rate, CFU TSA-SC,time is the number of colonies coated on the selective medium TSA-SC after repair (average value of 3 tests), CFU TSA-SC,start is the number of colonies coated on the selective medium TSA-SC with the same bacterial liquid without any repair (average value of 3 tests), and CFU TSA,start is the number of colonies coated on the TSA medium with the same bacterial liquid without any repair (average value of 3 tests).
According to the calculation result, the existing best TSB repair culture solution can only repair about 37.68% of escherichia coli sub-injury bacteria in 20 minutes, but the repair culture medium provided by the invention can efficiently repair about 100% of escherichia coli sub-injury bacteria in 20 minutes, and the escherichia coli viable bacteria are not obviously amplified in the repair time, so that the requirements of escherichia coli detection in accurate quantitative foods are met.
Preferably, the lactic acid sub-damage escherichia coli solution in the step one has an initial bacterial load of od600=0.1. Od600=0.1, corresponding to 10 8 CFU/ml of bacteria.
Preferably, the 1 ml of the lactic acid sub-damage escherichia coli solution in the step one comprises 0.02 mmol of L-type lactic acid solution, and the pH value of the L-type lactic acid solution is 5.82.
Preferably, the preparation process of the L-lactic acid solution comprises the steps of adding L-lactic acid into double distilled water, and filtering and sterilizing the double distilled water by using a 0.22-micrometer bacterial filter after vortex oscillation for 1 minute.
The method is characterized in that 0.5 ml of fresh escherichia coli culture solution is added into 5 ml of L-shaped lactic acid solution, the bacteria are damaged by shaking at 37 ℃ and 150 revolutions per minute in a constant temperature incubator for 60 minutes, 1ml of the lactic acid sub-damage escherichia coli solution is immediately sucked into a 1.5 ml centrifuge tube, 8000g of the lactic acid sub-damage escherichia coli solution is centrifuged for 5 minutes, and the supernatant is removed to stop the lactic acid sub-damage process.
Example 1
1. The culture medium is prepared by taking 100ml 15% spirulina protease degradation product, adjusting pH to 7 with sodium hydroxide, and sterilizing with high pressure steam at 121deg.C for 15 min. After cooling, 1g of glucose, 0.6513g of dipotassium hydrogen phosphate and 0.0355 of potassium dihydrogen phosphate are added in a sterile environment, finally 0.5 ml of Tween 80 is added, and after shaking and mixing, a bacterial filter of 0.22 microns is used for filtration and sterilization, so that the final culture medium is obtained.
2. The subthreshold and the subthreshold rate are calculated by taking a glass test tube with 10ml of specification, adding 5 ml of 0.02 millimole L-shaped lactic acid solution, and adding 0.5 ml of escherichia coli bacterial liquid with an OD600 value of 0.1. The bacteria were injured by shaking at 37℃in a constant temperature incubator at 150 rpm for 60 minutes, and immediately pipetting the 1 ml of lactic acid sub-injured E.coli liquid into a 1.5 ml centrifuge tube, centrifuging at 8000g for 5 minutes to terminate the lactic acid sub-injured process and obtain sub-injured E.coli. 1 ml of sterile water was added and diluted to 10 -5 plating tryptone soy agar (TRYPTICASE SOY AGAR, TSA) plates and 10 -4 plating selective media TSA-SC plates after shaking. The sub-damage rate of E.coli damaged by lactic acid was calculated from the colony count by culturing at 37℃for 16 hours in a constant temperature incubator (results are shown in FIG. 1). The results showed that the sub-damage rate of lactic acid-damaged E.coli was 94.35%.
3. Comparative evaluation of repair effect the culture medium described herein and the reference repair culture medium TSB were added to a centrifuge tube containing a precipitate of E.coli, and the repair was stopped by centrifugation at 8000g for 5 minutes immediately at 37℃in a constant temperature shaking incubator at 150 rpm, followed by 1 ml of sterile water and shaking to a uniform dilution of 10 -5, and the culture medium was spread on a selective culture medium TSA-SC plate, and after 16 hours of incubation at 37℃in the constant temperature incubator, the number of colonies was counted (results are shown in FIG. 2) and the repair rate was calculated (results are shown in FIG. 3). The results showed that the 20 minute repair rate at 37 ℃ was 102.53% (note that the repair rate was slightly higher than 100% and the main reason was that plate coating had some handling contingency), the TSB repair medium was only 37.68%, and the repair rate of the repair medium described herein was much higher than that of the TSB repair medium.
4. During the repair period, the live bacteria are not amplified, namely 1ml of undamaged escherichia coli is directly diluted to the original 10 -6 ml and coated on a tryptone soybean agar (TRYPTICASE SOY AGAR, TSA) plate, 1ml of undamaged escherichia coli is further taken, 8000g of the undamaged escherichia coli is centrifuged for 5 minutes to remove supernatant, the culture medium is added, 150 revolutions per minute is carried out in a constant-temperature shaking incubator for 20 minutes at 37 ℃, immediately 8000g of the culture medium is centrifuged for 5 minutes to remove supernatant to terminate incubation, 1ml of sterile water is added, and the solution is diluted to the original 10 -6 after shaking uniformly and coated on the tryptone soybean agar (TRYPTICASE SOY AGAR, TSA) plate. The number of colonies after 16 hours of incubation at 37℃in a constant temperature incubator (results shown in FIG. 4) were observed and compared (results shown in FIG. 5). The results showed that the number of colonies before and after incubation was substantially the same, indicating that no significant amplification of E.coli viable bacteria was seen during the 20 minute repair time.
As a result, the repair culture medium provided by the invention is far better than the best TSB repair culture medium, and can efficiently repair about 100% of E.coli subinjury bacteria in 20 minutes, and the E.coli live bacteria are not obviously amplified in the repair time.
Comparative example 1
A10 ml glass test tube was taken, 5ml of 0.02 mmol L-lactic acid solution was added, and 0.5 ml of E.coli bacteria liquid having an OD600 value of 0.1 was further added. And (3) vibrating the injured bacteria at 37 ℃ for 60min in a constant temperature incubator at 150 rpm, immediately sucking 1 ml of lactic acid sub-injury escherichia coli liquid into a 1.5 ml centrifuge tube by using a pipetting gun, centrifuging at 8000g for 5min, and stopping the lactic acid sub-injury process to obtain sub-injury escherichia coli.
The TSB repair culture solution and the repair culture medium provided by the invention are respectively added into centrifuge tubes of different sub-injured escherichia coli, 150 revolutions per minute are repaired for 20 minutes in a constant temperature shaking incubator at 37 ℃, immediately 8000g are centrifuged for 5 minutes, the supernatant is removed to stop repair, 1 ml of sterile water temperature is added, shaking is uniform, and then the culture medium is diluted to 10 -5 selective culture medium TSA-SC plates, and after 16 hours of culture in the constant temperature incubator at 37 ℃, the count is carried out (the result is shown in FIG. 2), and the repair rate is calculated (the result is shown in FIG. 3).
The third graph shows that the repair rate of the repair medium provided by the invention is 102.53% under the same condition, and the traditional optimal TSB repair medium is 37.68%, so that the repair efficiency of the repair medium provided by the invention is far better than that of the TSB repair medium.
While embodiments of the present application have been shown and described above, it should be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives, and variations of the above embodiments, within the scope of the information available to those of ordinary skill in the art in light of the disclosure herein, may be made without departing from the scope of the application.
Claims (9)
1. A lactic acid sub-injury escherichia coli repair culture medium is characterized by comprising the following components of a spirulina protease degradation product, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, glucose, tween 80 and double distilled water;
The double distilled water is added with 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.
2. The lactic acid sub-injury escherichia coli repair medium according to claim 1, wherein 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 are added per liter of double distilled water.
3. The lactic acid sub-injury escherichia coli repair medium according to claim 2, wherein 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 are added per liter of double distilled water.
4. A preparation method of a lactic acid sub-injury escherichia coli repair culture medium based on claim 1 is characterized by comprising the steps of firstly adding 100-180 g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.0-8.0, sterilizing by high-pressure steam with the temperature of at least 121 ℃ for at least 15 minutes, cooling, adding 5-15 g of glucose, 3.74-8.51 g of dipotassium hydrogen phosphate and 0.1-0.7 g of potassium dihydrogen phosphate into a sterile environment, finally adding 1-10 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
5. The method for preparing the lactic acid sub-injury escherichia coli repair culture medium according to claim 4, which is characterized by comprising the steps of adding 110-170 g of spirulina protease degradation product per liter of double distilled water, adjusting the pH value to 6.3-7.7, sterilizing by high-pressure steam with the temperature of at least 121 ℃ for at least 15 minutes, cooling, adding 7-13 g of glucose, 4.24-8.01 g of dipotassium hydrogen phosphate and 0.2-0.6 g of potassium dihydrogen phosphate into a sterile environment, finally adding 2.5-8.5 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
6. The method for preparing the lactic acid sub-injury escherichia coli repair culture medium according to claim 5, which is characterized by comprising the steps of adding 120-160 g of spirulina protease degradation product into each liter of double distilled water, adjusting the pH value to 6.6-7.4, sterilizing by high-pressure steam with the temperature of at least 121 ℃ for at least 15 minutes, cooling, adding 9-11 g of glucose, 4.74-7.51 g of dipotassium hydrogen phosphate and 0.3-0.5 g of potassium dihydrogen phosphate into a sterile environment, adding 4-7 ml of tween 80, shaking and uniformly mixing, and filtering and sterilizing by a bacterial filter with the thickness of 0.22 microns.
7. A repair method of lactic acid sub-injury escherichia coli is characterized by comprising the following steps:
Transferring the lactic acid sub-injury escherichia coli liquid into a centrifuge tube, centrifuging 8000g for 5 minutes, removing supernatant, and adding the supernatant into the lactic acid sub-injury escherichia coli repair medium according to any one of claims 1-3;
And step two, repairing for 20 minutes at a temperature of 37 ℃ in a constant-temperature shaking incubator at 150 revolutions per minute, immediately centrifuging for 5 minutes at 8000g, and removing the supernatant to stop repairing to obtain the escherichia coli repairing liquid.
8. The method for repairing a lactic acid sub-injury escherichia coli according to claim 7, wherein the initial bacterial load of the lactic acid sub-injury escherichia coli solution obtained in the step one is OD600 = 0.1.
9. The method of repairing a sub-injured E.coli cell by lactic acid according to claim 7, wherein the sub-injured E.coli cell by lactic acid in step one comprises E.coli cells which are sub-injured by L-type lactic acid solution.
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| US7326558B2 (en) * | 2005-03-16 | 2008-02-05 | Dainippon Ink And Chemicals, Inc. | Process for treating spirulina |
| CN112715964B (en) * | 2020-12-16 | 2022-04-19 | 中国科学院南海海洋研究所 | A kind of spirulina oligosaccharide and its application in preparing preparations for regulating intestinal health function |
| KR102392018B1 (en) * | 2021-05-24 | 2022-04-27 | 한국해양과학기술원 | Medium coposition for culturing cells comprising spirulina hydrolysate and preparing method of the same |
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Non-Patent Citations (1)
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| Developing a de novo designed broth to rapidly recover lactic acid-injured Escherichia coli to ensure almost no multiplication during repair for precise enumeration;Miao Zeng等;Food Control;20230617;第153卷;全文 * |
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