Method for improving heat resistance of lactic acid bacteria through directed evolution and obtained high-temperature stable strain

Abstract

The invention provides a method for improving heat resistance of lactic acid bacteria through directed evolution, which comprises the following steps: S1, selecting an original strain with application potential, directly using a target application matrix as a domestication culture medium in combination with a used industrial scene, and adding glucose, maltose and enzymolysis casein into the domestication culture medium. According to the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain, glucose, maltose, enzymolysis casein, compound amino acid, pyruvic acid, betaine, vitamin E and tween-80 are added into a domestication culture medium, so that the strain can easily utilize nutrient and thermal protection substances in the heated domestication culture solution; according to the present invention, the growth under the heat stress is accelerated, the passage efficiency is increased, and the temperature rising time is shortened by using the low-dose NTG to perform mutagenesis on the strain so as to make the strain adapt to the high temperature in advance, such that the culture efficiency is increased;

Description

Technical Field

[0001] This invention relates to the field of lactic acid bacteria production, and more particularly to a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains. Background Technology

[0002] Lactic acid bacteria are a collective term for a class of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. Based on their biochemical mechanisms, they can be divided into two types: positive lactic acid fermentation and negative lactic acid fermentation.

[0003] Lactic acid bacteria reproduce by binary fission. Their growth and reproduction depend on a variety of nutrients such as carbon sources, nitrogen sources, inorganic salts and nutrient factors. At the same time, they must continuously interact with the outside world for matter and energy during the growth process and are affected by external environmental factors such as temperature and pH.

[0004] Directed evolution relies on continuous passage and mutation accumulation of strains at different temperatures. However, the growth period of lactic acid bacteria is usually 1-4 hours per generation, and it takes 100-500 generations to achieve significant improvement in heat resistance, resulting in a long overall cycle and reduced work efficiency.

[0005] Therefore, it is necessary to provide a method for improving the heat resistance of lactic acid bacteria through directed evolution and to obtain high-temperature stable strains to solve the above-mentioned technical problems. Summary of the Invention

[0006] This invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains, which solves the problem that current directed evolution relies on continuous passage and mutation of strains at different temperatures, resulting in a long overall cycle.

[0007] To solve the above-mentioned technical problems, the present invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution, characterized by comprising the following steps: S1: Select a starting strain with application potential, and use the target application matrix directly as the acclimatization medium in combination with the industrial scenario. Add glucose, maltose, enzymatic casein hydrolysis, complex amino acids, pyruvate, betaine, vitamin E, and Tween-80 to the acclimatization medium. S2: After heating the strain with a heating device to determine the heat resistance critical value of the starting strain, the acclimatization culture is heated to the desired temperature. S3: Mutagenesis of the strain was induced by using a low dose of NTG; S4: Add the mutagenized strain to the heated acclimatization culture medium and heat it in a stepwise manner; S5: Under the target high temperature, the strains are initially screened and then screened again to select single colonies and select strains with normal colony morphology, fast growth rate, good heat resistance, functional preservation and genetic stability. S6: The strains are stored in containers after being screened.

[0008] Preferably, in the industrial scenario where step 1 is used, such as fermented dairy products, the target application matrix, such as milk or soy milk, is directly used as the acclimatization culture medium.

[0009] Preferably, the initial screening in step 5 is to screen single colonies and select strains with normal colony morphology and fast growth rate.

[0010] Preferably, the secondary screening in step 5 involves verifying the primary screening strains using multiple indicators, such as heat resistance, functional retention, and genetic stability.

[0011] Preferably, the heating device has a support frame that is slidably connected inside, and the support frame has two moving devices inside its fixing device.

[0012] Preferably, the support frame is provided with a clamping device inside. The clamping device includes multiple limiting plates, a threaded rod, and a rotating handle. The multiple limiting plates are slidably connected to the inside of the support frame, the threaded rod is threadedly connected to the inside of the multiple limiting plates, and the rotating handle is fixedly connected to the surface of the threaded rod.

[0013] Preferably, the support frame is provided with a sliding device inside, the sliding device including two sliding grooves and multiple sliding blocks, the multiple sliding grooves are all opened inside the support frame, and the multiple sliding blocks are respectively slidably connected to the inside of the two sliding grooves.

[0014] Preferably, the limiting plates are provided with multiple protective devices inside, and the limiting plates are provided with multiple sets of limiting devices inside.

[0015] Preferably, the limiting device includes two limiting grooves and two limiting rings, with the two limiting grooves being formed inside the limiting plate and the two limiting rings being slidably connected to the inside of the two limiting grooves respectively.

[0016] A high-temperature stable strain obtained by a method for improving the heat resistance of lactic acid bacteria through directed evolution, wherein the strain is Lactobacillus casei.

[0017] Compared with related technologies, the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains provided by this invention have the following beneficial effects: This invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains. By adding glucose, maltose, enzymatically hydrolyzed casein, complex amino acids, pyruvic acid, betaine, vitamin E, and Tween-80 to the acclimatization culture medium, the strains can easily utilize nutrients and heat-protective substances in the heated acclimatization culture medium, accelerating growth under heat stress and increasing passage efficiency. At the same time, by using a low dose of NTG to induce mutagenesis in the strains, the heating time is shortened, thereby enabling the strains to adapt to high temperatures in advance and increasing culture efficiency. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of a first embodiment of a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain, provided by the present invention. Figure 2 This is a schematic diagram of the structure of a second embodiment of a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain, provided by the present invention. Figure 3 for Figure 2 The enlarged schematic diagram of part A shown below; Figure 4 This is a schematic diagram of the structure of a third embodiment of a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain provided by the present invention. Figure 5 for Figure 4 The enlarged schematic diagram of part B is shown.

[0019] The following are the labels in the diagram: 1. Heating device, 2. Support frame, 3. Moving device, 4. Clamping device, 41. Limiting plate, 42. Threaded rod, 43. Rotating handle, 5. Sliding device, 51. Sliding groove, 52. Sliding block, 6. Protective device, 7. Limiting device, 71. Limiting groove, 72. Limiting ring. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] First Embodiment Please refer to the following: Figure 1 ,in, Figure 1 This is a schematic diagram of the structure of a method for improving the heat resistance of lactic acid bacteria through directed evolution and the first embodiment of the obtained high-temperature stable strain provided by the present invention. A method for improving the heat resistance of lactic acid bacteria through directed evolution is characterized by comprising the following steps: S1: Select a starting strain with application potential, and use the target application matrix directly as the acclimatization medium in combination with the industrial scenario. Add glucose, maltose, enzymatic casein hydrolysis, complex amino acids, pyruvate, betaine, vitamin E, and Tween-80 to the acclimatization medium. S2: After heating the strain using heating device 1 to determine the critical heat resistance value of the starting strain, the acclimatization culture is heated to the desired temperature. S3: Mutagenesis of the strain was induced by using a low dose of NTG; S4: Add the mutagenized strain to the heated acclimatization culture medium and heat it in a stepwise manner; S5: Under the target high temperature, the strains are initially screened and then screened again to select single colonies and select strains with normal colony morphology, fast growth rate, good heat resistance, functional preservation and genetic stability. S6: The strains are stored in containers after being screened.

[0022] A mixture of glucose and maltose is used as a carbon source. Maltose can be slowly decomposed at high temperatures, avoiding growth stagnation caused by rapid depletion of glucose.

[0023] Pyruvate directly replenishes intermediate products of the tricarboxylic acid cycle, reducing energy metabolism losses at high temperatures.

[0024] Betaine is used to stabilize protein structure and cell membrane integrity, and to reduce cell rupture caused by high temperatures.

[0025] By using low-dose NTG to mutate the strain, the mutated bacterial solution was directly inoculated into a culture medium "slightly higher than the initial heat tolerance threshold (T0+1℃)". The strain was acclimatized at a rate of "0.5℃ increase every 5 generations". It only takes 50-100 generations to achieve the heat tolerance effect of the traditional 200 generations, allowing the strain to adapt to high temperatures in advance and increasing culture efficiency.

[0026] Step 1 is used in industrial scenarios such as fermented dairy products, where the target application matrix, such as milk or soy milk, is directly used as the acclimatization culture medium.

[0027] The initial screening in step 5 involves screening single colonies and selecting strains with normal colony morphology and fast growth rate.

[0028] Initial screening: Under the target high temperature, single colonies are screened using the "streaking method" to select strains with normal colony morphology and fast growth rate.

[0029] The secondary screening in step 5 is to verify the primary screening strains using multiple indicators, such as heat resistance, functional retention, and genetic stability.

[0030] Heat resistance: The survival rate after heat shock treatment at 50-60℃ (e.g., 10, 20, 30 minutes) was determined.

[0031] Function retention: Determination of acid production and viable bacteria count at high temperatures.

[0032] Genetic stability: The selected strains were passaged for 10-20 generations, and the heat resistance was measured in each generation to ensure that there was no significant decrease.

[0033] The working principle of the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain provided by this invention is as follows: When using it, S1: Select a starting strain with application potential, and use the target application matrix directly as the acclimatization medium in combination with the industrial scenario. Then, add glucose, maltose, enzymatically hydrolyzed casein, complex amino acids, pyruvate, betaine, vitamin E, and Tween-80 to the acclimatization medium. S2: After heating the strain using heating device 1 to determine the critical heat resistance value of the starting strain, the acclimatization culture is heated to the desired temperature. S3: Mutagenesis of the strain was induced by using a low dose of NTG; S4: Add the mutagenized strain to the heated acclimatization culture medium and heat it in a stepwise manner; S5: Under the target high temperature, the strains are initially screened and then screened again to select single colonies and select strains with normal colony morphology, fast growth rate, good heat resistance and functional retention. S6: The strains are stored in containers after being screened.

[0034] Compared with related technologies, the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains provided by this invention have the following beneficial effects: This invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains. By adding glucose, maltose, enzymatically hydrolyzed casein, complex amino acids, pyruvic acid, betaine, vitamin E, and Tween-80 to the acclimatization culture medium, the strains can easily utilize nutrients and heat-protective substances in the heated acclimatization culture medium, accelerating growth under heat stress and increasing passage efficiency. At the same time, by using a low dose of NTG to induce mutagenesis in the strains, the heating time is shortened, thereby enabling the strains to adapt to high temperatures in advance and increasing culture efficiency.

[0035] Second Embodiment Please refer to the following: Figure 2 and Figure 3 Based on the first embodiment of this application, which provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the resulting high-temperature stable strain, the second embodiment of this application proposes another method for improving the heat resistance of lactic acid bacteria through directed evolution and the resulting high-temperature stable strain. The second embodiment is merely a preferred embodiment of the first embodiment, and the implementation of the second embodiment will not affect the separate implementation of the first embodiment.

[0036] Specifically, the second embodiment of this application provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain, which differs in that it also includes a support frame 2, which is disposed inside the heating device 1, and the internal fixing device of the support frame 2 has two moving devices 3.

[0037] Heating device 1 is a constant temperature water bath in the prior art, used to heat the strains in the storage container.

[0038] The moving device 3 is a moving handle, which is used to facilitate the movement of the support frame 2.

[0039] The support frame 2 is used to store the storage container, thereby heating the storage container by placing it inside the heating device 1.

[0040] The support frame 2 is provided with a clamping device 4 inside. The clamping device 4 includes multiple limiting plates 41, threaded rods 42 and rotating handles 43. The multiple limiting plates 41 are slidably connected to the inside of the support frame 2. The threaded rods 42 are threadedly connected to the inside of the multiple limiting plates 41. The rotating handles 43 are fixedly connected to the surface of the threaded rods 42.

[0041] The handle 43 is used to facilitate the rotation of the threaded rod 42.

[0042] The surface of the threaded rod 42 is provided with two sets of opposing threads, and the interior of the multiple limiting plates 41 is provided with threaded holes that are compatible with the threaded rod 42, so that when the threaded rod 42 rotates to one side, the multiple limiting plates 41 move on the surface of the threaded rod 42.

[0043] The support frame 2 is provided with a sliding device 5 inside. The sliding device 5 includes two sliding grooves 51 and multiple sliding blocks 52. The multiple sliding grooves 51 are all opened inside the support frame 2, and the multiple sliding blocks 52 are respectively slidably connected to the inside of the two sliding grooves 51.

[0044] One side of each of the multiple sliding blocks 52 is fixedly connected to both ends of the multiple limiting plates 41, so as to drive the multiple sliding blocks 52 to move to one side inside the two sliding grooves 51 when the multiple limiting plates 41 move.

[0045] By using multiple sliding blocks 52 to limit the two limiting plates 41, the stability of the limiting plates 41 is increased when they move.

[0046] The working principle of the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain provided by this invention is as follows: When in use, the storage container is placed on the support frame 2.

[0047] By rotating the handle 43 to one side, the threaded rod 42 is driven to rotate to one side, causing multiple limiting plates 41 to move towards the center simultaneously. When the multiple limiting plates 41 move, they drive multiple sliding blocks 52 to move inside the multiple sliding grooves 51 respectively. After the multiple limiting plates 41 move to one side to both sides of the storage container, they clamp and limit the storage container.

[0048] Compared with related technologies, the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains provided by this invention have the following beneficial effects: This invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain. The clamping device 4 and the sliding device 5 are used to clamp test tubes or other storage containers placed in the heating device, thereby increasing their stability during heating and facilitating the storage of test tubes.

[0049] Third Embodiment Please refer to the following: Figure 4 and Figure 5 Based on the first embodiment of this application, which provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the resulting high-temperature stable strain, the second embodiment of this application proposes another method for improving the heat resistance of lactic acid bacteria through directed evolution and the resulting high-temperature stable strain. The third embodiment is merely a preferred embodiment of the first embodiment, and the implementation of the third embodiment will not affect the separate implementation of the first embodiment.

[0050] Specifically, the difference between the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain provided in the third embodiment of this application is that it also includes multiple protective devices 6, which are respectively disposed inside the multiple limiting plates 41, and the limiting plates 41 are provided with multiple sets of limiting devices 7.

[0051] The protective device 6 is a protective sleeve used to protect the test tube from the limiting plate 41. The protective device 6 can be replaced so that the limiting plate 41 can better limit the test tubes of different sizes.

[0052] The limiting device 7 includes two limiting grooves 71 and two limiting rings 72. The two limiting grooves 71 are both opened inside the limiting plate 41, and the two limiting rings 72 are slidably connected to the inside of the two limiting grooves 71 respectively.

[0053] Both limiting grooves 71 are annular grooves that are adapted to the limiting ring 72, and are used to limit the protective device 6 after the limiting ring 72 is inserted into the limiting groove 71.

[0054] The working principle of the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain provided by this invention is as follows: In use, by pulling the protective device 6 to one side, the two limiting rings 72 move to one side and separate inside the two limiting grooves 71 respectively.

[0055] After inserting the two limiting rings 72 of the new protective device 6 into the two limiting grooves 71 respectively, the protective device 6 is installed.

[0056] Compared with related technologies, the method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strains provided by this invention have the following beneficial effects: This invention provides a method for improving the heat resistance of lactic acid bacteria through directed evolution and the obtained high-temperature stable strain. The protective device 6 protects the storage container and the limiting plate 41, and the limiting device 7 is used to replace the protective device 6, so that the limiting plate 41 can better limit the test tubes of different sizes.

[0057] A high-temperature stable strain obtained by a method for improving the heat resistance of lactic acid bacteria through directed evolution, wherein the strain is Lactobacillus casei.

[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for improving the heat resistance of lactic acid bacteria through directed evolution, characterized in that, Including: the following step: S1: Select a starting strain with application potential, and use the target application matrix directly as the acclimatization medium in combination with the industrial scenario. Add glucose, maltose, enzymatic casein hydrolysis, complex amino acids, pyruvate, betaine, vitamin E, and Tween-80 to the acclimatization medium. S2: After heating the strain with a heating device to determine the heat resistance critical value of the starting strain, the acclimatization culture is heated to the desired temperature. S3: Mutagenesis of the strain was induced by using a low dose of NTG; S4: Add the mutagenized strain to the heated acclimatization culture medium and heat it in a stepwise manner; S5: Under the target high temperature, the strains are initially screened and then screened again to select single colonies and select strains with normal colony morphology, fast growth rate, good heat resistance, functional preservation and genetic stability. S6: The strains are stored in containers after being screened.

2. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 1, characterized in that, Step 1 is used in industrial scenarios such as fermented dairy products, where the target application matrix, such as milk or soy milk, is directly used as the acclimatization culture medium.

3. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 1, characterized in that, The initial screening in step 5 involves screening single colonies and selecting strains with normal colony morphology and fast growth rate.

4. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 1, characterized in that, The secondary screening in step 5 is to verify the primary screening strains using multiple indicators, such as heat resistance, functional retention, and genetic stability.

5. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 1, characterized in that, The heating device has a support frame that slides inside, and the support frame has two moving devices inside its fixing device.

6. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 5, characterized in that, The support frame is equipped with a clamping device, which includes multiple limiting plates, a threaded rod, and a rotating handle. The multiple limiting plates are slidably connected to the inside of the support frame, the threaded rod is threadedly connected to the inside of the multiple limiting plates, and the rotating handle is fixedly connected to the surface of the threaded rod.

7. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 6, characterized in that, The support frame is provided with a sliding device, which includes two sliding grooves and multiple sliding blocks. The multiple sliding grooves are all opened inside the support frame, and the multiple sliding blocks are slidably connected to the interior of the two sliding grooves respectively.

8. The method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 6, characterized in that, The limiting plates are equipped with multiple protective devices inside, and the limiting plates are equipped with multiple sets of limiting devices inside.

9. A method for improving the heat resistance of lactic acid bacteria through directed evolution according to claim 8, characterized in that, The limiting device includes two limiting grooves and two limiting rings. The two limiting grooves are both opened inside the limiting plate, and the two limiting rings are slidably connected to the inside of the two limiting grooves respectively.

10. A high-temperature stable strain obtained by a method for improving the heat resistance of lactic acid bacteria through directed evolution, wherein the high-temperature stable strain obtained by the method for improving the heat resistance of lactic acid bacteria through directed evolution as described in any one of claims 1-9 is characterized in that... The strain is Lactobacillus casei.

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