Screening flat plate for high-yield benzene lactic acid bacteria strain as well as preparation method and application of screening flat plate

Abstract

The invention provides a screening flat plate for high-yield benzene lactic acid bacteria as well as a preparation method and application thereof, and relates to the technical field of microbial breeding. The preparation method comprises the following steps: pretreating calcium carbonate to obtain pretreated calcium carbonate, adding the pretreated calcium carbonate into a culture medium, sterilizing, and pouring on a flat plate to obtain the screening flat plate, the pretreatment comprises grinding treatment and plasma treatment. Transparent calcium rings formed by neutralization of the screening flat plate and phenyllactic acid are clearer.

Description

Technical Field

[0001] The invention relates to the technical field of microbial breeding, and in particular to a screening plate for high-yield phenyllactic acid strains and a preparation method and application thereof. Background Art

[0002] Phenyllactic acid is a natural preservative discovered in recent years. It has an inhibitory effect on the growth and development of harmful microorganisms, and the antibacterial spectrum of phenyllactic acid is very wide. It is different from bacteriocins such as Nisin. Most bacteriocins only have effects on similar bacteria, while phenyllactic acid still has an inhibitory effect on G-bacteria, yeasts, and molds. Phenyllactic acid itself also has good solubility and stability, and has good diffusion performance in food systems. At present, phenyllactic acid is mainly used in food, medicine and cosmetics. It has an inhibitory effect on pathogenic bacteria that harm the dairy industry and molds that are easily spread in the baking industry; it can replace tanshinone in clinical practice, or be used as a drug for clinical treatment of bleeding with IUDs; and it can be used in cosmetics to remove wrinkles and brighten the skin.

[0003] Phenyllactic acid is mainly produced by lactobacillus. In the screening of lactobacillus, the most commonly used primary screening method is the calcium dissolution circle method. This experimental study used the calcium dissolution circle method to screen lactobacillus. The samples were separated on a plate containing calcium carbonate to obtain lactobacillus that can produce calcium dissolution circles. However, traditional calcium carbonate-containing plates are prone to aggregation and precipitation during the production process, which makes it inconvenient to observe the diameter of the calcium dissolution circle. Therefore, how to further improve the dispersibility of calcium carbonate on solid separation plates has become a scientific problem and technical bottleneck that needs to be solved in the process of selecting high-yield phenyllactic acid strains. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes a screening plate for high-producing phenyllactic acid strains and a preparation method and application thereof.

[0005] The invention provides a method for preparing a screening plate for high-yield phenyllactic acid strains, comprising the following steps: pre-treating calcium carbonate to obtain pre-treated calcium carbonate, adding the pre-treated calcium carbonate into a culture medium for sterilization, and pouring the plate to obtain the screening plate; the pre-treatment comprises grinding treatment and plasma treatment.

[0006] Preferably, the grinding process comprises grinding the calcium carbonate to a particle size of 0.1-0.3 mm.

[0007] Calcium carbonate easily absorbs water and agglomerates. After sufficient grinding, its powder can be more dispersed, which is convenient for the next step of screening and plasma treatment.

[0008] Preferably, the plasma treatment includes performing a dielectric barrier discharge treatment in a plasma discharge region.

[0009] Modification of calcium carbonate by plasma can effectively enhance its dispersibility in solid separation plates. After plasma treatment, the increase in the number of charges on the surface of calcium carbonate powder, the activation of chemical groups, and the change in physical structure significantly increase the uniformity of calcium carbonate dispersion in the culture medium.

[0010] Preferably, the plasma power supply for the plasma treatment is a sinusoidal AC power supply with continuously adjustable frequency and voltage, and the reactor is made of quartz material and is located between upper and lower metal electrodes to form a dielectric barrier.

[0011] Preferably, the plasma atmosphere of the plasma treatment is helium, the gas flow rate is 0.2 L / min, the input voltage is 50-200 V, the current is 0.8-1.5 A, and the temperature is kept below 40° C. during the discharge process.

[0012] Preferably, the plasma treatment time is 0-3 min.

[0013] More preferably, the plasma treatment time is 3 minutes.

[0014] Preferably, the culture medium is MRS culture medium, and the agar concentration is 2%.

[0015] Preferably, the sterilization is high-pressure steam sterilization at a temperature of 121° C. for 25-35 minutes.

[0016] Preferably, the mass concentration of the pretreated calcium carbonate in the culture medium is 2-4 g / L.

[0017] More preferably, the mass concentration of the pretreated calcium carbonate in the culture medium is 3 g / L.

[0018] The present invention also provides a screening plate for high-producing phenyllactic acid strains prepared by the above preparation method.

[0019] An application of the above-mentioned screening plate for high-phenyllactic acid-producing strains in screening high-phenyllactic acid-producing strains.

[0020] Preferably, the application comprises: applying a bacterial solution containing a high-producing phenyllactic acid strain to the above-mentioned screening plate for cultivation to obtain plate colonies, and observing the state of the plate colonies.

[0021] More preferably, the high-producing phenyllactic acid strain is lactic acid bacteria.

[0022] More preferably, the culture temperature is 28-35°C and the culture time is 3-5 days.

[0023] More preferably, the method for preparing the bacterial solution containing the high-yield phenyllactic acid strain is as follows: taking the lactic acid bacteria solution after fermentation and diluting it with 0.9% physiological saline to a bacterial concentration of 10 3 Pieces / mL.

[0024] The beneficial effects of the present invention are:

[0025] The present invention pre-treats calcium carbonate, which helps to evenly disperse calcium carbonate in the culture medium, so that phenyllactic acid is timely neutralized by the evenly distributed calcium carbonate in the plate, and the formed transparent calcium ring of the lactic acid bacteria screening plate is clearer.

[0026] The increase in the number of charges on the surface of calcium carbonate after plasma treatment, the activation of chemical groups, and the change in physical structure significantly increase the uniformity of the dispersion of calcium carbonate in the culture medium. Since the phenyllactic acid produced by the bacteria is promptly neutralized by the evenly distributed calcium carbonate powder in the plate, the transparent calcium ring formed on the lactic acid bacteria screening plate is clearer. At the same time, the bacteria are less inhibited by the product phenyllactic acid, and the colony morphology is more complete and clear. Therefore, the present invention significantly improves the screening efficiency of high-yield phenyllactic acid strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a screening plate diagram of Example 1 of the present invention.

[0028] Figure 2 This is a screening plate diagram of Example 2 of the present invention.

[0029] Figure 3 This is a screening plate diagram of Example 3 of the present invention.

[0030] Figure 4 This is the plate colony diagram of Example 1 of the present invention.

[0031] Figure 5 This is the plate colony diagram of Example 2 of the present invention.

[0032] Figure 6 This is the plate colony diagram of Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail through specific embodiments.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] A method for preparing a screening plate for high-producing phenyllactic acid strains comprises the following steps:

[0037] Step 1. Prepare MRS medium (g / 500mL): peptone 5, beef extract 5, K2HPO4 1, yeast extract powder 2.5, diammonium citrate 1, sodium acetate 2.5, glucose 10, Tween 80 0.5mL, MgSO4·7H2O0.29, MnSO4·4H2O 0.125, and add agar to make the final concentration of 2%, heat at 80℃ and mix thoroughly, then take 50mL into blue-capped bottles for later use;

[0038] Step 2. Take calcium carbonate and grind it in a mortar. After grinding, pass it through a 100-mesh sieve to obtain calcium carbonate powder with a particle size of 0.15 mm;

[0039] Step 3. Take 2g of calcium carbonate powder and treat it with plasma (CTP-2000K, Nanjing Suman Plasma Technology Co., Ltd.), wherein the plasma power supply is a sinusoidal AC power supply with continuously adjustable frequency and voltage, the reactor is made of quartz material and is located between the upper and lower metal electrodes to form a dielectric barrier layer, and helium is used as the gas source during DBD discharge treatment, the gas flow rate is 0.2L / min, the input voltage is 150V, the current is 1A, and the temperature is kept below 40°C during the discharge process; after 0min of plasma treatment, pretreated calcium carbonate is obtained;

[0040] Step 4. Weigh 0.15 g of pretreated calcium carbonate and add it to 50 mL of MRS medium to obtain a medium with a pretreated calcium carbonate mass concentration of 3 g / L;

[0041] Step 5. Place the culture medium in a sterilizer for high-pressure steam sterilization at 121°C for 30 minutes. After sterilization, take out the culture medium and pour it into a plate.

[0042] Application: Take the fermented lactic acid bacteria liquid and dilute it with 0.9% physiological saline to a bacterial concentration of 10 3 Take 0.1 mL of lactic acid bacteria liquid and spread it evenly on the above screening plate, place it in an incubator at 30°C and culture it for 3 days to obtain lactic acid bacteria plate colonies.

[0043] Example 2

[0044] A method for preparing a screening plate for high-producing phenyllactic acid strains comprises the following steps:

[0045] Step 1. Prepare MRS medium according to Example 1, add agar to a final concentration of 2%, heat at 80°C and mix thoroughly, then dispense 50 mL into blue-capped bottles for later use;

[0046] Step 2. Take calcium carbonate and grind it in a mortar. After grinding, pass it through a 50-mesh sieve to obtain calcium carbonate powder with a particle size of 0.3 mm;

[0047] Step 3. Take 2g of calcium carbonate powder and treat it with plasma (CTP-2000K, Nanjing Suman Plasma Technology Co., Ltd.), wherein the plasma power supply is a sinusoidal AC power supply with continuously adjustable frequency and voltage, the reactor is made of quartz material and is located between the upper and lower metal electrodes to form a dielectric barrier layer, and helium is used as the gas source during DBD discharge treatment, the gas flow rate is 0.2L / min, the input voltage is 50V, the current is 0.8A, and the temperature is kept below 40°C during the discharge process; after plasma treatment for 1min, pretreated calcium carbonate is obtained;

[0048] Step 4. Weigh 0.15 g of pretreated calcium carbonate and add it to 50 mL of MRS medium to obtain a medium with a pretreated calcium carbonate mass concentration of 3 g / L;

[0049] Step 5. Place the culture medium in a sterilizer for high-pressure steam sterilization at 121°C for 30 minutes. After sterilization, take out the culture medium and pour it into a plate.

[0050] Application: Take the fermented lactic acid bacteria liquid and dilute it with 0.9% physiological saline to a bacterial concentration of 10 3 Take 0.1 mL of lactic acid bacteria liquid and spread it evenly on the above screening plate, place it in an incubator at 30°C and culture it for 3 days to obtain lactic acid bacteria plate colonies.

[0051] Example 3

[0052] A method for preparing a screening plate for high-producing phenyllactic acid strains comprises the following steps:

[0053] Step 1. Prepare MRS medium according to Example 1, add agar to a final concentration of 2%, heat at 80°C and mix thoroughly, then dispense 50 mL into blue-capped bottles for later use;

[0054] Step 2. Take calcium carbonate and grind it in a mortar. After grinding, pass it through a 120-mesh sieve to obtain calcium carbonate powder with a particle size of 0.125 mm;

[0055] Step 3. Take 2g of calcium carbonate powder and treat it with plasma (CTP-2000K, Nanjing Suman Plasma Technology Co., Ltd.), wherein the plasma power supply is a sinusoidal AC power supply with continuously adjustable frequency and voltage, the reactor is made of quartz material and is located between the upper and lower metal electrodes to form a dielectric barrier layer, and helium is used as the gas source during DBD discharge treatment, the gas flow rate is 0.2L / min, the input voltage is 200V, the current is 1.5A, and the temperature is kept below 40°C during the discharge process; after plasma treatment for 3min, pretreated calcium carbonate is obtained;

[0056] Step 4. Weigh 0.15 g of pretreated calcium carbonate and add it to 50 ml of MRS medium to obtain a medium with a pretreated calcium carbonate mass concentration of 3 g / L;

[0057] Step 5. Place the culture medium in a sterilizer for high-pressure steam sterilization at 121°C for 30 minutes. After sterilization, take out the culture medium and pour it into a plate.

[0058] Application: Take the fermented lactic acid bacteria liquid and dilute it with 0.9% physiological saline to a bacterial concentration of 10 3 Take 0.1 mL of lactic acid bacteria liquid and spread it evenly on the above screening plate, place it in an incubator at 30°C and culture it for 3 days to obtain lactic acid bacteria plate colonies.

[0059] like Figure 1-3 As shown in the figure, they are the screening plates prepared in Examples 1-3, that is, the calcium carbonate powders were sterilized after being treated with plasma for 0min, 1min and 3min and then poured into the plates. It can be seen from the figure that Figure 3 The distribution of calcium carbonate in the experimental group treated with plasma for 3 minutes was relatively uniform. It can be seen that after plasma treatment, the uniformity of calcium carbonate distribution in the culture medium was significantly improved due to the increase in the number of charges on the surface of calcium carbonate powder, the activation of chemical groups, and the change in physical structure.

[0060] like Figure 4-6 As shown, they are the plate colony diagrams of Examples 1-3, i.e., after the calcium carbonate powder was sterilized by plasma treatment for 0min, 1min and 3min, the plate was poured, and then lactic acid bacteria were taken to coat the plate, and the state of the plate colony after culturing at 30° C. for 3 days. It can be seen that the plate after the plasma treatment for 3min has good dispersibility of calcium carbonate, and the calcium ring formed by the colony growing on this plate is obvious, which makes it easier to screen the strains of high-yielding phenyl lactic acid Lactobacillus.

[0061] In summary, the screening plate provided by the present invention significantly improves the screening efficiency of high-producing phenyllactic acid strains.

[0062] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a screening plate for high-producing phenyllactic acid strains, characterized in that: The following steps are involved: The calcium carbonate is pretreated to obtain pretreated calcium carbonate, and the pretreated calcium carbonate is added to a culture medium for sterilization and poured into a plate to obtain; The pretreatment includes grinding treatment and plasma treatment.

2. The preparation method according to claim 1, characterized in that: The grinding process comprises grinding the calcium carbonate to a particle size of 0.1-0.3 mm.

3. The preparation method according to claim 1, characterized in that: The plasma atmosphere of the plasma treatment is helium, the gas flow rate is 0.2L / min, the input voltage is 50-200V, the current is 0.8-1.5A, and the temperature is kept below 40°C during the discharge process.

4. The preparation method according to claim 1, characterized in that: The plasma treatment time is 0-3 minutes.

5. The preparation method according to claim 1, characterized in that: The culture medium is MRS culture medium, and the agar concentration is 2%.

6. The preparation method according to claim 1, characterized in that: The sterilization is high pressure steam sterilization, the temperature is 121°C, and the time is 25-35 minutes.

7. A screening plate for high-producing phenyllactic acid strains, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the screening plate for high-phenyllactic acid producing strains according to claim 7 or the screening plate for high-phenyllactic acid producing strains prepared by the preparation method according to any one of claims 1 to 6 in screening high-phenyllactic acid producing strains.

Images