A method for screening lactic acid bacteria with high phenyllactic acid production

Abstract

The invention discloses a method for screening lactic acid bacteria with high phenyllactic acid production, wherein lactic acid bacteria are separated from kimchi, and the lactic acid bacteria are added to a liquid MRS culture medium for culture to obtain a bacterial suspension; the bacterial suspension is coated on an MRS agar calcium carbonate culture medium for culture; an MRS agar calcium carbonate culture medium plate containing a colony is separated from a culture dish, and the plate is placed on an MRS agar culture medium plate containing a neutral red indicator to form a double plate, which is sealed and cultured; and high phenyllactic acid production bacteria are screened out according to the size of a red transparent circle formed around the colony. The invention adopts a double plate method to screen lactic acid bacteria, because the phenyllactic acid formed by the colony is distributed in the transparent calcium circle of the upper plate, and the neutral red permeated from the lower plate here becomes darker when encountering phenyllactic acid, which makes the transparent circle of calcium carbonate more obvious, and the production of phenyllactic acid can be judged more easily and accurately, and the target strain can be screened more accurately and quickly in the process of strain mutation breeding of high phenyllactic acid production.

Description

Technical Field

[0001] The invention relates to the field of rapid screening of high-yield strains in microbial breeding technology, and in particular to a method for screening lactic acid bacteria with high phenyllactic acid production. Background Art

[0002] Phenyllactic acid has many important functions such as antibacterial, antiseptic and fresh-keeping, so it has broad prospects in the processing and storage of agricultural products. At present, the production of phenyllactic acid mainly comes from chemical synthesis and microbial fermentation synthesis. The chemical synthesis of phenyllactic acid is technically cumbersome, the product is impure, there are many by-products, and it is easy to pollute the environment; microbial fermentation dough mainly uses lactic acid bacteria as production bacteria to synthesize phenyllactic acid, but the phenyllactic acid isolated from nature generally has the disadvantages of low phenyllactic acid yield, slow growth rate, and high culture conditions, which is difficult to meet the needs of large-scale industrial production, which requires the improvement and screening of strains. At present, the method of primary screening of lactic acid bacteria is mainly to screen lactic acid bacteria using the calcium dissolution circle method. The principle is that the acidic substances produced by lactic acid bacteria react with calcium carbonate to form a dissolution circle, and the colonies with large calcium dissolution circles are selected; however, this method has problems such as unclear phenomenon and easy to produce calcium carbonate precipitation. Summary of the invention

[0003] Based on the technical problems existing in the background technology, the present invention proposes a method for screening lactic acid bacteria with high phenyllactic acid production.

[0004] The invention provides a method for screening lactic acid bacteria with high phenyllactic acid production. The method comprises the following steps: separating lactic acid bacteria from kimchi by adopting a microbial separation and purification technology, adding the lactic acid bacteria into a liquid MRS culture medium for cultivation to obtain a bacterial suspension; applying the bacterial suspension onto an MRS agar calcium carbonate culture medium for cultivation to obtain a lactic acid bacteria colony; separating an MRS agar calcium carbonate culture medium plate containing the colony from a culture dish, placing the culture medium plate onto an MRS agar culture medium plate containing a neutral red indicator to form a double plate, sealing the plate, and culturing the plate; and screening the high phenyllactic acid production bacteria according to the size of a red transparent circle formed around the colony.

[0005] Preferably, the lactic acid bacteria are added to a liquid MRS culture medium and cultured at 27-32° C. for 1-3 days to obtain a bacterial suspension.

[0006] Preferably, the bacterial suspension is spread on MRS agar calcium carbonate medium and cultured at 27-32° C. for 2-3 days to obtain lactic acid bacteria colonies.

[0007] Preferably, before applying the bacterial suspension onto the MRS agar calcium carbonate medium, the method further comprises diluting the bacterial suspension by ten-fold dilution method with physiological saline to prepare a 10 -5 ~10 -6 Dilution of multiple dilution.

[0008] Preferably, in the MRS agar medium plate containing the neutral red indicator, the content of the neutral red indicator is 0.1500-0.1750 g / L; the preparation thereof is obtained by adding the neutral red indicator to the MRS agar medium.

[0009] Preferably, after sealing, the mixture is cultured at 27-32° C. for 2-3 days.

[0010] In the prior art, the calcium dissolution ring method is often used for lactic acid bacteria screening. The method uses the phenyllactic acid produced by lactic acid bacteria to dissolve the calcium carbonate in the plate to form a transparent calcium ring. However, the size of the calcium ring, especially the boundary of the calcium ring, is not very obvious, especially in the middle and late stages of bacterial growth. Although the bacteria synthesize a large amount of phenyllactic acid at this time, the calcium ring will be more blurred, which often affects the judgment of phenyllactic acid production.

[0011] If the neutral red color development method is used, after neutral red is added to the plate, a deep red bacterial circle begins to appear around the colonies due to the formation of phenyllactic acid, but the boundary between the bacterial circle and the surrounding culture medium is not obvious. At the same time, the early addition of neutral red will inhibit the growth of the bacteria and the formation of phenyllactic acid, which will also affect the judgment of phenyllactic acid production.

[0012] Compared with the prior art, the present invention adopts a double plate method to screen lactic acid bacteria, that is, a calcium carbonate culture medium plate containing lactic acid bacteria colonies and a culture medium plate containing a neutral red indicator are stacked together in an upper and lower order, and the neutral red indicator in the lower plate will penetrate into the upper plate, produce an obvious color reaction with the phenyllactic acid in the upper plate and appear red, and then the phenyllactic acid produced by the lactic acid bacteria can dissolve the calcium carbonate in the plate to form a transparent calcium ring, thereby finally forming an obvious red transparent ring around the colony on the upper plate, and the size of the red transparent ring can be used to judge the ability of the lactic acid bacteria to produce phenyllactic acid.

[0013] The present invention establishes a new method for screening lactic acid bacteria based on the transparent calcium circle method and the color reaction method. The method is simple and easy to operate. Since the phenyllactic acid formed by the colony is distributed in the transparent calcium circle, and the neutral red here becomes darker in color when it encounters phenyllactic acid, it makes the transparent circle of calcium carbonate more obvious, and the production of phenyllactic acid is judged more easily and accurately. In the process of mutagenesis breeding of high-yield phenyllactic acid strains, the target strains can be screened more accurately and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a bacterial colony image of the MRS agar calcium carbonate plate culture in Example 1 of the present invention;

[0015] Figure 2 This is a colony image of the MRS agar calcium carbonate neutral red plate culture in Example 1 of the present invention;

[0016] Figure 3This is a colony diagram of double plate culture in Example 1 of the present invention. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is described in detail below through specific embodiments.

[0018] Example 1

[0019] 1. Isolation of lactic acid bacteria: Take kimchi purchased from the market, measure 5ml of kimchi juice with a pipette, place it in a pre-sterilized liquid MRS medium, and culture it in a constant temperature incubator at 30°C for 3 days. When the liquid in the entire conical flask becomes turbid, first observe the growth of the bacteria in the culture medium, select the bacteria with good growth, and use a pipette to transfer 5ml of bacterial liquid in a conical flask containing 100ml MRS liquid culture medium in the clean bench. Culture it at 30°C for 3 days to obtain a bacterial enrichment solution. Centrifuge the enrichment solution and dilute it with sterile physiological saline gradients, take the dilution solution to plate, and culture it at 30°C for 48h. Select a single colony with an obvious calcium carbonate dissolution circle around the colony, pick the corresponding single colony with an inoculation needle and streak it on the MRS solid culture medium until a milky white, uniform-sized single colony appears.

[0020] 2. Preparation of bacterial suspension: Add the obtained colonies into a Erlenmeyer flask containing 200 mL of MRS liquid culture medium at an inoculum volume of 1.5 vt%, and culture at 30° C. for 48 h to obtain a bacterial suspension.

[0021] 3. Lactic acid bacteria culture:

[0022] 1) MRS agar calcium carbonate culture: Use a pipette to draw 0.4 mL of bacterial suspension from the flask and inject it into a test tube containing 3.6 mL of sterile water. Repeat this process to make 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 The solutions of different dilutions were spread on MRS agar calcium carbonate medium plates and cultured at 30°C for 2-3 days. -6 The colonies grown from the diluted bacterial solution are evenly distributed on the culture medium, and the experimental phenomenon can be better observed. -6 The growth of the diluted bacterial solution on MRS agar calcium carbonate medium is as follows Figure 1 As shown. Figure 1 It can be seen that the phenyllactic acid produced by lactic acid bacteria can dissolve calcium carbonate in the plate to form a transparent calcium ring, but the size of the calcium ring, especially the boundary of the calcium ring, is not very obvious, especially in the middle and late stages of bacterial growth. Although the bacteria synthesize a large amount of phenyllactic acid at this time, the calcium ring is more blurred, which often affects the judgment of phenyllactic acid production.

[0023] 2) MRS agar calcium carbonate neutral red culture: Add a certain amount of neutral red indicator to the MRS agar calcium carbonate medium to prepare neutral red plates with neutral red concentrations of 0.1250, 0.1500, 0.1750, 0.2000, and 0.2250 g / L; use a pipette to draw 0.4 mL of bacterial suspension from the conical flask and inject it into a test tube containing 3.6 mL of physiological saline for gradient dilution until 10 -6 The diluted bacterial solution was spread on neutral red plates (1L culture medium) with different gradients and cultured at 30℃ for 2-3 days. After experimental determination, the experimental phenomenon can be better observed when the neutral red concentration is 0.1750g / L. The growth of lactic acid bacteria on MRS agar medium containing neutral red (neutral red concentration is 0.1750g / L) is as follows Figure 2 As shown. Figure 2 It can be seen that after neutral red is added to the plate, dark red bacterial circles begin to appear around the colonies due to the formation of phenyllactic acid, but the boundary between the bacterial circle and the surrounding culture medium is not obvious. At the same time, the early addition of neutral red will inhibit the growth of bacteria and the formation of phenyllactic acid, which will also affect the judgment of phenyllactic acid production.

[0024] 4. Double plate method screening, the specific operation is: dilute the lactic acid bacteria suspension (dilution degree is 10 -6 ) and then spread it on an MRS agar calcium carbonate plate, and then culture it in an incubator at 30°C for 60 hours. After the lactic acid bacteria have grown well, move the cultured lactic acid bacteria colonies and the plate on which the lactic acid bacteria were cultured to an MRS agar plate containing neutral red (concentration 0.1750g / L) and culture it for 60 hours. Since neutral red reacts with the phenyllactic acid produced by the lactic acid bacteria to produce a color reaction and appears red, and calcium carbonate reacts with phenyllactic acid to produce a dissolution circle, the surrounding colonies will form a red transparent circle. The size of the transparent circle and the depth of the red color can be used to determine the ability of the lactic acid bacteria to produce phenyllactic acid. The growth of lactic acid bacteria on the double plate is shown in the figure. Figure 3 As shown. Figure 3 It can be seen that using the double plate method, the phenyllactic acid formed by the colony is distributed in the transparent calcium circle, and the neutral red here becomes darker in color when it encounters phenyllactic acid, which makes the transparent circle of calcium carbonate more obvious, making it easier and more accurate to judge the production of phenyllactic acid.

[0025] The preparation of the MRS agar plate containing neutral red is as follows: 0.1750 g of neutral red is added to 1 L of MRS agar medium. If the concentration of neutral red is too low, the effect is not obvious, and if the concentration of neutral red is too high, the activity of lactic acid bacteria will be affected. Therefore, in order to avoid direct contact between early lactic acid bacteria and neutral red and inhibit their growth, a double plate culture of neutral red is used instead of directly adding neutral red to the MRS agar calcium carbonate medium.

[0026] Example 2

[0027] A method for screening lactic acid bacteria with high phenyllactic acid production, comprising the following steps:

[0028] 1. Isolation of lactic acid bacteria: Same as Example 1.

[0029] 2. Preparation of bacterial suspension: The obtained colonies were added to kimchi at an inoculum rate of 1vt%, and the lactic acid bacteria were isolated by microbial culture technology. The bacteria were placed in a conical flask containing 200 mL of MRS liquid culture medium and cultured at 27°C for 72 hours to obtain a bacterial suspension.

[0030] 3. Double plate screening: dilute the lactic acid bacteria suspension (dilution is 10 -6 ) and then applied to an MRS agar calcium carbonate plate, and then placed in an incubator for 72 hours at 27°C. After the lactic acid bacteria have grown well, the cultured lactic acid bacteria colonies and the plate for culturing the lactic acid bacteria are moved together to an MRS agar plate containing neutral red (concentration 0.1750 g / L) (the preparation method is the same as in Example 1) and cultured for 72 hours. Since neutral red reacts with the phenyllactic acid produced by the lactic acid bacteria to produce a color reaction and presents red, and calcium carbonate reacts with phenyllactic acid to produce a dissolution circle, the surrounding colonies will form a red transparent circle. According to the size of the transparent circle and the depth of the red color, the ability of the lactic acid bacteria to produce phenyllactic acid can be judged.

[0031] Example 3

[0032] A method for screening lactic acid bacteria with high phenyllactic acid production

[0033] 1. Isolation of lactic acid bacteria: Same as Example 1.

[0034] 2. Preparation of bacterial suspension: The obtained colonies were added to kimchi at an inoculum rate of 2vt%, and the lactic acid bacteria were isolated by microbial culture technology. The bacteria were placed in a conical flask containing 200 mL of MRS liquid culture medium and cultured at 32°C for 24 hours to obtain a bacterial suspension.

[0035] 3. Double plate screening: dilute the lactic acid bacteria suspension (dilution is 10 -5 ) and then applied to an MRS agar calcium carbonate plate, and then placed in an incubator at 32°C for 48 hours. After the lactic acid bacteria have grown well, the cultured lactic acid bacteria colonies and the plate for culturing the lactic acid bacteria are moved together to an MRS agar plate containing neutral red (concentration 0.1750 g / L) (the preparation method is the same as in Example 1) and cultured for 60 hours. Since neutral red reacts with the phenyllactic acid produced by the lactic acid bacteria to produce a color reaction and presents red, and calcium carbonate reacts with phenyllactic acid to produce a dissolution circle, the surrounding colonies will form a red transparent circle. According to the size of the transparent circle and the depth of the red color, the ability of the lactic acid bacteria to produce phenyllactic acid can be judged.

[0036] 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 screening lactic acid bacteria with high phenyllactic acid production, characterized in that: The invention adopts microbial separation and purification technology to separate lactic acid bacteria from kimchi, adds the lactic acid bacteria into liquid MRS culture medium for cultivation, and obtains bacterial suspension; spreads the bacterial suspension onto MRS agar calcium carbonate culture medium for cultivation, and obtains lactic acid bacteria colonies; separates the MRS agar calcium carbonate culture medium plate containing the colonies from the culture dish, and places the culture medium plate onto an MRS agar culture medium plate containing a neutral red indicator to form a double plate, seals the plate, and cultivates the plate; selects high-yield phenyl lactic acid bacteria according to the size of the red transparent circle formed around the colonies; the MRS agar culture medium plate containing the neutral red indicator is prepared by adding the neutral red indicator into the MRS agar culture medium; the content of the neutral red indicator in the MRS agar culture medium plate containing the neutral red indicator is 0.1500-0.1750 g / L; and after sealing, cultivates the plate at 27-32° C. for 2-3 days.

2. The method according to claim 1, characterized in that Add lactic acid bacteria to liquid MRS culture medium and culture at 27-32°C for 1-3 days to obtain a bacterial suspension.

3. The method according to claim 1, characterized in that The bacterial suspension was spread onto MRS agar calcium carbonate medium and cultured at 27-32°C for 2-3 days to obtain lactic acid bacteria colonies.

4. The method according to claim 1, characterized in that Before applying the bacterial suspension onto the MRS agar calcium carbonate medium, the bacterial suspension is diluted with physiological saline according to the ten-fold dilution method to make 10 -5 ~10 -6 Dilution of multiple dilution.

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