CN114381396A

CN114381396A - Microorganism culture medium and method for separating Lujie bacillus

Info

Publication number: CN114381396A
Application number: CN202111682444.4A
Authority: CN
Inventors: 罗海伟; 罗丹丽; 谢媚
Original assignee: Shenzhen Research Institute of CUHK
Current assignee: Shenzhen Research Institute of CUHK
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-22

Abstract

The application relates to the technical field of microbial separation, in particular to a microbial culture medium and a separation method of Rujie's bacteria. The nitrogen source of the microorganism culture medium of the present application includes at least one of betaine and choline, and N-acetylglucosamine. Experiments prove that the Lujie bacteria can specifically and efficiently utilize the nitrogen source (comprising at least one of betaine and choline and N-acetylglucosamine) as a culture substrate, and can realize the efficient growth of the Lujie bacteria, so that the microbial culture medium has good application prospect in the screening and separation of the Lujie bacteria.

Description

Microorganism culture medium and method for separating Lujie bacillus

Technical Field

The application belongs to the technical field of microorganism separation, and particularly relates to a microorganism culture medium and a separation method of Rujie's bacteria.

Background

The bacterium rougheria (Ruegeria) is a microorganism of the genus rougheria, which is now found to have some research value. There have been reports in the literature on methods for isolating the bacterium lurgesii using taurine (taurine) rich in coral mucus as a carbon source and ammonium chloride as a nitrogen source in a medium.

Taurine is a small molecular organic acid, can be efficiently utilized by various marine microorganisms, and particularly relates to a large class of bacteria including Roseobacter (Roseobacter) including the bacterium Lujie, so that the Roseobacter can be enriched to a certain extent by taking the taurine as a carbon source of a culture medium. Ammonium chloride or ammonium salt is a nitrogen source that can be widely used by microorganisms and generally does not have a screening effect. Therefore, although the culture medium can efficiently separate the roseobacter from the coral sample, the culture medium cannot enrich the lurgeella and has limited efficiency in separating the lurgeella.

Disclosure of Invention

The application aims to provide a microbial culture medium and a method for separating the Rujjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj _.

In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides a microbial culture medium having a nitrogen source comprising at least one of betaine and choline, and N-acetylglucosamine.

The nitrogen source in the microbial culture medium provided by the application is a compound nitrogen source, and comprises at least one of betaine and choline and N-acetylglucosamine; experiments prove that the Lujie bacteria can specifically and efficiently utilize the nitrogen source (comprising at least one of betaine and choline and N-acetylglucosamine) as a culture substrate, and can realize the efficient growth of the Lujie bacteria, so that the microbial culture medium has good application prospect in the screening and separation of the Lujie bacteria.

In a second aspect, the present application provides a method for isolating zygonella, comprising the steps of:

collecting a coral sample;

the coral samples were spread on the microbial culture medium described in the present application and subjected to a culture treatment.

According to the method for separating the Rujie's bacteria, collected coral samples are processed and then coated on a special microorganism culture medium for culture. The microorganism culture medium can realize the high-efficiency growth of the Rujjie bacteria, thereby realizing the high-efficiency separation of the Rujjie bacteria from the coral sample.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a colony diagram of a microorganism culture medium provided in the examples of the present application for isolating Lujie's bacteria;

FIG. 2 is another colony plot of a microorganism culture medium provided in the examples of the present application for isolation of Lujiella bacteria;

FIG. 3 is a further colony plot of a microbial culture medium isolated from Lujie's bacteria provided in the examples herein.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

In the present application, "at least one" means one or more, "plural" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items.

It should be understood that, in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the execution sequence, some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its function and inherent logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weight of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the mass described in the specification of the embodiments of the present application may be a mass unit known in the chemical industry field such as μ g, mg, g, kg, etc.

The terms "first", "second", etc. are used for descriptive purposes only and are used for distinguishing purposes such as substances from one another, and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

In a first aspect, embodiments of the present application provide a microbial culture medium, wherein a nitrogen source of the microbial culture medium comprises at least one of betaine and choline, and N-acetylglucosamine.

The nitrogen source in the microorganism culture medium provided by the embodiment of the application is a compound nitrogen source, and comprises at least one of betaine and choline and N-acetylglucosamine; experiments in the embodiment of the application prove that the Lujie bacteria can specifically and efficiently utilize the nitrogen source (comprising at least one of betaine and choline and N-acetylglucosamine) as a culture substrate and can realize the efficient growth of the Lujie bacteria, so that the microbial culture medium has a good application prospect in the screening and separation of the Lujie bacteria.

The composite nitrogen source in the microbial culture medium of the embodiment of the application is obtained by analyzing based on the colony genomics of the Lujiella and verifying the growth characteristics of the Lujiella under different substrates through physiological experiments. The applicant has surprisingly found that the growth rate and the maximum growth of the bacterium belonging to the genus of. Meanwhile, N-acetylglucosamine is a monomer of macromolecular organic substances (e.g., chitin) which are easily utilized by gamma-proteobacteria (e.g., Vibrio) growing rapidly, while N-acetylglucosamine is a monomer substance which is more easily utilized by alpha-proteobacteria (including Lujie bacteria). The previous major difficulty in separating and screening the Rugey bacteria from the coral samples is that the gamma-Proteus grows fast and has a large base number, often dominates in a screening culture medium, and the growth of the Rugey bacteria is limited. Therefore, the embodiment of the application provides a special microbial culture medium, and the special microbial culture medium is used for improving the growth advantage of the luregeria through combining N-acetylglucosamine as a composite nitrogen source on the basis of betaine and/or choline, so that the separation efficiency of the luregeria can be improved, and the effect is obvious.

In some embodiments, the microbial culture medium comprises: buffer solution, sea salt solution, ethylene diamine tetraacetic acid ferric sodium salt, vitamin solution and carbon nitrogen source solution; wherein the carbon-nitrogen source solution contains at least one of betaine and choline, and N-acetylglucosamine. The microbial culture medium has the nutrient components required by the growth of the Lujie bacteria. When the buffer solution, the sea salt solution, the ethylene diamine tetraacetic acid ferric sodium salt, the vitamin solution and the carbon-nitrogen source solution are used for culturing and separating the Rujie bacteria, the buffer solution, the sea salt solution, the ethylene diamine tetraacetic acid ferric sodium salt, the vitamin solution and the carbon-nitrogen source solution can be uniformly mixed to form a microorganism culture medium.

Furthermore, the microorganism culture medium contains taurine as a carbon source, namely, the carbon-nitrogen source solution contains taurine.

In some embodiments, the molar ratio of carbon to nitrogen in the carbon-nitrogen source solution is 0.8 to 1: 0.8 to 1, for example 1: 1. Under the conditions of the molar mass ratio of the carbon to the nitrogen source, better separation efficiency of the Rujie bacteria can be obtained. The actual concentrations of carbon and nitrogen sources may need to be properly learned and adjusted depending on the health of the test sample and the storage conditions and time from sample collection to sample processing.

In some embodiments, the volume ratio of the buffer solution, the sea salt solution, the iron sodium ethylenediaminetetraacetate solution, the vitamin solution and the carbon-nitrogen source solution is: 220-270: 660-740: 0.9-1.1: 0.9-1.1: 48 to 52; wherein the buffer solution comprises: 0.08-0.5 mg/ml K₂HPO₄And a Tris-HCl solution with the volume fraction of 27-30%; the sea salt solution contains: 0.014-0.09 g/ml sea salt and 0-0.06 g/ml agar; the ethylenediaminetetraacetic acid iron sodium salt solution contains: 0.03-0.2 g/ml of ethylenediaminetetraacetic acid ferric sodium salt; the vitamin solution contains: 0.01-0.06 mg/ml biotin, 0.01-0.06 mg/ml folic acid, 0.1-0.6 mg/ml pyridoxine hydrochloride, 0.025-0.15 mg/ml riboflavin, 0.025-0.15 mg/ml thiamine, 0.025-0.15 mg/ml nicotinic acid, 0.025-0.15 mg/ml pantothenic acid, 0.0005-0.003 mg/ml cyanocobalamin, and 0.025-0.15 mg/ml p-aminobenzoic acid; the carbon-nitrogen source solution comprises: 0.002-0.012 g/ml taurine, 0.0015-0.009 g/ml betaine or choline and 0.0008-0.005 g/ml N-acetyl glucosamine. The microorganism culture medium formed in the concentration range can obtain better separation efficiency of the Lujiella. Therefore, the microorganism culture medium of the embodiment can be used for separating and/or culturing the Lujie bacteria.

Specifically, the culture medium is obtained by the following steps:

preparing a first solution: 150mL 1M Tris-HCl pH 7.5, 87mg K₂HPO₄375mL of water;

preparing a second solution: 20g of sea salt, 15g of agar and 699mL of water;

preparing a third solution: 1g of ethylenediaminetetraacetic acid ferric sodium salt, 15mL of water;

preparing a fourth solution: 2mg biotin, 2mg folic acid, 10mg pyridoxine hydrochloride, 5mg riboflavin, 5mg thiamine, 5mg nicotinic acid, 5mg pantothenic acid, 0.1mg cyanocobalamin, 5mg p-aminobenzoic acid, 100mL water;

preparing a fifth solution: 0.23g of taurine, 0.15g of betaine, 0.08g N-acetylglucosamine and 49mL of water;

taking 250mL from the first solution as a buffer solution, taking 699mL from the second solution as a sea salt solution, taking 1mL from the third solution as an iron sodium ethylenediaminetetraacetate solution, taking 1mL from the fourth solution as a vitamin solution, and taking 49mL from the fifth solution as a carbon-nitrogen source solution; mixing the buffer solution, the sea salt solution and the ethylene diamine tetraacetic acid ferric sodium salt solution, performing damp-heat sterilization, and then mixing the solution with the vitamin solution and the carbon nitrogen source solution which are subjected to filtration sterilization by a 0.22 micron filter membrane.

In a second aspect of the embodiments of the present application, there is provided a method for separating a bacterium belonging to genus lactobacillus, comprising the steps of:

s01: collecting a coral sample;

s02: coral samples were spread on the microbial culture medium of the examples of the present application and subjected to culture treatment.

According to the separation method of the Rujie's bacteria, the collected coral samples are processed and then coated on the special microorganism culture medium for culture. The microorganism culture medium can realize the high-efficiency growth of the Rujjie bacteria, thereby realizing the high-efficiency separation of the Rujjie bacteria from the coral sample.

The Brucella may play an important role in coral symbionts, and the Brucella separation method provided by the embodiment of the application can successfully realize high-efficiency separation of the Brucella bacteria from various coral samples, and the separation efficiency can reach more than 60%, so that a foundation is laid for the subsequent application of the Brucella as coral probiotics and the like.

In the above step S01: the collected coral sample can include at least one of a coral mucus sample, a coral tissue sample, and a coral skeleton sample. Specifically, the step of collecting a coral sample comprises: collecting coral pieces from sea, cleaning the surfaces of the coral pieces with sterile seawater, and collecting the mucus on the surfaces of the coral pieces to obtain coral mucus samples; stripping the coral blocks from which the mucus is removed to obtain coral tissue samples; and grinding the bone blocks left after stripping treatment, and blowing and beating the bone blocks by using seawater to obtain a coral bone sample. The embodiment of the application can separate the Lujie bacteria from various coral samples.

Further, before the step of peeling off, the step of processing the coral piece with a magnesium chloride solution is also included. In order to obtain a purer coral tissue, if the coral mass is continuously secreted and mucus cannot be removed, the coral mass is anesthetized with a magnesium chloride solution (such as a sterile magnesium chloride solution with a mass fraction of 1.5-2.5%) for 1-5 minutes, so that mucus secretion can be better inhibited.

In the above step S02: the temperature of the culture treatment is 25-27 ℃, and the time is 2-4 weeks. Under the condition, the Lujie bacillus can grow better.

The following description will be given with reference to specific examples.

Example 1

(1) Treatment of different components of coral samples

1) Taking coral blocks with diameter of 2-8cm, packaging in sterilized self-sealing bag under water, and penetrating with tying bag. 500mL of in-situ seawater is taken in a sampling bottle and packaged under water.

2) Mucus: shaking and cleaning the surface of the coral piece with sterile seawater for three times to remove seawater pollution, pouring the cleaned coral piece into a sterile culture dish in a superclean bench, placing under low-speed sterile wind, collecting coral surface mucus, and preserving in a 2mL centrifuge tube to obtain a coral mucus sample.

3) Organizing: the coral pieces were washed three times with sterile seawater by vorex shaking, inverted in a 50mL centrifuge tube, and centrifuged at 4500rpm for 3 minutes until the mucus was removed (if the coral continued to secrete mucus and could not be removed, the coral pieces were anesthetized with 2% sterile magnesium chloride solution for 2 minutes, and the above operations were repeated until removed). The coral pieces from which the mucus was removed were detached from the coral tissues by Waterpik, and the tissue fluid was collected and stored in a 50mL centrifuge tube to obtain a sample of the coral tissues.

4) Bone: the coral tissue layer and superficial bone were dissected with a sterile scalpel and dissecting scissors, taking care that the dissection sequence is carefully protected against cross contamination, leaving a core bone mass of about 1cm in diameter. And (3) placing the bone blocks in a sterilized mortar for mashing, properly grinding, adding 2mL of sterilized artificial seawater for uniformly blowing, filtering the homogenate by using a sterilized bolting silk, and collecting 2mL of filtrate in a centrifuge tube to obtain a coral bone sample.

(2) Preparation of bacterial isolation Medium

A first solution: base substrate

·150mL 1M Tris-HCl，pH 7.5

·87mg K₂HPO₄

375mL of pure water.

A second solution: sea salt solution

20g sea salt (sea salt, Sigma)

15g agar (agar, Bacto) (added only when solid media were prepared, liquid media did not need to be added)

699mL of pure water.

A third solution: stock solution of ferric ethylenediaminetetraacetic acid sodium salt (FeNaEDTA)

1g iron sodium ethylenediaminetetraacetate (FeNaEDTA)

15mL of pure water.

A fourth solution: vitamin solution

2mg Biotin (biotin)

2mg Folic acid (folic acid)

10mg pyridoxine hydrochloride (pyridoxine-HCl)

5mg riboflavin (riboflavin)

5mg thiamine (thiamine)

5mg nicotinic acid (nicotinic acid)

5mg pantothenic acid (pantothenic acid)

0.1mg of cyanocobalamin (cyanocobalamin)

5mg of p-aminobenzoic acid (p-aminobenzoic acid)

100mL of pure water;

filtering and sterilizing with 0.2 μm filter membrane, subpackaging into 1ml each tube, and freezing at-20 deg.C for use.

A fifth solution: carbon nitrogen source solution

0.23g taurine (taurine)

0.15g betaine (glycine betaine)

0.08g N-acetylglucosamine (N-acetylglucosamine)

49mL of pure water;

filter sterilized using a 0.2 micron filter and kept at 4 ℃ until use.

Respectively preparing the five solutions, performing high-pressure steam sterilization on 250mL of the first solution, 699mL of the second solution and 1mL of the third solution (total 950mL), cooling the mixed solution to 50 ℃, adding 1mL of the fourth solution and 49mL of the fifth solution, and uniformly mixing to obtain the plate culture medium.

(3) Isolation and identification of bacteria

1) Diluting the coral sample in a gradient manner, coating the diluted coral sample on a plate culture medium, and culturing the diluted coral sample at room temperature (about 25-27 ℃) for 2-4 weeks.

2) Single colonies were picked and purified three times or more in 2216E medium.

3) Taking a proper amount of thalli, extracting DNA by using a bacterial genome extraction kit, detecting the quality of the DNA by agarose gel electrophoresis and NanoDrop, amplifying a 16S rRNA gene by PCR, sequencing, and identifying the bacteria after comparing with a database.

Analysis of results

The colony of the Brucella obtained in this example is round, slightly convex in the middle, and white to yellowish, as shown in FIGS. 1-3, and corresponds to the colony of the Brucella isolated from the coral mucus sample, the coral tissue sample, and the coral skeleton sample, respectively. And finally identified as the Lujie bacteria through 16S rRNA gene amplification and sequencing.

In addition, in comparison with the screening medium using ammonium chloride as a nitrogen source (the nitrogen source is ammonium chloride, and the rest is the same as in example 1), the separation efficiency of the medium using ammonium chloride as a nitrogen source to the rujgeria is only 7%, whereas the separation efficiency of the medium using ammonium chloride as a nitrogen source can reach 60%, and the specific data is shown in table 1.

TABLE 1

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A microbial culture medium, wherein a nitrogen source of the microbial culture medium comprises at least one of betaine and choline, and N-acetylglucosamine.

2. The microbial culture medium of claim 1, comprising: buffer solution, sea salt solution, ethylene diamine tetraacetic acid ferric sodium salt, vitamin solution and carbon nitrogen source solution; wherein the content of the first and second substances,

the carbon-nitrogen source solution contains the at least one of betaine and choline, and the N-acetylglucosamine.

3. The microorganism culture medium according to claim 2, wherein the molar ratio of carbon to nitrogen in the carbon-nitrogen source solution is 0.8 to 1: 0.8 to 1.

4. The microbial culture medium of claim 2, wherein the volume ratio of the buffer solution, the sea salt solution, the ferric ethylenediaminetetraacetic acid sodium salt solution, the vitamin solution, and the carbon-nitrogen source solution is: 220-270: 660-740: 0.9-1.1: 0.9-1.1: 48 to 52; wherein the content of the first and second substances,

the buffer solution contains: 0.08-0.5 mg/ml K₂HPO₄And a Tris-HCl solution with the volume fraction of 27-30%;

the sea salt solution contains: 0.014-0.09 g/ml sea salt and 0-0.06 g/ml agar;

the solution of the sodium iron ethylenediaminetetraacetate contains: 0.03-0.2 g/ml of ethylenediaminetetraacetic acid ferric sodium salt;

the vitamin solution contains: 0.01-0.06 mg/ml biotin, 0.01-0.06 mg/ml folic acid, 0.1-0.6 mg/ml pyridoxine hydrochloride, 0.025-0.15 mg/ml riboflavin, 0.025-0.15 mg/ml thiamine, 0.025-0.15 mg/ml nicotinic acid, 0.025-0.15 mg/ml pantothenic acid, 0.0005-0.003 mg/ml cyanocobalamin, and 0.025-0.15 mg/ml p-aminobenzoic acid;

the carbon-nitrogen source solution contains: 0.002-0.012 g/ml taurine, 0.0015-0.009 g/ml betaine or choline and 0.0008-0.005 g/ml N-acetyl glucosamine.

5. The microbial culture medium of any one of claims 1-4, wherein the microbial culture medium is used for culturing and/or isolating Lujie bacteria.

6. A method for separating the Lujie bacteria is characterized by comprising the following steps:

collecting a coral sample;

coating the coral sample on the microbial culture medium according to any one of claims 1 to 5, and culturing.

7. The separation method according to claim 6, wherein the temperature of the culture treatment is 25 to 27 ℃ and the time is 2 to 4 weeks.

8. The separation method of claim 6, wherein the coral sample comprises at least one of a coral mucus sample, a coral tissue sample, and a coral skeleton sample.

9. The separation method of claim 6, wherein the step of collecting a sample of coral comprises: collecting coral blocks from sea, cleaning the surfaces of the coral blocks with sterile seawater, and collecting the mucus on the surfaces of the coral blocks to obtain coral mucus samples; stripping the coral blocks from which the mucus is removed to obtain coral tissue samples; and grinding the bone blocks left after the stripping treatment, and blowing and beating the bone blocks by using seawater to obtain a coral bone sample.

10. The separation method of claim 9, wherein the step of peeling further comprises treating the coral cake with a magnesium chloride solution.