Lactobacillus plantarum for producing polysialic acid and application of lactobacillus plantarum

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum for producing polysialic acid and application of the lactobacillus plantarum. The lactobacillus plantarum capable of producing the polysialic acid, which is obtained by separating and screening from pickled vegetables of a Chongqing peasant family, is preserved in Guangdong Province Microbial Culture Collection Center, the culture and fermentation conditions of the strain are simple, the genetic property is stable, and the polysialic acid can be produced and secreted outside cells through liquid fermentation. The lactobacillus plantarum is used for producing the polysialic acid through fermentation, the fermentation efficiency is high, and the content of the polysialic acid in the obtained fermentation liquid is high. The risk of toxin generation is avoided in the fermentation process, and safety guarantee is achieved when the strain is applied to the fields of food, medicine and the like; the fermentation process is simple to operate, low in production cost and easier for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a polysialic acid-producing lactobacillus plantarum and an application thereof. Background Art

[0002] Polysialic acid (PSA) is a homopolymer of linear polyanions with a degree of polymerization (DP) of 8-400, which is formed by direct or indirect connection of N-acetylneuraminic acid (Neu5Ac) monomers with α-2,8 glycosidic bonds and / or α-2,9 glycosidic bonds. It exists in mammals and is attached to the surface of bacteria in the form of capsules. It is a subcellular structure that has a protective function for bacteria. It is of great significance in animal and medical applications. In nature, some bacteria can synthesize polysialic acid by themselves, such as Escherichia coli (E.coli), Neisseria meningitides, Campylobacter jejuni, Haemophilus influenzae, Haemophilus donovani, Pasteurella multocida, Neisseria gonorrhoeae and Streptococcus agalactiae. Polysialic acid exists in the form of capsules on the cell surface of a few bacterial species. When these bacteria are cultured on solid, polysialic acid is attached to the cell surface in the form of capsules. When they are cultured in liquid, polysialic acid is released into the fermentation broth in the form of mucus. The monomer that makes up polysialic acid is N-acetylneuraminic acid. After acid hydrolysis or enzymatic hydrolysis, polysialic acid can be separated and purified to obtain sialic acid.

[0003] At present, PSA is obtained by microbial fermentation. Microbial fermentation can directly use glucose, glycerol or other carbon sources as substrates, and can produce polysialic acid without adding any other precursor substances. Low production cost and wide research field play an important role in the production process of polysialic acid. At present, the main research is to use Escherichia coli for fermentation to produce polysialic acid, and there are few reports on the production of polysialic acid by Lactobacillus plantarum. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a polysialic acid-producing Lactobacillus plantarum and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a polysialic acid-producing Lactobacillus plantarum; the Lactobacillus plantarum has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC No. 63560.

[0007] There are few reports on the research of polysialic acid production by Lactobacillus plantarum. The present invention separates and screens a natural polysialic acid-producing Lactobacillus plantarum from pickled vegetables of Chongqing farmers, and stores it in the Guangdong Microbial Culture Collection Center. The strain has simple culture and fermentation conditions, stable genetic properties, and can produce polysialic acid through liquid fermentation and secrete it outside the cell.

[0008] Preferably, the 16S rDNA base sequence of the Lactobacillus plantarum is as shown in SEQ ID NO.1.

[0009] In a second aspect, the present invention provides the use of the polysialic acid-producing Lactobacillus plantarum in producing polysialic acid.

[0010] The present invention uses Lactobacillus plantarum to ferment and produce polysialic acid, has high fermentation efficiency, and the obtained fermentation liquid has a high polysialic acid content. There is no risk of toxins being produced during the fermentation process, and the application in the fields of food, medicine, etc. has safety assurance; the fermentation process is simple to operate, has low production cost, and is easier to industrialize.

[0011] In a third aspect, the present invention provides a method for producing polysialic acid by fermentation, wherein the polysialic acid-producing Lactobacillus plantarum is used as the strain to produce a fermentation liquid containing polysialic acid.

[0012] The method for producing polysialic acid by fermentation comprises the following steps:

[0013] (1) inoculating the Lactobacillus plantarum into a seed culture medium and culturing to obtain a seed solution;

[0014] (2) The seed liquid is inoculated into a fermentation medium and fermented to obtain a fermentation liquid containing polysialic acid.

[0015] Preferably, in step (1), the seed culture medium comprises the following components in mass concentrations: 10-40 g / L glucose, 5-12 g / L yeast powder, 0.5-5 g / L magnesium sulfate heptahydrate, 0.5-1.5 g / L iron sulfate, and 0.5-3 g / L potassium dihydrogen phosphate; and the pH value of the seed culture medium is 6.0-7.0.

[0016] Preferably, in step (1), the culture conditions are: culture at 32-37°C for 24-36 hours.

[0017] Preferably, in step (2), the inoculation amount of the seed liquid is 5%.

[0018] Preferably, in step (2), the fermentation medium comprises the following components in mass concentrations: sucrose 20-80 g / L, corn pulp powder 5-15 g / L, ammonium chloride 5-11 g / L, anhydrous calcium chloride 0.5-2 g / L, cobalt chloride hexahydrate 0.5-1.5 g / L, sodium acetate 2-5 g / L, dimethyl sulfoxide 1-5 g / L, ammonium ferrous sulfate hexahydrate 0.1-0.3 g / L, magnesium sulfate heptahydrate 0.7-2 g / L, potassium dihydrogen phosphate 1-3 g / L, and PEG 0.005-0.01 g / L; the pH value of the fermentation medium is 6.5-7.2.

[0019] Preferably, in step (2), the fermentation conditions are: culturing at 32-37°C for 24-48h.

[0020] The beneficial effects of the present invention are:

[0021] The present invention separates and screens a natural polysialic acid-producing Lactobacillus plantarum obtained from pickled vegetables of Chongqing farmers, and is stored in the Guangdong Provincial Microbial Culture Collection Center. The strain culture and fermentation conditions are simple, the genetic properties are stable, and polysialic acid can be produced through liquid fermentation and secreted outside the cell. The present invention uses the plant lactobacillus (Lactiplantibacillus plantarum) for fermentation and production of polysialic acid, with high fermentation efficiency and high polysialic acid content in the obtained fermentation liquid. There is no risk of toxins being produced during the fermentation process, and the application in the fields of food, medicine, etc. has safety assurance; the fermentation process is simple to operate, the production cost is low, and it is easier to industrialize production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the colony morphological characteristics of Lactiplantibacillus plantarum BOEN-980.

[0023] Figure 2These are the microscopic morphological characteristics of Lactiplantibacillus plantarum BOEN-980. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] The Lactobacillus plantarum BOEN-980 strain of the present invention is isolated from pickled vegetables of Chongqing farmers and is stored in the strain bank of Bowen Group Co., Ltd. and the Guangdong Microbial Strain Collection Center.

[0026] Example 1: Isolation and identification of strains

[0027] (1) Strain isolation and morphological observation

[0028] The polysialic acid-producing Lactobacillus plantarum of the present invention is isolated from pickled kimchi of Chongqing farmers, and the strain is named as strain BOEN-980.

[0029] The isolated strain BOEN-980 was subjected to colony morphology and strain microscopic observation.

[0030] The results of colony morphology and microscopic morphology observation of strain BOEN-980 are as follows Figure 1 , 2 As shown. The results of colony morphology observation showed that ( Figure 1 ), the colonies of strain BOEN-980 were round, white, with neat edges, moist and smooth surface, sticky and easy to pick up. Microscopic examination results showed that strain BOEN-980 was a bacillus ( Figure 2 ), arranged singly or in short chains with blunt ends.

[0031] (2) Molecular biological characteristics

[0032] The 16S rRNA gene of strain BOEN-980 was PCR amplified and sequenced, and the length of the 16SrRNA gene fragment was measured to be 1407bp, and its nucleotide sequence is shown in SEQ ID NO.1. The gene sequence was compared with the gene sequence logged in Genbank by Blast analysis, and it was found that the strain was Lactobacillus (lactobacillus), and had 99% similarity with the 16S rRNA sequence of the strain Lactiplantibacillus plantarum in the database. Combining this result with the morphological and physiological and biochemical identification characteristics of the strain, the bacterium was identified as Lactobacillus plantarum (Lactiplantibacillusplantarum). The bacterium was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 13, 2023, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No.63560.

[0033] Embodiment 2:

[0034] The method for producing polysialic acid by fermentation using polysialic acid-producing Lactobacillus plantarum of the present invention comprises the following steps:

[0035] (1) picking up the Lactobacillus plantarum isolated and obtained in Example 1 and stored on the slant, inoculating it into a seed culture medium, and culturing it at 37° C. for 24 h to obtain a seed solution;

[0036] (2) The seed liquid is inoculated into a fermentation medium at an inoculum rate of 5%, and fermented at 37° C. for 24 hours to obtain a fermentation liquid containing polysialic acid.

[0037] The seed culture medium comprises the following components in mass concentrations: 10 g / L glucose, 5 g / L yeast powder, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L iron sulfate, 0.5 g / L potassium dihydrogen phosphate, and pH 6.0-7.0; the seed culture medium is prepared with distilled water.

[0038] The fermentation medium comprises the following components in mass concentrations: 20 g / L sucrose, 5 g / L corn pulp powder, 5 g / L ammonium chloride, 0.5 g / L anhydrous calcium chloride, 0.5 g / L cobalt chloride hexahydrate, 2 g / L sodium acetate, 1 g / L dimethyl sulfoxide (DMSO), 0.1 g / L ammonium ferrous sulfate hexahydrate, 0.7 g / L magnesium sulfate heptahydrate, 1 g / L potassium dihydrogen phosphate, 0.005 g / L PEG, pH 6.5-7.2; sterilized at 121° C. for 20 min.

[0039] Embodiment 3:

[0040] The method for producing polysialic acid by fermentation using polysialic acid-producing Lactobacillus plantarum of the present invention comprises the following steps:

[0041] (1) picking up the Lactobacillus plantarum isolated and obtained in Example 1 and stored on the slant, inoculating it into a seed culture medium, and culturing it at 35° C. for 24 h to obtain a seed solution;

[0042] (2) The seed liquid was inoculated into the fermentation medium at an inoculum rate of 5%, and fermented at 35° C. for 42 h to obtain a fermentation liquid containing polysialic acid.

[0043] The seed culture medium comprises the following components in mass concentrations: 30 g / L glucose, 8 g / L yeast powder, 2 g / L magnesium sulfate heptahydrate, 1 g / L iron sulfate, 1.5 g / L potassium dihydrogen phosphate, and pH 6.0-7.0; the seed culture medium is prepared with distilled water.

[0044] The fermentation medium comprises the following components in mass concentrations: 40 g / L sucrose, 12 g / L corn pulp powder, 7 g / L ammonium chloride, 1 g / L anhydrous calcium chloride, 1 g / L cobalt chloride hexahydrate, 3 g / L sodium acetate, 2.5 g / L dimethyl sulfoxide (DMSO), 0.15 g / L ammonium ferrous sulfate hexahydrate, 1.2 g / L magnesium sulfate heptahydrate, 2 g / L potassium dihydrogen phosphate, and 0.006 g / L PEG; pH 6.5-7.2; sterilization at 121° C. for 20 min.

[0045] Embodiment 4:

[0046] The method for producing polysialic acid by fermentation using polysialic acid-producing Lactobacillus plantarum of the present invention comprises the following steps:

[0047] (1) picking up the Lactobacillus plantarum isolated and obtained in Example 1 and stored on the slant, inoculating it into a seed culture medium, and culturing it at 32° C. for 24 h to obtain a seed solution;

[0048] (2) The seed liquid was inoculated into the fermentation medium at an inoculum rate of 5%, and fermented at 32° C. for 48 h to obtain a fermentation liquid containing polysialic acid.

[0049] The seed culture medium comprises the following components in mass concentrations: 40 g / L glucose, 12 g / L yeast powder, 5 g / L magnesium sulfate heptahydrate, 1.5 g / L iron sulfate, 3 g / L potassium dihydrogen phosphate, and pH 6.0-7.0; the seed culture medium is prepared with distilled water.

[0050] The fermentation medium comprises the following components in mass concentrations: 80 g / L sucrose, 15 g / L corn pulp powder, 11 g / L ammonium chloride, 2 g / L anhydrous calcium chloride, 1.5 g / L cobalt chloride hexahydrate, 5 g / L sodium acetate, 5 g / L dimethyl sulfoxide (DMSO), 0.3 g / L ammonium ferrous sulfate hexahydrate, 2 g / L magnesium sulfate heptahydrate, 3 g / L potassium dihydrogen phosphate, 0.01 g / L PEG, pH 6.5-7.2; sterilized at 121° C. for 20 min.

[0051] Test Example 1: Determination of polysialic acid content in fermentation broth

[0052] The content of polysialic acid in the polysialic acid-containing fermentation broth prepared in Example 2-4 was detected by high performance liquid chromatography, and the detection conditions were: high performance liquid chromatography (Waters, e2695) was used for determination, chromatographic column: Bio-Rad Aminex HPX-87H chromatographic column (300 mm×7.8 mm, 9 μm), column temperature: 50°C, mobile phase: a mixture of 3.5 mmol / L sulfuric acid and acetonitrile (sulfuric acid: acetonitrile volume ratio = 99:1), flow rate: 0.6 mL / min, detection wavelength: 210 nm, detector: ultraviolet detector, injection volume: 5 μL.

[0053] Table 1 Determination of polysialic acid content in fermentation broth

[0054] Example Example 2 Example 3 Example 4 Polysialic acid content (g / L) 14.5 16.2 15.7

[0055] The test results are shown in Table 1. The polysialic acid content in the polysialic acid-containing fermentation broth prepared in Example 2-4 can reach up to 16.2 g / L, indicating that the polysialic acid content of Lactobacillus plantarum BOEN-980 is relatively high when liquid fermented under the culture conditions of the present invention.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A polysialic acid-producing Lactobacillus plantarum, characterized in that: The Lactobacillus plantarum was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 13, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 63560.

2. The polysialic acid-producing Lactobacillus plantarum according to claim 1, characterized in that The 16S rDNA base sequence of the Lactobacillus plantarum is shown in SEQ ID NO.

1.

3. Use of the polysialic acid-producing Lactobacillus plantarum as claimed in claim 1 or 2 in producing polysialic acid.

4. A method for producing polysialic acid by fermentation, characterized in that: The polysialic acid-producing Lactobacillus plantarum according to any one of claims 1 to 2 is used as a strain to produce a fermentation liquid containing polysialic acid.

5. The method for producing polysialic acid by fermentation according to claim 4, characterized in that: The steps include: (1) inoculating the Lactobacillus plantarum according to any one of claims 1 to 2 into a seed culture medium and culturing to obtain a seed solution; (2) The seed liquid is inoculated into a fermentation medium and fermented to obtain a fermentation liquid containing polysialic acid.

6. The method for producing polysialic acid by fermentation according to claim 5, characterized in that: In the step (1), the seed culture medium comprises the following components in mass concentrations: 10-40 g / L glucose, 5-12 g / L yeast powder, 0.5-5 g / L magnesium sulfate heptahydrate, 0.5-1.5 g / L iron sulfate, and 0.5-3 g / L potassium dihydrogen phosphate; and the pH value of the seed culture medium is 6.0-7.

0.

7. The method for producing polysialic acid by fermentation according to claim 5, characterized in that: In the step (1), the culture conditions are: culture at 32-37°C for 24h-36h.

8. The method for producing polysialic acid by fermentation according to claim 5, characterized in that: In the step (2), the inoculation amount of the seed liquid is 5%.

9. The method for producing polysialic acid by fermentation according to claim 5, characterized in that: In the step (2), the fermentation medium comprises the following components in mass concentrations: 20-80 g / L sucrose, 5-15 g / L corn pulp powder, 5-11 g / L ammonium chloride, 0.5-2 g / L anhydrous calcium chloride, 0.5-1.5 g / L cobalt chloride hexahydrate, 2-5 g / L sodium acetate, 1-5 g / L dimethyl sulfoxide, 0.1-0.3 g / L ammonium ferrous sulfate hexahydrate, 0.7-2 g / L magnesium sulfate heptahydrate, 1-3 g / L potassium dihydrogen phosphate, and 0.005-0.01 g / L PEG; the pH value of the fermentation medium is 6.5-7.

2.

10. The method for producing polysialic acid by fermentation according to claim 5, characterized in that: In the step (2), the fermentation conditions are: culturing at 32-37°C for 24-48 hours.

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