Lactobacillus plantarum strain and application thereof

Abstract

The invention discloses a lactobacillus plantarum strain and application thereof, the lactobacillus plantarum strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is July 29, 2024, and the preservation number is CGMCC NO.31485. The lactobacillus plantarum strain is named as lactobacillus plantarum, and the lactobacillus plantarum strain is named as lactobacillus plantarum. The lactobacillus plantarum C0502 can effectively realize degradation of xylan, not only provides a new way for realizing high-value utilization of xylan, but also can promote growth and metabolism of lactic acid bacteria, so that the lactobacillus plantarum C0502 can greatly reduce the production cost of probiotics and metabolites thereof, and has great application value and economic benefit.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum strain and application thereof. Background Art

[0002] Xylan is the main hemicellulose component in plant cell walls, accounting for about 15% to 35% of the dry weight of plant cells. It is the most abundant polysaccharide in nature besides cellulose. Therefore, the development and utilization of xylan is one of the important measures to achieve high-value utilization of renewable biomass resources - lignocellulose.

[0003] Although xylan is a type of renewable sugar that is widely distributed and abundant in nature, and is a potential cheap carbon source for microbial biotransformation, its comprehensive utilization in biotransformation is seriously restricted due to its stable structure, complex composition, and slow self-degradation process. At present, the research on microbial transformation of xylan mainly focuses on two aspects. On the one hand, chemical hydrolysis is first used to hydrolyze xylan into fermentable sugars that can be efficiently utilized by microorganisms, and then microorganisms are used for biotransformation. However, the process of chemical hydrolysis of lignin is accompanied by the production of certain fermentation inhibitors, such as 4-hydroxybenzaldehyde, hydroxymethylfurfural, etc., which will have a greater inhibitory effect on subsequent biotransformation reactions. On the other hand, enzymatic hydrolysis is first used to hydrolyze xylan into fermentable sugars that can be efficiently utilized by microorganisms, and then the microorganisms are used for bioconversion. However, due to the complex composition of xylan, the hydrolysis of xylan generally requires the efficient synergistic action of multiple enzymes such as endo-β-l, 4-D-xylanase, β-D-xylosidase, α-L-furanarabinosidase, α-D-glucuronidase, α-D-glucuronidase, acetylxylan esterase and phenolic acid esterase, which undoubtedly increases the bioconversion cost of xylan.

[0004] Probiotics are common dietary supplements in the field of nutrition and health. Screening potential probiotics whose growth and metabolism can be promoted by xylan can not only achieve high-value utilization of xylan in biotransformation, but also greatly reduce the production cost of probiotics and their metabolites, which has great application value and economic benefits. Summary of the invention

[0005] 1. Purpose of the invention The purpose of the present invention is to provide a Lactobacillus plantarum strain having excellent xylan degradation ability, and application of the Lactobacillus plantarum strain in efficiently degrading xylan.

[0006] (II) Technical solution To solve the above problems, the first aspect of the present invention provides a plant lactobacillus strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of July 29, 2024 and a deposit number of CGMCC NO.31485. The plant lactobacillus strain is named Lactiplantibacillus plantarum.

[0007] Hereinafter, the Lactobacillus plantarum with the deposit number of CGMCC NO.31485 will be referred to as Lactobacillus plantarum C0502.

[0008] According to another aspect of the present invention, the present invention also provides the use of the above-mentioned Lactobacillus plantarum C0502 in degrading xylan.

[0009] Preferably, the application comprises the following steps: Adding 0.5wt%-10wt% of xylan and a bacterial suspension of Lactobacillus plantarum C0502 to the MRS medium; The formula of the MRS culture medium is: 10 g / L peptone, 20 g / L glucose, 10 g / L beef extract, 5 g / L yeast powder, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.054 g / L manganese sulfate pentahydrate.

[0010] Preferably, the application comprises the following steps: Adding 5wt%-60wt% of xylan and a bacterial suspension of Lactobacillus plantarum C0502 to a sugar-free MRS medium; The formula of the sugar-free MRS medium is: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.054 g / L manganese sulfate pentahydrate.

[0011] Preferably, the bacterial suspension of Lactobacillus plantarum C0502 is obtained by the following method: The glycerol cryopreserved solution of Lactobacillus plantarum C0502 was activated and cultured twice in MRS medium until the end of the logarithmic growth phase. The obtained bacterial solution was centrifuged to remove the supernatant, and after washing, sterile water was added to prepare a bacterial suspension with an OD600 of 0.6-0.8.

[0012] Preferably, the glycerol cryopreserved solution of Lactobacillus plantarum C0502 is activated and cultured twice in MRS medium until the end of the logarithmic growth phase, specifically comprising: The glycerol frozen solution of Lactobacillus plantarum C0502 was added to the MRS medium, and cultured at 35°C to 40°C for 20 to 30 hours. The obtained bacterial solution was added to the MRS medium, and cultured at 35°C to 40°C for 10 to 15 hours.

[0013] Preferably, the glycerol cryopreservation solution of Lactobacillus plantarum C0502 is obtained by the following method: The Lactobacillus plantarum C0502 is cultured to the end of the logarithmic growth phase, the obtained bacterial solution is mixed with a glycerol solution with a mass fraction of 45% to 55%, and the mixture is frozen and stored at -80°C to obtain the glycerol cryopreservation solution of the Lactobacillus plantarum C0502.

[0014] Preferably, the volume ratio of the bacterial solution to the glycerol solution is 1:1-1.2.

[0015] (III) Beneficial effects The above technical solution of the present invention has the following beneficial technical effects: Xylan is a hemicellulose that is widely found in hardwood, grass, cereals, herbaceous plants and algae. However, xylan has a stable structure and complex composition, and requires the joint action of a series of enzymes to degrade it in order to achieve its high-value bioconversion utilization.

[0016] The experiment of the present application shows that compared with other plant lactobacillus of the same genus, xylan has shown the effect of promoting the growth of plant lactobacillus C0502 whose deposit number is CGMCC NO.31485, and the output of lactic acid has been improved along with the improvement of xylan concentration, and the screening result of carbohydrate active enzyme of plant lactobacillus C0502 shows that 4 kinds of xylanases are annotated in the plant lactobacillus C0502 genome, including acetyl xylan esterase, ferulic acid esterase, beta-xylosidase and alpha-D-glucuronidase, which are all xylanases needed for xylan degradation. This fully illustrates that plant lactobacillus C0502 can effectively realize the degradation of xylan, not only provides a promising approach for realizing the high value utilization of xylan, but also can promote the growth and metabolism of lactic acid bacteria, so that the present invention can greatly reduce the production cost of probiotics and metabolites thereof, and has greater application value and economic benefit.

[0017] Strain deposit information The plant lactobacillus strain was named Lactiplantibacillus plantarum and was deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.31485 and the deposit date is July 29, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the growth curve of Lactobacillus plantarum C0502 in MRS medium and MRS medium supplemented with 1 g / L xylan; Figure 2 This is a cell morphology diagram of Lactobacillus plantarum C0502; Figure 3 is the optical morphology of Lactobacillus plantarum C0502; Figure 4 is the phylogenetic tree of Lactobacillus plantarum C0502; Figure 5 is the effect of adding different concentrations of xylan in MRS medium on the growth of Lactobacillus plantarum C0502; Figure 6 The effect of adding different concentrations of xylan to MRS medium on the lactic acid metabolism of Lactobacillus plantarum C0502, where different letters on the bars of different groups indicate significant differences between the groups; Figure 7 The effect of removing glucose and adding different concentrations of xylan in MRS medium on the growth of Lactobacillus plantarum C0502; Figure 8 The effect of removing glucose and adding different concentrations of xylan in MRS medium on lactic acid metabolism of Lactobacillus plantarum C0502, where different letters on the bars of different groups indicate significant differences between the groups; Fig. 9 These are the main structural features of xylan and the action sites of different xylanases (xylanases annotated in the genome of Lactobacillus plantarum C0502 have gray bottom boxes, including acetylxylan esterase, α-D-glucuronidase, β-xylosidase and ferulic acid esterase). DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0020] The application finds that compared to other plant lactobacilli of the same genus, xylan has shown the effect of promoting the growth of plant lactobacillus C0502 with a deposit number of CGMCC NO.31485, and the output of lactic acid has been increasing with the increase of xylan concentration, and the screening result of carbohydrate active enzyme of plant lactobacillus C0502 shows that 4 kinds of xylanases are annotated in the plant lactobacillus C0502 genome, including acetyl xylan esterase, ferulic acid esterase, beta-xylosidase and alpha-D-glucuronidase, which are all xylanases required for xylan degradation. This fully illustrates that plant lactobacillus C0502 can effectively realize the degradation of xylan, not only provides a promising way for realizing the high-value utilization of xylan, but also can promote the growth and metabolism of lactic acid bacteria, thereby can greatly reduce the production cost of probiotics and their metabolites, with greater application value and economic benefit.

[0021] Based on the above findings, on the one hand, the present invention provides a plant lactobacillus strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a preservation date of July 29, 2024 and a preservation number of CGMCC NO.31485. The plant lactobacillus strain is named Lactiplantibacillus plantarum.

[0022] On the other hand, the present invention also provides the use of Lactobacillus plantarum strain C0502 in degrading xylan.

[0023] According to the present invention, the bacterial suspension formed after activation of Lactobacillus plantarum C0502 can be added to the MRS medium added with xylan to achieve the degradation of xylan.

[0024] According to the present invention, the bacterial suspension formed after activation of Lactobacillus plantarum C0502 can also be added to the sugar-free MRS medium added with xylan to achieve the degradation of xylan. The sugar-free MRS medium described in the present invention refers to the MRS medium without glucose.

[0025] The following examples will further illustrate the present invention, but are not intended to limit the present invention.

[0026] The culture medium used in the following examples is as follows: Lactic acid bacteria liquid medium (MRS): peptone 10 g / L, glucose 20 g / L, beef extract 10 g / L, yeast powder 5 g / L, Tween 80 1 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, sodium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate pentahydrate 0.054 g / L, used for the culture and counting of lactic acid bacteria; MRS-X medium: 1 g / L of xylan is added to MRS medium for screening lactic acid bacteria that can be proliferated by xylan.

[0027] Example 1: Screening and identification of lactic acid bacteria that can be proliferated by xylan 1. Screening, identification and preservation of strains Xylan is a hemicellulose widely found in hardwoods, grasses, cereals and herbaceous plants, as well as algae. Screening for lactic acid bacteria that can be significantly proliferated by xylan not only provides a promising approach to achieve high-value utilization of xylan, but also can promote the growth and metabolism of lactic acid bacteria.

[0028] The present application obtains a batch of lactic acid bacteria from the dilution of fermented fermented bean curd through enrichment culture, separation and screening. The specific process of enrichment culture, separation and screening is as follows: Add 1 g of fermented bean curd into 10 mL of normal saline and shake to mix, dilute in a 10-fold gradient, select the appropriate dilution and spread it on MRS solid culture medium, culture it at 37°C for 48 h, pick several typical single colonies, streak them on MRS solid culture medium for purification, and enrich and culture them in MRS liquid culture medium, and separate and screen to obtain a batch of lactic acid bacteria.

[0029] By comparing the growth curves of these lactic acid strains in MRS medium containing 1g / L xylan and in MRS medium, strains that may be proliferated by xylan were screened. The specific process of screening strains that may be proliferated by xylan is as follows: The glycerol frozen solution (0.2 mL) of various lactic acid bacteria obtained by the above screening was inoculated into a test tube containing 4.5 mL of MRS and cultured at 37°C and 200 rpm for 24 h for pre-culture.

[0030] Then, 0.5 mL of the pre-cultured bacterial solution was added to 4.5 mL of MRS medium and cultured at 37°C and 200 rpm for 12 h until the end of the logarithmic growth phase. After washing twice, sterile water was added to make an OD 600 =0.6, and then inoculated into ordinary MRS medium and MRS medium containing 1 g / L xylan at an inoculum size of 1% to obtain bacterial suspensions in two culture media.

[0031] Then the bacterial suspension was added to a 96-well plate, and the growth curves of each lactic acid bacteria in the two culture media were monitored using a microbial growth characteristic instrument (BioTek LogPhase 600, Agilent, USA). Finally, it was found that one strain had a significant proliferation effect of xylan, such as Figure 1 Shown is a comparison of the growth curves of the strain in the lactic acid bacteria MRS medium and the MRS medium supplemented with 1 g / L xylan. The strain was named C0502.

[0032] 2. Identification of strains 2.1 Morphological characteristics of strains: Streak strain C0502 on an MRS plate and incubate at 37°C for 24 h before observation. Figure 2 As shown in the figure, the colonies on the MRS medium are milky white, round, with neat edges, opaque, raised, and easy to pick. The strain was gram-stained and observed under an optical microscope. Figure 3 As shown, the bacteria are rod-shaped and are Gram-positive bacteria.

[0033] 2.2 Molecular identification Strain C0502 was cultured to the end of logarithmic growth, washed twice, and then PCR amplified 16s rDNA. The quantity and purity of the PCR amplification product were determined by Nanodrop (Nanodrop 2000 spectrophotometer, Thermo Scientific). The quality of the amplified product was then detected by 1.5% agarose gel electrophoresis. Sequencing was performed at Sangon Biotechnology Co., Ltd. (Shanghai, China). Subsequently, the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) was used to identify the 16S rDNA gene sequence at the species level. It was found that the strain C0502 had a similarity of 99% with Lactiplantibacillus plantarum; therefore, C0502 was identified as Lactobacillus plantarum and named Lactiplantibacillus plantarum. Its phylogenetic tree is shown in the figure below. Figure 4 shown.

[0034] 3. Preservation of strains After culturing Lactobacillus plantarum C0502 to the end of the logarithmic growth phase, 0.5 mL of the bacterial solution was mixed with 0.5 mL of 50% sterile glycerol solution in a cryopreservation tube and stored at -80°C for later use.

[0035] Example 2: Verification of the promotion of growth and lactic acid metabolism of Lactobacillus plantarum C0502 by xylan To further evaluate the effects of different concentrations of xylan on the growth and metabolism of Lactobacillus plantarum C0502, glycerol frozen solution (0.2 mL) of Lactobacillus plantarum C0502 was inoculated into a test tube containing 4.5 mL of MRS and cultured at 37°C, 200 rpm for 24 h for pre-culture. 0.5 mL of the pre-cultured bacterial solution was added to 4.5 mL of MRS medium and cultured at 37°C, 200 rpm for 12 h. After washing twice, sterile water was added to make a bacterial suspension with OD600=0.6, and then inoculated into different culture media at an inoculum size of 1% and cultured at 37°C for 48 h. These culture media included MRS medium with different concentrations of xylan (0, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%), and completely replacing glucose in MRS medium with different concentrations of xylan (0, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%).

[0036] The growth curve of Lactobacillus plantarum C0502 in different culture media was measured by a microbial growth characteristic instrument (BioTek LogPhase 600). After 48 h of culture, the metabolite lactic acid of Lactobacillus plantarum C0502 was determined by high performance liquid chromatography (HPLC, Thermo Fisher Scientific, USA).

[0037] Sample treatment: 0.25 mL of 150 g / L K4Fe(CN)6 and 0.25 mL of 300 g / LZnSO4 were added to 1 mL of sample, mixed thoroughly and allowed to stand for 30 min. Subsequently, the mixture was centrifuged at 10000g for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane.

[0038] Liquid phase conditions: chromatographic column: organic acid column (250 mm× 4.6 mm, 5μm, Avantor, New Jersey, USA); mobile phase: 25 mmol / L K2HPO4 (pH adjusted to 2.5 with phosphoric acid); flow rate: 0.8 mL / min; column temperature: 40℃; detector: ultraviolet detector (UV, Thermo Fisher Scientific); detection wavelength: 210 nm.

[0039] The results showed that compared with MRS medium, MRS medium supplemented with 0.5%-10% xylan was more conducive to the growth of Lactobacillus plantarum C0502, especially the addition of 0.5% low-concentration xylan, while high-concentration xylan showed an inhibitory effect on the growth of Lactobacillus plantarum C0502, such as Figure 5 As shown in Figure 2, the production of lactic acid increases with the increase of xylan concentration, as shown in Figure 2. Figure 6 As shown. Figure 7 As shown in the figure, adding xylan to the MRS medium without glucose also showed the effect of promoting the growth of Lactobacillus plantarum C0502, among which the addition of 5% xylan had the best effect. As the xylan concentration increased, it gradually showed the phenomenon of inhibiting the growth of C0502, especially the xylan concentration of 40%-60% had the most obvious inhibitory effect. However, the production of lactic acid increased with the increase of xylan concentration, such as Figure 8 shown.

[0040] Example 3: Xylan metabolizing enzymes annotated by genome analysis of Lactobacillus plantarum C0502 Xylan degradation requires the combined action of a series of enzymes, including (1) endo-1,4-β-xylanase (EC 3.2.1.8), which mainly hydrolyzes the β-1,4-glycosidic bonds of the xylan backbone to generate oligosaccharides; (2) β-xylosidase (EC 3.2.1.37), which mainly acts on the reducing end of xylo-oligosaccharides to generate xylose; (3) α-L-arabinofuranosidase (EC 3.2.1.55), which is responsible for cleaving α-L-arabinose bonds; (4) α-D-glucuronidase (EC 3.2.1.131), which mainly acts on α-1,2-glycosidic bonds to release glucuronic acid side chains; (5) acetyl xylan esterase (EC 3.2.1.60), which mainly acts on α-1,2-glycosidic bonds to release glucuronic acid side chains; Esterase (EC 3.1.1.72) removes the terminal acetate group by cleaving the ester bond; (6) ferulic acid esterase (EC 3.1.1.73) and p-coumaric acid esterase (EC 3.2.1.x).

[0041] Lactobacillus plantarum C0502 was cultured to the end of the logarithmic growth phase and centrifuged (14,000g, 4℃, 5 min) to collect the bacterial sludge. The samples were sent to Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) for DNA extraction and sequencing analysis to obtain the whole genome sequence, which has been uploaded to the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), GenBank No. GCA 040717425.1. Then, the carbohydrate-active enzyme of Lactobacillus plantarum C0502 was predicted using the Carbohydrate-Active Enzyme database (CAZY, http: / / www.cazy.org / ), and xylanase was manually screened. Fig. 9As shown in Table 1, a total of four xylanases were annotated in the genome of Lactobacillus plantarum C0502, including acetylxylan esterase, ferulic acid esterase, β-xylosidase and α-D-glucuronidase.

[0042] Table 1 All xylanases annotated in the genome of Lactobacillus plantarum C0502

[0043] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A plant lactobacillus strain, characterized in that The plant lactobacillus strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 29, 2024, the deposit number is CGMCC NO.31485, and the plant lactobacillus strain is named Lactiplantibacillus plantarum.

2. Use of the plant lactobacillus strain as claimed in claim 1 in degrading xylan.

3. The use according to claim 2, characterized in that: The application comprises the following steps: Adding 0.5wt%-10wt% of xylan and a suspension of Lactobacillus plantarum with a preservation number of CGMCC NO.31485 to MRS medium; The formula of the MRS medium is: 10 g / L peptone, 20 g / L glucose, 10 g / L beef extract, 5 g / L yeast powder, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.054 g / L manganese sulfate pentahydrate.

4. The use according to claim 2, characterized in that: The application comprises the following steps: Adding 5wt%-60wt% of xylan and a suspension of Lactobacillus plantarum with a preservation number of CGMCC NO.31485 to a sugar-free MRS medium; The formula of the sugar-free MRS medium is: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L sodium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.054 g / L manganese sulfate pentahydrate.

5. The use according to claim 3 or 4, characterized in that: The bacterial suspension of Lactobacillus plantarum with the deposit number of CGMCC NO.31485 is obtained by the following method: The glycerol frozen solution of Lactobacillus plantarum with the deposit number of CGMCC NO.31485 was activated and cultured twice in MRS medium until the end of the logarithmic growth phase. The obtained bacterial solution was centrifuged to remove the supernatant, and after washing, sterile water was added to prepare a bacterial suspension with an OD600 of 0.6-0.

8.

6. The use according to claim 5, characterized in that: The method of activating and culturing the frozen glycerol solution of Lactobacillus plantarum with the deposit number of CGMCC NO.31485 twice in MRS medium until the end of the logarithmic growth phase specifically comprises: The glycerol frozen solution of Lactobacillus plantarum with the preservation number of CGMCC NO.31485 is added to the MRS medium, and cultured at 35°C to 40°C for 20 to 30 hours. The obtained bacterial solution is added to the MRS medium, and cultured at 35°C to 40°C for 10 to 15 hours.

7. The use according to claim 5, characterized in that: The glycerol cryopreservation solution of Lactobacillus plantarum with the deposit number of CGMCC NO.31485 is obtained by the following method: The plant lactobacillus with a preservation number of CGMCC NO.31485 is cultured to the end of the logarithmic growth phase, the obtained bacterial solution is mixed with a glycerol solution with a mass fraction of 45% to 55%, and is frozen and stored at -80°C to obtain a glycerol frozen solution of the plant lactobacillus with a preservation number of CGMCC NO.31485.

8. The use according to claim 7, characterized in that: The volume ratio of the bacterial solution to the glycerol solution is 1:1-1.2.

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