Phytobacterium plantarum WKLPP0041 with high BSH activity as well as mutagenesis screening method and application of phytobacterium plantarum WKLPP0041

Abstract

The invention discloses a plant lactobacillus WKLPP0041 strain with high BSH activity as well as a mutagenesis screening method and application of the plant lactobacillus WKLPP0041 strain, and belongs to the technical field of microorganisms. The plant lactobacillus WKLPP0041 is preserved in the China Center for Type Culture Collection on December 30, 2024, the preservation address is Wuhan, China, and the preservation number is CCTCC NO: M 20242958. According to the invention, gradient mutagenesis treatment is carried out on the screened plant lactobacillus WKLPP0040 with the BSH activity advantage through a physical mutagenesis-ultraviolet induction method, and a mutant strain, namely the plant lactobacillus WKLPP0041, of which the BSH activity is remarkably improved is obtained through screening. The BSH (bile salt hydrolase) activity and cholesterol degradation rate of the strain are remarkably improved, and the strain can improve blood fat, reduce inflammation and improve immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus plantarum WKLPP0041 with high BSH activity, a mutagenesis screening method and an application thereof. Background Art

[0002] Studies have shown that intestinal microorganisms are closely related to metabolism, health, and the occurrence and development of diseases. Bile salt hydrolase (BSH) is a metabolite produced during the growth and reproduction of microorganisms. This enzyme is mainly produced by lactic acid bacteria. Studies have found that BSH has an important regulatory effect on intestinal microorganisms and host physiological metabolism. On the one hand, BSH helps microorganisms to colonize and adhere to the intestine. As a survival and self-defense mechanism of probiotics, BSH can help bacteria resist the toxic effects of bile acids in the host, thereby improving the survival rate of strains in the gastrointestinal tract. In addition, since glycine may be metabolized by probiotics into ammonia and carbon dioxide, taurine can be metabolized into ammonia, carbon dioxide, and sulfate. Therefore, probiotics that produce BSH can use the amino acids released by hydrolysis-coupled bile acids as a source of available carbon, nitrogen, and energy, promote the rapid proliferation and effective colonization of probiotics, effectively regulate the intestinal flora microenvironment, and improve the body's nutrition and inflammatory response; on the other hand, BSH regulates bile acid metabolism, thereby affecting the host's digestion and absorption of lipid substances, cholesterol metabolism, energy metabolism, and inflammatory response.

[0003] At present, the research on BSH active strains is mainly focused on two aspects. On the one hand, functional strains with BSH activity are obtained by mutagenesis or construction of genetically engineered bacteria. On the other hand, relevant functional research is carried out based on the BSH production function of the strains. The existing technology has confirmed that strains containing BSH activity have anti-inflammatory and lipid-improving functions. However, genetically engineered bacteria prepared by bioengineering cannot be used in food and health foods due to their biosafety and regulatory restrictions. At present, the functional probiotics industry has a relative shortage of strains with high BSH activity. Physical or chemical mutagenesis methods are used to induce mutations in isolated natural strains to obtain high-performance strains that produce BSH, and to study and expand their applications in the field of functional foods and health foods, which are of great value and significance to the development of the functional probiotics industry. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art and promote the development and application of strains with high BSH activity, the present invention provides a strain of Lactobacillus plantarum WKLPP0041 with high BSH activity, a mutagenesis screening method and application thereof.

[0005] The present invention provides a plant lactobacillus (Lactiplantibacillus plantarum) WKLPP0041, which is subjected to gradient mutagenesis by a physical mutagenesis-ultraviolet induction method, and the bile salt hydrolase (BSH) activity and cholesterol degradation rate of the strain are significantly improved. The present invention also provides the application of the plant lactobacillus WKLPP0041 powder and its postbiotics in improving blood lipids, regulating intestinal flora imbalance, improving intestinal microenvironment, reducing inflammation and enhancing immunity.

[0006] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a high BSH activity Lactobacillus plantarum, wherein the Lactobacillus plantarum is obtained by mutagenesis screening of the naturally selected strain Lactobacillus plantarum WKLPP0040, and is named Lactobacillus plantarum WKLPP0041, with a preservation number of CCTCC NO: M 20242958, and a classification name of Lactiplantibacillus plantarum WKLPP0041. Lactobacillus plantarum WKLPP0041 was deposited in the China Center for Type Culture Collection on December 30, 2024, with a preservation address of Wuhan, China, and a preservation number of CCTCC NO: M 20242958.

[0008] In a second aspect, the present invention provides a Lactobacillus plantarum (Lactiplantibacillus plantarum) WKLPP0040, characterized in that it was deposited in the China Center for Type Culture Collection on December 30, 2024, with the preservation address in Wuhan, China, and the preservation number is CCTCC NO: M 20242957.

[0009] In a third aspect, the present invention provides a method for mutagenesis screening of a strain of Lactobacillus plantarum WKLPP0041 with high BSH activity, comprising the following steps:

[0010] Step (1), activate and culture the strain WKLPP0040 to be induced twice, so that it enters the logarithmic growth phase, and the number of viable bacteria is ≥3.0*10 9 cfu / mL, dilute the bacterial solution concentration to obtain a bacterial suspension;

[0011] Step (2), evenly coating the diluted bacterial suspension on a culture dish containing MRS agar medium, placing it under a 20-25W ultraviolet lamp and irradiating it at a distance of 15-20cm for gradient mutagenesis treatment, and after the mutagenesis irradiation is completed, placing the plate in a light-proof environment for static culture; picking a single colony on the plate with a bacterial lethality rate of more than 95%, performing liquid culture, and detecting its bile salt hydrolase activity, and screening to obtain the first generation of optimal mutant strains;

[0012] Preferably, the gradient mutagenesis treatment is performed by irradiating for 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, and 180s respectively;

[0013] Step (3), secondary mutagenesis of the second-generation optimal mutant strain: the mutagenesis method of the bacterial solution of the first-generation mutant strain is used to select a single colony on a plate with a bacterial lethality rate of more than 95%, carry out liquid culture, and detect its bile salt hydrolase activity to screen and obtain the second-generation optimal mutant strain;

[0014] Step (4), three mutagenesis of the third-generation optimal mutant strain: the mutagenesis method of the bacterial solution of the first-generation mutant strain is followed, a single colony on a plate with a bacterial lethality rate of more than 95% is selected, liquid culture is performed, and the bile salt hydrolase activity is detected to screen and obtain the third-generation optimal mutant strain, i.e., WKLPP0041.

[0015] Furthermore, the in vitro cholesterol degradation rate of the induced mutant strain Lactobacillus plantarum WKLPP0041 was significantly improved.

[0016] In a fourth aspect, the present invention provides a postbiotic of Lactobacillus plantarum WKLPP0041 with high BSH activity.

[0017] Furthermore, the postbiotics are broken bacteria and their contents prepared by fermentation and ultrasonic disruption. 600 Value ≥ 0.6; further, the ultrasonic crushing conditions are: intermittent ultrasonic crushing 5s / 5s, continuously 60-80 times.

[0018] Furthermore, postbiotics are rich in lipoteichoic acid, with a content of ≥6.11 mg / g.

[0019] In a fifth aspect, the present invention provides an application of high BSH activity Lactobacillus plantarum WKLPP0041 to hypercholesterolemia. Based on this, the present invention provides an application of the Lactobacillus plantarum WKLPP0041 in the preparation of a product for maintaining a healthy level of blood lipids, wherein the blood lipids include cholesterol and triglycerides. The product is a health food or a medicine. Further, the present invention provides an application of the Lactobacillus plantarum WKLPP0041 in the preparation of a drug for treating hypercholesterolemia.

[0020] Furthermore, Lactobacillus plantarum WKLPP0041 can significantly reduce the levels of total cholesterol and low-density lipoprotein, while increasing the content of high-density lipoprotein; it can regulate the balance of intestinal flora and improve metabolism to reduce the level of inflammation in the body. Based on this, the present invention also provides the use of Lactobacillus plantarum WKLPP0041 in the preparation of products for regulating intestinal flora, including health foods and medicines.

[0021] Furthermore, the plant lactobacillus WKLPP0041 is freeze-dried bacterial powder, and the number of viable bacteria is not less than 4.5*10 11 cfu / g.

[0022] In a sixth aspect, the present invention provides the effect of Lactobacillus plantarum WKLPP0041 on improving immunity. Based on this, the present invention also provides the use of Lactobacillus plantarum WKLPP0041 in preparing products that enhance immunity, including health foods and medicines.

[0023] Furthermore, the postbiotics of Lactobacillus plantarum WKLPP0041 can enhance the cellular immunity and monocyte-macrophage function of mice.

[0024] In a seventh aspect, the present invention provides a microbial composition comprising Lactobacillus plantarum WKLPP0041.

[0025] Furthermore, the microbial composition is a food, a health product or a medicine.

[0026] Furthermore, the microbial composition comprises freeze-dried bacterial powder of Lactobacillus plantarum WKLPP0041 or postbiotics of Lactobacillus plantarum WKLPP0041 as a main functional component.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The BSH activity and cholesterol degradation rate of the Lactobacillus plantarum WKLPP0041 obtained by gradient ultraviolet mutagenesis treatment of the present invention are significantly improved, which are increased by 72.26-86.38% and 47.14% respectively compared with WKLPP0040.

[0029] (2) The Lactobacillus plantarum WKLPP0041 provided by the present invention can significantly improve the blood lipid status of hypercholesterolemia mice, and has a good regulatory effect on the intestinal flora of hypercholesterolemia mice. It can improve the intestinal environment, reduce the level of inflammatory factors, and improve the inflammatory condition of the body by regulating the intestinal flora.

[0030] (3) The Lactobacillus plantarum WKLPP0041 postbiotic provided by the present invention contains rich teichoic acid components, which can significantly improve the cellular immunity level of mice and the phagocytic ability of macrophages, and has the effect of enhancing immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1:WKLPP0040 microscopic image;

[0033] Figure 2 : Phylogenetic tree of 16S rDNA gene sequences of strain WKLPP0040 and related species;

[0034] Figure 3 : BSH activity detection of strain WKLPP0040 and comparison strains;

[0035] Figure 4 : Determination of the lethality of bacteria induced by single irradiation and the BSH activity of the selected UV mutant strains;

[0036] Figure 5 : Determination of the lethality of secondary irradiation-induced bacteria and BSH activity of the selected UV mutant strains;

[0037] Figure 6 :Determination of the lethality of three-irradiation-induced bacterial strains and the BSH activity of the selected UV mutant strains;

[0038] Figure 7 :Determination of the lethality of the four-time irradiation-induced bacteria and the BSH activity of the selected UV mutant strains;

[0039] Figure 8 : Cholesterol degradation rate of different strains in vitro;

[0040] Fig. 9 : Phylogenetic tree of 16S rDNA gene sequences of strain WKLPP0041 and related species;

[0041] Fig.10 : Survival rate of acid resistance test of strain WKLPP0041;

[0042] Fig.11 : The survival rate of strain WKLPP0041 in bile salt tolerance test, wherein (a) is the survival rate of strain WKLPP0041 in 0.2% bile salt environment, and (b) is the survival rate of strain WKLPP0041 in 0.2% bile salt environment;

[0043] Fig.12 :WKLPP0041 BSH activity genetic stability;

[0044] Fig.13 :The content of postbiotic lipoteichoic acid in different strains, * indicates comparison with CK group, and * indicates P < 0.05, i.e. significant difference;

[0045] Fig.14:Detection chart of four blood lipid indexes of mice (TC, TG, HDL, LDL), Notes * and ** indicate comparison with the model group, and * indicates P < 0.05, that is, significant difference, ** indicates P < 0.01, that is, extremely significant difference; # indicates comparison with 0040, and # indicates P < 0.05, that is, significant difference;

[0046] Fig.15 : Detection chart of mouse inflammatory factors (TNF-α, IL-6), *, ** indicate comparison with the model group, and * indicates P < 0.05, that is, significant difference, ** indicates P < 0.01, that is, extremely significant difference; & indicates comparison with the positive group, and & indicates P < 0.05, that is, significant difference;

[0047] Fig.16 :The delayed allergic reaction degree detection diagram of different groups, the notes * and ** indicate comparison with the blank group, and * indicates P < 0.05, i.e. significant difference, and ** indicates P < 0.01, i.e. extremely significant difference;

[0048] Fig.17 : Phagocytic index detection diagram of different groups, notes *, ** indicate comparison with the blank group, and * represents P < 0.05, i.e. significant difference, ** represents P < 0.01, i.e. extremely significant difference. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is described by the following specific embodiments, but is by no means limited thereto. The following are preferred embodiments of the present invention, which are only used to describe the present invention and cannot be understood as limiting the present invention. It should be pointed out that any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0050] Example 1 Mutagenesis screening of a strain of Lactobacillus plantarum WKLPP0041 with high BSH activity

[0051] 1. Isolation and purification of the original strain WKLPP0040

[0052] (1) Under sterile conditions, 2 g of Jilin Yanji sauerkraut sample was placed in a 100 mL sterile saline conical flask with glass beads, and cultured in a 37°C constant temperature shaker at 180 r / min for 20 min. The sample dilution was picked up with an inoculation loop for MRS solid plate streaking, and then placed in a 37°C constant temperature incubator for 24-48 h.

[0053] (2) After the culture is completed, a single lactic acid bacteria colony is picked from the plate, further streaked on an MRS solid plate containing CaCO3, and cultured at 37°C for 24-48 hours. A single colony with a calcium dissolution zone is selected, and after microscopic examination and purification, the bacteria are freeze-dried to prepare freeze-dried tubes, which are stored in a -80°C refrigerator for later use.

[0054] 2. Identification of WKLPP0040 strain

[0055] (1) Analysis of strain morphological characteristics

[0056] MRS medium, cultured at 37℃ for 24h, the colonies are white, with neat round edges, moist and opaque; microscopic examination shows that the bacteria are short rods, arranged singly or in pairs, and are Gram-positive bacteria ( Figure 1 ).

[0057] (2) 16S rDNA sequencing analysis

[0058] The 16S rDNA sequence of strain WKLPP0040 (SEQ ID NO: 1) was compared with the professional database based on the NCBI database, and the phylogenetic tree of the gene sequences of WKLPP0040 and related species was displayed by the proximity joining method ( Figure 2 ). The results of 16SrDNA analysis showed that strain WKLPP0040 was consistent with Lactiplantibacillus plantarum. 16SrDNA gene sequence analysis and identification showed that the strain was Lactiplantibacillus plantarum and was named WKLPP0040.

[0059] It was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit address in Wuhan, China, and the deposit number is CCTCC NO: M 20242957.

[0060] 3. Mutagenesis screening of Lactobacillus plantarum WKLPP0041 with high BSH activity

[0061] (1) Activation of bacterial strains: Under sterile conditions, use an inoculation loop to take the freeze-dried bacterial strain WKLPP0040 and inoculate it on a solid MRS slant medium and culture it at 37°C for 16-24 h;

[0062] (2) Liquid culture: Take the activated slant and inoculate it into MRS liquid culture medium under sterile conditions. Incubate it at 37°C for 16-24 hours to obtain bacterial culture liquid.

[0063] (3) Liquid secondary activation: Take the bacterial liquid, 2-5% inoculation amount, inoculate it into MRS liquid culture medium, and culture it at 37°C for 16-24 hours to obtain the secondary activated bacterial liquid.

[0064] (4) Gradient mutagenesis treatment and BSH activity detection:

[0065] 1) Bacterial solution dilution: The strain WKLPP0040 to be induced by activation culture was diluted to a concentration of 1*10 5 cfu / mL.

[0066] 2) Primary mutagenesis treatment: evenly spread the diluted bacterial suspension on a culture dish containing MRS agar medium, place it under a 20-25W ultraviolet lamp at 15-20cm and irradiate for 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, and 180s. After the mutagenesis irradiation, place the plate in a dark environment at 37°C and culture for 48h. Pick a single colony on a plate with a bacterial lethality rate of more than 95%, culture it in liquid, and detect its bile salt hydrolase activity. The first-generation optimal mutant strain UVP1 was screened and obtained.

[0067] 3) UVP1 secondary mutagenesis, according to the mutagenesis method of the first generation mutant strain, pick a single colony with a bacterial lethality rate of more than 95%, carry out liquid culture, and detect its bile salt hydrolase activity. The second generation optimal mutant strain UVP2 is screened and obtained.

[0068] 4) UVP2 was mutagenized three times according to the mutagenization method of the first generation mutant strain, and single colonies with a bacterial lethality rate of more than 95% were selected for liquid culture and the bile salt hydrolase activity was tested to obtain the third generation optimal mutant strain UVP3.

[0069] 5) UVP3 was induced to mutagenesis four times according to the mutagenesis method of the first generation mutant strain, and single colonies with a lethality rate of more than 95% were selected for liquid culture and the bile salt hydrolase activity was tested to obtain the fourth generation optimal mutant strain UVP4.

[0070] 6) BSH activity detection method

[0071] Take 1mL of bacterial solution (WKLPP0040\WKLPP0041) after 16-24h of culture, centrifuge at 3000rpm at 4℃ for 10min; discard the supernatant, resuspend the precipitate with 1mL 0.1M phosphate buffer, centrifuge at 12000rpm for 2min, discard the supernatant, repeat the operation once; resuspend the precipitate with 1mL Vc phosphate buffer, transfer to a 4mL Ep tube, add 500μL 50mg / mL lysozyme and 2mL phosphate buffer, add ice bath ultrasound for 4min, centrifuge at 12000rpm at 4℃ for 3min, and discard the supernatant; add 1mL of water to the precipitate, resuspend it, and centrifuge at 12000rpm at 4℃ for 3min, discard the supernatant, and resuspend the precipitate with 1mL phosphate buffer; take 100μL of the resulting solution, add 100μL bile salt solution (taurodeoxycholate, glycodeoxycholate), 800μL pH 6.0 sodium phosphate buffer was incubated at 37°C for 30 min, and then immediately placed in an ice bath; after cooling, 500 μL of 15% trichloroacetic acid was added, mixed and allowed to stand for 3 min, and then centrifuged at 4°C 12000 rpm for 3 min, and the supernatant was retained for later use; 1.5 mL of color developing solution was added to 1 mL of the obtained supernatant, vortexed, and placed in a boiling water bath for 15 min, and then quickly cooled in an ice bath for 3 min, and then 1 mL of 95% ethanol was added to dilute and mix, and allowed to stand for 5 min, and the absorbance was measured at 570 nm.

[0072] The results are as follows Figure 3 As shown: Taurodeoxycholate and glycodeoxycholate were used as substrates for enzyme activity determination. The BSH activity of the original strain WKLPP0040 obtained by screening was significantly better than that of other control strains. The enzyme activities for the two substrates were 0.23μmol / min and 0.31μmol / min, respectively. WKLPP0040 was subjected to gradient irradiation to pick out a total of 10 UV mutant strains with a lethality of more than 95% of the plate single colonies. After BSH activity comparison analysis, UV mutant strains 1-3 were the first-generation optimal mutant strain UVP1. UVP1 was screened and tested by the same screening and detection method, and mutant strains 2-5 were screened as the second-generation optimal mutant strain UVP2. After mutagenesis screening and BSH activity comparison analysis of UVP2 strain, the third-generation optimal mutant strain UVP3 was obtained. After UVP3 was mutagenized 4 times, the colonies were picked for BSH activity detection and it was found that the BSH activity of the bacteria did not continue to increase after mutagenesis or a reverse mutation occurred, so the UV mutation was terminated. UVP3 was the optimal mutant strain obtained by ultraviolet mutagenesis screening, that is, WKLPP0041. After three mutagenesis treatments, the optimal UV mutant strain was obtained, and the enzymatic activity of taurodeoxycholate and glycodeoxycholate could reach 0.429μmol / min and 0.534μmol / min. The BSH enzyme activity increased by 86.38% and 72.26% respectively. The lethality of irradiated mutagenesis bacteria and the BSH activity of the optimal UV mutant strain were determined as shown in the following figure. Figure 4 to Figure 7 shown.

[0073] 4. Detection of cholesterol degradation ability of mutant strain WKLPP0041

[0074] Take 0.5mL of bacterial solution (WKLPP0040\WKLPP0041) after culturing for 24h in a test tube, add 3mL of anhydrous ethanol and 2mL of 0.5mol / L KOH solution, vortex shake for 1min, place in a 60℃ water bath for saponification for 10min, take out and shake for 1min, continue saponification for 10min, shake again for 1min, continue saponification for 30-60min, then take out the test tube and quickly cool it with cold water. After complete cooling, add 3mL of 50g / L sodium chloride solution and 5mL of n-hexane, vortex and shake for 2min, let stand for stratification (at least 1h) for extraction, take 2.5mL of the upper solution into a test tube, place it in an 80℃ water bath to evaporate until no liquid remains, take out the test tube, add 4mL of o-phthalaldehyde color developer, vortex and shake for 1min, then let stand for 10min, add 2mL of sulfuric acid, vortex and shake for 1min, let stand in the dark for 10min, measure the absorbance at a wavelength of 550nm with a spectrophotometer, and calculate the mass concentration of cholesterol according to the cholesterol and absorbance standard curve equation.

[0075] Cholesterol removal rate / % = (1-A / B) × 100, where A is the mass concentration of cholesterol in the supernatant of the bacterial culture after culture.

[0076] Data such as Figure 8 As shown, the high BSH activity strain WKLPP0041 obtained by UV mutagenesis screening had a significantly improved cholesterol degradation rate, which was increased by 47.14% compared with the original strain WKLPP0040.

[0077] 5. Analysis of 16S rDNA sequence of mutant strain WKLPP0041

[0078] The 16S rDNA sequence of strain WKLPP0041 (SEQ ID NO: 2) was compared with a professional database based on the NCBI database, and the proximity joining method showed the phylogenetic tree of the gene sequences of WKLPP0041 and related species ( Fig. 9 ). The results of 16SrDNA analysis showed that strain WKLPP0041 was consistent with Lactiplantibacillus plantarum. Analysis and identification showed that the strain was Lactobacillus plantarum. However, after UV mutagenesis, its base sequence was significantly different from the original source strain WKLPP0040. It can be seen that mutagenesis can induce mutations in the base sequence of key enzymes, thereby enhancing the activity of BSH.

[0079] 6. Preservation

[0080] The plant lactobacillus WKLPP0041 was deposited in the China Center for Type Culture Collection on December 30, 2024, with the preservation address in Wuhan, China, and the preservation number is CCTCC NO: M20242958.

[0081] Example 2 Evaluation of the physiological and biochemical characteristics of Lactobacillus plantarum WKLPP0041 with high BSH activity

[0082] 1. Analysis of acid resistance characteristics

[0083] The pH value of physiological saline was adjusted to 2.0 with HCl, sterilized at 115°C for 30 minutes, and the bacterial solution fermented for 24 hours was aseptically inoculated with 1% inoculum. The number of viable bacteria was determined at 1 hour, 1.5 hours, and 2 hours at 37°C, with physiological saline as the blank control and WKLPP0040 as the negative control. The survival rate was determined.

[0084] The results are as follows Fig.10 As shown in the figure, under the condition of pH 2.0, the strain can survive and grow after being cultured for 1h, 1.5h, and 2h respectively; the survival rate of the strain after 1h is 75.26%; the survival rate after 1.5h is 53.37%, and the survival rate after 2h is still 46.01%, indicating that the strain has strong acid resistance. And there is no significant difference in acid resistance compared with WKLPP040. UV mutagenesis treatment does not affect the acid resistance of the strain.

[0085] 2. Analysis of bile salt tolerance characteristics

[0086] The bacterial liquid was inoculated at a 1% (v / v) inoculum into MRS medium with bile salt concentrations of 0.2% and 0.3% (w / v). MRS medium without bile salts was used as a positive control, and WKLPP0040 was used as a negative control. After culturing at 37°C for 0h, 1h, 2h, and 3h, samples were taken for plate colony counts, and the survival rate was calculated.

[0087] like Fig.11 As shown in (a), the survival rate of strain WKLPP0041 can reach 74.46% after being cultured for 3 hours in a 0.2% bile salt environment. Fig.11 As shown in (b), under the condition of bile salt concentration of 0.3%, the survival rate of the strain decreased after culturing for 3 hours, and the survival rate was 35.93%. The bile salt tolerance was good, and the tolerance to bile salt was better than that of WKLPP0040. This shows that after the strain WKLPP0041 was treated with ultraviolet mutagenesis, its BSH activity increased, and its tolerance to bile salt was improved.

[0088] Example 3 Evaluation of drug sensitivity of Lactobacillus plantarum WKLPP0041 with high BSH activity

[0089] The drug sensitivity test was carried out by paper diffusion method with different antibiotic concentrations. 1 mL of fermented bacteria was added to the MRS solid culture medium plate, evenly coated, placed for 1 hour for liquid absorption, and then placed in different gradient MIC quantitative drug sensitivity test strips. The plate was placed in a 37°C incubator and cultured for 16-24 hours. After the bacteria covered the plate, the inhibitory concentration (MIC value) of the drug was read to judge the sensitivity. The MIC values ​​were compared according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) of the United States. The results of drug sensitivity determination are shown in Table 1. WKLPP0041 was sensitive to the other 6 antibiotics except for vancomycin (VA). There was no significant difference with WKLPP0040, indicating that the ultraviolet mutagenesis treatment did not change the antibiotic sensitivity of the strain.

[0090] Table 1 Drug sensitivity test results of strain WKLPP0041

[0091]

[0092] Example 4 Analysis of Genetic Stability of BSH Activity of Lactobacillus plantarum WKLPP0041

[0093] The UV-mutated mutant WKLPP0041 obtained by mutagenesis in Example 1 was inoculated into MRS liquid culture medium with an inoculation amount of 2%, subcultured at 37° C. for 24 h, and subcultured 10 times in succession. The BSH activity of bacterial broths of different subcultures was detected.

[0094] Depend on Fig.12 It can be seen that after continuous subculture, the BSH activity of the strain did not decline, weaken, or show any reversion mutation. The mutagenic strain had good subculture stability.

[0095] Example 5 Preparation of postbiotics from Lactobacillus plantarum WKLPP0041 and analysis of active ingredients 1. Preparation of postbiotics from Lactobacillus plantarum WKLPP0041

[0096] (1) Activation of bacterial strains: freeze-dried bacterial strains were inoculated on solid MRS slant medium and cultured at 37°C for 16-24 h;

[0097] (2) Liquid culture: Take the activated slant and, under sterile conditions, scrape a small amount of bacteria with an inoculation loop, inoculate it into MRS liquid culture medium, and culture it at 30-37°C for 16-24 hours. If no foreign bacteria are found under a microscope, the seed solution is obtained.

[0098] (3) Expanding the culture: Inoculate the bacterial culture medium at a rate of 1-5% into MRS liquid culture medium, and culture at 35-37°C for 16-24 hours to obtain a fermentation liquid; OD 600 ≥0.6, number of live bacteria in fermentation liquid ≥3*10 9cfu / mL. (4) Take 1 mL of the fermentation liquid of the mutagenic strain, centrifuge at 4800 rpm for 10 min, discard the supernatant, wash the bacteria repeatedly with PBS for 3 times, then add 2 mL of PBS to resuspend the bacteria, place them in an ultrasonic crusher, and crush them (ultrasonic crushing conditions: 5S / 5S, repeated ultrasonication 60-80 times), and check under a microscope that the bacteria are completely broken. The crushed bacteria are freeze-dried to obtain solid WKLPP0041 postbiotics.

[0099] 2. Detection of postbiotic lipoteichoic acid

[0100] Lipoteichoic acid (LTA) was detected using a kit.

[0101] The results are as follows Fig.13 As shown, the lipoteichoic acid content of WKLPP0041 was significantly higher than that of the control strains (CK1, CK2), but there was no significant difference in the lipoteichoic acid content compared with WKLPP0040.

[0102] Example 6 Intervention effect of Lactobacillus plantarum WKLPP0041 on hypercholesterolemia mice

[0103] (1) Modeling and intervention: SPF male Kunming mice with a body weight of 20±2 g were used as experimental animals. After 7 days of pre-feeding, they were divided into blank group, model group, positive group and different sample groups, with 12 mice in each group. The blank group was fed with basic feed, and the model group, positive group and sample group were fed with high-cholesterol feed for 30 consecutive days. After successful modeling, the blank group continued to be fed with basic feed, and the other groups were fed with high-cholesterol feed. The positive group (Xuezhikang capsule) and different sample groups were gavaged with different test samples, and the blank group and model group were gavaged with the same dose of normal saline. After 30 days of gavage, the intervention ended, and experimental treatment and index detection were carried out.

[0104] (2) Grouping and dosage of oral gavage

[0105]

[0106]

[0107] (3) Blood lipid index detection

[0108] After the intervention, the eyeballs were removed and blood was collected. An appropriate amount of whole blood was collected from each mouse and placed in an anticoagulant centrifuge tube (anticoagulant was added to a 1.5 mL centrifuge tube in advance), incubated at 37°C for 30 min-1 h, 3000 r / min, and centrifuged for 10 min. The supernatant was taken as serum, and a blood lipid detection kit was used to detect blood lipid indicators (TC, TG, HDL, LDL). The results are shown in Fig.14As shown in the results, compared with the model control group, WKLPP0041 can significantly reduce the serum total cholesterol and low-density lipoprotein levels of mice, and the intervention effect on total cholesterol and low-density lipoprotein is better than WKLPP0040, and there is a significant difference; WKLPP0041 can significantly increase the level of high-density lipoprotein, compared with the model group, the difference is extremely significant, and compared with WKLPPP0040, there is no significant difference. However, WKLPP0041 and WKLPP0040 have no effect on improving triglycerides. It shows that WKLPP0041 can improve hypercholesterolemia by reducing total cholesterol, low-density lipoprotein, and increasing high-density lipoprotein.

[0109] (4) Intestinal flora count analysis

[0110] After the blood was collected, the experimental animals were killed, and the cecal feces were collected under sterile conditions. 1.0g was accurately weighed and placed in a 100mL glass bead sterile saline triangular bottle, placed in a constant temperature shaker, and the speed was 180r / min. The mixture was shaken and mixed for 30min, and different gradient dilutions were performed. The plate counting method was used to select the selective culture medium suitable for different types of bacteria to quantitatively count the live bacteria of bifidobacteria, lactobacilli, enterococci, enterobacteria, and Clostridium perfringens. The experimental results are shown in Table 2. Long-term high cholesterol diet leads to imbalance of intestinal flora in mice, the number of bifidobacteria and lactobacilli is significantly reduced, and the number of Clostridium perfringens is significantly increased. After different intervention evaluations, WKLPP0041 and WKLPP0040 can significantly increase the number of intestinal bifidobacteria and lactobacilli, reduce the number of Clostridium perfringens, and effectively improve the imbalance of intestinal flora. However, the positive group had no improvement on the intestinal flora because it did not contain probiotic components.

[0111] Table 2: Mouse intestinal flora detection results

[0112]

[0113]

[0114] Note: * and ** indicate comparison with the model group, and * represents P < 0.05, i.e., significant difference, and ** represents P < 0.01, i.e., extremely significant difference.

[0115] (5) Detection of inflammatory factors

[0116] The serum preparation method is the same as (3) and is used for the detection of inflammatory factors tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6).

[0117] like Fig.15As shown: WKLPP0041 can significantly downregulate the levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), which is extremely significantly different from the model group. And compared with the positive group, there is a significant difference. There is no significant difference with WKLPP0040. It shows that the improvement of intestinal flora has a significant improvement effect on mouse inflammation.

[0118] Example 7 Effect of Lactobacillus plantarum WKLPP0041 on Immunity Enhancement

[0119] (1) Pre-feeding and intervention of mice: SPF male Kunming mice were used as experimental animals, weighing 20±2g. After 7 days of pre-feeding, they were divided into groups, totaling 12 groups, 6 groups for mouse cellular immune function determination (delayed-type hypersensitivity reaction DTH); 6 groups for monocyte-macrophage function determination (carbon clearance experiment). Blank group, positive group and different sample groups were set up, with 12 mice in each group, all fed with basic feed, and different sample groups were gavaged with different strains of postbiotics, the blank group was given the same dose of normal saline, and the positive group was given a bottle of positive health food (Ganoderma lucidum spore powder capsule). After 30 days of gavage, the intervention ended, and experimental treatment and index detection were carried out.

[0120] (2) Grouping and dosage of oral gavage

[0121]

[0122]

[0123] (3) Index detection

[0124] Delayed hypersensitivity reaction (DTH): After the intervention, the abdominal skin of each mouse was depilated with barium sulfide, with an area of ​​about 3cm×3cm, and 50μL of DNFB solution was evenly applied to induce sensitization. Five days later, 10μL of DNFB solution was evenly applied to the right ear (both sides) of the mouse for attack. 24h after the attack, the mouse was killed by cervical dislocation, and the left and right ear shells were cut off. The ear piece with a diameter of 8mm was removed with a punch and weighed. The difference was calculated. The results are as follows Fig.16 As shown, through comparative analysis of the degree of delayed hypersensitivity, the degree of DTH in WKLPP0041 was significantly higher than that in the control group, but there was no significant difference compared with WKLPP0040.

[0125] Carbon clearance experiment in mice: After the intervention, the mice in the five groups of the carbon clearance experiment were weighed and injected with diluted Indian ink (10 mL / kg) according to their body weight through the tail vein. The injection was timed immediately. 20 μL of blood was collected from the medial canthal venous plexus of the mice at 2 minutes and 10 minutes, and immediately added to 2 mL of 0.1% Na2CO3 solution. The Na2CO3 solution was used as a blank control, and the optical density (OD) was measured at a wavelength of 600 nm using an enzyme marker. After the blood collection was completed, the mice were killed by breaking the neck, and the liver and spleen were dissected and the blood stains on the surface of the organs were absorbed with filter paper and weighed separately. The phagocytic index was used to represent the carbon clearance ability of mice.

[0126] like Fig.17 As shown, the phagocytic index of WKLPP0041 was significantly higher than that of the control group, and there was no significant difference compared with WKLPP0040. According to the "Health Food Function Test and Evaluation Method (2023 Edition)", it is helpful to enhance immunity: if the results are positive in any two of the four aspects of cellular immune function, humoral immune function, monocyte-macrophage function, and NK cell activity, it can be determined that the test sample has the effect of enhancing immunity. Therefore, it can be judged that WKLPP0041 has the effect of enhancing immunity.

[0127] Example 6 Application of Lactobacillus plantarum WKLPP0041 in improving blood lipids, reducing inflammation and enhancing immunity

[0128] The bacterial powder and postbiotics prepared based on Example 1 and Example 3 can be used to develop food, health products or medicines.

[0129] Lactobacillus plantarum WKLPP0041 is used in food, health food or medicine. The active bacteria or postbiotics of Lactobacillus plantarum WKLPP0041 are used as the main functional ingredients of the product, which can be combined with other excipients or auxiliary functional factors to develop its functional products.

[0130] The above-mentioned embodiments are only preferred implementations of the present application, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the technical solution concept of the present application, which all belong to the protection scope of the present application.

Claims

1. A Lactobacillus plantarum strain with high BSH activity WKLPP0041, characterized in that It was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit address in Wuhan, China, and the deposit number is CCTCC NO: M 20242958.

2. The Lactobacillus plantarum WKLPP0041 with high BSH activity according to claim 1, characterized in that: The plant lactobacillus is obtained by mutagenesis screening of the naturally selected strain plant lactobacillus (Lactiplantibacillus plantarum) WKLPP0040. The plant lactobacillus (Lactiplantibacillus plantarum) WKLPP0040 was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit address in Wuhan, China, and the deposit number is CCTCC NO: M 20242957.

3. A Lactobacillus plantarum WKLPP0040, characterized in that It was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit address in Wuhan, China, and the deposit number is CCTCCNO: M 20242957.

4. A method for mutagenesis screening of a strain of Lactobacillus plantarum WKLPP0041 with high BSH activity, characterized in that: The following steps are involved: Step (1), activate and culture the strain WKLPP0040 to be induced twice, so that it enters the logarithmic growth phase, and the number of viable bacteria is ≥3.0*10 9 cfu / mL, dilute the bacterial solution concentration to obtain a bacterial suspension; Step (2), evenly coating the diluted bacterial suspension on a culture dish containing MRS agar medium, placing it under a 20-25W ultraviolet lamp and irradiating it at a distance of 15-20cm for gradient mutagenesis treatment, and after the mutagenesis irradiation is completed, placing the plate in a light-proof environment for static culture; picking a single colony on the plate with a bacterial lethality rate of more than 95%, performing liquid culture, and detecting its bile salt hydrolase activity, and screening to obtain the first generation of optimal mutant strains; Step (3), secondary mutagenesis of the second-generation optimal mutant strain: the mutagenesis method of the bacterial solution of the first-generation mutant strain is used to select a single colony on a plate with a bacterial lethality rate of more than 95%, carry out liquid culture, and detect its bile salt hydrolase activity to screen and obtain the second-generation optimal mutant strain; Step (4), three mutagenesis of the third-generation optimal mutant strain: the mutagenesis method of the bacterial solution of the first-generation mutant strain is followed, a single colony on a plate with a bacterial lethality rate of more than 95% is selected, liquid culture is performed, and the bile salt hydrolase activity is detected to screen and obtain the third-generation optimal mutant strain, i.e., WKLPP0041.

5. A postbiotic with high BSH activity of Lactobacillus plantarum WKLPP0041, characterized in that: The postbiotics are broken bacteria and their contents prepared by fermentation and ultrasonic disruption of Lactiplantibacillus plantarum WKLPP0041, and the OD of the bacterial fermentation liquid is 600 Value ≥ 0.

6.

6. The postbiotic with high BSH activity of Lactobacillus plantarum WKLPP0041 according to claim 5, characterized in that: The lipoteichoic acid content of postbiotics is ≥6.11 mg / g.

7. Use of the plant lactobacillus (Lactiplantibacillus plantarum) WKLPP0041 according to claim 1 in the preparation of products for maintaining healthy blood lipid levels, wherein the blood lipids include cholesterol and triglycerides, and the products include foods, health foods and medicines.

8. Use of the Lactiplantibacillus plantarum WKLPP0041 according to claim 1 in the preparation of products for regulating intestinal flora, wherein the products include health foods and medicines.

9. Use of the Lactiplantibacillus plantarum WKLPP0041 according to claim 1 in the preparation of products for enhancing immunity, wherein the products include health foods and medicines.

10. A microbial composition, characterized in that The composition comprises Lactiplantibacillus plantarum WKLPP0041 or Lactiplantibacillus plantarum WKLPP0041 postbiotics as a main functional component.

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