Lactobacillus plantarum HP-B1280 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum HP-B1280 and application thereof. The lactobacillus plantarum HP-B1280 provided by the invention is preserved in the China Center for Type Culture Collection on September 2, 2024, and the preservation number of the lactobacillus plantarum HP-B1280 is CCTCC (China Center for Type Culture Collection) NO: M 20241891. The strain is excellent in acid production capacity, has good reproductive capacity, has high tolerance to temperature, can well tolerate gastric acid and cholate, has an inhibition effect on pathogenic bacteria, can enhance the resistance of a body to pathogens, and has a certain immunoregulation effect. In addition, the lactobacillus plantarum HP-B1280 disclosed by the invention can also participate in adjuvant therapy of the non-alcoholic fatty liver disease (NAFLD), and has good development and application prospects in the aspects of adjuvant therapy of the non-alcoholic fatty liver disease (NAFLD), treatment of early microbial infection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a plant lactobacillus HP-B1280 and an application thereof. Background Art

[0002] Lactobacillus plantarum belongs to the genus Lactobacillus, a round-ended straight bacillus, usually 0.9-1.2vtm×3.0-8.0μm, single, paired or short chain. Usually lacks flagella, but can move. Gram-positive, no spores. The surface colony is about 3mm in diameter, convex, round, smooth, dense, white, occasionally light yellow or dark yellow. It is a chemoheterotrophic bacterium, which requires a nutrient-rich medium for growth, calcium pantothenate and niacin, but does not require thiamine, pyridoxal or pyridoxamine, folic acid, vitamin B12. It can ferment pentose or gluconate, and more than 85% of the final product is lactic acid. It usually does not reduce nitrates, does not liquefy gelatin, and is negative for catalase and oxidase. It can produce DL-lactic acid, has the activity of 1,6-diphosphate fructose aldolase and monophosphate hexose pathway, can grow in gluconate, and produces carbon dioxide. Fermentation of 1 molecule of ribose or other pentose produces 1 molecule of lactic acid and 1 molecule of acetic acid. It can grow at 15℃, and the optimum growth temperature is usually 30-37℃.

[0003] As a kind of probiotic, Lactobacillus plantarum can tolerate the body's defense mechanisms, including enzymes in the mouth, low pH in gastric juice, and bile acids in the small intestine. Therefore, it can survive in large numbers in the intestine, regulating the balance of intestinal flora and promoting the digestion and absorption of nutrients. At the same time, Lactobacillus plantarum has the functions of alleviating lactose intolerance, reducing serum cholesterol levels, preventing cardiovascular diseases, enhancing human immunity, and inhibiting tumor formation. Therefore, it can be used in many industries such as medical care, industrial production, animal feed, and functional foods. However, there are still only a few products that use Lactobacillus plantarum today. Due to the large-scale use of antibiotics today, many microorganisms have developed drug resistance mutations, and drug residues in food have also brought difficult-to-solve problems to the human gastrointestinal tract.

[0004] Therefore, it is extremely important to discover a probiotic that has good antibacterial effect, can regulate the human gastrointestinal environment, and assist in the treatment of non-alcoholic fatty liver disease (NAFLD). Summary of the invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide a plant lactobacillus (Lactiplantibacillus plantarum) HP-B1280 and its application. The plant lactobacillus HP-B1280 provided by the present invention has excellent acid production ability, good reproduction ability, high tolerance to temperature, can well tolerate gastric acid and bile salts, and has an inhibitory effect on pathogenic bacteria, can enhance the body's resistance to pathogens, and has a certain immunoregulatory effect. In addition, the plant lactobacillus HP-B1280 of the present invention can also participate in the auxiliary treatment of non-alcoholic fatty liver disease (NAFLD), and has good development and application prospects in auxiliary treatment of non-alcoholic fatty liver disease (NAFLD) and treatment of early microbial infections.

[0006] The technical solution of the present invention is:

[0007] The present invention provides a strain of Lactobacillus plantarum HP-B1280, which has been deposited in the China Center for Type Culture Collection on September 2, 2024, with the address of the depository unit being Wuhan University, Wuhan, China, with a deposit number of CCTCC NO:M 20241891, and a taxonomic name of Lactobacillus plantarum. The 16S rRNA sequence of this strain is shown in SEQ ID NO: 1, has been uploaded to the GenBank database, and the received sequence number is PP813579.

[0008] Furthermore, the present invention provides an application of the Lactobacillus plantarum HP-B1280 in the preparation of medicines for preventing and treating non-alcoholic fatty liver disease.

[0009] Furthermore, the present invention provides an application of the Lactobacillus plantarum HP-B1280 in the preparation of foods and health products for improving non-alcoholic fatty liver disease.

[0010] Furthermore, the present invention also provides a use of the Lactobacillus plantarum HP-B1280 in the preparation of medicines with antibacterial effects.

[0011] Furthermore, the pathogens inhibited by the antibacterial effect include Acinetobacter baumannii, Escherichia coli, and Shigella.

[0012] Furthermore, the pathogens inhibited by the antibacterial effect include Acinetobacter baumannii (strain number BNCC 337173), resistant Escherichia coli (strain number CICC 10372), resistant Acinetobacter baumannii (strain number CICC 10980), resistant Shigella (strain number CICC 21535), Escherichia coli DH5α (strain number CPCC 100512), and Shigella (strain number BNCC 186377).

[0013] It can be seen that the plant lactobacillus HP-B1280 of the present invention can inhibit the growth of various pathogenic bacteria, has the ability to regulate intestinal flora, and can reduce the colonization of pathogenic bacteria by competitive exclusion and production of antibacterial substances, thereby maintaining the balance of intestinal microecology, promoting digestion and nutrient absorption, promoting the production of immunomodulatory factors and enhancing intestinal barrier function and other mechanisms, thereby regulating and enhancing the body's immune function.

[0014] Furthermore, the present invention provides an application of the Lactobacillus plantarum HP-B1280 in the preparation of food, medicine or health care product for regulating and enhancing the immune function of an organism.

[0015] Another technical solution of the present invention is to provide a bacterial agent, wherein the bacterial agent comprises the Lactobacillus plantarum HP-B1280 or its fermentation liquid according to claim 1.

[0016] The bacterial liquid and fermentation liquid of Lactobacillus plantarum HP-B1280 of the present invention are both prepared by the following method: a single colony is picked from a solid MRS culture medium and aerobically cultured in an MRS broth liquid culture medium.

[0017] Furthermore, the number of viable bacteria of Lactobacillus plantarum HP-B1280 in the bacterial agent is 50 to 300 billion CFU / g.

[0018] Furthermore, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0019] In the present invention, plant lactobacillus HP-B1280 is observed for bacterial growth state, temperature tolerance, acid production, acid resistance, bile resistance, adjuvant treatment of non-alcoholic fatty liver disease (NAFLD) effect evaluation, and its bacterial liquid and fermentation product are studied and analyzed for the antibacterial effect of various pathogenic bacteria, and plant lactobacillus HP-B1280 has strong growth and reproduction ability, high temperature tolerance, acid production, acid resistance, and bile resistance, can improve liver lipid metabolism, reduce liver damage and inflammation, and its bacterial liquid and fermentation product have a certain resistance to early microbial infection, and have good inhibitory effect on pathogenic bacteria such as Acinetobacter baumannii, drug-resistant Escherichia coli, drug-resistant Acinetobacter baumannii, drug-resistant Shigella, Escherichia coli DH5α, and Shigella. Finally, the present invention carries out whole genome sequencing to plant lactobacillus HP-B1280, and provides a basis for the in-depth study of subsequent plant lactobacillus HP-B1280. Through bioinformatics comparison and analysis, the genome of the Lactobacillus plantarum HP-B1280 of the present invention does not contain resistance genes, drug-resistant genes and virulence factors, and is a probiotic with relatively high safety.

[0020] Compared with the prior art, the Lactobacillus plantarum HP-B1280 provided by the present invention has the following advantages:

[0021] (1) The plant lactobacillus HP-B1280 provided by the present invention is screened from raw cow milk purchased from Mudan District, Heze City, Shandong Province. The strain has strong reproductive ability, can tolerate high temperature environment, has excellent gastric acid resistance, and has good bile salt resistance.

[0022] (2) The Lactobacillus plantarum HP-B1280 provided by the present invention can improve liver lipid metabolism, reduce fat accumulation in liver cells, reduce liver damage and inflammation, and can assist in the treatment of non-alcoholic fatty liver disease (NAFLD).

[0023] (3) The bacterial liquid of Lactobacillus plantarum HP-B1280 and its fermentation products provided by the present invention can be used in regulating and enhancing the immune function of the body, and can be used in antibacterial products and can be used for the treatment of symptoms in the early stage of general microbial infection.

[0024] Biological Deposit Description

[0025] Lactobacillus plantarum HP-B1280 was deposited in the China Center for Type Culture Collection on September 2, 2024. The depository address is Wuhan University, Wuhan, China. The deposit number is CCTCCNO: M20241891, and the taxonomic name is Lactobacillus plantarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the growth morphology diagram of Lactobacillus plantarum HP-B1280 strain;

[0027] Figure 2 The agarose gel electrophoresis diagram of the PCR product obtained by PCR amplification reaction of Lactobacillus plantarum HP-B1280 16S rRNA; wherein, M: 2K plus II DNA marker; 1, 2, 3, 4: PCR amplification products of Lactobacillus plantarum HP-B1280 16S rRNA;

[0028] Figure 3 The figure is an agarose gel electrophoresis diagram of the PCR product of 16S rRNA monoclonal bacteria of Lactobacillus plantarum HP-B1280; wherein, M: 2K plus II DNA marker; 1: monoclonal antibody PCR product;

[0029] Figure 4 is the genetic evolution tree of Lactobacillus plantarum HP-B1280;

[0030] Figure 5 This is a color change diagram of carbohydrate fermentation test of Lactobacillus plantarum HP-B1280; wherein 1-11 use esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon sources respectively;

[0031] Figure 6 HE staining of liver tissue sections of mice with non-alcoholic fatty liver disease (NAFLD) in which Lactobacillus plantarum HP-B1280 participated in the adjuvant treatment;

[0032] Figure 7 The antibacterial effect diagram of Lactobacillus plantarum HP-B1280 fermentation liquid on different bacterial species, where A to F are Acinetobacter baumannii, resistant Escherichia coli, resistant Acinetobacter baumannii, resistant Shigella, Escherichia coli DH5α, and Shigella, respectively;

[0033] Figure 8 This is the complete genome map of Lactobacillus plantarum HP-B1280;

[0034] Fig. 9Statistical graphs of common and unique annotations for the ENCODE gene general database;

[0035] Fig.10 It is the statistical diagram of PFAM domain;

[0036] Fig.11 This is a classification diagram of GO annotation results;

[0037] Fig.12 This is the classification diagram of KEGG pathway results;

[0038] Fig.13 This is the classification diagram of COG results;

[0039] Fig.14 This is the NR species distribution map;

[0040] Fig.15 This is the classification diagram of TCDB results. DETAILED DESCRIPTION

[0041] The present invention is further illustrated below through the description of specific implementation methods, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0042] In the following examples, reagents not specially described are conventional reagents and can be purchased from conventional reagent production and sales companies. The methods used are all prior arts unless otherwise specified.

[0043] The plant lactobacillus (Lactiplantibacillus plantarum) HP-B1280 described in the present invention is isolated from raw cow milk purchased from Mudan District, Heze City, Shandong Province. The MRS solid culture medium (model CM188), MRS liquid culture medium (model CM187), and LB solid culture medium (model CM158) are all purchased from Beijing Luqiao Technology Co., Ltd. Example 1 Isolation, purification and identification of plant lactobacillus (Lactiplantibacillus plantarum) HP-B1280

[0044] (1) Isolation and purification of Lactobacillus plantarum HP-B1280

[0045] This strain was isolated from raw milk purchased from Mudan District, Heze City, Shandong Province. The specific steps are as follows: 1 mL of raw milk was dissolved in 9 mL of normal saline and diluted to a concentration of 10 -5 ~10 -7CFU / mL was spread on MRS solid medium and inverted at 37℃ for 20h to obtain single colonies; single colonies were picked and three-zoned in MRS solid medium, inverted at 37℃ for 20h, and 16S rRNA sequencing was performed on the single colonies finally obtained to obtain purified strains. The purified strains were identified as Lactobacillus plantarum HP-B1280. The growth morphology of the strain is shown in the figure below. Figure 1 .

[0046] (2) Identification of Lactobacillus plantarum HP-B1280 by 16S rRNA sequencing

[0047] The purified strains were picked and the DNA fragments of the strains were obtained by the modified SDS-alkaline lysis method. The 16S rRNA of the strains was amplified by PCR using the 16S primers 27F-PBACS and 1541R-PBACS (as shown in the primer sequence I in Table 1), and the conditions were: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles, 72°C extension for 10 min, and 16°C storage.

[0048] Table 1 Primer sequence I

[0049]

[0050] The obtained PCR products were identified by agarose gel electrophoresis. Figure 2 As shown, the gene of the product recovered from the post-gel was recombined into GB05-dir, and the monoclonal antibody was picked after being cultured in solid culture medium for a period of time. The monoclonal antibody obtained was subjected to PCR amplification reaction using 16S primers T7-Elong and T7-TermElong (as shown in primer sequence II in Table 2). The gene amplification of the strain used universal primers 27F and 1541R, and the conditions were: 95℃ pre-denaturation for 10min, 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1.5min, 30 cycles, 72℃ extension for 10min, and 16℃ storage. The obtained PCR product was identified by agarose gel electrophoresis, as shown Figure 3 shown.

[0051] Table 2 Primer sequence II

[0052] Target Primer sequences T7-Elong 5′-TTAATACGACTCACTATAGGGGAATTG-3′ T7-TermElong 5′-TTATGCTAGTTATTGCTCAGCGG-3′

[0053] The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the 16S rRNA of the strain is shown in SEQ ID NO: 1:

[0054] GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTC

[0055] GAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCG

[0056] AACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAAC

[0057] AGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTC

[0058] GGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCA

[0059] CCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGA

[0060] CACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAG

[0061] TCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGT

[0062] TAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAA

[0063] GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCC

[0064] GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTT

[0065] CGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACA

[0066] GTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCG

[0067] AAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACA

[0068] GGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTT

[0069] TCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGC

[0070] AAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTT

[0071] TAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGA

[0072] GATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCG

[0073] TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCA

[0074] GCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA

[0075] TGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGG

[0076] TACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCG

[0077] GATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGC

[0078] ATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGT

[0079] TTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGA

[0080] CAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATC

[0081] ACCTCCTT

[0082] Blast gene comparison showed that the similarity with Lactobacillus plantarum DSM 16365 in GenBank was 100.00%, indicating that it was a strain of Lactobacillus plantarum, named HP-B1280. The 16S rRNA sequence had been uploaded to NCBI with the sequence number PP813579.

[0083] The genetic evolution tree was used to compare Lactobacillus plantarum HP-B1280. Figure 4 shown.

[0084] (3) Carbohydrate fermentation experiment with Lactobacillus plantarum HP-B1280

[0085] HBI lactic acid bacteria biochemical identification strips (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) were used to identify the response of Lactobacillus plantarum HP-B1280 to 11 carbohydrates. Figure 5 As shown in Table 3, among which, 1-11 respectively use esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon sources. The results are shown in Table 3. It can be seen from Table 3 that plant lactobacillus HP-B1280 can utilize 8 carbohydrates such as cellobiose, maltose, mannitol, salicin, sucrose, raffinose, inulin, and lactose in the 11 carbohydrate fermentation tests.

[0086] Table 3 Carbohydrate fermentation experiment of Lactobacillus plantarum HP-B1280

[0087]

[0088] Example 2: Evaluation of the growth status of Lactobacillus plantarum HP-B1280

[0089] Use a bamboo stick to inoculate a single colony of Lactobacillus plantarum HP-B1280 on MRS solid medium into fresh sterilized MRS liquid medium, and culture aerobically at 37°C and 180rpm for 18-24h. Use a pipette to draw 1mL of fermentation liquid, centrifuge at 8300rpm for 1min, discard the supernatant, keep the bacteria, weigh and calculate the wet weight. After drying at 55°C for 12h, weigh again and calculate the dry weight. The test results are shown in Table 4.

[0090] Table 4 Study on the growth status of Lactobacillus plantarum HP-B1280

[0091] Cultivation time (h) Wet weight / (g / mL) Dry weight / (g / mL) 24 <![CDATA[2.70×10 -2 ±0.0053]]> <![CDATA[3.967×10 -3 ±0.00013]]>

[0092] From Table 4, we can see that the dry weight of the bacteria is 3.967×10 -3 ±0.00013 g / mL, which shows that the Lactobacillus plantarum HP-B1280 of the present invention has a strong reproductive ability and can be reproduced in large quantities.

[0093] Example 3: Evaluation of temperature tolerance of Lactobacillus plantarum HP-B1280

[0094] Use a bamboo stick to inoculate a single colony of Lactobacillus plantarum HP-B1280 on MRS solid medium into fresh sterilized MRS liquid medium and culture aerobically at 37°C and 180rpm for 18-24h. Heat the bacterial solution at 37°C, 50°C, and 65°C for 1min, 2min, and 3min respectively, and dilute the bacterial solution to a concentration of 10 -6 and 10 -7 After the culture medium was plated with 1000 CFU / mL, it was spread on solid MRS medium and cultured aerobically at 37°C for 18-24 hours. The single colonies on the plate were counted, and the test results are shown in Table 5.

[0095] Table 5 Study on temperature tolerance of Lactobacillus plantarum HP-B1280

[0096]

[0097] As shown in Table 5, a large number of live bacteria still exist in the Lactobacillus plantarum HP-B1280 of the present invention after being heated at 65° C. for 1 min. It can be seen that the Lactobacillus plantarum HP-B1280 of the present invention has good high temperature resistance.

[0098] Example 4: Evaluation of acid resistance of Lactobacillus plantarum HP-B1280

[0099] Prepare artificial gastric juice (1.64mL dilute hydrochloric acid, 80mL double distilled water, 1g pepsin, shake well and add double distilled water to make up to 100mL, take a certain amount of solution to adjust the pH to 2.80, 3.52, and 4.86. Sterilize with a 0.44μm filter membrane and store in a 4℃ refrigerator for later use). Use a bamboo stick to inoculate a single colony of Lactobacillus plantarum HP-B1280 on MRS solid culture medium into freshly sterilized MRS liquid culture medium, and culture aerobically at 37℃, 180rpm for 18-24h. Take 60mL of bacterial solution and centrifuge at 3580rpm for 15min, pour off the supernatant, and resuspend the bacteria with 1.5mL of normal saline. Take 3 centrifuge tubes, add 6mL of artificial gastric juice to each, and then add 200μL of resuspended bacterial solution to each and mix well. Dilute to a concentration of 10 at 0min and 150min. -6 and 10 -7 After the CFU / mL was plated on solid MRS medium and cultured for 18-24 hours, the single colonies on the plate were counted and the survival rate was calculated. The test results are shown in Table 6.

[0100] Table 6 Study on acid resistance of Lactobacillus plantarum HP-B1280

[0101]

[0102] As shown in Table 6, a large number of strains of the plant lactobacillus HP-B1280 of the present invention still survived in the artificial gastric juice of pH 2.80 after 150 minutes, so it has a strong acid resistance.

[0103] Example 5: Evaluation of the Acid Production Capacity of Lactobacillus plantarum HP-B1280

[0104] A single colony of Lactobacillus plantarum HP-B1280 on MRS solid medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick and cultured aerobically at 37°C and 180 rpm for 18-24 hours. The pH of the supernatant was measured after centrifugation at 3580 rpm for 15 minutes at 8, 12, and 18 hours. The test results are shown in Table 7.

[0105] Table 7 Study on the acid production ability of Lactobacillus plantarum HP-B1280

[0106] Cultivation time (h) pH 8 3.51 12 3.26 18 3.21

[0107] As shown in Table 7, the plant lactobacillus HP-B1280 of the present invention can reach strong acidity after anaerobic culture for 8 hours, with a pH value of 3.51. It can be seen that the plant lactobacillus HP-B1280 of the present invention has a strong acid production ability.

[0108] Example 6: Evaluation of bile salt tolerance of Lactobacillus plantarum HP-B1280

[0109] Prepare artificial intestinal fluid (0.68g potassium dihydrogen phosphate, 50mL double distilled water, adjust pH to 6.8 with 0.1mol / L sodium hydroxide solution, add 0.5g trypsin, shake well, add double distilled water to 100mL, take a certain amount of solution and add a certain amount of ox bile salt to prepare artificial intestinal fluid with bile salt concentrations of 0.03%, 0.1%, 0.2%, and 0.3%, respectively. Sterilize with a 0.44μm filter membrane and store in a refrigerator at 4℃ for later use). Use a bamboo stick to inoculate a single colony of Lactobacillus plantarum HP-B1280 on MRS solid medium into freshly sterilized MRS liquid medium, and culture aerobically at 37℃, 180rpm for 18-24h. Take 60mL of bacterial solution and centrifuge at 3580rpm for 15min, pour off the supernatant, and resuspend the bacteria with 1.5mL of saline. Take 4 centrifuge tubes, add 6mL artificial intestinal fluid to each tube, and then add 200μL resuspended bacterial solution to mix well. Dilute to a concentration of 10 at 0min and 150min. -6 and 10 -7 After the CFU / mL was plated on solid MRS medium and cultured for 18-24 hours, the single colonies on the plate were counted and the survival rate was calculated. The test results are shown in Table 8.

[0110] Table 8 Study on bile salt tolerance of Lactobacillus plantarum HP-B1280

[0111]

[0112] As shown in Table 8, when the bile salt concentration was 0.3%, the survival rate of the Lactobacillus plantarum HP-B1280 of the present invention could still reach 88.24% after 150 minutes, indicating that the plantarum HP-B1280 had a high tolerance to bile salt.

[0113] Example 7: Evaluation of the effect of Lactobacillus plantarum HP-B1280 in the adjuvant treatment of non-alcoholic fatty liver disease (NAFLD)

[0114] Fifty male Kunming mice were purchased from Beijing Guanshi Company and stored in the Experimental Animal Center of Heze University. All animal experimental procedures were approved by the Ethics Committee of Heze University. All mice were allowed to adapt to the new environment for one week before the formal experiment began.

[0115] HE staining is the hematoxylin-eosin staining method, which is one of the commonly used staining methods in paraffin section technology. It is also the most basic and widely used technical method in teaching and scientific research in histology, embryology, and pathology. The alkaline hematoxylin staining solution can make cells blue, and the acidic eosin stains cells red. As a result, the nucleus is blue and the cytoplasm is red. The two different staining solutions make the cells change the refractive index through color, presenting a cell image under a light microscope. HE staining is related to the intracellular lipid content. The lower the lipid content, the darker the color; the higher the lipid content, the lighter the color.

[0116] The steps of HE staining are as follows:

[0117] (1) Dewaxing: Use xylene to remove the paraffin from the sections.

[0118] (2) Moisturizing: Hydrate the tissue sections using baths of gradually decreasing alcohol concentrations (100%, 90%, 80%, 70%) and water.

[0119] (3) Nuclear staining: staining with hematoxylin.

[0120] (4) Cleaning: Clean under running water until part of it turns “blue”.

[0121] (5)Difference: Dye is removed in 1% acid alcohol.

[0122] (6) Bluing: Rinse with running tap water and then immerse in ammonia water until part of it turns blue.

[0123] (7) Counterstaining: Staining in 10% Eosin Y.

[0124] (8) Wash with distilled water.

[0125] The mice were randomly divided into 5 groups: control group, HFD group, HFD+Lactobacillus plantarum low-dose group, HFD+Lactobacillus plantarum medium-dose group, HFD+Lactobacillus plantarum high-dose group, with 10 mice in each group. Control group: fed with ordinary feed; HFD group: fed with high-fat feed containing 45% fat energy; HFD+Lactobacillus plantarum low-, medium-, and high-dose groups: fed with high-fat feed containing 45% fat, and oral administration of low-dose (100 billion CFU), medium-dose (200 billion CFU), and high-dose (300 billion CFU) of Lactobacillus plantarum HP-B1280 daily. The experiment was carried out for 12 weeks. On the last day of the experiment, mice in each group were anesthetized with ether and killed. Serum and liver were taken for the determination of various indicators. The liver was fixed with 4% paraformaldehyde, and the fixed liver tissue was dehydrated with gradient alcohol, embedded in paraffin, and then the liver tissue was made into a thickness of 4μm. The tissue sections were stained with HE, and the coloring of the liver tissue sections of mice in different groups was observed after staining.

[0126] The results of HE staining of liver tissue sections are as follows Figure 6 As shown in the figure, the results of liver pathological examination showed that there was no obvious abnormality in the liver of the normal control group. Long-term high-fat diet leads to fat accumulation in liver cells and inflammatory cell infiltration between cells, indicating the presence of NAFLD in the liver. After the intervention of Lactobacillus plantarum HP-B1280, the fat accumulation in liver cells and inflammatory cell infiltration between liver cells of mice fed a high-fat diet were significantly reduced.

[0127] Compared with the staining of liver tissue sections of normal (control) mice, the staining depth of liver tissue sections of mice with fatty liver (HFD group) was significantly lighter, the staining of liver tissue sections of low-dose (HFD + Lactobacillus plantarum low-dose group) mice was darker than that of mice with fatty liver (HFD group), the staining depth of liver tissue sections of medium-dose (HFD + Lactobacillus plantarum medium-dose group) mice was significantly deeper than that of mice with fatty liver (HFD group), and the staining depth of liver tissue sections of high-dose (HFD + Lactobacillus plantarum high-dose group) mice was even close to that of liver tissue sections of normal (control) mice.

[0128] The results show that the Lactobacillus plantarum HP-B1280 of the present invention can improve liver lipid metabolism, reduce fat accumulation in liver cells, reduce liver damage and inflammation, and can treat NAFLD induced by a high-fat diet.

[0129] Example 8: Evaluation of the antibacterial effect of Lactiplantibacillus plantarum HP-B1280 fermentation broth

[0130] Use a bamboo stick to inoculate a single colony of Lactobacillus plantarum HP-B1280 on the MRS solid medium into a freshly sterilized MRS liquid medium, and culture aerobically at 37°C and 180rpm for 18-24h. Take a certain amount of bacterial solution, centrifuge at 8300rpm for 1min, and collect the supernatant. Take another certain amount of bacterial solution for later use. Mix 200μL of each of Acinetobacter baumannii, resistant Escherichia coli, resistant Acinetobacter baumannii, resistant Shigella, Escherichia coli DH5α, and Shigella with 50mL LB solid medium, pour it onto the LB solid medium, and evenly place 4 Oxford cups with a diameter of 8.0mm after the mixed bacterial plate is slightly dry. Let it stand for 5min, add 200μL of supernatant to two Oxford cups, and add 200μL of uncentrifuged bacterial solution to two Oxford cups. After standing in a 4°C refrigerator for 4h, culture it upright at 37°C for 24h and observe and record the antibacterial diameter. The test results are shown in Table 9 and Figure 7 As shown. Figure 7 In the figure, Figure A is Acinetobacter baumannii BNCC337173, Figure B is resistant Escherichia coli CICC 10372, Figure C is resistant Acinetobacter baumannii CICC 10980, Figure D is resistant Shigella CICC 21535, Figure E is Escherichia coli DH5α CPCC 100512, and Figure F is Shigella BNCC 186377. The above strains are all purchased from conventional markets. In Figures A to F, the inhibition diameter S represents the supernatant of the Lactobacillus plantarum HP-B1280 fermentation broth after centrifugation (fermentation broth supernatant), and F represents the Lactobacillus plantarum HP-B1280 fermentation broth (fermentation broth containing bacteria) that has not been centrifuged.

[0131] Table 9 Evaluation of antibacterial effect of Lactobacillus plantarum HP-B1280 fermentation broth

[0132]

[0133] Depend on Figure 7 It can be seen from the results in Table 9 that the plant lactobacillus HP-B1280 fermentation broth of the present invention and the supernatant of the fermentation broth have a strong antibacterial effect on Acinetobacter baumannii, resistant Escherichia coli, resistant Acinetobacter baumannii, resistant Shigella, Escherichia coli DH5α, and Shigella, and have a strong inhibitory effect on the growth of these strains.

[0134] Example 9: Determination of the whole genome of Lactobacillus plantarum HP-B1280

[0135] The genome of Lactobacillus plantarum HP-B1280 was obtained by sequencing and assembling the genome based on Nanopore third-generation sequencing technology and second-generation sequencing technology platform. Figure 8This is a visualized genome map of the bacterium. The genome size of the bacterium is 2,718,849bp, the GC ratio is 44.58%, and its genome contains 3,001 CDS, 68 tRNAs, 1 tmRNA and 16 rRNAs, and does not contain plasmids.

[0136] Example 10 Functional Annotation Analysis of the Lactobacillus plantarum HP-B1280 Database Genome

[0137] (1) Statistics of annotation ratios in general databases

[0138] In order to obtain comprehensive gene function information, the present invention has performed gene function annotation of eight major databases, including: UniProt, KEGG and KEGG Pathway, GO, Pfam, COG, TIGERfams, RefSeq, and NR. The predicted gene sequence was compared with the functional databases such as COG, KEGG, Uniprot, and Refseq by BLAST+ (Version: 2.11.0+) to obtain the gene function annotation results. The software hmmer (Version: 3.3.2) was used to perform functional annotation based on the Pfam and TIGERFAM databases. The annotation statistical results are shown in Table 10. The common and unique annotations of the general database of coding genes are as follows Fig. 9 As shown, Fig. 9 In the figure, the left side shows the gene annotation statistics of the general database; the right side shows the common and unique statistics. For example, the first column shows the common statistics of all general databases, the top part shows the number statistics, and the bottom part shows the annotations of the common or unique databases.

[0139] From Table 10 and Fig. 9 It can be seen that the gene fragments contained in the Lactobacillus plantarum HP-B1280 of the present invention can obtain comprehensive gene function information in major general databases, and the gene function information that can be obtained is relatively large, which provides a certain basis for the research on each gene of the strain.

[0140] Table 10 Statistics of coding gene annotation results

[0141]

[0142]

[0143] (2) Pfam domain classification

[0144] The Pfam database is a homologous protein family database, a collection of protein domain families, including more than 16,000 protein families. Proteins are generally composed of one or more functional regions, which are usually called domains. Domains appear in different combinations in different proteins, forming the diversity of proteins. Identifying domains in proteins is important for understanding the function of proteins.

[0145] Based on the annotation of Pfam domains, the present invention statistically summarizes the genes annotated in each domain, and displays the top 20 domains with the most annotations. The results are as follows: Fig.10 As shown in the figure, the horizontal axis is the name of the protein family, and the vertical axis is the number of genes mapped to the protein family.

[0146] (3) Gene Ontology Classification

[0147] Gene Ontology (GO) is an internationally standardized gene function classification system that provides a set of dynamically updated controlled vocabularies to comprehensively describe the properties of genes and gene products in organisms.

[0148] The present invention simplifies GO (Gene Ontology) annotation information to obtain GOslim classification, summarizes and statistics the functions of genes from three aspects: cell components, molecular functions, and biological processes, and selects the top 20 GOslim secondary classifications with the most annotations in each classification for mapping. The results are shown in the figure. Fig.11 As shown in the figure, the horizontal axis is the content of each GO classification, and the vertical axis is the number of genes. Fig.11 It shows the gene enrichment of each secondary function of GO in the context of all genes, reflecting the status of each secondary function in this context.

[0149] (4) KEGG Pathway classification

[0150] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a major public database that systematically analyzes the metabolic pathways of gene products in cells and the functions of these gene products. KEGG can be used to further study the complex biological behaviors of genes.

[0151] After KEGG annotation of the genes, the present invention classified them according to the KEGG metabolic pathways in which they participated. Fig.12 As shown in the figure, the horizontal axis is the number of genes annotated under the Pathway classification; the vertical axis is the Pathway classification, and different colors represent different major classifications. Fig.12 The metabolic pathways of each gene product of Lactobacillus plantarum HP-B1280 in the cell are shown in FIG.

[0152] (5) COG classification

[0153] COG (Cluster of Orthologous Groups of proteins) is a database for orthologous classification of gene products. Each COG protein is assumed to be derived from an ancestral protein. The COG database is constructed based on the coding proteins and phylogenetic relationships of bacteria, algae, and eukaryotes with complete genomes. Unigene and COG databases are compared to predict the possible functions of genes and perform functional classification statistics on them, so as to understand the gene function distribution characteristics of the species from a macro perspective. COG is divided into 26 groups.

[0154] The present invention classifies the COG annotated genes according to the COG group, and the results are as follows: Fig.13 As shown in the figure, the horizontal axis is the content of each COG classification, and the vertical axis is the number of genes. In different functional categories, the number of genes reflects the metabolism or physiological bias in the corresponding period and environment, and can be scientifically explained in combination with the distribution of the research object in each functional category.

[0155] (6) Uniprot annotation

[0156] UniProt Archive is a comprehensive, non-redundant database that contains protein sequences from all major, publicly available databases. Since proteins may exist in different databases and there may be multiple versions in the same database,

[0157] The number of genes annotated to the Uniprot database of the whole genome of Lactobacillus plantarum HP-B1280 described in the present invention is 2985, accounting for 99.47%.

[0158] (7) Refseq annotation

[0159] The Refseq (Reference Sequences) database is a non-redundant transcript, protein and genome sequence database that has been annotated. It is a database that has been corrected by NCBI and other organizations. It uses the terms defined by the Human Gene Nomenclature Committee and includes official gene symbols and optional symbols. RefSeq sequences are non-redundant databases screened by NCBI and are generally more credible.

[0160] The number of genes annotated to the Refseq database in the whole genome of Lactobacillus plantarum HP-B1280 described in the present invention is 2962, accounting for 98.70%.

[0161] (8) NR Notes

[0162] NR database: a non-redundant protein amino acid sequence database, including non-redundant data from SwissProt, PIR (Protein Information Resource), PRF (Protein Research Foundation), PDB (Protein Data Bank) protein databases, as well as protein data translated from CDS data from GenBank and RefSeq.

[0163] The NR species distribution diagram of Lactiplantibacillus plantarum HP-B1280 of the present invention is as follows Fig.14 As shown, different colors in the figure represent different species.

[0164] (9)TIGRFAMs annotation

[0165] TIGRFAMs is a database consisting of multiple sequence alignments, hidden Markov models (HMMs) and related information. It is a member of interPro for protein sequence classification and supports automatic annotation of (mostly prokaryotic) proteins. Starting from v10.0, the TIGRFAMs model uses HMMER 3, which has excellent search speed and sensitivity.

[0166] The number of genes annotated to the Tigrfams database of the whole genome of Lactobacillus plantarum HP-B1280 described in the present invention is 1648, accounting for 54.92%.

[0167] Example 11: Lactiplantibacillus plantarum HP-B1280 proprietary database annotation

[0168] The present invention is a personalized analysis of Lactobacillus plantarum HP-B1280, and a proprietary database annotation is performed on it, including ARDB database annotation, carbohydrate enzyme annotation based on CAZy database, pathogen-host interaction analysis, drug resistance gene annotation, cytochrome P450 annotation, signal peptide prediction, type III secretion system effector protein annotation, and TCDB transporter protein annotation.

[0169] After the whole genome of Lactobacillus plantarum HP-B1280 described in the present invention was annotated with a proprietary database, it was found that: the resistance gene results annotated in the ARDB database are shown in Table 11; 1295 completely matched gene sequences were annotated in the pathogen-host interaction database; no resistance genes were annotated in the CARD database; 287 completely matched gene sequences were annotated in the type III secretion system effector protein database; the carbohydrate enzyme annotation results are shown in Table 12; the signal peptide prediction results of the whole genome of Lactobacillus plantarum HP-B1280 are shown in Table 13, and the transmembrane protein and secretory protein prediction results are shown in Table 14; the cytochrome P450 annotation results are shown in Table 15; the TCDB membrane transporter annotation results are shown in Table 16. Fig.15 As shown in the figure, the horizontal axis is the content of each classification of TCDB, and the vertical axis is the number of genes.

[0170] Table 11

[0171]

[0172] Table 12 CAZy annotation

[0173]

[0174]

[0175] Table 13 Signal peptide prediction statistics

[0176] Protein Type quantity Protein containing information peptide 226

[0177] Table 14 Prediction statistics of transmembrane proteins and secretory proteins

[0178] Protein Type quantity Transmembrane proteins 843 Secretory proteins 76

[0179] Table 15 Cytochrome P450 annotations

[0180]

[0181] According to the above data analysis, the cytochrome P450 of Lactobacillus plantarum HP-B1280 of the present invention is a type of monooxygenase widely present in organisms, mainly involved in the metabolic process of endogenous and exogenous substances, including drugs, hormones, fatty acids, cholesterol, etc. Based on the above data, the study found that the cytochrome P450 of the present invention may have the following characteristics:

[0182] 1. Sequence diversity: The ctg_01702 and ctg_01372 sequences differ in consistency, coverage, and homologous families, indicating that they may have different functions and characteristics.

[0183] 2. Potential application value: Cytochrome P450 plays an important role in drug metabolism, biotransformation, etc.

[0184] Therefore, these sequences may have potential biotechnological applications, such as drug development, biocatalysis, etc. Species specificity: Since they are derived from different species, they may have species-specific functions and properties, which is of great significance for studying the differences and evolutionary relationships between species.

[0185] In summary, the cytochrome P450 sequence of Lactobacillus plantarum HP-B1280 of the present invention has diversity and potential application value, and is worthy of further in-depth study.

[0186] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum HP-B1280, characterized in that: It was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC NO:M 20241891.

2. Use of Lactobacillus plantarum HP-B1280 according to claim 1 in the preparation of medicines for preventing and treating non-alcoholic fatty liver disease.

3. Use of Lactobacillus plantarum HP-B1280 according to claim 1 in preparing foods and health products for improving non-alcoholic fatty liver disease.

4. Use of Lactobacillus plantarum HP-B1280 according to claim 1 in the preparation of medicines with antibacterial effects.

5. The use according to claim 4, characterized in that: The pathogens inhibited in the antibacterial effect include Acinetobacter baumannii, Escherichia coli, and Shigella.

6. Use of the Lactobacillus plantarum HP-B1280 according to claim 1 in preparing food, medicine or health product for regulating and enhancing the immune function of an organism.

7. A bacterial agent, characterized in that The bacterial agent comprises the Lactobacillus plantarum HP-B1280 or its fermentation liquid according to claim 1.

8. The bacterial agent according to claim 7, characterized in that The number of viable bacteria of Lactobacillus plantarum HP-B1280 in the bacterial agent is 50 to 300 billion CFU / g.

9. The bacterial agent according to claim 7, characterized in that The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

Images