CN116836868B - Lactobacillus casei BIGTREE-B101142, and microbial inoculum and application thereof - Google Patents
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus casei BIGTREE-B101142, a microbial inoculum and application thereof. The Lactobacillus casei (Lacticaseibacillus casei) BIGTREE-B101142 provided by the invention is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2023463, the strain has good reproductive capacity and acid production capacity, strong gastric acid tolerance, good heat tolerance and extremely high bile salt tolerance, can adapt to complex and changeable gastrointestinal tract environments, and has the purposes of enhancing the immunity of human bodies and regulating the balance of intestinal flora. Meanwhile, the strain has good inhibition effect on shigella flexneri, acinetobacter baumannii, drug-resistant acinetobacter baumannii, bacillus cereus and other strains, and has good development and application prospects in the aspect of treating early microbial infection.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus casei BIGTREE-B101142, a microbial inoculum and application thereof.
Background
Lactobacillus casei belongs to the genus Lactobacillus, is gram positive bacteria, does not produce spores, does not have flagella, does not move, and can ferment various sugars. Together with lactobacillus acidophilus and bifidobacterium are called "healthy triple probiotics".
Lactobacillus casei is used as one of probiotics and can resist the defense mechanism of organisms, including enzymes in the oral cavity, low pH in gastric juice, bile acid of small intestine and the like. So that the compound can survive in a large amount in intestinal tracts, and has the functions of regulating the balance of intestinal flora, promoting digestion and absorption and the like. Meanwhile, the lactobacillus casei also has the effects of relieving lactose intolerance, efficiently reducing blood pressure, reducing cholesterol, enhancing human immunity, inhibiting tumor growth and the like.
The lactobacillus casei has good acid resistance and bile resistance, so the lactobacillus casei can be used in a plurality of industries such as medical care, livestock breeding and functional foods, but the number of products using the lactobacillus casei is still small, and due to the large amount of antibiotics used today, a plurality of microorganisms generate drug resistance variation, and the drug residues in the foods also bring problematic problems to human gastrointestinal tracts, so the probiotics which have good antibacterial effect and can regulate the human gastrointestinal tract environment are extremely important.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide lactobacillus casei BIGTREE-B101142, and a microbial inoculum and application thereof. The Lactobacillus casei BIGTREE-B101142 provided by the invention has good gastric acid tolerance and high temperature tolerance, has good acid production capacity and physiological and biochemical capacity suitable for gastrointestinal tracts of organisms, has good inhibition effect on strains such as Shigella flexneri, acinetobacter baumannii, drug-resistant Acinetobacter baumannii, bacillus cereus and the like, can enhance the immunity of organisms, and has very obvious effect on early treatment and prevention of pathogenic bacteria.
The technical scheme of the invention is as follows:
the invention provides a lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 is preserved in China center for type culture Collection (China, with a preservation number of CCTCC NO: m2023463, taxonomically named Lactobacillus casei (Lactobacilli)casei) The method comprises the steps of carrying out a first treatment on the surface of the The 16S rRNA is shown as a nucleotide sequence shown as SEQ ID NO. 1, the 16S rRNA complete sequence is uploaded to a GenBank database, and the receiving sequence number is as follows: OQ608442.1.
Further, the invention provides application of lactobacillus casei BIGTREE-B101142 in preparation of medicines with antibacterial effect.
Further, the pathogenic bacteria inhibited in the antibacterial effect comprise shigella flexneri, acinetobacter baumannii and bacillus cereus.
Further, the Shigella flexneri has a deposit number of BNCC186377, bacillus cereus has a deposit number of BNCC192974, acinetobacter baumannii has a deposit number of BNCC337173, and Acinetobacter baumannii has a deposit number of ATCC BAA-1605.
Furthermore, the invention also provides an application of the lactobacillus casei BIGTREE-B101142 in preparing medicines for reducing triglyceride.
Furthermore, the Lactobacillus casei BIGTREE-B101142 has strong reproductive capacity, can resist high-temperature environment, has strong acid production capacity, has pH of 4.37 when being cultured in MRS culture medium for 25 hours at 37 ℃, and has excellent gastric acid resistance and bile salt resistance.
According to another technical scheme, the invention provides a microbial inoculum, which comprises the lactobacillus casei BIGTREE-B101142.
Further, the viable count of the lactobacillus casei BIGTREE-B101142 in the microbial inoculum is 500-3000 hundred million CFU/g.
Further, the microbial inoculum is a solid microbial inoculum or a liquid microbial inoculum.
According to the invention, bacterial growth state observation, temperature tolerance evaluation, acid production capability evaluation, acid resistance evaluation, bile salt tolerance evaluation and bacterial liquid and fermentation products of the Lactobacillus casei BIGTREE-B101142 are carried out to research and analysis on the antibacterial effect of various pathogenic bacteria, so that the Lactobacillus casei BIGTREE-B101142 has strong growth and propagation capability, strong high temperature resistance, strong acid production capability, strong acid resistance and strong bile salt tolerance, and the bacterial liquid and fermentation products thereof have a certain effect on early microbial infection and have good inhibition effects on Shigella flexneri, acinetobacter baumannii, drug-resistant Acinetobacter baumannii, bacillus cereus and the like. Finally, the invention carries out whole genome sequencing on lactobacillus casei BIGTREE-B101142, and provides a foundation for the subsequent deep research on lactobacillus casei HP-B1142.
Compared with the prior art, the invention has the following beneficial effects:
(1) The Lactobacillus casei BIGTREE-B101142 sieve provided by the invention is selected from Xinjiang milk pimple (purchased from Xinjiang Wulu-Muzu-Tou-Tu river region), grows well on MRS solid culture medium, has strong growth and reproduction capacity, strong acid production capacity and acid resistance, has very high tolerance to bile salt, can effectively improve the tolerance to gastrointestinal tract environment, can adapt to and regulate gastrointestinal tract flora balance, regulate and strengthen the immune function of organism, and promote organism health.
(2) The lactobacillus casei BIGTREE-B101142 screened by the method has excellent antibacterial effect and strong inhibition effect on shigella flexneri, acinetobacter baumannii, drug-resistant acinetobacter baumannii and bacillus cereus. Can be used for early treatment and prevention of pathogenic bacteria.
The bacterial liquid and the fermentation product of the lactobacillus casei BIGTREE-B101142 screened by the invention can effectively reduce triglyceride, and experiments prove that the degradation rate of the bacterial liquid of the lactobacillus casei BIGTREE-B101142 on the triglyceride is 43.5 percent. Meanwhile, through bioinformatics comparison and analysis, the lactobacillus casei BIGTREE-B101142 genome does not contain resistance genes, drug resistance genes and virulence factors, is a probiotic with high safety, and can be widely used in industries such as medical care and the like.
Biological preservation description:
lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 is preserved in China center for type culture Collection (China, with a preservation number of CCTCC NO: m2023463, taxonomically named Lactobacillus casei (Lactobacilli)casei)。
Drawings
FIG. 1 is a growth morphology of Lactobacillus casei BIGTREE-B101142;
FIG. 2 is an agarose gel electrophoresis chart of a PCR amplification reaction of 16S rRNA of Lactobacillus casei BIGTREE-B101142, wherein M:2K plus II DNA marker;1,2,3,4: 16S rRNA PCR amplification product of Lactobacillus casei BIGTREE-B101142;
FIG. 3 is an agarose gel electrophoresis of a PCR product of Lactobacillus casei BIGTREE-B10114 monoclonal thallus, wherein M:2K plus II DNA marker;1: monoclonal antibody PCR products;
FIG. 4 is a genetic evolutionary tree of Lactobacillus casei BIGTREE-B101142;
FIG. 5 is a graph showing the color change in a Lactobacillus casei BIGTREE-B10114 carbohydrate fermentation test; wherein, 1-11 respectively take esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose and 1% sodium hippurate as carbon sources;
FIG. 6 is a graph showing the bacteriostatic effect of a Lactobacillus casei BIGTREE-B101142 broth, wherein A is Shigella flexneri BNCC186377, B is Bacillus cereus BNCC192974, C is Acinetobacter baumannii BNCC337173, and D is Acinetobacter baumannii ATCC BAA-1605;
FIG. 7 is a full genome map of Lactobacillus casei BIGTREE-B101142;
FIG. 8 is a statistical graph of common and unique annotations of coding gene general database annotations;
FIG. 9 is a statistical diagram of PFAM domains;
FIG. 10 is a GO annotation result classification diagram; wherein A is a gene function classification diagram of a biological process, B is a gene function classification diagram of a cell component, and C is a gene function classification diagram of a molecular function;
FIG. 11 is a KEGG path result classification diagram;
FIG. 12 is a chart of classification of COG results;
FIG. 13 is a NR species profile;
fig. 14 is a TCDB result classification diagram.
Description of the embodiments
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention as long as they do not depart from the basic idea of the invention.
In the following examples and comparative examples, the reagents not specifically described were conventional reagents, which were purchased from conventional reagent manufacturing and selling companies, and the methods used, unless otherwise specified, were all prior art.
The strain of the invention is separated from Xinjiang milk lump (purchased from Xinjiang Uruku Jiuzu Toku river region), and the MRS solid culture medium (model CM 188), MRS liquid culture medium (model CM 187) and LB solid culture medium (model CM 159) are all purchased from Beijing land bridge technology Co.
Example 1: lactobacillus casei (Lactobacilli)casei) Isolation, purification and identification of BIGTREE-B101142
(1) Lactobacillus casei (Lactobacilli)casei) Isolation and purification of BIGTREE-B101142
The strain is derived from Xinjiang milk lump. The separation and purification process is as follows: dissolving 1 g fresh Xinjiang milk lump in 9 ml normal saline, and diluting to concentration of 10 -5 ~10 -9 CFU∙ml −1 Then coating the mixture in a solid MRS culture medium for culturing 24 h at 37 ℃ to obtainSingle colony; and (3) picking single bacterial colonies, culturing at 37 ℃ for 24 h in a three-region line in a solid MRS culture medium, and then picking single bacterial colonies for strain identification to obtain purified bacterial strains.
Photographs of the growth of lactobacillus casei bigtre-B101142 of the present invention on MRS solid medium are shown in figure 1,
the colony is milky white and round in shape and has a diameter of 1-3 mm when observed by naked eyes.
(2) Lactobacillus casei (Lactobacilli)casei) 16S rRNA full sequencing identification of BIGTREE-B101142
The purified strain was selected and modified SDS-base lysis was performed to obtain a strain DNA fragment. The strain was subjected to PCR amplification of 16S rRNA using the 16S primers 27F-PBACS and 1541R-PBACS. The PCR amplification reaction system comprises: pre-denaturation (95 ℃,10 min), denaturation (95 ℃,30 s), annealing (55 ℃,30 s), extension (72 ℃,90 s), 31 cycles, post-extension (72 ℃,10 min). The primer sequences I are shown in Table 1.
The resulting PCR products were subjected to agarose gel electrophoresis as shown in FIG. 2. Then, carrying out gel recovery, recombining gel recovery product genes into GB05-dir, reversely culturing 12 h in a solid LB culture medium containing kanamycin at 37 ℃, picking up monoclonal bacteria, and carrying out PCR amplification reaction on the obtained monoclonal antibody by using 16S primers T7-Elong and T7-TermElong, wherein the reaction system is as follows: pre-denaturation (95 ℃,10 min), denaturation (95 ℃,30 s), annealing (55 ℃,30 s), extension (72 ℃,90 s), 31 cycles, post-extension (72 ℃,10 min). The primer sequences II are shown in Table 2. The PCR products were identified by agarose gel electrophoresis as shown in FIG. 3.
The PCR product was sent to Suzhou gold intellectual Biotech Co.Ltd for sequencing, and the nucleotide sequence of the strain 16S rRNA was SEQ ID NO:1: GAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTTTGGTCGATGAACGGTGCTTGCACTGAGATTCGACTTAAAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGCAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTGAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT.
The similarity of the strain with Lactobacillus casei DSM 20011 in GenBank reaches 99.80% after BLAST gene comparison, and the strain is a Lactobacillus casei strain named BIGTREE-B101142, and the 16S rRNA sequence is uploaded with NCBI and the sequence number is OQ608442.1.
Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 was aligned and the results are shown in FIG. 4.
(3) Lactobacillus casei (Lactobacilli)uscasei) BIGTREE-B101142 carbohydrate fermentation test
Identification of Lactobacillus casei (Lactobacilli) by using HBI G11 lactobacillus biochemical identification strip (purchased from Qingdao sea Bo Biotechnology Co., ltd.)casei) BIGTREE-B101142 response to 11 carbohydrates. The color change is shown in FIG. 5, wherein 1-11 respectively uses esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon source. As shown in Table 3, it is clear from Table 3 that Lactobacillus casei BIGTREE-B101142 was able to utilize, decompose and metabolize 10 carbohydrates including esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, lactose, 1% sodium hippurate in a11 carbohydrate fermentation test.
Example 2: lactobacillus casei (Lactobacilli)casei) Evaluation of growth State of Strain of BIGTREE-B101142
Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into freshly sterilized fully cooled MRS liquid medium and anaerobically cultured at 37℃at 100 rpm for 24 h. The pipette sucks 1 ml fermentation broth, centrifugates at 8000 rpm for 1 min, discards the supernatant, leaves thalli, and calculates the wet weight. The dry weight was calculated again after drying at 55℃for 12 h, and the test results are shown in Table 4. The dry weight of the obtained cells was still 1.915×10 -3 0.002.+ -. G/mL, from which Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 has strong growth ability and can be propagated in large quantities.
Example 3: lactobacillus casei (Lactobacilli)casei) Evaluation of temperature tolerance of BIGTREE-B101142
Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into freshly sterilized fully cooled MRS liquid medium and anaerobically cultured at 37℃at 100 rpm for 24 h. Respectively heating the bacterial solutions at 37deg.C, 45deg.C, 60deg.C and 75deg.C for 1 min, 2 min and 3 min, and diluting the bacterial solutions to concentration of 10 -6 、10 -7 、10 -8 、10 -9 CFU∙ml −1 100 mu L of the culture medium is respectively coated on a solid MRS culture medium for anaerobic culture for 18-24 hours at 37 ℃. The plate single colonies were counted and the test results are shown in Table 5.
As can be seen from Table 5, the Lactobacillus casei BIGTREE-B101142 of the present invention still had a large amount of viable bacteria when heated at 60℃for 3 min, and it was found that Lactobacillus casei BIGTREE-B101142 had good high temperature tolerance.
Example 4: lactobacillus casei (Lactobacilli)casei) Evaluation of acid generating ability of BIGTREE-B101142
Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into freshly sterilized completely cooled MRS liquid medium, anaerobically cultured at 37℃and 100 rpm, and the pH of the supernatant was measured after centrifugation at 3580 rpm for 15 min at 25 h, 43 h, 50 h, respectively, and the test results are shown in Table 6.
As can be seen from Table 6, lactobacillus casei (Lactobacillicasei) BIGTREE-B101142 can reach strong acidity during anaerobic culture of 25 h, pH can reach 4.37, and Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 has a strong acid-producing ability.
Example 5: lactobacillus casei (Lactobacilli)casei) Evaluation of acid resistance of BIGTREE-B101142
Deployment of artificial stomachThe liquid (8.2 ml dilute hydrochloric acid, 400 ml double distilled water and 5 g pepsin) is added with double distilled water to constant volume to 500 ml after shaking, a certain amount of solution is taken to adjust pH to 2.48, 3.53 and 4.59, a 0.45 mu m filter membrane is used for sterilization, and the mixture is put into a refrigerator at 4 ℃ for standby. Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into freshly sterilized fully cooled MRS liquid medium and anaerobically cultured at 37℃at 100 rpm for 18-24 h. The bacterial liquid of 60 ml was centrifuged at 3580 rpm for 15 min, and the supernatant was removed, and the bacterial cells were resuspended in 1.5 ml physiological saline. Taking 4 branch separation tubes, respectively adding 6 ml artificial gastric juice, respectively adding 200 mu l of heavy suspension bacteria liquid, and uniformly mixing. Diluting for 0 min and 150 min to concentration of 10 -6 、10 -7 、10 -8 、10 -9 CFU∙ml −1 After 100. Mu.L of the solution was applied to the solid MRS medium for 18-24 h, plate single colonies were counted and survival rate was calculated, and the test results are shown in Table 7.
As is clear from Table 7, lactobacillus casei (Lactobacillicasei) The survival rate of BIGTREE-B101142 at 150 min of artificial gastric juice with pH of 2.48 reaches 117.6%. It can be seen that Lactobacillus casei (Lactobacillicasei) BIGTREE-B101142 has strong acid resistance and strong acid resistance to gastric juice.
Example 6: lactobacillus casei (Lactobacilli)casei) Bile salt tolerance evaluation of BIGTREE-B101142
Preparing artificial intestinal juice (3.4 g potassium dihydrogen phosphate, 250 ml double distilled water for dissolving, adjusting pH to 6.8 with 0.1 mol/L sodium hydroxide solution, adding 2.5 g trypsin, shaking, adding double distilled water to volume of 500 ml, adding a certain amount of ox gall salt into the solution, preparing into artificial intestinal juice with concentration of 0.03%, 0.1%, 0.2% and 0.3%, sterilizing with 0.45 μm filter membrane, and storing in refrigerator at 4deg.C for use). Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into freshSterilizing completely cooled MRS liquid culture medium, and anaerobic culturing at 37deg.C and 100 rpm for 16-20 h. The bacterial liquid of 60 ml was centrifuged at 3580 rpm for 15 min, and the supernatant was removed, and the bacterial cells were resuspended in 1.5 ml physiological saline. Taking 4 branch separation tubes, respectively adding 6 ml artificial intestinal juice, respectively adding 200 mu l of heavy suspension bacteria liquid, and uniformly mixing. Diluting for 0 min and 150 min to concentration of 10 -6 、10 -7 、10 -8 、10 -9 CFU∙ml −1 100 mu L of the solution is respectively coated on a solid MRS culture medium for 16-20 hours, single colonies of the flat plate are counted, the survival rate is calculated, and the test results are shown in Table 8.
As can be seen from Table 8, lactobacillus casei (Lactobacillicasei) BIGTREE-B101142 has a survival rate as high as 188.88% at a bile salt concentration of 0.3%. Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 has high resistance to bile salts.
Example 7: lactobacillus casei (Lactobacilli)casei) Evaluation of bacteriostasis effect of BIGTREE-B101142 fermentation liquor
Lactobacillus casei (Lactobacilli) on MRS solid medium with bamboo stickscasei) Single colonies of BIGTREE-B101142 were inoculated into fresh sterilized completely cooled MRS liquid medium at 37℃and 100 rpm min -1 Anaerobic culture 16 h. Taking a certain amount of bacterial liquid, centrifuging at 8000 rpm for 1 min, and collecting supernatant. And taking a certain amount of bacterial liquid for standby. Respectively taking Shigella flexneri (BNCC 186377), bacillus cereus (BNCC 192974), acinetobacter baumannii (BNCC 337173), drug-resistant Acinetobacter baumannii (ATCC BAA-1605), mixing with LB solid culture medium according to 5% bacterial concentration (namely adding 5 ml bacterial liquid into every 100 ml solid culture medium), pouring onto LB solid culture medium, uniformly placing 4 oxford cups with diameter of 8.0 mm after the mixed bacterial plates are slightly dried, and standing for 5 min; 200. Mu.l of supernatant (supernatant S of fermentation broth) was added to two oxford cups, 200. Mu.l of non-centrifuged bacterial liquid (bacterial F in fermentation broth) was added to two oxford cups, and the mixture was left to stand in a refrigerator at 4℃for 4 h, and then was cultivated at 37℃in a normal positionAntibacterial diameters were observed and recorded after 24-h-cultivation. The test results are shown in fig. 6 and table 9, fig. 6 is a diagram of the bacteriostatic effect of lactobacillus casei BIGTREE-B101142 fermentation broth, wherein A is shigella flexneri BNCC186377, B is bacillus cereus BNCC192974, C is Acinetobacter baumannii BNCC337173, and D is drug-resistant Acinetobacter baumannii ATCC BAA-1605; in FIG. 6A to D, F represents the undiffracted Lactobacillus casei BIGTREE-B101142 fermentation broth (the fermentation broth contains bacteria), and S represents the supernatant of the centrifuged Lactobacillus casei BIGTREE-B101142 fermentation broth (the fermentation broth supernatant).
The results showed that Lactobacillus casei (Lactobacilli)casei) The BIGTREE-B101142 fermentation product shigella flexneri, bacillus cereus, acinetobacter baumannii and drug-resistant Acinetobacter baumannii have strong antibacterial effect.
Example 8: lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 whole genome assay
Sequencing and genome assembly based on Nanopore third-generation sequencing technology and second-generation sequencing technology platform to obtain Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 genome completion map, FIG. 7 is a visual genome map of the bacterium, which is sequentially from outside to inside: first circle, genome sequence information; in the second circle, the GC content curve of the genome sequence slides on the genome by taking 2000bp as a sliding window, and the average GC content is counted; the dashed line shows the average GC content of the reference genome; third, sliding a GC skew curve of the genome sequence on the genome by taking 2000bp as a sliding window, and counting average GC content; the dashed line shows a reference line with GC skew of 0; fourth circle, second generation sequencing depth and coverage information, sliding on genome by using 2000bp as a sliding window, and counting average sequencing depth to reflect reads coverage conditions of different areas; the dashed line shows the overall level of average reads coverage; fifth circle, third generation sequencing depth and coverage information, sliding on genome with 2000bp as a sliding window, counting average sequencing depth, reflecting different regionsReads coverage cases of (2); the dashed line shows the overall level of average reads coverage; the sixth circle, in which the gene coding region (CDS) and the non-coding RNA region (rRNA, tRNA) in the reference genome are shown, is represented by the inner and outer two layers, the outer layer representing the positive strand and the inner layer representing the negative strand. The genome of the strain has the size of 292063 bp and the GC ratio of 47.91 percent, contains 2873 CDSs, 59 tRNA and 15 rRNA, and does not contain plasmids.
Example 9: lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 general database genome functional annotation analysis
(1) General database annotation scale statistics
In order to obtain comprehensive gene function information, the invention carries out gene function annotation of eight databases, and comprises the following steps: uniProt, KEGG and KEGG path, GO, pfam, COG, TIGERfams, refSeq, NR. And (3) comparing the predicted gene sequence with a COG, KEGG, uniprot, refseq functional database to obtain a gene function annotation result. Functional annotations were made using a software hmmer (Version: 3.3.2) based on the Pfam, TIGERFAM database. The annotation statistics are shown in table 10. Common and special annotation of the general database annotation of the coding genes is shown in fig. 8, and statistics of the general database annotation are shown on the left side of the figure; the right side is the consensus and characteristic statistics, wherein the abscissa is the database on the left side corresponding to the position of the black spot, and the ordinate represents the number of genes contained in each database combination of the strain on the abscissa. For example, the first column is statistics common to all general databases, the top is statistics for numbers, and the bottom is labeling of common or unique databases. From the data, lactobacillus casei (Lactobacillicasei) The BIGTREE-B101142 contains more gene fragments which can obtain comprehensive gene function information in various large general databases, and the obtained gene function information is more, thus providing a certain foundation for the research of various genes of the strain.
(2) Pfam domain classification
The Pfam database is a homologous protein family database, which is a collection of protein domain families, comprising over ten thousand protein families. Proteins generally consist of one or more functional regions, commonly referred to as domains (domains). The domains in different proteins appear in different combinations, forming a diversity of proteins, and identifying the domains in proteins is important for understanding the function of the proteins.
The invention is based on the annotation of Pfam domains, the statistics of genes annotated by each domain are summarized, and the top 20 domains with the highest annotation are graphically displayed, and the result is shown in figure 9, wherein the abscissa of the graph is the name of a protein family, and the ordinate is the number of genes aligned to the protein family.
By comparison with the Pfam homologous protein family database, lactobacillus casei (Lactobacillicasei) BIGTREE-B101142 contains many domains and is shown by plotting the top 20 domains with the highest annotation, and the domains provide research basis for the research of the functions of protein molecules.
(3) Gene Ontology classification
Gene Ontology (GO) is an internationally standardized system of Gene functional classification that provides a set of dynamically updated standard vocabulary (controlled vocabulary) to comprehensively describe the attributes of genes and Gene products in organisms.
According to the invention, GO (Gene Ontology) annotation information is simplified to obtain GOslim classification, the functions of genes are summarized and counted from three aspects of cell components, molecular functions and biological processes, and then the two-stage classification of GOslim with the highest annotation of the first 20 pieces of each classification is selected to be plotted, the result is shown in fig. 10, the abscissa of the graph is the content of each classification of GO, the ordinate is the number of genes, in fig. 10, A is a gene function classification chart of the biological processes, B is a gene function classification chart of the cell components, and C is a gene function classification chart of the molecular functions. FIG. 10 shows the gene enrichment of each secondary function of GO in the context of the whole gene, embodying the status of each secondary function in this contextThe method comprises the steps of carrying out a first treatment on the surface of the From FIG. 10, lactobacillus casei (Lactobacilli)casei) The functions of the genes and gene products in BIGTREE-B101142 and the number of the functional genes are displayed, the strength of each function is displayed, and the research direction is provided for subsequent deep research.
(4) KEGG Pathway classification
KEGG (Kyoto Encyclopedia of Genes and Genomes) is a major public database for the systematic analysis of the metabolic pathways (Pathway) of gene products in cells and the function of these gene products, and the use of KEGG can be used to further study the biologically complex behaviour of genes.
The invention classifies the KEGG annotated genome according to the KEGG metabolic Pathway they participate in, and the result is shown in FIG. 11, wherein the abscissa of the graph is the number of annotated genes under Pathway classification; the ordinate is Pathway class, different colours representing different large classes to which they belong; FIG. 11 shows Lactobacillus casei (Lactobacilli)casei) Ways of metabolizing the respective gene products of BIGTREE-B101142 in cells.
(5) COG classification
COG (Cluster of Orthologous Groups of proteins) is a database of 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 encoded proteins, phylogenetic relations of bacteria, algae, eukaryotes with complete genomes, unigene and COG databases are aligned, the possible functions of genes are predicted and the functional classification statistics of the genes are performed, and the gene function distribution characteristics of the species are macroscopically known. COG is divided into 26 groups.
The present invention classifies COG annotated genes according to the groups of COG, and the results are shown in FIG. 12, wherein the abscissa indicates the content of each classification of COG and the ordinate indicates the number of genes. In different functional classes, the content such as metabolism or physiological bias in corresponding period and environment is reflected by the gene, and scientific explanation can be made by combining the distribution of the study objects in each functional class.
(6) Uniprot annotation
UniProt Archive is a comprehensive, non-redundant database that contains all the major, published database protein sequences. Since proteins may exist in different databases and there may be multiple versions in the same database.
Lactobacillus casei (Lactobacilli)casei) The number of genes annotated to the Uniprot database by the HP-B1142 whole genome was 2842, accounting for 98.92%.
(7) Refseq annotation
Refseq (Reference Sequences) database is a database of annotated non-redundant transcripts, proteins and genomic sequences, is a database of NCBI and other organizational corrections, uses terminology defined by the Committee for human gene nomenclature, and includes official genetic symbols and optional symbols. The RefSeq sequence is a non-redundant database screened by NCBI, and has higher general reliability.
Lactobacillus casei (Lactobacilli)casei) The gene number annotated to the Refseq database by the HP-B1142 whole genome was 2827, accounting for 98.40%.
(8) NR annotation
NR database: the amino acid sequence databases of non-redundant proteins include the non-redundant data of the SwissProt, PIR (Protein Information Resource), PRF (Protein Research Foundation), PDB (Protein Data Bank) protein databases and protein data translated from the CDS data of GenBank and RefSeq.
Lactobacillus casei (Lactobacilli)casei) The NR species profile of HP-B1142 is shown in FIG. 13. Different colors in the figure represent different species.
(9) TIGRFAMs annotation
TIGRFAMs is a database of multiple sequence alignments, hidden Markov Models (HMMs) and related information, is a member of the interPro class, is used for protein sequence classification, and supports automatic annotation (mostly prokaryotes) of proteins. Starting from v10.0, the tigrofams model uses HMMER 3, which has excellent search speed and sensitivity.
The invention is characterized in thatLactobacillus casei (Lactobacilli)casei) The HP-B1142 whole genome annotated to the Tigrfams database had a gene number of 1419, accounting for 49.39%.
Example 10: lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142 proprietary database annotation
The invention relates to a personalized analysis method of Lactobacillus casei (Lactobacilli)casei) BIGTREE-B101142, which is annotated with proprietary databases including ARDB database annotation, CAZy database-based carbohydrate enzyme annotation, pathogen-host interaction analysis, drug resistance gene annotation, cytochrome P450 annotation, VFDB annotation, signal peptide prediction, TCDB transporter annotation.
Lactobacillus casei (Lactobacilli)casei) The whole genome of BIGTREE-B101142 was annotated with the proprietary database to find: annotating the ARDB database with resistance gene results as shown in Table 11; a completely matched gene sequence is not annotated in the pathogen-host interaction database; no drug resistance genes were annotated in the CARD database; cytochrome P450 notes that the gene sequences were not perfectly matched; no exactly matching virulence factors were annotated to the VFDB database. The results of the carbohydrate enzyme annotation are shown in table 12; the predicted results of the whole genome signal peptide of Lactobacillus casei BIGTREE-B101142 are shown in Table 13, and the predicted results of the transmembrane protein and the secretory protein are shown in Table 14; the TCDB membrane transporter annotation results are shown in FIG. 14, wherein the abscissa indicates the contents of each class of TCDB and the ordinate indicates the number of genes.
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Example 11: lactobacillus casei triglyceride reducing (TG) in vitro experiments
(1) Preparation of experimental strains
The bamboo sticks are used for dipping glycerinum bacteria in an MRS culture medium to carry out three-area streak culture for 24 h, and then bacterial colonies with good growth state are picked and inoculated in the MRS liquid culture medium and the triglyceride culture medium for standby.
Preparation of triglyceride medium formulation: mixing 2% of polyvinyl alcohol aqueous solution with 100% of soybean vegetable oil according to a volume ratio of 3:1, mixing uniformly to prepare vegetable oil emulsion which is used as a source of triglyceride. Adding the above vegetable oil emulsion into MRS culture medium at a ratio of 5%, adjusting pH to 6.4, 121 deg.C, sterilizing for 15 min, making into triglyceride culture medium, and storing at 4deg.C in refrigerator for use.
(2) Triglycerides content determination experiment by single reagent method (GPO-PAP method)
The culture medium of lactobacillus casei BIGTREE-B101142 and 10 mL inoculated with no bacteria liquid after culturing 24 h in 10 mL was centrifuged at 4℃and 4000r/min for 10 min with a refrigerated centrifuge, and the supernatant was collected and the OD value at 510 nm was measured using a triglyceride kit (Nanjing Biochemical Co. A110-1-1). TG (mmol/L) = (sample OD) 510 Blank OD 510 ) /(calibration OD) 510 Blank OD 510 ) Concentration of X standard (mmol/L)
(3) Lactobacillus casei TG degradation rate calculation
The degradation rate of lactobacillus casei of the present invention on TG was calculated according to the formula TG degradation rate (%) = (total TG content-residual TG content)/total TG content x 100% to be 43.5%.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (6)
1. Lactobacillus casei strainLacticaseibacillus casei ) BIGTREE-B101142, which is characterized in that the BIGTREE-B is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023463.
2. Use of lactobacillus casei bigtre-B101142 as claimed in claim 1 for the manufacture of a medicament having a bacteriostatic effect, wherein the pathogenic bacteria inhibited in the bacteriostatic effect comprise shigella flexneri, acinetobacter baumannii and bacillus cereus.
3. Use of lactobacillus casei BIGTREE-B101142 as claimed in claim 1 in the manufacture of a medicament for lowering triglycerides.
4. A microbial agent comprising lactobacillus casei BIGTREE-B101142 as claimed in claim 1.
5. The microbial inoculum according to claim 4, wherein the viable count of lactobacillus casei BIGTREE-B101142 in the microbial inoculum is 500-3000 hundred million CFU/g.
6. The microbial inoculant of claim 4, wherein the microbial inoculant is a solid microbial inoculant or a liquid microbial inoculant.
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