CN117946911A - Lactobacillus plantarum and application thereof in degradation of intestinal indole - Google Patents
Lactobacillus plantarum and application thereof in degradation of intestinal indole Download PDFInfo
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- CN117946911A CN117946911A CN202311860737.6A CN202311860737A CN117946911A CN 117946911 A CN117946911 A CN 117946911A CN 202311860737 A CN202311860737 A CN 202311860737A CN 117946911 A CN117946911 A CN 117946911A
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- lactobacillus plantarum
- indole
- vhprobi
- intestinal
- strain
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- 235000021574 pickled cabbage Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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- 238000012163 sequencing technique Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
- 229940046307 sodium thioglycolate Drugs 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to lactobacillus plantarum and application thereof in degradation of intestinal indole. The preservation number of the lactobacillus plantarum is CCTCCNO: m20232619. The lactobacillus plantarum VHProbi O03 provided by the invention has a very strong degradation effect on intestinal indole, and can significantly degrade indole in vitro. The strain has stronger tolerance to artificial intestinal juice; is sensitive to common antibiotics such as erythromycin and ampicillin, does not generate hemolysin, can not dissolve blood cells, and has good biological safety; has strong antioxidant and cholesterol degrading abilities. The lactobacillus plantarum VHProbi O can be used for preparing health-care products or medicines for clearing urine toxins and relieving chronic kidney diseases, and has a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus plantarum and application thereof in degradation of intestinal indole.
Background
Chronic kidney disease is a common chronic kidney disease characterized by progressive kidney damage, and the treatment thereof requires a lot of sanitary resources, so that it is very important to prevent or delay the entry of chronic kidney disease into end-stage kidney disease. Studies have shown that the protein-binding small molecule compound indoxyl sulfate (Indoxyl sulfate, IS) IS one of the uremic toxins. IS the formation of dietary tryptophan by the catabolism of tryptophan-containing bacteria in the intestine to produce indole, which IS oxidized and sulfonated in the liver after intestinal absorption. Indoxyl sulfate IS in excess of 90% of the blood binds to albumin, which IS difficult to remove by conventional hemodialysis and peritoneal dialysis after binding, and IS mainly excreted through the kidney. The indoxyl sulfate concentration in serum of normal population is almost zero. In the case of chronic kidney diseases, the kidney excretion capacity is reduced, and indoxyl sulfate is accumulated, and the existing researches prove that when the kidney function is not complete, especially when the blood creatinine is more than or equal to 265 mu mol/L, the serum indoxyl sulfate content of a patient can reach 0.8-1.0mg/dl. Indolol sulfate accumulated in the body enters various tissue organs of the body along with blood circulation, thereby causing damage to kidneys and organs outside the kidneys, such as promotion of glomerulosclerosis and tubular interstitial fibrosis, and acceleration of renal failure; causing cardiovascular diseases, heart failure and coronary atherosclerosis; resulting in damage to the central nervous system, cognitive dysfunction, etc.
Studies have shown that intestinal indole-producing bacteria catabolize food tryptophan to be the only source of IS in vivo. Thus, reducing the production of protein-bound toxins during the progression of end-stage renal failure is critical to delay disease progression. Limiting protein intake and regulating intestinal flora are two alternative approaches. However, limiting protein intake is not a long-term option for chronic kidney disease patients to reduce protein-bound toxin production due to side effects such as malnutrition and poor compliance. And modulation of intestinal flora is a more widespread and effective approach, with prebiotics and probiotics as the primary choices.
Probiotics are a class of active microorganisms that improve the balance of flora and are beneficial to the body, and can colonize the intestinal tract of the human body. Because of the close relationship between intestinal flora and chronic renal failure, students begin to reduce IS production by intervening in pathogenic bacteria growth and reducing uremic toxin absorption and the like, and aim at interfering metabolism and production of IS and PCS by intestinal bacterial preparations. The study proves that the probiotics fermented camel milk can effectively relieve pathological injury of nephrons, slow down development of chronic renal failure of rats and improve vitality of rats. For example, by administering a bifidobacteria formulation, the growth of E.coli can be inhibited, and the tryptophan breakdown by E.coli can be reduced to significantly reduce IS levels in patients with chronic renal failure. The wakoku team found that inulin-type levan reduced intestinal indole production in peritoneal dialysis patients by modulating intestinal flora and reducing species abundance of indole-producing bacteria. Therefore, the metabolic pathway in the intestinal bacteria is targeted, available probiotics are screened, indole in the intestinal tract is degraded, so that the production level of in-vivo urotoxin indoxyl sulfate is reduced, the damage of indoxyl sulfate to the organism is lightened, and the method is an important strategy for effectively relieving symptoms of chronic renal failure patients and prolonging the life of the patients.
Disclosure of Invention
The invention aims to provide a novel lactobacillus plantarum (Lactiplantibacillus plantarum) and application thereof. The provided lactobacillus plantarum is separated from a natural fermented acid cabbage fermentation liquor, has strong degradation capability on a tryptophan metabolite indole in intestinal tracts, wherein the indole is a precursor substance of protein-bound urotoxin indoxyl sulfate, and the content of the indole is reduced, so that the indoxyl sulfate level in a patient is reduced, the symptoms of the patient suffering from renal failure are relieved, and the lactobacillus plantarum can be applied to the adjuvant therapy of the patient suffering from renal failure, prevent more complications and prolong the service life of the patient.
The lactobacillus plantarum provided by the invention is VHProbi O (Lactiplantibacillus plantarum VHProbi O03), and is preserved in China Center for Type Culture Collection (CCTCC) in the year 2023, month 12 and 21, and the preservation number is CCTCC NO: m20232619.
The Riboprinter fingerprint of the Lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is shown in figure 3; the RAPD fingerprint is shown in FIG. 4, the rep-PCR fingerprint is shown in FIG. 5, and the MALDI-TOF-MS protein fingerprint is shown in FIG. 6.
The lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is applied to the preparation of foods or health-care products.
The lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is applied to preparation of medicines with indole degradation function.
The invention also provides application of the lactobacillus plantarum VHProbi O strain VHProbi O in preparing medicines for clearing urea toxins and relieving chronic kidney diseases.
The lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is applied to preparation of medicines with cholesterol reducing function.
The lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is applied to preparation of medicines with an antioxidant function.
The invention also provides a product for degrading intestinal indole and relieving chronic kidney disease, which comprises lactobacillus plantarum VHProbi O live bacteria and/or fermentation products thereof.
The product is a health product or a medicine.
The lactobacillus plantarum VHProbi O03 provided by the invention has a very strong degradation effect on intestinal indole, can significantly degrade indole in vitro, and the degradation rate of 48 hours is 51.58 +/-4.15%. The strain has stronger tolerance to artificial intestinal juice; is sensitive to common antibiotics such as erythromycin and ampicillin, does not generate hemolysin, can not dissolve blood cells, and has good biological safety. In addition, lactobacillus plantarum VHProbi O03 has strong antioxidant capacity and cholesterol degradation capacity, and DPPH clearance rate reaches 25.57%; the clearance rate of the bacterial suspension and the fermentation supernatant to the HRS reaches 58.92 percent and 91.02 percent respectively; the anti-lipid peroxidation inhibition rate of the fermentation supernatant is 41.48%; the degradation rate of cholesterol reaches 20.40 percent. The lactobacillus plantarum VHProbi O can be widely applied to the production of foods, health-care products and medicines for removing urinary toxins and relieving chronic kidney diseases, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the growth of O03 strain in indole-containing medium;
FIG. 2 is a carbon source metabolism map of O03 strain;
FIG. 3 is a fingerprint of strain O03 Riboprinter;
FIG. 4 shows RAPD finger-prints of O03 strain;
FIG. 5 shows rep-PCR fingerprint of O03 strain;
FIG. 6 shows the MALDI-TOF-MS protein fingerprint of O03 strain.
Detailed Description
The safety of the lactobacillus plantarum VHProbi O03 meets the requirement of regulations, can be used as a food raw material source, and has no side effect and excessive risk after long-term administration. The lactobacillus plantarum VPHrobi O is a newly discovered strain through heterogeneous taxonomy identification. The lactobacillus plantarum VHProbi O03 provided by the invention has a very strong degradation effect on intestinal indole, and can significantly degrade indole in vitro. The strain can be singly used, can play a role in relieving chronic kidney disease without being compounded with prebiotics and/or other probiotics, and has important application value.
Applicant preserved lactobacillus plantarum VHProbi O (Lactiplantibacillus plantarum VHProbi O) at 2023, 12 and 21 at chinese typical culture collection at university of armed chinese with the preservation number cctccc NO: m20232619.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of strains
1. Isolation of strains
Preparing MRS (Man Rogosa Sharpe) broth: 1L of pure water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of tween 80, 2.0g of diamine citrate, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate and pH adjustment of 6.2-6.5.
Preparing MRS agar culture medium: 1LMRS broth 15g agar was added.
Taking 1g of pickled cabbage juice, diluting with sterile normal saline, placing into a sterile sample bag, and beating and mixing uniformly with a homogenizer; and (3) taking 100 mu L of mixed solution for gradient dilution, coating the mixed solution on an MRS agar medium, and performing anaerobic culture at 37 ℃ for 48 hours, and performing microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant screens 28 potential lactobacillus strains which are respectively named as O01, O02, … … and O28.
2. Bacterial strain screening of in vitro degradable indole
Preparing an indole solution: preparing indole mother liquor of 30mg/ml with 70% ethanol, and sterilizing with 0.22um filter membrane. The strain culture medium is diluted to the required concentration when in use.
Strain preparation: lactic acid bacteria streak activation. Taking the frozen and preserved lactobacillus glycerin tube, carrying out aseptic operation, streaking to an MRS plate, and culturing for 48-72 h at 37 ℃.
Preparing a bacterial solution to be screened: after single colony is grown on the plate, the plate is aseptically picked up to MRS broth, and the plate is subjected to stationary culture at 37 ℃ for 24 hours. The fresh MRS broth was inoculated at 1% inoculum size, about 6ml per strain.
(1) Resistance test of strains to different concentrations of indole.
Indole was diluted to 0, 50, 100, 150, 200, 300, 400, 600ug/ml with strain medium, and the strains were inoculated at 1% inoculum size, respectively, and the growth curves of the strains were monitored with a microplate reader.
The results show that the activity of the strain is not affected, even slightly promoted, when the indole content in the culture medium is close to 300ug/ml, and the activity of the strain is inhibited, and according to the data, 0.25-2.6mM of human intestinal or fecal indole is shown to be about 45-468ug/ml, so that 300ug/ml can be used as the indole addition concentration for verifying the indole degradation capacity of the strain.
(2) Detection of indole metabolizing ability of the strain:
inoculating lactobacillus obtained by primary screening into fresh culture medium with indole concentration of 300ug/ml according to inoculum size of 1%, culturing at 37deg.C for 48 hr, respectively taking 1.5ml fermentation liquor to new EP tube at 0 hr, 24 hr and 48 hr, centrifuging for 3min, collecting supernatant, and detecting indole content in supernatant by using indole detection kit. The operation method and the result judgment are specifically described in Indole Assay Kit specifications.
Indole degradation rate calculation: indole degradation rate = (initial concentration-final concentration)/initial concentration × 100%.
The result shows that at 24h, 11 strains of 28 potential lactobacillus obtained by preliminary screening have the degradation rate of indole more than 30%, wherein the degradation rate of O03 strain is highest and reaches 31.13+/-1.70%; at 48h, the degradation rate of 5 strains to indole exceeds 50%, wherein the degradation rate of the O03 strain is highest and reaches 51.58 +/-4.15%.
The growth curves of O03 strains in different concentrations of indole are shown in FIG. 1.
Example 2 identification of strains
1. Colony morphology identification
O03 strain is inoculated on MRS agar culture medium, after anaerobic culture for 24h at 37 ℃, O03 single colony is seen to be milky white, the surface is smooth, the thallus is in a short rod shape or a bent rod shape under a microscope, and sometimes is in a chain shape, and no spores or flagellum exist.
2. Identification of physiological and biochemical characteristics
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh O03 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are diluted by 50 times after the same volume of PBS buffer is used as an inoculation liquid.
2.1 Salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results showed that the O03 strain had a maximum tolerated salt concentration of 7%.
2.2 Carbon Source metabolism test
And C, carrying out carbon source metabolism experiments on the O03 strain by using an API 50CHL kit, wherein the concrete reference of the description of the API 50CHL kit is the experimental method and the result interpretation. The identification result of the O03 strain is as follows: % id=99.1 and T value=1, api results are lactobacillus plantarum, see figure 2.
2.3 Glucose acid and gas production test
The medium formulation used in this example is as follows:
Peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; 0.5ml of saline solution A; 0.5ml of salt solution B; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100ml; the pH is 6.8-7.0.
The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.
Salt solution A: KH 2PO4 10g、K2HPO4 1.0.0 g was dissolved in distilled water to a volume of 100mL.
Salt solution B: mgSO 4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2 O0.68 g was dissolved in distilled water and the volume was set to 100mL.
Under aseptic condition, inoculating the inoculating solution with 10% inoculating amount, inoculating the culture medium without inoculating bacteria as control, sealing the top with 2mL sterile liquid paraffin, culturing at 37deg.C for 24 hr, and observing whether the color of the culture medium changes.
The results show that: after 24h of culture at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the small inverted tube, which indicates that O03 strain ferments glucose to produce acid and does not produce gas.
3. Molecular biological identification
3.1, 16S rDNA Gene sequence analysis
3.1.1 Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
3.1.2, 16S rDNA Gene amplification
Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
The 16s rDNA sequence (SEQ ID NO: 1) of the O03 strain was obtained by sequencing, and the sequences were aligned in the NCBI database to preliminarily determine that the O03 strain was Lactobacillus plantarum.
SEQ ID NO. 1 sequence is as follows:
GGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCAAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTGC.
3.2, riboprinter finger print
The purified single colony is dipped from an agar culture medium plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then a sample frame is placed into a heater for inactivation and then placed into a Riboprinter system, and a bacterial identification result is obtained after DNA preparation, film transfer, imaging detection and data processing are carried out on the sample. The identification result shows that the O03 strain is lactobacillus plantarum, and the Riboprinter fingerprint result is shown in figure 3.
3.3, RAPD and rep-PCR fingerprint identification
3.3.1RAPD fingerprint identification
Primer sequence: GAGGGTGGCGGTTCT.
Table 1: RAPD reaction System Table
1.5% Agarose gel plates were prepared, DL2000DNA MARKER was used as a result control, 100V was regulated for 80min, and finally the electropherograms were detected using a gel imaging system. RAPD finger-prints of O03 strain are shown in FIG. 4.
3.3.2Rep-PCR finger print
Primer sequence: CTACGGCAAGGCGACGCTGACG.
Table 2: table of the reaction System of rep-PCR
DL2000 DNA MARKER served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of O03 strain is shown in FIG. 5.
3.4 MALDI-TOF-MS detection of strain ribosomal protein expression
Inoculating fresh bacterial liquid into MRS liquid culture medium according to 0.1% inoculum size, culturing at 37deg.C and 150rpm for 48 hr, collecting bacterial cells, washing with sterile water for 4 times, and air drying surface water. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. The co-crystallization film formed by the sample and the matrix is irradiated by laser to ionize the protein in the sample, the ions are accelerated to fly through a flight pipeline under the action of an electric field of 10 KV to 20KV, and the molecular weight of the protein is detected according to different flight time reaching a detector. Protein fingerprint is obtained by Autofms 1000,1000 analysis software Autof Analyzer v 1.0.0, and the ion peaks of the main ribosomal proteins of the O03 strain are as follows: m/z5178.112, 5193.559, 7843.814. The results of the identification are shown in FIG. 6.
3.5 Whole genome sequencing
Fresh bacterial liquid was inoculated into MRS liquid medium at an inoculum size of 0.1%, cultured at 37℃for 20 hours, centrifuged at 8000rpm for 10 minutes, and the bacterial cells were collected. The bacterial cells are sent to a sequencing center to obtain the whole genome sequence of the bacterial cells, the genome sequence is uploaded to NCBI gene database, the genome contains 1 chromosome and 2 plasmids, and GenBank accession numbers are CP134217, CP134218 and CP134219 respectively.
The colony morphology, the physiological and biochemical characteristics and the molecular biological identification result of the O03 strain are combined, the O03 strain is determined to be a new lactobacillus plantarum, and the new lactobacillus plantarum is named as lactobacillus plantarum VHProbi O (Lactiplantibacillus plantarum VHProbi O03).
Example 3 antibiotic resistance test of Lactobacillus plantarum VHProbi O03
The minimum inhibitory concentration MIC value of the antibiotic for Lactobacillus plantarum VHProbi O was determined by a micro broth dilution method, and the specific results are shown in Table 3.
TABLE 3 antibiotic MIC values for Lactobacillus plantarum VHProbi O03
MIC units μg/mL.
From the results shown in Table 3, the lactobacillus plantarum VHProbi O provided by the invention is sensitive to common antibiotics such as erythromycin and ampicillin Lin Heke, and has good biological safety.
EXAMPLE 4 tolerance test 1 of Lactobacillus plantarum VHProbi O03 against Artificial gastric juice and Artificial intestinal juice, preparation of artificial gastric juice
5G of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
2. Preparation of artificial intestinal juice
5G of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH 2PO4 and 3.0g of ox gall salt are respectively weighed, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3. Experimental method
2ML of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial gastric juice or intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after being digested by artificial gastric juice or intestinal juice is shown in Table 4.
TABLE 4 viable bacteria scale after digestion of Artificial gastrointestinal fluids
As shown in Table 4, the viable bacteria amount of the lactobacillus plantarum VHProbi O screened by the method is basically unchanged after the lactobacillus plantarum VHProbi O is digested by artificial gastric juice, and the higher viable bacteria amount can be still detected after the lactobacillus plantarum VHProbi O is digested by artificial intestinal juice for 3 hours, which proves that the strain has strong tolerance to gastrointestinal juice.
EXAMPLE 5 antioxidant function assay of Lactobacillus plantarum VHProbi O03
1. Determination of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) removal ability
1ML of lactobacillus plantarum VHProbi O strain suspension is taken, 1mL of 0.4mM of the existing DPPH free radical solution is added, after uniform mixing, the mixture is placed at room temperature for shading reaction for 30min, then an absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution, and with equal volumes of Lactobacillus plantarum VHProbi O bacterial suspension and ethanol mixed solution.
The clearance was calculated according to the following formula, and the results are shown in Table 5.
Clearance% = [1- (a Sample of -A Blank space )/A Control ] ×100%).
TABLE 5 effect of Lactobacillus plantarum VHProbi O bacterial suspension on DPPH radical scavenging
2. HRS clearance capability assay
After mixing 100. Mu.L of 5mM sodium salicylate-ethanol solution, 100. Mu.L of 5mM ferrous sulfate, 500. Mu.L of deionized water and 200. Mu.L of Lactobacillus plantarum VHProbi O.sp.03 suspension, 100. Mu.L of hydrogen peroxide solution (3 mM) was added, and after 15min in a 37℃water bath, the absorbance of the sample was measured at a wavelength of 510 nm.
The hydroxyl radical scavenging rate was calculated according to the following formula.
Clearance% = (a Sample of -A Control of )/(A Blank space -A Control of ) ×100%.
Wherein: a Control of is a deionized water substitution sample, a Blank space is a deionized water substitution sample and H 2O2.
The specific results are shown in Table 6.
TABLE 6 scavenging effect of Lactobacillus plantarum VHProbi O03 on HRS free radicals
3. Determination of lipid peroxidation resistance
Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water. Adding 1mL of linoleic acid emulsion, 1mLFeSO 4 (1%) and 0.5mL of sample into 0.5mL of PBS solution (pH 7.4), carrying out water bath at 37 ℃ for 1.5 hours, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30 minutes, rapidly cooling, centrifuging at 4000rpm/min for 15 minutes, and collecting supernatant, wherein absorbance at 532nm is measured to obtain A; the control group was A 0 as a substitute for the sample with 0.5mL distilled water.
Inhibition (%) = (a 0－A)/A0 ×100%.
Note that: a is absorbance of a sample group; a 0 is the absorbance of the control group and the results are shown in Table 7.
TABLE 7 inhibition of lipid peroxidation by Lactobacillus plantarum VHProbi O03
Example 6 Lactobacillus plantarum VHProbi O03 in vitro cholesterol degradation test
1. Qualitative assay of bile salt enzyme Activity
Preparation of bile salt-containing MRS solid plates: to freshly prepared MRS broth was added 0.2% TCA, 0.2% sodium thioglycolate, 0.37g/L CaCl 2, and 1.5% agar. Sterilizing at 121deg.C for 15min, pouring into flat plate, and condensing.
Preparation of MRS solid plates: fresh MRS culture medium is sterilized at 121deg.C for 15min, and poured into flat plate for condensation.
The Lactobacillus plantarum VHProbi O strain VHProbi O is streaked on an MRS plate for separation and purification, single bacterial colonies are taken for identification and streaked on the MRS plate and the MRS plate containing bile salts respectively, after the culture is carried out for 72 hours at 37 ℃, the results are observed, and the photographs are taken and archived.
The results showed that the two plates streaked colonies were indistinguishable and no other material was produced beside the colonies, indicating that Lactobacillus plantarum VHProbi O had negative bile salt enzyme activity.
2. Cholesterol degradation test
Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.
10.0G of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salt and 1000mL of distilled water are weighed, the pH value is regulated to 7.3, sterilization is carried out for 30min at 115 ℃, and then a cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1%.
Inoculating Lactobacillus plantarum VHProbi O03 fresh bacterial liquid according to 0.1% inoculum size, standing at 37 ℃ for 48 hours, taking 0.2mL bacterial liquid, adding 1.8mL absolute ethanol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 rpm for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003< measurement of cholesterol in food >.
The results show that: the degradation rate of the lactobacillus plantarum VHProbi O03 provided by the invention on cholesterol reaches 20.40% (which is data without bile salts).
EXAMPLE 7 measurement of hydrophobicity of Lactobacillus plantarum VHProbi O03 on cell surface
After the lactobacillus plantarum VHProbi O strain VHProbi O is activated in MRS liquid culture medium, 1ml of bacterial liquid is sucked, 6000rmp is centrifuged for 2min, and after bacterial bodies are collected, PBS solution is used for washing 2 times. The cells were resuspended in sterilized 0.1MKNO 3 ml solution. The OD600 was recorded as a 0 after three-fold dilution of the bacterial suspension. One third of the volume of xylene was added to the bacterial suspension, and after mixing, the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase was carefully aspirated and the absorbance A1 was measured at OD600 nm. Cell hydrophobicity was calculated according to the following formula and the average was obtained from three experiments.
Hydrophobicity (%) = (a 0 -A1)/a1×100%.
The result shows that the surface hydrophobicity of the lactobacillus plantarum VHProbi O strain VHProbi O provided by the invention is 38.95%.
Example 8 intestinal cell adhesion test of Lactobacillus plantarum VHProbi O03
Caco-2 cells were inoculated in six-well plates at an inoculum size of 2X 10 6 cells/well, and cultured in a carbon dioxide incubator for 24 hours for cell adhesion experiments; strains at stationary phase were resuspended to 5X 10 7 CFU/mL with MRS medium; 1mL of lactobacillus plantarum VHProbi O03 bacterial liquid is added into a six-hole plate with the existing cell wall, and the culture is carried out for 2 hours in a carbon dioxide incubator; repeatedly washing with PBS for 3 times to remove non-adhering bacteria; digestion was stopped by adding 500ul of pancreatin for 3 min, followed by 1.5mL of cell culture broth, repeated pipetting, and collecting the resulting solution into sterile EP tubes, and plating counts by 10-fold, 100-fold, 1000-fold, 10000-fold gradient dilution of the collected solution. Cells from the blank group were counted simultaneously.
The adhesion ability of the test strain was calculated according to the following formula:
Adhesion capacity (CFU/cells) =total number of bacteria adhered per culture well/total number of cells per culture well.
The results show that: the adhesion capacity of the lactobacillus plantarum VHProbi O is 1.72, and the standard deviation is 0.15%.
In conclusion, the lactobacillus plantarum VHProbi O03 provided by the invention is sensitive to common antibiotics, does not produce hemolysin, and has good biological safety. In vitro experiments prove that the lactobacillus plantarum VHProbi O has stronger degradation capability to indole, is favorable for removing urinary toxins and relieving chronic kidney diseases. The lactobacillus plantarum VHProbi O can be widely used for preparing health-care products or medicines with the functions of degrading intestinal indole and relieving chronic kidney diseases.
Claims (10)
1. The lactobacillus plantarum is characterized in that the preservation number of the lactobacillus plantarum is CCTCC NO: m20232619.
2. The lactobacillus plantarum according to claim 1, wherein the sequence of the 16s rDNA of the lactobacillus plantarum is SEQ ID NO. 1.
3. The lactobacillus plantarum of claim 1, wherein the Riboprinter fingerprint of the lactobacillus plantarum is shown in figure 3; RAPD finger print is shown in FIG. 4, rep-PCR finger print is shown in FIG. 5, MALDI-TOF-MS protein finger print is shown in FIG. 6.
4. Use of lactobacillus plantarum according to claim 1 for the preparation of a food or a health product.
5. Use of lactobacillus plantarum according to claim 1 for the preparation of a medicament with indole degrading function.
6. Use of lactobacillus plantarum according to claim 1 for the manufacture of a medicament for the removal of urinary toxins and alleviation of chronic kidney disease.
7. Use of lactobacillus plantarum according to claim 1 for the preparation of a medicament having cholesterol lowering function.
8. Use of lactobacillus plantarum according to claim 1 for the preparation of a medicament with antioxidant function.
9. A medicine for degrading intestinal indole and relieving chronic kidney disease, which is characterized by comprising the lactobacillus plantarum of claim 1.
10. The pharmaceutical product of claim 8, wherein the product comprises the lactobacillus plantarum fermentation product of claim 1.
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