CN113957021B - Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof - Google Patents
Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof Download PDFInfo
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- CN113957021B CN113957021B CN202111496871.3A CN202111496871A CN113957021B CN 113957021 B CN113957021 B CN 113957021B CN 202111496871 A CN202111496871 A CN 202111496871A CN 113957021 B CN113957021 B CN 113957021B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof, lactobacillus plantarum (A)Lactobacillus plantarum) grx401 has been deposited in the general microbiological culture collection center of the chinese committee for culture collection on 8/10/2021, address: the collection number of the microorganism research institute of Chinese academy of sciences, no. 3 Xilu No. 1 of Beijing, chaoyang, and the collection number is CGMCC NO.23544. The lactobacillus plantarum grx401 can be used for preparing fermented milk, fermented milk beverages and lactobacillus solid beverages containing lactobacillus with digestion stress resistance and high intestinal adhesion. The strain is derived from intestinal tracts of longevity people of Guangxi Bama, the digestion stress resistance and the capability of adhering intestinal mucoprotein and intestinal epithelial Caco-2 cells of the strain are researched, and the drug resistance and the change of the adhesion capability of the strain under the saliva-gastric juice-intestinal juice stress are researched.
Description
Technical Field
The invention relates to the technical field of biology, in particular to lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof.
Background
The lactobacillus is used as an important probiotic in human intestinal tracts, can regulate the micro-ecological balance of the intestinal tracts of organisms, and has the probiotic functions of improving immunity, resisting oxidation and the like. And the content of lactobacillus is more than 10 6 The precondition that CFU/mL viable bacteria reach and adhere to the intestinal tract is that the CFU/mL viable bacteria play a probiotic role; before reaching the intestinal tract, the lactic acid bacteria need to pass through a series of digestive stresses of the oral cavity, the stomach, the intestinal tract and the like, lysozyme in the oral cavity can destroy the cell wall structure of the lactic acid bacteria body by hydrolyzing N-acetylglucosamine and N-acetylmuramic acid, the protein maintaining normal physiological functions in the bacteria body cells is denatured by the acid environment (pH is 2.0-5.0) in the stomach, pepsin and the like, and bile salt (0.1% -0.3%) and pancreatic enzyme (0.1%) in the intestinal tract can also cause the damage and even death of the bacteria body.
The adhesion of lactic acid bacteria to intestinal tract can prolong the stay time of lactic acid bacteria in intestinal tract, enhance the information exchange between lactic acid bacteria and intestinal epithelial cells, and promote bacterial strain to improve the health of organism by regulating intestinal microbiota and its metabolites. A series of adverse environments such as oral cavity, stomach and intestinal tract also have great influence on the intestinal adhesion capability of the strain. Therefore, the strong digestion stress resistance and good intestinal adhesion ability after sequentially passing through saliva, gastric juice and intestinal juice are important standards for screening probiotic lactic acid bacteria.
Disclosure of Invention
The invention aims to solve the problems and provides lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof.
The purpose of the invention is realized as follows: a Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity is preserved in China general microbiological culture Collection center at 10-8.2021, address: no. 3 of Xilu No. 1 of Beijing, chaoyang, beijing, china academy of sciences microbial research institute, with the collection number of CGMCC NO.23544.
A Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity has low drug resistance, and the intestinal adhesion capacity has no significant change after digestion stress.
Application of Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity in production of fermented milk, fermented milk beverage and Lactobacillus solid beverage food.
The invention provides a Lactobacillus plantarum (Lactobacillus plantarum) which has digestion stress resistance and high intestinal adhesion capacity, is preserved in China general microbiological culture Collection center (CGMCC) at 10/8/2021, and has the address: no. 3 of Xilu No. 1 of Beijing, chaoyang, beijing, china academy of sciences microbial research institute, with the collection number of CGMCC NO.23544. The lactobacillus plantarum grx401 is obtained through the following steps:
separating lactobacillus from intestinal tracts of longevity people of Guangxi Bama by a culture medium;
sequentially carrying out a resistance test simulating saliva-gastric juice-intestinal juice on the separated strain to obtain a digestion stress resistant strain;
testing the ability of the isolate to adhere to intestinal mucoprotein and intestinal epithelial Caco-2 cells to obtain a strain with good intestinal adhesion ability;
carrying out antibiotic resistance research on the separated strains to obtain a strain with low resistance, namely grx401;
changes in intestinal adhesion ability of grx401 after saliva-gastric fluid-intestinal fluid stress were studied.
The lactobacillus plantarum grx401 can be used for preparing fermented milk, fermented milk beverages and lactobacillus solid beverages containing lactobacillus with digestion stress resistance and high intestinal adhesion. The strain is derived from intestinal tracts of longevity people of Guangxi Bama, the digestion stress resistance and the capability of adhering intestinal mucoprotein and intestinal epithelial Caco-2 cells of the strain are researched, and the drug resistance and the change of the adhesion capability of the strain under the saliva-gastric juice-intestinal juice stress are researched.
Drawings
FIG. 1 is a morphological diagram of the strain of the invention;
FIG. 2 is an electrophoretogram of 16S rDNA PCR amplification products of the strain of the present invention;
Detailed Description
The research adopts the simulated in vivo ionic environment and the existing part of digestive enzyme to prepare simulated saliva-gastric juice-intestinal juice, screens out the lactobacillus tolerant to digestive stress, screens out the lactobacillus with high intestinal adhesion capacity through intestinal mucoprotein and intestinal epithelium Caco-2 cell tests, and researches the drug resistance and the change of the intestinal adhesion capacity after the digestive stress.
Related reagent and culture medium formula
(1) Configuration of simulated saliva: preparing CaCl with 0.75mmol/L by deionized water 2 、3.70mmol/L KH 2 PO 4 、0.15mmol/L MgCl 2 (H 2 O) 6 、15.10mmol/L KCl、0.06mmol/L(NH 4 ) 2 CO 3 、13.60mmol/L NaHCO 3 Adding lysozyme to the solution of (1), adjusting pH to 7.0 with 1mol/L HCl, filtering with 0.22 μm filter membrane to remove bacteria, and preparing.
(2) Configuration of simulated gastric fluid: prepared by deionized water and contains 0.075mmol/L CaCl 2 、0.90mmol/L KH 2 PO 4 、0.1 0mmol/L MgCl 2 (H 2 O) 6 、6.9 0mmol/L KCl、0.50mmol/L(NH 4 ) 2 CO 3 、25mmol/L NaHCO 3 And 47.20mmol/L NaCl solution, adding pepsin to the final concentration of 3.0g/L, adjusting pH to 3.0 with 1mol/L HCl, filtering and sterilizing with 0.22 μm filter membrane, and preparing the final product.
(3) Preparation of simulated intestinal fluid: prepared with 0.30mmol/L CaCl in PBS 2 、0.80mmol/L KH 2 PO 4 、0.33mmol/L MgCl 2 (H 2 O) 6 、6.90mmol/L KCl、0.50mmol/L(NH 4 ) 2 CO 3 、85mmol/L NaHCO 3 And 38.40mmol/L NaCl solution, adding trypsin and bile salt to final concentrations of 0.1% and 0.3%, respectively, adjusting pH to 8.0 with 1mol/L HCl, filtering with 0.22 μm filter membrane for sterilization, and preparing for use.
(4) MRS liquid culture medium: 10.0g of peptone, 20.0g of glucose, 10.0g of beef extract, 5.0g of yeast extract, 1mL of Tween-80 and C 2 H 3 NaO 2 5.0g、K 2 HPO 4 7H 2 O 0.2g、MnSO 4 ·4H 2 O0.05 g, deionized water to 1000mL, sterilization at 121 ℃ for 15min, placing at room temperature for cooling and using. MRS solid culture medium is prepared, namely 15.0g of agar is added.
Establishment of intestinal adhesion model
Building a mucin model: weighing a certain amount of mucin, dissolving the mucin in PBS to prepare a mucin solution with the concentration of 1mg/mL, filtering and sterilizing the solution through a 0.22-micron filter membrane, taking 0.5mL to a 24-hole cell culture plate, culturing the cell culture plate at 37 ℃ for 1h, then culturing the cell culture plate at 4 ℃ overnight, adding the mucin with the same volume for 2h at 37 ℃ to make up blank sites, and washing the cell culture plate for 2 times by using PBS (pH 7.2) after the mucin with the same volume is added.
Establishing a Caco-2 cell monolayer model: and taking out the Caco-2 cell cryopreservation tube from liquid nitrogen, quickly putting the tube in a water bath at 37 ℃ for unfreezing, and wiping the exterior of the cryopreservation tube with 75% alcohol after thawing. Transferring the cell suspension to a 15mL centrifuge tube, adding 3mL of MEM complete medium (20% high-quality fetal bovine serum, 1% MEM non-essential amino acid solution, 1% Gluta MAX glutamine additive, 1% sodium pyruvate solution), centrifuging at 1000 Xg for 5min, discarding the supernatant after centrifugation, adding 1mL of MEM complete medium to resuspend the cells, and transferring to 25cm 2 Cell culture flask, 9mL of MEM complete culture medium added thereto, 37 ℃ C., 5% CO 2 Cultured in a cell culture box. When the cells grow to 80% -90% fusion, 0.25% pancreatin cell digestive juice is used for digestion, and the ratio of 1: and 3, subculturing. When the cell generation number is between 22 and 30 generations, the cell can be used for subsequent experiments. After digestion of Caco-2 cells, the suspension was adjusted to 2X 10 5 cell/mL, 0.5mL aliquot was plated in 24-well cell culture plates at 37 ℃ and 5% CO 2 Culturing in a cell incubator, and changing the culture solution 1 time per day until the culture reaches a monolayer.
Detailed description of the invention
1. Collecting a sample;
respectively collecting feces samples of Guangxi Bama longevity people who do not take antibiotics within one week and Yunnan traditional fermented foods such as pickles, cheeses, pickled vegetables and the like, adding 1mL of sterile liquid paraffin oil into the feces samples, sealing, storing the feces samples and the traditional fermented foods together at 4 ℃, and separating lactic acid bacteria in the feces samples as soon as possible.
2. Separating lactic acid bacteria;
smashing the collected sample into paste, homogenizing with a vortex oscillator, diluting with sterile sample diluent on a sterile operating platform to corresponding gradient, inoculating appropriate amount of the diluted sample into solid culture medium such as MRS, LBS and the like, culturing in an anaerobic jar at 37 ℃ for 48h, selecting typical bacterial colony on a plate, and streaking and separating to obtain pure bacterial colony. Inoculating the pure bacterial colony into MRS liquid culture medium, performing anaerobic culture at 37 ℃ until the culture reaches logarithmic phase, and freeze-drying and preserving for later use.
3. Physiological and biochemical tests of lactic acid bacteria;
the isolated strains were gram-stained and subjected to physiological and biochemical tests such as catalase, nitrate reduction, indole, and the like.
46 strains are obtained by separating from collected samples, 37 strains are positive in gram staining and rod-shaped or spherical in shape, can grow under the environment of 15 ℃ and 45 ℃, and are negative in catalase, nitrate reduction and indole tests, so that 37 strains are preliminarily identified as lactic acid bacteria.
4. A digestion stress tolerance test of lactic acid bacteria;
centrifuging MRS activated lactobacillus at 8000 Xg for 10min, collecting thallus PBS, washing, suspending in simulated saliva, shake culturing at 37 deg.C (200 rpm) for 5min, and centrifuging at 8000 Xg for 10min; collecting thallus, re-suspending in simulated gastric juice, shake culturing at 37 deg.C (200 rpm) for 3h, and centrifuging at 8000 Xg for 10min; the collected cells were resuspended in simulated intestinal fluid and shake-cultured (200 rpm) at 37 ℃ for 2h. After each stress, a proper dilution gradient is selected, the viable count is determined by using a plate colony counting method, and the survival rate (%) is calculated according to the formula (1).
Survival rate (%) = number of live bacteria after stress/number of initial live bacteria × 100% (1)
The ability of 37 strains of lactic acid bacteria to withstand digestive stress is shown in Table 1.
TABLE 1 lactic acid bacteria ability to tolerate digestive stress
Note: different upper and lower case letters in the same column indicate significant difference (p < 0.05), the same below.
As can be seen from Table 1, the strains have different tolerance capacities at different digestion stress stages, 30 of the 37 lactic acid bacteria have stronger tolerance in the oral cavity, and the survival rate is more than 70%; after the 16 strains are stressed by simulated saliva-gastric juice, the survival rate is more than 50 percent; after simulated stress of saliva, gastric juice and intestinal juice, the strains L73, L167, e113, grx401 and L160 have stronger tolerance capability, the survival rates are all more than 46 percent and are obviously higher than other strains (p is less than 0.05).
5. The adhesion ability of lactic acid bacteria to intestinal mucin and intestinal epithelial Caco-2 cells;
centrifuging the activated lactobacillus at 8000 Xg for 10min, collecting thallus, suspending in PBS, and adjusting viable count to 10 8 CFU/mL, adding 0.5mL into mucin model, culturing at 37 deg.C for 2h, washing with PBS for 3 times to remove non-adhered lactobacillus, adding 0.5mL/L Triton-100 solution, and culturing at 37 deg.C for 30min to remove adhered lactobacillus. And lightly scraping by using a gun head, diluting by 10 times of gradient, detecting the number of viable bacteria by using a flat plate bacterial colony counting method, and calculating the adhesion rate of the lactic acid bacteria according to a formula (2).
Take 0.5mL of 2X 10 5 cell/mL Caco-2 cells were seeded in 24-well cell culture plates, cultured to a monolayer, washed 2 times with PBS and then added with 0.5mL 10 8 Culturing the CFU/mL lactobacillus suspension at 37 ℃ for 2h, washing with PBS for 3 times to remove non-adhered lactobacillus, adding 0.15mL pancreatin cell digestive juice, adding 0.35mL complete cell culture solution after the cells completely fall off to stop digestion, diluting by 10 times of gradient, and detecting the viable count by using a plate colony counting method. And the adhesion rate of the lactic acid bacteria was calculated according to the formula (2).
Adhesion rate (%) = number of viable lactic acid bacteria adhered/number of viable initial lactic acid bacteria × 100% (2)
TABLE 2 adhesion of lactic acid bacteria to the intestinal tract
As can be seen from Table 2, the adhesion rates of the strains grx401 and L73 to mucin are both greater than 2.78%, which is significantly higher than that of other strains (p is less than 0.05), and the adhesion rates to Caco-2 cells are also significantly higher than that of other strains (p is less than 0.05), which are both greater than 10%; the adhesion rates of the strain grx401 to mucin and Caco-2 cells are respectively 22.89% and 37.55%, which are obviously higher than that of the rest 4 lactic acid bacteria (p is less than 0.05).
6. Susceptibility of lactic acid bacteria to antibiotics;
the sensitivity of lactic acid bacteria to antibiotics was studied using the drug sensitive paper method. Suspending the activated lactobacillus in sterile physiological saline, and adjusting thallus concentration to 1 × 10 8 And (3) absorbing 1.0mL of CFU/mL into the melted MRS solid culture medium, uniformly mixing, quickly pouring into a sterilized dish, solidifying, attaching a standard medicine paper sheet, inversely culturing in a constant-temperature incubator at 37 ℃, measuring and recording the diameter of an inhibition zone after 48 hours, and judging the drug resistance of the lactobacillus to the antibiotics according to an antibacterial drug susceptibility test execution standard (2019 edition) formulated by CLSI.
TABLE 3 resistance of lactic acid bacteria to antibiotics
Note: "S" indicates that the strain is sensitive to antibiotics, "I" indicates that the strain is moderately sensitive to antibiotics, and "R" indicates that the strain is moderately sensitive to antibiotics
Resistance to antibiotics.
As can be seen from table 3, 5 strains of lactic acid bacteria have strong resistance to ciprofloxacin and vancomycin, and have low resistance to rifampin, penicillin, cefazolin (pioneer), cefazolin, and clarithromycin; the strain grx401 is sensitive to all 7 antibiotics tested and less resistant than the remaining 4 lactic acid bacteria.
7. The effect of digestive stress on the adhesion ability of lactic acid bacteria;
suspending the strain grx401 after saliva-gastric juice-intestinal juice stress by PBS, and researching the adhesion rate of the strain after digestion stress on intestinal mucoprotein and intestinal epithelial Caco-2 cells according to the method in the step 5.
TABLE 4 Effect of digestive stress on intestinal adhesion Capacity of Strain grx401
As can be seen from table 4, compared with the strain without digestion stress, the adhesion ability of the strain grx401 to mucin and Caco-2 cells after sequential saliva-gastric juice-intestinal juice stress has no significant change (p > 0.05), which indicates that the intestinal adhesion ability of the strain grx401 has stronger tolerance to digestion stress.
8. Identifying lactic acid bacteria;
(1) Identifying a strain API;
the identification of the strain grx401 was carried out with reference to the API 50CHL series identification reagent strip protocol, and the results are shown in tables 5 and 6.
TABLE 5 API 50CHL systems identification results
Note: "+" indicates positive; "-" indicates negative
TABLE 6 API comparison results
As can be seen from tables 5 and 6, the strain grx401 of the present invention is Lactobacillus plantarum (Lactobacillus plantarum), and the identification rate was 99%.
(2) Sequencing and identifying 16S rDNA of the strain;
taking the genome DNA of the strain grx401 as a PCR amplification template, and performing PCR amplification by adopting a universal 16S rDNA primer; after the amplification product is detected by electrophoresis, the product is sent to Biotechnology engineering (Shanghai) GmbH for sequencing. The sequences obtained by sequencing were subjected to BLAST for alignment analysis, and the results are shown in Table 7.
TABLE 7 1696 rDNA alignment results
As can be seen from table 7, strain grx401 is lactobacillus plantarum (l.plantarum), with 99% homology; the strain grx401 of the invention is identified as lactobacillus plantarum (l.plantarum) by combining the API 50CHL alignment result and the 16S rDNA sequence alignment result.
Application examples
1 a method for producing fermented milk containing the strain of the present invention;
standardizing whole milk (11% -13%), adding 6% -7% sugar, preheating to 60-65 deg.C, homogenizing under 15-20Mpa, heat treating at 95 deg.C for 5-8min, cooling to 42 deg.C, and inoculating Lactobacillus plantarum grx401 at 3%. Fermenting at 42 deg.C to pH 4.2-4.5, cooling, and storing at 4 deg.C to obtain fermented milk containing digestion stress resistant and high intestinal adhesion lactobacillus.
2 a method for preparing fermented milk beverage containing the strain;
mixing and dissolving 25-45% of the fermented milk prepared in the application example 1 with sterilized and cooled water, syrup and a compound emulsion stabilizer (0.4-0.6%), homogenizing at the temperature of 20MPa and 60 ℃, cooling, and performing aseptic filling to prepare the fermented milk beverage containing the active lactobacillus plantarum grx 401.
3 the preparation method of the solid beverage containing the bacterial strain of the invention;
a freeze-drying protective agent for preparing strains, which mainly contains 30 to 60 percent of skim milk, 3.5 to 6.5 percent of cane sugar, 3 to 7.5 percent of inulin, 3 to 6.5 percent of sodium glutamate, 3.5 to 7 percent of maltodextrin, 4.5 to 8.5 percent of glycerin and 0.05 to 0.25 percent of gelatin, and ionized water is added to 1Kg; the strain is subjected to high-density culture and then centrifuged to obtain thalli, the thalli and a protective agent are uniformly mixed according to the mass ratio of 1.
Sequence listing
<110> Yangzhou university
<120> lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1477
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
cccgtggggg ggtgccctat acatgcaagt cgaacgaact ctggtattga ttggtgcttg 60
catcatgatt tacatttgag tgagtggcga actggtgagt aacacgtggg aaacctgccc 120
agaagcgggg gataacacct ggaaacagat gctaataccg cataacaact tggaccgcat 180
ggtccgagct tgaaagatgg cttcggctat cacttttgga tggtcccgcg gcgtattagc 240
tagatggtgg ggtaacggct caccatggca atgatacgta gccgacctga gagggtaatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 420
taaaactctg ttgttaaaga agaacatatc tgagagtaac tgttcaggta ttgacggtat 480
ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600
agccttcggc tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga 660
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctgt ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat 780
tagataccct ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc 840
cttcagtgct gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaaggctg 900
aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960
ctacgcgaag aaccttacca ggtcttgaca tactatgcaa atctaagaga ttagacgttt 1020
cccttcgggg acatggatac aggtggtgca tggattgtcg tcagctcgtg tcgtgagatg 1080
ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat taagttgggc 1140
actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat 1200
gccccttatg acctgggcta cacacgtgct acaatggatg gtacaacgag ttgcgaactc 1260
gcgagagtaa gctaatctct taaagccatt ctcagttcgg attgtaggct gcaactcgcc 1320
tacatgaagt cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg 1380
ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag tcggtggggt 1440
aaccttttag gaaccagccg cctaaggtgg atcaggt 1477
Claims (4)
1. Lactobacillus plantarum (II)Lactobacillus plantarum) grx401, characterized by: has been preserved in China general microbiological culture Collection center (CGMCC) at 10/8/2021, address: the collection number of the microorganism research institute of Chinese academy of sciences, no. 3 Xilu No. 1 of Beijing, chaoyang, and the collection number is CGMCC NO.23544.
2. The Lactobacillus plantarum (F) (A) of claim 1Lactobacillus plantarum) Use of grx401 in fermented milk.
3. The Lactobacillus plantarum (F) (as defined in claim 1)Lactobacillus plantarum) Application in fermented milk beverage.
4. Such as rightA Lactobacillus plantarum (L) according to claim 1Lactobacillus plantarum) Application in lactobacillus solid beverage.
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| CN105255752A (en) * | 2015-04-27 | 2016-01-20 | 南昌大学 | Lactobacillus plantarum ZDY2013 with high acid resistance |
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| CN113699071A (en) * | 2021-08-30 | 2021-11-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicine for treating infectious vaginosis |
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| CN102533618A (en) * | 2012-02-28 | 2012-07-04 | 江南大学 | Lactobacillus plantarum CCFM8724 and application thereof |
| CN105255752A (en) * | 2015-04-27 | 2016-01-20 | 南昌大学 | Lactobacillus plantarum ZDY2013 with high acid resistance |
| CN111778180A (en) * | 2020-06-12 | 2020-10-16 | 北京三元食品股份有限公司 | Breast milk source lactobacillus plantarum and application thereof |
| CN113699071A (en) * | 2021-08-30 | 2021-11-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicine for treating infectious vaginosis |
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