CN113957021A

CN113957021A - Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof

Info

Publication number: CN113957021A
Application number: CN202111496871.3A
Authority: CN
Inventors: 陈大卫; 任晨瑜; 顾瑞霞; 程月; 瞿恒贤; 梁雅婷; 燕宪涛; 关成冉; 张臣臣; 马文龙
Original assignee: Yangzhou University
Current assignee: Zhongkelijun Co ltd
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-01-21
Anticipated expiration: 2041-12-09
Also published as: CN113957021B

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 at the china general microbiological culture collection center 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, researches the digestion stress resistance and the capability of adhering intestinal mucoprotein and intestinal epithelial Caco-2 cells of the strain, and researches the drug resistance and the saliva spitting of the strainChange in adhesion under liquid-gastric-intestinal fluid stress.

Description

Lactobacillus plantarum grx401 with digestion stress resistance and high intestinal adhesion capacity and application thereof

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 ratio of lactobacillus is more than 10⁶The precondition that the viable count of CFU/mL reaches and adheres to the intestinal tract is that the CFU/mL plays 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: 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.

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: 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 of the invention is obtained by the following steps:

separating lactobacillus from intestinal tracts of Guangxi Bama longevity people by using 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 isolate to obtain a strain with low resistance, namely grx 401;

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 a 16S rDNA PCR amplification product 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 water₂、3.70mmol/L KH₂PO₄、0.15mmol/L MgCl₂(H₂O)₆、15.10mmol/L KCl、0.06mmol/L(NH₄)₂CO₃、13.60mmol/L NaHCO₃The lysozyme is added into the solution of (1) to the final concentration of 100mg/L, the pH value is adjusted to 7.0 by using 1mol/L HCl, and the solution is filtered and sterilized by a 0.22 mu m filter membrane and is prepared just before use.

(2) Configuration of simulated gastric fluid: prepared by deionized water and contains 0.075mmol/L CaCl₂、0.90mmol/L KH₂PO₄、0.1 0mmol/L MgCl₂(H₂O)₆、6.9 0mmol/L KCl、0.50mmol/L(NH₄)₂CO₃、25mmol/L NaHCO₃47.20mmol/L NaCl, adding pepsin to a final concentration of 3.0g/L, adjusting pH to 3.0 with 1mol/L HCl, filtering with 0.22 μm filter membrane for sterilization, and preparing for use.

(3) Preparation of simulated intestinal fluid: prepared with 0.30mmol/L CaCl in PBS₂、0.80mmol/L KH₂PO₄、0.33mmol/L MgCl₂(H₂O)₆、6.90mmol/L KCl、0.50mmol/L(NH₄)₂CO₃、85mmol/L NaHCO₃Adding trypsin and bile salt into 38.40mmol/L NaCl solution until the final concentration is 0.1% and 0.3%, adjusting pH to 8.0 with 1mol/L HCl, filtering with 0.22 μm filter membrane for sterilization, and preparing.

(4) MRS liquid medium: 10.0g of peptone, 20.0g of glucose, 10.0g of beef extract, 5.0g of yeast extract, Tween-801 mL and C₂H₃NaO₂ 5.0g、K₂HPO₄ 7H₂O 0.2g、MnSO₄·4H₂0.05g of O, adding deionized water to 1000mL, sterilizing at 121 ℃ for 15min, standing at room temperature, and cooling for use. 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 in PBS to prepare 1mg/mL mucin solution, filtering and sterilizing by a 0.22 mu m filter membrane, taking 0.5mL to a 24-hole cell culture plate, culturing at 37 ℃ for 1h, then culturing at 4 ℃ overnight, adding the same volume of mucin, continuing to culture at 37 ℃ for 2h to make up blank sites, and washing with PBS (pH 7.2) for 2 times.

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. The cell suspension was transferred to a 15mL centrifuge tube, 3mL of MEM complete medium (20% high-quality fetal bovine serum, 1% MEM nonessential amino acid solution, 1% Gluta MAX glutamine additive, 1% sodium pyruvate solution) was added, and the mixture was centrifuged at 1000 Xg to 5min, centrifuging, removing supernatant, adding 1mL MEM, resuspending cells in complete culture medium, transferring to 25cm²The cell culture flask was then charged with 9mL of MEM complete medium and placed at 37 ℃ under 5% CO₂Cultured 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 10⁵cell/mL, 0.5mL of the cell was pipetted into a 24-well cell culture plate at 37 ℃ in 5% CO₂Culturing in a cell culture box, and changing the culture solution 1 time every 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 Chinese cabbage and the like, adding 1mL of sterile liquid paraffin oil into the feces samples, sealing, storing the feces samples and the traditional fermented foods at the temperature of 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, wherein the gram stain of 37 strains is positive, the shape of the strain is rod-shaped or spherical, the strain can grow under the environment of 15 ℃ and 45 ℃, and the catalase, nitrate reduction and indole tests are negative, so that the 37 strains are preliminarily identified as the 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 (200rpm) for 5min, and centrifuging at 8000 Xg for 10 min; collecting thallus, re-suspending in simulated gastric juice, shake culturing at 37 deg.C (200rpm) for 3h, and centrifuging at 8000 Xg for 10 min; the collected cells were resuspended in simulated intestinal fluid and cultured with a shaker (200rpm) at 37 ℃ for 2 h. 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 (%). viable count after stress/initial viable count × 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), as follows.

As can be seen from Table 1, the strains have different tolerance capacities at different digestion stress stages, and 30 of the 37 lactic acid bacteria have stronger tolerance in the oral cavity and the survival rate is more than 70 percent; after the 16 strains are stressed by simulated saliva-gastric juice, the survival rate is more than 50 percent; after stress simulating 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, resuspending in PBS, and adjusting viable count to 10⁸CFU/mL，Adding 0.5mL of the mixture into a mucin model, culturing at 37 ℃ for 2h, washing with PBS 3 times to remove non-adhered lactobacillus, and adding 0.5mL of 5mL/L Triton-100 solution, and culturing at 37 ℃ 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 10⁵cell/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 10⁸Culturing the CFU/mL lactobacillus suspension at 37 ℃ for 2h, washing with PBS for 3 times to remove non-adhered lactobacillus, adding 0.15mL of pancreatin cell digestive juice, adding 0.35mL of complete cell culture solution after the cells completely fall off to stop digestion, diluting in a 10-fold gradient manner, and detecting the viable count by adopting 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 lactic acid bacteria initially adhered X100% (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% and significantly higher than those of other strains (p is less than 0.05), and the adhesion rates to Caco-2 cells are also significantly higher than those of other strains (p is less than 0.05) and are both greater than 10%; the adhesion rates of the strain grx401 to mucin and Caco-2 cells are 22.89% and 37.55%, respectively, which are significantly higher than that of the rest 4 strains of 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 the thallus concentration to 1 × 10⁸Absorbing 1.0mL of CFU/mL into a melted MRS solid culture medium, mixing uniformly, quickly pouring into a sterilized dish, sticking a standard medicine paper sheet after solidification, performing inverted culture in a constant-temperature incubator at 37 ℃, measuring and recording the diameter of a bacteriostatic circle and the root diameter after 48 hoursThe resistance of the lactic acid bacteria to antibiotics is judged according to the execution standard of antibacterial drug susceptibility test (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 low resistance to rifampicin, 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 System identification results

Note: "+" indicates positive; "-" indicates negative

TABLE 6 API comparison results

As is clear 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 the 16S rDNA of the strain;

taking the genomic DNA of the strain grx401 as an amplification template of PCR, and performing PCR amplification by adopting a universal 16S rDNA primer; after the amplified products were detected by electrophoresis, they were sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing. The sequenced sequences were submitted to BLAST for alignment analysis, and the results are shown in Table 7.

TABLE 716S 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 was identified as lactobacillus plantarum (l.plantarum) by combining the API 50CHL alignment and the 16S rDNA sequence alignment.

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 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;

preparing a freeze-drying protective agent of a strain, which mainly comprises 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 adding ionized water to 1 Kg; the strain is cultured in high density and then centrifuged to obtain thalli, the thalli and a protective agent are mixed uniformly according to the mass ratio of 1:1, freeze-dried powder with the water content not higher than 3% is obtained after vacuum freeze-drying, and the freeze-dried powder, maltodextrin and xylooligosaccharide are mixed uniformly according to a certain proportion under the aseptic condition, so that the lactobacillus plantarum grx401 solid beverage is obtained.

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

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<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

1. Lactobacillus plantarum (Lactobacillus plantarum) with digestion stress resistance and high intestinal adhesion capacityLactobacillus 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 (Lactobacillus plantarum) with digestion stress resistance and high intestinal adhesion capacity according to claim 1Lactobacillus plantarum) grx401, characterized by: the drug resistance is low, and the intestinal adhesion capability has no significant change after digestive stress.

3. The Lactobacillus plantarum (A) with digestion stress resistance and high intestinal adhesion capacity as claimed in claim 1Lactobacillus plantarum) Application of grx401 in production of fermented milk, fermented milk beverage and lactobacillus solid beverage and food.