Lactobacillus reuteri with antibiotic post-effect prevention and control and Chinese herbal medicine synergistic interaction functions and application of lactobacillus reuteri

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus reuteri with antibiotic post-effect prevention and control and Chinese herbal medicine synergistic interaction functions, the lactobacillus reuteri is named as HBNK11 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M20251479. The lactobacillus reuteri provided by the invention has a remarkable inhibition effect on gram-positive bacteria and gram-negative bacteria, and breaks through the limitation of narrow antibacterial spectrum of traditional probiotics. Meanwhile, the survival rate in simulated gastric juice with the pH value of 2.5 reaches 112.4%, it is ensured that the strain stably survives and plays a role in the strong acid environment of the gastrointestinal tract of livestock and poultry, and the problem that the gastrointestinal tract tolerance of traditional probiotics is poor is solved. When the lactobacillus reuteri is coordinated with a Chinese herbal medicine monomer, the monomer dosage can be reduced by 128 times. The lactobacillus reuteri provides a high-quality resource for developing a novel microbial feed additive, and has important application value for reducing antibiotic dependence in a breeding link and preventing and controlling propagation of multiple drug-resistant bacteria.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus reuteri strain with antibiotic post-effect prevention and control and Chinese herbal medicine synergistic enhancement functions and an application thereof. Background Art

[0002] With the large-scale development of livestock and poultry farming, the problem of the proliferation of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus MRSA and multidrug-resistant Escherichia coli) caused by the abuse of antibiotics is becoming increasingly serious. At the same time, the imbalance of intestinal flora caused by the use of antibiotics (the so-called "post-antibiotic effect") can lead to digestion and absorption disorders, weight loss and even secondary infections in animals, causing huge economic losses to the farming industry. Although traditional probiotics can partially alleviate the above problems, they have defects such as a narrow antibacterial spectrum (only targeting Gram-positive or Gram-negative bacteria), poor tolerance to the gastrointestinal environment (insufficient resistance to gastric acid and bile salts), and a single function, which makes it difficult to meet the needs of actual applications. At the same time, Chinese herbal medicines have shown potential in antibacterial and immune regulation due to their natural, low-toxicity and residue-free characteristics. However, the efficacy of some Chinese herbal medicines is weak and larger doses are required to achieve the ideal effect, which limits their application.

[0003] The present invention obtains a strain of Lactobacillus reuteri that has broad-spectrum antibacterial properties, high gastrointestinal tolerance, antibiotic post-effect prevention and control, and synergistic enhancement functions of Chinese herbal medicine by isolating and screening from the intestines of healthy breeding pigs fed with medicinal and edible materials (tea residues and Houttuynia cordata). This provides a new microbial resource for solving the problems of drug-resistant bacteria prevention and control and antibiotic dependence in livestock and poultry farming. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides a Lactobacillus reuteri strain having the functions of preventing and controlling post-antibiotic effects and synergistic enhancement of Chinese herbal medicines and its application.

[0005] To achieve the above object, the present invention is implemented by the following technical solution: a Lactobacillus reuteri strain, which is Lactobacillus reuteri HBNK11 strain, which has been deposited in the China Center for Type Culture Collection with the deposit number: CCTCC M20251479.

[0006] Preferably, the isolation and identification of the strain

[0007] (1) Isolation source: The Lactobacillus reuteri HBNK11 was isolated from the intestinal contents of healthy sows that were fed with tea dregs (3% by mass) and houttuynia cordata powder (1% by mass) for a long time.

[0008] (2) Isolation method: Take 1 g of the jejunal contents of the sow and add 9 mL of sterile saline to dilute it to 10 -6100 μL was spread on MRS solid medium (containing 2% calcium carbonate), cultured anaerobically at 37°C for 48 h, and single colonies with obvious calcium dissolution rings were picked for purification to obtain pure culture strain HBNK11.

[0009] (3) Identification method:

[0010] Morphological identification: Gram staining is positive, and under the microscope it is short or long rod-shaped, without spores or flagella.

[0011] Molecular biological identification: Genomic DNA of the strain was extracted, and the 16S rDNA sequence was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The sequencing result is shown in SEQ ID NO. 1. NCBI BLAST comparison showed 100% homology with the sequence of the model strain of Lactobacillus reuteri (GenBank: MT707268.1), confirming that the strain was Lactobacillus reuteri.

[0012] Preferably, the functional properties of the strain

[0013] (1) Broad-spectrum antibacterial activity: Lactobacillus reuteri HBNK11 exhibits significant inhibitory effects against both Gram-negative and Gram-positive bacteria. As determined by the double-layer agar assay, the inhibition zone radius against multidrug-resistant Escherichia coli B2 (producing extended-spectrum β-lactamases) was 20.0 mm, and the inhibition zone radius against methicillin-resistant Staphylococcus aureus (MRSA) T144 was 16.8 mm.

[0014] (2) Strong acid production ability: HBNK11 was inoculated into MRS liquid culture medium at a 1% inoculum size and cultured anaerobically at 37°C for 48 h. The pH value of the fermentation liquid could be reduced to 3.0±0.1.

[0015] (3) Acid resistance: HCl was used to adjust the pH of MRS culture medium to 2.5, and 10 6 CFU / mL strain, after 4 h of incubation at 37°C, survival rate = 112.4%.

[0016] Preferably, the application of the strain in preventing and controlling the post-antibiotic effect of livestock and poultry

[0017] The present invention provides a method for using Lactobacillus reuteri HBNK11 in preventing and controlling the post-antibiotic effect of livestock and poultry, which specifically comprises the following steps:

[0018] (1) HBNK11 was inoculated into MRS liquid medium and cultured anaerobically at 37°C until the logarithmic growth phase (OD 600=1.2-1.5), collect the cells by centrifugation, resuspend them in sterile PBS, and adjust the number of viable cells to ≥1×10 9 CFU / mL;

[0019] (2) C57BL / 6 mice were gavaged with 0.2 mL of 200 mg / mL streptomycin, and 24 h later, 0.2 mL of the above bacterial suspension was gavaged;

[0020] (3) After 7 days of continuous gavage, the weight loss and intestinal flora imbalance caused by the use of antibiotics can be significantly alleviated, as shown by: an increase in the number of beneficial bacteria (Akk bacteria) and a decrease in the abundance of pathogenic bacteria (Escherichia coli-Shigella, Clostridium difficile); the weight of mice remained stable, with an average weight gain of 2.007g compared with the simple antibiotic group.

[0021] Preferably, the strain is used in synergistic antibacterial Chinese herbal medicine

[0022] When used synergistically with a Chinese herbal monomer, the Lactobacillus reuteri HBNK11 strain can reduce the monomer dosage by 128 times. In a synergistic enhancement test, a variety of Chinese herbal monomers were selected and combined with the strain for drug sensitivity testing. The antibacterial effect of the two combined use was determined by bacterial counts. The results showed that the strain and the various Chinese herbal monomers exhibited significant synergistic antibacterial effects, significantly reducing the dosage of the herbal monomers and improving the utilization efficiency of the Chinese herbal medicines.

[0023] The present invention provides a strain of Lactobacillus reuteri that has the functions of preventing and controlling post-antibiotic effects and synergizing with Chinese herbal medicines, and its application. It has the following beneficial effects:

[0024] 1. The Lactobacillus reuteri HBNK11 provided by the present invention exhibits significant inhibitory effects against both Gram-positive and Gram-negative bacteria, overcoming the narrow antibacterial spectrum limitations of traditional probiotics. Furthermore, its survival rate in simulated gastric fluid at pH 2.5 reached 112.4%, ensuring the strain's stable survival and effectiveness in the highly acidic environment of the livestock and poultry gastrointestinal tract, addressing the poor gastrointestinal tolerance of traditional probiotics.

[0025] 2. When the Lactobacillus reuteri HBNK11 provided by the present invention is used in combination with Chinese herbal monomers such as cyperus ulforin and quercetin, the monomer dosage can be reduced by 128 times, significantly improving the utilization efficiency of the Chinese herbal medicines through synergistic antibacterial effects. This feature not only reduces the dosage of the Chinese herbal monomers, but also avoids the toxicity issues that may arise from high-dose medications, providing a new approach for the efficient application of Chinese herbal medicines in livestock and poultry farming.

[0026] 3, The mouse experiment proves that, after intragastric administration of streptomycin, the use of HBNK11 can make the average weight gain of mice 2.007g higher than that of the pure antibiotic group, significantly alleviating the digestive and absorptive disorders caused by antibiotics. At the same time, it can also significantly increase the number of beneficial bacteria (such as Akk bacteria), reduce the abundance of pathogenic bacteria such as Escherichia coli-Shigella, Clostridium difficile and other pathogenic bacteria, and restore the diversity of intestinal flora, thereby fundamentally solving the risk of secondary infection caused by antibiotic aftereffect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of gram staining of the lactobacillus reuteri strain of the present application;

[0028] Figure 2 It is a schematic diagram of the phylogenetic tree of the lactobacillus reuteri strain of the present application;

[0029] Figure 3 It is a schematic diagram of acid production and gastric acid tolerance experiment of the lactobacillus reuteri strain of the present application, wherein A is acid production test, and B is acid tolerance test;

[0030] Figure 4 It is a schematic diagram of the inhibition zone of the lactobacillus reuteri strain of the present application on drug-resistant bacteria;

[0031] Figure 5 It is a graph of weight gain change of mice under antibiotic intervention and given probiotics of the present application, wherein A is a grouping schematic diagram, and B is a graph of weight change of experimental mice;

[0032] Figure 6 It is a 16S analysis graph of feces of mice under antibiotic intervention and given probiotics of the present application;

[0033] Figure 7 It is a schematic diagram of the synergistic inhibition of lactobacillus reuteri HBNK11 and Chinese herbal medicine monomers. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0035] Example 1:

[0036] As shown in the drawings, Figure 1-2 The present application provides a separation and identification method of lactobacillus reuteri with antibiotic aftereffect prevention and control and Chinese herbal medicine synergistic effect function, comprising the following steps:

[0037] (1) Take 1 g of ileal contents from a healthy pig, add 9 mL of sterile normal saline, shake well, and then dilute to 10 -6 A 100 μL aliquot was spread on MRS solid medium (containing 2% calcium carbonate) and incubated anaerobically at 37°C for 48 h. Single colonies with smooth surface, milky white color, and neat edges were picked and purified by streaking three times to obtain pure strain HBNK11.

[0038] (2) Gram staining showed that the strain was purple (positive), and microscopic observation showed long rod shape; 16S rDNA sequencing results (SEQ ID NO. 1) had 100% homology with Lactobacillus reuteri type strain (GenBank: MT707268.1, CP170358.1), confirming that it was Lactobacillus reuteri.

[0039] SEQ ID NO. 1 is shown below:

[0040]

[0041] Example 2:

[0042] like Figure 4 As shown, the embodiment of the present invention provides a method for determining the antibacterial activity of Lactobacillus reuteri HBNK11, as follows:

[0043] Pick a single HBNK11 colony in MRS medium and grow it to the logarithmic phase. Adjust the bacterial turbidity to 1×10 8 CFU / mL, fermentation supernatant (48h culture), antibacterial activity was determined by Oxford cup method: 10mL of bacterial solution (1×10 6 LB agar (containing 500 CFU / mL) was poured onto MRS agar plates that had been incubated for 24 hours at 37°C. The inhibition zone radius was measured after incubation for 24 hours at 37°C. The results showed that the inhibition zone radius for E. coli B2 was 20.0 mm, and the inhibition zone radius for MRSA T144 was 16.8 mm.

[0044] Example 3:

[0045] like Figure 3 As shown, the embodiment of the present invention provides an acid production and acid resistance experiment of Lactobacillus reuteri HBNK11, which is as follows:

[0046] (1) Acid production test: HBNK11 was inoculated into MRS liquid culture medium at a 1% inoculum size and cultured anaerobically at 37°C for 48 h. The pH value of the fermentation liquid could be reduced to 3.0.

[0047] (2) Acid resistance: HBNK11 bacterial suspension (1×10 8 CFU / mL) was added to simulated gastric fluid (0.3% pepsin, pH 2.5), incubated at 37°C for 4 h, and spread on MRS plates after gradient dilution. The plates were anaerobically cultured at 37°C for 48 h, and the number of surviving bacteria was counted. The survival rate = (number of bacteria after treatment / initial number of bacteria) × 100% = 112.4%.

[0048] Example 4:

[0049] like Figure 5-6 As shown, the embodiment of the present invention provides an animal experiment for preventing and controlling the post-antibiotic effect of Lactobacillus reuteri HBNK11, which is as follows:

[0050] Thirty SPF-grade C57BL / 6 mice (6 weeks old) were randomly divided into three groups (10 mice in each group);

[0051] Blank control group: fed with normal basal feed;

[0052] Antibiotic group: 200mg / mL 0.2mL streptomycin by gavage, normal feeding of basic feed;

[0053] Experimental group: 20mg / mL 0.2mL streptomycin by gavage for 24h, 1x10 9 CFU / mL 0.2mL HBNK11 bacterial suspension, normal feeding of basic feed;

[0054] The results show that the weight gain of the experimental group mice (0.465±0.39g) is higher than that of the blank control group (0.033±0.58g), and is significantly higher than that of the antibiotic treatment group (-1.524±0.16g); the alpha diversity analysis of 16S data shows that the chao1 and shannon values of the experimental group increase, increasing the diversity of the flora; the abundance of Escherichia-Shigella and Clostridium in the feces of the experimental group mice is significantly lower than that of the antibiotic control group, and the abundance of AKK bacteria is significantly increased, indicating that the probiotics reduce the load of harmful bacteria and promote the reproduction of beneficial bacteria.

[0055] Example Five

[0056] As shown in Figure 7 , the embodiment of the present application provides an experiment of Lactobacillus reuteri HBNK11 and Chinese herbal medicine monomer synergistic antibiosis, specifically as follows:

[0057] (1) Preparation of culture medium:

[0058] MRS culture medium: weigh 10g of proteose peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of potassium phosphate dibasic, 2g of triammonium citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, and 10mL of Tween-80, dissolve in 1000mL of distilled water, and sterilize at 121℃ for 20min.

[0059] MHB culture medium: weigh 2g of beef powder, 1.5g of soluble starch, and 17.5g of acid hydrolyzed casein, dissolve in 1000mL of distilled water, and sterilize at 121℃ for 20min.

[0060] (2) Bacterial culture

[0061] Take a ring from the preserved Lactobacillus reuteri HBNK11 strain, inoculate it in 5mL of MRS liquid medium, and incubate it at 37℃ for 24h, then inoculate it again in a 100mL, 250mL flask at a 1% inoculation amount, and incubate it at 37℃ for 24h, and adjust the concentration of lactic acid bacteria to 2x10 8 CFU / mL.

[0062] (3) Chinese herbal medicine monomer treatment

[0063] Select Chinese herbal medicine monomers such as cyperus rotundus and quercetin, dissolve them in appropriate amount of distilled water, and prepare mother solutions with a concentration of 10 mg / mL for later use.

[0064] (4) Synergistic antibacterial test

[0065] The resuspended Lactobacillus reuteri culture (the concentration of the culture was adjusted to 2×10 8 CFU / mL) and 64 μg / mL quercetin or 1 μg / mL cypermethrin were mixed at a volume ratio of 1:1. 100 μL of the mixture was added to each well of a 96-well plate, and then 100 μL of a 1×10 6 CFU / mL of multidrug-resistant Escherichia coli B2 solution. 100μL MHB+100μL B2 was the blank control group, 100μL 1×10 8 Control groups consisted of a Lactobacillus reuteri suspension (CFU / mL) supplemented with 100 μL of B2, or 100 μL of 64 μg / mL quercetin or 1 μg / mL linalool supplemented with 100 μL of B2. After 24 hours of incubation at 37°C, bacterial growth was observed. Wells with the MIC of the synergistic group and wells of the corresponding HBNK11-treated group were counted using a 10-fold serial dilution. The results showed that the number of B2 bacteria in the synergistic treatment group was significantly lower than that in the HBNK11-treated group.

[0066] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A Lactobacillus reuteri, characterized in that: The Lactobacillus reuteri is the Lactobacillus reuteri HBNK11 strain, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCM20251479.

2. Lactobacillus reuteri according to claim 1, characterized in that: The Lactobacillus reuteri is isolated from the intestinal contents of healthy sows that have been fed with tea dregs and houttuynia cordata powder for a long time.

3. Lactobacillus reuteri according to claim 1, characterized in that: The 16S rDNA sequence of the Lactobacillus reuteri is shown in SEQ ID NO.

1.

4. The microbial agent according to claim 3, characterized in that: The fermentation product contains metabolites of the Lactobacillus reuteri.

5. Use of the Lactobacillus reuteri according to any one of claims 1 to 3 in the preparation of a feed additive for preventing and controlling the post-antibiotic effect of livestock and poultry.

6. The use according to claim 5, characterized in that: The following steps are involved: S1. The Lactobacillus reuteri HBNK11 was inoculated into MRS liquid medium and cultured anaerobically at 37°C until the logarithmic growth phase. The cells were collected by centrifugation and resuspended in sterile PBS to adjust the viable cell count to ≥1×10 9 CFU / mL; S2. 24 hours after mice were gavaged with streptomycin, the bacterial suspension was gavaged orally for 7 consecutive days.

7. Use of the Lactobacillus reuteri according to any one of claims 1 to 3 in the preparation of a feed additive having synergistic antibacterial properties with Chinese herbal medicine monomers.

8. The use according to claim 7, characterized in that: When the Lactobacillus reuteri HBNK11 is used in combination with a Chinese herbal medicine monomer, the dosage of the Chinese herbal medicine monomer can be reduced by 128 times.

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