A strain of Lactobacillus gasseri and its application

Abstract

The present invention discloses a strain of Lactobacillus gasseri, which is classified and named as Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, which has been deposited in the China Center for Type Culture Collection on June 1, 2020, with the deposit number: CCTCC NO: M 2020167. The present invention also discloses the use of the Lactobacillus gasseri in the preparation of a drug for inhibiting Vibrio vulnificus and Actinobacillus actinomycetemcomitans. Therefore, the Lactobacillus gasseri BXP11 provided by the present invention has a high social application value.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains and their applications, and in particular to a Lactobacillus gasseri strain and its application. Background Art

[0002] Vibrio vulnificus ( Vibriovulnificus ), or marine vibrio, is a bacterium that lives in the ocean. Vibrio vulnificus is widely distributed in seawater and can be isolated from seafood such as oysters. The bacteria mainly cause infection through wounds contacting seawater, and can also be infected through the mouth. Wound infection can cause various inflammations such as cellulitis and osteomyelitis. Oral infection often quickly leads to bacteremia or sepsis. If infection with this bacteria is not treated in time, the mortality rate is very high. Actinobacillus actinomycetemcomitans ( Aggregatibacteractinomycetem-comitans Aa) is a Gram-negative short rod, a facultative anaerobic bacterium with pili, no spores, and no motility. In recent years, it has been recognized to be closely related to periodontitis, especially aggressive periodontitis, and has become one of the most studied bacteria in the bacterial etiology of periodontitis. More and more studies have found that Aa may also cause other diseases, such as endocarditis, pericarditis, pneumonia, bacteremia, sepsis, osteomyelitis, skin infection, urinary tract infection and various abscesses. At present, antibiotics are mainly used to treat infections caused by the above pathogens, but antibiotics have the problem of drug resistance. Therefore, it is of great significance to develop non-antibiotic products that can effectively inhibit the growth of the above pathogens.

[0003] Lactobacillus gasseri ( Lactobacillus gasseri ) is also known as Lactobacillus gasseri. It is an important species in the genus Lactobacillus. It is a Gram-positive, rod-shaped, facultative anaerobic bacterium that is widely found in the gastrointestinal tract, vagina or breast milk. Lactobacillus gasseri can exert its probiotic properties by producing bacteriocins, organic acids, hydrogen peroxide, etc., including inhibiting the growth of pathogens, relieving intestinal inflammation, and anti-oxidation. So far, there have been no reports in the industry on the development of Lactobacillus gasseri for drugs that inhibit specific pathogens (Vibrio vulnificus and Actinobacillus actinomycetemcomitans). Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus gasseri and application thereof, and to develop a strain capable of significantly inhibiting Vibrio vulnificus, Vibrio vulnificus and Actinobacillus actinomycetemcomitans.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] A strain of Lactobacillus gasseri, the strain is classified and named as Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, was deposited in the China Center for Type Culture Collection on June 1, 2020, with the deposit number: CCTCC NO: M2020167, and the deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072.

[0007] The Lactobacillus gasseri BXP11 provided by the present invention is separated from human feces, and has the following colony characteristics: after being cultured on an MRS plate culture medium for 24 to 48 hours, the colony morphology is milky white, round, smooth, moist and shiny; scanning electron microscope observation shows that the bacteria are nearly spherical short rods with a size of 0.6 to 0.8 × 0.8 to 1.0 μm; the pH value suitable for growth is 4.0 to 8.0, and the optimal pH for growth is 7.0; the temperature suitable for growth is 30 to 45° C., and the optimal growth temperature is 40° C.; the strain has a significant inhibitory effect on the growth of pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetemcomitans.

[0008] The present invention also provides the use of the Lactobacillus gasseri BXP11 in the preparation of a drug for inhibiting Vibrio vulnificus; the number of viable bacteria of the Lactobacillus gasseri BXP11 contained in the drug is 1.0×10 6 -5.0×10 11 cfu / mL or 1.0×10 6 -5.0×10 11 cfu / g; the preferred viable count is 2.0×10 7 cfu / mL or 2.0×10 7 cfu / g.

[0009] The present invention also provides the use of the Lactobacillus gasseri BXP11 in the preparation of a drug for inhibiting Actinobacillus actinomycetemcomitans; the number of viable bacteria of the Lactobacillus gasseri BXP11 contained in the drug is 1.0×10 6 -5.0×10 11 cfu / mL or 1.0×10 6 -5.0×10 11 cfu / g, preferably 2.0×10 6 -2.0×10 8 cfu / mL or 2.0×10 6 -2.0×10 8 cfu / g, the most preferred viable count is 2.0×10 7 cfu / mL or 2.0×10 7 cfu / g.

[0010] The Lactobacillus gasseri BXP11 provided by the present invention is separated from human feces, and the strain has an excellent inhibitory effect on the growth of pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetemcomitans. Therefore, the Lactobacillus gasseri BXP11 provided by the present invention has a high social application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the colony morphology of Lactobacillus gasseri BXP11.

[0012] Figure 2 This is a scanning electron micrograph of Lactobacillus gasseri BXP11.

[0013] Figure 3 This is the growth curve of Lactobacillus gasseri BXP11 at different temperatures.

[0014] Figure 4 This is the growth curve of Lactobacillus gasseri BXP11 at different pH values. DETAILED DESCRIPTION

[0015] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, the culture medium and experimental conditions in the examples are conventional culture medium and experimental conditions. The experimental materials, reagents, etc. used in the examples, unless otherwise specified, can all be obtained from commercial sources.

[0016] Example 1 Lactobacillus gasseri ( Lactobacillus gasseri ) Isolation and identification of BXP11

[0017] 1. Strain Isolation

[0018] Take 0.1 mL of culture medium from the culture medium of healthy human feces, distribute it to MRS liquid culture medium, and culture it anaerobically at a constant temperature of 37°C until colonies appear. Use an inoculation needle to pick up bacteria with good growth on the edge and inoculate them on a new MRS plate culture medium. After the newly inoculated bacteria grow into colonies, pick up the endophytic bacteria on the edge for streak culture. Repeat this process to purify until a single colony is obtained, which is named strain BXP11.

[0019] The formula of MRS liquid culture medium is as follows: tryptone 10.0 g / L, beef powder 5.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, K2HPO4•3H2O 1.5 g / L, anhydrous sodium acetate 3.0 g / L, ammonium citrate 2.0 g / L, MgSO4•7H2O 0.2 g / L, MnSO4•H2O 0.04 g / L. The pH value is 6.0, and it is sterilized at 121℃ for 20 min.

[0020] 2. Strain identification

[0021] (1) Observation of colony and bacterial morphology: The strain BXP11 was streaked on MRS plate medium and cultured anaerobicly at 37°C for 24-48 h. Figure 1 ) is milky white, round, smooth, moist and shiny. Under a scanning electron microscope, the bacteria are short rods that are nearly spherical, with a size of 0.6~0.8 × 0.8~1.0 μm. The scanning electron microscope image is Figure 2 .

[0022] (2) Observation of the growth characteristics of strain BXP11: The strain BXP11 was inoculated into MRS liquid culture medium at a volume ratio of 3.0% and cultured anaerobically at different temperatures for 24 h. It was found that the strain did not grow well at 10°C or 50°C, but grew well at 30°C to 45°C. The optimal growth temperature was 40°C (see Figure 3 ); placed in different initial pH environments for anaerobic culture for 24 h, it was found that the strain could grow in an environment with a pH range of 2.5-10.0, and grew fastest in an environment with a pH of 6.0-8.0, with the optimal growth pH being 7.0 (see Figure 4 ).

[0023] (3) Molecular biological identification of strain BXP11: The whole genome of strain BXP11 was extracted using a bacterial whole genome rapid extraction kit, and PCR was performed using bacterial 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'), followed by sequencing analysis. The sequencing results were compared by BLAST and the strain was identified as Lactobacillus gasseri. The gene sequence is shown in SEQ ID NO: 1 and named: Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, deposited in the China Center for Type Culture Collection on June 1, 2020, with the deposit number: CCTCC NO: M 2020167, and the deposit address: China Center for Type Culture Collection, Wuhan University, Wuhan, China, Postal Code: 430072.

[0024] Example 2 Inhibition test of Lactobacillus gasseri BXP11 on the growth of Vibrio vulnificus

[0025] Experimental method: Vibrio vulnificus (concentration: 1.0×10 6 cfu / mL) as indicator bacteria, spread on LB agar plates, punch wells with a diameter of 6 mm on the plates with a sterile hole puncher, and set up 3 replicates for each group; add 100 μL of BXP11 bacterial solution (concentration of 2.0×10 7 cfu / mL), and cultured in a 37℃ biochemical incubator for 24 h. Observe and record the size of the inhibition zone.

[0026] The experimental results are as follows:

[0027] Table 1 Antibacterial activity of Lactobacillus gasseri BXP11 of the present invention against Vibrio vulnificus

[0028]

[0029] The results in Table 1 show that the size of the inhibition zone of BXP11 bacterial solution against Vibrio vulnificus is 2.5 cm, indicating that Lactobacillus gasseri BXP11 has a very obvious inhibitory effect on the growth of Vibrio vulnificus.

[0030] Example 3 Inhibition test of Lactobacillus gasseri BXP11 on the growth of Actinobacillus actinomycetemcomitans

[0031] Experimental method: Actinobacillus actinomycetemcomitans (concentration: 1.0×10 6 cfu / mL) were used as indicator bacteria and spread on Columbia blood agar plates (CA-B). A sterile hole puncher was used to punch wells with a diameter of 6 mm on the plate. Three replicates were set up for each group. 100 μL of BXP11 bacterial solution (concentration of 2.0×10 7 cfu / mL), and cultured in a 37℃ biochemical incubator for 24 h. Observe and record the size of the inhibition zone.

[0032] The experimental results are as follows:

[0033] Table 2 Antibacterial activity of Lactobacillus gasseri BXP11 of the present invention against Actinobacillus actinomycetemcomitans

[0034]

[0035] The results in Table 2 show that the size of the inhibition zone of BXP11 bacterial solution against Actinobacillus actinomycetemcomitans is 1.7 cm, indicating that Lactobacillus gasseri BXP11 has a significant inhibitory effect on the growth of Actinobacillus actinomycetemcomitans.

[0036] In summary, the Lactobacillus gasseri BXP11 provided by the present invention has a significant inhibitory effect on the growth of pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetemcomitans; and given that Lactobacillus gasseri is one of the strains approved by my country for use in ordinary food and is a strain recognized in the industry as having high safety, the Lactobacillus gasseri BXP11 provided by the present invention has a very high practical application value.

[0037] The above are only some specific embodiments of the present invention. The present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A strain of Lactobacillus gasseri BXP11, characterized in that Lactobacillus gasseri ( Lactobacillus gasseri ), which was deposited in the China Center for Type Culture Collection on June 1, 2020, with the deposit number: CCTCC NO: M2020167, and the deposit address is: Wuhan University, Wuhan, China.

2. Use of the Lactobacillus gasseri BXP11 as claimed in claim 1 in the preparation of a medicament for inhibiting Vibrio vulnificus.

3. The use of Lactobacillus gasseri BXP11 according to claim 2 in the preparation of a drug for inhibiting Vibrio vulnificus, characterized in that: The viable count of Lactobacillus gasseri BXP11 contained in the drug is 1.0×10 6 -5.0×10 11 cfu / mL or 1.0×10 6 -5.0×10 11 cfu / g.

4. Use of the Lactobacillus gasseri BXP11 as claimed in claim 1 in the preparation of a medicament for inhibiting Actinobacillus actinomycetemcomitans.

5. Use of Lactobacillus gasseri BXP11 according to claim 4 in the preparation of a medicament for inhibiting Actinobacillus actinomycetemcomitans, characterized in that: The viable count of Lactobacillus gasseri BXP11 contained in the drug is 1.0×10 6 -5.0×10 11 cfu / mL or 1.0×10 6 -5.0×10 11 cfu / g.

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