CN115927127A - Lactobacillus gasseri and application thereof - Google Patents

Abstract

The invention discloses a strain of lactobacillus gasseri, which is classified and named as lactobacillus gasseri (L.) (Lactobacillus gasseri) BXP11, which has been deposited at the China center for type culture Collection on 6/1/2020 with the deposit number: CCTCC NO: m2020167. The invention also discloses application of the lactobacillus gasseri in preparing medicaments, health-care foods or common foods for inhibiting vibrio vulnificus and actinobacillus actinomycetemcomitans. Therefore, the Lactobacillus gasseri BXP11 provided by the invention has higher social application value.

Description

A strain of Lactobacillus gasseri and its application

technical field

The invention relates to the technical field of microbial strains and applications thereof, in particular to a strain of Lactobacillus gasseri and applications thereof.

Background technique

Vibrio vulnificus , or marine vibrio, is a bacterium that inhabits the ocean. Vibrio vulnificus is widely distributed in seawater, and Vibrio vulnificus can be isolated from seafood such as oysters. The bacteria mainly cause infection through wound contact with seawater, and can also be infected through the mouth. Infection through the wound can lead to various inflammations such as cellulitis and osteomyelitis, and oral infection often quickly leads to bacteremia or sepsis. If not treated in time after infection with this bacterium, the fatality rate is very high. Aggregatibacter actinomycetem-comitans (Aa) is a Gram-negative short bacillus, which is a facultative anaerobic bacterium with pili, no spores, and no motility. In particular, aggressive periodontitis is closely related, becoming 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, the infection of the above pathogenic bacteria is mainly treated with antibiotics, but there is a problem of drug resistance in antibiotics. Therefore, it is of great significance to develop non-antibiotic products that can effectively inhibit the growth of the above pathogenic bacteria.

Lactobacillus gasseri ( Lactobacillus gasseri ), also known as Lactobacillus gasseri, is an important species of Lactobacillus, belonging to Gram-positive, rod-shaped, facultative anaerobic bacteria, widely present in the gastrointestinal tract, vagina or breast milk middle. Lactobacillus gasseri can produce bacteriocins, organic acids, hydrogen peroxide, etc. to exert its probiotic properties, including inhibiting the growth of pathogenic bacteria, relieving intestinal inflammation, and anti-oxidation. So far, there are no relevant reports in the industry on the development of Lactobacillus gasseri for the inhibition of specific pathogenic bacteria (Vibrio vulnificus and Actinobacillus actinomycetes), health food or general food.

Contents of the invention

The object of the present invention is to provide a strain of Lactobacillus gasseri and its application, and develop a bacterial strain capable of significantly inhibiting Vibrio vulnificus, Vibrio vulnificus and Actinobacillus actinomycetes.

The purpose of the present invention is achieved by the following technical solutions:

A strain of Lactobacillus gasseri, the strain is classified as Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, which has been preserved in the China Center for Type Culture Collection on June 1, 2020, with the preservation number: CCTCC NO: M2020167, preserved Unit address: Wuhan University, Wuhan, China, postcode: 430072.

Lactobacillus gasseri BXP11 provided by the present invention is isolated from human feces, and the characteristics of the colony are: after being cultured on the MRS plate medium for 24 to 48 hours, the colony form is milky white, round, smooth, moist and shiny; observation by scanning electron microscope It shows that the bacteria are close to spherical brevibacteria with a size of 0.6~0.8 × 0.8~1.0 μm; the suitable pH value for growth is 4.0~8.0, and the optimum growth pH is 7.0; the suitable growth temperature is 30~45℃, the most suitable The growth temperature is 40°C; the strain has significant inhibitory effect on the growth of pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetes.

The present invention also provides the application of the Lactobacillus gasseri BXP11 in the preparation of medicines, health food or common food for inhibiting Vibrio vulnificus; the living bacteria of Lactobacillus gasseri BXP11 contained in the medicine, health food or common food The count 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.

The present invention also provides the application of the Lactobacillus gasseri BXP11 in the preparation of medicines, health food or common food for inhibiting Actinobacillus actinomycetes; the Lactobacillus gasseri BXP11 contained in the medicine, health food or common food The number of viable bacteria is 1.0×10 ⁶ -5.0×10 ¹¹ cfu/mL or 1.0×10 ⁶ -5.0×10 ¹¹ cfu/g, and the preferred number of viable bacteria is 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.

The Lactobacillus gasseri BXP11 provided by the invention is isolated from human feces, and the bacterial strain has excellent inhibitory effects on the growth of pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetes. Therefore, Lactobacillus gasseri BXP11 provided by the present invention has high social application value.

Description of drawings

Figure 1 is the colony morphology of Lactobacillus gasseri BXP11.

Fig. 2 is a scanning electron micrograph of Lactobacillus gasseri BXP11.

Fig. 3 is the growth curve of Lactobacillus gasseri BXP11 at different temperatures.

Figure 4 is the growth curve of Lactobacillus gasseri BXP11 at different pH values.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. For the experimental methods in the following examples, unless otherwise specified, the medium and experimental conditions in the examples are conventional medium and experimental conditions. The test materials and reagents used in the examples can be obtained from commercial sources unless otherwise specified.

Example 1 Isolation and identification of Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11

1. Strain isolation

Take 0.1 mL culture solution from the culture solution of healthy human feces, distribute it into the MRS liquid medium, and incubate it anaerobically at a constant temperature at 37°C until colonies appear. On the MRS plate medium, after the newly inoculated bacteria grow into colonies, the endophytic bacteria on the edge are picked and cultured, and purified repeatedly until a single colony is obtained, which is named strain BXP11.

The formula of MRS liquid medium is: 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, K ₂ HPO ₄ •3H ₂ O 1.5 g/L, anhydrous sodium acetate 3.0 g/L, ammonium citrate 2.0 g/L, MgSO ₄ •7H ₂ O 0.2 g/L, MnSO ₄ •H ₂ O 0.04 g/L. The pH value was 6.0, and it was sterilized at 121°C for 20 min.

2. Strain identification

(1) Observation of the morphology of colonies and bacteria: Streak culture of the strain BXP11 on the MRS plate medium, after 24-48 hours of constant temperature anaerobic culture at 37°C, the colony morphology (Figure 1) is milky white, round , the surface is smooth, wet and shiny. Observed under a scanning electron microscope, the bacteria are close to spherical brevibacteria with a size of 0.6-0.8 × 0.8-1.0 μm. The scanning electron microscope picture is shown in Figure 2.

(2) Observation of the growth characteristics of the strain BXP11: The strain BXP11 was inoculated in the MRS liquid medium at an inoculum volume of 3.0% by volume, and cultured anaerobically at different temperatures for 24 hours. The growth is not good, but it grows well in the environment of 30°C~45°C, and the optimum growth temperature is 40°C (see Figure 3). After anaerobic culture for 24 hours under different initial pH values, it is found that the strain is in the pH range of 2.5 The strain can grow in an environment of ~10.0, and grows fastest in an environment with a pH value of 6.0-8.0, and the optimum growth pH value is 7.0 (see Figure 4).

(3) Molecular biology identification of the strain BXP11: The whole genome of the strain BXP11 was extracted using the bacterial whole genome rapid extraction kit, and the bacterial 16S rDNA universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') for PCR 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, named: Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, which was preserved in China on June 1, 2020 Typical Culture Collection Center, the deposit number is: CCTCC NO: M 2020167, the address of the depository unit: Wuhan University, Wuhan, China, postcode: 430072.

Embodiment 2 Lactobacillus gasseri BXP11 inhibits the growth of Vibrio vulnificus

Experimental method: Vibrio vulnificus (concentration: 1.0×10 ⁶ cfu/mL) was used as the indicator bacteria, spread on the LB agar plate, and punched a sample hole with a diameter of 6 mm on the plate with a sterile puncher. Three replicates were set up for each group; 100 μL of BXP11 bacterial solution (concentration: 2.0×10 ⁷ cfu/mL) was added to the wells, and cultured in a biochemical incubator at 37°C for 24 h. Observe and record the size of the inhibition zone.

The experimental results are as follows:

Table 1 The antibacterial activity of Lactobacillus gasseri BXP11 of the present invention to Vibrio vulnificus

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

Example 3 Inhibition test of Lactobacillus gasseri BXP11 on the growth of Actinobacillus actinomycetes

Experimental method: use Actinobacillus actinomycetes (concentration: 1.0×10 ⁶ cfu/mL) as the indicator bacteria, spread it on the Columbia blood plate (CA-B), and use a sterile puncher to make a diameter on the plate 6 mm wells were used, and three replicates were set for each group; 100 μL of BXP11 bacterial solution (concentration: 2.0×10 ⁷ cfu/mL) was added to the wells, and cultured in a biochemical incubator at 37°C for 24 h. Observe and record the size of the inhibition zone.

The experimental results are as follows:

Table 2 The antibacterial activity of Lactobacillus gasseri BXP11 of the present invention to Actinobacillus actinomycetes

The results in Table 2 showed that the BXP11 bacterial solution had a significant inhibition zone size of 1.7 cm, indicating that Lactobacillus gasseri BXP11 had a significant inhibitory effect on the growth of Actinobacillus.

In summary, the Lactobacillus gasseri BXP11 provided by the present invention has obvious inhibitory effect on the growth of the pathogenic bacteria Vibrio vulnificus and Actinobacillus actinomycetes; It is one of the bacterial strains recognized in the industry as having high safety, so the Lactobacillus gasseri BXP11 provided by the present invention has high practical application value.

The above are only some specific embodiments of the present invention. The present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (5)

1. A strain of Lactobacillus gasseri, characterized in that the strain is classified as Lactobacillus gasseri ( Lactobacillus gasseri ) BXP11, which has been preserved in the China Center for Type Culture Collection on June 1, 2020, and the preservation number is: CCTCCNO: M 2020167, deposit address: Wuhan University, Wuhan, China. 2. an application of Lactobacillus gasseri as claimed in claim 1 in the preparation of medicines, health food or common food for inhibiting Vibrio vulnificus. 3. the application of Lactobacillus gasseri according to claim 2 in the preparation of medicines, health food or common food for inhibiting Vibrio vulnificus, it is characterized in that the milk of gravidarum contained in the medicine, health food or common food The viable count of Bacillus BXP11 is 1.0×10 ⁶ -5.0×10 ¹¹ cfu/mL or 1.0×10 ⁶ -5.0×10 ¹¹ cfu/g. 4. The application of a Lactobacillus gasseri as claimed in claim 1 in the preparation of medicines, health food or common food for inhibiting Actinobacillus actinomycetes. 5. the application of Lactobacillus gasseri according to claim 4 in the preparation of medicines, health food or common food inhibiting Actinobacillus actinomycetes, it is characterized in that, contained in the medicine, health food or common food The viable count of Lactobacillus gasseri BXP11 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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