Lactobacillus crispatus CML521 and application thereof

Abstract

The invention discloses lactobacillus crispatus CML521 and application thereof, and relates to the technical field of microorganisms, and the lactobacillus crispatus CML521 is technically characterized in that the lactobacillus crispatus CML521 is obtained through separation and culture from poultry craw contents, and the lactobacillus crispatus CML521 has remarkable biological characteristics and can be effectively used for preparing medicines for treating poultry candidiasis; the discovery of the lactobacillus crispatus CML521 provides a brand new treatment thought and method for poultry to treat candidiasis, and the newly discovered microbial strain not only can help poultry to resist the infringement of candidiasis, but also is expected to play an important role in the veterinary medicine field, and provides guarantee for poultry health.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to Lactobacillus crispatus CML521 and its application. Background Art

[0002] Candidiasis is an infection caused by fungi of the genus Candida (mainly Candida albicans). Candida is usually part of the normal flora of the human body and exists in parts such as the oral cavity, intestine, and vagina, usually causing no problems. However, when the body's immunity declines or the environment changes, Candida may overgrow and cause infection. Common types include oral candidiasis (commonly known as thrush), manifested as white spots on the oral mucosa, vaginal candidiasis, manifested as vaginal itching and increased discharge, and systemic candidiasis, which occurs when Candida enters the blood or other organs, commonly seen in immunocompromised patients. The treatment of candidiasis usually uses antifungal drugs such as fluconazole and itraconazole. There are many drugs that inhibit Candida albicans, but to avoid drug resistance, probiotics are an alternative active microorganism. Microorganisms that inhibit Candida in humans are relatively mature, but there are few reports in poultry.

[0003] Candidiasis in poultry is an infection caused by Candida such as Candida albicans, commonly seen in poultry such as chickens and ducks, mainly affecting the digestive system, respiratory tract, and other internal organs. Typical symptoms include reduced appetite, weight loss, diarrhea, lethargy, and dry feathers, and in severe cases, it can lead to acute death. Currently, the treatment of poultry candidiasis mainly relies on antifungal drugs such as fluconazole and itraconazole, but there are some disadvantages in the existing technology: long-term use of antifungal drugs may cause Candida to develop drug resistance and reduce the treatment effect; antifungal drugs may burden the organs such as the liver and kidneys of poultry.

[0004] Therefore, the present invention aims to provide a Lactobacillus crispatus CML521 and its application to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems, and provide a Lactobacillus crispatus CML521 and its application. Lactobacillus crispatus CML521 is isolated and cultured from the crop contents of poultry. Lactobacillus crispatus CML521 can be used to prepare a drug for treating poultry candidiasis, providing a new idea for treating poultry candidiasis.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The present invention provides a Lactobacillus crispatus CML521, which is isolated and cultured from the crop contents of poultry.

[0008] The present invention also provides an application of Lactobacillus crispatus CML521, which can be used to prepare a drug for treating avian candidiasis, and the Lactobacillus crispatus CML521 has an antibacterial effect on the pathogenic fungus Candida albicans with consistency.

[0009] Compared with the prior art, the beneficial effects of the present solution are as follows:

[0010] In the present invention, Lactobacillus crispatus CML521 is isolated and cultured from the crop contents of poultry. The Lactobacillus crispatus CML521 has significant biological characteristics and can be effectively used to prepare a drug for treating avian candidiasis; the discovery of Lactobacillus crispatus CML521 provides a brand-new treatment idea and method for treating avian candidiasis. This newly discovered microbial strain can not only help poultry resist the invasion of candidiasis, but also is expected to play an important role in the field of veterinary medicine and provide protection for the health of poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the phylogenetic tree diagram of Lactobacillus crispatus CML 521 in the embodiment of the present invention;

[0012] Figure 2 is the schematic diagram of the genome circle of Lactobacillus crispatus CML 521 in the embodiment of the present invention;

[0013] Figure 3 is the schematic diagram of the growth curve of Lactobacillus crispatus in the embodiment of the present invention;

[0014] Figure 4 is the schematic diagram of the acidity of Lactobacillus crispatus in the embodiment of the present invention;

[0015] Figure 5 is the schematic diagram of the growth curves of Lactobacillus crispatus in eggs (a) and Candida albicans from pigeons (b) under the interference of strain CLM 521 in the embodiment of the present invention;

[0016] Figure 6 is the schematic diagram of the inhibitory effect of strain CLM 521 on Candida albicans in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0019] Embodiment:

[0020] I. Detailed steps for the isolation, screening, and identification of Lactobacillus crispatus CML521;

[0021] The steps for isolating, culturing, and identifying lactic acid bacteria from the crop contents of poultry are as follows. Take an appropriate amount of the contents and mix it with sterile PBS at a ratio of 1:9. Sterilize and enrich the lactic acid bacteria by water bath at 80°C for 10 minutes, and perform gradient dilution with PBS to reduce the microbial concentration. Take 100 - 200 μL of the diluted solution and spread it on the prepared sterile MRS plate, and incubate it upside down at 37°C for 12 - 24 hours. Select suspected lactic acid bacteria colonies and purify them by the quadrant streak method. Inoculate the purified colonies with an inoculation loop into the MRS liquid medium in a laminar flow hood and incubate it at 37°C and 200 rpm on a shaker for 12 - 24 hours. Extract the bacterial DNA and identify the lactic acid bacteria species by 16S rRNA gene sequencing. The universal primers are as follows:

[0022] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0023] 1492R: 5'-ACGGCTACCTTGTTACGACTT-3

[0024] Perform a PCR reaction. The total volume of the PCR amplification system is 30 μL, and the specific configuration is as follows: GoldMix (Beijing Tsingke, China) is 25.5 μL, the 27F upstream primer is 1.5 μL, the 1492R downstream primer is 1.5 μL, and the DNA template is 1.5 μL.

[0025] The specific steps are as follows: First, perform pre-denaturation treatment at 98°C for 10 minutes to promote the separation of the DNA double strands and prepare for the subsequent reaction. Enter the cycling stage: First, perform denaturation at 98°C for 10 seconds to separate the DNA double strands; then perform annealing at 48°C for 15 seconds to enable the primers to effectively bind to the template DNA; then perform extension at 72°C for 25 seconds to promote the synthesis of new DNA strands. Such a cycling process is repeated 10 times. After that, raise the annealing temperature to 52°C and cycle 20 more times according to the same operating steps. Finally, perform an extension reaction at 72°C for 5 minutes to ensure that the DNA strands can be fully extended; finally, terminate the reaction at 4°C and properly store the amplification product.

[0026] Separation by agarose gel electrophoresis: The molecular sieve effect of the agarose gel enables DNA fragments of different sizes to migrate at different speeds in an electric field. Small fragments move fast, and large fragments move slow, thus separating the PCR products for subsequent observation.

[0027] Sequencing and identification: With the help of gel electrophoresis technology, the product with the target band was sent to Beijing Sunbiotech Co., Ltd. for sequencing, and then the corresponding DNA sequence information was obtained. Sequence alignment was performed on the EzBioCloud platform, which has rich 16S rRNA and genomic sequence data. By alignment, the similarity between the sample and known strains was determined to complete the identification of Lactobacillus and judge its specific classification.

[0028] II. Relevant results of the performance test of Lactobacillus crispatus CML521;

[0029] 1. Gene-related information of Lactobacillus crispatus

[0030] A phylogenetic tree of strain CML521 was constructed on MEGA ( Figure 1 ). In terms of genetic relationship, CML352 and CP92301 are the closest; and the phylogenetic tree results of the genus Lactobacillus show that Lactobacillus circulans is relatively close.

[0031] As shown in Table 1, the genome size of Lactobacillus crispatus is 1,263,775 bp, the average G+C content is 38.3%, the N 50 size is 67,897 bp, and the number of coding sequences is 1,937. The circular map of its genome is as Figure 2 shown.

[0032] Table 1 Basic genomic information of strain CML521

[0033]

[0034] 2. Growth curve and pH of Lactobacillus crispatus

[0035] Figure 3 The growth curve of Lactobacillus crispatus (CML 521) within 24 hours is presented. In the initial 0 to 2 hours, the growth rate of Lactobacillus crispatus (CML 521) was relatively slow. After 2 hours, Lactobacillus crispatus (CML 521) quickly entered the exponential growth phase with rapid growth, and this stage could last for up to 6 hours. Around 8 hours, its growth rate significantly slowed down and entered the plateau phase. After 14 hours, its growth state gradually stabilized. This growth pattern is very similar to the results of the study by Kaur et al. Figure 4It shows that within 0 to 10 hours, the pH value of this strain drops sharply. Within 10 to 14 hours, the pH value continues to gradually decrease. However, after 14 hours, the pH value stabilizes at around 4.49, indicating a certain acid-producing ability.

[0036] 3. Acid resistance and bile salt resistance of Lactobacillus crispatus

[0037] As shown in Table 2 and Table 3, in a low pH environment (e.g., pH values of 3 and 4 respectively), after culturing for 1 hour, 2 hours, and 3 hours, the survival magnitude of CML 521 is the same as that in an environment with a pH value of 5 and 6, all above 10 7 CFU / mL, which indicates that CML 521 has a relatively obvious resistance to a low pH environment, and the survival rate of CML 521 reaches more than 50.00%.

[0038] As shown in Table 4 and Table 5, Lactobacillus crispatus CML 521 was cultured in 0.1% and 0.2% porcine bile salts for 1, 2, and 3 hours, and the viable cell magnitude was the same as that of the control group (0.00% bile salt), and all were 10 6 CFU / mL, but the survival rate was lower than 50.00%. After culturing in 0.3% bile salt for 1 hour and plating, it was found that there were almost no viable bacteria, and the survival rate was zero, indicating its weak resistance to bile salt. Existing research has shown that after culturing in 0.1% bile salt for 1 hour, the survival rate is about 20%, indicating that this strain has a certain bile salt tolerance.

[0039] Table 2 Tolerance of Lactobacillus crispatus CML521 to different pH values (n = 6)

[0040]

[0041] Table 3 Survival rate of Lactobacillus crispatus CML521 at different pH values (n = 6)

[0042]

[0043] Table 4 Tolerance of Lactobacillus crispatus CML521 to different concentrations of porcine bile salts (n = 6)

[0044]

[0045]

[0046] Table 5 Survival rate of Lactobacillus crispatus CML521 at different concentrations of porcine bile salts (n = 6)

[0047]

[0048] 4. Hydrophobicity, auto-aggregation and co-aggregation of Lactobacillus crispatus

[0049] Previous related research results have shown that when the hydrophobicity of a certain strain reaches ≥20%, its hydrophobicity can be considered good. Further detection of the hydrophobic activity on the cell surface of the strain found that the strain has high hydrophobicity, and its hydrophobicity value reached 28.93% after measurement (see Table 6 for details).

[0050] We evaluated the auto-aggregation and co-aggregation abilities of the strain. These strains showed significant auto-aggregation phenomena, and this ability varied at different times of the day. Specifically, the auto-aggregation rates at 2 h, 4 h, 12 h, and 24 h were 23.16%, 44.9%, 69.2%, and 58.76% respectively, and the details are shown in Table 6. Notably, the target strain showed extremely high co-aggregation with Candida, and the co-aggregation rate reached 78.81% (Table 6).

[0051] Table 6 Auto-aggregation, co-aggregation and hydrophobicity of Lactobacillus crispatus (n = 6)

[0052]

[0053] 5. Lactobacillus crispatus in artificial gastric juice and artificial pancreatic juice

[0054] According to the data in Table 7 and Table 8, the colony counts (10 6 CFU / mL) of strain CML 521 cultured in artificial gastric juice for 0 h, 1 h, 2 h, and 3 h were 1.64 ± 0.06, 1.53 ± 0.05, 1.24 ± 0.03, and 0.54 ± 0.02 respectively, and its relative survival rates at 1 h, 2 h, and 3 h were 94.0%, 80.0%, and 42.0% respectively. Similarly, the colony counts (10 6 CFU / mL) of strain CML 521 cultured in artificial pancreatic juice for 0 h, 1 h, 2 h, and 3 h were 0.55 ± 0.09, 0.48 ± 0.04, 0.31 ± 0.01, and 0.25 ± 0.03 respectively, and its relative survival rates at 1 h, 2 h, and 3 h were 86.70%, 68.20%, and 56.40% respectively.

[0055] Table 7 Tolerance of strain CML521 to artificial gastric juice and artificial pancreatic juice (n = 6)

[0056]

[0057] Table 8 Tolerance of strain CML 521 to artificial gastric juice and artificial pancreatic juice (n = 6)

[0058]

[0059] III. Application examples of Lactobacillus crispatus CML521 in antibacterial aspects and provide relevant experimental data.

[0060] Figure 5 It was shown that Lactobacillus crispatus entered the stationary phase after 0 to 2 hours, the rapid growth phase after 2 hours, and the stable phase after 14 hours. This is similar to the research of Kaur et al. Candida albicans grew relatively slowly within 0 to 2 hours, then entered the logarithmic phase of rapid growth after 2 hours, and reached the stable phase after 18 hours. Lactobacillus and Candida were co-cultured for 12 hours at night (at this time, Candida was in the logarithmic phase), then inoculated into the fermentation medium, and its antibacterial activity was detected.

[0061] Oxford cup and microplate methods:

[0062] Figure 6 It shows the antibacterial effect of strain CLM 521 against Candida albicans, and the change trend of the inhibition of Candida albicans by the supernatant CFS of Lactobacillus crispatus at different concentrations. The data in Table 9 indicate that the diameter of the antibacterial zone of the supernatant of Lactobacillus crispatus CML 521 against Candida albicans is (20.75±0.54) mm. Combining the analysis of the chart, it can be seen that Lactobacillus crispatus CML 521 has a significant antibacterial effect on the consistent pathogenic bacterium Candida albicans, and this finding has important research significance.

[0063] Table 9 Diameter of the antibacterial zone of the supernatant (CFS) of strain CML521 against Candida albicans

[0064]

[0065] Detection of the hemolytic activity of the strain

[0066] As shown in Table 9, Lactobacillus crispatus CML521 showed γ-hemolysis phenomenon and low toxicity.

[0067] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. Those skilled in the art can make modifications to this embodiment without creative contributions according to needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Lactobacillus crispatus CML521, characterized in that: The Lactobacillus crispatus CML521 was isolated and cultured from the crop contents of poultry.

2. Use of Lactobacillus crispatus CML521 according to claim 1, characterized in that: The Lactobacillus crispatus CML521 can be used to prepare a drug for treating avian candidiasis.

3. Use of Lactobacillus crispatus CML521 according to claim 2, characterized in that: The Lactobacillus crispatus CML521 has an antibacterial effect on the consistent pathogen Candida albicans.

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