Alpaca source bacillus subtilis and application thereof

Abstract

The invention provides alpaca source bacillus subtilis and application thereof, and belongs to the technical field of probiotics. The bacillus subtilis L29K provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 20, 2024, and the preservation number is CGMCC NO.32017. The bacillus subtilis L29K has the advantages that the preservation number is CGMCC NO.32017; the bacillus subtilis provided by the invention has the characteristics of pathogenic bacterium resistance, high protease activity, high amylase activity and low drug resistance, and is suitable for development of novel animal feed additives.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to alpaca-derived Bacillus subtilis and applications thereof. Background Art

[0002] Bacillus subtilis is a Gram-positive facultative anaerobic bacterium that is widely distributed in nature and is generally recognized to be safe for animals and humans. Many Bacillus subtilis strains have been used to produce commercial probiotics, such as BioPlusR 2B, BaoZyme-Aqua, and BiostartR (Cutting, 2011).

[0003] Bacillus subtilis grows rapidly and has low nutritional requirements, so it can easily survive, colonize and reproduce in a variety of environments. It secretes a variety of active enzymes, including amylase, protease and cellulase, which can effectively decompose starch, protein and fiber, and promote the utilization of nutrients by organisms. In addition, it can also produce a variety of antibiotic substances to inhibit the proliferation of harmful microorganisms. It is worth noting that surfactin is one of the most important categories of antimicrobial active substances synthesized by Bacillus subtilis, with strong antibacterial, antiviral, antitumor and antimycoplasma activities.

[0004] In harsh environments, Bacillus subtilis can produce metabolically dormant endospores that are extremely resistant to high temperatures, acidity, alkali, desiccation, radiation, and mechanical extrusion. Therefore, Bacillus subtilis is a probiotic strain often added to animal feed in the form of spores. After resuscitation and proliferation in the gastrointestinal tract (GI) of animals, it can exert its probiotic properties, including improving intestinal flora, enhancing immunity, inhibiting pathogenic microorganisms, and promoting nutrient absorption.

[0005] Alpaca, belonging to the Camelidae family, is a species native to the extreme environment of the Andes Plateau. This species exhibits a series of excellent biological characteristics, including but not limited to excellent cold resistance, drought resistance, strong disease resistance and the ability to produce natural single-chain antibodies. These unique physiological advantages exhibited by alpacas speculate that they may have a close ecological and genetic relationship with the intestinal microbial flora. However, up to now, systematic research on the structure of alpaca intestinal microbial communities is still relatively scarce. Early research work has revealed that there are probiotic Bacillus strains with high bacteriocin production capacity in the alpaca intestine. Therefore, the technical problem to be solved by the present invention is to isolate and culture strains with strong stress resistance, good stability, anti-pathogenic bacteria, high protease activity, high amylase activity and low drug resistance from alpaca feces, so as to determine the feasibility of using alpaca-derived Bacillus subtilis. Summary of the invention

[0006] The invention aims to provide a strain of Bacillus subtilis L29K, which has the characteristics of anti-pathogenic bacteria effect, high protease activity, high amylase activity and low drug resistance.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a Bacillus subtilis L29K, wherein the Bacillus subtilis L29K is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms, with the address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being September 20, 2024, and the deposit number being CGMCCNO.32017.

[0009] The invention also provides the use of the Bacillus subtilis as an animal feed additive.

[0010] Preferably, the animal feed additive is a fermentation broth of Bacillus subtilis L29K.

[0011] Preferably, the fermentation broth is obtained by activating Bacillus subtilis L29K and then inoculating it into LB medium for cultivation.

[0012] The present invention also provides an animal feed additive in the application, wherein the animal feed additive is a fermentation liquid of Bacillus subtilis L29K.

[0013] The Bacillus subtilis provided by the invention has gastrointestinal fluid resistance, enzyme production performance and antibacterial activity. It has been identified that the Bacillus subtilis strain L29K is relatively sensitive to penicillin, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, norfloxacin, ciprofloxacin, co-trimoxazole and chloramphenicol, has high safety and is suitable for the development of new animal feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the colony morphology of Bacillus subtilis in Example 1;

[0015] Figure 2 The bacterial morphology of Bacillus subtilis in Example 1;

[0016] Figure 3 The results of DNA agarose gel electrophoresis in Example 1 are as follows;

[0017] Figure 41 is the growth curve of the three strains of Bacillus subtilis in Example 1;

[0018] Figure 5 These are the tolerance test results of the three strains of Bacillus subtilis in Example 1.

[0019] Collection Instructions

[0020] Bacillus subtilis L29K is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is: September 20, 2024, and the deposit number is CGMCC NO.32017. DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0022] Experimental equipment and reagents: clean bench (Beijing Lianhe Keyi Technology Co., Ltd.); autoclave (Shanghai Boxun Medical Biological Instrument Co., Ltd.); constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.); electronic balance (Shanghai Precision Instrument Co., Ltd.); optical microscope (Changzhou Wantai Balance Instrument Co., Ltd.); PCR instrument, multifunctional microplate reader (Hangzhou Langji Scientific Instrument Co., Ltd.); UV gel imager (Yishike (Shanghai) Enterprise Development Co., Ltd.); -80℃ refrigerator (Shanghai Maumo Scientific Instrument Co., Ltd.). Gram staining solution, nutrient agar, nutrient broth, red fluorescent nucleic acid dye, PCRMasterMix, DNAMarker (D2000), bacterial genomic DNA small-scale extraction kit / centrifugal column type (Beijing Solebold Technology Co., Ltd.); soluble starch (Shandong Fengtai Biotechnology Co., Ltd.); skim milk powder (Tianjin Tianli Chemical Reagent Co., Ltd.); crystal violet (Shanghai Aladdin Biochemical Technology Co., Ltd.); Lugol's iodine solution (Yonghua Chemical Co., Ltd.).

[0023] Example 1

[0024] 1.1 Sample collection and rough processing

[0025] Fresh alpaca feces is collected from the China Pet Alpaca Breeding Base. The collected samples are promptly placed in sterile sealed bags, numbered and marked, and the sealed bags are promptly placed in ice boxes. After sampling, they are sent to the laboratory within 24 hours and stored in a -80℃ refrigerator.

[0026] 1.2 Isolation and purification of strains

[0027] Weigh 5g of the alpaca feces sample stored in the refrigerator and put it into a conical flask containing 45mL of distilled water. Shake and mix well. Heat the conical flask on low heat until it boils and then maintain it for 15min. Let the conical flask stand and cool. After the supernatant in the flask is clarified, take 0.1mL of the supernatant and add it to a test tube containing 9.9mL of distilled water to make a 1:100 concentration suspension. Dilute the concentration gradient by 10 2 and 10 3 , apply 0.2mL to LB medium (do 3 parallel controls for each dilution), and place the medium in a 37℃ constant temperature incubator for 24h. Pick a single colony with an inoculation loop and inoculate it in 10mL LB liquid medium, and incubate it in a sterile box at 37℃ for 24h. Then inoculate the bacterial liquid on the LB solid medium and incubate it in a sterile box at 37℃ for 24h.

[0028] 1.3 Morphological identification Observe the morphological characteristics of the colonies on the culture dish and keep a record. Pick a single colony suspected of Bacillus subtilis for Gram staining and observe the bacterial morphology under an oil microscope.

[0029] 1.4 Molecular Biological Identification DNA was extracted using a bacterial genomic DNA extraction kit.

[0030] result

[0031] The isolation and identification test of probiotics found that yellow-white, wrinkled, and rough colonies ( Figure 1 ), which is consistent with the colony characteristics of Bacillus subtilis on LB medium. Pick a single colony that meets the colony characteristics of Bacillus subtilis, which is a purple rod-shaped bacterium ( Figure 2 ).

[0032] The molecular identification results of Bacillus subtilis were Figure 3 (In the figure, "1" is 21k, "2" is 25k, and "3" is 29k) It can be seen that the band size of the DNA after 16S rDNA amplification in the agarose gel is about 1500bp, which is consistent with the size of the Bacillus subtilis PCR band. The sequencing results were compared on NCBI and it was found that the three probiotics were all Bacillus subtilis, named 21k, 25k, and 29k respectively.

[0033] The sequence of 21k (SEQ ID NO.1) is as follows:

[0034]

[0035] The sequence of 25k (SEQ ID NO.2) is as follows:

[0036]

[0037] The sequence of 29k (SEQ ID NO.3) is as follows:

[0038]

[0039] Biological characteristics of strains

[0040] 1.5 Growth curve determination After the strain was activated (a single colony was picked and placed in a glass test tube containing 3 mL of LB liquid medium, and cultured overnight at 37°C, 220 r / min), it was inoculated into LB liquid medium at a rate of 5%, and cultured at 37°C, 220 r / min. Samples were taken every 2 hours from the start of inoculation, and the OD600 values ​​were determined. The growth curve of the strain was drawn with the time after inoculation as the horizontal axis and the OD600 value as the vertical axis.

[0041] The growth curves of the three strains are shown in Figure 4 As shown in the figure, the results showed that 21k and 29k were in the lag phase or adaptation phase of the strain in the first 2 hours; as time went on, the strain multiplied rapidly from 2 to 32 hours and entered the logarithmic phase of the strain. The strain entered the stable phase at 32 hours, and after 48 hours of cultivation, 21k, 25k, and 29k began to enter the decline phase.

[0042] 1.6 Determination of antibacterial ability The antibacterial ability of three probiotics against three pathogens was determined by agar diffusion method. The concentration of probiotics was 1×10 8 CFU / mL, the concentration of pathogenic bacteria is 1×10 6 CFU / mL. The pathogenic indicator bacteria are Escherichia coli, Salmonella, and Staphylococcus aureus. After culturing in a constant temperature incubator for 24 hours, the diameter of the transparent antibacterial zone of each well is measured and recorded. The larger the diameter, the stronger the antibacterial ability.

[0043] The diameters of the inhibition zones of 21k, 25k and 29k against pathogenic bacteria are shown in Table 1.

[0044] Table 1 Diameters of inhibition zones of 21k, 25k, and 29k

[0045]

[0046] The results showed that the antibacterial ability of 29k against Staphylococcus aureus was stronger than that of 25k and 21k, and the three probiotics had a certain inhibitory effect on pathogenic bacteria Escherichia coli and Salmonella.

[0047] 1.7 Drug sensitivity test The sensitivity of the strain to antibiotics was determined by the drug sensitivity paper agar diffusion method. The activated strain was cultured overnight for 12 hours and the concentration of the bacterial solution was adjusted to about 10 8CFU / mL. Use a sterilized cotton swab to evenly apply the culture solution of the strain on the LB plate. After natural drying in the clean bench, evenly stick 20 kinds of drug-sensitive paper sheets on the surface of the LB plate. Use tweezers to gently press the drug-sensitive paper sheets so that they are not easy to fall off. After culturing at 37°C for 24 hours, measure the diameter of the inhibition zone of each drug-sensitive sheet, and take the average value of three measurements. The drugs include: β-lactams (penicillin, ampicillin, cefazolin), aminoglycosides (amikana, gentamicin), macrolides (erythromycin), quinolones (ciprofloxacin, norfloxacin), sulfonamides (cotrimoxazole), and chloramphenicol (chloramphenicol), a total of 6 categories and 10 kinds of antibacterial drugs.

[0048] The drug sensitivity results are shown in Table 2. By comparing the CLSI drug sensitivity breakpoints, it can be seen that 21k and 25k are sensitive to β-lactams, aminoglycosides, macrolides, sulfonamides and amides, and are resistant to quinolones: ciprofloxacin and norfloxacin; 29k is sensitive to all types of antimicrobial drugs in the test. In summary, the three probiotics are highly sensitive to most of the antimicrobial drugs commonly used in clinical practice.

[0049] Table 2 Drug sensitivity of 21k, 25k, and 29k

[0050]

[0051] 1.8 Determination of enzyme production capacity: Take the bacterial solution after probiotic inoculation (adjust the concentration of the bacterial solution after overnight culture for 12 hours to about 10% with PBS). 6 CFU / mL) was inoculated into amylase and protease screening medium (amylase screening medium: soluble starch ratio is 0.2%, protease screening medium: skimmed milk powder ratio is 2%), respectively. After culturing in a constant temperature incubator at 37°C for 24 h, the amylase medium was stained with 2 mL of iodine solution, and the colony diameter d (mm) and the diameter of the transparent zone around the colony D (mm) were measured by the cross method. The starch decomposition ability and protease production ability of the strain were determined according to the Hc value (Hc=D / d), and the average value was taken after three measurements.

[0052] As shown in Table 3, in the enzyme production test, the ability of strain 29K to produce protease and amylase was higher than that of the other two strains of Bacillus subtilis, and strains 21K, 25K and 29K all had obvious ability to produce protease and amylase.

[0053] Table 3 Ratio of the diameter of the enzyme hydrolysis circle to the bacterial lawn of strains 21k, 25k and 29k

[0054]

[0055]

[0056] 1.9 Tolerance determination Preparation of artificial simulated gastric fluid: Adjust the PBS buffer to pH 2.5 with 1mol / L hydrochloric acid. Then add 0.3% pepsin and dissolve it completely. Sterilize the solution by filtering it through a 0.22μm microporous membrane and store it for later use. Preparation of artificial simulated intestinal fluid: Add 10g / L pancreatic enzyme and 3g / L ox bile salt powder to the PBS buffer. Adjust the pH to 8.0 with 0.1mol / LNaOH and dissolve the solution completely. After filtering through a 0.22μm microporous membrane, keep it for later use. Add 100ul bacterial suspension (3×10 4 CFU / ml) was plated for viable count as a control, and 1 mL of bacterial suspension was inoculated into 9 mL of pH 2.5 sterilized artificial gastric juice for tolerance test. After sufficient mixing, incubate at 37°C for 3 hours and measure the viable count. Then 1 mL of sterile gastric juice treated for 3 hours was inoculated into 9 mL of pH 8.0 sterilized artificial intestinal juice. After sufficient mixing, incubate in a 37° constant temperature incubator. The viable count was measured at 3, 6, 9, and 21 hours, and the survival rate was calculated.

[0057] The ability of three strains of Bacillus subtilis to resist gastrointestinal fluid is as follows Figure 5 As shown, after growing in simulated gastric fluid for 3 hours, the survival rates of 21k, 25k, and 29k were all maintained above 90% compared with the initial growth rate; 21k, 25k, and 29k still had high activity after 21 hours of culture, indicating that the three probiotics have a certain tolerance to gastrointestinal fluid.

[0058] In summary, after a comprehensive evaluation of the tolerance, stability, stress resistance, enzyme production activity, drug sensitivity, and ability to inhibit pathogenic bacteria of the three strains, it was found that 29k was sensitive to all types of antimicrobial drugs in the test, and the three strains of Bacillus subtilis were sensitive to most of the antimicrobial drugs commonly used in clinical practice. In addition, the 29K strain was particularly outstanding in terms of tolerance, and its stability and stress resistance were better than the other two strains. At the same time, it showed significant enzyme production ability and the potential to inhibit pathogenic bacteria. Therefore, the 29K strain has significant application prospects as a Bacillus subtilis probiotic preparation.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis L29K, characterized in that: The Bacillus subtilis L29K is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is: September 20, 2024, and the preservation number is CGMCCNO.32017.

2. Use of the Bacillus subtilis according to claim 1 as an animal feed additive.

3. The use according to claim 2, characterized in that The animal feed additive is the fermentation liquid of Bacillus subtilis L29K.

4. The use according to claim 3, characterized in that The fermentation liquid is obtained by activating Bacillus subtilis L29K and then inoculating it into LB culture medium for cultivation.

5. An animal feed additive for use according to any one of claims 2 to 4, characterized in that: The animal feed additive is the fermentation liquid of Bacillus subtilis L29K.

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