A new bacillus strain and its application

Abstract

The present invention discloses a new bacillus and its application, which relate to the field of microbial technology. The new bacillus is named Neobacillus sp. GZUAS791019, which was deposited in Guangdong Microbial Culture Collection Center on May 13, 2024, with a deposit number of GDMCC NO: 64616. Feeding Bacillus subtilis GZUAS561008 with the metabolites of the new bacillus provided by the present invention can significantly increase the biomass of Bacillus subtilis GZUAS561008.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a new bacillus strain and application thereof. Background Art

[0002] Microbial communities are not simple aggregations of individual microorganisms, but ecosystems formed by different species through various forms of collaboration. Resource competition and cross-feeding mechanisms between populations are the basis for the balance and stability of community structure. Nutritional cross-feeding refers to the use of metabolites of one bacterium by another. This phenomenon affects the composition, stability, evolution, and antibiotic sensitivity of microbial communities. When nutrients are deficient, it can promote cooperative cross-feeding between different species, thereby improving community stability. For example, studies have shown that microorganisms in cheese rely on chemicals secreted by fungal mycelium to find habitats; amino acids and other metabolites released by Saccharomyces cerevisiae can stimulate the growth of Lactobacillus plantarum; Lactobacillus bulgaricus provides peptides and amino acids to Streptococcus thermophilus, which in turn produces purines and folic acid to promote cross-feeding between the two.

[0003] Bacillus subtilis is widely distributed in nature, with many varieties, strong adaptability, fast growth and other advantages. It can not only synthesize enzymes such as α-amylase, protease, lipase, cellulase, but also synthesize vitamin B1, B2, B6, niacin and other B vitamins. It has a wide range of applications in aquaculture, biological control, animal feed and aquatic products. With the in-depth study of Bacillus subtilis, the existing bacterial culture method can no longer meet the demand. At present, there is no report on the cross-feeding phenomenon between Bacillus subtilis and Bacillus neoformans.

[0004] The present invention provides a new bacillus strain and application of its metabolites in feeding Bacillus subtilis GZUAS561008, aiming to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a new bacillus Neobacillus sp. GZUAS791019.

[0006] Another object of the present invention is to provide an application of the metabolites of the new Bacillus in feeding Bacillus subtilis GZUAS561008.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] The new Bacillus described in the present invention is named Neobacillus sp. GZUAS791019, which was deposited in Guangdong Microbiological Collection Center on May 13, 2024, with a deposit number of GDMCC NO: 64616.

[0009] The Bacillus subtilis described in the present invention is named Bacillus subtilis GZUAS561008, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 1, 2023, with a deposit number of GDMCC No: 64097.

[0010] The metabolites of the new bacillus of the present invention are used in feeding Bacillus subtilis GZUAS561008.

[0011] The method for preparing the metabolites of the new Bacillus sp. of the present invention comprises the following steps:

[0012] 1) Activate the new Bacillus on an LB medium plate for later use;

[0013] 2) inoculating the activated new Bacillus into LB liquid medium and culturing in a shake flask for 48 hours;

[0014] 3) Take the culture medium, centrifuge it, filter and sterilize the supernatant, and prefreeze it twice at -20℃ and -80℃ respectively, each time for 3 hours. After each prefreezing, fully thaw it at room temperature and then prefreeze it again.

[0015] The specific process of activation in step 1) of the preparation method of the new Bacillus of the present invention is: taking the bacterial culture preservation solution stored at -80°C and thawing it at room temperature; taking 10 μL of the thawed bacterial culture preservation solution to streak on the LB plate, and inverting and culturing at 30°C for 24 hours.

[0016] The shaking flask culture conditions in step 2) of the method for preparing the new Bacillus sp. of the present invention are 30° C. and 180 r / min.

[0017] The centrifugation in step 3) of the method for preparing the new Bacillus of the present invention is specifically: centrifugation at 4° C. and 6000 r / min for 10 min.

[0018] In the method for preparing the new Bacillus sp. of the present invention, the filtration sterilization in step 3) is sterilization through a 0.22 μm filter.

[0019] Beneficial effects of the present invention:

[0020] The invention provides a new bacillus Neobacillus sp. GZUAS791019, whose metabolites can be utilized by Bacillus subtilis GZUAS561008, thereby significantly improving the biomass of Bacillus subtilis GZUAS561008.

[0021] Through the cross-feeding experiment of the metabolites of Neobacillus sp. and Bacillus subtilis GZUAS561008, the results show that under environmental conditions with certain nutrition, different concentrations of metabolites of Neobacillus sp. can promote the growth of Bacillus subtilis GZUAS561008 to varying degrees. In an environment without nutrition, its metabolites can still support the growth of Bacillus subtilis GZUAS561008 and enable it to reach a certain degree of biomass. This shows that the Neobacillus sp. GZUAS791019 and its metabolites provided by the present invention can help Bacillus subtilis GZUAS561008 to exert its function to a greater extent when mixed with Bacillus subtilis GZUAS561008.

[0022] Storage information description:

[0023] Strain name: Neobacillus sp.GZUAS791019

[0024] Preservation unit: Guangdong Microbiological Culture Collection Center

[0025] Storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0026] Date of deposit: May 13, 2024

[0027] Deposit number: GDMCC NO: 64616 BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Morphological identification of new Bacillus

[0029] Figure 2 Gram staining of new bacillus

[0030] Figure 3 Phylogenetic tree of new Bacillus

[0031] Figure 4 Growth curves of Bacillus subtilis GZUAS561008 in different treatment groups DETAILED DESCRIPTION

[0032] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solution of the present invention.

[0033] Example 1

[0034] Neobacillus sp. GZUAS791019 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC NO: 64616.

[0035] Example 2

[0036] Feeding Bacillus subtilis GZUAS561008 with new Bacillus metabolites

[0037] (1) Activate Bacillus subtilis GZUAS561008 and Bacillus neoformans respectively: take the culture stock solution stored at -80°C and thaw it at room temperature; take 10 μL of the thawed culture stock solution and streak it on an LB plate, and incubate it upside down at 30°C for 24 h.

[0038] (2) The activated bacteria were inoculated into LB liquid medium and cultured in a shaking flask at 30°C and 180 r / min. Bacillus subtilis GZUAS561008 was cultured until the sporulation rate was above 90%, and the new Bacillus was cultured for 48 hours.

[0039] (3) The culture broth of the new Bacillus was centrifuged at 4°C and 6000 r / min for 10 min, the supernatant was filtered and sterilized with a 0.22 μm filter, and pre-frozen twice at -20°C and -80°C for 3 h each time. After each pre-freezing, the culture broth was fully thawed at room temperature and then pre-frozen again to obtain the sterile fermentation broth of the new Bacillus.

[0040] (4) The sterile fermentation liquid of the new Bacillus prepared above was mixed with LB liquid culture medium, inoculated with Bacillus subtilis GZUAS561008, and cultured under shaking conditions of 30° C. and 180 rpm.

[0041] In order to further verify the reliability of the present invention and screen out the best solution, the inventor conducted a series of experiments, as follows:

[0042] Unless otherwise specified, the materials and reagents used in the experiments can be purchased from commercial channels.

[0043] 1. Isolation, purification, screening and identification of strains

[0044] 1.1 Sample collection

[0045] Forest soil samples were collected in Qiandongnan area of ​​Guizhou Province. When collecting, leaf litter on the soil surface was pushed aside and samples were taken in the 5-10 cm soil layer. The samples were stored in sterile sampling bags at room temperature and away from light.

[0046] 1.2 Isolation and purification of strains

[0047] Take 1g of soil sample and add it to 100mL of sterile water. Soak it in a water bath at 80℃ for 30min, shaking the bottle every 5min. After the incubation, let it stand for 30min, and then draw 1mL of supernatant into LB liquid culture medium. Culture it at 30℃ and 180rpm for 48h. Transfer 1mL of culture supernatant to LB culture medium diluted by 50% volume and culture it with shaking for 24h. Transfer 1mL of culture solution to LB culture medium diluted by 75% volume and culture it for 24h. Dilute the culture solution to 10 with sterile water. -2 , 10 -3 , 10 -4 times, spread them on LB solid plates respectively, and culture them at 30℃ for 48h; select single colonies on the plates and streak them on new LB solid plates for purification, and repeat the purification operation until only a single form of strain exists on the plates.

[0048] 1.3 Identification of strains

[0049] 1.3.1 Morphological identification of strains

[0050] The isolated single colony was streaked on the LB plate and cultured at 30°C for 24 hours. The colony morphology was observed. A single colony was picked from the plate and placed on a slide with a drop of water. The cover was covered with a coverslip and observed after removing bubbles. The strain was morphologically identified according to the Bergey's Manual of Bacterial Identification.

[0051] 1.3.2 Gram staining

[0052] Drop a drop of water in the center of the slide, pick a single colony and disperse it in the water. Add 1 drop of crystal violet and stain for 1 minute. Rinse off the excess crystal violet liquid with water. Cover the surface with iodine solution and stain for about 1 minute. Rinse with water and absorb the water with absorbent paper. Add a few drops of 95% alcohol and shake gently to decolorize. After 20 seconds, wash with water and absorb the water. Stain with safflower color solution (dilute) for 2 minutes, then rinse with water. Dry and examine under a microscope.

[0053] 1.3.3 Molecular identification of strains

[0054] The 16S rDNA of the isolated new Bacillus was sequenced, and the phylogenetic tree was constructed using MEGA X software to determine its species relationship.

[0055] The primer sequences are as follows:

[0056] 27F:5-AGAGTTTGATCCTGGCTCAG-3

[0057] 1492R:5-GGTTACCTTGTTACGACTT-3

[0058] 2. Metabolites of the new Bacillus cross-feeding Bacillus subtilis GZUAS561008

[0059] 2.1 Isolation, screening and identification of Bacillus subtilis GZUAS561008

[0060] Bacillus subtilis GZUAS561008 was isolated, screened and identified by referring to the method in our invention patent (application number: CN117925454A) "A multifunctional Bacillus subtilis strain and its separation and purification method and application".

[0061] 2.2 Preparation of new Bacillus metabolites and cross-feeding experimental setup

[0062] (1) Activate Bacillus subtilis GZUAS561008 and Bacillus neoformans on LB medium plates: Take the culture stock solution stored at -80°C and thaw it at room temperature; take 10 μL of the thawed culture stock solution and streak it on the LB plate, and incubate it upside down at 30°C for 24 hours. Set aside.

[0063] (2) The activated strains were inoculated into LB liquid medium and cultured in a shaking flask at 30°C and 180 r / min. Bacillus subtilis GZUAS561008 was cultured until the sporulation rate was above 90%, and the new Bacillus was cultured for 48 hours.

[0064] (3) The culture medium of the new Bacillus was centrifuged at 4°C and 6000 r / min for 10 min. The supernatant was sterilized by filtering with a 0.22 μm filter and pre-frozen twice at -20°C and -80°C for 3 h each time. After each pre-freezing, the culture medium was fully thawed at room temperature and then pre-frozen again to obtain a sterile fermentation medium.

[0065] (4) Cross-feeding experimental settings, as shown in Table 1:

[0066] Table 1 Cross-feeding experimental setup

[0067]

[0068] 2.3 Plotting the growth curve of Bacillus subtilis GZUAS561008

[0069] (1) Take 25 g of LB broth, add 1 L of distilled water to fully dissolve it, and sterilize it by high pressure at 121°C to prepare LB liquid culture medium.

[0070] (2) Inoculate a single colony of Bacillus subtilis GZUAS561008 in 10 mL LB liquid medium until the OD 600 =1.0, as seed solution.

[0071] (3) Perform group processing according to the experimental settings in Table 1.

[0072] (4) Add 0.1 mL of seed solution into the liquid culture medium of different treatment groups and culture at 30°C and 180 rpm with shaking.

[0073] (5) Measure the OD of each treatment group every 2 hours 600 Values ​​were used to draw the growth curves of Bacillus subtilis GZUAS561008 under different treatments.

[0074] 3. Results and Discussion

[0075] 3.1 Identification of strains

[0076] 3.1.1 Morphological identification of strains

[0077] According to Bergey's Manual of Bacterial Identification, it was initially identified as Bacillus. Figure 1 .

[0078] 3.1.2 Gram staining

[0079] Gram staining results showed that the strain was rod-shaped, Gram-positive bacteria. Figure 2 .

[0080] 3.1.3 Molecular identification of strains

[0081] Phylogenetic tree of the new Bacillus Figure 3 .

[0082] The 16S rDNA sequencing results were compared with the NCBI database, and the results showed that it belonged to Bacillus subtilis. The 16S region sequence was obtained as follows:

[0083] TCAATAGGAGCTTGCTCCTGTTGGTTAGCGGCGGACGGGTGAGTAACACG

[0084] TGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGGAGCTAAT

[0085] ACCGGATAATCCTTTTCCTCACATGAGGGAAAACTGAAAGTCGGTTTCGGC

[0086] TGACACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACG

[0087] GCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACA

[0088] CTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAAT

[0089] CTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGCGATGAA

[0090] GGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTATCGGAGT

[0091] AACTGCCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACG

[0092] TGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATT

[0093] GGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCAC

[0094] GGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGA

[0095] GGAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGA

[0096] ACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCG

[0097] AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAA

[0098] ACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAA

[0099] CGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAA

[0100] GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA

[0101] GCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAG

[0102] ATAGGACGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTC

[0103] GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC

[0104] CTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGT

[0105] GACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATG

[0106] ACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAAACC

[0107] GCAAGGTTTAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTG

[0108] CAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGC

[0109] CGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGA

[0110] GAGTTTGTAACACCCGAAGTCGGTGGGGTAACCGTAAGGAGCCA

[0111] 3.2 Cross-feeding of Bacillus subtilis GZUAS561008 by metabolites of Bacillus neo-sporogenes

[0112] The test results show that under environmental conditions with certain nutrients, different concentrations of the new Bacillus metabolites can promote the growth of Bacillus subtilis GZUAS561008 to varying degrees. In an environment without nutrients, its metabolites can still support the growth of Bacillus subtilis GZUAS561008 and enable it to reach a certain level of biomass. This shows that the strains and their metabolites provided by the present invention can help Bacillus subtilis GZUAS561008 to function to a greater extent when mixed with Bacillus subtilis GZUAS561008. The growth curves of Bacillus subtilis GZUAS561008 in different treatment groups are shown in Figure 2. Figure 4 .

Claims

1. A new Bacillus strain, characterized in that: The new Bacillus sp. was named Neobacillus sp. GZUAS791019, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 13, 2024, with the deposit number GDMCCNO: 64616.

2. The method for preparing the metabolites of the new Bacillus according to claim 1, characterized in that: The preparation method comprises the following steps: 1) Activate the new Bacillus on an LB medium plate for later use. The specific process is as follows: take the strain preservation solution stored at -80°C and thaw it at room temperature; take 10 μL of the thawed strain preservation solution to streak on the LB plate, and invert and culture at 30°C for 24 hours. 2) The activated new Bacillus was inoculated into LB liquid culture medium and cultured in a shaking flask at 30°C and 180 rpm for 48 h; 3) Take the culture medium, centrifuge it at 4°C and 6000 r / min for 10 min, sterilize the supernatant through a 0.22 μm filter, and prefreeze it twice at -20°C and -80°C, each time for 3 h. After each prefreezing, fully thaw it at room temperature and then prefreeze it again.

3. Use of a metabolite of the new Bacillus prepared by the preparation method of claim 2 in feeding Bacillus subtilis GZUAS561008 with a deposit number of GDMCC NO.64097.

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