Bacillus coagulans t-21, a bacterial agent prepared therefrom and use thereof

Abstract

This invention discloses a strain of *Weizmannia coagulans* T-21, its prepared inoculum, and its applications. The T-21 strain was deposited on October 16, 2023, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou—Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 63889. The strain provided by this invention exhibits high efficiency in degrading nitrite and ammonia nitrogen compounds, and can be used as a feed additive in aquaculture where nitrite and ammonia nitrogen levels exceed standards. Simultaneously, it can be applied to the remediation of environments with excessive nitrite and ammonia nitrogen levels, demonstrating significant application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus coagulans T-21, its preparation agents, and its applications. Background Technology

[0002] Bacillus coagulans is a facultative anaerobic, spore-forming, lactic acid-producing Gram-positive bacterium belonging to the genus Bacillus. It lacks flagella on its surface, and its cells are rod-shaped, resembling human hair, approximately 2.5-5.0 μm long and 0.6-1.0 μm wide, with blunt, rounded ends. It occurs singly, in pairs, or in short chains in some cases. The spores are terminal, resembling a "hammer." Its optimal growth pH is 6.0-7.0, and its optimal temperature is 37-45℃. As a facultative anaerobic probiotic, Bacillus coagulans multiplies by utilizing free oxygen in the animal's intestines, thus creating a more anaerobic intestinal environment that favors the survival of anaerobic lactic acid bacteria and bifidobacteria, regulating intestinal flora balance, and thereby protecting the body's health. Therefore, developing beneficial strains for animal health to enrich the Bacillus coagulans resource pool, along with effective probiotic preparation processes and agents, has become a major issue of high concern nationwide.

[0003] Excessive levels of harmful substances in the environment can severely impact the quality of life and production. Among these, excessive levels of nitrite and ammonia nitrogen compounds in water bodies pose a particularly serious problem that needs to be addressed. Currently, there is increasing attention being paid to Bacillus coagulans, and its performance is a key factor influencing its applicability and effectiveness. However, different strains of Bacillus coagulans exhibit significant differences in performance and effectiveness. Therefore, treating and improving the environment through microbial degradation is a promising approach. Summary of the Invention

[0004] The purpose of this invention is to provide a strain that degrades nitrite and ammonia nitrogen compounds, which has significant implications for environmental improvement and aquaculture.

[0005] To achieve the above objectives, the present invention provides a *Weizmannia coagulans* T-21 strain, which was deposited on October 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No: 63889.

[0006] The Bacillus coagulans T-21 provided by this invention can be used to degrade nitrite and / or ammonia nitrogen compounds.

[0007] The present invention also provides a bacterial agent prepared from the above-mentioned Bacillus coagulans T-21.

[0008] Preferably, the above-mentioned microbial agent is a powder, liquid, or granular microbial agent.

[0009] More preferably, when the microbial agent is in the form of a powder, the number of viable T-21 bacteria in it is not less than 4 × 10⁻⁶. 10 CFU / g.

[0010] The microbial agent provided by this invention can be applied in aquaculture where nitrite and / or ammonia nitrogen levels exceed the standards.

[0011] The microbial agent provided by this invention can be applied to improve environments with excessive nitrite and / or ammonia nitrogen.

[0012] The Bacillus coagulans T-21 provided by this invention has the following advantages:

[0013] The Bacillus coagulans T-21 provided by this invention was isolated from the intestinal feces of Asian elephants. It was identified as different from existing Bacillus coagulans strains. The discovery of this strain has supplemented the strain resource bank of Bacillus coagulans.

[0014] The Bacillus coagulans T-21 provided by this invention can efficiently degrade nitrite and ammonia nitrogen compounds. It can completely degrade 10 mg / L of nitrite and ammonia nitrogen compounds within 10 hours. This bacterium can be used to prepare biological agents and has significant application effects and social value in aquaculture and environmental improvement. Attached Figure Description

[0015] Figure 1 This is a colony morphology diagram of strain T-21 in this invention.

[0016] Figure 2 The results of microscopic examination of the spore morphology of strain T-21 in this invention are shown.

[0017] Figure 3 This is a phylogenetic tree diagram of T-21 in this invention. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Note: Methods not described in this invention are all conventional techniques in the field, and reagents and consumables not described are all conventional reagents and consumables in the field.

[0020] Example 1: Isolation and Screening of Strains

[0021] 1. Fecal sample collection:

[0022] Fresh intestinal fecal samples were collected from three Asian elephants in the Wild Elephant Valley of Xishuangbanna National Nature Reserve, Yunnan Province, China. The elephants were tracked until they defecated, and samples were immediately collected from the middle portion of the feces. The fresh fecal samples were flash-frozen in liquid nitrogen and transported to the laboratory, where they were then preserved at -80°C.

[0023] 2. Enrichment culture and isolation:

[0024] Weigh approximately 1g of feces into an Erlenmeyer flask, add 100mL of sterile physiological saline (0.9% NaCl), shake thoroughly to disperse the feces, and let stand for 1 hour for extraction. Then, take 1mL of the fecal extract and add it to 100mL of MRS-enriched solution. + The enrichment medium was statically cultured in an anaerobic workstation at 40°C until the enrichment medium became turbid. The enrichment medium was then diluted to 10⁻⁶ using a serial dilution method. -1 ~10 -5 Take 1 mL of each dilution gradient and spread it onto the MRS enrichment plate. + On solid plates (containing 0.4 g / L bromocresol green), anaerobic culture was carried out at 37°C for 48 h. After colonies grew, representative colonies with yellow circles were selected.

[0025] 3. Secondary screening of bacterial strains:

[0026] Representative colonies with yellow circles obtained in the above steps were selected and inoculated into shake-flask fermentation medium (4% corn starch, 1.0% beef extract, 3% enzymatically hydrolyzed soybean meal, 3% glucose, 0.2% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.1% magnesium sulfate heptahydrate, 0.2% manganese sulfate monohydrate, and 0.2% anhydrous calcium chloride). The medium was shaken and cultured at 200 rpm for 48 hours. The fermentation broth was examined under a microscope to observe whether each representative colony had teliospores. For fermentation broth capable of forming teliospores, an inoculation loop was used to collect the bacterial solution, and MRS-enriched medium was added. + Stranding was performed on solid culture medium. The plates were then inverted and incubated at 37°C until distinct single colonies appeared. The morphology of colonies grown on one plate was as follows: Figure 1 As shown, the morphology of the spores of this strain was examined under a microscope, and the results are as follows. Figure 2 As shown, the spore morphology of this strain, after primary staining with malachite green and secondary staining with safranin, was observed under a microscope at 100x magnification. Green represents spores, and red represents bacterial cells. The strain was also preserved using ultra-low temperature glycerol freeze-dried tubes.

[0027] Identification of strain in Experiment Example 2

[0028] The culture seed solution of the strain preserved in Experiment 1 was inoculated into MRS enrichment at an inoculum volume of 1‰. +After culturing in liquid culture medium at 37℃ and 200 rpm for 16 h, the bacterial cells were collected by centrifugation, and DNA was extracted according to the Tiangen reagent kit instructions. The DNA size was detected by 1.0% agarose gel electrophoresis. Using the extracted total DNA of the strain as a template, the 16S rDNA sequence of the strain was amplified by PCR using universal 16S rDNA primers (27F and 1492R, specific sequences as follows).

[0029] The primer sequences are as follows:

[0030] 27F (SEQ ID NO. 1): 5'-AGAGTTTGATCCTGGCTCAG-3';

[0031] 1492R (SEQ ID NO. 2): 5'-TACGGCTACCTTGTTACGACTT-3'.

[0032] The PCR amplification system consisted of: a 40 μL PCR reaction system containing 0.4 μL rTaq enzyme, 1 μL template DNA, primers 27F and 1492R (20 μmol·L⁻¹). -1 ) 1 μL each, 10×Buffer 4 μL, dNTPs 2 μL, ddH2O 30.6 μL.

[0033] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 0.5 min, 52℃ annealing for 0.5 min, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 5 min.

[0034] The PCR amplification product was recovered and purified using 1.0% agarose gel electrophoresis. The recovered fragment was ligated into the pMD19-T vector and introduced into *E. coli* DH5α. After positive verification of the vector, the positive vector was sent to Qingke Biotechnology Co., Ltd. for sequencing, obtaining the 16S rDNA gene sequence of the strain, as shown in SEQ ID NO.3. The sequencing results of the 16S rDNA gene fragment of the strain were compared with the determined sequences using the online database Blast / blastp (http: / / www.ncbi.nlm.nih.gov / Blast). Similar sequences were selected and multiple alignments were performed using BioEdit software. The alignment results were used to construct a phylogenetic tree using the Neighbor-Joining method in MEGA 5.1 software. The results are shown below. Figure 3As shown, based on the clustering results, the isolated strain was identified as Bacillus coagulans and named Bacillus coagulans T-21, with the taxonomic name Bacillus coagulans. This strain was deposited on October 16, 2023, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No: 63889.

[0035] Experimental Example 3: Whole Genome Determination of Bacillus coagulans T-21

[0036] 1. The genome of this strain was sequenced using high-throughput sequencing (Illumina and Nanopore) technology. The specific steps are as follows:

[0037] (1) High-quality genomic DNA was extracted from Bacillus coagulans T-21 and its purity, concentration and integrity were tested by Nanodrop, Qubit and 0.35% agarose gel electrophoresis.

[0038] (2) The BluePippin fully automated nucleic acid recovery system recovers large DNA fragments;

[0039] (3) Library construction (SQK-LSK109 ligation kit), including the following:

[0040] a. After the samples pass quality inspection, large DNA fragments are enriched and purified using magnetic beads;

[0041] b. Repairing DNA fragment damage;

[0042] c. Perform end repair on both ends of the DNA fragment and add A;

[0043] d. Perform the ligation reaction using the adapters in the LSK109 ligation kit;

[0044] (4) Use Qubit to perform quantitative detection on the constructed DNA library.

[0045] (5) Sequencing.

[0046] 2. Bioinformatics analysis of sequencing results mainly includes the following steps:

[0047] (1) Raw data quality control: This step filters out low-quality reads and retains high-quality reads. The filtered data becomes Clean Data.

[0048] (2) Genome assembly: The genome is assembled to obtain sequence information that reflects the genome of the sample;

[0049] (3) Average Nucleic Acid Similarity (ANI) analysis: Average Nucleic Acid Similarity (ANI) is one of the most powerful indicators for determining the closeness of genomic relationships among bacteria. It is based on the average value of all orthologous protein sequences compared in genome quality control to reflect the evolutionary distance between genomes. When ANI > 95%, it indicates that the two genomes belong to the same species.

[0050] Table 1. Statistical analysis results of average nucleic acid similarity

[0051]

[0052] Note: Reference ID is the reference genome number; Mapped_fragment is the number of homologous fragments; Query_fragment is the total number of aligned fragments; ANI is the ANI value.

[0053] It is known that the whole genome sequence of Bacillus coagulans T-21 has an average nucleic acid similarity of only 94.73% with known strains. The original sequence data has been stored in the genome sequence archive (genomics, proteomics and bioinformatics 2021) of the National Genomics Data Center (Nucleic Acid Research Center 2022) of the China Center for Biotechnology Information / Beijing Institute of Genomics, Chinese Academy of Sciences, and can be publicly accessed at https: / / ngdc.cncb.ac.cn / gsa.

[0054] Experimental Example 4: Fermentation of Bacillus coagulans T-21, Preparation and Application of Inoculum

[0055] 1. T-21 Fermentation and Preparation of Microbial Agents

[0056] (1) Strain activation: The Bacillus coagulans T-21 strain preserved in freeze-dried tubes was streak-inoculated onto a plate and then onto a plate containing MRS. + The preserved strain was activated by incubating the following ingredients on a plate: tryptone 1.0%, yeast extract 0.5%, beef extract 0.5%, glucose 0.5%, sodium chloride 0.25%, anhydrous calcium chloride 0.015%, manganese sulfate monohydrate 0.01%, L-cysteine ​​hydrochloride 0.05%, and tomato powder 0.05%.

[0057] (2) Preparation of seed culture: Pick a milky white single colony from the activated plate and inoculate it into 100 mL of MRS-enriched solution. + The culture medium was placed in an Erlenmeyer flask and incubated at 40°C with a shaker at 200 rpm for 16 hours. The culture was then transferred again to a flask containing 2500 mL of medium for further incubation to allow the OD to... 600 Seed culture was obtained with a pH value ≥ 2.0 and a pH ≤ 5.0.

[0058] (3) Scale-up of primary fermentation tank: The prepared seed culture was inoculated into a 50L fermenter containing 35L of sterilized enriched MRS at a volume ratio of 5%. + Culture medium, fermentation conditions controlled as follows: temperature 40℃, tank pressure 0.05 MPa, aeration 0.7 V / V·m, stirring 35 Hz, waiting for OD. 600 If the value is ≥10.0, the first-stage seed liquid is obtained and can be transferred to the second-stage fermentation tank;

[0059] (4) Secondary fermenter scale-up: The prepared seed culture was inoculated into a 500L fermenter containing 350L of sterilized enriched MRS at a volume ratio of 10%. + Culture medium, fermentation conditions controlled as follows: temperature 40℃, tank pressure 0.05 MPa, aeration 15V / V·m, stirring 35HZ, waiting for OD. 600 If the value is ≥25.0, a secondary seed culture is obtained, which can then be transferred to a tertiary fermentation tank;

[0060] (5) Scale-up of the three-stage fermenter: The prepared seed culture was inoculated at a volume ratio of 5% into a 10T fermenter containing 7.5T of sterilized fermentation medium (4% corn starch, 1.0% beef extract, 3% enzymatically hydrolyzed soybean meal, 3% glucose, 0.2% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.1% magnesium sulfate heptahydrate, 0.2% manganese sulfate monohydrate, 0.2% anhydrous calcium chloride, and the remainder being water; the percentages are by mass and volume, pH 6.5-7.0, autoclaved at 121℃ for 40 minutes). Fermentation conditions were controlled as follows: temperature 40℃, tank pressure 0.05 MPa, aeration 180V / V·m, and stirring 120HZ. Every 6 hours, 10L of growth factors (composed of 5% methionine, 4% cysteine, and 2% glutamic acid) were added. Every 4 hours, the fermentation broth was sampled for microscopic examination and relevant fermentation parameters were measured. The OD was calculated. 600 After the pH value is ≥70.0 and the spore formation rate is ≥99%, the fermentation broth is obtained;

[0061] (6) Add 20% starch to the fermentation broth, mix well, and spray dry to obtain a product with a moisture content ≤7% and a viable cell count ≥4×10⁻⁶. 10 CFU / g of bacterial agent.

[0062] 2. Applied Research

[0063] The prepared bacterial agent was dissolved and then subjected to MRS. +Activation on agar plates. *Bacillus coagulans* CGMCC No. 21621 was selected as a control strain and activated in the same manner. Single, milky-white colonies were picked and inoculated into liquid culture medium to prepare seed culture solutions. These solutions were then inoculated into MRS+ liquid culture medium (containing nitrite and ammonium chloride at a final concentration of 10 mg / L, denoted as A0) at a volume ratio of 5%. The cultures were incubated at 40℃ and 200 rpm in a shaker. Cultures were collected every 60 minutes, and the contents of nitrite and ammonia nitrogen were determined using a kit (denoted as A1). The degradation rates of nitrite and ammonia nitrogen were calculated using the formula [(A0-A1) / A0]*100%. The effects of the bacterial agent on the contents of nitrite and ammonia nitrogen compounds at different time points are shown in Tables 2 and 3.

[0064] Table 2. Nitrite content (%) at different time points

[0065]

[0066]

[0067] Table 3. Ammonia nitrogen content (%) at different time points

[0068]

[0069] As shown in Table 2, Bacillus coagulans T-21 can completely eliminate 10 mg / L of nitrite within 10 hours. Bacillus coagulans CGMCC No. 21621 can only degrade 22.31% of nitrite within 10 hours, with a residual rate as high as 80%. This demonstrates that the Bacillus coagulans T-21 provided by this invention has a significant degradation effect on nitrite.

[0070] As shown in Table 3, Bacillus coagulans T-21 can completely eliminate 10 mg / L of ammonia nitrogen compounds within 10 hours. However, Bacillus coagulans CGMCC No. 21621 has virtually no degradation effect on ammonia nitrogen.

[0071] Application Example 1: Research on the Application of Bacillus coagulans T-21 Inoculant in Litopenaeus vannamei (South American Shrimp) Farming

[0072] Experimental Design: 360 healthy juvenile Litopenaeus vannamei shrimp of uniform size with an initial average weight of (1.05±0.05) g were randomly selected and cultured in 12 250L aquariums (with 2 mg / L of nitrite and ammonium chloride added), with 30 shrimp per tank. They were divided into 3 groups, with 4 replicates per group. The levels of the microbial agent prepared in Example 4 added to the experimental diets were 0% (control group), 0.3% (experimental group), and 0.05% (w / w) of the agent to the shrimp mass. The three isonitrogenous and isolipid experimental diets (crude protein content approximately 42.0%, crude fat content approximately 6.5%) maintained consistent nutritional levels.

[0073] Animal husbandry and performance indicator measurement: Shrimp were fed three times daily. During the experiment, the aquaculture system was set with a micro-flow. The initial feed amount was calculated as 4-6% of the initial body weight, and the amount was adjusted promptly based on feeding behavior. Feed amounts were recorded. Uneaten feed and feces were removed daily, and uneaten feed was collected, dried, and weighed. The culture period was 42 days. Water quality was measured regularly during the experiment: water temperature 25-29℃, salinity 6‰-7‰, dissolved oxygen concentration 6-7 mg / L, and pH 7.5-7.9. Feeding behavior, mortality, and responses to external stimuli were recorded during the experiment. After the 42-day culture period, each group was starved for 24 hours. The shrimp in each aquarium were then weighed and counted to calculate weight gain rate, feed conversion ratio, and survival rate.

[0074] The weight gain rate (%) was calculated as follows: 100 × (final average weight - initial average weight) / initial average weight; the survival rate (%) was calculated as follows: 100 × final number of shrimp tails / initial number of shrimp tails; the feed conversion ratio was calculated as: (dry weight of feed given - dry weight of uneaten feed) / (final body weight - initial body weight). The experimental results are expressed as mean ± standard deviation. SPSS 21.0 software was used. First, a one-way ANOVA was performed. If significant differences were found, Duncan's method was used for multiple comparison tests between groups. P < 0.05 was considered significant. The effects of this microbial agent on shrimp growth performance are shown in Table 4 below.

[0075] Table 4 Growth performance of Litopenaeus vannamei

[0076]

[0077] As shown in Table 4, the final body weight, weight gain rate, and feed conversion ratio of the 0.03% and 0.05% groups were significantly higher than those of the control group (P<0.05). The weight gain rate increased by 31.43% and 45.60%, respectively, while the feed conversion ratio decreased by 19.73% and 22.45%, respectively. The survival rate was significantly higher than that of the control group by 81.42% and 94.27%, respectively (P<0.05).

[0078] In summary, the Bacillus coagulans T-21 or the bacterial agent prepared therefrom provided by this invention has the ability to efficiently degrade nitrite and ammonia nitrogen compounds, and can be used as a feed additive in aquaculture where nitrite and ammonia nitrogen levels exceed the standards, thus having significant economic value. Simultaneously, this bacterium or bacterial agent can also be used in the environmental remediation of excessive nitrite and ammonia nitrogen levels, showing significant environmental improvement effects and substantial social value.

[0079] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Bacillus coagulans (Bacillus coagulans (T-21), characterized in that, Weizmannia coagulans The T-21 strain was deposited on October 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No: 63889. ​ 2. The use of Bacillus coagulans T-21 as described in claim 1 in the degradation of nitrite and / or ammonia nitrogen compounds.

3. The bacterial agent prepared from Bacillus coagulans T-21 as described in claim 1.

4. The bacterial agent of claim 3, characterized in that, The microbial agent is a powder, liquid, or granular form.

5. The bacterial agent of claim 4, characterized in that, The powder has the viable cell count of T-21 not less than 4 x 10 10 CFU / g.

6. The use of the bacterial agent according to any one of claims 3 to 5 in the field of aquaculture with nitrite or / and ammonia nitrogen exceeding the standard, characterized in that, The application is for degrading nitrite and / or ammonia nitrogen compounds in aquaculture systems.

7. The use of the bacterial agent according to any one of claims 3-5 in the improvement of the environment with excessive nitrite or / and ammonia nitrogen, characterized in that, The application is to degrade nitrite and / or ammonia nitrogen compounds in the environment.

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