A bacterial strain of the genus brenneria and use thereof

Abstract

This invention discloses a strain of *Bacillus spp.* from soil, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO.66967. This strain exhibits good heat resistance, and the mannanase it produces maintains stable enzyme activity under high-temperature conditions. Compared to hydrolases under ordinary conditions, it has greater plasticity and is suitable for the production of products or by-products in multiple fields. In addition, this strain can also be applied to the degumming process of hemp fibers, demonstrating excellent degumming effects on hemp fibers.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of *Bacillus spp.* from soil and its applications. Background Technology

[0002] Mannan is a plant polysaccharide, primarily found in plant cell walls, and belongs to the hemicellulose family. Mannan is abundant in sources, including plant-derived konjac glucomannan and hemimannan, mannan derived from marine bacteria and fungi, and mannan from brewer's yeast. Its hydrolysis products, mannan oligosaccharides or mannose, can improve human health in the medical field, regulating the immune system to promote wound healing, reduce inflammation, inhibit tumor growth and metastasis, and play an important role in increasing cancer survival rates. Mannan oligosaccharides or mannose are mainly produced by the hydrolysis of mannan by mannanase.

[0003] β-Mannanase is a key enzyme in the hydrolysis of mannan, breaking it down into oligosaccharides or monosaccharides. It is an important hemicellulase. Mannanase has a wide range of applications, including industrial degumming in textiles, oil and gas extraction, papermaking, detergents, feed additives, food processing, and pharmaceuticals. Mannanase is abundant in sources, including plant-based, animal-based, and microbial-based enzymes. Microbial mannanase is the primary source, offering advantages such as abundant resources, low cost, simple cultivation conditions, and easy operation.

[0004] Currently, some applications of mannanase require high temperatures during processing, such as feed production and material production. This necessitates that the mannanase used has good heat resistance, which is currently difficult to achieve. Summary of the Invention

[0005] Based on the aforementioned technical problems in the existing technology, the present invention provides a soil brevicorbacterium strain, named M7, which has good heat resistance and produces mannanase whose enzyme activity is stable under high temperature conditions, and has stronger plasticity than hydrolases under ordinary conditions.

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

[0007] A strain of the genus *Brevibacillus agri* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO. 66967.

[0008] The present invention also provides the application of the above-mentioned soil-borne Bacillus strain in the production of mannanase.

[0009] In some embodiments, the method for the *Bacillus spp.* strain to produce mannanase is as follows: the *Bacillus spp.* strain is inoculated into a fermentation medium in which mannan is the sole carbon source, and fermentation is carried out at a temperature of 55-65°C.

[0010] In some embodiments, the inoculation amount of the strain is 1%-10%.

[0011] In some embodiments, the fermentation medium contains konjac flour, yeast extract, tryptone, NaCl, K2HPO4, and has a pH of 7.4-7.6.

[0012] In some embodiments, the fermentation medium comprises, by mass percentage, 0.5%-2.0% konjac flour, 0.3%-0.5% yeast extract, 0.5%-1.5% tryptone, 0.2%-1.0% NaCl, and 0.02%-0.05% K2HPO4, prepared by adding water and adjusting the pH to 7.4-7.6.

[0013] In some embodiments, the culture medium comprises, by mass percentage, 1% konjac flour, 0.3-0.5% yeast extract, 0.5-1.5% tryptone, 0.5% NaCl, 0.03% K2HPO4, and pH 7.4-7.6.

[0014] The present invention also provides a biological agent comprising the above-mentioned *Bacillus spp.* strains and / or their fermentation broth, wherein the fermentation broth is obtained by fermentation using the method of any of the above embodiments.

[0015] In some embodiments, the mannanase in the biological agent has an enzyme activity of 5.0 U / ml or higher at 55-65°C.

[0016] The present invention also provides the application of the above-mentioned soil brevicorbacterium strain in the degumming of hemp.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides a strain of *Bacillus spp.* that exhibits high growth activity at relatively high temperatures (55-65℃), good heat resistance, and produces mannanase with stable enzyme activity at higher temperatures. Compared to hydrolases under ordinary conditions, this strain demonstrates greater plasticity and exhibits high enzyme activity and stability at the higher temperatures required for product processing in its application fields. This allows for processing without temperature reduction, thereby improving production efficiency. Furthermore, it reduces energy consumption for cooling and lowers production costs to some extent. Attached Figure Description

[0019] Figure 1Gram staining results for some strains;

[0020] Figure 2 The growth of each strain at different temperatures;

[0021] Figure 3 Standard curves for mannanase activity assays of each strain were prepared. Detailed Implementation

[0022] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] A strain of *Bacillus brevis*, designated M7, was deposited on September 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.66967.

[0025] To further understand the present invention, the technical solution of the present invention will now be described in detail with reference to preferred embodiments.

[0026] The reagents and culture media used in the following examples are as follows:

[0027] DNS reagents:

[0028] Preparation of solution A: Dissolve 6.9g of crystalline phenol in 15.2ml of 10wt% NaOH solution, dilute with distilled water to 69ml, and then add 6.9g of sodium bisulfite to the solution.

[0029] Preparation of solution B: Dissolve 255g of potassium sodium tartrate in 300ml of 10wt% NaOH solution, and then add 880ml of 1wt% 3,5-dinitrosalicylic acid solution.

[0030] Preparation of the yellow reagent: Mix solution A and solution B to obtain the yellow reagent, store it in a brown bottle for 7-10 days before use; it is effective for one year when stored in a brown bottle.

[0031] Preparation of citric acid-NaH2PO4 buffer solution: Take 45.23g of NaH2PO4·12H2O and 8.07g of citric acid, dissolve them in 900ml of distilled water, adjust the pH to 6, and make up to 1L to obtain citric acid-NaH2PO4 buffer solution.

[0032] Preparation of mannan substrate: Konjac flour was added to citric acid-NaH2PO4 buffer solution to prepare a 1wt% konjac flour solution.

[0033] Preparation of mannose standard solution: Add D-mannose to citric acid-NaH2PO4 buffer solution to prepare a D-mannose standard solution with a concentration of 10 mg / ml.

[0034] Culture medium:

[0035] Solid screening medium: by mass percentage, it includes: 0.3% beef extract, 1% peptone, 0.5% NaCl, 1.5%-2.5% agar powder, 4% konjac powder, 0.03% tribenzene blue, pH: 7.4-7.6;

[0036] Seed culture medium: by weight percentage, it includes 1% glucose, 0.5% yeast extract, 1% tryptone, and 0.5% NaCl, prepared with water, pH: 7.4-7.6;

[0037] Fermentation medium: by mass percentage, it includes 1% konjac flour, 0.3%-0.5% yeast extract, 0.5%-1.5% tryptone, 0.5% NaCl, and 0.03% K2HPO4, prepared with water, pH: 7.4-7.6.

[0038] Both the solid and liquid culture media were sterilized at 121℃ for 20-30 minutes.

[0039] Unless otherwise specified, all other reagents or medicines are commercially available.

[0040] Example 1: Isolation and Screening of Strains

[0041] I. Strain Screening

[0042] The inventors collected hot spring water samples from the hot springs in Wentang Town, Yichun, and added them to conical flasks containing raw ramie fibers, making the water-to-ramie fiber bath ratio 10:1. The samples were then cultured at 55℃ in a shaker at 160 r / min for 48 h to obtain an enriched culture medium. The enriched culture medium was then diluted and spread onto a solid screening medium and cultured at 55℃ for 24 h to observe the size of the hydrolysis zone.

[0043] During the screening process, strains that grew well in 55℃ solid screening medium and had hydrolysis zones were selected, while colonies without hydrolysis zones or with slow growth were discarded. Finally, 19 strains were obtained, as shown in Table 1 below.

[0044] Table 1. Ratio of hydrolysis zone to colony diameter for each strain

[0045]

[0046] The results in Table 1 show that, through initial screening, strain MII 7-5 had a D / d value of 3.33 and the largest clear zone, suggesting that it may have the highest mannanase activity.

[0047] II. Morphological Identification

[0048] Colonies of the 19 strains in Table 1 were picked and further isolated and purified on solid culture medium. The colony morphology on solid culture medium was observed. Single colonies were picked for Gram staining to determine whether the selected strains were Gram-positive or Gram-negative. Single colonies were stained with malachite green staining solution and tomato red staining solution to observe whether there were spores.

[0049] The results of spore staining and Gram staining of some strains are as follows: Figure 1 As shown in the figure. Testing revealed that all 19 bacterial strains possessed spores and were Gram-positive bacteria. The colony morphology and culture medium status of each strain are described in Table 2 below.

[0050] Table 2 Colony morphology of each strain

[0051]

[0052] Example 2: Determination of the optimal growth temperature for the strain

[0053] The purified 19 strains were inoculated into fermentation medium at a 2% inoculum, and then cultured overnight for 12 hours at 160 rpm in a shaker at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃, respectively. Uninoculated fermentation medium was provided as a blank control at each temperature. The OD value of the fermentation broth at 600 nm was measured using a spectrophotometer. The fermentation results are shown below. Figure 2 As shown.

[0054] like Figure 2Under pH 7.4 conditions, the optimal growth temperature for strains MA-1, MA-2, and MI 7-2 is 45℃; the optimal growth temperature for MA-3, MI6-1, MI 6-2, MI 7-4, MII 6-2, MII 6-3, MII 6-4, MII 6-5, MII 7-3, MII 7-4, MII 7-5, and MII 7-6 is 50℃; and the optimal temperature for MI 7-3, MI 7-5, MI 7-6, and MI 7-7 is 55℃.

[0055] This indicates that under conditions where konjac flour is the sole carbon source, the OD of strain MII 7-5... 600 The largest detection range suggests that it has the strongest ability to utilize konjac flour (mannan), which is consistent with the result that the transparent zone of the initial screening strain MII 7-5 was the largest.

[0056] Example 3: Determination of the enzyme activity of extracellular mannanase produced by the strain

[0057] Preparation of extracellular crude enzyme solution: Inoculate the enrichment liquid culture medium with an inoculum of 2%, and incubate at 55℃ and 160r / min for 12h; collect the bacterial solution and centrifuge at 5000r / min for 10min at 4℃, and take the supernatant as the extracellular crude enzyme solution.

[0058] Preparation of the standard curve: According to Table 3, pipette the mannose standard solution, buffer solution, and DNS reagent into separate test tubes, shake well, place in boiling water, incubate for 5 minutes, remove and cool to room temperature, then dilute to 25 ml. Measure the absorbance at 540 nm. Plot the standard curve with the mannose solution concentration on the x-axis and the OD value at 540 nm on the y-axis. Results are as follows: Figure 3 As shown.

[0059] Table 3. Standard Curve of Mannose

[0060]

[0061] Mannanase activity assay: Four stoppered colorimetric tubes were set up in a temperature gradient. 1 ml of crude enzyme solution was added to each of the four tubes. One tube was boiled in a water bath for 10 min to inactivate the enzyme, serving as a blank control. 1 ml of mannan substrate buffer was added to each of the four tubes simultaneously. The mixture was incubated in water at the corresponding temperatures (35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃) for 10 min. Then, 3 ml of DNS reagent was added, and the mixture was thoroughly mixed to terminate the enzyme reaction. The mixture was then boiled in a water bath for 5 min, cooled to room temperature, and diluted to 25 ml with ultrapure water. The OD value was measured at 540 nm.

[0062] Mannanase activity calculation: Record the OD value of each strain and calculate the amount of mannose reduced by each strain y (in mg / ml) using the formula in the mannose standard curve.

[0063] The mannanase activity (in U / ml) of each strain was calculated using the following formula:

[0064] Mannanase activity = (1000 × C × V1 × n) / (V × T × 180.155)

[0065] C is the concentration of mannose (mg / ml), V1 is the volume of the substrate after dilution (ml), V is the volume of crude enzyme solution used (ml), T is the reduction time (min), 180.155 is the molecular weight of mannose (g / mol), and n is the dilution factor.

[0066] The enzyme activities of each strain at different temperatures are shown in Table 5.

[0067] Table 5. Enzyme activity (U / ml) of crude enzyme solutions of various strains at different temperatures.

[0068]

[0069] Table 5 shows that the strains with the optimal temperature for the extracellular crude enzyme solution below 45℃ are MA-1, MI 7-4, MI 7-6, and MII 7-3; those below 50℃ are MA-2, MA-3, MI 6-1, MI 7-3, MI 7-5, MI 7-7, MII 6-2, MII 6-3, and MII 6-4; those below 55℃ are MI 6-2, MI 7-2, MII 7-4, and MII 7-6; and those below 60℃ are MII 6-5 and MII 7-5. In the identification of optimal growth temperatures, several strains showed relatively slow growth. Furthermore, enzymatic studies revealed consistently low enzyme activities in strains MI 7-2 and MI 7-6. This indicates that while these two strains can produce extracellular mannanase, their enzyme activity is low. It is speculated that this is because, under a single carbon source, the strains utilize mannan substrates inefficiently, resulting in insufficient production of mannan oligosaccharides or monosaccharides. Insufficient nutrients for bacterial growth ultimately affect the strain's growth and reproduction. Therefore, the optimal temperature for strain MII 7-5 is the highest at 60℃, with a mannanase activity reaching 8.75 U / ml.

[0070] Example 4 Molecular identification of the strain

[0071] Using universal bacterial primers 27F and 1492R as upstream and downstream primers, the 16S rDNA gene of strain MII 7-5 was amplified. The purified 16S rDNA was sent to Changsha Qingke Biotechnology Co., Ltd. in Hunan Province for sequencing, and the sequence was submitted to the NCBI website, obtaining the GenBank accession number PX471726. Comparison with 16S rDNA from other bacterial species showed that it had 99.93% sequence homology with Brevibacillus agri FW9 (GenBank accession number: CP139100.1), meeting the Kuttzman & Robnett standard that the difference between different strains within the same species should not exceed 1%. Thus, a strain of *Brevibacillus* was obtained, designated M7, and deposited at the Guangdong Provincial Microbial Culture Collection Center on September 15, 2025, with accession number GDMCC NO. 66967.

[0072] Example 5: Fermentation expansion of strain M7 and mannanase activity

[0073] (1) Seed propagation

[0074] Take one loopful of strain M7 and inoculate it into 100 ml of seed culture medium. Incubate at 60℃ and 160 r / min for 18 h. Then transfer 100 ml of seed culture medium to 5 bottles of seed culture medium (100 ml / bottle) and incubate for 20-24 h.

[0075] (2) High-density culture in fermenters

[0076] After cleaning the fermenter, add 20L of fermentation medium, adjust the pH to 7.5 with NaOH, then sterilize at 121℃ for 20-30 minutes. Cool to 35℃ and inoculate with 500ml of seed culture. Maintain the temperature at approximately 60℃, pH between 7.0 and 7.5, pressure below 0.12MPa, and dissolved oxygen above 30%. After a period of cultivation, the dissolved oxygen will gradually decrease. When the dissolved oxygen drops to 25%, begin adding 1000ml of a 50% glucose and sucrose complex carbon source (m...). 葡萄糖 :m 蔗糖 =1:2), to maintain carbon source stability; when dissolved oxygen rises again to 40%, begin supplementing with a composite nitrogen source (m 酵母膏 :m 蛋白胨 :m 硫酸铵 The nitrogen source ratio was 5:10:2, which satisfied the needs of cell growth and spore formation. The pH was adjusted to 7.0, and the dissolved oxygen was controlled at 20%-30% by adjusting the tank pressure, rotation speed, and flow rate. Fermentation was terminated when the dissolved oxygen level dropped below 20%. The growth activity and extracellular mannanase activity of strain M7 were measured from the fermentation broth, and the results are shown in Table 6 below.

[0077] Table 6. Growth viability and extracellular mannanase activity of fermentation broth of strain M7

[0078]

[0079] As shown in Table 6, strain M7 obtained in this application has high growth activity at higher temperatures, and the mannanase obtained from its fermentation has high activity.

[0080] Example 6: Degumming effect of strain M7 on ramie

[0081] S1, Raw material pretreatment

[0082] The raw ramie fibers are pre-treated by mechanically rolling them with rollers to crack them and initially expose the fibers.

[0083] S2, Soaking and Degumming

[0084] Add 2% of the fermented M7 strain fermentation broth to preheated tap water at 60℃ and mix thoroughly to obtain degumming solution; then soak the pretreated ramie raw material in the prepared degumming solution at a bath ratio of 1g:10ml, mix well, and degumm at 60℃ for 24 hours.

[0085] S3, Inactivation

[0086] Boil to inactivate microorganisms and stop degumming, add caustic soda and boil for 20-30 minutes; then wash with water, add hydrogen peroxide and boil for 20-30 minutes to break up the fibers;

[0087] S5, Post-processing

[0088] Ramie fibers are washed, soaked in oil, de-oiled, shaken, and dried to obtain refined ramie hemp.

[0089] The residual gum content of refined ramie was determined according to the "Quantitative Analysis Method of Chemical Composition of Ramie" (GB / T 5889-1986).

[0090] The test results showed that the residual gum rate of the obtained ramie dried hemp was 8.1%, which meets the requirements for practical application. This indicates that strain M7 has excellent degumming effect and is an excellent strain for degumming hemp.

[0091] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A soil-borne short-spore bacillus ( Brevibacillus agri The M7 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO. 66967.

2. The application of the soil brevicorbacterium M7 strain according to claim 1 in the production of mannanase.

3. Use according to claim 2, characterized in that, The application method is as follows: the soil brevicorbacterium M7 strain is inoculated into a fermentation medium in which mannan is the only carbon source, and fermentation is carried out at a temperature of 55-65℃.

4. Use according to claim 3, characterized in that, The inoculation amount of the M7 strain is 1%-10%.

5. Use according to claim 3 or 4, characterized in that, The fermentation medium contains konjac flour, yeast extract, tryptone, NaCl, and K2HPO4, with a pH of 7.4-7.

6.

6. Use according to claim 4, characterized in that, The fermentation medium, by mass percentage, includes 0.5%-2.0% konjac flour, 0.3%-0.5% yeast extract, 0.5%-1.5% tryptone, 0.2%-1.0% NaCl, and 0.02%-0.05% K2HPO4, prepared by adding water and adjusting the pH to 7.4-7.

6.

7. Use according to claim 5, characterized in that, The culture medium, by mass percentage, comprises 1% konjac flour, 0.3%-0.5% yeast extract, 0.5%-1.5% tryptone, 0.5% NaCl, 0.03% K2HPO4, and has a pH of 7.4-7.

6.

8. A biological agent, characterized in that, Includes the M7 strain of claim 1 and / or its fermentation broth, wherein the fermentation broth is obtained by fermentation using the method described in any one of claims 3-7.

9. The biological preparation of claim 8, wherein, In the biological agent, the mannanase has an enzyme activity of 5.0 U / ml or higher at 55-65℃.

10. The application of the soil brevicorbacterium M7 strain according to claim 1 in the degumming of hemp.

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