Brevibacillus sp. Strain and application thereof

Abstract

The invention discloses a brevibacillus sp. Strain, which is preserved in Guangdong Microbial Culture Collection Center on September 15, 2025, and has a preservation number of GDMCC NO.66967. The brevibacillus sp. Strain is preserved in the Guangdong Microbial Culture Collection Center on September 15, 2025. The strain is good in high-temperature resistance, the produced mannase is stable in enzyme activity under the high-temperature condition, and compared with hydrolase under the common condition, the mannase is higher in plasticity and suitable for production of products or by-products in multiple fields; in addition, the strain can also be applied to a hemp degumming process, and has an excellent degumming effect on hemp.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and more particularly to a Brevibacillus agri strain and its application. BACKGROUND

[0002] Mannan is a plant polysaccharide mainly existing in plant cell walls and belongs to hemicellulose. Mannan is abundant in sources, mainly including konjac glucomannan, semi-mannan, mannans from marine bacteria and fungi, and mannans from beer yeast. The hydrolysis products of mannans, manno-oligosaccharides or mannose, can improve human health in the medical field, can regulate the human immune system, and thus promote wound healing, anti-inflammation, inhibit tumor growth and metastasis, and play an important role in increasing cancer survival rate. The source of manno-oligosaccharides or mannose is mainly mannans hydrolyzed by mannanase.

[0003] Beta-mannanase is a key enzyme for hydrolyzing mannans into oligosaccharides or monosaccharides, and is an important hemicellulase. Mannanase is widely used in the fields of industrial degumming textile industry, oil and gas exploitation, papermaking industry, washing industry, feed addition, food processing, pharmaceutical industry, etc. Mannanase is abundant in sources and can be divided into plant-derived, animal-derived and microbial-derived mannans, and the main source is microorganism. Microbial mannanase has the advantages of wide resource, low cost, simple culture condition and easy operation.

[0004] At present, some application fields of mannanase require high temperature during processing, such as feed production, material production, etc., which requires the used mannanase to have good heat resistance. At present, such heat-resistant enzymes are difficult to meet the requirements. SUMMARY

[0005] Based on the above technical problems existing in the prior art, the present application provides a Brevibacillus agri strain, named M7, which has good high-temperature resistance, and the mannase produced by the strain has stable enzyme activity under high-temperature conditions and stronger plasticity than that of the hydrolytic enzyme under ordinary conditions.

[0006] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0007] A Brevibacillus agri strain, which was preserved in the Guangdong Microbial Culture Collection Center on September 15, 2025, and the preservation number is GDMCC NO. 66967.

[0008] The present application also provides the application of the above-mentioned Brevibacillus agri strain in the production of mannanase.

[0009] In some embodiments, the method for producing mannanase by the soil Brevibacillus strain is as follows: the soil Brevibacillus is inoculated into a fermentation medium with mannans as the sole carbon source, and fermentation culture is carried out at a temperature of 55-65℃.

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

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

[0012] In some embodiments, the fermentation medium contains, by mass percentage, 0.5%-2.0% konjac powder, 0.3%-0.5% yeast extract, 0.5%-1.5% tryptone, 0.2%-1.0% NaCl, and 0.02%-0.05% K2HPO4, is prepared by adding water, and has a pH of 7.4-7.6.

[0013] In some embodiments, the medium contains, by mass percentage, 1% konjac powder, 0.3-0.5% yeast extract, 0.5-1.5% tryptone, 0.5% NaCl, and 0.03% K2HPO4, and has a pH of 7.4-7.6.

[0014] The application also provides a biological agent containing the soil Brevibacillus strain described above and / or a fermentation liquor of the soil Brevibacillus strain, wherein the fermentation liquor is obtained by the method of any one of the embodiments described above.

[0015] In some embodiments, the mannanase in the biological agent has an enzyme activity of 5.0 U / ml or more at a temperature of 55-65℃.

[0016] The application also provides the use of the soil Brevibacillus strain described above in hemp degumming.

[0017] Compared with the prior art, the application has the following advantages:

[0018] The application provides a soil Brevibacillus strain, which has high growth activity at a high temperature (55-65℃), is good at resisting high temperature, and produces mannanase with stable enzyme activity at a high temperature. Compared with hydrolytic enzymes under ordinary conditions, the mannanase has higher plasticity, higher enzyme activity and stability at a high temperature required in the product processing process in its application field, can make the product not need to reduce the temperature in the processing process, thereby improving the production efficiency, and to some extent, reducing the energy consumption required for temperature reduction and the production cost. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0021] Figure 3 Standard curve for determination of mannanase activity of each strain. DETAILED DESCRIPTION

[0022] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order to avoid obscuring the present application.

[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 application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0024] A soil Brevibacillus strain, numbered M7, was preserved in the Guangdong Microbial Culture Collection Center on September 15, 2025, at an address of 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard; the preservation number is GDMCC NO.66967.

[0025] In order to facilitate further understanding of the present application, the technical solutions of the present application will be described in detail in combination with preferred embodiments.

[0026] Some reagents and culture solutions used in the following examples are as follows:

[0027] DNS reagent:

[0028] Preparation of the first solution: 6.9 g of crystalline phenol was dissolved in 15.2 ml of 10 wt% NaOH solution, diluted with distilled water to 69 ml, and then 6.9 g of sodium bisulfite was added to the solution.

[0029] Preparation of the second solution: 255 g of potassium sodium tartrate was dissolved in 300 ml of 10 wt% NaOH solution, and then 880 ml of 1 wt% 3,5-dinitrosalicylic acid solution was added.

[0030] Preparation of the yellow reagent: the yellow reagent was obtained by mixing the first solution and the second solution, and was stored in a brown bottle and used after being placed for 7-10 days; it was effective for one year when stored in the brown bottle.

[0031] Preparation of citric acid-NaH2PO4 buffer solution: take NaH2PO4-12H2O 45.23g and citric acid 8.07g, dissolve in 900ml distilled water, adjust pH to 6, constant volume to 1L, citric acid-NaH2PO4 buffer solution is obtained.

[0032] Preparation of mannose substrate: konjac powder is added to citric acid-NaH2PO4 buffer solution to prepare a 1wt% konjac powder solution.

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

[0034] Culture medium:

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

[0036] Seed culture medium: 1% glucose, 0.5% yeast extract, 1% tryptone, 0.5% NaCl, water, pH: 7.4-7.6 by mass percentage;

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

[0038] The above solid and liquid culture media are sterilized at 121℃ for 20-30min.

[0039] Other reagents or agents are commercially available unless otherwise specified.

[0040] Example 1 Isolation and screening of strains

[0041] I. Strain screening

[0042] The inventors collected water samples from hot springs in Wenshang Town, Yichun, added them to conical flasks containing ramie raw jute, so that the bath ratio of water sample to ramie raw jute was 10:1, and cultured them in a shaking bed at 55℃ and 160r / min for 48h to obtain an enriched culture solution; the enriched culture solution was diluted and plated on solid screening medium, and cultured at 55℃ for 24h, and the size of the hydrolysis ring was observed.

[0043] In the screening process, the strains growing well and having hydrolysis ring in the solid screening medium at 55℃ were selected, and the colonies without hydrolysis ring or growing slowly were discarded, and finally 19 strains were obtained, as shown in Table 1 below.

[0044] Table 1 Hydrolysis ring and colony diameter ratio of each strain

[0045]

[0046] The results of Table 1 show that through the preliminary screening, the D / d value of strain MII 7-5 is 3.33, and the transparent ring is the largest, and it is speculated that the mannanase activity may be the largest.

[0047] II. Morphological identification

[0048] The colonies of the 19 strains in Table 1 above were further separated and purified on solid medium, and the colony morphology on the solid medium was observed, and single colonies were selected for Gram staining to determine whether the screened strains were Gram-positive or negative bacteria, and the single colonies were stained with peacock green dye solution and tomato red dye solution to observe whether there were spores.

[0049] The spore staining and Gram staining results of some strains are shown in Table 2. Figure 1 After detection, the 19 strains all had spores and were Gram-positive bacteria. The colony morphology and cell culture solution state of each strain are described in Table 2 below.

[0050] Table 2 Colony morphology of each strain

[0051]

[0052] Example 2 Determination of optimum growth temperature of strains

[0053] The purified 19 strains above were inoculated into the fermentation medium at an inoculation amount of 2%, and then cultured at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃ and 65℃, respectively, at 160r / min on a shaking bed overnight for 12h. The fermentation medium without inoculation was set as a blank control at each temperature. The OD value of the fermentation broth at 600nm wavelength was measured by spectrophotometer, and the fermentation results are shown in Table 3. Figure 2

[0054] As shown in Table 3, the OD value of the fermentation broth at 600nm wavelength was measured by spectrophotometer, and the fermentation results are shown in Table 3. Figure 2 ​The optimum growth temperature of strains MA-1, MA-2 and MI 7-2 was 45℃ at pH 7.4; the optimum growth temperature of MA-3, MI 6-1, 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 was 50℃; the optimum temperature of MI 7-3, MI 7-5, MI 7-6 and MI 7-7 was 55℃.

[0055] It was shown that the OD of strain MII 7-5 was the highest under the condition of konjac powder as the only carbon source 600 The maximum in the detection range was predicted to have the strongest ability to utilize konjac powder (mannan), which was consistent with the result that the transparent circle of strain MII 7-5 was the largest in the previous primary screening.

[0056] Example 3 Determination of enzyme activity of extracellular mannanase produced by strains

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

[0058] Preparation of standard curve: according to Table 3, respectively take the mannose standard solution, buffer and DNS reagent into each test tube, shake well, place in boiling water, water bath for 5min, take out and cool to room temperature, constant volume to 25ml, measure the absorbance at 540nm. Draw the standard curve with the concentration of mannose solution as the abscissa and the OD value at 540nm as the ordinate. The results are shown in Figure 3

[0059] Table 3 Mannose standard curve

[0060]

[0061] Determination of enzyme activity of mannanase: take 4 stoppered colorimetric tubes as 1 temperature gradient, and add 1ml of crude enzyme solution into each of the 4 colorimetric tubes, in which 1 is subjected to boiling water bath for 10min to inactivate the enzyme, as a blank control. Add 1ml of mannose substrate buffer into the above 4 colorimetric tubes at the same time, and react in the water bath at the corresponding temperature (35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃) for 10min, then add 3ml of DNS reagent, mix well, terminate the enzyme reaction, boiling water bath for 5min, take out and cool to room temperature, constant volume to 25ml with ultrapure water, and measure the OD value at 540nm.

[0062] ​Mannase activity calculation: The OD value of each strain was recorded, and the amount of reduced mannose y (unit: mg / ml) of each strain was calculated using the formula in the mannose standard curve.

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

[0064] Mannase activity = (1000 x C x V1 x n) / (V x T x 180.155)

[0065] C is the concentration of mannose (mg / ml), V1 is the amount of substrate after dilution (ml), V is the amount 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 activity of each strain at each temperature is shown in Table 5.

[0067] Table 5 Enzyme activity of crude enzyme solution of each strain at different temperatures (U / ml)

[0068]

[0069] The results in Table 5 show that the strains with optimal temperature of extracellular crude enzyme solution within 45°C are MA-1, MI 7-4, MI 7-6 and MII 7-3; within 50°C 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; within 55°C are MI 6-2, MI 7-2, MII 7-4 and MII 7-6; and within 60°C are MII 6-5 and MII 7-5. In the optimal growth temperature identification, the growth of several strains was relatively slow, and through the study of enzyme properties, it was found that the enzyme activity of MI 7-2 and MI 7-6 strains was always low, indicating that although these two strains can produce extracellular mannase, the enzyme activity is not high. It is speculated that the reason is that under the condition of single carbon source, the efficiency of the strain in utilizing mannose substrate is low, so that the amount of mannose oligosaccharide or monosaccharide produced is small, and the growth of the strain is insufficient in nutrition, which ultimately affects the growth and reproduction of the strain. Therefore, the optimal temperature of strain MII 7-5 is the highest at 60°C, and the mannase activity reaches 8.75 U / ml.

[0070] Example 4 Molecular identification of the strain

[0071] The 16S rDNA gene of the strain MII 7-5 was amplified by using the bacterial universal primers 27F and 1492R as the upstream and downstream primers. The purified 16S rDNA was sent to Hunan Changsha Qianke Biological Co., Ltd. for sequencing, and the measured sequence was submitted to the NCBI website to obtain the Genbank accession number PX471726. The 16S rDNA of other strains was compared, and the results showed that the sequence homology with Brevibacillus agri FW9 was 99.93% (Genbank accession number: CP139100.1), which met the standard of not more than 1% difference between different strains within the same species defined by Kuttzman & Robnett. Thus, the soil Brevibacillus strain was obtained, which was numbered M7 and preserved in the Guangdong Microbial Culture Collection Center on September 15, 2025, with the preservation number GDMCC NO. 66967.

[0072] Example 5 Fermenter expansion and mannanase enzyme activity of strain M7

[0073] (1) Seed expansion

[0074] Take one ring of strain M7 and inoculate into 100 ml of seed culture medium, and cultivate at 60°C and 160 r / min on a shaking table for 18 h. Then, transfer 100 ml of the seed culture solution into 5 bottles of seed culture medium (100 ml / bottle) and cultivate for 20-24 h.

[0075] (2) High-density cultivation in a fermenter

[0076] After the fermenter is cleaned, add 20 L of fermentation medium, adjust the pH to 7.5 with NaOH, and then sterilize at 121°C for 20-30 min. When the temperature is cooled to 35°C, inoculate 500 ml of the seed culture solution. Control the temperature at about 60°C, the pH at 7.0-7.5, the tank pressure below 0.12 MPa, and the dissolved oxygen rate above 30%. After a period of cultivation, the dissolved oxygen will gradually decrease. When the dissolved oxygen decreases to 25%, start to flow add 1000 ml of 50% glucose and sucrose compound carbon source (m 葡萄糖 : m 蔗糖 =1:2), to maintain the stability of the carbon source. When the dissolved oxygen increases to 40% again, start to supplement the compound nitrogen source (m 酵母膏 : m 蛋白胨 : m 硫酸铵 =5:10:2) to meet the nitrogen source for the growth and spore formation of the bacterial cells. Adjust the pH to 7.0, and then control the dissolved oxygen at 20%-30% by adjusting the tank pressure, rotation speed, and flow rate. When the dissolved oxygen is lower than 20%, terminate the fermentation. Measure the growth activity and extracellular mannanase enzyme activity of strain M7 from the fermentation liquor, and the results are shown in Table 6.

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

[0078]

[0079] As shown in Table 6, the strain M7 obtained in the application has higher growth activity at higher temperature, and the mannanase obtained by fermentation thereof has higher activity.

[0080] Example 6 Ramie degumming effect of strain M7

[0081] S1, raw material pretreatment

[0082] The raw ramie is physically rolled and pressed by a roller machine to make the raw ramie crack and preliminarily expose the fibers;

[0083] S2, soaking degumming

[0084] The fermentation broth of the above-mentioned fermentation mature M7 strain is added into 60°C preheated tap water at 2%, and is mixed thoroughly to obtain a degumming liquid; the prepared degumming liquid is soaked in the pretreated raw ramie at a bath ratio of 1 g:10 ml, and is mixed uniformly, and is degummed at 60°C for 24 h;

[0085] S3, inactivation

[0086] Boiling inactivation of microorganisms stops degumming, and caustic soda is added for boiling and scouring for 20-30 min; after water washing, hydrogen peroxide is added for boiling and scouring for 20-30 min, and fiber is beaten;

[0087] S5, post-treatment

[0088] The ramie fiber is washed, dipped, deoiled, fiber shaken, and dried to obtain the refined raw ramie.

[0089] The residual gum rate of the refined raw ramie is detected according to the Quantitative Analysis Method of Chemical Components of Ramie (GB / T 5889-1986).

[0090] Through detection, the residual gum rate of the obtained refined raw ramie is 8.1%, which meets the actual application requirements, and indicates that the strain M7 has excellent degumming effect and is an excellent strain for degumming of bast fibers.

[0091] The technical features of the above-described embodiments can be combined arbitrarily, and in order to make the description simple, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, it should be considered that it is within the scope of the present application.

[0092] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the scope of the application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

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

2. The use of the Brevibacillus agri strain of claim 1 in the production of mannanase.

3. Use according to claim 2, characterized in that, The application method is: inoculating the Brevibacillus agri strain into a fermentation medium with mannans as the sole carbon source, and carrying out fermentation culture at a temperature of 55-65°C.

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

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

6.

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

6.

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

6.

8. A biological agent, characterized in that, The strain of claim 1 and / or the fermentation broth obtained by the method of any one of claims 2-7 are included.

9. The biological preparation of claim 8, wherein, The mannanase in the biological preparation has an enzyme activity of 5.0 U / ml or more at 55-65°C.

10. The use of the Brevibacillus agri strain of claim 1 in the degumming of hemp.

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