Bacillus amylophilus R4.1 strain and preparation method thereof

Abstract

The invention discloses a bacillus thermoamylophilus R4.1 strain which grows under a high-temperature condition and has a proteolysis function and a preparation method of the bacillus thermoamylophilus R4.1 strain. The bacillus thermoamylophilus R4.1 strain is classified and named as Caldibacillus thermoamylophilus R4.1 and is preserved in China Center for Type Culture Collection (CCTCC), the address of the preservation unit is Wuhan University, Wuhan, China, and the preservation date is November 20, 2024. The preservation number of the strain is CCTCC (China Center For Type Culture Collection) No. M 20242601. The invention discloses a bacillus thermoamylophilus R4.1 strain which is a microbial agent with an efficient protein degradation function under a high-temperature condition.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically relates to a strain of Bacillus thermoamylovorans R4.1 and a preparation method thereof. Background Art

[0002] Bacillus thermoamylovorans ( Caldibacillus thermoamylovorans ) is a thermophilic bacterium, first isolated from palm wine in 1995, and has the ability to hydrolyze starch. Subsequently, it was found to have the ability to degrade cellulose and lignin from plant biomass compost; Bacillus thermoamylovorans isolated from hot springs and soybean oil environments can secrete lipase; it was found to have the ability of biogasification degradation during the drying degradation process of sludge, and gene annotation found that it is mainly related to the transport metabolism of amino acids and the transport metabolism of carbohydrates, indicating the potential to degrade proteins and carbohydrates; Bacillus thermoamylovorans found in milk proved to have the ability to hydrolyze casein.

[0003] The rendering method is a harmless treatment method for intensively treating dead and diseased livestock and poultry carcasses during the breeding process. After the carcasses are broken by a rendering processor, pathogenic microorganisms are killed at high temperature and high pressure, and then an industrial grease or biodiesel and solid product meat and bone meal can be obtained through a solid-liquid separation system. The meat and bone meal is rich in protein, and the crude protein content is as high as 63.59 - 73.35%. After fermentation, it can be made into organic fertilizer for resource recycling. Currently, the degradation of proteins is mainly accomplished by protease catalyzing the hydrolysis of peptide bonds in proteins or polypeptides into small peptides or amino acids. Microorganisms are the main producers of proteases, and microorganisms with high protease-producing performance have been developed at the commercial level to assist in protein degradation.

[0004] There is no reported preparation method for a Bacillus thermoamylovorans bacterial agent with high protease-producing performance. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Bacillus thermoamylovorans R4.1 that grows under high-temperature conditions and has high protease-producing performance, and a preparation method thereof.

[0006] To solve the problems of the existing technology, the present invention provides the following technical solutions: In the first aspect, the present application provides a strain of Bacillus thermoamylovorans R4.1 that can grow under high-temperature conditions and has the performance of producing protease; this strain is classified and named as Bacillus thermoamylovorans Caldibacillus thermoamylovorans strain R4.1, deposited with the China Center for Type Culture Collection (CCTCC), the address of the depository is Wuhan University, Wuhan, China, the deposit date: November 20, 2024; the deposit number: CCTCC No. M 20242601.

[0007] Furthermore, the Bacillus thermoamylovoransCaldibacillus thermoamylovorans The 16S rRNA gene sequence of strain R4.1 is the nucleotide sequence shown in SEQ ID No.1.

[0008] Furthermore, the strain is stained blue-violet by Gram staining, is a Gram-positive bacterium, has a long rod-shaped cell microscopic morphology, and after purification culture by streaking on an LB plate, the colony is dry and irregularly milky white.

[0009] In a second aspect, the present application provides a method for preparing a thermophilic Bacillus amyloliquefaciens R4.1 bacterial agent with high protease production performance; Furthermore, its active ingredient is at least one of the following (a), (b), and (c): (a) The fermentation culture of thermophilic Bacillus amyloliquefaciens; (b) The spore suspension of the obtained thermophilic Bacillus amyloliquefaciens; (c) The ultrasonic lysis precipitate of the obtained thermophilic Bacillus amyloliquefaciens cells.

[0010] Furthermore, the preparation method of the high protease-producing bacterial agent of the present invention includes the following steps: (1) The compost fermentation product of pig carcass rendering meat and bone meal is inoculated into an LB liquid medium and enriched by shaking culture at 50 °C for 24 h; the bacterial suspension is spread on a protease screening culture medium and continued to be shaken at 50 °C for 24 h, and the strains showing a proteolysis zone around are screened; the screened strains are inoculated into an LB solid medium and shaken at 50 °C for 24 h to obtain solid seed strains.

[0011] (2) The strain is inoculated into a culture dish and subjected to ultraviolet mutagenesis under the irradiation of a 20 W ultraviolet lamp for different times, and the surviving strains are subcultured; repeated twice for three times of ultraviolet mutagenesis. After the strain obtained by ultraviolet mutagenesis is cultured to the logarithmic growth phase, the culture temperature is increased. When the survival rate of the strain reaches more than 90%, it is subcultured; in this cycle, the culture temperature is increased step by step by 5 °C to carry out high-temperature domestication on the strain until its high-temperature tolerance reaches 65 °C.

[0012] (3) The strain after high-temperature domestication is cultured in an LB medium with an initial pH of 8.02, a NaCl concentration of 0.59%, and a Ca 2+ concentration of 1.11% at 65 °C for 96.36 h to prepare a thermophilic Bacillus amyloliquefaciens bacterial agent with the highest protease production performance.

[0013] Beneficial effects: The present invention discloses a thermophilic Bacillus amyloliquefaciens R4.1 strain, which can grow under high-temperature conditions and can secrete protease to exert proteolysis function.

[0014] Compared with the prior art, the present invention has the following advantages: In the present invention, a thermophilic Bacillus amyloliquefaciens strain capable of producing protease under high-temperature conditions was screened from the solid meat and bone residues obtained from the rendering process of pig carcasses. Caldibacillus thermoamylovorans Strain R4.1. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 Schematic diagram of the thermophilic Bacillus amyloliquefaciens R4.1 of the present invention: having casein hydrolysis function ( Figure 1 A), morphological structure of irregular milky white colonies ( Figure 1 B), long rod-shaped cell morphology of Gram-positive ( Figure 1 C), and phylogenetic tree clustering in the branch of Bacillus amyloliquefaciens thermophilus ( Figure 1 D).

[0017] Figure 2 Changes in protease activity of the thermophilic Bacillus amyloliquefaciens R4.1 strain of the present invention under ultraviolet mutagenesis (A) and high-temperature acclimation (B).

[0018] Figure 3 Growth curve ( Figure 3 A) and changes in protease activity ( Figure 3 B) of the thermophilic Bacillus amyloliquefaciens R4.1 of the present invention Figure 4 Changes in protease activity of the thermophilic Bacillus amyloliquefaciens R4.1 of the present invention at different pH values (A), NaCl concentrations (B), 1% of different metal ions (C), and different Ca 2+ concentrations (D). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] To make the technical problems to be solved, technical solutions, and beneficial effects of the present application clearer, the following further details the present application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0020] In the present application, the term "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0021] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or a similar expression means any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can each be single or multiple.

[0022] It should be understood that in various embodiments of this application, the magnitude of the serial numbers of the above - mentioned processes does not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0023] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms "a", "the", and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The weight of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the specification of the embodiments of this application is scaled up or down proportionally, it is within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass described in the specification of the embodiments of this application can be mass units well - known in the chemical industry such as μg, mg, g, kg, etc.

[0025] The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of this application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.

[0026] The present invention provides a Bacillus amyloliquefaciens Caldibacillus thermoamylovorans strain R4.1, classified and named as Caldibacillus thermoamylovorans R4.1 , deposited in the China Center for Type Culture Collection (CCTCC), the address of the depositary institution is Wuhan University, China, the deposit date: November 20, 2024; the deposit number: CCTCC No. M 20242601.

[0027] PCR amplification was performed using the universal primers 27F (5’- AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’- CGGTTACCTTGTTACGACTT-3’) of the bacterial 16S rRNA gene to obtain the 16S rRNA gene of the strain, and the sequence was the nucleotide sequence shown in SEQ ID No.1, which could be used as the basis for molecular identification of the strain.

[0028] The strain is a Gram-positive bacterium, the cells are long rod-shaped, cultured on an LB plate, and the colonies are dry and irregular milky white.

[0029] Bacillus thermoamylovorans of the present invention Caldibacillus thermoamylovorans The Bacillus thermoamylovorans agent prepared from strain R4.1 has the function of growing under high temperature conditions and secreting protease.

[0030] In some embodiments, its active ingredient is at least one of the following (a), (b), and (c): (a)The fermentation culture of Bacillus thermoamylovorans; (b)The spore suspension of the obtained Bacillus thermoamylovorans; (c)The ultrasonic lysis precipitate of the obtained Bacillus thermoamylovorans cells.

[0031] Bacillus thermoamylovorans with high protease production performance Caldibacillus thermoamylovorans The preparation method of the agent of R4.1 includes the following steps: (1)The compost fermentation product of the carcass rendering product of live pigs, meat and bone meal, was inoculated into LB liquid medium and enriched by shaking culture at 50 °C for 24 h; the bacterial suspension was spread on a protease screening medium and continued to be shaken at 50 °C for 24 h to screen the strains showing a proteolytic zone around them; the screened strains were inoculated into LB solid medium and shaken at 50 °C for 24 h to obtain solid seed strains.

[0032] (2)The strain was inoculated into a petri dish and subjected to ultraviolet mutagenesis under the irradiation of a 20 W ultraviolet lamp for different times, and the surviving strains were subcultured; repeated twice for three times of ultraviolet mutagenesis. After the strain obtained by ultraviolet mutagenesis was cultured to the logarithmic growth phase, the culture temperature was increased. When the survival rate of the strain reached more than 90%, it was subcultured; in this cycle, the culture temperature was gradually increased by 5 °C to perform high-temperature domestication on the strain until its high-temperature tolerance reached 65 °C.

[0033] (3)The strain after high-temperature domestication was cultured in an LB medium with an initial pH of 8.02, a NaCl concentration of 0.59%, and a Ca 2+ concentration of 1.11% at 65 °C for 96.36 h to prepare the Bacillus thermoamylovorans agent with the highest protease production performance.

[0034] The present invention will be further described in detail below in conjunction with embodiments. The microbial inoculum in the embodiments is prepared according to the conventional methods of microbial fermentation and the preparation of microbial inoculum.

[0035] Test Example 1 1. Thermophilic Bacillus amyloliquefaciens Caldibacillus thermoamylovorans Isolation and identification of strain R4.1 1.1 Materials Meat and bone meal residue after the rendering of livestock and poultry carcasses, LB liquid medium (peptone 1%, yeast powder 0.5%, NaCl 1%), LB solid medium added with 2% agar powder); protease screening medium (casein 1%, NaCl 0.5%, agar 2%), protease activity detection medium (peptone 0.5%, beef extract 0.1%, yeast powder 0.25%, casein 0.25%, NaCl 0.5%, pH 7.0).

[0036] 1.2 Isolation of strains Under sterile conditions, take 1 g of meat and bone meal sample and shake it in 10 mL of sterile water for 1 h. After standing for 1 h, take the supernatant and inoculate it into LB liquid medium, and culture it at 50 °C for 24 h. Take 5 mL of the bacterial suspension and spread it on a plate prepared with 100 mL of protein screening medium, and culture it in a constant temperature incubator at 50 °C for 24 h. Select the strains with a protein hydrolysis zone around the colonies and purify them by streaking on LB solid medium. The purified strains are inoculated into the protease detection medium, and the protease activity is measured every 12 h using the Folin-Ciocalteu colorimetric method.

[0037] 1.3 Strain identification Morphological identification: Gram stain the strains prepared in the examples and observe the cell morphology and color under a microscope. Molecular identification: Use the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 to extract the total DNA of the strains, and use the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-CGGTTACCTTGTTACGACTT-3’) of the 16S rRNA gene for PCR amplification. The PCR amplification program: 95 °C for 5 min; 95 °C for 30 s, 56 °C for 15 s, 72 °C for 20 s, for a total of 30 cycles; 72 °C for 7 min; store at 4 °C. After detecting the PCR products by 1.2% agarose gel electrophoresis, send them to Sangon Biotech Co., Ltd. for sequencing. The sequencing results are compared by BLAST in the GenBank database, and multiple sequence alignment analysis is performed using the MEGA X software to determine the molecular type of the strains.

[0038] 1.4 Results A strain R4.1 was able to produce a clear zone on the protease medium ( Figure 1 A), and the protease activity was 24.7 U / mL; the colony was dry and irregular milky white in shape ( Figure 1 B); Gram staining showed that the strain was a long rod-shaped with blue-violet color, and it was a Gram-positive bacterium ( Figure 1 C); the phylogenetic tree of 16S rRNA constructed by molecular biology methods showed that the strain clustered with multiple strains of Bacillus thermoamylovorans in the same branch ( Figure 1 D). This strain R4.1 was named Bacillus thermoamylovorans ( Caldibacillus thermoamylovorans ) R4.1. The 16S rRNA gene sequence of Bacillus thermoamylovorans R4.1 is shown in the sequence listing.

[0039] Test Example 2

[0040] 2. Optimization of protease production ability and high-temperature tolerance of Bacillus thermoamylovorans R4.1 by combining ultraviolet mutagenesis and high-temperature acclimation 2.1 Test Method Ultraviolet mutagenesis: Select the bacterial liquid cultured to the logarithmic growth phase, spread and inoculate it in a culture dish, and irradiate it under a 20 W ultraviolet lamp for 0 s, 30 s, 60 s, 90 s, 120 s, 150 s. After culturing at 50 °C for 48 h, colony counting was carried out to calculate the lethality rate. Select the mutant strains with a lethality rate of 90 - 98%, and repeatedly subculture until the strain is stable. Repeat the steps of ultraviolet mutagenesis of the strain more than twice. Measure the protease activity of the strain after ultraviolet mutagenesis.

[0041] High-temperature acclimation: Select the mutagenized strain with the strongest protease activity, culture it in 50 °C LB liquid medium to the logarithmic growth phase, and transfer it to a 55 °C environment for culture. At the same time, inoculate the bacterial liquid into the protease screening medium to calculate the survival rate. Select the strains with a survival rate of more than 90%, and carry out high-temperature acclimation by gradually increasing the temperature step by step, with the temperature increasing by 5 °C each time. At the same time, measure the enzyme production ability of the strain after high-temperature acclimation to determine the optimal maximum culture temperature for protease production of the strain.

[0042] 2.2 Results The results of ultraviolet mutagenesis of Bacillus thermoamylovorans R4.1 strain showed ( Figure 2 A), after three mutageneses, the protease activity of the strain reached the maximum value of 41.30 U / mL at 96 h, and compared with the original strain, the protease activity increased by 67%. The results of high-temperature acclimation showed ( Figure 2 B), Bacillus thermoamylovorans R4.1 strain obtained growth ability in the range of 55 - 75 °C, and the enzyme production ability was the strongest at 65 °C for 96 h, and the enzyme activity was 45.56 U / mL.

[0043] Test Example 3

[0044] 3. Preparation Conditions of Thermophilic Bacillus amyloliquefaciens R4.1 Bacterial Agent with High Temperature Tolerance and High Protease Yield 3.1 Test Method Single-factor optimization of the preparation conditions of the high-temperature-tolerant and high-protease-yield bacterial agent: The strain was inoculated into the protease-producing medium at an inoculation amount of 1%. The bacterial liquid at different culture times (sampling once every 12 h after inoculation of the strain, lasting until 108 h) was selected to measure the protease activity, and the optimal protease-producing time was determined. Adjust the initial pH value (6.0, 7.0, 8.0, 9.0, 10.0), NaCl concentration (0.25%, 0.5%, 1%, 2%, 4%), add 1% of different metal ions (Fe 2+ 、Cu 2+ 、Ca 2+ 、Mn 2 + 、Mg 2+ 、K + ), and different concentrations (0%, 0.5%, 1%, 2%, 4%) of the metal ion with the best promoting effect were added. The protease activity of the strain at the optimal enzyme-producing time was measured and expressed by the relative protease activity of the strain. Each group of tests was designed with 3 replicates.

[0045] Optimization of the preparation conditions of the high-temperature-tolerant and high-protease-yield bacterial agent by the response surface method: Based on the test results of the single factor, with the enzyme activity as the response value, the culture time, initial pH value, NaCl concentration, and Ca 2+ concentration were selected as independent variables. Three variable levels were designed for each variable, and the Box-Behnken test of four factors and three levels was designed using Design-Expert 13 software. Based on the enzyme activity response value data of four factors and three levels, a quadratic multiple regression fitting was performed to establish a quadratic polynomial regression model of protease activity (Y) against A (time), B (pH), C (NaCl concentration), and D (Ca 2+ concentration).

[0046] Verification of the optimal enzyme-producing bacterial agent preparation conditions for Bacillus amyloliquefaciens R4.1: Based on the optimal enzyme-producing preparation conditions for Bacillus amyloliquefaciens R4.1 obtained by the response surface analysis, 3 repeated verifications were carried out.

[0047] 3.2 Results Determination of the optimal culture time for the protease performance of the strain: The growth results of Bacillus amyloliquefaciens R4.1 showed that the strain concentration showed a trend of first increasing and then stabilizing. The strain growth reached the maximum value at 26 h ( Figure 3A); The change in protease activity showed that with the passage of time, the enzyme activity continued to increase and reached a maximum value of 42.98 U / mL at 96 h, after which the enzyme activity decreased ( Figure 3 B), indicating that the optimal enzyme production time of the strain was 96 h.

[0048] Determination of the optimal preparation conditions of the strain under single factor: As the culture conditions changed, the protease activity of the strain changed as Figure 4 shown. After culturing for 96 h, the strain could grow within the range of pH 7.0 - 9.0. The enzyme activity of the strain reached the peak at pH 8.0, and the protease activity increased by 9.36% (46.51 U / mL) compared with the initial fermentation medium ( Figure 4 A). The protease activity of the strain was the highest (46.95 U / mL) in the protease production medium containing 0.5% NaCl. Too high or too low NaCl concentration in the medium would significantly inhibit the protease activity of the strain (P < 0.01) ( Figure 4 B). The addition of Fe 2+ , Mn 2+ , Mg 2+ , K + , Cu 2+ had a certain inhibitory effect on the protease activity of the strain (P < 0.01). Only the addition of Ca 2+ significantly enhanced the protease activity of the strain (P < 0.01) ( Figure 4 C). Further exploration of the effect of Ca2+ concentration on the protease activity of the strain found that Ca 2+ could enhance the protease activity of the strain within the concentration range of 0.5 - 2.0%. Among them, the addition of 1% Ca 2+ had the most significant enhancement effect on the protease activity of the strain (P < 0.01). The protease activity increased by 18.80% (55.24 U / mL) ( Figure 4 D).

[0049] Determination of the optimal preparation conditions of the strain under multiple factors: Using the software Design Expert 13, based on the enzyme activity response value data of four factors and three levels of culture time (84 h, 96 h, 108 h), pH (7, 8, 9), NaCl (0.25%, 0.5% and 1%) concentration and Ca2+ (0.5%, 1% and 2%) concentration, quadratic multiple regression fitting was carried out to obtain the quadratic polynomial regression model of protease activity (Y) on A (time), B (pH), C (NaCl concentration), D (Ca2+ concentration) as: (R 2 = 0.9840; Radj 2 = 0.9620).

[0050] Under this model, the optimal conditions for enzyme production are a time of 96.36 h, an initial pH of 8.02, an NaCl concentration of 0.59%, and a Ca 2+ concentration of 1.11%, with an enzyme activity of 56.49 U / mL.

[0051] Verification of the optimal conditions for preparing the enzyme-producing microbial agent: Under the optimal conditions, after three repeated experiments, the actual protease activity was detected to be 55.08 U / mL, with a relatively small relative error from the predicted value (2.56%), thus determining the optimal conditions for preparing the enzyme-producing microbial agent.

[0052] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification, and their equivalents.

Claims

1. A thermoamylophilic Bacillus R4.1 strain, characterized in that: The strain is a thermoamylophilic Bacillus R4.1 strain, which is classified and named Caldibacillus thermoamylovorans R4.1 , deposited in the China Center for Type Culture Collection CCTCC, the depository address is Wuhan University, Wuhan, China, the deposit date is November 20, 2024; the deposit number is CCTCC No. M 20242601.

2. The thermoamylophilic Bacillus R4.1 strain according to claim 1, characterized in that: The 16S rRNA gene sequence of the thermoamylophilic Bacillus R4.1 strain is the nucleotide sequence shown in SEQ ID No.

1.

3. The thermoamylophilic Bacillus agent prepared by the thermoamylophilic Bacillus R4.1 strain according to claim 1.

4. The thermoamylophilic Bacillus agent according to claim 3, characterized in that: The active ingredient is at least one of the following (a), (b), and (c): (a) a fermentation culture of the thermoamylophilic Bacillus according to claim 1; (b) a spore suspension of thermoamylophilic Bacillus obtained according to claim 1; (c) The ultrasonic lysis precipitate of the thermoamylophilic Bacillus cells obtained according to claim 1.

5. The method for preparing the thermoamylophilic bacillus agent according to claim 3, characterized in that The steps include: (1) The fermented product of the meat and bone residue of the pig carcass was inoculated with LB liquid medium and cultured in a shaking incubator at 50°C for 24 hours; the bacterial suspension was spread on a protease screening medium and cultured in a shaking incubator at 50°C for 24 hours to screen strains with proteolytic circles around them; the selected strains were inoculated with LB solid medium and cultured in a shaking incubator at 50°C for 24 hours to obtain solid seed strains; (2) The strain was inoculated into a culture dish and irradiated with a 20 W ultraviolet lamp for different periods of time for ultraviolet mutagenesis. The surviving strains were subcultured. This process was repeated twice and three times for ultraviolet mutagenesis. After the strains obtained by ultraviolet mutagenesis were cultured to the logarithmic growth phase, the culture temperature was increased. When the survival rate of the strains reached more than 90%, the strains were subcultured. The culture temperature was increased step by step by 5°C in this cycle to acclimate the strains to high temperatures until their high temperature resistance reached 65°C. (3) After high temperature acclimation, the strain was at an initial pH of 8.02, NaCl concentration of 0.59%, and Ca 2+ The thermoamylophilic Bacillus inoculum with the highest protease production performance was obtained by using LB medium with a concentration of 1.11% and culturing at 65°C for 96.36 h.

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