Bacillus subtillis for producing amylase, protease and cellulase and application of bacillus subtillis

Abstract

The invention provides bacillus subtillis capable of producing amylase, protease and cellulase and application of the bacillus subtillis, the bacillus subtillis is bacillus subtillis FJAT-10612, the bacillus subtillis is preserved in China General Microbiological Culture Collection Center on August 6, 2025, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.35537. The amylase production activity of the bacillus subtillis disclosed by the invention is 166.12 U / mL, the cellulase production activity of the bacillus subtillis is 88.05 U / mL, and the protease production activity of the bacillus subtillis is 18.36 U / mL; certain tolerance to artificial gastric juice, artificial intestinal juice and cholate is achieved; when the strain fermentation liquor is used for pomace micro-storage, the content of pomace crude protein and total sugar can be remarkably increased, the cellulase activity is improved, meanwhile, the content of crude fibers, neutral detergent fibers, lignin and ammoniacal nitrogen is remarkably reduced, and an effective strain resource is provided for pomace micro-storage fermented feed.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus subtilis species that produces amylase, protease, and cellulase, and its applications. Background Technology

[0002] Fruit pomace silage boasts advantages such as low cost, high efficiency, high digestibility, good palatability, high feed intake, long production season, long shelf life, non-toxicity, ease of production, and enhanced disease resistance and immunity in livestock and poultry, as well as increased livestock product yield. Micro-storage by thoroughly mixing fruit pomace with fermentation agents and cellulase can significantly improve its nutritional value and digestibility. A common method involves crushing the fruit pomace, mixing it evenly with soybean meal, adding fermentation agents, and fermenting in a sealed container for a period of time to obtain fruit pomace roughage. Fruit pomace silage is a natural and green feed. Micro-storing waste fruit pomace not only turns waste into treasure, reduces environmental pollution, and increases the added value of agricultural by-products, but also expands the feed sources for cattle and sheep, alleviating the conflict between forestry and animal husbandry. However, current research on fruit pomace fermentation agents is limited. Screening for suitable microbial agents for fruit pomace fermentation will promote the development of the fruit pomace silage industry.

[0003] Bacillus strains can produce spores, are highly tolerant of environmental changes, and can secrete various functional enzymes and produce antibacterial substances, such as Bacillus subtilis and Bacillus cereus. They are characterized by short fermentation cycles, low fermentation costs, and high enzyme production. Therefore, further screening of more potential candidate enzyme-producing Bacillus strains is needed, especially those producing amylase, protease, and cellulase, in order to provide more probiotic strains for fruit pomace microstorage. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a Bacillus subtilis strain that produces amylase, protease and cellulase and its application. This strain can simultaneously produce high levels of amylase, protease and cellulase, and has good tolerance to the gastrointestinal environment. When the fermentation broth of this strain is used for fruit pomace micro-storage, it can significantly increase the crude protein and total sugar content of fruit pomace, enhance cellulase activity, and significantly reduce the content of crude fiber, neutral detergent fiber, lignin and ammonia nitrogen, thus providing an effective strain resource for fruit pomace micro-storage fermented feed.

[0005] This invention is implemented as follows: A type of Bacillus subtilis that produces amylase, protease, and cellulase. Bacillus subtle FJAT-10612 was deposited on August 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35537.

[0006] Furthermore, the Bacillus subtilis ( Bacillus subtilis FJAT-10612 has the ability to produce amylase, protease and cellulase.

[0007] Furthermore, this strain is tolerant to artificial gastric juice, artificial intestinal juice, and bile salts.

[0008] Furthermore, a method for culturing the aforementioned Bacillus subtilis, the method comprising the following steps: (1) Strain activation: Bacillus subtilis (Bacillus subtilis) was inoculated with an inoculation loop. Bacillus subtilis FJAT-10612 was streaked onto LB medium and placed in a constant temperature incubator for 48±10 h at 30±3℃. (2) Preparation of fermentation liquid: The single colony of FJAT-10612 obtained in step (1) was inoculated into LB medium and placed in a constant temperature shaking shaker. It was cultured for 48±10h at 30±3℃ and 180rpm / min.

[0009] Furthermore, a microbial preparation comprising the aforementioned Bacillus subtilis.

[0010] Furthermore, the microbial preparation is Bacillus subtilis (Bacillus subtilis). Bacillus subtilis Fermentation broth of FJAT-10612.

[0011] Furthermore, the fermentation broth is used for fruit pomace fermentation.

[0012] Furthermore, the fruit pomace includes citrus fruit pomace, including citrus and navel orange.

[0013] The present invention has the following advantages: 1. Enzyme production activity: The Bacillus subtilis ( Bacillus subtilis The FJAT-10612 strain exhibited amylase activity of 166.12 U / mL, cellulase activity of 88.05 U / mL, and protease activity of 18.36 U / mL, demonstrating efficient secretion of these three key hydrolytic enzymes, providing an enzymatic basis for the degradation of starch, cellulose, and protein in fruit pomace. When the fermentation broth of this strain was used for fruit pomace fermentation, the crude protein and total sugar content of the pomace treated with the addition of strain FJAT-10612 were significantly higher than those of the control group, as was the cellulase activity. Conversely, the content of crude fiber, neutral detergent fiber, lignin, and ammonia nitrogen was significantly lower in the pomace treatment group.

[0014] 2. Environmental tolerance: The Bacillus subtilis strain exhibits a certain degree of tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The survival rate is 14% in artificial gastric juice, 29% in artificial intestinal juice, and 55% in bile salts (0.3% w / v), ensuring that the strain can maintain a certain level of activity after entering the digestive tract of livestock and poultry. Attached Figure Description

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Figure 1 This is a colony morphology diagram of strain FJAT-10612 in an embodiment of the present invention. Detailed Implementation

[0017] The following will be combined with the appendix Figure 1 The technical solution of the present invention will be clearly and completely described in detail with specific 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Example

[0018] The Bacillus subtilis of the present invention ( Bacillus subtilis FJAT-10612 is a strain of Bacillus subtilis isolated from soil collected in Xinjiang Uygur Autonomous Region, China, capable of producing amylase, protease, and cellulase. Bacillus subtilis FJAT-10612 exhibits protease, amylase, and cellulase activity, and shows some tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. When the fermentation broth of this strain was used for micro-fermentation of fruit pomace, the crude protein and total sugar content of the pomace treated with FJAT-10612 were significantly higher than the control group, as was the cellulase activity. The content of crude fiber, neutral detergent fiber, lignin, and ammonia nitrogen was significantly lower in the pomace treatment, making it suitable for use in micro-fermented fruit pomace feed.

[0019] 1. Isolation and screening of strain FJAT-10612 (1) Take 10g of soil and put it into 90mL of sterile water. Shake well and then take 1mL for serial dilution. Select a dilution of 10. -4 10 -5 Or 10 -6 ; (2) Spread the diluted solution obtained in step (1) onto LB medium plates (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15-20 g agar, pH 7.0-7.2), and then incubate the LB medium plates at 30°C for 2 days. (3) Streak each strain obtained in step (2) onto nutrient agar medium and incubate at 30°C for 48 h.

[0020] (4) The strains obtained in step (3) were inoculated onto a selective medium for amylase production (10 g soluble starch, 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 17 g agar, pH 7.2, and water was added to bring the volume to 1 L) using a sterile toothpick. After culturing at 30 °C for 2-3 days, the culture was removed and iodine solution was added. A transparent zone appeared around the colony. The diameter of the colony and the diameter of the transparent zone were measured and the ratio was calculated to obtain the amylase-producing strain (the strain with the highest ratio).

[0021] 2. Identification of strain FJAT-10612 (1) Morphological identification The main morphological observation of strain FJAT-10612 on LB medium revealed the following: After cultivation on LB medium, the colonies were pale yellowish-brown, smooth, uneven, and slightly transparent. Specifically... Figure 1 As shown.

[0022] (2) 16S rDNA sequence determination and analysis of strain FJAT-10612 Genomic DNA was extracted from strain FJAT-10612 following the instructions in the kit provided by Shanghai Jierui Biotechnology Co., Ltd. Universal 16S rRNA primers were synthesized by Shanghai Boshan Biotechnology Co., Ltd. Using genomic DNA extracted from strain FJAT-10612 as a template, PCR amplification was performed using universal 16S rRNA primers 27F and 1492R. The reaction mixture consisted of 25 μL: 12.5 μL Taq MIX, 0.5 μL each of primers 27F and 1492R, 1 μL DNA, and deionized water to a final volume of 20 μL. The reaction program was as follows: 94 °C pre-denaturation for 4 min; 94 °C denaturation for 1 min, 50 °C annealing for 1 min, 72 °C annealing for 1 min, repeated for 35 cycles, followed by a final extension at 72 °C for 10 min.

[0023] PCR product detection and sequencing analysis: 2 μL of PCR product was spotted onto a 1.5% agarose gel. Electrophoresis was performed at 100 V for 30 min using a 100 bp marker as the standard molecular weight, followed by EB staining. The PCR products were then separated and tested by gel electrophoresis. Sequencing of the PCR products was then performed by Shanghai Platinum Biotechnology Co., Ltd. After comparing the 16S rRNA sequence of strain FJAT-10612 on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), it was confirmed that strain FJAT-10612 belongs to *Bacillus subtilis* (…). Bacillus subtilisThe 16S rDNA sequence of strain FJAT-10612 is shown in SEQ ID NO:1.

[0024] Based on the above 16S rRNA sequence determination and analysis, combined with morphological characteristics, strain FJAT-10612 was finally determined to belong to Bacillus subtilis (Bacillus subtilis). Bacillus subtilis A strain of Bacillus subtilis was named Bacillus subtilis (B. subtilis). Bacillus subtle )FJAT-10612.

[0025] 3. Determination of amylase, protease and cellulase activities in strain FJAT-10612 (1) Activation of the strain: The strain FJAT-10612 was streaked onto LB medium with an inoculation loop and cultured in a constant temperature incubator for 48 h at a temperature of 30℃. (2) Preparation of fermentation liquid: The single colony of FJAT-10612 obtained in step (1) was inoculated into 100 ml of LB medium (the components of the LB liquid medium are: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 1000 mL water, pH 7.0), and placed in a constant temperature shaking shaker for 48 h at 30 °C and 180 rpm / min. (3) Collect 1 mL of bacterial solution from step (2) to determine amylase activity. At pH 6, the amount of enzyme required to hydrolyze the substrate to produce 1 µmol of reducing sugar in 1 min of 1 mL of enzyme solution is defined as 1 unit of enzyme activity (U / mL). The amylase activity of this strain is 166.12 U / mL. Determine cellulase activity. The cellulase activity of this strain is determined by the method of determining cellulase activity of this strain. An enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of glucose in 1 min of 1 mL of enzyme solution, i.e., U / mL. The cellulase activity of this strain is 88.08 U / mL. Determine protease activity. At 40 °C, the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine in 1 min of 1 mL of enzyme solution is defined as 1 unit of enzyme activity (U / mL). The protease activity of this strain is 18.36 U / mL.

[0026] 4. Determine the tolerance of strain FJAT-10612 to artificial gastrointestinal fluid and bile salts. (1) Activation of the strain: The strain FJAT-10612 was streaked onto LB medium with an inoculation loop and cultured in a constant temperature incubator for 48 h at a temperature of 30℃. (2) Preparation of fermentation liquid: The single colony of FJAT-10612 obtained in step (1) was inoculated into 100 ml of LB medium (the components of the LB liquid medium are: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 1000 mL water, pH 7.0), and placed in a constant temperature shaking shaker for 48 h at 30 °C and 180 rpm / min. (3) Collect the bacterial suspension from step (2), place it in an 80 ℃ water bath for 15 min, centrifuge at 7000 r / min for 10 min, and collect the spores; wash the bacterial cells twice with PBS buffer, and reconstitute them in PBS buffer to obtain the spore suspension. Artificial gastrointestinal fluid: Take 1 mL of the spore suspension and add it to 9 mL of preset artificial gastric fluid and preset artificial intestinal fluid. After mixing thoroughly, incubate at 37 ℃ and 170 r / min for 3 h with shaking. Take samples at 0 and 3 h respectively, dilute them with sterile physiological saline at a serial ratio of 1:10, and then perform plate colony counting; This strain has a certain tolerance to artificial gastric fluid and artificial intestinal fluid. The survival rate in artificial gastric fluid is 14%, and the survival rate in artificial intestinal fluid is 29%.

[0027] Bile salts: 0.3% (w / v) bile salts were added to LB liquid medium and sterilized at 121℃ for 20 min. 1 mL of spore suspension was added to the medium and cultured at 37℃ with shaking at 170 r / min for 24 h. Samples were taken at 0 and 24 h, serially diluted with sterile physiological saline at a 1:10 ratio, and then plate colony counting was performed to calculate the survival rate. This strain showed some tolerance to bile salts, with a survival rate of 55% in bile salts.

[0028] 5. Determine the effect of fermentation broth from strain FJAT-10612 on micro-storage fermentation of fruit pomace. (1) Activation of the strain: The strain FJAT-10612 was streaked onto LB medium with an inoculation loop and cultured in a constant temperature incubator for 48 h at a temperature of 30℃. (2) Preparation of fermentation broth: The single colony of FJAT-10612 obtained in step (1) was inoculated into 100 ml of LB medium (the components of the LB liquid medium are: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 1000 mL water, pH 7.0), and placed in a constant temperature shaking incubator for 48 h at 30 °C and 180 rpm / min to obtain the fermentation broth. The viable count in the fermentation broth was 2*108 CFU / mL. (3) The pomace was harvested in Wuping, Longyan. It was mixed with 40% navel orange pomace, 30% corn flour, 7.5% wheat bran and 22.5% soybean meal. The fermentation liquid in step (2) was added at 5% and mixed evenly with the pomace mixture. 5 kg was put into a large micro storage bag and sealed. The mixture was repeated 3 times. After 10 days of storage in a dark storage room, the bag was opened and samples were taken for analysis. The crude protein content was determined by the Kjeldahl method, the total sugar content was determined by the national food safety standard GB5009.8-2023, the crude fiber content was determined by GB / T6434-2022, the acid detergent fiber content was determined by NY / T1459-2022, the neutral detergent fiber content was determined by GB / T20806-2022, and the lignin content was determined by GB / T20805-2006. The results are shown in Table 3. The crude protein and total sugar content of the fruit pomace treated with strain FJAT-10612 were significantly higher than those of the control group (with an equal amount of sterile water added, but no strain added). The cellulase activity was also significantly higher than that of the control group, while the content of crude fiber, neutral detergent fiber, lignin and ammonia nitrogen was significantly lower than that of the control group.

[0029] Table 3. Effects of strain FJAT-10612 on the quality of pomace micro-storage fermentation

[0030] As can be seen, the present invention contains Bacillus subtilis ( Bacillus subtilis The fermentation broth of strain FJAT-10612 can be used for fruit pomace fermentation, which can increase the crude protein and total sugar content of fruit pomace, significantly increase cellulase activity, and significantly reduce the content of crude fiber, neutral detergent fiber, lignin and ammonia nitrogen. It can provide an effective strain resource for fruit pomace micro-storage fermented feed.

[0031] Therefore, the present invention has the following advantages: Excellent strain performance: Bacillus subtilis FJAT-10612 simultaneously produces high levels of amylase, protease and cellulase, and has good tolerance to the gastrointestinal environment, balancing enzymatic hydrolysis efficiency and intestinal colonization ability, making it suitable for fruit pomace fermentation and the digestive tract environment of livestock and poultry.

[0032] Fruit pomace fermentation has significant effects: it can significantly increase the crude protein and total sugar content and cellulase activity of fruit pomace feed, reduce the content of anti-nutritional factors such as crude fiber, neutral detergent fiber, and lignin, and at the same time reduce ammonia nitrogen (an indicator of excessive protein decomposition), thus improving the nutritional quality and digestibility of feed.

[0033] High application value: On the one hand, it provides efficient strain resources for micro-fermented fruit pomace feed, promoting the industrial application of "fruit pomace to feed"; on the other hand, it reduces environmental pollution from fruit pomace, increases the added value of agricultural and sideline products, and has both economic and ecological benefits.

[0034] In summary, the Bacillus subtilis FJAT-10612 of this invention and its practical application are of great significance to promoting the development of the fruit pomace micro-silage industry and have broad application prospects.

[0035] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A Bacillus subtilis species producing amylase, protease, and cellulase, characterized in that: The Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis FJAT-10612 was deposited on August 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35537.

2. The Bacillus subtilis strain producing amylase, protease, and cellulase according to claim 1, characterized in that: The Bacillus subtilis ( Bacillus subtilis FJAT-10612 has the ability to produce amylase, protease and cellulase.

3. The Bacillus subtilis strain producing amylase, protease, and cellulase according to claim 1, characterized in that: This strain is tolerant to artificial gastric juice, artificial intestinal juice, and bile salts.

4. A method for culturing Bacillus subtilis as described in claim 1, characterized in that: The method includes the following steps: (1) Strain activation: Bacillus subtilis (Bacillus subtilis) was inoculated with an inoculation loop. Bacillus subtilis FJAT-10612 was streaked onto LB medium and placed in a constant temperature incubator for 48±10 h at 30±3℃. (2) Preparation of fermentation liquid: The single colony of FJAT-10612 obtained in step (1) was inoculated into LB medium and placed in a constant temperature shaking shaker. It was cultured for 48±10h at 30±3℃ and 180rpm / min.

5. A microbial preparation comprising the Bacillus subtilis of claim 1.

6. The microbial preparation according to claim 5, characterized in that: The microbial preparation is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis Fermentation broth of FJAT-10612.

7. The microbial preparation according to claim 6, characterized in that: The fermentation broth is used for fruit pomace fermentation.

8. The microbial preparation according to claim 7, characterized in that: The fruit pomace includes the pomace of citrus fruits.

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