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

Abstract

The invention provides bacillus velezensis capable of producing amylase, protease and cellulase and application of the bacillus velezensis, the bacillus velezensis is bacillus velezensis FJAT-10502 and is preserved in China General Microbiological Culture Collection Center on August 6, 2025, the preservation address is No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The amylase production activity of the bacillus velezensis is 181.39 U / mL, the cellulase production activity of the bacillus velezensis is 28.4 U / mL, and the protease production activity of the bacillus velezensis is 18.48 U / mL; the antibacterial activity on escherichia coli and salmonella is achieved, and certain tolerance on artificial gastric juice, artificial intestinal juice and cholate is achieved; when the strain fermentation liquor is used for pomace micro-storage, the content of crude protein and total sugar of pomace can be remarkably increased, meanwhile, the content of crude fiber, acid detergent fiber, neutral detergent fiber and lignin 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 bellis 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 bellis strain that produces amylase, protease and cellulase and its application. This strain can simultaneously produce high levels of amylase, protease and cellulase, and has strong antibacterial activity and 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, while significantly reducing the content of crude fiber, acid detergent fiber, neutral detergent fiber and lignin, thus providing an effective strain resource for fruit pomace micro-storage fermented feed.

[0005] This invention is implemented as follows: A type of Bacillus belye that produces amylase, protease, and cellulase, wherein the Bacillus belye is Bacillus belye ( Bacillus velezensis FJAT-10502 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. 35538.

[0006] Furthermore, the Bacillus belye ( Bacillus velezensis FJAT-10502 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, an application of the aforementioned Bacillus belyssus, wherein the Bacillus belyssus ( Bacillus velezensis FJAT-10502 is used for the prevention and control of Escherichia coli and Salmonella.

[0009] Furthermore, a method for culturing the aforementioned Bacillus belye includes the following steps: (1) Activation of the strain: Use an inoculation loop to inoculate Bacillus belye ( Bacillus velezensis FJAT-10502 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-10502 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.

[0010] Furthermore, a microbial preparation comprising the aforementioned Bacillus belye.

[0011] Furthermore, the microbial preparation is Bacillus belyssus (B. belyssus). Bacillus velezensis Fermentation broth of FJAT-10502.

[0012] Furthermore, the fermentation broth is used for fruit pomace fermentation.

[0013] Furthermore, the fruit pomace includes pomace from citrus fruits, including citrus fruits, navel oranges, etc.

[0014] The present invention has the following advantages: 1. Enzyme Activity: The *Bacillus belyssioides* FJAT-10502 strain exhibits amylase activity of 181.39 U / mL, cellulase activity of 28.4 U / mL, and protease activity of 18.48 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 is used for fruit pomace fermentation, it can increase crude protein and total sugar content while reducing the content of crude fiber, acid detergent fiber, neutral detergent fiber, and lignin in the fermented fruit pomace, resulting in good performance in micro-fermented fruit pomace feed.

[0015] 2. Antibacterial activity: The Bacillus berberis FJAT-10502 exhibits significant antibacterial activity against Escherichia coli and Salmonella. The inhibition zone diameter against Escherichia coli was 28.71 mm and the inhibition zone diameter against Salmonella was 34.24 mm, as determined by the double-layer plate method.

[0016] 3. Environmental tolerance: The Bacillus berberis FJAT-10502 strain exhibits a certain degree of tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The survival rate is 46% in artificial gastric juice, 65% in artificial intestinal juice, and 8% 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

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 This is a colony morphology diagram of strain FJAT-10502 in an embodiment of the present invention.

[0019] Figure 2 This is a 16S rDNA diagram of strain FJAT-10502 in an embodiment of the present invention. Detailed Implementation

[0020] The following will be combined with the appendix Figures 1-2 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

[0021] The Bacillus berberis of the present invention ( Bacillus velezensis FJAT-10502 is a strain of *Bacillus belyssus* isolated from fermented bedding material used in pig farming in Fuzhou, Fujian Province. It possesses the ability to produce amylase, protease, and cellulase. Bacillus velezensis FJAT-10502 exhibits protease, amylase, and cellulase activity, and shows antibacterial activity against Escherichia coli and Salmonella. It also demonstrates some tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The fermentation broth of this strain can be used for micro-fermentation of fruit pomace, increasing crude protein and total sugar content while reducing the content of crude fiber, acid detergent fiber, neutral detergent fiber, and lignin in the fermented fruit pomace. It can be applied to micro-fermented fruit pomace feed.

[0022] 1. Isolation and screening of strain FJAT-10502 (1) Take 10g of bedding material 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.

[0023] (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).

[0024] 2. Identification of strain FJAT-10502 (1) Morphological identification The main morphological observation of strain FJAT-10502 was performed, and the main morphology of this strain on LB medium was as follows: After cultivation on LB medium, the colonies were light yellowish-brown, smooth, uneven, and slightly transparent. Specifically... Figure 1 As shown.

[0025] (2) 16S rDNA sequence determination and analysis of strain FJAT-10502 Genomic DNA was extracted from strain FJAT-10502 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-10502 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.

[0026] 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-10502 on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), it was confirmed that strain FJAT-10502 belongs to *Bacillus belyesense* (B. belyesense). Bacillus velezensis The 16S rDNA diagram of strain FJAT-10502 is shown below. Figure 2 As shown.

[0027] Based on the above 16S rRNA sequence determination and analysis, combined with morphological characteristics, strain FJAT-10502 was finally determined to belong to Bacillus belyssae (B). Bacillus velezensis A strain of Bacillus belye was named (Bacillus belye). Bacillus velezensis )FJAT-10502.

[0028] 3. Determination of amylase, protease and cellulase activities in strain FJAT-10502 (1) Activation of the strain: The strain FJAT-10502 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-10502 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 181.39 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 28.4 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 18.48 U / mL.

[0029] 4. Determine the antibacterial activity of strain FJAT-10502 against Escherichia coli and Salmonella. (1) Activation of the strain: The strain FJAT-10502 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-10502 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 the bacterial culture from step (2). *E. coli* and *Salmonella* were inoculated into LB medium and fermented for 24 h in a constant temperature incubator at 37 ℃ and 170 r / min. The double-layer plate method was used: a layer of solid medium was poured into a plate, dried, and then a semi-solid medium containing *E. coli* and *Salmonella* was poured in. After the medium solidified, wells (7 mm in diameter) were evenly punched, and approximately 100 μL of FJAT-10502 bacterial culture was added to each well. The plates were incubated at 30 ℃ for 1-2 days, and the diameter of the inhibition zone (mm) was measured. This strain showed good inhibitory effects on *E. coli* and *Salmonella*, with inhibition zone diameters of 28.71 and 34.24 mm, respectively.

[0030] 5. Determine the tolerance of strain FJAT-10502 to artificial gastrointestinal fluid and bile salts. 1) Activation of the strain: The strain FJAT-10502 was streaked onto LB medium with an inoculation loop and incubated in a constant temperature incubator for 48 h at a temperature of 30℃. (2) Preparation of fermentation liquid: The single colony of FJAT-10502 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, mix thoroughly, and 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 46%, and the survival rate in artificial intestinal fluid is 65%.

[0031] 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 incubated 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 counts were performed to calculate the survival rate. This strain showed some tolerance to bile salts, with a survival rate of 8% in bile salts.

[0032] 6. Determine the effect of fermentation broth from strain FJAT-10502 on micro-storage fermentation of fruit pomace. (1) Activation of the strain: The strain FJAT-10502 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 liquid fermentation: The single colony of FJAT-10502 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℃ and 180 rpm / min to obtain the fermentation broth. The viable count in the fermentation broth was 2*10^6 cells / min. 8 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. 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 1. The crude protein and total sugar content of the fruit pomace treated with strain FJAT-10502 was significantly higher than that of the control group (with an equal amount of sterile water added, but no strain added), while the crude fiber, acid detergent fiber, neutral detergent fiber and lignin content were significantly lower than those of the control group.

[0033] Table 1. Effects of strain FJAT-10502 on the quality of pomace micro-storage fermentation

[0034] In summary, this invention provides a novel Bacillus belesiensis (B. belesiensis) Bacillus velezensis FJAT-10502 strain exhibits amylase activity of 181.39 U / mL, cellulase activity of 28.4 U / mL, and protease activity of 18.48 U / mL. This strain demonstrates antibacterial activity against *Escherichia coli* and *Salmonella*, and shows some tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The fermentation broth of this strain, when used for fruit pomace fermentation, can increase crude protein and total sugar content while reducing the content of crude fiber, acid detergent fiber, neutral detergent fiber, and lignin in fermented fruit pomace, providing an effective strain resource for fruit pomace micro-storage fermented feed.

[0035] Therefore, the present invention has the following advantages: Excellent strain performance: Bacillus berberis FJAT-10502 simultaneously produces high levels of amylase, protease and cellulase, and has strong antibacterial activity and good tolerance to the gastrointestinal environment. It balances enzymatic hydrolysis efficiency and intestinal colonization ability, and is suitable for fruit pomace fermentation and the digestive tract environment of livestock and poultry.

[0036] Fruit pomace fermentation has significant effects: it can significantly increase the crude protein and total sugar content of fruit pomace feed, reduce the content of anti-nutritional factors such as crude fiber, acid detergent fiber, neutral detergent fiber and lignin, and improve the nutritional quality and digestibility of feed.

[0037] 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.

[0038] In summary, the Bacillus berberis FJAT-10502 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.

[0039] 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 belye that produces amylase, protease, and cellulase, characterized in that: The Bacillus belesii is Bacillus belesii ( Bacillus velezensis FJAT-10502 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. 35538.

2. The *Bacillus belyceae* strain producing amylase, protease, and cellulase according to claim 1, characterized in that: The Bacillus berleis ( Bacillus velezensis FJAT-10502 has the ability to produce amylase, protease and cellulase.

3. The *Bacillus belyceae* 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. An application of Bacillus belye as described in claim 1, characterized in that: The Bacillus berleis ( Bacillus velezensis FJAT-10502 is used for the prevention and control of Escherichia coli and Salmonella.

5. A method for culturing Bacillus belye as described in claim 1, characterized in that: The method includes the following steps: (1) Activation of the strain: Use an inoculation loop to inoculate Bacillus belye ( Bacillus velezensis FJAT-10502 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-10502 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.

6. A microbial preparation comprising the Bacillus belye as described in claim 1.

7. The microbial preparation according to claim 6, characterized in that: The microbial preparation is Bacillus bellis (Bacillus). Bacillus velezensis Fermentation broth of FJAT-10502.

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

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

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