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

Abstract

The invention provides bacillus licheniformis capable of producing amylase, protease and cellulase and application of the bacillus licheniformis. The bacillus licheniformis is bacillus licheniformis FJAT-27214 and is preserved in the 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. 35536. The bacillus licheniformis disclosed by the invention can be used for simultaneously producing amylase, protease and cellulase at high yield; the bacillus subtilis has antibacterial activity to escherichia coli and has certain tolerance to artificial gastric juice, artificial intestinal juice and cholate, the survival rate in the artificial gastric juice is 58.11%, the survival rate in the artificial intestinal juice is 82.58%, and the survival rate in the cholate is 28.03%. 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 neutral detergent fiber 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 licheniformis that produces amylase, protease, and cellulase, and its applications. Background Technology

[0002] Citrus and navel orange pomace, a byproduct of fruit processing, is rich in nutrients but has a low utilization rate. Large-scale accumulation leads to resource waste, and the putrefaction and ammonification of pomace cause environmental pollution, making it a critical issue that urgently needs to be addressed. Currently, microbial microstorage is one of the effective ways to utilize pomace resources. After pomace is mixed with other raw materials and microorganisms for microstorage, substances such as fruit acids, pectin, and starch are degraded into small molecules that are easily digested and absorbed by livestock and poultry, as well as a large amount of microbial protein. This effectively balances the amino acid composition, increases the number of probiotics, reduces cellulose and hemicellulose content, and resists nutrient degradation, thereby improving the nutritional value of the pomace and the digestibility and absorption rate of livestock and poultry. Fermented pomace has a positive promoting effect on animal growth and health.

[0003] Bacillus strains are capable of producing spores, exhibit strong environmental tolerance, and secrete various enzymes such as cellulase, amylase, and protease. Examples include Bacillus subtilis and Bacillus cereus. They are characterized by rapid growth, short fermentation cycles, 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 strain resources for the fermentation of probiotic preparations from fruit pomace feed. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a Bacillus licheniformis 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 neutral detergent fiber and ammonia nitrogen, thus providing an effective strain resource for fruit pomace micro-storage fermented feed.

[0005] This invention is implemented as follows: A Bacillus licheniformis that produces amylase, protease, and cellulase, wherein the Bacillus licheniformis is Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 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. 35536.

[0006] Furthermore, the Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 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 licheniformis, wherein the Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 is used for the prevention and control of Escherichia coli.

[0009] Furthermore, a method for culturing the aforementioned Bacillus licheniformis includes the following steps: (1) Activation of strain: Bacillus licheniformis (Bacillus licheniformis) was inoculated with an inoculation loop. Bacillus licheniformis FJAT-27214 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-27214 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 of the aforementioned Bacillus licheniformis.

[0011] Furthermore, the microbial preparation is Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis Fermentation broth of FJAT-27214.

[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 production activity: The Bacillus licheniformis ( Bacillus licheniformis The FJAT-27214 strain exhibits amylase activity of 38.87 U / mL, cellulase activity of 20.03 U / mL, and protease activity of 13.91 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-27214 was significantly higher than that of the control group, while the neutral detergent fiber and ammonia nitrogen content were significantly lower.

[0015] 2. Antibacterial activity: The Bacillus licheniformis exhibits significant antibacterial activity against Escherichia coli. The inhibition zone diameter against Escherichia coli was 17.92 mm, as determined by the double-layer plate method.

[0016] 3. Environmental tolerance: The Bacillus licheniformis strain exhibits a certain degree of tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The survival rate in artificial gastric juice is 58.11%, in artificial intestinal juice it is 82.58%, and in bile salts (0.3% w / v) it is 28.03%, 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-27214 in an embodiment of the present invention.

[0019] Figure 2 This is a 16S rDNA diagram of strain FJAT-27214 in an embodiment of the present invention. Detailed Implementation

[0020] The following will be combined with the appendix Figure 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 licheniformis of the present invention ( Bacillus licheniformis FJAT-27214 is a strain of Bacillus licheniformis isolated from soil in Wudalianchi, Heihe City, Heilongjiang Province, capable of producing amylase, protease, and cellulase. Bacillus licheniformis FJAT-27214 exhibits protease, amylase, and cellulase activity, and shows antibacterial activity against Escherichia coli. It also demonstrates some tolerance to artificial gastric and intestinal fluids and porcine 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 neutral detergent fiber and ammonia nitrogen content in the fermented fruit pomace. It can be applied to micro-fermented fruit pomace feed.

[0022] 1. Isolation and screening of strain FJAT-27214 (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.

[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-27214 (1) Morphological identification The main morphological observation of strain FJAT-27214 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.

[0025] (2) 16S rDNA sequence determination and analysis of strain FJAT-27214 Genomic DNA was extracted from strain FJAT-27214 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-27214 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-27214 with data from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), it was confirmed that strain FJAT-27214 belongs to *Bacillus licheniformis* (…). Bacillus licheniformis The 16S rDNA diagram of strain FJAT-27214 is shown below. Figure 2 As shown.

[0027] Based on the above 16S rRNA sequence determination and analysis, combined with morphological characteristics, strain FJAT-27214 was finally determined to belong to Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis A strain of Bacillus licheniformis was named Bacillus licheniformis. Bacillus licheniformis )FJAT-27214.

[0028] 3. Determination of amylase, protease and cellulase activities in strain FJAT-27214 (1) Activation of the strain: The strain FJAT-27214 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-27214 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 38.87 U / mL. Determine cellulase activity. The 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 20.03 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 13.91 U / mL.

[0029] 4. Determine the antibacterial activity of strain FJAT-27214 against Escherichia coli. (1) Activation of the strain: The strain FJAT-27214 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-27214 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* was inoculated into LB medium and fermented for 24 h in a constant temperature incubator at 30 ℃ 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* was poured in. After the medium solidified, wells (7 mm in diameter) were evenly punched, and approximately 100 μL of FJAT-27214 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*, with an inhibition zone diameter of 17.92 mm.

[0030] 5. Determine the tolerance of strain FJAT-27214 to artificial gastrointestinal fluid and porcine bile salts. 1) Activation of the strain: The strain FJAT-27214 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-27214 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, and dilute them with sterile physiological saline at a serial ratio of 1:10. 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 58.11%, and the survival rate in artificial intestinal fluid is 82.58%.

[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 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 counts were performed to calculate the survival rate. This strain showed some tolerance to bile salts, with a survival rate of 28.03% in bile salts.

[0032] 5. Determining the effect of fermentation broth from strain FJAT-27214 on micro-storage fermentation of fruit pomace. (1) Activation of the strain: The strain FJAT-27214 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-27214 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 from step (2) was added at 5% and mixed evenly with the pomace. 5 kg of the mixture was placed in a large micro-storage bag and sealed. This process was repeated 3 times. The pomace was stored in a dark storage room for 10 days. After that, the bags were 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 neutral detergent fiber content was determined by the standard GB / T20806-2022, and the ammonia nitrogen content was determined by the micro-method using an ammonia nitrogen reagent kit. The results are shown in Table 1. The crude protein and total sugar content of the pomace treated with strain FJAT-27214 were significantly higher than those of the control group (with an equal amount of sterile water added, but no strain added), while the neutral detergent fiber and ammonia nitrogen content were significantly lower than those of the control group.

[0033] Table 1. Effects of strain FJAT-27214 on the quality of pomace micro-storage fermentation

[0034] Therefore, this invention provides a novel Bacillus licheniformis (B. licheniformis) Bacillus licheniformis The strain FJAT-27214 exhibits amylase activity of 38.87 U / mL, cellulase activity of 20.03 U / mL, and protease activity of 13.91 U / mL. This strain shows antibacterial activity against *Escherichia coli* and exhibits some tolerance to artificial gastric and intestinal fluids, as well as porcine bile salts. The survival rate in artificial gastric fluid is 58.11%, in artificial intestinal fluid 82.58%, and in bile salts 28.03%. The fermentation broth of this strain can be used for fruit pomace fermentation, increasing crude protein and total sugar content while reducing neutral detergent fiber and ammonia nitrogen content 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 licheniformis ( Bacillus licheniformis FJAT-27214 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, making it 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 neutral detergent fiber content, and reduce ammonia nitrogen (an indicator of excessive protein decomposition), thereby improving the nutritional quality and digestibility of the feed.

[0037] High application value: On the one hand, it provides efficient strain resources for micro-storage fermented feed of fruit pomace, promoting the industrial application of "turning fruit pomace into 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 licheniformis FJAT-27214 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. 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 invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A Bacillus licheniformis that produces amylase, protease, and cellulase, characterized in that: The Bacillus licheniformis is Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 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. 35536.

2. The Bacillus licheniformis producing amylase, protease, and cellulase according to claim 1, characterized in that: The Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 has the ability to produce amylase, protease and cellulase.

3. The Bacillus licheniformis 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 licheniformis as described in claim 1, characterized in that: The Bacillus licheniformis ( Bacillus licheniformis FJAT-27214 is used for the prevention and control of Escherichia coli.

5. A method for culturing Bacillus licheniformis as described in claim 1, characterized in that: The method includes the following steps: (1) Activation of strain: Bacillus licheniformis (Bacillus licheniformis) was inoculated with an inoculation loop. Bacillus licheniformis FJAT-27214 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-27214 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 licheniformis of claim 1.

7. The microbial preparation according to claim 6, characterized in that: The microbial preparation is Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis Fermentation broth of FJAT-27214.

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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