Multifunctional bacillus licheniformis and derivative product and application thereof

Abstract

The invention provides multifunctional bacillus licheniformis as well as a derivative product and application thereof, and belongs to the technical field of microbial agents. A multifunctional bacillus licheniformis strain is separated and screened from a chicken manure fermentation pile in a chicken farm in Jianyang City in Sichuan province, is named as Bacillus licheniformis NCF and is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the number of the preservation center is CCTCC No.M 20242510, and the preservation time is November 11, 2024. The NCF bacterium has multiple functions, can be added into feed as an additive, helps animals to better digest and absorb nutritional ingredients in the feed, and increases the weight of the animals; organic matters in the excrement can be decomposed through high-temperature fermentation, the fermentation process is accelerated, and the composting fermentation period is shortened; the odor value and the ammonia gas concentration in different livestock and poultry manure / biogas slurry can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a multifunctional Bacillus licheniformis strain and its derivative products and applications. Background Technology

[0002] Probiotics have garnered significant attention in animal husbandry due to their potential to improve animal health and productivity. They can not only improve gut health and boost immunity but also promote feed digestion and absorption, reducing the frequency of antibiotic use. However, the development of new probiotic strains is ongoing.

[0003] At the same time, with the booming development of the livestock industry, a large amount of livestock and poultry manure has become a significant source of environmental pollution. How to effectively treat this manure, develop new microbial fermentation agents, and transform it into valuable resources has become an urgent problem to be solved.

[0004] In addition, foul odors in the air, commonly referred to as odor pollution, mainly originate from gases such as hydrogen sulfide and ammonia produced during the decomposition of organic matter. These odors have multiple sources, including but not limited to garbage dumps, sewage treatment facilities, and livestock farms, which severely impact human living and working environments and physical and mental health. Therefore, developing new and highly effective deodorizing agents has also become a significant technical challenge.

[0005] More importantly, how to develop a microbial agent that can simultaneously possess the above-mentioned multiple functions has become an urgent technical problem to be solved. Summary of the Invention

[0006] This invention aims to solve the aforementioned technical problems, thereby providing a multifunctional Bacillus licheniformis strain and its derivatives and applications. This invention isolated and screened a multifunctional Bacillus licheniformis strain from chicken manure fermentation piles in a chicken farm in Jianyang City, Sichuan Province, and named it *Bacillus licheniformis* NCF. This NCF bacterium can effectively exert its multifunctional effects. It can be added to feed as an additive to help animals better digest and absorb nutrients, increasing animal weight. It can also decompose organic matter in manure through high-temperature fermentation, accelerating the fermentation process and shortening the composting cycle. Furthermore, it can effectively reduce odor levels and ammonia concentrations in various livestock and poultry manure / biogas slurry. This multifunctional strain breaks down the boundaries between fields, achieving comprehensive application from within the animal body to the external environment, representing a forward-looking application model.

[0007] This invention first provides a multifunctional Bacillus licheniformis strain, named Bacillus licheniformis NCF, deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with CCTCC No. M 20242510, and deposited on November 11, 2024.

[0008] Furthermore, the gene sequence of the Bacillus licheniformis is shown in SEQ NO.1.

[0009] The present invention provides a deodorant comprising Bacillus licheniformis as described above.

[0010] The present invention further provides a feed additive comprising Bacillus licheniformis as described above.

[0011] The present invention further provides a fermentation promoter comprising Bacillus licheniformis as described above.

[0012] The present invention also provides the application of Bacillus licheniformis in the preparation of deodorants as described above.

[0013] Specifically, the substances targeted for deodorization include hydrogen sulfide and ammonia.

[0014] The present invention also provides the use of Bacillus licheniformis as described above in the preparation of feed additives.

[0015] The present invention also provides the application of the aforementioned Bacillus licheniformis in the preparation of fermentation promoters.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) Most strains commonly found on the market have single or partial functions. For example, some strains can only deodorize, or can only withstand stomach acid to improve animal growth performance, or can only withstand high temperatures for fermenting feces. This strain, however, integrates multiple functions such as deodorization, acid resistance, and high temperature resistance. This multifunctional integration is rare in existing technologies and provides a brand-new solution for related fields, which can meet the needs of multiple application scenarios at the same time, showing significant innovation. These functions are not simply superimposed, but have potential synergistic effects. For example, while the strain survives in the animal intestine and improves animal growth performance, its metabolites may help improve the properties of animal feces and reduce fecal odor, which is beneficial for subsequent high-temperature fermentation treatment and improves fermentation efficiency. This synergistic effect further enhances the comprehensive application value of the strain.

[0018] (2) Breaking through the boundaries of application fields: Traditional research and application of strains are mostly concentrated in specific fields, such as animal feed additives, waste resource utilization, or environmental management. This strain, however, transcends these fields. It can be used as a probiotic additive in animal husbandry to improve animal growth performance, and it can also be used for manure treatment, transforming manure into valuable resources through fermentation. Furthermore, it can reduce ammonia and odor levels in different environments, achieving comprehensive application from the animal's internal environment to the external environment. This breaks down the boundaries between fields and expands new ideas and directions for the application of strains.

[0019] (3) Optimizing manure fermentation effect: In manure fermentation applications, this strain has excellent heat resistance characteristics, such as improving fermentation efficiency, shortening the fermentation cycle, and reducing energy consumption during high-temperature composting or industrial fermentation. In addition, its fermentation process can reduce the generation of odors, reduce environmental pollution, and achieve harmless and resource-based treatment of manure, which is of great practical significance for solving the environmental pollution problems faced by the livestock industry. Attached Figure Description

[0020] Figure 1 The colony morphology of Bacillus licheniformis NCF is shown in the figures: A) NCF colony plate morphology; B) NCF colony morphology under Gram staining microscopy (×100).

[0021] Figure 2 This is the growth curve of NCF.

[0022] Figure 3 The effect of NCF on the fermentation cycle of sheep manure compost.

[0023] Figure 4 The study aimed to evaluate the deodorization effects of Bacillus licheniformis NCF on different types of livestock and poultry manure / biogas slurry. The results included: A. (a) Odor value of broiler manure, (b) Ammonia value of broiler manure; B. (a) Odor value of pig manure, (b) Ammonia value of pig manure; C. (a) Odor value of pig biogas slurry, (b) Ammonia value of pig biogas slurry; D. (a) Odor value of sheep manure, (b) Ammonia value of sheep manure. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0025] Example 1

[0026] I. Experimental Materials and Methods

[0027] 1. Main materials and instruments

[0028] Source of microbial strains: The deodorizing bacteria used in this invention for isolation and screening were derived from chicken manure fermentation piles at a chicken farm in Jianyang City, Sichuan Province. Source of manure used in the deodorization experiment: The samples used in the deodorization experimental group in this invention included broiler manure, pig manure and biogas slurry, and sheep manure, which were respectively derived from a chicken farm in Jianyang City, Sichuan Province, a pig farm in Qionglai City, Sichuan Province, and a sheep farm in Dayi County, Chengdu City, Sichuan Province.

[0029] NH3 selective medium: sucrose 50.0 g / L, ammonia 10.0 mL / L, KH2PO4 2.0 g / L, MgSO4 0.5 g / L, FeSO4 0.1 g / L, 1 wt% ZnSO4 5.0 mL / L, pH 7.0, sterilized at 121℃ for 20 min. TSB and TSA media were used for the culture of bacterial NCF.

[0030] SPF-grade mice were 14 weeks old and purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0031] Odor detection uses the Japanese Shinei OMX-SRM odor detector, which can detect odor intensity ranging from 0 to 999.

[0032] Ammonia gas detection uses the EDKORS X-1 ammonia gas detector, with a gas detection range of 0-100 PPM.

[0033] 2. Screening of deodorizing bacteria

[0034] (1) Enrichment of NH3 strains

[0035] To prepare NH3, use 100 mL of liquid culture medium, add 2 mL of concentrated ammonia, inoculate with 5 g of fermented chicken manure, and incubate in a shaker at 30℃ and 150 r / min for 72 h. Repeat this enrichment process 5 times.

[0036] (2) Isolation, purification, staining and microscopic examination of NH3 strains

[0037] The enrichment solution was spread onto TSA plates, and single colonies were picked and purified using the streak plate method. Well-grown single colonies were picked from the purified bacterial culture medium and Gram-stained. The morphological characteristics of the bacteria were observed under a light microscope.

[0038] 3. Molecular identification and preservation of bacteria

[0039] After the strain was sent to BGI Genomics Co., Ltd. for 16S rRNA sequence identification, it was sent to the China Center for Type Culture Collection (CCTCC) at Wuhan University for biological preservation. The Bacillus licheniformis was named Bacillus licheniformis NCF, the depositary institution was China Center for Type Culture Collection (CCTCC), the depositary address was Wuhan University, Wuhan, China, the depositary number was CCTCC No. M 20242510, and the deposit date was November 11, 2024.

[0040] 4. Determination of growth curve

[0041] Fresh single colonies were picked from the solid culture medium and inoculated into bacterial bottles containing TSB liquid culture medium. The bottles were then placed in a shaker at 30°C and the rotation speed was 180 rpm / min. The bottles were taken out every 2 hours, and bacterial growth curves were plotted based on the number of bacterial CFUs.

[0042] 5. Acid resistance test

[0043] Adjust the pH of the liquid TSB medium to 2.0 and sterilize at 121℃ for 15 min. Add 100 ml of the logarithmic growth phase bacterial culture to 900 ml of TSB medium at the corresponding pH and incubate for 3 h. Then, serially dilute the culture and spread it on TSA solid medium and incubate at 30℃ for 24 h. The number of viable strains is determined by plate counting the next day.

[0044] 6. Bile salt tolerance test

[0045] Prepare a 0.2 wt% TSB liquid medium and filter it through a 0.22 μm filter into a sterile wide-mouth bottle. Use TSB liquid medium without porcine bile salts as a negative control. Incubate 100 ml of the logarithmic growth phase bacterial culture in 900 ml of TSB medium with porcine bile salt concentration for 2 h, then serially dilute and plate onto TSA medium. Incubate at 30°C for 24 h. The next day, determine the number of viable strains by plate counting.

[0046] 7. High temperature resistance test

[0047] The NCF strain was treated in water baths at 15℃, 35℃, 55℃, 75℃ and 95℃ respectively. After 30 minutes, the bacteria were plated and their growth and survival were observed.

[0048] 8. Animal feeding trials

[0049] Forty mice with no significant difference in initial weight were randomly divided into two groups: a control group (Control) and an experimental group (NCF), with four replicates in each group. The control group received no microbial inoculant, while the experimental group received NCF bacteria (1 × 10⁻⁶ live bacteria per kilogram of feed). 9(cfu). Mice were allowed free access to food. The pre-feeding period was 4 days, and the main trial period was 20 days.

[0050] 9. Composting Cycle Comparison Experiment

[0051] In the harmless treatment area of ​​the sheep farm, compost windrows were built with each treatment group measuring 3m long, 1m high, and 1.5m wide. The experiment consisted of two treatment groups, each replicated three times. A control group without added microbial agents was used, while the experimental group included NCF. The experimental period was 24 days, with the windrow temperature recorded every two days, and a sheep manure composting cycle curve plotted. NCF treatment group: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 11 cfu / m 3 Apply the fungicide evenly and adjust the moisture content to between 60-65%. Reapply every 7 days.

[0052] 10. Deodorization tests in different environments

[0053] (1) Deodorization test of fresh broiler feces

[0054] 30 kg of fresh chicken manure was collected and divided into two treatment groups, each replicated three times. The control group received no microbial inoculant, while the experimental group received NCF bacteria. The experiment lasted for three weeks, with odor levels and ammonia release measured weekly. NCF treatment group: [Information missing - likely a dosage or dosage] per 1×10 [units unclear]. 9 Apply the fungicide evenly at a rate of cfu / kg, and repeat the application once after 7 days.

[0055] (2) Deodorization test of fresh pig manure and biogas slurry

[0056] 60 kg of fresh pig manure was collected, with two treatment groups, each replicated three times. The control group (without added microbial agents) served as the blank control, and the experimental group (with added NCF bacteria) served as the experimental group. 30 L of pig biogas slurry was also collected, with two treatment groups, each replicated three times. The control group (without added microbial agents) served as the blank control, and the experimental group (with added NCF bacteria) served as the experimental group. The experiment lasted for two weeks, with odor levels and ammonia release measured weekly using instruments. NCF treatment group: 1 × 10⁻⁶ mg / L of NCF bacteria was added to the biogas slurry. 9 Apply the fungicide evenly at a rate of cfu / kg, and repeat the application every 7 days.

[0057] (3) Deodorization test of fresh sheep manure

[0058] 60 kg of fresh sheep manure was collected. The experiment consisted of two treatment groups, each replicated three times. The control group received no microbial inoculant, while the experimental group received NCF bacteria. The experiment lasted three weeks, with odor levels and ammonia release measured weekly. NCF treatment group: [Information missing - likely a dosage or dosage]. 9 Apply the fungicide evenly at a rate of cfu / kg, and repeat the application every 7 days.

[0059] II. Experimental Results

[0060] 1. Colony morphology

[0061] The isolated and purified Bacillus licheniformis NCF was cultured on TSA plates at 30°C for 24 hours, and its morphology was as follows. Figure 1 As shown in Figure A, the colonies are yellowish-white, 1-3 mm in diameter, flat, opaque, and have rough edges. Microscopic examination reveals that NCF is a Gram-positive bacterium, arranged in a rod-like shape. Figure 1 As shown in B.

[0062] 2. Gene 16S rRNA sequence

[0063] The gene sequence of the NCF bacterium of the present invention is shown in SEQ NO.1.

[0064] 3. Growth curve

[0065] Fresh NCF culture entered its growth phase after 2 hours of incubation in TSB medium, and reached a plateau phase after 10 hours, with colony counts reaching 1.4 × 10⁻⁶. 9 cfu, such as Figure 2 As shown.

[0066] 4. Results of acid, choline, and high temperature resistance tests

[0067] The colony count of NCF strain grown in a medium with a pH of 7.0 was used as a positive control, and the bacterial count and survival rate of the strain after 3 hours of cultivation in a medium with a pH of 2.0 were compared. The colony count of NCF strain grown in a medium without added bile salts was also used as a positive control, and the bacterial count and survival rate of the strain after 2 hours of cultivation in a medium with a bile salt concentration of 0.2% were compared. Table 1 shows that the survival rate of the strain was 60.27% at pH 2.0 and 28.44% in a medium with a bile salt concentration of 0.2%. Table 2 shows that strain NCF can grow in environments ranging from 15 to 95°C. This indicates that NCF can survive in extreme environments, including high temperatures, low pH, and high concentrations of bile salts.

[0068] Table 1. Effects of low pH and 0.2% bile salts on the survival rate of strain NCF.

[0069]

[0070] Table 2 shows the suitable growth temperature of NCF.

[0071]

[0072] Note: + indicates normal growth.

[0073] 5. Animal growth status

[0074] As can be seen from Table 3, there were no significant differences in the initial weights and average daily feed intakes of the mice in the Control group and the NCF group (P > 0.05). The final weight of the mice in the NCF group was 98.33 ± 1.82 g, which was significantly increased by 5.89 g compared with the control group (0.01 < P < 0.001), and the average daily weight gain was significantly increased by 0.22 g (P < 0.05). The results showed that NCF could improve the growth performance of mice.

[0075] Table 3 Effects of NCF on the growth performance of mice

[0076]

[0077]

[0078] 6. Sheep manure compost fermentation

[0079] From Figure 3 it can be seen that on the 4th day of composting, the temperature of the compost treated with NCF reached ^{\circ}C, while the temperature of the Control group was around 40^{\circ}C. On the 6th day of composting, the temperature of the NCF group reached 70^{\circ}C, while the compost of the Control group only reached 70^{\circ}C on the 10th day. The composting fermentation cycle of the NCF group was about 20 days, while that of the Control group was about 24 days. The results showed that after adding NCF bacteria, the fecal fermentation process was accelerated and the fermentation cycle was shortened.

[0080] 7. Deodorization tests in different environments

[0081] From Figure 4 A, it can be seen that the odor value and ammonia value of fresh broiler manure began to decrease significantly from the 7th day. After 21 days, the odor value and ammonia value of the NCF group were 137 OU / m 3 and 3.7 PPM respectively, while those of the control group were > 999 OU / m 3 and > 100 PPM. The odor reduction rate reached more than 86%, and the ammonia reduction rate reached more than 96.3%. From Figure 4 B, it can be seen that the odor value and ammonia value of fresh pig manure began to decrease significantly from the 7th day. After 14 days, the odor value and ammonia value of the NCF group were 17.7 OU / m 3 and 4.3 PPM respectively, while those of the control group were 161.7 OU / m 3 and 31.7 PPM respectively. The odor value decreased by 89.05%, and the ammonia value decreased by 86.44%. From Figure 4 C, it can be seen that the odor value and ammonia value of pig biogas slurry began to decrease significantly from the 7th day. After 14 days, the odor value and ammonia value of the NCF group were 98.7 OU / m 3 and 21 PPM respectively, while those of the control group were 161.33 OU / m3 And 73 PPM. Odor level decreased by 38.82%, and ammonia level decreased by 71.23%. From Figure 4 According to data from the study, the odor and ammonia levels of fresh sheep feces decreased significantly starting from day 7. After 21 days, the odor and ammonia levels in the NCF group were 250.3 U / m³. 3 The odor level was 0.33 PPM, while the odor and ammonia levels in the control group were 848 U OU / m³. 3 And 8.33 PPM. Odor level decreased by 70.48%, and ammonia level decreased by 96.04%.

Claims

1. A multifunctional Bacillus licheniformis strain, characterized in that, The Bacillus licheniformis species described is named Bacillus licheniformis NCF. It is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with CCTCC No. M 20242510, on November 11, 2024.

2. The Bacillus licheniformis according to claim 1, characterized in that, The gene sequence of the Bacillus licheniformis is shown in SEQ NO.

1.

3. A deodorant, characterized in that, The deodorant contains Bacillus licheniformis as described in claim 1 or 2.

4. A feed additive, characterized in that, The feed additive contains Bacillus licheniformis as described in claim 1 or 2.

5. A fermentation promoter, characterized in that, The fermentation promoter comprises Bacillus licheniformis as described in claim 1 or 2.

6. The use of Bacillus licheniformis as described in claim 1 or 2 in the preparation of deodorants.

7. The application according to claim 6, characterized in that, The deodorizer targets substances including hydrogen sulfide and ammonia.

8. The use of Bacillus licheniformis as described in claim 1 or 2 in the preparation of feed additives.

9. The use of Bacillus licheniformis as described in claim 1 or 2 in the preparation of fermentation promoters.

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