Application of intestinal source bacillus licheniformis in preparation of feed for improving growth performance of piglets or reducing diarrhea

Abstract

The invention discloses application of intestinal source bacillus licheniformis in preparation of feed for improving growth performance of piglets or reducing diarrhea, and is characterized in that the intestinal source bacillus licheniformis is bacillus licheniformis RD508, and the preservation number is GDMCC No: 64606. Test results show that compared with a control group, the average daily gain of the piglets in the group B and the group C is remarkably increased, the feed conversion ratio is remarkably reduced, and the diarrhea rate of the piglets in the test group added with the bacillus licheniformis RD508 is remarkably reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology; in particular to the application of Bacillus licheniformis of intestinal origin in the preparation of feed for improving the growth performance or reducing diarrhea of piglets. BACKGROUND

[0002] With the continuous development of livestock breeding industry, the production performance and intestinal health of piglets after early weaning have been increasingly concerned. Weaning stress often leads to decreased feed intake, growth retardation, reduced feed conversion rate, and easily causes intestinal diseases such as diarrhea, which seriously affects the breeding efficiency. In order to solve this problem, antibiotics are often used as growth-promoting and disease-preventing means in the breeding industry, but long-term abuse of antibiotics can easily lead to increased bacterial drug resistance, drug residues and environmental pollution, which has been strictly limited.

[0003] In recent years, probiotics as a green and safe feed additive show good potential in improving intestinal health, enhancing immunity and improving production performance of animals. Bacillus licheniformis as a common probiotic has the characteristics of high temperature resistance, acid resistance and bile salt resistance, and can colonize in the intestinal tract of animals and produce various enzymes, antibacterial substances and vitamins, thereby regulating intestinal flora balance, enhancing barrier function and promoting nutrient digestion and absorption.

[0004] At present, some studies have shown that Bacillus licheniformis has certain application effect in piglet feeding. For example, some strains can improve the average daily gain of piglets, reduce the feed conversion ratio, reduce the incidence of diarrhea, and improve the intestinal microbial composition. However, different Bacillus licheniformis strains have different effects on animals due to their genetic characteristics, metabolic products and action mechanisms. Therefore, it is of great significance to screen efficient, stable and suitable Bacillus licheniformis strains for piglet physiological characteristics, and to determine the appropriate additive amount, for the development of new probiotic products and the promotion of healthy breeding.

[0005] In the prior art, there is no research report on the systematic application of Bacillus licheniformis RD508 in piglet feed, and the specific effects of Bacillus licheniformis RD508 on piglet growth performance, feed utilization and diarrhea control are not clear. Therefore, it is necessary to carry out in-depth experiments on this strain to provide a basis for its scientific application in piglet production. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the present application aims to explore the effect of bacillus licheniformis RD508 added in feed on the production performance and diarrhea of piglets. 240 piglets of similar weight of "Du × Chang × Da" three-way cross 35-day-old weaned piglets were randomly selected and divided into 4 groups, including a control group, test group A, test group B and test group C, 5 replicates in each group, 12 pigs in each replicate. The control group is fed with the basic diet, group A is added with 200g / t bacillus licheniformis RD508 in the basic diet, group B is added with 400g / t bacillus licheniformis RD508 in the basic diet, and group C is added with 600g / t bacillus licheniformis RD508 in the basic diet. The test results show that: compared with the control group, the average daily gain of piglets in groups B and C is significantly increased, the feed conversion ratio is significantly reduced, and the diarrhea rate of piglets in the test groups with bacillus licheniformis RD508 is significantly reduced.

[0007] One of the purposes of the present application is achieved by adopting the following technical solutions:

[0008] The present application provides the application of enteric bacillus licheniformis in preparing feed for improving the growth performance of piglets or reducing diarrhea, wherein the enteric bacillus licheniformis is bacillus licheniformis RD508 with a preservation number of GDMCC No: 64606.

[0009] Preferably, the feed comprises a basic diet and bacillus licheniformis RD508; the bacillus licheniformis RD508 is live bacteria.

[0010] Preferably, the sequence of the housekeeping gene gyrB of the bacillus licheniformis RD508 is shown in SEQ ID No. 8.

[0011] Preferably, 200-600g of bacillus licheniformis RD508 is added per ton of basic diet.

[0012] Preferably, the basic diet comprises the following components by mass percentage:

[0013] Corn 63%

[0014] Puffed soybean 12%

[0015] Soybean meal 15%

[0016] Fish meal 3.2%

[0017] Soybean oil 2%

[0018] Stone powder 0.9%

[0019] Calcium hydrogen phosphate 1%

[0020] Salt NaCl 0.4%

[0021] L-lysine HCl 0.5%

[0022] Methionine 0.28%

[0023] Choline chloride 0.1%

[0024] Antioxidant 0.02%

[0025] Mold inhibitor 0.1%

[0026] Mineral premix 1%

[0027] Montmorillonite 0.5%.

[0028] The present application has the following beneficial effects, but is not limited to:

[0029] The present application provides an intestinal source bacillus licheniformis strain, which has the effects of temperature tolerance and bile salt tolerance, and greatly improves the yield of lipopeptide active substances metabolized by the strain, especially lichenin; the second purpose of the present application is to provide an application of the intestinal source bacillus licheniformis strain, and the strain and its metabolites can be used for preparing microecological and metagenomic preparations, and can be used as a substitute for feed antibiotics; the third purpose of the present application is to provide a method for breeding an intestinal source bacillus licheniformis strain with high lichenin yield, which comprises the following steps: directionally isolating and screening a temperature-tolerant and bile salt-tolerant bacillus licheniformis strain from healthy piglet feces, combining protoplast ARTP mutagenesis, breeding a functional bacillus licheniformis strain with high product tolerance and lichenin metabolism, and a chromatographic preparation method of lichenin lipopeptide compounds.

[0030] The bacillus licheniformis RD508 can improve the production performance of piglets, increase the average daily weight gain, reduce the feed-meat ratio, and thus improve the feed efficiency. In addition, the bacillus licheniformis RD508 can also reduce the diarrhea rate of piglets, which is beneficial to the intestinal health of piglets. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to clearly show the specific embodiments of the present application and certain detection techniques used in the experiments, the embodiments and the techniques used will be described below, mainly in the form of drawings.

[0032] Figure 1 . Phylogenetic tree diagram of strain BL02.

[0033] Figure 2 . Inhibition zone diagram of supernatant culture solution of different strains in 96-well plates (part).

[0034] Figure 3 . Evolution tree of RD508 strain based on housekeeping gene gyrB gene.

[0035] DETAILED DESCRIPTION

[0036] The application will be further described in detail below in connection with specific examples. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples were carried out according to conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources. In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged.

[0037] Example 1

[0038] Isolation, screening and identification of the starting strain BL02

[0039] 1. Pretreatment of healthy piglet fecal samples and isolation of strains

[0040] Sample pretreatment: In a sterile operation table, 5-10 g of feces of healthy piglets at different time periods was taken and added to a sterile PBS (0.1 mol / L, pH 7.4, purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.) containing 50 mL triangular flask, 5 sterile glass beads were added, and the mixture was shaken at 150 rpm for 30 min, then filtered with gauze, and the filtrate was taken to a new sterile triangular flask. In order to specifically screen strains that can adapt to high temperature, the content-containing triangular flask was further shaken at 150 rpm for 60 min in a 40-45℃ water bath.

[0041] Isolation of strains: After the end of the shaking incubation, the water bath liquid was diluted 10-fold with sterile PBS buffer to 10 -3 The original solution at different dilution ratios was spread on a 0.2% bile salt-tolerant medium, and incubated at 40-50℃ for 1-3 days until obvious single colonies grew. Single strains with obvious Bacillus colony characteristics were selected and purified by restreaking on the isolation medium.

[0042] The above bile salt-tolerant medium is an isolation medium with additional 0.25% pig bile salt, i.e., tryptone 12.0 g, yeast extract 8.0 g, potassium dihydrogen phosphate 2.0 g, magnesium sulfate 0.1 g, pig bile salt 2.5 g, agar powder 20.0 g, deionized water 1000 mL, pH 7.0±0.2, sterilized at 121℃ for 20 min.

[0043] Bacteriostatic confrontation preliminary screening: Bacillus licheniformis is a facultative anaerobic bacteria, its metabolic mechanism can utilize oxygen for aerobic respiration, and can also obtain energy and metabolize bacteriostatic substances (such as lichenin) under anaerobic conditions through fermentation or nitrate respiration. However, some Bacillus are strict aerobes, such as Bacillus subtilis, which is a strict aerobe, and its growth and spore germination are highly dependent on the presence of free oxygen. In an anaerobic environment, its metabolic activity is inhibited, and the vegetative cells can only partially survive and cannot complete reproduction or metabolic activity. Therefore, further use Clostridium perfringens ATCC13124 as the indicator strain, prepare the indicator strain suspension by anaerobic confrontation culture, then prepare the confrontation plate containing the indicator strain suspension, and then inoculate the single colonies purified by streaking on the confrontation plate, and culture at 42°C in the anaerobic box. According to the difference in the ability of different strains to reproduce and metabolize under anaerobic conditions, pick the strains that can grow well under 40°C, anaerobic conditions, and can form a confrontation bacteriostatic circle, and make a preliminary ranking of the ratio of the diameter of the bacteriostatic circle to the diameter of the (B) colony (C) of the strains, and the larger the ratio, the stronger the bacteriostatic activity.

[0044] The above-mentioned indicator strain suspension refers to inoculating the indicator strain ATCC13124 into a 150 mL brain heart infusion broth medium, and incubating at 30°C in an anaerobic box for 24 h.

[0045] The above-mentioned confrontation plate refers to using a modified Clostridium perfringens culture medium (CW culture medium), which contains 10.0 g / L proteose peptone, 52.0 g / L brain heart infusion broth medium (purchased from Guangdong Huan Kai Microbial Science and Technology Co., Ltd.), 5.0 g / L glucose, 20.0 g / L agar powder, and 1000 mL deionized water, and the pH is 7.2. Take 100 μL of the test bacterial culture solution in the anaerobic operation room and evenly coat it on the surface of the CW culture medium. After fully absorbing, place it in a 4°C refrigerator for 30 min, which is the confrontation plate.

[0046] The above-mentioned confrontation culture refers to inoculating the isolated and purified strain on the surface of the confrontation plate, and placing the plate containing the indicator strain and the isolated strain in an anaerobic culture box at 42°C overnight, observing and measuring the bacteriostatic ratio.

[0047] Table 1. Bacteriostatic activity of different strains in confrontation - bacteriostatic ratio (part)

[0048] Strain No. Bacteriostasis ratio B / C 01 1.2 02 2.0 09 1.5 15 1.3 26 1.5 28 1.6 40 1.6 54 1.3 95 1.6 102 1.4

[0049] Note: If there is no bacteriostatic activity, the bacteriostatic ratio H / C is 1.0.

[0050] The results show that 120 strains of bacteria were selected for confrontation culture, most of which had a bacteriostatic ratio of 1.0, and 10 strains had obvious bacteriostatic circles. Among them, the bacteriostatic ratio of strain No. 02 was the best.

[0051] Shaking flask fermentation re-screening: 10 strains with high H / C ratio were transferred to test tube slant (containing LB medium, 18x180 mm test tube) and cultured at 42°C for 24 h, then 5 mL sterile normal saline was added for elution to prepare bacterial suspension (bacterial number was 1x10 9 CFU / mL), finally the bacterial suspension was transferred to a shaking flask for fermentation culture, wherein the shaking flask fermentation conditions were as follows: 50 mL of shaking flask fermentation medium was added into a 250 mL triangular flask, and the flask was cultured at 42°C and 220 rpm for 48 h.

[0052] The above fermentation medium contained (g / L): 50.0 of sugarcane molasses, 3.0 of sucrose, 60.0 of corn syrup, 2.0 of ammonium sulfate, 2.5 of potassium dihydrogen phosphate, 0.5 of magnesium sulfate, 1.0 of sodium chloride, pH 7.0±0.5, sterilized at 121°C for 20 min.

[0053] After fermentation, the biomass (wet weight of the strain) was calculated and the supernatant was collected by centrifugation at 12 000 rpm for 5 min, and the agar diffusion method and HPLC method were used to detect the antibacterial performance of the fermentation supernatant on the indicator strain ATCC13124 and the content of lichenysin cyclic lipopeptide, respectively.

[0054] The above agar diffusion method refers to that 200 μL of the indicator strain culture solution was uniformly coated on the surface of the CW medium in an anaerobic operating room, punched (6.0 mm), 50 μL of the supernatant was added, quickly placed in an anaerobic culture box, and placed in a 4°C refrigerator for 2 h, and then the sample was fully absorbed into the medium, and then the anaerobic culture box containing the glass flat plate was placed in a 42°C incubator overnight, and the inhibition zone was observed and measured.

[0055] The above HPLC method for detecting the content of lichenysin lipopeptide is briefly described as follows:

[0056] ① Sample pretreatment: 8 mL of fermentation broth was centrifuged at 12 000 rpm for 8 min to remove the bacteria; the supernatant was adjusted to pH 2.0 with 6 mol / L hydrochloric acid and statically placed at 4°C overnight; then the supernatant was removed by centrifugation at 12 000 rpm for 8 min, and the precipitate was vacuum dried to obtain a crude lipopeptide product.

[0057] The crude product was resuspended with 1.6 mL of methanol, extracted for 1-2 h, and finally centrifuged at 12 000 rpm for 2 min, and the supernatant was filtered through a 0.45 μm organic filter membrane for HPLC detection.

[0058] ② HPLC detection method: Shimadzu LC2030C was used for detection of the lichenysin lipopeptide. The chromatographic column was C18 (4.6 μm x 250 mm, 5 μm);

[0059] Mobile phase A: 0.1% trifluoroacetic acid (TFA) in water, 1 ml of TFA was taken and made up to 1000 ml with ultrapure water, and filtered through a 0.45 μm filter membrane.

[0060] Mobile phase B: 0.1% trifluoroacetic acid (TFA) in acetonitrile,

[0061] Injection volume 20 μL, column temperature 40ºC, flow rate 1.0 mL / min, UV detection wavelength 215 nm;

[0062] The mobile phase gradient was set as follows:

[0063] Time / min Mobile phase A Mobile phase B 0~5 90% 10% 5~35 90%→50% 10%→50% 35~45 50% 50% 45~50 50%→90% 50%→10%

[0064] Note: Lichenysin is a multi-component homologous series. Self-prepared lichenysin samples were used as controls. Its structure is similar to surfactin (Surfactin), which is also a cyclic lipopeptide. Surfactin (CAS: 24730-31-2) can also be used as a basic control.

[0065] Table 2. Inhibition zone diameter and lichenysin content of supernatant of different strains

[0066] Strain No. Biomass (wet weight of bacteria / 100 mL) Inhibition zone diameter (mm) Crude lipopeptide (mg / 100 mL) Lichenins (mg / 100 mL) 01 10.5 6.3 105.2 12.3 02 13.6 13.5 212.2 33.5 09 10.3 8.3 115.6 10.5 15 10.6 7.5 108.4 12.6 26 11.5 8.2 121.1 15.3 28 10.5 9.8 133.3 13.1 40 10.1 8.6 123.0 12.3 54 10.8 11.6 155.5 14.1 95 11.4 10.2 138.6 11.1 102 11.1 11.0 142.1 12.8

[0067] Note: Inhibition zone diameter (mm) = Inhibition zone outer diameter (mm) - Hole diameter (6.0 mm)

[0068] The 02 strain with the best biomass, the largest agar inhibition zone diameter, and the highest content of lichenysin cyclic lipopeptide was selected for preservation.

[0069] 2. Molecular identification of the strain

[0070] The colony of the 02 strain separated and screened above was used as a template, and the 16S rDNA partial sequence thereof was amplified using a universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') sequence as shown in SEQ ID No. 1 / 1492R (5'-TACGGCTACCTTGTTACGACTT-3') sequence as shown in SEQ ID No. 2. The reaction system was as follows: Pre-mix Ex Taq (Takara Company) 12.5 μL, 27F primer (10 μmol / L) 1 μL, 1492R primer (10 μmol / L) 1 μL, LA18 genomic DNA template 0.5 μL, ultrapure water 10 μL. The PCR conditions were as follows: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 40 s, 57 ℃ annealing for 40 s, 72 ℃ extension for 1 min 30 s, 30 cycles, finally 72 ℃ extension for 10 min, and preservation at 4 ℃. After obtaining the specific amplification product, the PCR recovery kit (purchased from Sheng Wu Sheng Gong (Shanghai) Company Limited) was used to recover the target fragment, and the sample was sent to Sheng Wu Sheng Gong (Shanghai) Company Limited for sequencing. The sequencing results were subjected to Blast analysis and comparison in the NCBI nucleic acid library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and a phylogenetic tree was constructed.

[0071] >BL02_16S rDNA, sequence as shown in SEQ ID No. 3

[0072]

[0073] Results as Figure 1 The results show that the closest relative of the strain 02 is Bacillaceae, and the 16S rDNA sequence of the homologous strain with high similarity is selected for phylogenetic analysis. The phylogenetic tree is constructed by N-J method (Neighbor-Joining method) in MEGA4.0. The strain 02 is closest to Bacillus licheniformis, and is far from Hyndrikkia (formerly Weizmannia). Moreover, it is clustered with Bacillus subtilis and Bacillus spizizenii, indicating that the strain 02 and Bacillus licheniformis are closely related species in evolution. Combined with the morphological characteristics of the colony and the bacterial body, it can be confirmed that the strain 02 is Bacillus licheniformis, which is renumbered as BL02, and is referred to as Bacillus licheniformis BL02.

[0074] This is the starting strain Bacillus licheniformis BL02 isolated and obtained.

[0075] Example 2:

[0076] Screening and preservation of excellent mutant strain Bacillus licheniformis RD508

[0077] 1. Preparation of protoplasts

[0078] 1. Preparation of protoplasts

[0079] (1) Activation of the starting strain

[0080] The strain BL02 isolated above is taken as the starting strain, which is activated by LB slant culture medium for two times.

[0081] (2) Preparation of the starting strain suspension

[0082] The strain suspension is prepared by washing the above activated strain on the slope with sterile normal saline, and fully vortexing and mixing. The bacterial cells are collected by centrifugation at 12000 r / min at 4℃ for 5 min, and washed twice with sterile normal saline, and then washed twice with high osmotic solution SMM. The supernatant is removed by centrifugation, and the bacterial cells are finally resuspended in SMM solution to obtain the bacterial cell suspension, and the number of bacterial colonies is adjusted to about 1.0×10 8 CFU / mL (SMM high osmotic solution consists of: sucrose 0.5 mol / L, MgCl2·6H2O 0.02 mol / L, maleic acid 0.02 mol / L, prepared with distilled water, pH 7.0, sterilized at 121℃ for 22 min, and stored at 4℃ for standby).

[0083] (3) Preparation of protoplasts

[0084] The enzyme digestion of cells is carried out in the form of combined enzymes. Specifically, the combined digestion is carried out by using lysozyme and snail enzyme. The above bacterial suspension is added with a certain amount of filtered and sterilized enzyme solution (SMM solution prepared), to prepare a bacterial suspension containing enzymes, and placed in a shaking bed for reaction at 50-100 r / min. The formation of protoplasts is observed by microscope every 5 min. When about 90% of the cells are converted into protoplasts, the cells are collected by centrifugation at 2000-3000 rpm, washed and centrifuged twice with SMM, and resuspended with an appropriate amount of SMM. The use concentration of lysozyme (purchased from Shengong Bioengineering (Shanghai) Co., Ltd.) is 0.05%-0.5%, and the optimal concentration is 0.05%-0.15%. The use concentration of snail enzyme (purchased from Shengong Bioengineering (Shanghai) Co., Ltd.) is 0.05%-0.5%, and the optimal concentration is 0.05%-0.15%. The enzyme digestion temperature ranges from 24-35℃, and the optimal enzyme digestion temperature is 26-30℃. The enzyme digestion time is 10-90 min, and the optimal enzyme digestion time is 10-30 min. Under the optimized conditions, the final protoplast concentration reaches more than 0.9 x 10 8 protoplasts / mL, the protoplast formation rate reaches 95.0% or more, and the regeneration rate of the protoplasts is also the best, reaching 24.3%.

[0085] 2. ARTP mutagenesis of protoplasts

[0086] (1) Determination of ARTP mutagenesis parameters

[0087] 10-20 μL of the protoplast suspension is uniformly coated on the upper surface of the metal slide, dried, and then transferred to the loading table with a pair of tweezers. High-purity helium gas is used as the working gas of the plasma, the power of the power source is set to 80 W, the irradiation distance is 4 mm, the temperature of the plasma is 26-30℃, the gas flow is 10 L / min, the bacterial material slide is treated, different treatment groups are set, and the treatment time of each group is 0 (control), 10, 20, 30, 40, 50, and 60 s, respectively, and each group is set with three repeats. After treatment, the slide is transferred to an EP tube containing SMM solution, shaken to elute to form a new protoplast suspension, coated on a regeneration plate, and placed in a 42℃ incubator for culture until single colonies grow, and counted. The mortality rate is calculated as follows:

[0088] Mortality rate% = (number of untreated bacteria - number of treated bacteria) / number of untreated bacteria x 100%

[0089] The results showed that, compared with the untreated strain (treatment time 0 s), the lethality rates of BL02 protoplast suspension after ARTP mutagenesis were 60.6% (treatment time 10 s), 80.5% (treatment time 20 s), and 90.9% (treatment time 30 s), respectively. Furthermore, the lethality rate almost reached 100% after treatment of 40 s or more. Therefore, to ensure sufficient cell survival while preventing mutagenesis damage, a lethality rate of approximately 90% was selected for ARTP treatment. Thus, the optimal treatment time for ARTP on the BL02 strain was determined to be 30 s.

[0090] (3) Screening of mutagenic strains

[0091] Primary screening using 96-well plates: From the ARTP-mutated regenerated plates, select 280 colonies with larger, lighter blue to lighter red circles (approximately 380 colonies in total) and transfer them to 96-well microplates containing microplate fermentation medium. Incubate at 37-42℃ and 250 rpm for 72 h with shaking. After incubation, centrifuge the 96-well plates at 6000 rpm for 15 min, and use the supernatant to detect their antibacterial activity using the agar diffusion method.

[0092] The microplate culture medium composition includes: glucose 10 g / L, corn steep liquor 10 g / L, KH2PO4 5 g / L, Na2HPO4 5.0 g / L, (NH4)2HPO4 8.0 g / L, FeSO4·4H2O 0.01 g / L, MgSO4·7H2O 0.21 g / L, pH 7.0±0.5, sterilized at 121℃ for 20 min, ready for use.

[0093] like Figure 2 As shown, using the starting strain BL02 as a control, 12 strains with an inhibition zone increase of more than 10% and good clarity of the transparent zone were selected for further shake-flask fermentation and screening. The fermentation medium and culture conditions were the same as described above.

[0094] Table 3. Shake-flask fermentation secondary screening of different mutant strains

[0095] Strain No. Crude lipopeptide (mg / 100 mL) Lichenins (mg / 100 mL) BL02 215.3 32.5 02 315.6 30.5 06 515.5 90.6 21 501.0 45.6 36 369.0 32.0 49 355.6 31.5 65 358.4 36.6 76 321.1 35.1 101 333.3 41.1 124 323.3 33.2 144 345.5 34.1 215 338.6 40.1 242 342.1 40.8

[0096] The results, as shown in Table 3, indicate that the crude lipopeptide yield of strain 5-06 was 139.4% higher than that of the original strain, and significantly higher than that of other strains. Simultaneously, its total lichenin content and its proportion of total crude lipopeptides were also higher than those of the original strain. Strain 06 was preserved by slant culture and glycerol culture, and designated RD508.

[0097] 3. Identification of RD508 strain based on the housekeeping gene gyrB

[0098] Firstly, the genomic DNA of RD508 was extracted by using bacterial genomic DNA extraction kit (purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.), and the partial sequence of the gyrB gene of RD508 was amplified by using the housekeeping gene gyrB primer. The sequence of the PCR amplification primer is as follows:

[0099] gyrB-F: the sequence is shown as SEQ ID No. 4

[0100] 5-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA

[0101] gyrB-R: the sequence is shown as SEQ ID No. 5

[0102] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'

[0103] The reaction system is as follows:

[0104] Pre-mix Ex Taq (Takara Company) 12.5 μL, gyrB-F / R primer 1 μL each, genomic template 0.5 μL, ultrapure water 10 μL.

[0105] PCR conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57℃ annealing for 40 s, 72℃ extension for 90 s, 30 cycles, finally 72℃ extension for 10 min, and storage at 4℃. After obtaining the specific amplification product, the PCR recovery kit (purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.) was used to recover the target fragment, and the sample was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing.

[0106] The sequence primer is as follows:

[0107] S-gyrB-F: 5-GAAGTCATCATGACCGTTCTGCA-3, shown as SEQ ID No. 6

[0108] S-gyrB-R: 5-AGCAGGGTACGGATGTGCGAGCC-3, shown as SEQ ID No. 7

[0109] The sequencing results of the RD508 strain are as follows:

[0110] >RD508_gyrB, shown as SEQ ID No. 8

[0111]

[0112] The sequencing results were subjected to Blast analysis and alignment in the NCBI nucleotide library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the results showed that the closest relative of the RD508 strain was Bacillus in the family Bacillaceae, and the homologous strain sequence with high similarity was selected for phylogenetic analysis, and a phylogenetic evolution tree was constructed using the N-J method (Neighbor-Joining method) in MEGA4.0 (as shown in FIG. 1). Figure 3

[0113] The RD508 strain was clustered with the strains of Bacillus, and the evolutionary distance with some B. licheniformis branches was the closest, and the similarity was as high as 99.91%, indicating that RD508 and B. licheniformis were closely related species in evolution. Combined with the colony and cell morphological characteristics, it can be determined that RD508 is B. licheniformis, numbered as Bacillus licheniformis RD508, and referred to as B. licheniformis RD508.

[0114] 4. Verification of genetic stability

[0115] The excellent mutant strain B. licheniformis RD508 obtained above was subcultured every 3 days, and subcultured for 10 times. The fermentation broth was fermented every other generation, and the contents of crude lipopeptide and lichenin were measured to investigate the stability of the strain during subculture. The results showed that the contents of crude lipopeptide and lichenin in the fermentation broth of B. licheniformis RD508 did not change significantly during subculture, and the strain had good genetic stability.

[0116] The genetically stable excellent mutant strain B. licheniformis RD508 was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on May 11, 2024 (address: No. 59, Building 5, Guangdong Institute of Microbiology, 100, Martyrs' Road, Guangzhou, Guangdong, China, postcode: 510075), and the preservation number of the RD508 strain was GDMCC No: 64606.

[0117] Example 3

[0118] Fermentation application of B. licheniformis RD508

[0119] 1. Seed culture

[0120] (1) Strain activation

[0121] ​First, inoculate RD508 glycerol bacteria to LB medium slope (18 x 180 mm), incubate in the incubator at 42°C for 24 h, which is F1 generation. Then, wash the slope strain with 5 mL sterile normal saline to prepare the bacterial suspension, transfer to the flask culture medium containing LB medium, incubate in the incubator at 42°C for 12 h, which is F2 generation activated strain.

[0122] (2) Seed culture in shake flask

[0123] The activated flask seed is added with 20 mL sterile water to prepare a bacterial suspension, which is transferred to a 5 L shake flask containing 2000 mL nutrient broth medium, and incubated in a constant temperature shaking incubator at 220 r / min, 42°C for 16 h, to further improve the total number of colonies and strengthen the strain activity.

[0124] (3) Seed expansion culture

[0125] Seed expansion culture is carried out in seed tank using seed shake flask, and the seed shake flask is transferred to the seed tank containing seed culture medium at 5% inoculation amount, and the process is controlled as follows: tank pressure: 0.06 Mpa, tank temperature: 37-42°C, initial stirring: 250 r / min, by controlling stirring, the dissolved oxygen (DO) in the tank is adjusted to 20-50%, and the culture is aerated for 12 h, and the seed transfer standard is colony number > 2 x 10 9 CFU / mL.

[0126] The above seed culture medium includes the following components (g / L): glucose 25, yeast extract 20, ammonium sulfate 5.0, ammonium dihydrogen phosphate 2.0, ferrous sulfate 0.05, magnesium sulfate 0.02, zinc sulfate 0.01, and the pH value is adjusted to 7.0 ± 0.5. 121°C high pressure sterilization for 20 min.

[0127] 2. Fermentation production of lichenysin cyclic lipopeptide

[0128] The seed culture solution is transferred to the fermentation tank containing the fermentation medium, and the transfer amount is 20%. In the initial stage of fermentation culture, the aeration amount of the fermentation tank is controlled at 150-200 mL / min, the initial stirring is maintained at 250 r / min, the temperature is controlled at 37-42°C, the initial total sugar concentration is controlled at 100-160 g / L, and the dissolved oxygen (DO) is controlled at 20-50% by stirring. The whole process is controlled by controlling the flow of 20% ammonia water to control the pH value of the fermentation broth to 6.0-6.5, and by supplementing molasses, the total sugar content in the fermentation tank is maintained at 10-20 g / L. Fermentation for 48 h, stop fermentation.

[0129] The fermentation medium composition includes the following components (g / L): molasses 100, sucrose 20, yeast extract 10, corn steep liquor 50, ammonium sulfate 20, potassium dihydrogen phosphate 25, sodium hydrogen phosphate 15, ferrous sulfate 0.05, magnesium sulfate 0.02, zinc sulfate 0.01, folic acid 10 mg / L, biotin 2 mg / L, and deionized water to make up the balance to 1000 L. The medium is autoclaved at 121°C for 20 min.

[0130] 3. Fermentation of the control strain

[0131] The starting strain BL02 is used instead of RD508 to perform fermentation under the same conditions, and the same method is used to detect the production of orsellin.

[0132] 4. Comparison of the content of orsellin cyclic lipopeptide

[0133] The results show that the content of orsellin in the fermentation broth of RD508 strain reaches 1235.3 mg / L, while the orsellin production of the starting strain BL02 is only 355.3 mg / L, which reflects a very good ability to synthesize and accumulate orsellin.

[0134] 5. Preparation of high orsellin cyclic lipopeptide active product postbiotic preparation

[0135] After fermentation, the above fermentation broth is adjusted to pH 2.0-2.5 with 6 mol / L hydrochloric acid, acidified for 3-6 h, then adjusted to pH 5.5-6.5 with sodium hydroxide, and then heated to 80-100°C for 30 min to completely inactivate the bacterial cells. Then, it is evaporated and concentrated to a dry matter content of 35-45%, and then spray dried to obtain the high orsellin cyclic lipopeptide active product postbiotic preparation.

[0136] Example 4:

[0137] Chromatographic preparation of orsellin cyclic lipopeptide

[0138] High-pressure liquid chromatography is used to purify and prepare orsellin.

[0139] First, the fermentation broth is pretreated: centrifugation to obtain the supernatant, then adjust the pH to 2.0 with 6 mol / L hydrochloric acid, and stand overnight at 4°C; then centrifuge for 8 min under the same conditions, remove the supernatant, and vacuum dry the precipitate to obtain the crude lipopeptide. The subsequent two-step hydrophobic purification is carried out as follows:

[0140] 1. Primary purification and preparation of orsellin (crude purification)

[0141] Chromatography column: 50*250mm DAC50 chromatography column (purchased from Jiangsu Hanbang Technology Co., Ltd.)

[0142] Filler: kromasil Phenyl 10μm (purchased from Guangzhou Rui Ba Instrument Technology Co., Ltd.)

[0143] Detection wavelength: 215nm

[0144] Flow rate: 60mL / min

[0145] Take 0.4g of the crude product, dissolve it in 18mL of DMSO, filter it through a 0.45μm filter membrane, and inject it.

[0146] Mobile phase A: acetonitrile

[0147] Mobile phase B: 30mM sodium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid)

[0148] Elution method:

[0149] Time (min) A% B% 0 20 80 15 20 80 75 50 50 80 60 40 80 60 40

[0150] Collect the HPLC detection solution and combine the fractions containing the target product.

[0151] Thus, repeat the preparation 5 times.

[0152] 2. Secondary purification of lichenysin (refinement)

[0153] Chromatographic column: 50*250mm DAC50 chromatographic column, filler: kromasil C8 10μm

[0154] Detection wavelength: 215nm

[0155] Flow rate: 60mL / min

[0156] Take 1 / 3 of the target product fraction obtained from the first purification, dilute it with pure water to double the volume, and then inject it using a pump

[0157] Mobile phase A: acetonitrile

[0158] Mobile phase B: 0.1% acetic acid aqueous solution

[0159] Elution method:

[0160] Time (min) A% B% 0 27 73 10 27 73 80 47 53 85 60 40 100 60 40

[0161] Collect the HPLC detection solution and combine the fractions containing the target product with a purity greater than 95%. For fractions with a purity less than 95%, repeat the purification using this method until the purity is greater than 95%.

[0162] 3. Concentration and drying:

[0163] Combine the target product fractions with a purity greater than 95% and remove the acetonitrile by rotary evaporation under reduced pressure. Then freeze-dry to obtain the refined lichenysin product.

[0164] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

[0165] Example 5

[0166] Effects of Bacillus licheniformis RD508 on growth performance and diarrhea of piglets

[0167] In this experiment, 240 weaned piglets at 35 days of age were used as experimental subjects, and Bacillus licheniformis RD508 was added to the piglet diet to explore the effects of Bacillus licheniformis RD508 on the growth performance and diarrhea of piglets and provide a scientific basis for piglet feeding.

[0168] 1 Test time and place

[0169] Test time: February 25, 2024 to March 28, 2024, pre-test period 5 days, test period 28 days.

[0170] Test site: A pig farm in Huadu District, Guangzhou City, Guangdong Province.

[0171] 2 Materials and methods

[0172] 2.1 Test materials

[0173] Bacillus licheniformis RD508 used in the experiment was provided by Guangdong Rongda Biological Co., Ltd.; piglets and feed were provided by a pig farm in Guangzhou City, Guangdong Province.

[0174] 2. 2 Test animals and grouping

[0175] Test animals: 240 three-way crossbred piglets of "Du × Chang × Da" at 35 days of age with uniform body condition, good body condition, and similar body weight were selected, including 60 in the control group and a total of 180 in the test group.

[0176] 2.3 Test diet and test design

[0177] 2.3.1 Test diet

[0178] The feed used in the experiment was mainly based on corn and soybean meal, and the feed formula was formulated according to GB / T 39235—2020 Pig Nutrient Requirements. The composition and nutritional level of the basic feed are shown in Table 5.1.

[0179] Table 5.1 Composition and nutritional level of the basic diet (dry basis)

[0180] Table 1 Composition and nutrient level of the basal diet (air-drybasis)

[0181] Ingredients Content % Nutrient level 2） Nutrient level Content % Content % 63 Corn 14.21 Digestible energy DE / (MJ / kg) 12 Extruded soybean 17.82 Crude protein CP 15 Soybean meal 0.76 Calcium Ca 3.2 Fish meal 0.36 Available phosphorus AP 2 Soybean oil 0.15 Sodium Na 0.9 Limestone 0.35 Chlorine Cl 1 Calcium hydrogen phosphate CaHPO4 0.4 Sodium chloride NaCl 0.5 L-lysine hydrochloride L-lysine·HCl 0.28 Methionine 0.1 Choline chloride 0.02 Antioxidant 0.1 Mineral premix 1) ]] 1 Mildew-proof agent 0.5 Smectite 100

[0182] Note: 1. The premix provided per kg of the diet was as follows: Fe (FeSO4) 80 mg, Zn (ZnSO4) 80 mg, Cu (CuSO4) 5 mg, Mn (MnSO4) 3 mg, I (CaI2) 0.14 mg, VA 2 250 IU, VD3 220 IU, VE 16 IU, VK3 0.5 mg, VC 20 mg, VB1 2 mg, VB2 4.5 mg, VB6 7 mg, VB12 0.03 mg, biotin 0.2 mg, folic acid 0.3 mg, nicotinic acid 30 mg, and pantothenic acid 25 mg. 2) Crude protein, calcium, total phosphorus, and selenium are measured values; other nutrient levels are calculated values.

[0183] 2.3.2 Experimental Design

[0184] The experimental design groups are shown in Table 5.2.

[0185] Table 5.2 Experimental Design

[0186] Total Group Diet composition Control group Basic diet Test group A Basic diet + 200 g / t Bacillus licheniformis RD508 Test group B Basic diet + 400 g / t Bacillus licheniformis RD508 Test group C

[0187] 2.4 Feeding and Management

[0188] The feeding trial was conducted at a large-scale pig farm. The temperature in the nursery pigsty was controlled at 25-28℃ and the relative humidity was 55%-65%. The piglets had free access to water and feed. The daily condition of the piglets was carefully observed, and the occurrence of diarrhea was recorded. During the trial, the farm was cleaned and the manure was removed every day. Disinfection, disease prevention, and deworming were carried out in a routine manner.

[0189] 2.5 Measurement Indicators and Methods

[0190] 2.5.1 Growth performance

[0191] The weight was measured at the beginning (1 d) and the end (28 d) of the formal period, respectively, with repetition as the unit. The animals were fasted and deprived of water for 12 h before weighing; the feed intake and residual feed were accurately recorded every day during the experiment, and the average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (F / G) were calculated.

[0192] The calculation formula is as follows:

[0193] ADG = (final weight - initial weight) / test days

[0194] ADFI = (feed intake - residual feed) / test days

[0195] F / G = ADFI / ADG

[0196] 2.5.2 Diarrhea rate and diarrhea index

[0197] During the experiment, fecal scores were recorded at 10:00 and 16:00 every day according to the appearance of piglet feces, with 1 point for hard, 2 points for slightly soft, 3 points for soft, 4 points for semi-liquid, and 5 points for water or mucus; a fecal score > 2 was considered diarrhea, and the number of diarrhea episodes was recorded to calculate the diarrhea rate and diarrhea index.

[0198] The calculation formula is as follows:

[0199] Diarrhea rate (%) = total number of first diarrhea / (total number of test piglets x total test days) x 100%

[0200] Diarrhea index = sum of fecal scores / number of test piglets.

[0201] 2.6 Data processing

[0202] The experimental data were processed using Excel, and single-factor variance analysis was performed using SPSS 25.0 statistical software, with P < 0.05 indicating significant differences, and multiple comparisons were made using the Duncan method. The results are expressed as "mean ± standard error".

[0203] 3 Results

[0204] 3.1 Effect of Bacillus licheniformis RD508 on the growth performance of piglets

[0205] Table 5.3 Effect of Bacillus licheniformis RD508 on the growth performance of piglets

[0206] Basic diet + 600 g / t Bacillus licheniformis RD508 Index Control group Test group A Test group B Test group C 10.08±1.04 10.23±1.11 9.89±1.54 10.11±1.24 Initial weight / kg 22.57 ± 2.21 b ]] 23.11 ± 2.52 a ]] 23.92 ± 2.41 a ]] 24.27 ± 2.76 a ]] Final weight / kg 934.15±15.42 944.32±16.22 938.64±15.01 957.56±17.02 Average daily feed intake ADFI / g 446.54 ± 8.54 b ]] 460.81 ± 7.92 b ]] 500.37 ± 8.61 a ]] 505.64 ± 9.11 a ]] Average daily gain ADG / g 2.10 ± 0.04 b ]] 2.06 ± 0.01 ab ]] 1.88 ± 0.07 a ]] 1.89 ± 0.05 a ]]

[0207] Note: The same row data with different letters are significantly different (P < 0.05); no letters or the same letters indicate no significant difference, and the same below.

[0208] From Table 5.3, it can be seen that there was no significant difference in initial weight and average daily feed intake of piglets among different groups (P > 0.05). Compared with the control group, the final weight of piglets in the test groups A, B and C was significantly increased (P < 0.05), and the average daily gain of piglets in the test groups B and C was significantly improved (P < 0.05). In addition, the feed conversion ratio of piglets in the test groups B and C was significantly lower than that in the control group (P < 0.05), and the feed conversion rate was improved.

[0209] 3.2 Effect of Bacillus licheniformis RD508 on diarrhea of piglets

[0210] Table 5.4 Effect of Bacillus licheniformis RD508 on diarrhea rate of piglets

[0211] Feed conversion ratio F / G Index Control group Test group A Test group B Test group C 2.84 ± 0.51 a ]] 2.31 ± 0.43 b ]] [2.12 ± 0.42 bc ]] 2.01 ± 0.28 c ]] Diarrhea rate / % Diarrhea index 7.01 ± 0.94 a ]] 6.54 ± 0.78 ab ]] 6.10 ± 0.85 b ]] 5.92 ± 0.63 b ]]

[0212] Note: The same row with different letters indicates significant difference (P < 0.05).

[0213] From Table 5.4, it can be seen that compared with the control group, the diarrhea rate of piglets in the test groups supplemented with Bacillus licheniformis RD508 was significantly reduced (P < 0.05). In addition, compared with the control group, the diarrhea index of piglets in the test groups B and C was significantly reduced (P < 0.05)

[0214] 4 DISCUSSION

[0215] The use of probiotics in livestock production to improve intestinal health and growth performance of animals has made some progress, which has attracted widespread attention in the livestock industry [1] . A large number of studies have shown that supplementing Bacillus licheniformis in feed can promote animal growth and improve animal health [2] . Sun et al. [3] found that adding Bacillus licheniformis S6 strain in the diet could increase the average daily gain of piglets, improve the intestinal mucosal integrity, and reduce the incidence of diarrhea in piglets. Yu et al. [4] found that adding Bacillus licheniformis in the diet could improve the intestinal microbiota of weaned piglets, increase the relative abundance of Clostridium_sensu_stricto_1 (narrow-sense Clostridium 1 type) and Oscillospira (Oscillospira), thereby increasing the average daily gain and reducing the incidence of diarrhea. In addition, Bacillus licheniformis can also increase the content of short-chain fatty acids and promote intestinal development. Lin et al. [5]Studies have shown that adding Bacillus licheniformis to feed can reduce the incidence of diarrhea in weaned piglets, and that the relative abundance of Prevotella 9, Prevotellaceae_unclassified, Blautia, and Subdoligranulum is negatively correlated with the incidence of diarrhea in weaned piglets. The results of this experiment indicate that adding 400 g / t and 600 g / t of Bacillus licheniformis RD508 to the feed significantly improved the average daily weight gain of weaned piglets and reduced the feed conversion ratio and diarrhea rate.

[0216] 5. Conclusion

[0217] In conclusion, the use of Bacillus licheniformis RD508 in this experiment can improve the production performance of piglets, increase average daily weight gain, and reduce the feed conversion ratio, thereby improving feed efficiency. Furthermore, Bacillus licheniformis RD508 can also reduce the diarrhea rate in piglets, which is beneficial to their intestinal health. 6 References

[0218] [1] Gao Fuxian, Shang Enci, Yue Zhiguang, et al. Research progress on probiotic preparation processing technology and its application in animal husbandry production [J]. Journal of Animal Nutrition, 2025, 37(07):4243-4255.

[0219] [2] Muras A, Romero M, Mayer C, Otero A. Biotechnological applications of Bacillus licheniformis. Crit Rev Biotechnol. 2021 Jun;41(4):609-627. doi: 10.1080 / 07388551.2021.1873239. Epub 2021 Feb 16. PMID:33593221.

[0220] [3] Sun W, Chen W, Meng K, Cai L, Li G, LiX, Jiang X. DietarySupplementationwith Probiotic Bacillus licheniformis S6 lmproves IntestinalIntegrity via ModulatingIntestinal Barrier Function and Microbial Diversityin Weaned Piglets. Biology (Basel)2023,12

[0221] [4] Yu X, Cui Z, Qin S, Zhang R, Wu Y, Liu J, Yang C. Effects of Bacillus licheniformis on Growth Performance, Diarrhea Incidence, Antioxidant Capacity, Immune Function, and Fecal Microflora in Weaned Piglets. Animals (Basel). 2022.12

[0222] [5] Lin KH, Yu YH, Evaluation of Bacillus licheniformis-Fermented Feed Additive as an Antibiotic Substitute: Effect on the Growth Performance, Diarrhea Incidence, and Cecal Microbiota in Weaning Piglets. Animals (Basel), 2020, 10.

Claims

1. The application of *Bacillus licheniformis* of enterogenic origin in the preparation of feed to improve the growth performance of piglets or reduce diarrhea, characterized in that... The enteric Bacillus licheniformis is Bacillus licheniformis RD508, with accession number GDMCC No: 64606.

2. The application as described in claim 1, characterized in that: The feed includes a basal diet and Bacillus licheniformis RD508; the Bacillus licheniformis RD508 is a live bacterium.

3. The application as described in claim 1, characterized in that: The sequence of the housekeeping gene gyrB of Bacillus licheniformis RD508 is shown in SEQ ID No.

8.

4. The application as described in claim 2, characterized in that: Add 200-600g of Bacillus licheniformis RD508 to each ton of basal diet.

5. The application as described in claim 4, characterized in that: The basal diet comprises the following components by weight percentage: Corn 63% 12% puffed soybeans 15% soybean meal Fish meal 3.2% 2% soybean oil Stone powder 0.9% 1% Dicalcium Phosphate 0.4% NaCl (table salt) L-Lysine hydrochloride 0.5% Methionine 0.28% Choline chloride 0.1% Antioxidant 0.02% 0.1% antifungal agent Mineral premix 1% Montmorillonite 0.5%.

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