Lactobacillus gallus, micro-ecological additive and application of micro-ecological additive

Abstract

The invention discloses lactobacillus gallinarum, a micro-ecological additive and application of the lactobacillus gallinarum and the micro-ecological additive, the lactobacillus gallinarum is named as lactobacillus gallinarum CD-13 and is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 20241172, and the nucleotide sequence of 16SrDNA of the lactobacillus gallinarum is SEQ ID NO.1. The lactobacillus gallinarum is named as lactobacillus gallinarum CD-13 and is preserved in the China Center for Type Culture Collection. The lactobacillus gallus CD-13 provided by the invention is good in stress resistance and can tolerate a strong acid environment, the survival rate of the lactobacillus gallus CD-13 in a solution with the pH value of 2.5 is 107.62%, and the lactobacillus gallus CD-13 has very strong tolerance to acidity. And the survival rate in a 0.3% cholate solution is 12.97%. In a high bile salt environment, the resistance is relatively poor, but a small amount of the strain still can survive, the stress resistance is relatively good, and the strain can adapt to a gastrointestinal tract environment. When the lactobacillus gallus CD-13 provided by the invention is added into drinking water as a micro-ecological additive, the lactobacillus gallus CD-13 has an obvious growth promoting effect on broiler chickens.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganism application, and specifically relates to the screening of a probiotic Lactobacillus gallinaceus strain and application thereof. Background Art

[0002] Growth promotion is an eternal theme in the broiler farming industry, and modern microecological studies have shown that intestinal microorganisms are the host's "second set of genomes." There is a complex and large microbial system in the digestive tract of broilers, with billions of microorganisms gathering in the nutrient-rich intestine. These microorganisms co-evolve with the host to form a dynamic and balanced microbial flora. The complex and diverse intestinal flora plays a vital role in broiler food digestion and absorption, inhibiting the reproduction of pathogens, enhancing immune function and protecting the health of the body. Therefore, it is expected to discover beneficial microorganisms to promote broiler growth. Lactobacillus johnsonii, Lactobacillus fragilis and Enterococcus faecalis have all been proven to promote broiler growth and intestinal development. Studies have shown that through fecal microbiota transplantation (FMT) technology, using fecal microorganisms from high-weight chickens, the jejunal flora can be reconstructed, the abundance of jejunal lactic acid bacteria can be increased, the tryptophan metabolic pathway can be regulated, the Th17 / Treg ratio can be downregulated to reduce intestinal inflammation, and ultimately the growth performance of recipient chickens can be improved.

[0003] Inspired by the scientific idea of ​​modern microecology that "intestinal microorganisms are the host's second set of genomes", the high-weight phenotype of broiler chickens is linked to their intestinal microorganisms. On the basis of reproducing the "high-weight" phenotype using fecal microbiota transplantation technology, the team targeted the defects of existing technologies and screened beneficial microorganisms from the intestines of fecal microbiota transplant-positive chickens, mainly based on the activity of digestive enzymes, to obtain strains that aid digestion (protein, etc.) and promote growth, based on the fact that broiler feed contains high protein and the digestive tract of broilers is shorter than that of economic animals other than poultry, resulting in relatively insufficient food digestion. A comprehensive investigation of the probiotic properties, stress resistance, and antibiotic resistance of the strains laid the foundation for the final acquisition of excellent strains. Summary of the invention

[0004] Based on the fact that broiler feed contains high protein and the digestive tract of broilers is shorter than that of economic animals other than poultry, so that food digestion is relatively insufficient, the present invention isolates and screens a strain of Lactobacillus gallinarum CD-13 from fecal microbiota transplantation-positive recipient chickens. The strain has strong digestive enzyme (especially protease) production ability and can promote broiler growth, and is applied to broiler breeding industry to improve its production efficiency. According to 16S rDNA identification, the probiotic strain isolated by the present invention is Lactobacillus gallinarum, and the enzyme production (protease, lipase) performance, safety, stress resistance of the isolated strain and the growth-promoting effect as a broiler feed additive of the isolated strain are studied.

[0005] In order to achieve the above-mentioned purpose, the technical solution of the present invention is as follows:

[0006] According to the screening objectives and the physiological, biochemical and genetic characteristics of probiotics, a strain of Lactobacillus gallinarum CD-13 was obtained from the intestinal contents of high-weight and low-abdominal fat fecal bacteria transplant recipient broilers after large-scale sorting using classical physiological and biochemical methods and modern molecular biological methods. It was sent to the China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, China on June 7, 2024 for preservation, with the preservation number CCTCC M 20241172.

[0007] The mycological characteristics of Lactobacillus gallinarum CD-13 strain are as follows:

[0008] The CD-13 strain of Lactobacillus gallinarum is a Gram-positive bacterium, a facultative anaerobe, and is short rod-shaped, elongated and short rod. On the MRS plate of the lactic acid bacteria screening medium, it grows into a round, milky white protrusion, a smooth surface, and a colony of about 1 mm in diameter. The 16S rRNA gene of the strain was cloned and sequenced, and a Blast comparison was performed in NCBI, and it was found that the 16S rDNA sequence of the strain of the present invention had the highest homology with that of Lactobacillus gallinarum (KU311639.1), which was 99.73%.

[0009] Lactobacillus chicken CD-13 strain was added with CaCO 3 It was isolated and screened on MRS plates, and there was an obvious calcium dissolution circle. It was cultured in an anaerobic environment on the MRS plate, and its reproduction ability was strong. It was in the delayed phase from 0 to 3 hours, entered the logarithmic growth phase after 3 hours, and entered the stable phase after 9 hours. Chicken Lactobacillus CD-13 has good stress resistance and can tolerate strong acid and weak bile salt environments. After incubation in a pH 2.5 solution for 2 hours, the survival rate was 107.62%. After incubation in a 0.3% bile salt solution for 2 hours, the survival rate was 12.97%. This strain has a strong tolerance to acidity, and its resistance is relatively poor in a high bile salt environment, but it can still survive in small amounts. It has relatively good stress resistance and can adapt to the gastrointestinal environment.

[0010] Lactobacillus chicken CD-13 grows on a milky white LB plate containing 2.5% skim milk powder, and a transparent circle forms around the colony, indicating that the strain produces protease but not lipase. CD-13 has a significant inhibitory effect on Escherichia coli K88, Salmonella C78-1, and Staphylococcus aureus ATCC25923. It is sensitive to commonly used veterinary antibiotics such as penicillin, erythromycin, cephalexin, chloramphenicol, and tetracycline, but insensitive to streptomycin, gentamicin, norfloxacin, and lincomycin.

[0011] The applicant conducted a broiler feeding experiment using the Lactobacillus chicken CD-13 strain of the present invention by feeding it with drinking water. The strain had a significant growth-promoting effect on broilers, significantly increased the weight of the breast and leg muscles, and significantly increased the length of the cecum, achieving the expected effect.

[0012] Compared with the prior art, the present invention has the following advantages and effects:

[0013] (1) The Lactobacillus chicken CD-13 strain isolated from high-weight and low-abdominal fat receptor-positive broilers can produce protease with a clearing zone of 14.14 mm.

[0014] (2) The CD-13 strain of the present invention has strong reproductive ability and good stability; Lactobacillus chicken CD-13 has good stress resistance and can tolerate acidic environment. After incubation in a pH 2.5 solution for 2 hours, the survival rate is 107.62%, which is conducive to adapting to the gastrointestinal environment of broilers and can be used as a microecological feed additive.

[0015] (3) The CD-13 strain of the present invention has a strong inhibitory effect on both pathogenic Gram-positive and Gram-negative bacteria, which is beneficial for antagonizing harmful bacteria in the intestines of broiler chickens.

[0016] (4) Animal experiments have shown that the microecological preparation CD-13 of the present invention can be used as an additive for livestock and poultry. It is very simple to produce and use. It can not only improve the production performance of livestock and poultry, but also has the advantages of being environmentally friendly and safe for humans and animals.

[0017] It can be prepared into a microecological additive and used as a broiler feed additive to promote the digestion and absorption of broiler feed and promote the growth of broilers; it can improve feed conversion rate and production efficiency, and is beneficial to the economic development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The strain Lactobacillus chicken CD-13 of the present invention was isolated in a screening medium (MRS+CaCO 3 ) The situation of calcium dissolution circle;

[0019] Figure 2 The growth state of the strain Lactobacillus chicken CD-13 of the present invention on the MRS agar plate;

[0020] Figure 3 The Gram staining result of the strain Lactobacillus chicken CD-13 of the present invention (oil immersion lens; ×1000);

[0021] Figure 4 The PCR electrophoresis results of 16SrDNA of the strain Lactobacillus chicken CD-13 of the present invention, Note: X: negative control; CD-13: isolated CD-13 strain; M: DL2000 Plus;

[0022] Figure 5 This is the bacterial phylogenetic tree of the neighbor-joining method of the strain Lactobacillus gallinarum CD-13 of the present invention. Note: D13 in the figure is the strain CD-13 of the present invention;

[0023] Figure 6 The result of the transparent zone of protease produced by the strain Lactobacillus chicken CD-13 of the present invention (LYS: Lactobacillus salivarius, a positive control bacterium);

[0024] Figure 7 The results of the lipase clearing zone produced by the strain Lactobacillus chicken CD-13 of the present invention (LYS: Lactobacillus salivarius, a positive control bacterium);

[0025] Figure 8 The results of in vitro antibacterial activity of the fermented liquid of the strain Lactobacillus gallinarum CD-13 of the present invention. The indicator strains used from left to right are: Escherichia coli K88ac, Salmonella C78-1, Staphylococcus aureus ATCC25923, and D13 in the figure is the strain CD-13 of the present invention;

[0026] Fig. 9 is the growth curve of the strain Lactobacillus chicken CD-13 of the present invention;

[0027] Fig.10 The results of the 40-day body weight and average daily weight gain of broiler chickens subjected to the strain Lactobacillus gallinarum CD-13 of the present invention: CD-13 group: fed with Lactobacillus gallinarum CD-13 in drinking water; Ctrl group: fed with sterile water in drinking water;

[0028] Fig.11 The growth of breast and leg muscles of broiler chickens tested for 40 days by the strain Lactobacillus gallinarum CD-13 of the present invention is shown in Figure 1;

[0029] Fig.12 The second is the growth of breast and leg muscles of broiler chickens tested for 40 days by the strain Lactobacillus gallinarum CD-13 of the present invention;

[0030] Fig.13 This is the cecal index result of the 40-day broiler experiment on the strain Lactobacillus gallinarum CD-13 of the present invention. Specific implementation plan

[0031] For more detailed technical solutions, please see the following contents.

[0032] Example 1: Isolation and identification of Lactobacillus chicken CD-13 strain

[0033] 1. Strain Isolation

[0034] A method for screening chicken-derived probiotic lactic acid bacteria that promotes broiler growth, the steps of which are: A. Samples are taken from the experimental chicken farm of Huazhong Agricultural University. Among the fecal microbiota transplantation group, 1-2 g of intestinal contents and feces of each segment of the largest recipient chicken (FMT 49d Cobb broiler) are selected and temporarily stored in a 4°C refrigerator for later use. B. 1 mL of the collected sample is diluted 10 times in a gradient (100 μL of culture solution is added to 900 μL of sterile water for gradient dilution) to 10 -7 .

[0035] C. From 10 -4 , 10 -5 , 10 -6 , 10 -7 Take 200 μL of bacterial suspension from each of the four dilution gradient samples and spread it evenly on MRS solid medium (10 g peptone, 8 g beef extract, 4 g yeast extract, 20 g glucose, 20 g K 2 HPO 4 2g, sodium acetate 5g, diammonium citrate 2g, Tween-80 1mL, MgSO 4 7H 2 0 0.58g, MnSO 4 7H 2 O 0.25g, then add 1.5% agar powder and distilled water to 1000mL).

[0036] D. Place in a 37°C anaerobic incubator, invert and culture for 48 hours, and pick out single colonies of different morphologies for pure culture.

[0037] E. The pure culture strain was inoculated into the CaCO 3 MRS plates were used for screening.

[0038] F. Inoculate pure culture strains in a culture medium supplemented with CaCO 3 The isolated bacteria were screened on MRS plates with the addition of CaCO 3 The colonies grew into milky white, smooth, round colonies with a diameter of about 3 mm on the MRS plate, with a transparent calcium-dissolving ring ( Figure 1 ), pick a single colony with a transparent calcium-dissolving ring around the colony for purification and culture;

[0039] G. Gram staining of the purified strains yielded Gram-positive bacteria, facultative anaerobic bacteria, short rod-shaped, elongated rods (see Figure 2 ).

[0040] H. The pure culture strain was inoculated on the lactic acid bacteria screening medium MRS plate, and it grew into round, milky white protrusions with a smooth surface and a diameter of about 1 mm (see Figure 3 ).

[0041] 2. Identification of strain species

[0042] On the basis of the above identification, the 16S rRNA gene sequence of the above strains was further detected to confirm the species to which the strains belonged. The specific steps are as follows:

[0043] A. Target strain genome extraction:

[0044] (1) Take 1 mL of culture medium and centrifuge at 10,000 rpm for 30 seconds. Discard the supernatant as much as possible and collect the bacteria.

[0045] (2) Add 200 μL of buffer RB to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant.

[0046] (3) Add 120 μL of lysozyme (20 mg / mL in 10 mM Tris-HCl, pH 8.0) and mix by inversion. Incubate at 37°C for 30-60 min. Centrifuge at 12,000 rpm for 2 min, discard the supernatant, and resuspend in 180 μL of buffer RB. (4) Add 20 μL of proteinase K (20 mg / mL) solution, mix thoroughly, then add 200 μL of binding buffer CB, vortex and mix thoroughly, and place at 70°C for 10 min.

[0047] (5) After cooling, add 100 μL of isopropanol and immediately vortex to mix thoroughly. Flocculent precipitation may appear at this time.

[0048] (6) Add the mixture from the previous step (including any precipitate) to an adsorption column AC (the adsorption column is placed in a collection tube) and centrifuge at 13,000 rpm for 30-60 seconds. Discard the waste liquid in the collection tube.

[0049] (7) Add 500 μL of inhibitor removal solution IR, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0050] (8) Add 700 μL of rinse solution WB (with anhydrous ethanol), centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. (9) Add 500 μL of rinse solution WB (with anhydrous ethanol), centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. (10) Place the adsorption column AC back into the empty collection tube and centrifuge at 13,000 rpm for 2 min to remove the rinse solution as much as possible to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction.

[0051] (11) Take out the adsorption column AC and put it into a clean centrifuge tube. Add 50 μL of elution buffer EB (preheat the elution buffer in a 67-70°C water bath) to the middle of the adsorption membrane, leave it at room temperature for 3-5 minutes, and centrifuge it at 12,000 rpm for 1 minute. Add the resulting solution back to the centrifugal adsorption column, leave it at room temperature for 2 minutes, and centrifuge it at 12,000 rpm for 1 minute.

[0052] (12) Store the DNA sample at -20°C until use.

[0053] B. Amplification of 16S rRNA gene:

[0054] The 16S rRNA sequence of the isolated strain was amplified using bacterial universal primers. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The DNA sequences of the primers are as follows:

[0055] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3,

[0056] Reverse primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′;

[0057] The PCR amplification system is shown in Table 1. The amplification conditions are: 94°C pre-denaturation for 5 min, 94°C for 1 min, 50°C for 30 s, 72°C for 1.5 min, 30 cycles, and 72°C extension for 10 min. The PCR amplification product was electrophoresed on a 0.8% agarose gel (containing ethidium bromide). The size of the amplified fragment was consistent with the expectation, about 1500 bp (see Figure 4 ).

[0058] Table 1 PCR amplification system of strains

[0059]

[0060] C. Construct bacterial evolutionary tree to determine species:

[0061] The bacterial genomic DNA was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were subjected to Blast analysis on NCBI. The bacterial phylogenetic tree was constructed by the neighbor-joining method using MEGA7.0 software. It was found that the 16S rDNA sequence of the strain of the present invention had the highest homology with that of Lactobacillus gallinarum (KU311639.1), which was 99.73% (see Figure 5 ).

[0062] It was named Lactobacillus gallinarum CD-13 and sent to the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on June 7, 2024, with the collection number CCTCCM 20241172.

[0063] Example 2: Detection of probiotic properties of Lactobacillus gallinarum CD-13 strain

[0064] 1. Enzyme production test

[0065] (I) Protease activity detection

[0066] Take 100 μL of the fermentation liquid of Lactobacillus chicken CD-13 strain and inoculate it on a plate with skimmed milk powder as the only carbon source, and place it in an anaerobic incubator at 37°C for 24 hours. If protease is produced, the protein around the colony is decomposed, and a transparent circle appears on the plate, indicating that the strain produces protease (see Figure 6 The results of the protease activity test of the strains are shown in Table 2.

[0067] Table 2 Protease detection results of Lactobacillus chicken CD-13 strain

[0068]

[0069] (II) Lipase activity detection

[0070] 100 μL of the fermentation liquid of Lactobacillus chicken CD-13 strain was inoculated on the tributyrin plate and placed in a 37°C anaerobic incubator for 24 hours. If lipase is produced, the fat around the colony is decomposed and a transparent circle appears on the plate. Figure 7 As shown, Lactobacillus gallinaria CD-13 strain does not produce lipase.

[0071] II. Antibacterial activity test of Lactobacillus chicken CD-13 strain (Escherichia coli K88ac, Salmonella C78-1, Staphylococcus aureus ATCC25923)

[0072] Using Escherichia coli K88ac, Salmonella C78-1, and Staphylococcus aureus ATCC25923 as indicator bacteria and the fermentation supernatant of strain CD-13 as an antibacterial agent, the in vitro antibacterial activity of the strains was detected. The specific operation steps are as follows:

[0073] A. Preparation of indicator bacteria (Escherichia coli K88ac, Salmonella C78-1, Staphylococcus aureus ATCC25923) suspension: Streak indicator bacteria on a plate and culture at 36℃ for 20h; pick a single colony and place it in LB liquid culture medium and culture it in a shaking incubator at 37℃ for 16h to revive the indicator bacteria. Adjust the cultured indicator bacteria solution to a bacterial count of 1.0×10 7CFU / mL. (Note: 0.01mol / L PBS is required for dilution)

[0074] B. Preparation and treatment of bacterial fermentation broth: Streak the strain plate of the present invention and culture it at 37°C for 16 hours. Pick the isolated strain in 50mL of LB liquid culture medium and culture it at 37°C and 200r / min for 24 hours as seed liquid. Inoculate the seed liquid into 100mL of LB liquid culture medium at an inoculum of 1%, and culture it anaerobically at 37°C and 200r / min for 48 hours as fermentation broth. Centrifuge the prepared fermentation broth at 10000r / min for 5 minutes, and filter the supernatant after centrifugation with a 0.22μm sterile filter for later use.

[0075] C. Antibacterial test: Take 200μL indicator bacteria suspension, drop it on the LB plate, spread it evenly with a coating rod until no water droplets are visible, then use sterile tweezers to take a sterilized Oxford cup, gently place it on the surface of the LB solid culture medium, and evenly place 3 Oxford cups on each culture medium. Use a micropipette to absorb 200μL of the supernatant of the strain fermentation liquid, and inject it into the Oxford cup placed steadily (add carefully, do not drip on the culture medium outside the Oxford cup); then replace the original glass plate cover with a sterilized ceramic cover (to absorb the water evaporated from the culture medium during the subsequent test process).

[0076] D. Cultivation: Place the culture medium with supernatant in a 4℃ refrigerator for 8 hours, then place the plate after diffusion treatment in a 37℃ constant temperature incubator. After 9 hours of cultivation, observe and record the test results.

[0077] The test results show that the strain of the present invention has a strong inhibitory ability against pathogenic bacteria, among which CD-13 has a very strong inhibitory effect on Escherichia coli K88ac, and highly inhibits Staphylococcus aureus ATCC25923 and Salmonella C78-1. Figure 8 and Table 3.

[0078] Table 3 In vitro antibacterial test of fermentation supernatant of strains

[0079]

[0080] Example 3: Safety evaluation of Lactobacillus gallinarum CD-13 strain

[0081] 1. Drug sensitivity test

[0082] Nine drug sensitivity papers (purchased from Hangzhou Microbiological Reagent Co., Ltd.), including penicillins, cephalosporins, quinolones, aminoglycosides, tetracyclines, and sulfonamides, were selected for drug sensitivity testing. The test judgment standard was based on the latest version of NCCLS provided by the World Health Organization (WHO) (2020 edition). The specific steps are as follows:

[0083] A. Inoculate 1% of the bacterial suspension into LB liquid culture medium and culture overnight at 37°C with a shaker at 200 rpm / min.

[0084] B. Using white paper with black words as the background, adjust the turbidity to 0.5 McFarland standard turbidimetric tube turbidity. If the bacterial solution is too concentrated, dilute it with physiological saline.

[0085] C. Dip a sterile cotton swab in the bacterial solution, press it against the tube wall to squeeze out the excess bacterial solution, and then apply it on the LB agar plate. Rotate the plate 60 degrees each time, and finally apply two circles on the inner edge of the plate. Repeat several times to ensure uniform application. D. After the moisture on the plate is completely absorbed by the agar, use sterile tweezers to pick up the drug-sensitive paper and stick it on the surface of the plate. Once the paper is attached, it cannot be picked up again. Stick 5 pieces of paper on each plate, with a distance of no less than 24mm between each piece of paper, and the center of the paper is no less than 15mm from the edge of the plate.

[0086] E. Place the plate in a 37°C constant temperature incubator and incubate for 12 hours to observe the results.

[0087] The test results are shown in Table 4. The Lactobacillus gallinarum CD-13 strain of the present invention is sensitive to commonly used veterinary antibiotics such as penicillin, erythromycin, cephalexin, tetracycline, and chloramphenicol, but is insensitive to streptomycin, gentamicin, norfloxacin, and lincomycin.

[0088] Table 4 Antibiotic sensitivity test results of Lactobacillus gallinarum CIM-3 strain

[0089]

[0090] Note: S stands for sensitive, M stands for moderately sensitive, and R stands for resistant.

[0091] Example 4: Stress resistance and growth characteristics of Lactobacillus chicken CD-13

[0092] 1. Stress resistance test

[0093] (1) Acid resistance test

[0094] A. Prepare an acidic solution and adjust the pH to 2.5 using sodium hydroxide solution and dilute hydrochloric acid.

[0095] B. Prepare bacterial liquid: Use a sterile inoculation loop to pick a small amount of isolated and preserved strains and streak them on MRS agar plates. Incubate them in a 37°C incubator for 24 hours. Pick a single colony and inoculate it into MRS liquid culture medium. Incubate it anaerobically at 37°C for 18 hours.

[0096] C. Take 1 mL of the above cultured bacterial solution, centrifuge at 6000 r / min, 4°C for 20 min, discard the supernatant, and add 50 mmol / L K 2 HPO 4(pH 6.5) was resuspended twice, 1 mL of hydrochloric acid solution with pH = 2.5 was added, and PBS was used as a control. After resuspending, the mixture was incubated at 37°C for 2 h.

[0097] D. Take 200 μL of the incubated bacterial solution and add it to 5 mL of MRS medium. Incubate anaerobically at 37°C for 16 h.

[0098] E. Measure the OD of the strain using an ELISA reader 600 The absorbance value below.

[0099] (2) Bile salt tolerance test

[0100] A. Prepare a 0.3% bile salt solution.

[0101] B. Prepare bacterial liquid: Use a sterile inoculation loop to pick a small amount of isolated and preserved strains and streak them on MRS agar plates. Incubate them in a 37°C incubator for 24 hours. Pick a single colony and inoculate it into MRS liquid culture medium. Incubate it anaerobically at 37°C for 18 hours.

[0102] C. Take 1 mL of the above cultured bacterial solution, centrifuge at 6000 r / min, 4°C for 20 min, discard the supernatant, and add 50 mmol / L K 2 HPO 4 (pH 6.5) and resuspended twice, 1 mL of 0.3% bile salt solution was added, and PBS was used as a control. After resuspending, the cells were incubated at 37°C for 2 h.

[0103] D. Take 200 μL of the incubated bacterial solution and add it to 5 mL of MRS medium. Incubate anaerobically at 37°C for 16 h.

[0104] E. Measure the OD of the strain using an ELISA reader 600 The absorbance value below.

[0105] The acid / bile salt resistance survival rate of the strain was calculated according to the following formula: Survival rate (%) = OD of the bacterial solution treated with hydrochloric acid solution / bile salt solution 600 Value / OD of bacterial solution treated with PBS 600 Value × 100%. The results are shown in Table 5. This shows that the strain has a strong tolerance to acidity, has relatively poor resistance in a high bile salt environment but can still survive in small quantities, has relatively good stress resistance, and can adapt to the gastrointestinal environment.

[0106] Table 5 Results of strain acid / bile salt resistance test

[0107]

[0108] (3) Determination of growth curve

[0109] A. Preparation of seed solution: Pick a single colony and inoculate it into 3 mL of MRS liquid medium, shake and culture at 37°C for 24 hours to obtain seed solution;

[0110] B. Preparation of fermentation broth: inoculate the seed liquid into 100 mL of MRS liquid medium at a 1% inoculum amount, and culture at 37° C. with shaking for 24 h to obtain fermentation broth;

[0111] C. Determination of growth curve: Inoculate the fermentation broth into 50mL of MRS liquid culture medium at a 1% inoculum volume, take out the bacterial suspension at 0h, 3h, 5h, 7h, 9h, 11h, 13h, 15h, 17h, 19h, 21h, 23h, and 24h, and count the plates. After 10-fold gradient dilution, measure the growth curve of each strain by pouring method, and perform three replicates for each group of experiments. With the culture time as the horizontal axis, the colony count CFU / mL*10 8 The value is the vertical axis, and the growth curve of the strain is drawn.

[0112] The experiment showed that the isolated strain of the present invention has a strong reproductive capacity, is in a slow phase from 0 to 3 hours, enters a logarithmic growth phase at 3 hours and lasts until 9 hours, and then enters a stable phase. Fig. 9 .

[0113] Example 5: Broiler feeding trial

[0114] 1. Experimental Grouping and Methods

[0115] The liquid fermentation liquid of Lactobacillus gallinarum CD-13 of the present invention was fed to broiler chickens (Cobb broiler chickens) in drinking water, in order to verify the growth-promoting effect of the probiotic strain of the present invention on broiler chickens.

[0116] A. A total of 72 healthy broilers (Cobb broilers) at one day old were selected and randomly divided into two groups, a blank control group and a hen Lactobacillus group, with 3 replicates in each group and 12 chickens in each replicate. Group C was the blank control group, fed with a basic diet; the CD-13 group was the hen Lactobacillus group, fed with a basic diet + 1 ml of CD-13 liquid fermentation liquid / (bird / day). B. The number of viable bacteria in the liquid fermentation preparation (the culture liquid was centrifuged to remove the supernatant after 13 hours of culture, and the bacteria were resuspended in sterile water with an equal volume of supernatant) was 7×10 8 CFU / ml.

[0117] C. The chickens were raised for a total of 40 days, with the chick diet being fed from 1 to 21 days of age and the grower diet being fed from 22 to 40 days of age. The diet used was based on the basic broiler diet formula.

[0118] D. The broiler feeding experiment was conducted at the experimental chicken farm of Huazhong Agricultural University in Wuhan City, Hubei Province. The experimental broilers were raised in three-layer stacked chicken cages. The specific feeding time was from April 2, 2024 to May 12, 2024, a total of 40 days.

[0119] E. During the experiment, broiler chickens were free to drink water and eat food. Feed and water were changed twice a day (9:30 a.m. and 15:00 p.m.). Broiler chickens were adapted for three days, after which 1 mL of bacterial suspension was added to the drinking trough every morning.

[0120] F. When starting to raise the fish, keep the temperature at around 35°C, and lower it by 2-3°C every week until it reaches 25°C in the fourth week. Adjust the temperature and humidity accordingly based on the actual growth conditions.

[0121] 2. Measurement indicators

[0122] (1) Production performance

[0123] The production performance indicators of broilers, including average daily gain (ADG) and final body weight, were calculated at 1d, 14d and 42d.

[0124] (2) Organ index

[0125] Twelve chickens of similar body weight were selected from each group for dissection, sampling and weighing.

[0126] 3. Results Analysis

[0127] The results in Table 6 show that after 40 days of the experiment, the average daily weight gain of group C was 88.87 g, and the average daily weight gain of group CD-13 was 104.61 g; compared with group C, the daily weight gain of group CD-13 was extremely significantly increased (P<0.01); the pectoral muscle index of group C was 18.55%, and the pectoral muscle index of group CD-13 was 19.97%; compared with group C, the pectoral muscle of group CD-13 was significantly increased (P<0.05), and the cecal index was significantly increased (P<0.05).

[0128] Table 6 Comparison of broiler growth performance

[0129]

[0130] During the experimental period, it had a significant growth-promoting effect on broiler chickens (see Fig.10 ), significantly increased chest and leg muscle weight (see Fig.11 and Fig.12 ), significantly increasing the length of the cecum (see Fig.13 ), the addition of Lactobacillus gallinaceus increased the body weight of broilers by 17.17%, indicating that the addition of the strain CD-13 of the present invention to drinking water can significantly improve the growth performance of broilers.

Claims

1. A Lactobacillus gallinarum CD-13, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC M 20241172 and a nucleotide sequence of 16S rDNA of SEQ ID NO.

1.

2. A microecological additive, characterized in that: The microecological additive contains the Lactobacillus chicken CD-13 described in claim 1.

3. The microecological additive according to claim 2, characterized in that: The additive is in the form of liquid, powder or granule.

4. Use of the microecological additive as described in any one of claims 2-3 in livestock and poultry breeding.

5. The use according to claim 4, characterized in that: The application includes the application of the Lactobacillus chicken CD-13 or microecological additives in promoting the growth of livestock and poultry.

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