WO2023087944A1 - 一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用 - Google Patents

一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用 Download PDF

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WO2023087944A1
WO2023087944A1 PCT/CN2022/122539 CN2022122539W WO2023087944A1 WO 2023087944 A1 WO2023087944 A1 WO 2023087944A1 CN 2022122539 W CN2022122539 W CN 2022122539W WO 2023087944 A1 WO2023087944 A1 WO 2023087944A1
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animal
intestinal
pediococcus
bacterial
performance
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French (fr)
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李冲
邓雪娟
蔡辉益
李爽
王少龙
王腾飞
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天津博菲德科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/41—Pediococcus
    • A23V2400/413—Acidilactici
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of microorganisms, and in particular relates to a strain of Pediococcus lactis capable of improving the production performance and immune level of broiler chickens, a screening method and application thereof.
  • AGPs Antibiotics
  • Pediococcus lactis As a lactic acid bacteria, Pediococcus lactis has been proven to be a reliable bacteriocin producer with unique antibacterial, anti-inflammatory and anti-cancer properties.
  • a "disconnect" problem in the screening of Pediococcus lactis in vitro and in vivo which is specifically reflected in: 1High antibacterial efficacy in vitro does not necessarily produce probiotic effects in vivo; 2The stress resistance of most lactic acid bacteria is poor; 3Fresh See the research application of this bacterium in broiler production, and the strains of Pediococcus lactis that have positive effects on broiler chickens are even rarer.
  • patent CN112226389A discloses a method for colonization and cultivation of probiotic flora in the intestinal tract of three-yellow broiler chicks, in which the process of screening lactic acid bacteria mainly includes tolerance, enzyme production and acid production ability, but no in vitro anti-pathogen evaluation and in vivo evaluation. evaluate.
  • Patent CN111011590A discloses a compound probiotic preparation containing Pediococcus lactis, which can improve salpingitis and egg quality of laying hens, but does not clearly evaluate the effect on production performance. Wajda (Wajda S et al.
  • the technical problem to be solved by the present invention is: to provide a kind of chyme screened from the intestinal tract of healthy animals that has the following properties: (a) high yield of organic acid, remarkable antibacterial effect, (b) high safety and strong stress resistance, ( c) improving the growth performance of broiler chickens, (d) improving the immune level of broiler chickens, improving the health status of broiler chickens, and reducing the dead panning rate of Pediococcus lactis, and the strains obtained by screening through the method.
  • the present invention firstly provides a strain of Pediococcus acidilacticii (Pediococcus acidilacticii) LC-9-1.
  • the preservation number of Pediococcus acidilacticii LC-9-1 provided by the present invention is CGMCC No. 21345.
  • the strain has been deposited in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on December 10, 2020; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101 ).
  • the Pediococcus acidilacticii LC-9-1 provided by the present invention has gone through complex in vitro screening and in vivo evaluation procedures, and has the characteristics of safety, reliability, practicality and high efficiency.
  • the test results show that the strain has good stress resistance and acid production ability in vitro, and has a significant inhibitory effect on pathogenic bacteria such as Escherichia coli, Salmonella, and Staphylococcus aureus.
  • the strain showed good stress resistance and colonization ability, and has the probiotic function of improving the flora structure and intestinal development, repairing the pathological damage of the liver and spleen, and improving the immune level and growth performance of broiler chickens.
  • the present invention also provides a bacterial agent.
  • the active ingredient of the bacterial preparation provided by the present invention is the above-mentioned Pediococcus acidilacticii LC-9-1 or its bacterial suspension or its culture or its culture liquid or its fermentation liquid.
  • the concentration of the bacterial suspension is 1.0 ⁇ 10 7 -1.0 ⁇ 10 9 CFU/mL, preferably 1.0 ⁇ 10 8 CFU/mL.
  • the present invention also provides a new application of the above-mentioned Pediococcus acidilacticii LC-9-1 or bacterial agent.
  • the present invention provides the application of above-mentioned Pediococcus acidilacticii (Pediococcus acidilacticii) LC-9-1 or bacterial agent in any one of the following 1)-6):
  • the present invention also provides the method described in any one of the following d1)-d5):
  • a method for inhibiting pathogenic bacteria comprising the steps of: treating the pathogenic bacteria with the above-mentioned Pediococcus acidilacticii LC-9-1 or bacterial agent, so as to inhibit the pathogenic bacteria;
  • a method for improving animal growth performance or immune performance comprising the steps of: feeding the animal with the above-mentioned Pediococcus acidilacticii LC-9-1 or bacterial agent, so as to realize the improvement of animal growth performance or immune performance;
  • a method for promoting the development of animal intestinal tissue or maintaining the integrity of animal intestinal mucosal barrier function comprising the following steps: feeding the animal with the above-mentioned Pediococcus acidilacticii LC-9-1 or bacterial agent, to achieve Promote the development of animal intestinal tissue or maintain the integrity of animal intestinal mucosal barrier function;
  • a method for relieving or repairing pathological damage of animal liver and/or spleen tissue or protecting animal liver and/or spleen tissue comprising the following steps: using the above-mentioned Pediococcus acidilacticii LC-9-1 or bacteria The animal is fed with the medicine to relieve or repair the pathological damage of the liver and/or spleen tissue of the animal or protect the liver and/or spleen tissue of the animal;
  • a method for optimizing or improving the structure of the intestinal flora of an animal comprising the steps of: feeding the animal with the above-mentioned Pediococcus acidilacticii LC-9-1 or bacterial agent, so as to optimize the structure of the intestinal flora of the animal or improve.
  • the pathogenic bacteria include but not limited to Escherichia coli, Salmonella, Proteus mirabilis and Staphylococcus aureus.
  • the improvement of animal immune performance is reflected in the improvement of animal spleen index.
  • the present invention finally provides a screening method for Pediococcus lactis;
  • the Pediococcus lactis has the following properties: high safety, strong stress resistance, easy colonization in the intestinal tract of animals, improvement of animal intestinal bacteria Group structure, promote animal intestinal development, alleviate pathological damage to animal liver and/or spleen, improve animal growth performance and/or immune performance.
  • the screening method of Pediococcus lactis comprises the steps:
  • step X2 Evaluate the acid production of the strains obtained by the separation and purification of step X1), and screen the strains with good acid production performance;
  • step X3 carry out bacteriostasis evaluation to the bacterial strain screened in step X2), and screen the bacterial strain with good bacteriostasis performance;
  • step X4 evaluating the stress resistance of the bacterial strains screened in step X3), and screening the strains with good stress resistance;
  • step X5 performing an in vitro safety evaluation on the strains screened in step X4);
  • the bacteriostatic evaluation includes the following steps: measuring the inhibitory performance on pathogenic bacteria Escherichia coli, Salmonella, Proteus mirabilis and Staphylococcus aureus.
  • the stress resistance evaluation includes the following steps: measuring the tolerance to gastric juice and bile salt.
  • the in vitro safety evaluation includes the following steps: measuring antibiotic sensitivity and hemolysis.
  • the identification includes morphological identification, biochemical identification and molecular biological identification.
  • the in vivo evaluation of broiler chickens includes evaluation of growth performance of broiler chickens, evaluation of immune organ development of broiler chickens, evaluation of intestinal tissue development of broiler chickens, evaluation of liver and spleen tissue morphology of broiler chickens, and evaluation of intestinal flora structure of broiler chickens .
  • the preparation method of the bacterial suspension comprises the following steps: inoculate the isolated and purified LC-9-1 bacterial strain in the MRS solid medium, and after solid culture (cultivation time can be 24h), pick Single colonies are placed in the MRS liquid medium, and cultured at 37°C and 160r/min (the culture time can be 18-24h), so that the bacterial concentration in the culture system reaches 1.0 ⁇ 10 7 ⁇ 1.0 ⁇ 10 9 CFU/ mL, centrifuged (centrifugation condition can be 4000rpm centrifugal 10min), collects thalline, with sterile physiological saline, described thalline is washed successively (washing times can be three times) and resuspended, obtains described bacterial suspension (bacterial suspension The bacterial cell concentration in the solution is 1.0 ⁇ 10 7 ⁇ 1.0 ⁇ 10 9 CFU/mL).
  • the animal is chicken, specifically broiler chicken (such as AA broiler chicken).
  • the present invention provides a new lactic acid tablet by improving the screening program and adopting the "in vitro + in vivo" combination strategy
  • a method for screening cocci and Pediococcus lactis LC-9-1 obtained by screening the method.
  • Pediococcus lactis LC-9-1 has the following application effects: high safety, strong stress resistance, easy colonization in the intestinal tract, can improve the structure of intestinal flora, promote intestinal development, relieve liver and spleen pathological damage, improve growth performance and immune performance, and can replace AGPs to a certain extent.
  • Fig. 1 is the screening of Pediococcus lactis LC-9-1 (CaCO 3 -MRS solid medium) of the present invention.
  • Fig. 2 is a comparison of stress resistance characteristics of Pediococcus lactis LC-9-1 of the present invention.
  • Fig. 3 is the hemolysis experiment (24h) of Pediococcus lactis LC-9-1 of the present invention.
  • Fig. 4 is the strain characteristic of Pediococcus lactis LC-9-1 of the present invention.
  • A is the colony of LC-9-1;
  • B is the cell of LC-9-1 ( ⁇ 1000 oil lens);
  • C is the cell of LC-9-1 ( ⁇ 10000 electron microscope);
  • D is the growth curve of LC-9-1.
  • Fig. 5 is a phylogenetic tree of Pediococcus lactis LC-9-1 of the present invention.
  • Fig. 6 shows the effect of Pediococcus lactis LC-9-1 of the present invention on the number of broiler ileum goblet cells.
  • CON control group
  • PA2 1.0 ⁇ 10 8 CFU/kg of Pediococcus lactis LC-9-1 added to the basal diet
  • AGPs growth-promoting antibiotics. * indicates significant difference within the same index P ⁇ 0.05, ** indicates extremely significant difference within the same index P ⁇ 0.01.
  • Fig. 7 is the effect of Pediococcus lactis LC-9-1 of the present invention on the tissue morphology of broiler liver and spleen.
  • CON-G liver of control group
  • CON-P spleen of control group
  • PA2-G liver of P. lactis group
  • PA2-P spleen of P. lactis group
  • AGPs-G liver of antibiotic group
  • AGPs-P antibiotic group spleen.
  • Fig. 8 is the effect of Pediococcus lactis LC-9-1 of the present invention on the intestinal flora of broilers.
  • CON control group;
  • PA2 1.0 ⁇ 10 8 CFU/kg of Pediococcus lactis LC-9-1 added to the basal diet;
  • AGPs growth-promoting antibiotics. * indicates significant difference within the same index P ⁇ 0.05, ** indicates extremely significant difference within the same index P ⁇ 0.01.
  • the culture medium involved in the present invention (the solvents are all distilled water, all sterilized at 121°C for 20min before use) are as follows:
  • MRS solid medium (/L): peptone 10.0g, beef powder 5.0g, glucose 20.0g, yeast powder 4.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, triammonium citrate 2.0g , manganese sulfate 0.05g, Tween 80 1ml, agar powder 15.0g, pH adjusted to 7.2-7.4.
  • MRS liquid medium peptone 10.0g, beef powder 5.0g, glucose 20.0g, yeast powder 4.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, triammonium citrate 2.0g , manganese sulfate 0.05g, Tween 80 1ml, pH adjusted to 7.2-7.4.
  • CaCO 3 -MRS solid medium (/L): peptone 10.0g, beef powder 5.0g, glucose 20.0g, yeast powder 4.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, tricitrate Ammonium 2.0g, manganese sulfate 0.05g, Tween 80 1ml, CaCO 3 10.0g, pH adjusted to 7.2-7.4.
  • LB liquid medium tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, pH adjusted to 7.2-7.4.
  • LB solid medium tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, agar powder 15.0g, pH adjusted to 7.2-7.4.
  • Embodiment 1 have broad-spectrum bacteriostasis and stress-resistance characteristic Pediococcus lactis screening method
  • the antibacterial ability is expressed by the area ratio of the inhibition zone/inner pore diameter (H/C).
  • Strains LC-9-1, LC-3-9, LC-2-5 and LC-2-9 were evaluated for their tolerance to gastric juice and bile salts.
  • the evaluation of gastric juice tolerance refers to the method of Garcia-Ruiz et al. (2014), and the specific steps are as follows: the initial concentration of the strain to be tested is maintained at 10 8 to 10 9 CFU/mL, and 2 ⁇ SES solution is mixed with 0.6% (w/v) gastric The protease was mixed to simulate the gastric environment, cultured with shaking at 37°C, and the pH value was adjusted with hydrochloric acid (1mol/L).
  • the strains to be tested were inoculated in 0, 0.2%, 0.3% and 0.5% concentration of bile salt MRS medium, cultured anaerobically at 37°C for 24 hours, and the number of viable bacteria was measured.
  • the test adopts the drug-sensitive disc agar diffusion method. Take 100 ⁇ L of the activated LC-9-1 bacterial suspension and spread it on the MRS solid medium. After the bacterial solution is completely absorbed, put the drug-sensitive disc on the medium. Above, after stabilizing for 15 minutes, incubate upside down at 37°C for 20 hours, measure the diameter of the inhibition zone and calculate the area, and judge the results according to the "CLSI Antimicrobial Susceptibility Test Standard".
  • the preparation method of the above-mentioned LC-9-1 bacterial suspension is as follows: inoculate the isolated and purified strain LC-9-1 in MRS solid medium, and after solid culture for 24 hours, pick a single colony and inoculate it into 10 mL of MRS liquid medium , 37°C, 160r/min shaking culture for 18-24h, so that the concentration of bacteria in the culture system reached 1.0 ⁇ 10 9 CFU/mL, centrifuged at 4000rpm for 10min, collected the bacteria, washed 3 times with sterile normal saline and then washed with 10mL sterile The bacteria were resuspended in normal saline to obtain the bacteria suspension (the concentration of the bacteria was 1.0 ⁇ 10 9 CFU/mL).
  • the sensitivity of strain LC-9-1 to 20 kinds of antibiotics is shown in Table 3, and the strain showed sensitivity to many kinds of antibiotics.
  • the specific performance is that the overall sensitivity to aminoglycoside antibiotics is low, especially the sensitivity to amikacin and kanamycin is the lowest, followed by oxacillin of ⁇ -lactams and ceftazidime of cephalosporins a lower sensitivity.
  • Inhibition zone diameter insensitive: 0mm; low sensitive: ⁇ 10mm; medium sensitive: 10-14mm; high sensitive > 15mm.
  • the hemolysin produced by bacteria during the growth process is likely to cause the rupture and dissolution of red blood cells, thereby causing pathogenicity. Inoculate the activated LC-9-1 bacterial suspension on a 5% defibrinated sheep blood plate, and invert at 37°C Cultivate and observe whether hemolysis occurs at 24 hours. The results are shown in Figure 3. The strain LC-9-1 grew well on the blood plate without hemolysis, indicating that the strain is not hemolytic.
  • the strain characteristics of LC-9-1 are: facultative anaerobic, inoculated in MRS solid medium by streaking, the colony characteristics after 24 hours of culture are: small white colonies, smooth and opaque surface and edges, growth along the puncture line of agaric Filamentous (Fig. 4A); Gram-stained bacterial cells and observed under an optical microscope with an oil lens ( ⁇ 1000), they were Gram-positive cocci (Fig.
  • the LC-9-1 strain was analyzed using API 50CHL reagent strips, and the specific results are shown in Table 4.
  • strain LC-9-1 The DNA of strain LC-9-1 was extracted using a bacterial genome DNA extraction kit, and the 16S rDNA was amplified and sequenced using universal primers. The sequencing results showed that the 16S rDNA PCR product of strain LC-9-1 obtained a gene fragment with a size of 1476 bp. , the nucleotide sequence of which is shown in sequence 1.
  • the 16S rDNA gene sequence of strain LC-9-1 was homologously compared at NCBI, the phylogenetic relationship was analyzed, and a phylogenetic tree was constructed (Fig. 5). The results showed that the strain LC-9-1 and the standard strain Pediococcus acidilactici FJ905315.2 clustered into a branch with high similarity, and were initially identified as Pediococcus acidilactici.
  • strain LC-9-1 was Pediococcus (Pediococcus genus) and Pediococcus acidilactici (Pediococcus lactis). Its taxa are: Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; Pediococcus.
  • Pediococcus acidilactici (Pediococcus acidilactici) LC-9-1 has been preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures (CGMCC for short) on December 10, 2020; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing , Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), the deposit number is CGMCCNo.21345.
  • Embodiment 3 the impact of Pediococcus lactis LC-9-1 on broiler growth performance and immune performance
  • Grouping and dietary composition control group CON Feeding basal rations; Pediococcus lactis LC-9-1 groups PA1, PA2, and PA3 respectively added Pediococcus lactis LC-9-1 bacterial suspension to the basal rations, so that the bacterial concentrations in the basal rations were 1.0 ⁇ 10 7 CFU/kg, 1.0 ⁇ 10 8 CFU/kg, 1.0 ⁇ 10 9 CFU/kg; AGPs group added combination antibiotics (chlortetracycline 100 mg/kg, kitasarmycin 20 mg/kg) on the basis of basal diet.
  • the basal diet is a corn-soybean meal type diet, and the formula design refers to the "Chicken Feeding Standard" (NY/T 33-2004).
  • the experiment lasted for 42 days to analyze the effects of Pediococcus lactis LC-9-1 on the growth performance and immune organs of broilers. developmental impact.
  • indexes of the broilers in each group were counted in the early stage of the experiment (1-21 days) and the later stage of the experiment (22-42 days): final weight, average daily gain, average daily feed intake, feed-to-weight ratio and mortality rate.
  • Immune organ index (%) (organ weight/living body weight) ⁇ 100.
  • Pediococcus lactis LC-9-1 has a significant effect on the immune performance of broiler chickens. Specifically, the addition of Pediococcus lactis at 1.0 ⁇ 10 8 CFU/kg and 1.0 ⁇ 10 9 CFU/kg significantly improved the Broiler spleen index (P ⁇ 0.05), but no significant effect on bursa and thymus (P>0.05).
  • Example 4 Effect of Pediococcus lactis LC-9-1 on the development of broiler intestinal tissue and the morphology of liver and spleen tissue
  • PA2 group has The trend of increasing the villus height, villi ratio and intestinal wall thickness of the jejunum of 42-day-old broilers (P>0.05), significantly increased the villi height of ileum of 21-day-old and 42-day-old broilers (P ⁇ 0.05), while the 21-day-old ileum Intestinal wall thickness and chorionic ratio at 42 days were also significantly increased (P ⁇ 0.05).
  • 1 CON basal diet
  • 2 PA2 1.0 ⁇ 10 8 CFU/kg of Pediococcus lactis LC-9-1 added to the basal diet
  • 3 AGPs growth-promoting antibiotics.
  • 1 CON basal diet
  • 2 PA2 1.0 ⁇ 10 8 CFU/kg of Pediococcus lactis LC-9-1 suspension added to the basal diet
  • 3 AGPs growth-promoting antibiotics.
  • Goblet cells are specialized intestinal epithelial cells that can secrete a variety of proteins and participate in maintaining the integrity of the intestinal mucosa.
  • the number of goblet cells in the small intestine can reflect the integrity of the intestinal mucosal barrier function.
  • the sampling method is the same as that in step 2.
  • Goblet cells are present in the small intestinal villi in the field of view.
  • liver tissue Liver cell degeneration, hepatic cell necrosis and inflammatory cell infiltration were found in the liver tissue of each treatment group, but the necrotic area was found to be significantly reduced in the group supplemented with 1.0 ⁇ 10 8 CFU/kg of Pediococcus lactis in the diet , the hepatocytes were arranged radially around the central vein, the shape was normal, and the interlobular arteries, veins, and bile ducts in the portal area were relatively complete.
  • Spleen tissue The boundary between the white pulp and red pulp in each treatment group was not clear, but in the control group and the antibiotic group, obvious white pulp lymphocytes decreased and the cells were sparsely arranged, and the red pulp in the control group had obvious congestion.
  • Pediococcus lactis LC-9-1 has a certain protective effect on the liver and spleen of broilers.
  • Example 5 the influence of Pediococcus lactis LC-9-1 on the structure of broiler intestinal flora

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Abstract

本发明公开了一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用。本发明通过改良筛选程序，并采用"体外+体内"结合策略，提供了一种新的乳酸片球菌的筛选方法以及通过该方法筛选获得的乳酸片球菌LC-9-1，该乳酸片球菌LC-9-1经体内试验证明具有如下应用效果：安全性高、抗逆性强、易于在肠道中定植、可改善肠道菌群结构、促进肠道发育、缓解肝脏及脾脏的病理性损伤，提高生长性能和免疫性能，能够在一定程度上起到替代AGPs的作用。

Description

一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用 技术领域
本发明属于微生物技术领域，具体涉及一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用。
背景技术
在家禽饲养过程中，肠道功能尚未发育完善的雏鸡，免疫性能机能低下，极易受到致病菌、营养和环境等外部因素的干扰，是造成生长性能不佳和高死淘率的重要原因。抗生素(AGPs)曾作为促生长型添加剂被广泛应用到动物饲料中，在家禽养殖业中用以治疗和预防致病菌感染，有效提高了生产效率。然而，越来越多的研究表明，AGPs的滥用会导致耐药性问题的加剧并且造成环境中抗生素的残留，通过食物链传播进而危及人类健康，如耐药性大肠杆菌和沙门氏菌等，特别是家禽和水产养殖抗生素依赖性强的行业。欧盟成员国于2006年禁止了AGPs的使用；中国也在2020年7月1日起禁止了AGPs在动物饲料中的添加。然而，短期内也对养殖行业带来了巨大的挑战，如动物疾病感染增加、养殖生产效益降低。无抗养殖是今后畜禽业发展的必然趋势，开发寻找AGPs替代产品、减免使用已成为当务之急。
乳酸片球菌作为乳酸菌类，已被证实是一种可靠的细菌素生产菌，具有独特的抑菌性、抗炎性和抗癌性。然而目前针对乳酸片球菌在体外和体内的筛选存在“脱节”问题，具体体现为：①体外抑菌效能高并不一定在体内发生益生作用；②大部分乳酸菌的抗逆性较差；③鲜见该菌在肉鸡生产中的研究应用，且针对肉鸡有积极效果的乳酸片球菌菌株更为鲜见。如专利CN112226389A公开了一种三黄肉雏鸡肠道益生菌群定植培养方法，其中在筛选乳酸菌流程主要包括耐受性、产酶和产酸能力，但并未进行体外抗致病菌评价和体内评价。专利CN111011590A公开了一种含乳酸片球菌的复合益生菌制剂，可改善蛋鸡输卵管炎、提高蛋品质，也未明确对生产性能的效果评价。Wajda(Wajda S et al.The efficacy of lactic acid bacteria Pediococcus acidilactici,lactoseand formic acid as dietary supplements for turkeys[J].Pol J Vet Sci,2010)将其筛选的乳酸片球菌应用于火鸡，发现提高了前12周的平均日增重，但并未见对免疫性能和肠道健康进一步评价。王利红(鸡源乳酸菌的分离筛选及其对肉鸡生产性能的影响[D].2014)曾分离筛选出一株乳酸片球菌，发现菌株显著降低了肉鸡的平均日增重和饲料转化率。
发明内容
本发明要解决的技术问题是：提供一种从健康动物肠道食糜中筛选得到具有如下性能：(a)高产有机酸、抗菌效果显著，(b)安全性高、抗逆性强，(c)提高肉鸡生长性能，(d)提高肉鸡免疫水平、改善肉鸡健康状况、降低死淘率的乳酸片球菌的 方法、以及通过该方法筛选得到的菌株。
为了解决上述技术问题，本发明首先提供了一株乳酸片球菌(Pediococcusacidilacticii)LC-9-1。
本发明提供的乳酸片球菌(Pediococcus acidilacticii)LC-9-1的保藏编号为CGMCCNo.21345。该菌株已于2020年12月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮编：100101)。本发明提供的乳酸片球菌(Pediococcus acidilacticii)LC-9-1经过了复杂的体外筛选和体内评价流程，具备安全可靠、实用高效的特性。试验结果显示，该菌株在体外具有良好的抗逆性和产酸能力，对大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌等致病菌具有显著的抑制效果。此外，在肉鸡试验中该菌株表现出良好的抗逆性及定植能力，具备改善菌群结构及肠道发育，修复肝脏及脾脏的病理性损伤，提升肉鸡免疫水平和生长性能的益生功能。
为了解决上述技术问题，本发明还提供了一种菌剂。
本发明提供的菌剂的活性成分为上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或其菌悬液或其培养物或其培养液或其发酵液。
上述菌剂中，所述菌悬液的浓度为1.0×10 7～1.0×10 9CFU/mL，优选为1.0×10 8CFU/mL。
为了解决上述技术问题，本发明还提供了上述乳酸片球菌(Pediococcusacidilacticii)LC-9-1或菌剂的新用途。
本发明提供了上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或菌剂在如下1)-6)中任一种中的应用：
1)抑制致病菌；
2)提高动物生长性能；
3)提高动物免疫性能；
4)促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性；
5)缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织；
6)优化或改善动物肠道菌群结构。
为了解决上述技术问题，本发明还提供了如下d1)-d5)任一所述的方法：
d1)一种抑制致病菌的方法，包括如下步骤：用上述乳酸片球菌(Pediococcusacidilacticii)LC-9-1或菌剂处理病原菌，实现抑制病原菌；
d2)一种提高动物生长性能或免疫性能的方法，包括如下步骤：用上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或菌剂饲喂动物，实现动物生长性能或免疫性能 的提高；
d3)一种促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性的方法，包括如下步骤：用上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或菌剂饲喂动物，实现促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性；
d4)一种缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织的方法，包括如下步骤：用上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或菌剂饲喂动物，实现缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织；
d5)一种优化或改善动物肠道菌群结构的方法，包括如下步骤：用上述乳酸片球菌(Pediococcus acidilacticii)LC-9-1或菌剂饲喂动物，实现动物肠道菌群结构的优化或改善。
上述任一所述应用或方法中，所述致病菌包括但不限于大肠埃希氏菌、沙门氏菌、奇异变形杆菌和金黄色葡萄球菌。
上述任一所述应用或方法中，所述提高动物生长性能体现在如下a1)-a3)中的任一种：
a1)增加动物体重(如末重、平均日增重)；
a2)降低料重比；
a3)降低死淘率；
所述提高动物免疫性能体现在提高动物脾脏指数。
上述任一所述应用或方法中，所述促进动物肠道组织发育和/或维持动物肠道粘膜屏障功能的完整性体现在如下b1)-b5)中的任一种：
b1)提高动物肠道上皮细胞排列的整齐度；
b2)增加动物空肠和/或回肠的绒毛高度；
b3)增加动物空肠和/或回肠的绒隐比；
b4)增加动物空肠和/或回肠的肠壁厚度；
b5)增加动物回肠的杯状细胞数量。
上述任一所述应用或方法中，所述优化或改善动物肠道菌群结构体现在如下c1)-c3)中任一种：
c1)增加动物肠道的菌群丰度；
c2)增加动物肠道中有益菌的比例；
c3)降低动物肠道中有害菌的比例。
为了解决上述技术问题，本发明最后提供了一种乳酸片球菌的筛选方法；所述乳酸片球菌具有如下性能：安全性高、抗逆性强、易于在动物肠道中定植、改善动物肠 道菌群结构、促进动物肠道发育、缓解动物肝脏和/或脾脏的病理性损伤、提高动物生长性能和/或免疫性能。
本发明提供的乳酸片球菌的筛选方法包括如下步骤：
X1)从健康动物肠道食糜中进行菌株的分离纯化，得到分离纯化后的菌株；
X2)对步骤X1)分离纯化得到的菌株进行产酸评价，筛选产酸性能好的菌株；
X3)对步骤X2)筛选得到的菌株进行抑菌评价，筛选抑菌性能好的菌株；
X4)对步骤X3)筛选得到的菌株进行抗逆性评价，筛选抗逆性好的菌株；
X5)对步骤X4)筛选得到的菌株进行体外安全性评价；
X6)对步骤X4)筛选得到的菌株进行鉴定；
X7)对步骤X4)筛选得到的菌株进行肉鸡在体评价。
进一步的，所述抑菌评价包括如下步骤：对致病菌大肠埃希氏菌、沙门氏菌、奇异变形杆菌和金黄色葡萄球菌抑制性能的测定。
所述抗逆性评价包括如下步骤：对胃液和胆盐的耐受性进行测定。
所述体外安全性评价包括如下步骤：对抗生素敏感性和溶血性进行测定。
所述鉴定包括形态鉴定、生化鉴定和分子生物学鉴定。
所述肉鸡在体评价包括对肉鸡生长性能的评价、对肉鸡免疫器官发育的评价、对肉鸡肠道组织发育的评价、对肉鸡肝脏与脾脏组织形态的评价、对肉鸡肠道菌群结构的评价。
上述应用或方法中，所述菌悬液的制备方法包括如下步骤：将分离纯化后的LC-9-1菌株接种于MRS固体培养基中，固体培养(培养时间可为24h)后，挑取单菌落至MRS液体培养基中，在37℃、160r/min条件下进行培养(培养时间可为18-24h)，使培养体系中的菌体浓度达到1.0×10 7～1.0×10 9CFU/mL，离心(离心条件可为4000rpm离心10min)，收集菌体，用无菌生理盐水将所述菌体依次进行洗涤(洗涤次数可为三次)和重悬，得到所述菌悬液(菌悬液中菌体浓度为1.0×10 7～1.0×10 9CFU/mL)。
上述应用或方法中，所述动物为鸡，具体为肉鸡(如AA肉鸡)。
基于现有技术中存在的缺少应用于改善肉鸡生产性能和免疫水平的乳酸片球菌菌株的问题，本发明通过改良筛选程序，并采用“体外+体内”结合策略，提供了一种新的乳酸片球菌筛选方法以及通过该方法筛选获得的乳酸片球菌LC-9-1。经体内试验证明该乳酸片球菌LC-9-1具有如下应用效果：安全性高、抗逆性强、易于在肠道中定植、可改善肠道菌群结构、促进肠道发育、缓解肝脏及脾脏的病理性损伤，提高生长性能和免疫性能，能够在一定程度上起到替代AGPs的作用。
附图说明
图1为本发明乳酸片球菌LC-9-1的筛选(CaCO 3-MRS固体培养基)。
图2为本发明乳酸片球菌LC-9-1的抗逆特性对比。
图3为本发明乳酸片球菌LC-9-1的溶血实验(24h)。
图4为本发明乳酸片球菌LC-9-1的菌株特性。A为LC-9-1菌落；B为LC-9-1菌体(×1000油镜)；C为LC-9-1菌体(×10000电镜)；D为LC-9-1生长曲线。
图5为本发明乳酸片球菌LC-9-1的系统发育树。
图6为本发明乳酸片球菌LC-9-1对肉鸡回肠杯状细胞数量的影响。CON＝对照组；PA2＝在基础日粮基础上添加了1.0×10 8CFU/kg的乳酸片球菌LC-9-1；AGPs＝促生长型抗生素。*表示同一指标内差异显著P＜0.05，**表示同一指标内差异极显著P＜0.01。
图7为本发明乳酸片球菌LC-9-1对肉鸡肝、脾的组织形态影响。CON-G＝对照组肝；CON-P＝对照组脾；PA2-G＝乳酸片球菌组肝；PA2-P＝乳酸片球菌组脾；AGPs-G＝抗生素组肝；AGPs-P＝抗生素组脾。箭头：黄＝坏死，绿＝淋巴细胞减少，红＝淤血。
图8为本发明乳酸片球菌LC-9-1对肉鸡肠道菌群的影响。CON＝对照组；PA2＝在基础日粮基础上添加了1.0×10 8CFU/kg的乳酸片球菌LC-9-1；AGPs＝促生长型抗生素。*表示同一指标内差异显著P＜0.05，**表示同一指标内差异极显著P＜0.01。
保藏说明
中文名：乳酸片球菌
拉丁名：Pediococcus acidilacticii
菌株编号：LC-9-1
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2020年12月10日
保藏中心登记入册编号：CGMCC No.21345
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明所涉及到的培养基(溶剂均为蒸馏水，使用前均121℃灭菌20min)如下：
MRS固体培养基(/L)：蛋白胨10.0g，牛肉粉5.0g，葡萄糖20.0g，酵母粉4.0g， 乙酸钠5.0g，磷酸氢二钾2.0g，硫酸镁0.2g，柠檬酸三铵2.0g，硫酸锰0.05g，吐温80 1ml，琼脂粉15.0g，pH调整为7.2～7.4。
MRS液体培养基(/L)：蛋白胨10.0g，牛肉粉5.0g，葡萄糖20.0g，酵母粉4.0g，乙酸钠5.0g，磷酸氢二钾2.0g，硫酸镁0.2g，柠檬酸三铵2.0g，硫酸锰0.05g，吐温80 1ml，pH调整为7.2～7.4。
CaCO 3-MRS固体培养基(/L)：蛋白胨10.0g，牛肉粉5.0g，葡萄糖20.0g，酵母粉4.0g，乙酸钠5.0g，磷酸氢二钾2.0g，硫酸镁0.2g，柠檬酸三铵2.0g，硫酸锰0.05g，吐温80 1ml，CaCO 3 10.0g，pH调整为7.2～7.4。
LB液体培养基(/L)：胰蛋白胨10.0g，酵母提取物5.0g，氯化钠10.0g，pH调整为7.2～7.4。
LB固体培养基(/L)：胰蛋白胨10.0g，酵母提取物5.0g，氯化钠10.0g，琼脂粉15.0g，pH调整为7.2～7.4。
SES溶液(/L)：氯化钙0.22g，氯化钠6.2g，氯化钾2.2g，碳酸氢钠1.2g。
下面实施例将对本发明进一步阐述：
实施例1、具有广谱抑菌和抗逆特性的乳酸片球菌筛选方法
一、菌株的分离纯化
收集健康且生长状况良好的若干奶牛瘤胃、鸡盲肠、猪回肠、家兔盲肠食糜样品35例，用于筛选目的菌株。具体方法为：各取上述样品10g，溶于90mL无菌的PBS溶液中，在160rpm条件下恒温震荡20min，吸取500μL转入5.5mL MRS液体培养基中，37℃恒温160rpm富集培养24小时，随后再吸取100μL菌液，接种于MRS固体培养基，37℃静置培养24h，用平板稀释方法进行分离纯化，挑选单菌落在MRS固体培养基进行三代纯化后保存于50％甘油中，-80℃保藏。
二、菌株产酸性能的测定
1、将分离纯化的菌株分别点板于CaCO 3-MRS固体培养基中，37℃倒置培养，挑选有明显溶钙圈的单菌落，用平板划线法进行分离(图1)。
2、将上述分离的菌株，分别定量接种于MRS液体培养基中(pH均调为6.0)，37℃培养，每间隔2h，用酸度计测定培养液pH值并记录，直至48小时最后一次采集数据。最终选择pH较低的前20株菌进行下一步筛选，具体结果如表1所示。
表1、乳酸菌菌株的液体发酵产酸能力
序号 菌株 来源 终末pH值 序号 菌株 来源 终末pH值
1 LC-1-3 兔盲肠 4.20 11 LC-4-5 兔盲肠 4.16
2 LC-1-13 兔盲肠 4.23 12 LC-4-10 鸡盲肠 3.89
3 LC-2-5 兔盲肠 3.90 13 LC-7-10 兔盲肠 4.17
4 LC-2-2 牛瘤胃 4.22 14 LC-8-8 兔盲肠 4.13
5 LC-2-3 兔盲肠 4.36 15 LC-9-1 兔盲肠 3.75
6 LC-2-4 牛瘤胃 4.36 16 LC-9-5 兔盲肠 3.97
7 LC-2-9 牛瘤胃 3.98 17 LC-10-10 鸡盲肠 4.22
8 LC-3-2 兔盲肠 4.22 18 LC-11-11 猪回肠 4.14
9 LC-3-10 牛瘤胃 4.25 19 LC-11-18 猪回肠 3.98
10 LC-3-9 鸡盲肠 4.03 20 LC-12-17 兔盲肠 4.16
三、菌株体外抑菌性能的测定
采用指示菌：大肠杆菌埃希菌ATCC25922、沙门氏菌ATCC13076、奇异变形杆菌CMCCB49005、金黄色葡萄球菌ATCC6538，分别接种于LB液体培养基中，37℃震荡培养24h，调节浓度为0.5MCF(分光光度计OD 625nm＝0.08～0.13)作为指示菌悬液，按照牛津杯扩散法进行测定，计算抑菌圈面积，平行测定3次后取平均值。
经过分析对比，有9株具有广谱抑菌特性(表2)，取4株抑菌性能较佳的菌株LC-9-1、LC-3-9，LC-2-5及LC-2-9备以进一步筛选。
表2、乳酸菌菌株的体外抑菌能力
Figure PCTCN2022122539-appb-000001
*注：抑菌能力以抑菌圈/内孔径(H/C)的面积比表示。
四、菌株抗逆性评价
对菌株LC-9-1、LC-3-9、LC-2-5及LC-2-9分别进行胃液与胆盐的耐受性评价。胃液耐受性评价参考Garcia-Ruiz等(2014)的方法，具体步骤如下：待测菌株初始浓度保持在10 8～10 9CFU/mL，将2×SES溶液与0.6％(w/v)胃蛋白酶混合模拟胃环境，37℃内震荡培养，用盐酸(1mol/L)调节pH值，pH由6.2依次降低至5.0、4.0、3.0、2.0，每个间隔培养20min，各节点取样计数。胆盐耐受性评价是将待测菌株分别接种于0、0.2％、0.3％和0.5％浓度的胆盐MRS培养基中，37℃厌氧培养24h，测定活菌数。
菌株对胃液与胆盐的耐受性评价结果如图2所示，其中菌株LC-3-9与LC-9-1均表现了较佳的抗逆性，对比分析LC-9-1耐低pH胃液及高浓度胆盐的能力更强。
实施例2、具有广谱抑菌和抗逆特性的LC-9-1菌株的安全性评价与鉴定
一、安全性评价
1、抗生素敏感试验
试验采用药敏纸片琼脂扩散法，取100μL活化好的LC-9-1菌悬液，涂布于MRS固体培养基中，在菌液被完全吸收后，将药敏纸片放于培养基上，稳定15min后，于37℃中倒置培养20h，测量抑菌圈直径并计算面积，根据《CLSI抗菌药物敏感性试验标准》判定结果。
上述LC-9-1菌悬液的制备方法如下：将分离纯化后的菌株LC-9-1接种于MRS固体培养基中，固体培养24h后，挑取单菌落接种至10mL MRS液体培养基中，37℃、160r/min振荡培养18-24h，使培养体系中的菌体浓度达到1.0×10 9CFU/mL，4000rpm离心10min，收集菌体，用无菌生理盐水洗涤3次再用10mL无菌生理盐水重悬，得到菌悬液(菌体浓度为1.0×10 9CFU/mL)。
菌株LC-9-1对20种抗生素的敏感性如表3所示，该菌对多类抗生素均表现出敏感性。具体表现为对氨基糖苷类抗生素总体敏感性较低，尤其对丁胺卡那和卡纳霉素的敏感性最低，其次对β-内酰胺类的苯唑西林和头孢菌素类的头孢他啶也表现出较低的敏感性。
表3、菌株LC-9-1抗生素敏感试验
Figure PCTCN2022122539-appb-000002
注：Inhibition zone diameter：不敏感:0mm；低敏感：＜10mm；中敏感：10～14mm；高敏感＞15mm。
2、溶血实验
细菌在生长过程中产生的溶血素容易造成红细胞的破裂溶解，从而造成致病性， 将活化好的LC-9-1菌悬液，接种于5％脱纤维绵羊血平板，于37℃中倒置培养，于24h观察是否出现溶血现象。结果如图3所示，菌株LC-9-1在血平板上生长良好未发生溶血现象，说明该菌株不具有溶血性。
二、特性与形态特征
LC-9-1的菌株特性为：兼性厌氧，划线接种于MRS固体培养基中，培养24h后菌落特征为：白色小菌落，表面及边缘光滑不透明，沿洋菜穿刺线的生长物呈丝状(图4A)；菌体进行革兰氏染色并在光学显微镜下油镜(×1000)观察，为革兰氏阳性球菌(图4B)；电子显微镜下(×10000)观察，菌体一般成对生，非链状排列，少见单生细胞，在直角两个平面交替分裂形成四联状(图4C)；细菌在接种后4～6h进入对数期，12～14h后进入平台期(图4D)。
三、生化鉴定
采用API 50CHL试剂条对LC-9-1菌株进行分析，具体结果如表4所示。
表4、API 50CHL分析系统鉴定结果
Figure PCTCN2022122539-appb-000003
Figure PCTCN2022122539-appb-000004
*"+"反应阳性，"—"阴性反应。
四、分子生物学鉴定
采用细菌基因组DNA提取试剂盒提取菌株LC-9-1的DNA，采用通用引物进行16SrDNA扩增与测序，测序结果表明：菌株LC-9-1的16S rDNA PCR产物共得到大小为1476bp的基因片段，其核苷酸序列如序列1所示。
将菌株LC-9-1的16S rDNA基因序列在NCBI进行同源性比对，分析亲缘关系，构建系统发育树(图5)。结果表明：菌株LC-9-1与标准菌株Pediococcus acidilacticiFJ905315.2聚为一个分支，相似度高，初步鉴定为乳酸片球菌(Pediococcus acidilactici)。
综合菌落及菌体形态特征、API 50CHL的分析结果、分子生物学鉴定等手段，确定菌株LC-9-1为Pediococcus(片球菌属)，为Pediococcus acidilactici(乳酸片球菌)。其分类单元为：Bacteria；Firmicutes；Bacilli；Lactobacillales；Lactobacillaceae；Pediococcus。
五、菌株LC-9-1的保藏
乳酸片球菌(Pediococcus acidilactici)LC-9-1已于2020年12月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮编：100101)，保藏编号为CGMCCNo.21345。
实施例3、乳酸片球菌LC-9-1对肉鸡生长性能及免疫性能的影响
一、不同梯度乳酸片球菌LC-9-1对肉鸡饲养试验
选择300只1日龄的健康艾拔益加肉鸡(AA肉鸡)，随机分为5个处理组，每个处理组6个重复，每个重复10只鸡，分组及日粮组成：对照组CON饲喂基础日粮；乳酸片球菌LC-9-1组PA1、PA2、PA3分别在基础日粮中添加乳酸片球菌LC-9-1菌悬液，使基础日粮中的菌浓度分别为1.0×10 7CFU/kg、1.0×10 8CFU/kg、1.0×10 9CFU/kg；AGPs组在基础日粮基础上添加组合抗生素(金霉素100mg/kg、吉他霉素20mg/kg)。基础日粮为玉米-豆粕型日粮，配方设计参考《鸡饲养标准》(NY/T 33-2004)，试验前后期共42d，分析乳酸片球菌LC-9-1对肉鸡生长性能和免疫器官发育的影响。
二、乳酸片球菌LC-9-1对肉鸡生长性能的影响
分别在试验前期(1～21天)和试验后期(22～42天)统计各组肉鸡的如下指标：末重、平均日增重、平均日采食量、料重比和死淘率。
结果如表5所示，结果显示：添加1.0×10 8CFU/kg的乳酸片球菌组可显著改善肉鸡生长性能。具体表现为试验前期(1～21天)有改善饲料转化率和降低死淘率的趋势；后期(22～42天)，PA2组的末重、平均日增重、饲料转化率及死淘率方面显著优于对照组(P＜0.05)，总体应用效果与抗生素组无显著差异(P＞0.05)。
表5、饲粮添加乳酸片球菌LC-9-1对肉鸡生长性能的影响
Figure PCTCN2022122539-appb-000005
注：同行数据肩标不同小写字母表示差异显著(P＜0.05)，相同或无字母表示差异不显著(P＞0.05)，下同。
三、乳酸片球菌LC-9-1对肉鸡免疫器官发育的影响
在肉鸡42日龄时，每个重复取接近平均体重的个体，记录体重后处死，取免疫器官，去除脂肪组织并用滤纸吸去多余的水分后称重，计算免疫器官指数。免疫器官指数(％)＝(器官重量/活体重量)×100。
结果如表6所示，乳酸片球菌LC-9-1对肉鸡免疫性能有显著影响，具体表现为添加1.0×10 8CFU/kg和1.0×10 9CFU/kg的乳酸片球菌组显著提高了肉鸡的脾脏指数(P＜0.05)，但对法氏囊及胸腺的影响不显著(P＞0.05)。
表6、饲粮添加乳酸片球菌LC-9-1对免疫器官指数的影响
Figure PCTCN2022122539-appb-000006
实施例4、乳酸片球菌LC-9-1对肉鸡肠道组织发育及肝脏、脾脏组织形态的影响
一、不同梯度乳酸片球菌LC-9-1对肉鸡饲养试验
同实施例3中1。
二、乳酸片球菌LC-9-1对肉鸡肠道组织发育的影响
分别于试验21及42d时，每个重复取接近平均体重的个体，剖杀取空肠及回肠1.5cm用生理盐水冲洗干净后，于4％多聚甲醛溶液固定，制作切片并观察肠道组织形态。
镜下观察发现，添加1.0×10 8CFU/kg的乳酸片球菌组促进了肉鸡肠道粘膜的完整性，肠道上皮细胞排列更加整齐，测量数据如表7和表8所示，PA2组有提高42日龄肉鸡空肠的绒毛高度、绒隐比和肠壁厚度的趋势(P＞0.05)，显著提高了21日龄和42日龄肉鸡回肠绒毛高度(P＜0.05)，同时21日龄回肠肠壁厚度和42日龄绒隐比也显著升高(P＜0.05)。
表7、乳酸片球菌LC-9-1对21和42日龄肉鸡空肠绒毛高度、绒隐比和肠壁厚度的影响
Figure PCTCN2022122539-appb-000007
1CON＝基础日粮； 2PA2＝在基础日粮基础上添加了1.0×10 8CFU/kg的乳酸片球菌LC-9-1； 3AGPs＝促生长型抗生素。
表8、乳酸片球菌LC-9-1对21和42日龄肉鸡回肠绒毛高度、绒隐比和肠壁厚度的影响
Figure PCTCN2022122539-appb-000008
Figure PCTCN2022122539-appb-000009
1CON＝基础日粮； 2PA2＝在基础日粮基础上添加了1.0×10 8CFU/kg的乳酸片球菌LC-9-1悬液； 3AGPs＝促生长型抗生素。
三、乳酸片球菌LC-9-1对肉鸡回肠杯状细胞数量的影响
杯状细胞是一种特异化的肠上皮细胞，可分泌多种蛋白，参与维持肠黏膜的完整性，小肠杯状细胞的数量可以反映肠黏膜屏障功能的完整性。试验42d时，每个重复取接近平均体重的个体，取样方法同步骤二，制备5mm横切面并用过碘酸希夫染色，以每100个上皮细胞计数，每只鸡1个肠横截面上不同视野的小肠绒毛中存在杯状细胞。
结果如图6所示，较对照组和抗生素组，饲粮中添加了1.0×10 8CFU/kg的乳酸片球菌组肉鸡回肠中的杯状细胞数量显著增加(P＜0.05)。
四、乳酸片球菌LC-9-1对肉鸡肝脏、脾脏组织形态的影响
试验42d时，观察各组试验肉鸡的肝、脾组织形态。
结果如图7所示。肝脏组织：各处理组肝脏组织均发现肝细胞变性、肝细胞坏死和炎性细胞浸润的情况，但在饲粮中添加了1.0×10 8CFU/kg的乳酸片球菌组发现坏死面积明显减小，肝细胞围绕中央静脉呈放射状排列，形态正常，门管区小叶间动脉、静脉及胆管结构较为完整。脾脏组织：各处理组间白髓和红髓的分界均欠清，但在对照组和抗生素组还发现明显的白髓淋巴细胞减少，细胞排列稀疏现象，且对照组红髓部分有明显淤血。综上所述，乳酸片球菌LC-9-1对肉鸡的肝和脾均发挥了一定的保护作用。
实施例5、乳酸片球菌LC-9-1对肉鸡肠道菌群结构的影响
一、不同梯度乳酸片球菌LC-9-1对肉鸡饲养试验
同实施例3中1。
二、乳酸片球菌LC-9-1对肉鸡肠道菌群结构的影响
在肉鸡42日龄时，取回肠食糜，通过16S扩增子测序及生物信息学方法，分析肠道菌群结构。
结果如图8所示，结果表明，添加了1.0×10 8CFU/kg的乳酸片球菌组的肉鸡肠道菌群丰度较对照组有增高趋势。其中肠球菌属(Enterococcus)、罗氏菌属(Rothia)比例有增高趋势，乳杆菌属(Lactobacillus)比例降低(P＞0.05)；产志贺毒素大肠杆菌属(Escherichia-Shigella)的比例显著降低(P＜0.05)。抗生素组促进了天然生产抗生素的北里孢菌属(Kitasatospora)和库特氏菌属(Kurthia)的比例显著增高。综上所述，乳酸片球菌LC-9-1优化了肠道菌群结构，增加了有益菌的比例，减少了有害菌比例，相对抗生素组肠道耐药菌比例减少。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨 和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

Claims (10)

  1. 乳酸片球菌(Pediococcus acidilacticii)LC-9-1，其保藏编号为CGMCC No.21345。
  2. 一种菌剂，其活性成分为权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或其菌悬液或其培养物或其培养液或其发酵液。
  3. 根据权利要求2所述的菌剂，其特征在于：所述菌悬液的浓度为1.0×10 7～1.0×10 9CFU/mL。
  4. 权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂在如下1)-6)中任一种中的应用：
    1)抑制致病菌；
    2)提高动物生长性能；
    3)提高动物免疫性能；
    4)促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性；
    5)缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织；
    6)优化或改善动物肠道菌群结构。
  5. 根据权利要求4所述的应用，其特征在于：所述致病菌包括大肠埃希氏菌、沙门氏菌、奇异变形杆菌和金黄色葡萄球菌。
  6. 根据权利要求4所述的应用，其特征在于：所述提高动物生长性能体现在如下a1)-a3)中的任一种：
    a1)增加动物体重；
    a2)降低料重比；
    a3)降低死淘率；
    或，所述提高动物免疫性能体现在提高动物脾脏指数。
  7. 根据权利要求4所述的应用，其特征在于：所述促进动物肠道组织发育和/或维持动物肠道粘膜屏障功能的完整性体现在如下b1)-b5)中的任一种：
    b1)提高动物肠道上皮细胞排列的整齐度；
    b2)增加动物空肠和/或回肠的绒毛高度；
    b3)增加动物空肠和/或回肠的绒隐比；
    b4)增加动物空肠和/或回肠的肠壁厚度；
    b5)增加动物回肠的杯状细胞数量。
  8. 根据权利要求4所述的应用，其特征在于：所述优化或改善动物肠道菌群结构体现在如下c1)-c3)中任一种：
    c1)增加动物肠道的菌群丰度；
    c2)增加动物肠道中有益菌的比例；
    c3)降低动物肠道中有害菌的比例。
  9. 如下d1)-d5)任一所述的方法：
    d1)一种抑制致病菌的方法，包括如下步骤：用权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂处理病原菌，实现抑制病原菌；
    d2)一种提高动物生长性能或免疫性能的方法，包括如下步骤：用权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂饲喂动物，实现动物生长性能或免疫性能的提高；
    d3)一种促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性的方法，包括如下步骤：用权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂饲喂动物，实现促进动物肠道组织发育或维持动物肠道粘膜屏障功能的完整性；
    d4)一种缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织的方法，包括如下步骤：用权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂饲喂动物，实现缓解或修复动物肝脏和/或脾脏组织的病理性损伤或保护动物肝脏和/或脾脏组织；
    d5)一种优化或改善动物肠道菌群结构的方法，包括如下步骤：用权利要求1所述的乳酸片球菌(Pediococcus acidilacticii)LC-9-1或权利要求2或3所述的菌剂饲喂动物，实现动物肠道菌群结构的优化或改善。
  10. 一种乳酸片球菌的筛选方法，包括如下步骤：
    X1)从健康动物肠道食糜中进行菌株的分离纯化，得到分离纯化后的菌株；
    X2)对步骤X1)分离纯化得到的菌株进行产酸评价，筛选产酸性能好的菌株；
    X3)对步骤X2)筛选得到的菌株进行抑菌评价，筛选抑菌性能好的菌株；
    X4)对步骤X3)筛选得到的菌株进行抗逆性评价，筛选抗逆性好的菌株；
    X5)对步骤X4)筛选得到的菌株进行体外安全性评价；
    X6)对步骤X4)筛选得到的菌株进行鉴定；
    X7)对步骤X4)筛选得到的菌株进行肉鸡在体评价；
    所述乳酸片球菌具有如下性能：安全性高、抗逆性强、易于在动物肠道中定植、改善动物肠道菌群结构、促进动物肠道发育、缓解动物肝脏和/或脾脏的病理性损伤、提高动物生长性能和/或免疫性能。
PCT/CN2022/122539 2021-11-22 2022-09-29 一株改善肉鸡生产性能和免疫水平的乳酸片球菌及其筛选方法与应用 WO2023087944A1 (zh)

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