A composite probiotic for relieving diarrhea and soft stool in pets and its application

Abstract

The present invention provides a composite probiotic for relieving diarrhea and soft stool in pets and its application, wherein the composite probiotic comprises Lactobacillus acidophilus ZYpet‑009 (with a deposit number of CGMCC No. 31559) and Lactobacillus salivarius ZYpet‑010 (with a deposit number of CGMCC No. 31560). The present invention finds that the composite of the two bacteria can cooperate with each other, promote each other, and synergize in relieving diarrhea and soft stool in pets, which is specifically manifested in: significantly relieving animal diarrhea, improving animal anti-inflammatory and intestinal immune functions, significantly improving animal soft stool, and inhibiting the colonization and reproduction of pathogens in the intestine.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composite probiotic for relieving diarrhea and soft stools in pets and an application thereof. Background Art

[0002] Diarrhea and loose stools are common health problems in pets, especially in young and elderly pets. Such problems not only affect the daily quality of life of pets, but may also lead to more serious health problems such as severe dehydration and electrolyte imbalance. There are many reasons for pet diarrhea and loose stools, including dietary changes, infection with pathogens (such as bacteria, viruses, parasites), stress reactions, and side effects of drugs.

[0003] The methods currently on the market for relieving pet diarrhea and soft stools mainly include the following: using antibiotics to treat bacterial infections, applying probiotics to regulate intestinal flora, taking special dietary management, and using drugs to alleviate symptoms. However, each of these methods has its limitations. The widespread use of antibiotics is prone to drug resistance problems and may disrupt the normal balance of intestinal flora in pets. Although probiotics can improve intestinal health to a certain extent, most of the existing pet probiotics and related products are derived from other hosts, with varying quality, and the effects vary greatly between different strains. Dietary management requires long-term persistence and has limited effect on some acute diarrhea cases. The method of using drugs to alleviate symptoms may have side effects, posing potential risks to pet health.

[0004] Therefore, pet owners and veterinarians are in urgent need of an innovative technology to deal with pet diarrhea and soft stool problems that can effectively relieve symptoms without causing side effects or other health problems. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a composite probiotic for relieving pet diarrhea and soft stool and its application. The composite probiotic includes Lactobacillus acidophilus ZYpet-009 (with a preservation number of CGMCC No. 31559) and Lactobacillus salivarius ZYpet-010 (with a preservation number of CGMCC No. 31560). The present invention finds that the composite of the two bacteria can cooperate with each other, promote each other, and synergize in relieving pet diarrhea and soft stool.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a composite probiotic for relieving diarrhea and soft stool in pets, wherein the composite probiotic comprises Lactobacillus acidophilus ( 嗜酸乳杆菌 ) ZYpet-009 and Lactobacillus salivais ( 唾液利乳酸菌 )ZYpet-010;

[0008] Lactobacillus acidophilus ZYpet-009, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No. 31559;

[0009] Saliva-associated Lactobacillus ZYpet-010 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No. 31560;

[0010] The live bacteria content ratio of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010 in the composite probiotics is 1:5-5:1, preferably 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1.

[0011] The second aspect of the present invention provides a probiotic preparation for alleviating diarrhea and soft stools in pets, wherein the probiotic preparation comprises the composite probiotics described in the first aspect.

[0012] Furthermore, the 16S rDNA gene sequences of the Lactobacillus acidophilus ZYpet-009 and the Lactobacillus salivarius ZYpet-010 are shown in SEQ ID NO.: 1 and SEQ ID NO.: 2, respectively.

[0013] Furthermore, the effective viable count of the composite probiotics in the probiotic agent is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, preferably, the effective viable count of the composite probiotics in the probiotic agent is 1-5×10 9 CFU / mL or 1~5×10 9 CFU / g. More preferably 1×10 9 CFU / g (CFU / mL), 2×10 9 CFU / g (CFU / mL), 3×10 9 CFU / g (CFU / mL), 4×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL).

[0014] The third aspect of the present invention provides a method for preparing the probiotic agent according to the second aspect, comprising the following steps:

[0015] (1) Activating Lactobacillus acidophilus ZYpet-009 and Lactobacillus acidophilus ZYpet-010, amplifying the culture in liquid medium, collecting the cells by centrifugation, resuspending the cells in reconstituted skim milk, adjusting the effective live bacterial concentration, and freeze-drying the cells to obtain bacterial powder;

[0016] The liquid culture medium is preferably MRS culture medium; the mass concentration of the reconstituted skim milk is 10-20%, more preferably 10-15%, for example 10%, 11%, 12%, 13%, 14%, 15%.

[0017] (2) The bacterial powder is mixed with glucose to prepare the probiotic agent.

[0018] The fourth aspect of the present invention provides a feed additive, wherein the feed additive comprises the composite probiotics described in the first aspect or the probiotic agent described in the second aspect.

[0019] The fifth aspect of the present invention provides the use of the composite probiotic described in the first aspect, or the probiotic agent described in the second aspect, or the feed additive described in the fourth aspect in the preparation of a product that promotes animal intestinal health.

[0020] Furthermore, the product is any one of pet food, veterinary medicine or feed.

[0021] Furthermore, the application is one or more of the following:

[0022] (1) The application can increase the body weight of animals;

[0023] (2) The application can reduce the diarrhea rate of animals;

[0024] (3) The application can improve the intestinal immune function of animals;

[0025] (4) The application can reduce intestinal inflammation in animals;

[0026] (5) The application can reduce the level of TNF-α in animal serum;

[0027] (6) The application can increase the IL-10 level and IFN-γ level in animal serum;

[0028] (7) The application can increase the level of immunoglobulin in animal serum;

[0029] (8) The application can prevent and treat diarrhea and / or relieve soft stools in animals.

[0030] Furthermore, the animals include livestock, poultry and pets, preferably cats, dogs, rabbits, pigs, chickens, hedgehogs and hamsters.

[0031] Strain deposit information:

[0032] Lactobacillus acidophilus ZYpet-009, depository institution: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; deposit date: August 7, 2024; deposit number: CGMCC No. 31559; classification name: Lactobacillus acidophilus 嗜酸乳杆菌。

[0033] Saliva-associated Lactobacillus ZYpet-010, deposited by: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code: 100101; deposit date: August 5, 2024; deposit number: CGMCC No. 31560; classification name: Saliva-associated Lactobacillus 利乳酸菌属 唾液(菌)。

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention uses a composite probiotic agent composed of Lactobacillus acidophilus ZYpet-009 and saliva-combined Lactobacillus ZYpet-010 in the preparation of products that promote intestinal health in animals. It is found that the combination of the two bacteria can cooperate with each other, promote each other, and synergize in relieving pet diarrhea and soft stool (significantly better than a single strain), which is specifically manifested in: significantly relieving animal diarrhea, improving animal anti-inflammatory and intestinal immune functions, significantly improving animal soft stool, and inhibiting the colonization and reproduction of pathogens in the intestine;

[0036] 2. The Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010 described in the present invention can both tolerate the gastrointestinal tract, have strong intestinal colonization ability, and have high safety performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0038] Figure 1 Shown are the phylogenetic trees of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivarius ZYpet-010;

[0039] Figure 2 Shown are the survival rates of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivarius ZYpet-010 in artificial simulated gastrointestinal fluid;

[0040] Figure 3 The results of the hemolysis test of Lactobacillus acidophilus ZYpet-009 and Lactobacillus saliva-combined ZYpet-010 are shown; A: control strain Staphylococcus aureus ATCC 29213 T, B: strain ZYpet-009, C: strain ZYpet-010. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.

[0042] It should be noted that the experimental methods used in the following examples are all conventional methods in the art unless otherwise specified. Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates.

[0043] MRS medium: 10 g peptone, 10 g beef extract, 10 g yeast extract, 20 g glucose, 5 g sodium acetate, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, and 0.25 g manganese sulfate. Dissolve in deionized water and make up to 1 L. Adjust the pH to 6.0 and sterilize by high-pressure steam at 121°C for 15 min.

[0044] Example 1: Isolation and identification of bacterial species

[0045] 1. Isolation and purification of strains

[0046] (1) Isolation of strains: Feces of healthy cats were collected from catteries and animal rescue stations in Yancheng District, Luohe City. 1 g of fecal sample was weighed into a 50 mL centrifuge tube, 9 mL of sterile water was added, and the mixture was shaken and mixed. This was used as a 10-fold dilution solution, and then continuously diluted to 10 5 Take 100 μL of 10 times, 10 times 3 times and 10 5 The 10-fold dilutions were spread on MRS solid plates and incubated anaerobically at 30 ºC for 48 h.

[0047] (2) Purification of strains: Pick milky white raised colonies of different shapes and sizes for isolation and purification, pick a single colony and streak it on an MRS solid plate in three zones. After streaking, culture it at 37°C under anaerobic conditions for 48 h. Repeat the above operation for a total of three times to purify the pure strain.

[0048] (3) Morphological identification: Gram staining was used to identify the morphological characteristics of lactic acid bacteria. First, a drop of sterile water was added to the slide, a small amount of a single colony was picked and smeared on the slide, and air-dried on an alcohol lamp; staining was performed with ammonium oxalate crystal violet for 1 min, rinsed with sterile water, and air-dried on an alcohol lamp; iodine solution was added to the slide, mordanting was performed for about 1 min, and sterile water was rinsed and air-dried; 95% alcohol was added for decolorization, and after 20 s, the slide was rinsed and air-dried; staining was performed with safranin staining solution for 1 min, and sterile water was rinsed and air-dried; finally, microscopic examination was performed using an optical microscope.

[0049] (4) Physiological experiments: Catalase experiment: Drop 100 μL of 3% H2O2 enzyme on a blank culture dish, pick up a single colony with an inoculation loop and place it on the H2O2 enzyme to observe whether bubbles are produced. If bubbles are produced, it is catalase positive. If no bubbles are produced, it is catalase negative.

[0050] Glucose gas production experiment: First, culture lactic acid bacteria, pick a single colony of lactic acid bacteria and inoculate it in MRS liquid, and culture it at 30 ºC for 24 hours. Invert the Duroc tube into a test tube, inoculate the lactic acid bacteria liquid into the MRS liquid at a 1% inoculation rate, and culture it at 30 ºC for 7 days to observe whether bubbles are generated in the Duroc tube. If bubbles are generated, it is heterotypic fermentation, and if no bubbles are generated, it is homotypic fermentation.

[0051]

[0052] As can be seen from Table 1, strain ZYpet-009 and strain ZYpet-010 are both homofermentative lactic acid bacteria, with Gram staining results being positive and catalase negative.

[0053] 2. Strain identification

[0054] The strain was cultured anaerobically at 30°C on an MRS plate for 48 h, and a single colony was picked for PCR. The primers were 16S rRNA universal primers 27F and 1492R, and the PCR reaction conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 90 s, 35 cycles; 72°C final extension for 5 min. The obtained 16S rRNA was subjected to BLAST analysis with the sequences of known strains on the GenBank website, and a phylogenetic tree was constructed using MEGA-X software (see Figure 1 ). Phylogenetic tree analysis identified the ZYpet-009 strain as Lactobacillus acidophilus ( 嗜酸乳杆菌 ), named as Lactobacillus acidophilus ZYpet-009, and ZYpet-010 strain was identified as Lactobacillus salivarius ( 唾液利乳酸菌 ), named as Lactobacillus salivarius ZYpet-010.

[0055] The specific sequence is as follows:

[0056] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0057] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0058] ZYpet-009 strain 16S rRNA gene sequence:

[0059]

[0060] ZYpet-010 strain 16S rRNA gene sequence:

[0061]

[0062] Example 2: Experiment on simulated artificial gastrointestinal fluid using Lactobacillus acidophilus ZYpet-009 and saliva combined with Lactobacillus ZYpet-010

[0063] In order to explore the gastrointestinal tolerance of the two strains of bacteria isolated in Example 1, Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010, the following simulated artificial gastrointestinal fluid experiment was conducted in this example:

[0064] (1) Preparation of simulated gastric fluid: Dissolve 0.35 g of pepsin in 0.2% sterile saline and adjust the pH to 2.0 so that the total volume of the solution reaches 100 mL. Filter through a 0.22 µm filter membrane for later use.

[0065] (2) Preparation of simulated intestinal fluid: Dissolve 0.1 g of trypsin, 1.8 g of ox bile salts, and 1.1 g of sodium bicarbonate in 0.2% sterile saline and adjust the pH to 6.8 so that the total volume of the solution reaches 100 mL. Filter through a 0.22 µm filter membrane for later use.

[0066] (3) 2% 1×10 8 CFU / mL Lactobacillus acidophilus ZYpet-009 bacterial solution and Lactobacillus salivais ZYpet-010 were added to 20 mL of gastric juice and incubated for 3 h, 100 µL of the bacterial solution cultured for 3 h was added to 900 µL of intestinal fluid and incubated for 4 h. The dilution coating method was used to determine the number of viable lactic acid bacteria in gastric juice at 0 h, 3 h, intestinal juice at 0 h and 4 h on MRS plates, and the survival rate in the simulated gastrointestinal tract was calculated.

[0067] like Figure 2 As shown, the effective bacterial counts of Lactobacillus acidophilus ZYpet-009 before and after treatment with gastric juice for 0 and 3 hours were 8.17 and 6.99 CFU / mL, respectively, and the survival rate was 85.64%, while the effective bacterial counts before and after treatment with intestinal juice for 0 and 4 hours were 5.99 and 6.32 CFU / mL, respectively, and the survival rate was 88.8%. The effective bacterial counts of Lactobacillus salivais ZYpet-010 before and after treatment with gastric juice for 0 and 3 hours were 8.22 and 7.13 CFU / mL, respectively, and the survival rate was 86.74%, while the effective bacterial counts before and after treatment with intestinal juice for 0 and 4 hours were 6.13 and 5.47 CFU / mL, respectively, and the survival rate was 89.23%. Strains ZYpet-009 and ZYpet-010 still maintained a survival rate of more than 80% after treatment with gastric and intestinal juices.

[0068] The above experimental results confirm that both Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivais ZYpet-010 have good gastrointestinal tolerance.

[0069] Example 3: Hydrophobicity and self-aggregation experiments of Lactobacillus acidophilus ZYpet-009 and saliva-combined Lactobacillus ZYpet-010 cells

[0070] In order to explore the cell hydrophobicity and self-aggregation of the two strains of bacteria isolated in Example 1, Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivarius ZYpet-010, the following experiments were conducted in this example:

[0071] Select different lactic acid bacteria monoclonal colonies and inoculate them into 20 mL of MRS liquid medium, and culture them anaerobically at 30°C for about 16 hours. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, rinse twice with PBS, resuspend in PBS, and measure the absorbance at 600 nm as A0. Mix 3 mL of cell suspension with 1 mL of xylene, vortex the two-phase system for 2 minutes, and let stand at 37°C for 30 minutes to separate the aqueous phase and the organic phase. Carefully remove the aqueous phase and measure its absorbance at 600 nm as A1. The cell surface hydrophobicity (%) is calculated using the formula: (1-A1 / A0)×100%.

[0072] 3 mL of cell suspension was placed at 37°C for 0 and 8 h, and its absorbance at 600 nm was detected and recorded as At (the absorbance at each specific time was marked as a different At). The self-aggregation (%) was calculated using the formula: 1-(At / A0)×100, and each experiment was repeated three times.

[0073]

[0074] As can be seen from Table 2, the hydrophobicity of strain ZYpet-009 is 43.16, and the self-aggregation is 60.4, and the hydrophobicity of strain ZYpet-010 is 46.36, and the self-aggregation is 62.2, indicating that strain ZYpet-009 and strain ZYpet-010 have higher hydrophobicity and self-aggregation.

[0075] Example 4: Safety evaluation of Lactobacillus acidophilus ZYpet-009 and Lactobacillus saliva-combined ZYpet-010

[0076] In order to explore the safety of the two strains of bacteria isolated in Example 1, Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivarius ZYpet-010, the following experiments were conducted in this example:

[0077] 1. Antibiotic sensitivity test

[0078] The susceptibility of strains ZYpet-009 and ZYpet-010 to 10 common antibiotics, including gentamicin (GEN), ciprofloxacin (CIP), ceftriaxone (CTR), erythromycin (E), ampicillin (AMP), tetracycline (TET), sulfamethoxazole-trimethoprim (SXT), chloramphenicol (C), lincomycin (MY), and penicillin (PEN), was determined according to the method of Clinical and Laboratory Standards Institute (CLSI).

[0079] 5% inoculum of ZYpet-009 and ZYpet-010 bacterial liquid was added to MRS solid culture medium cooled to about 45 ℃, and antibiotic sensitivity tablets were placed on the surface. The strains cultured on MRS agar plates without antibiotics were used as controls. After culturing at 30 ℃ for 24 h, the diameters of the inhibition zones were measured with a vernier caliper. The sensitivity of strains ZYpet-009 and ZYpet-010 to 10 antibiotics were determined based on the diameters.

[0080]

[0081] As can be seen from Table 3, strain ZYpet-009 and strain ZYpet-010 were resistant to gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole, sensitive to erythromycin, ampicillin, chloramphenicol, and penicillin, and moderately resistant to ceftriaxone, tetracycline, and lincomycin.

[0082] 2. Hemolysis test

[0083] The hemolytic activity of strains ZYpet-009 and ZYpet-010 was analyzed according to the instructions for Columbia blood agar plates. After strains ZYpet-009 and ZYpet-010 were subcultured twice, single clones were streaked on blood agar plates. They were then anaerobically cultured at 30°C for 24 h to observe whether they had hemolytic activity. Staphylococcus aureus ATCC 29213 was used as the T As a positive control.

[0084] like Figure 3 As shown, Staphylococcus aureus ATCC 29213 TIt has obvious hemolytic activity, and the blood plate under the colonies is transparent, while strains ZYpet-009 and strains ZYpet-010 do not have hemolytic activity.

[0085] 3. Detection of virulence factor genes, biogenic amine synthesis genes and antibiotic resistance genes

[0086] PCR was used to amplify the common virulence factor genes, biogenic amine synthesis genes, and antibiotic resistance genes in strains ZYpet-009 and ZYpet-010. 粪肠球菌 ATCC 29212 T ) was used as a positive control, and ultrapure water (milli-Q) was used as a negative control.

[0087] The targeted genes and PCR primers are as follows:

[0088] Gelatinase (gelatinase, gelE):

[0089] F:5'-TATGACAATGCTTTTTGGGAT-3';

[0090] R:5'-AGATGCACCCGAAAATAATATA-3'.

[0091] Cytolisin (cylA):

[0092] F:5'-GAATTGAGCAAAAGTTCAATCG-3;

[0093] R:5'-GTCTGTCTTTTCACTTGTTTC-3'.

[0094] Adhesion of collagen (ACE):

[0095] F: 5'-ACTCGGGGATTGATAGGC-3'; R: 5'-GCTGCTAAAGCTGCGCTT-3'.

[0096] Vancomycin (vancomyc, vanA):

[0097] F:5'-TCTGCAATAGAGATAGCCGC-3';

[0098] R:5'-GGAGTAGCTATCCCGCATT-3'.

[0099] Tetracycline resistance (tetM):

[0100] F:5'-ATTACACTTCCGATTTCGG-3'; R:5'-GTTAAATAGTGTTCTTGGAG-3'.

[0101] Histidine decarboxylase (histidine decar-boxylase, hdc):

[0102] F: 5'-AGATGGTATTGTTTCTTATG-3';

[0103] R: 5'-AGACCATACACCATAACCTT-3'.

[0104] Tyrosine decarboxylase (TDC):

[0105] F:5'-GAYATNATNGGNATNGGNYTNGAYCARG-3';

[0106] R:5'-CCRTARTCNGGNATAGCRAARTCNGTRTG-3'.

[0107] Ornithine decarboxylase (odc):

[0108] F: 5'-GTNTTYAAYGCNGAYAARCANTAYTTYGT-3';

[0109] R: 5'-ATNGARTTNAGTTCRCAYTTYTCNGGG-3'.

[0110] The 16S rRNA gene of a single colony of the selected strain was amplified by PCR using universal primers 27F and 1492R.

[0111] The PCR reaction system was 50 μL: 25 μL 2×Taq Plus Master mix, 1 μL each of 27F and 1492R primers, and 23 μL ddH2O. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 50 s, 52°C annealing for 50 s, 72°C extension for 50 s, and after 33 cycles, a final extension at 72°C for 17 min.

[0112] PCR product detection: The PCR product was placed on a 1% agarose gel for electrophoresis. After electrophoresis, it was stained with ethidium bromide solution (EB) and the amplified product was observed and inspected in the UV gel imaging system Gel Doc™ XR +imaging system (Universal Hood II, BIO-RAD, USA).

[0113]

[0114] As can be seen in Table 4, the PCR results showed that there were no virulence factor genes in ZYpet-009 and ZYpet-010, such as the adhesin-related ace gene, gelatinase gelE gene and hemolysin cylA gene; biogenic amine synthesis genes, such as histidine decarboxylase hdc, tyrosine decarboxylase tdc and ornithine decarboxylase odc; antibiotic resistance gene vancomycin vanA and tetracycline resistance tetM gene.

[0115] In summary, it can be determined that Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivais ZYpet-010 have high safety performance.

[0116] Example 5: Probiotics of Lactobacillus acidophilus ZYpet-009 and saliva combined with Lactobacillus ZYpet-010

[0117] This embodiment provides a probiotic preparation for relieving diarrhea and soft stool in pets, wherein the probiotic preparation contains Lactobacillus acidophilus ( 嗜酸乳杆菌 ) ZYpet-009 and Lactobacillus salivais ( 唾液利乳酸菌 )ZYpet-010.

[0118] Lactobacillus acidophilus ZYpet-009 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 31559; Lactobacillus salivarius ZYpet-010 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 31560.

[0119] The preparation method of the probiotic agent is:

[0120] (1) Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivarius ZYpet-010 were activated on MRS plate culture medium, respectively, and then inoculated into MRS liquid culture medium at a 1% inoculation rate. The culture was cultured at 37°C for 24 h to obtain bacterial suspension.

[0121] (2) Centrifuge the bacterial solution, collect the cells, wash them with sterile saline, and resuspend them in 15% (w / w) reconstituted skim milk to obtain a suspension. The effective viable bacterial concentration of the suspension was adjusted to 2.0×10 9 CFU / mL, a bacterial suspension was obtained, and after freeze-drying the bacterial suspension, Lactobacillus acidophilus ZYpet-009 powder and Lactobacillus salivarius ZYpet-010 powder were obtained respectively.

[0122] (3) The above two bacterial powders were mixed with glucose to prepare a bacterial agent. The total number of cells of the two bacteria in the bacterial agent was 5.0×10 9 CFU / g.

[0123] Among them, the addition ratio of the two bacterial powders to glucose is 0.5:0.5:4.

[0124] Example 6: Effect of compound probiotics on diarrhea in mice

[0125] In order to explore the effects of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010 on the intestinal health of animals, the following animal experiments were conducted in this example:

[0126] Forty-eight 6-8 week old BALB / c male mice (clean grade) were selected and randomly divided into the following six groups, with eight mice in each group.

[0127] (1) Control group: fed with normal feed;

[0128] (2) Model group: normal feed + challenge (0.2 mL enteropathogenic Escherichia coli);

[0129] (3) Antibiotic group: normal feed + 0.2% chlortetracycline + challenge (0.2 mL enteropathogenic Escherichia coli);

[0130] (4) ZYpet-009 group: normal feed + 1 g Lactobacillus acidophilus ZYpet-009 + challenge (0.2 mL enteropathogenic Escherichia coli).

[0131] (5) ZYpet-010 group: normal feed + 1 g saliva combined with Lactobacillus ZYpet-010 + challenge (0.2 mL enteropathogenic Escherichia coli).

[0132] (6) Compound probiotic group: normal feed + 0.5 g Lactobacillus acidophilus ZYpet-009 + 0.5 g saliva combined with Lactobacillus ZYpet-010 + challenge (0.2 mL enteropathogenic Escherichia coli).

[0133] The light cycle was 12 h per day, the ambient temperature was 20-25℃, the relative humidity was 50-60%, and the mice were free to eat and drink. The feed and drinking water were changed every day. The feed was a standard commercial rodent feed. After all mice were adaptively fed with normal feed for one week, the formal experiment was carried out. The formal experimental period was 4 weeks. In the first three weeks, each group was fed with different diets (Table 5). In the fourth week, the model group, antibiotic group, single probiotic group and compound probiotic group were fed with 0.2 mL of enteropathogenic Escherichia coli every day, and the control group was fed with 0.2 mL of normal saline every day. At about 4 pm on the 0th, 7th, 14th, 21st and 28th day, the weight and diarrhea rate of each mouse were observed and recorded (diarrhea rate % = number of diarrheal mice in each group / total number of mice in this group × 100%).

[0134] Blood was collected from the mouse eyeballs and centrifuged at 3000 rpm for 10 min. The serum was collected and quickly frozen in liquid nitrogen and stored at -80°C for later use. The levels of immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and interleukin 10 (IL-10) in the serum were determined using Dogesce enzyme-linked immunosorbent assay kit (Beijing Dongge Boye Biotechnology Co., Ltd) according to the manufacturer's instructions.

[0135]

[0136]

[0137]

[0138] As shown in Tables 6 and 7, the weight gain of mice pre-fed for three weeks did not change significantly in the six groups, but after one week of challenge, the weight of the model group, antibiotic group, single probiotic group and compound probiotic group was significantly lower than that of the control group, and there was no significant difference between the probiotic group and the antibiotic group, all of which were better than the model group. The diarrhea rate of mice in the compound probiotic group was lower than that in the model group, antibiotic group and single probiotic group.

[0139]

[0140] As can be seen from Table 8, the level of tumor necrosis factor (TNF-α) in mice fed with the compound probiotic group was the lowest, and the compound probiotic group and the control group were significantly lower than the model group, the antibiotic group and the single probiotic group. TNF-α is the most common proinflammatory factor in animal serum, which can increase cell permeability by activating lymphocytes and neutrophils. The lower the TNF-α value, the milder the inflammatory response. Interleukin-10 (IL-10) is one of the most effective anti-inflammatory cytokines and plays an important role in regulating intestinal homeostasis during host defense. The IL-10 content in the serum of mice in the compound probiotic group and the control group was significantly higher than that in the model group, the antibiotic group and the single probiotic group. In addition, interferon (IFN-γ) is also a lymphokine that regulates immunity. The IFN-γ in the serum of mice in the control group, the antibiotic group and all probiotic groups was significantly lower than that in the model group.

[0141]

[0142] As can be seen from Table 9, immunoglobulins include IgG, IgM and IgA, which play a key role in the immune system. IgG is mainly responsible for chronic and memory immune responses, neutralizing toxins and viruses, activating the complement system, and providing passive immunity of placental transport in mammals. IgM is the main antibody in the primary immune response, rapidly activating the complement system and enhancing the efficiency of pathogen clearance. IgA mainly plays a role in mucosal immunity, neutralizing pathogens and toxins to prevent their invasion and spread on mucosal surfaces such as the respiratory tract and digestive tract. Compared with the model group, the control group and the single probiotic group, feeding compound probiotics can significantly increase the levels of IgG, IgM and IgA in mouse serum.

[0143] Example 7: Effect of compound probiotics on alleviating soft stool in pet cats

[0144] In order to explore the improvement effect of Lactobacillus acidophilus ZYpet-009 and Lactobacillus saliva-combined ZYpet-010 on soft stool in pet cats, the following animal experiments were conducted in this example:

[0145] Twenty cats with soft stools and diarrhea were divided into four groups: model control group, ZYpet-009 group, ZYpet-010 group and compound probiotic group. Another five healthy cats served as blank control groups. The ZYpet-009 group was fed 2 g of Lactobacillus acidophilus ZYpet-009 on top of the basic cat food daily. The ZYpet-010 group was fed 2 g of saliva-combined Lactobacillus ZYpet-010 on top of the basic cat food daily. The compound probiotic group was fed 1 g of Lactobacillus acidophilus ZYpet-009 and 1 g of saliva-combined Lactobacillus ZYpet-010 on top of the basic cat food daily. Cats in other groups ate ordinary cat food normally for 2 weeks. The fecal scores of the cats were recorded every 2 days and scored by professional doctors in a pet hospital according to a 5-point system. The scoring criteria are shown in Table 10 and the results are shown in Table 11. After the experiment, the veterinarian performed venous blood sampling, collected the blood, and centrifuged it at 3000rpm for 15 minutes to obtain serum. The Shanghai ELISA Biotechnology Co., Ltd. was used to determine the levels of D-lactic acid (D-LA) and diamine oxidase (DAO), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum.

[0146]

[0147]

[0148] As shown in Table 11, the stool score of the compound probiotic group improved from 4 points of soft stool to 3 points of normal after 4 days of feeding, while the stool of the model control group returned to normal after 8 days of feeding, and the stool of the single probiotic group also took 6 days to return to normal.

[0149]

[0150] As shown in Table 12, the D-lactic acid (D-LA) in the serum of cats in the single probiotic group, compound probiotic group and blank control group was significantly lower than that in the model control group, and the content of the compound probiotic group was the lowest among the five groups; the content of diamine oxidase (DAO) in the compound probiotic group was significantly lower than that in the other four groups. The compound probiotics composed of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivais ZYpet-010 can effectively improve intestinal health, reduce lactic acidosis, reduce intestinal permeability, and alleviate inflammatory response, thereby regulating the cat's soft stool problem and improving its overall health status.

[0151]

[0152] As shown in Table 13, the levels of immunoglobulin G (IgG) and immunoglobulin A (IgA) in the serum of cats in the compound probiotic group were significantly higher than those in the blank control group, the model control group and the single probiotic group. This is because Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010 can promote each other, regulate the balance of intestinal flora, enhance intestinal barrier function and immune response, and thus increase the levels of IgG and IgA. The increase in IgG and IgA helps to enhance the immune system function of cats, inhibit the colonization and reproduction of pathogens in the intestine, and reduce intestinal inflammation, thereby effectively improving the problem of soft stool and improving overall health.

[0153] Comparative ratio: Effects of other compound probiotic combinations on alleviating soft stool in pet cats

[0154] In order to explore the effects of different strains on animal intestinal health, this comparative example provides two composite probiotics, namely:

[0155] (1) Combination 1: Lactobacillus acidophilus ZYpet-009 + Lactobacillus salivais C326;

[0156] (2) Combined combination 2: Saliva combined with Lactobacillus ZYpet-010 + Lactobacillus acidophilus C55.

[0157] The same method as in Example 7 was used to conduct an animal experiment: another 10 cats with soft stools and diarrhea were divided into 2 groups: Compound combination 1: 1 g of Lactobacillus acidophilus ZYpet-009 and 1 g of saliva-combined Lactobacillus C326 were additionally fed on the basis of the basic cat food; Compound combination 2: 1 g of saliva-combined Lactobacillus ZYpet-010 and 1 g of Lactobacillus acidophilus C55 were additionally fed on the basis of the basic cat food. The control group was the composite probiotic group of Example 7. The test method refers to Example 7.

[0158]

[0159] As shown in Table 14, the stool score of the compound probiotic group (1g Lactobacillus acidophilus ZYpet-009 + 1g saliva combined Lactobacillus ZYpet-010) improved from 4 points of soft stool to a normal 3 points after the 4th day of feeding, while the compound combinations 1 and 2 both required 6 days of feeding for the kittens' stool status to improve to 3 points.

[0160]

[0161] As shown in Table 15, the levels of D-lactic acid (D-LA) and diamine oxidase (DAO) in the serum of cats in the compound probiotic group were significantly lower than those in compound combinations 1 and 2, indicating that the compound probiotics composed of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivais ZYpet-010 can effectively improve intestinal health, reduce lactic acidosis, reduce intestinal permeability, and improve overall health status.

[0162]

[0163] As shown in Table 16, the levels of immunoglobulin G (IgG) and immunoglobulin A (IgA) in the serum of cats in the compound probiotic group were significantly higher than those in compound combination 1, and the levels of immunoglobulin G in the serum of cats in the compound probiotic group were significantly higher than those in compound combination 2. There were no significant differences in other indicators. The increase of IgG and IgA helps to enhance the immune system function of cats, inhibit the colonization and reproduction of pathogens in the intestine, and reduce intestinal inflammation, thereby effectively improving the problem of soft stool and improving overall health.

[0164] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A composite probiotic for relieving diarrhea and soft stool in pets, characterized in that: The composite probiotics include Lactobacillus acidophilus ( Lactobacillus acidophilus ) ZYpet-009 and Lactobacillus salivais ( Ligilactobacillus salivarius )ZYpet-010; Lactobacillus acidophilus ZYpet-009, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No. 31559; Saliva-associated Lactobacillus ZYpet-010 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No. 31560; The ratio of the live bacteria content of Lactobacillus acidophilus ZYpet-009 and Lactobacillus salivae ZYpet-010 in the composite probiotics is 1:5-5:

1.

2. A probiotic agent for relieving diarrhea and soft stool in pets, characterized in that: The probiotic agent comprises the composite probiotic according to claim 1.

3. The probiotic agent according to claim 2, characterized in that The 16S rDNA gene sequences of the Lactobacillus acidophilus ZYpet-009 and the Lactobacillus salivarius ZYpet-010 are shown in SEQ ID NO.: 1 and SEQ ID NO.: 2, respectively.

4. The probiotic agent according to claim 2, characterized in that: The effective viable bacteria count of the composite probiotics in the probiotic agent is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. A method for preparing the probiotic agent according to any one of claims 2 to 4, characterized in that: The preparation method comprises the following steps: (1) Activating Lactobacillus acidophilus ZYpet-009 and Lactobacillus acidophilus ZYpet-010, amplifying the culture in liquid medium, collecting the cells by centrifugation, resuspending the cells in reconstituted skim milk, adjusting the effective live bacterial concentration, and freeze-drying the cells to obtain bacterial powder; (2) The bacterial powder is mixed with glucose to prepare the probiotic agent.

6. Use of the composite probiotic according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in the preparation of a product for promoting intestinal health of animals, wherein the product is a veterinary drug and the animal is a cat or a mouse.

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