Young cat source pediococcus pentosaceus JZ01 and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to kitten source pediococcus pentosaceus JZ01 and application thereof. The kitten source pediococcus pentosaceus JZ01 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 19, 2024, and the preservation number is CGMCC NO.31002. The kitten source pediococcus pentosaceus JZ01 has the advantages that the kitten source pediococcus pentosaceus JZ01 is The kitten source pediococcus pentosaceus JZ01 disclosed by the invention has relatively strong acid resistance, bile salt resistance and extremely strong intestinal adhesion capacity, has a good inhibition effect on common pathogenic bacteria, is sensitive to various antibiotics and has good probiotic potential, a new candidate strain is provided for pet food, health-care treatment products or probiotic preparations, and the kitten source pediococcus pentosaceus JZ01 has a good application prospect. Wide application prospects are realized in the aspects of preparing pet foods, health care products, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Pediococcus pentosaceus JZ01 derived from kittens and its applications. Background Art

[0002] In recent years, with the increasing demand for pet health, the research and development of probiotics targeting intestinal microecological balance has become a hot field. The animal intestinal flora plays a key role in pet health by regulating the host immune response, inhibiting the colonization of pathogenic bacteria, and maintaining the mucosal barrier function. Among them, lactic acid bacteria, as one of the dominant intestinal flora, can enhance the host immunity through mechanisms such as enhancing the activity of natural killer cells and promoting macrophage phagocytosis, becoming an important direction for probiotic development.

[0003] Currently, due to its natural property of being rich in lactic acid bacteria, cat milk has been regarded as an important source for probiotic isolation. However, the types and functions of existing cat milk-derived strains are still relatively limited and difficult to meet diverse application requirements. In addition, probiotics need to have acid tolerance, bile salt tolerance, and strong adhesion ability for colonization in the digestive tract, and there are few candidate strains with the above characteristics among existing strains, which limits their application in pet food and medical fields.

[0004] Pediococcus pentosaceus, as a Gram-positive lactic acid bacterium, has been proven to have the potential to regulate the intestinal flora, enhance immunity, and metabolize beneficial products. However, Pediococcus pentosaceus traditionally isolated from fermented foods or mammalian milk often shows insufficient intestinal adaptability and functional stability due to host-specific differences, and there is an urgent need to develop new strains with stronger host adaptability.

[0005] In addition, the safety assessment of probiotics is crucial, especially the risk of carrying antibiotic resistance genes needs to be avoided. Some existing strains have potential antibiotic resistance hazards, and the strain resources that meet the sensitive characteristics are scarce, further restricting their compliant application in pet health care products. Therefore, screening new strains of Pediococcus pentosaceus with strong tolerance, high adhesion, broad-spectrum antibacterial characteristics, and meeting the antibiotic sensitivity standard from the intestinal environment of healthy kittens will provide a better solution for the development of pet microecological agents.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a Pediococcus pentosaceus JZ01 derived from kittens and its applications. This strain is a strain with excellent probiotic properties selected after isolation, screening, and identification from the feces of healthy kittens, providing a new candidate strain for cat milk replacers, pet foods, and health care treatment products.

[0008] In the first aspect of the present invention, a strain of Pediococcus pentosaceus JZ01 derived from young cats is provided. The preservation number of this Pediococcus pentosaceus is CGMCC NO. 31002. It is preserved in the China General Microbiological Culture Collection Center, with the preservation date being June 19, 2024, and the preservation address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0009] Through a large number of experiments, the present invention screened the above-mentioned Pediococcus pentosaceus JZ01 from the feces of healthy young cats. Extracting probiotic groups from young cat feces has many significant advantages. The strain itself is derived from the pet intestine and can better adapt to the host intestinal environment compared with strains from other sources, and can precisely regulate the balance of the intestinal flora of young cats. Such probiotics can tolerate the gastrointestinal environment, have a close symbiotic relationship with young cats, are stably colonized, and have high safety, no toxic side effects, and rarely cause allergies, which is an excellent choice to promote the healthy growth of young cats.

[0010] The 16s rRNA gene sequence of the Pediococcus pentosaceus JZ01 derived from young cats of the present invention is as shown in SEQ ID NO: 01.

[0011] The Pediococcus pentosaceus JZ01 derived from young cats of the present invention has an inhibitory effect on Gram-positive bacteria and Gram-negative bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae.

[0012] Furthermore, the diameter of the inhibition zone of the Pediococcus pentosaceus JZ01 derived from young cats against Klebsiella pneumoniae and Escherichia coli is ≥ 17.5 mm; the diameter of the inhibition zone against Salmonella and Methicillin-resistant Staphylococcus aureus is ≥ 22.2 mm; the diameter of the inhibition zone against Staphylococcus aureus is ≥ 28 mm.

[0013] The Pediococcus pentosaceus JZ01 derived from young cats of the present invention has no hemolytic property and is a safe and non-toxic strain.

[0014] The Pediococcus pentosaceus JZ01 derived from young cats of the present invention is sensitive to ampicillin, penicillin, chloramphenicol, and cefazolin, can avoid the problem of antibiotic resistance, can prevent the imbalance of the intestinal flora, and has safety.

[0015] The Pediococcus pentosaceus JZ01 strain derived from young kittens of the present invention has acid tolerance, bile salt tolerance and intestinal adhesion ability, can stably survive and colonize in the host, and can play a probiotic role in the intestine to regulate gastrointestinal dysfunction caused by disordered intestinal flora structure.

[0016] Furthermore, the survival rate of Pediococcus pentosaceus JZ01 derived from young kittens is ≥92.33% after surviving for 3 h under the condition of pH 2.5, ≥97.17% after surviving for 3 h under the condition of pH 3, and ≥100.00% after surviving for 3 h under the condition of pH 3.5. The survival rate of Pediococcus pentosaceus JZ01 derived from young kittens is ≥81.28% after surviving for 3 h under the condition of 0.3% bile salt, ≥100.00% after surviving for 3 h under the condition of 0.1% bile salt, and ≥100.00% after surviving for 3 h under the condition of 0.2% bile salt.

[0017] Furthermore, Pediococcus pentosaceus JZ01 derived from young kittens has extremely strong cell adhesion ability, and the adhesion rate reaches 33.89%, indicating that this strain has good probiotic potential. Adhesion and colonization in the intestine are important prerequisites for lactic acid bacteria to exert their ecological effects and physiological functions, and are one of the important bases for evaluating their probiotic potential. The adhesion of lactic acid bacteria to epithelial cells is the main basis for their probiotic functions. The adhered lactic acid bacteria can exist in the intestine and resist the invasion of pathogenic bacteria, and produce metabolites such as antibacterial peptides to help effectively kill pathogenic bacteria, achieving functions such as avoiding intestinal infection by pathogenic bacteria and enhancing the immune ability of the body.

[0018] The Pediococcus pentosaceus JZ01 strain derived from young kittens of the present invention has antioxidant properties.

[0019] Furthermore, the scavenging rate of the bacterial suspension of Pediococcus pentosaceus JZ01 derived from young kittens against DPPH free radicals is greater than 51.60%, and the scavenging rate of the supernatant against ABTS free radicals is greater than 88.40%.

[0020] In the second aspect of the present invention, a bacterial agent is provided, and the bacterial agent contains Pediococcus pentosaceus JZ01.

[0021] In the third aspect of the present invention, there is provided an animal food, a health product, and a medicine, which contain Pediococcus pentosaceus JZ01 derived from young cats and / or its metabolites, or a bacterial agent containing Pediococcus pentosaceus JZ01 derived from young cats.

[0022] In the fourth aspect of the present invention, there is provided the use of the Pediococcus pentosaceus JZ01 derived from young cats in a medicine or food for treating, preventing or alleviating gastrointestinal dysfunction in animals.

[0023] Preferably, the animals include but are not limited to cats.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention provides a new strain of Pediococcus pentosaceus JZ01 derived from young cats, which has strong adhesion ability and good probiotic ability.

[0026] (2) The Pediococcus pentosaceus JZ01 provided by the present invention has no hemolytic property and high biosafety.

[0027] (3) The Pediococcus pentosaceus JZ01 provided by the present invention has good acid tolerance, bile salt tolerance and intestinal adhesion ability.

[0028] (4) The Pediococcus pentosaceus JZ01 provided by the present invention has certain antibacterial ability and has good inhibitory effects on common Gram-positive bacteria and Gram-negative bacteria.

[0029] (5) The Pediococcus pentosaceus JZ01 provided by the present invention has certain sensitivity to antibiotics, can avoid the problem of antibiotic resistance, prevent the imbalance of intestinal flora, and has safety.

[0030] (6) The Pediococcus pentosaceus JZ01 provided by the present invention is a strain with excellent probiotic properties selected after isolation, screening and identification from the feces of healthy young cats, provides a new candidate strain for pet foods, health care products or probiotic preparations, and has broad application prospects in the preparation of pet foods, health products, medicines, etc. Description of the Drawings

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 is the result of the acid resistance experiment with pH = 2.5 in Example 1;

[0033] Figure 2 is the result of the bile salt resistance experiment with a bile salt concentration of 0.3% in Example 1;

[0034] Figure 3 is the antibacterial ability result of Pediococcus pentosaceus JZ01 from kittens in Example 1;

[0035] Figure 4 is the BLAST database annotation result in Example 2;

[0036] Figure 5 is the colony morphology of Pediococcus pentosaceus JZ01 from kittens in Example 2;

[0037] Figure 6 is the growth curve of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0038] Figure 7 is the result of the in vitro acid resistance ability of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0039] Figure 8 is the result of the in vitro bile salt resistance ability of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0040] Figure 9 is the hemolytic ability result of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0041] Figure 10 is the result of the adhesion experiment of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0042] Figure 11 is the result of the DPPH free radical scavenging test of Pediococcus pentosaceus JZ01 from kittens in Example 3;

[0043] Figure 12 is the result of the ABTS free radical scavenging test of Pediococcus pentosaceus JZ01 from kittens in Example 3. Specific Embodiments

[0044] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0045] Example 1: Screening and isolation of Pediococcus pentosaceus JZ01 from young cats

[0046] 1. Cultivation and isolation of strains

[0047] Select fresh and healthy young cat fecal samples for screening strains. After gradient dilution with sterilized PBS buffer (purchased from Beijing Saiweier Biotechnology Co., Ltd.), coat them on MRS solid medium containing 1% CaCO3, and culture them in an anaerobic incubator at 37°C for 48 h. Pick white or milky white single colonies with obvious transparent circles for Gram staining and microscopic examination. Select rod-shaped and Gram-positive bacteria strains. Select the relatively well-growing Pediococcus pentosaceus strains JZ01, JZ60, JZ66, and JZ23 from among many strains, and conduct enrichment culture in an anaerobic incubator at 37°C; mix the bacterial cells with sterilized 50% glycerol and store them in a -80°C refrigerator for later use.

[0048] 2. Screening of strains

[0049] Inoculate the strains of JZ01, JZ60, JZ66, and JZ23 stored with glycerol under sterile conditions into MRS broth medium, and culture them anaerobically at 37°C for 24 h, and activate them continuously for three generations; after centrifuging the bacterial liquid, wash the medium with sterilized PBS 2-3 times.

[0050] Conduct acid tolerance, bile salt tolerance, and antibacterial tests on the above strains of JZ01, JZ60, JZ66, and JZ23.

[0051] The test steps for acid tolerance, bile salt tolerance, and adhesion are as follows:

[0052] (1) Acid tolerance test

[0053] Resuspend the washed medium in MRS broth medium with a pH of 2.5, culture it anaerobically at 37°C, take samples for gradient dilution at 0 h and 3 h respectively, coat them on MRS solid medium plates, and count after anaerobic culture at 37°C for 48 h.

[0054] Survival rate of the strain (%) = lg(N1 / N0) * 100%;

[0055] In the formula: N0 is the viable count (CFU / mL) at 0 h; N1 is the viable count (CFU / mL) of the strain after 3 h of acid tolerance.

[0056] The experimental results are as Figure 1 shown, and the survival rate of strain JZ01 is the highest, reaching as high as 92.33%.

[0057] (2) Bile salt tolerance test

[0058] The washed culture medium was resuspended in MRS broth medium containing 0.3% bile salt and cultured anaerobically at 37°C. Samples were taken at 0 h and 3 h for serial dilution, spread on MRS solid medium plates, and counted after anaerobic culture at 37°C for 48 h.

[0059] Survival rate of strain (%) = lg(N1 / N0) * 100%;

[0060] Where: N0 is the number of viable bacteria at 0 h (CFU / mL); N1 is the number of viable bacteria after 3 h of bile salt tolerance of the strain (CFU / mL).

[0061] The experimental results are as Figure 2 shown, and the survival rate of strain JZ01 is 81.28%.

[0062] (3) Determination of antibacterial ability of Pediococcus pentosaceus JZ01 from kittens

[0063] Pet foods contain a large amount of nutrients. In daily life, food spoilage and deterioration not only endanger the health of pets but also cause economic losses. Microbial contamination and growth are the main reasons for food spoilage and deterioration. Therefore, the antibacterial performance of JZ01 is one of the safety indicators for its application in food.

[0064] Using five common foodborne pathogenic bacteria, Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae, as indicator bacteria, after activating the indicator bacteria for three generations, the Oxford cup method was used for the preliminary determination of antibacterial activity.

[0065] Using the Oxford cup method, the specific method is as follows: Take the activated strain and inoculate it into MRS broth medium at an inoculation amount of 2% (V / V), and culture it anaerobically at 37°C for 24 h. Serial dilutions of 10-fold of the activated Escherichia coli, Staphylococcus aureus, and Salmonella bacterial suspensions were made, and the dilution factor of 10 2The bacterial suspension is ready for use. Add 5 mL of LB agar medium to the petri dish for bottom sealing. After cooling and solidifying, place Oxford cups evenly. Take 100 μL of the indicator bacterial suspension of each gradient and add it to 15 mL of LB agar at 50 - 60 °C. Mix well and slowly pour it into the plate with Oxford cups. After condensation, use forceps to pull out the Oxford cups to form round holes with a size of 8×6×10. Add 200 μL of the fermentation supernatant of the strain into the round holes. After diffusion in a 4 °C refrigerator for 12 h, perform anaerobic culture at 37 °C for 9 - 12 h. Observe whether an inhibition zone appears, and measure the diameter of the inhibition zone with a vernier caliper. Each group has three parallel experiments, and the results are expressed as x±s.

[0066] The experimental results are as Figure 3 shown. Strain JZ01 has strong inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Salmonella, Klebsiella pneumoniae), and the diameters of the inhibition zones are all above 17.5 mm. Among them, the inhibitory effect on Staphylococcus aureus is the strongest, and the diameter of the inhibition zone of the supernatant is 28 mm.

[0067] From Figure 1 , Figure 2 and Figure 3 the results, it can be seen that Pediococcus pentosaceus JZ01 from kitten sources has extremely strong antibacterial ability, and the inhibition zone can reach 28 mm, which is much higher than that of other Pediococcus pentosaceus from kitten sources; and after surviving for 3 h under the acid tolerance condition of pH = 2.5 and the bile salt tolerance condition of 0.3% bile salt concentration, the survival rates reach 92.33% and 81.28% respectively, and the acid tolerance and bile salt tolerance abilities are better.

[0068] Example 2: Identification of Pediococcus pentosaceus JZ01 from kitten sources

[0069] Perform whole-genome sequencing analysis on the screened strain JZ01. Use the Pacbio (10Kb SMRT Bell library) and Illumina PE150 (350bp small fragment library) sequencing platforms to perform 16s rRNA genome sequencing on it. The sequenced sequences are as follows:

[0070]

[0071] The sequencing results were used for homology search in the gene bank by applying BLAST on the NCBI website. The results showed the highest similarity with *Pediococcus pentosaceus*. The results are as Figure 4 shown, and 100.00% of the genes corresponded to the genus *Pediococcus pentosaceus*.

[0072] The strain JZ01 was diluted and spread on the plate to observe the colony state. The results are as Figure 5 shown. Combining with the morphological characteristics of the colonies of JZ01, the strain JZ01 was identified as *Pediococcus pentosaceus*.

[0073] Example 3: Determination of the probiotic properties of *Pediococcus pentosaceus* JZ01 from kittens

[0074] 1. Determination of the growth curve of *Pediococcus pentosaceus* JZ01 from kittens

[0075] The cultured JZ01 bacterial solution was activated for three generations and inoculated into MRS broth medium, then placed in an incubator at 37 °C for anaerobic culture. The viable counts of each bacterium were measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h respectively;

[0076] The growth curve of *Pediococcus pentosaceus* JZ01 from kittens was plotted. The results are shown in Figure 6 the figure.

[0077] 2. Determination of the in vitro acid and bile salt tolerance of *Pediococcus pentosaceus* JZ01 from kittens

[0078] The cultured JZ01 bacterial solution was activated for three generations and inoculated into MRS broth medium with pH values of 3, 3.5 and bile salt concentrations of 0.1%, 0.2% respectively. Then it was placed in an incubator at 37 °C for anaerobic culture. Samples were taken for plate counting at 0 h and 3 h respectively;

[0079] The experimental results are shown in Figure 7 and Figure 8 respectively; It can be seen from Figure 7 that when the bacteria were cultured in an environment with pH 3 and pH 3.5 for 0 - 3 h, the growth of the bacteria was stable, and the viable count was above 10 8 CFU / mL, indicating strong acid tolerance.

[0080] It can be seen from Figure 8 that the bacteria grew relatively stably in the medium with a bile salt concentration of 0.1%; the survival rate decreased by 2% after being cultured in the medium with a bile salt content of 0.2% for 3 h, indicating strong bile salt tolerance.

[0081] 3. Determination of the antibiotic sensitivity of *Pediococcus pentosaceus* JZ01 from kittens

[0082] The excessive use of antibiotics in the pharmaceutical industry can lead to an enhanced drug resistance of pathogenic strains, and the flora in the human intestine is prone to disorder and imbalance. Therefore, obtaining lactic acid bacteria sensitive to antibiotics has become one of the important indicators for their safety in food applications.

[0083] Adjust the concentrations of the fermentation broths of each strain cultured for 24 h to 10 8 CFU / mL. Add 100 μL of the bacterial solution to the MRS solid plate, spread it evenly, and let it dry. Place 5 - 6 drug sensitivity test discs in each plate on average. After diffusing in a 4°C refrigerator for 12 h, incubate anaerobically at 37°C for 9 - 12 h. The sensitivity of the strain to the drug was judged by referring to the instructions for using the drug sensitivity test discs.

[0084] The results are shown in Table 1. Pediococcus pentosaceus JZ01 was extremely sensitive to ampicillin, penicillin, chloramphenicol, and cefazolin (cephalosporins), and tolerant to gentamicin, ciprofloxacin, and norfloxacin.

[0085] Table 1 Antibiotic Sensitivity Test

[0086] Antibiotic Name Gentamicin Ampicillin Penicillin Ciprofloxacin Chloramphenicol Cefazolin Norfloxacin Sensitivity R S S R S S R

[0087] S: susceptible, sensitive; I: intermediate, moderately sensitive; R: resistance, tolerant

[0088] 4. Determination of the hemolytic ability of Pediococcus pentosaceus JZ01 from kittens

[0089] Dip a inoculation loop into a small amount of the bacterial solution and streak it on a Columbia blood agar plate. Incubate anaerobically at 37°C for 24 - 36 h, and observe whether there is a hemolytic zone on the plate. According to the hemolytic phenomenon, it can be divided into α-hemolysis (greenish hemolysis), β-hemolysis (completely transparent hemolytic ring), and γ-hemolysis (non-hemolysis).

[0090] The hemolysis experiment can detect the safety of the strain and determine whether it has hemolytic activity. The results are as Figure 9 shown. No hemolysis occurred on the blood plate for this strain, which was γ-hemolysis. Staphylococcus aureus was selected as the positive control, and a completely transparent hemolytic ring was found around Staphylococcus aureus, which was β-hemolysis. The above results indicate that this Pediococcus pentosaceus strain has no hemolytic activity and is a safe and non-toxic strain.

[0091] 5. Adhesion test

[0092] After resuscitating Caco-2 cells (human colon cancer cells), inoculate them into a cell culture flask, add fresh DMEM complete medium (DMEM high-glucose medium containing double antibodies purchased from Beijing Solarbio Science & Technology Co., Ltd., 20% fetal bovine serum), and culture them in an incubator at 37°C and 5% CO2. Change the cell culture medium every two days. When the cell adhesion and growth fusion state reaches about 70%-80%, use 0.25% trypsin-EDTA (purchased from Beijing Solarbio Science & Technology Co., Ltd.) mixed digestive solution for passage. The last medium used is DMEM incomplete medium.

[0093] Preparation of FITC solution: FITC (fluorescein isothiocyanate) labeling solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.): Place the FITC stored at 4°C at room temperature for 30 min before preparation. Accurately weigh 0.5 g and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 500 mg / mL. Dilute it 1000-fold (take 10 μL and add it to a 9.99 mL DMSO brown 15 mL EP tube) to obtain a labeling solution with a working concentration of 500 μg / mL, and store it at 4°C.

[0094] Fluorescent labeling: Take 1 mL of freshly cultured probiotic bacterial suspension and centrifuge it in a brown 1.5 mL EP tube (4°C, 4000 rpm, 5 min). Wash it continuously 3 times with sterile PBS. After the last washing, resuspend the bacteria with 1 mL of FITC solution that has been placed at room temperature for 30 min in advance, and incubate it with shaking in the dark at 37°C for 2 h. After the dark incubation, centrifuge it (4°C, 4000 rpm, 5 min) and wash the strain 3 times with sterile PBS to wash away the unbound FITC labeling solution, and resuspend it with 1 mL of DMEM cell culture medium. Take 200 μL and add it to a black 96-well plate to measure the relative fluorescence intensity value (RFU) of the strain under the conditions of wavelength 485 nm (absorption wavelength) and wavelength 530 nm (emission wavelength), which is recorded as the relative fluorescence intensity value R0 of the strain before adhesion.

[0095] Adhesion test: Passage Caco-2 cells continuously for more than 3 generations and prepare for the experiment.

[0096] Before the experiment, adjust the cell concentration to 5×10 5Cells / mL. 1 mL of cell suspension was evenly spread in each well of a 24-well cell culture plate and cultured further. After observing that the cells in the 24-well plate grew to a monolayer (about 24 h of culture), the cell culture medium was discarded. After washing three times with sterile PBS, 0.6 mL of the labeled probiotic suspension was added. Three replicate wells were set for each strain. The mixture was incubated at 37 °C and 5% CO2 in the dark for 2 h. After taking out, the culture medium was discarded, and the strains that did not adhere to the cells were removed by washing three times with sterile PBS; 0.3 mL of trypsin was added to each well to digest the cells for 5 min, and the digestion was terminated with DMEM cell culture medium. The relative fluorescence intensity value (RFU) of the cell suspension was measured under the same wavelength condition and recorded as the relative fluorescence intensity value R of the adhered strain.

[0097] Adhesion rate (%) calculation formula: R / R0×100%

[0098] CFU (colony-forming unit): The total number of bacterial communities in a unit volume; when counting viable bacteria in culture, the colonies formed by single bacteria or multiple bacteria aggregated into groups on solid medium are called colony-forming units, and the number of viable bacteria is expressed by them.

[0099] The experimental results are shown in Figure 10 As shown, the adhesion rate of strain JZ01 was as high as 33.89%.

[0100] 6. DPPH free radical scavenging assay

[0101] The cells were collected and the cell suspension concentration was adjusted to 10 8 CFU / mL. A DPPH free radical scavenging ability kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was used, and the specific operation was carried out according to the steps in the instruction manual.

[0102] Sample DPPH free radical scavenging rate (%) = (1 - (A measurement - A control) / A blank)×100%

[0103] The experimental results are shown in Figure 11 , and the DPPH free radical scavenging rate of the cell suspension of strain JZ01 was 51.60%, showing certain antioxidant properties.

[0104]

[0105]

[0106] 7. ABTS free radical scavenging assay

[0107] The cells were collected and the cell suspension concentration was adjusted to 10 8 CFU / mL. An ABTS free radical scavenging ability kit (Beijing Solarbio Science & Technology Co., Ltd.) was used, and the specific operation was carried out according to the steps in the instruction manual.

[0108] Sample ABTS radical scavenging rate (%) = (1 - (A measured - A control) / A blank) × 100%

[0109] The experimental results are shown in Figure 12 , and the ABTS radical scavenging rate of the supernatant of strain JZ01 was 88.40%, indicating certain antioxidant activity.

[0110]

[0111] Example 4: Application of Pediococcus pentosaceus JZ01 from young cats in preventing feline gastrointestinal dysfunction

[0112] To explore the effect of Pediococcus pentosaceus JZ01 on feline gastrointestinal function, 10 sterilized cats of similar body weight and about 1 year old were randomly divided into a control group (fed a basic nutritional baked food) and an experimental group (fed a diet based on the control group formula with an additional 1 billion CFU of Pediococcus pentosaceus JZ01 per kilogram of cat food) for 7 days. The formula of the basic nutritional baked food: 60 - 70 parts of dried chicken, 5 - 10 parts of chicken liver, 5 - 10 parts of chicken heart, 1 - 5 parts of potato starch, 1 - 5 parts of tapioca starch, 1 - 5 parts of chicken oil, 1 - 5 parts of fish oil, 1 - 5 parts of chicken liver powder, 1 - 5 parts of krill powder, 1 - 5 parts of cellulose, 1 - 5 parts of fruit and vegetable powder, 1 - 5 parts of vitamins and minerals. The fecal output of the test cats was collected for 7 days, and the fecal status and scoring were analyzed.

[0113] The scoring criteria and rules are as follows:

[0114] Score Status 1 Watery diarrhea 2 Viscous, lack of consistency, basically unformed 3 Higher water content, unformed 4 Well-formed, cylindrical 5 Dry and hard, spherical

[0115] The results are shown in the following table. Compared with the control group, the fecal scores of the cats in the experimental group were better than those in the basic baked food group. Therefore, adding Pediococcus pentosaceus JZ01 to the baked food helps to improve soft stools and diarrhea in cats and helps the feces to form. Strain JZ01 plays a certain role in protecting feline gastrointestinal function.

[0116]

[0117] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Pediococcus pentosaceus JZ01 derived from kitten, characterized in that, The preservation number of the Pediococcus pentosaceus is CGMCC NO. 31002. It is preserved in the China General Microbiological Culture Collection Center, with the preservation date being June 19, 2024, and the preservation address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

2. The Pediococcus pentosaceus JZ01 derived from young cats according to claim 1, characterized in that, The 16s rRNA gene sequence of the Pediococcus pentosaceus is as shown in SEQ ID NO:

01.

3. The Pediococcus pentosaceus JZ01 derived from young cats according to claim 2, characterized in that, The Pediococcus pentosaceus has an inhibitory effect on Gram-positive bacteria and Gram-negative bacteria, including Escherichia coli, Staphylococcus aureus, Salmonella, Methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae.

4. The Pediococcus pentosaceus JZ01 derived from young cats according to claim 2, characterized in that, The Pediococcus pentosaceus is non-hemolytic and is a safe and non-toxic strain.

5. The Pediococcus pentosaceus JZ01 derived from kittens according to claim 2, characterized in that, The Pediococcus pentosaceus is sensitive to ampicillin, penicillin, chloramphenicol, and cefazolin, can avoid the problem of antibiotic resistance, prevent the imbalance of the intestinal flora, and has safety.

6. The Pediococcus pentosaceus JZ01 from young cats as described in claim 2, characterized in that, The Pediococcus pentosaceus has acid tolerance, bile salt tolerance, and intestinal adhesion ability, can stably survive and colonize in the host body, can play a probiotic role in the intestine, and regulate the gastrointestinal dysfunction caused by the disorder of the intestinal flora structure.

7. The Pediococcus pentosaceus JZ01 derived from young kittens according to claim 2, characterized in that, The Pediococcus pentosaceus has antioxidant properties.

8. A bacterial agent, characterized in that, It contains the Pediococcus pentosaceus JZ01 from kitten sources described in claim 1.

9. An animal food, health product, or medicine, characterized in that, The animal food, health product, and medicine contain the Pediococcus pentosaceus JZ01 from kitten sources described in claim 1 and / or its metabolites or the bacterial agent described in claim 8.

10. Use of the Pediococcus pentosaceus JZ01 from kitten sources described in claim 1 or 2 in a drug or food for treating, preventing, or alleviating animal gastrointestinal dysfunction.

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