A probiotic preparation for pets, a preparation method and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a postbiotic preparation for pets, a preparation method and application thereof. The postbiotic preparation provided by the present application comprises inactivated bacteria and metabolites of Lactobacillus plantarum Ps-8, inactivated bacteria and metabolites of Lactobacillus casei C-9, inactivated bacteria and metabolites of Bifidobacterium animalis subsp. lactis BB-16, and inactivated bacteria and metabolites of Lactobacillus acidophilus LA-14, and can be applied to various food types of different breeds of cats and dogs, and has the effects of improving intestinal barrier function of pets, enhancing immunity, reducing incidence of diarrhea, reducing stress of changing food, promoting digestion of nutrients, optimizing mental and fur state, reducing odor of feces, reducing odor of oral cavity, and improving oral cavity and tooth health.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a postbiotic preparation for pets, its preparation method, and its application. Background Technology

[0002] Inactivated lactic acid bacteria cells and their metabolites belong to postbiotics, which have become a popular substance for improving health in recent years. In 2013, Tsilingiri K et al. defined postbiotics as factors released by the metabolic activity of probiotics that can have a beneficial effect on the host, either directly or indirectly. Since then, different researchers have redefined the concept of postbiotics three times. Finally, in 2021, the International Society for the Study of Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics, which for the first time proposed the official concept of postbiotics as "preparations of non-living microorganisms and / or components thereof that are beneficial to the health of the host." Compared to probiotics, which have stringent requirements for their living environment, whose efficacy is greatly affected by storage conditions, and which have poor stability, and whose resistance genes carried by live probiotics carry the risk of transfer, postbiotics, due to their "non-living" characteristics and their bioactivity being comparable to that of "live" probiotics with minimal side effects, have enormous application prospects and are currently regarded by the industry as the fourth generation of microecological products after probiotics, prebiotics, and synbiotic preparations.

[0003] Metabiotics primarily promote animal growth through mechanisms such as antioxidation, anti-inflammation, regulation of immunity and gastrointestinal function, and liver protection. Currently, key research areas for metabiotics focus on five main aspects: First, regulating immunity by controlling biofilm formation, modulating gene expression, and reducing inflammatory cytokines based on probiotics (such as Lactobacillus and Bifidobacterium); second, the role of extracellular polysaccharides as components of metabiotics in reducing inflammation and promoting barrier function through unclear signaling mechanisms; third, the therapeutic effects of inactivated lactic acid bacteria on obesity; fourth, the improvement of apoptosis, regulation of gut microbiota balance, promotion of interleukin production, and improvement of inflammatory bowel disease by metabiotics; and fifth, the health benefits of metabiotics in allergic dermatitis.

[0004] Studies show that lactobacilli and bifidobacteria are dominant bacteria in the gut of healthy cats and dogs. They are not only important components of the intestinal barrier but also enhance the immune system of cats and dogs by stimulating macrophages, inducing interferon production, promoting cell division, and producing antibodies. Data indicates that after cats and dogs ingest compound lactic acid bacteria preparations for a period of time, the numbers of Escherichia coli, Clostridium perfringens, and Salmonella in their intestines significantly decreased, while the number of beneficial bacteria significantly increased. Simultaneously, the apparent digestibility of nutrients in their diet showed a gradual upward trend. Furthermore, literature reports that feeding cats and dogs with intestinal disorders and weak constitutions with lactic acid bacteria and their metabolites for a period of time can significantly improve their gut microbiota, enhance their physical condition, and reduce oral and fecal odor. Therefore, feeding cats and dogs a certain amount of probiotic lactic acid bacteria and their metabolites can enhance immunity and improve gut microbiota, thereby reducing the use of chemical drugs and playing an important role in the health of cats and dogs.

[0005] Currently, most published patents related to postbiotics for promoting pet gut health involve products made by adding postbiotics and / or probiotics to a single pet food product, such as cat treats containing postbiotics and / or probiotics. This doesn't allow postbiotics to be adapted to a variety of pet food types simultaneously, or to be suitable for both cat and dog food. Furthermore, finished pet foods often contain a large amount of pure meat, and the unstable meat source can pose other risks, affecting the actual effectiveness of postbiotics. In reality, medications or health supplements used to treat, improve, or prevent diseases are often mixed into food to make it palatable for cats and dogs. Because cat and dog food is complex, and the staple foods for different ages and breeds of cats and dogs vary, whether postbiotic formulations can adapt to a variety of pet food types needs to be verified before use. However, currently published patents for postbiotic formulations contain relatively little detailed experimental data demonstrating product efficacy or adaptability to a variety of pet food types, leaving the product efficacy to be verified. In addition, the efficacy of currently published postbiotic products is relatively limited, only applicable to treating or improving a single symptom.

[0006] Therefore, there is a need for a postbiotic formulation that can be adapted to cats and dogs of different ages and breeds, as well as a variety of cat and dog foods, and has rich efficacy, as a preparation for animal food compositions or supplement compositions. Summary of the Invention

[0007] Technical issues

[0008] The purpose of this invention is to provide a prebiotic formulation that can adapt to a variety of pet food types and cats and dogs of different ages and breeds. This formulation also has the effects of promoting intestinal health in cats / dogs, reducing stress during food changes, and improving oral and dental health.

[0009] Technical solution

[0010] The first aspect of this invention provides a Lactobacillus plantarum Ps-8, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 5359 and deposit date of October 28, 2011.

[0011] The second aspect of this invention provides a Lactobacillus acidophilus LA-14, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number GMCC No. 31624 and deposit date of August 6, 2024.

[0012] The third aspect of this invention provides a Lactobacillus casei C-9, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 6734 and deposit date of October 29, 2012.

[0013] The fourth aspect of this invention provides a *Bifidobacterium animalis* subsp. *lactis* BB-16, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 31623 and deposit date of August 6, 2024.

[0014] The fifth aspect of this invention provides a postbiotic preparation for pets, the postbiotic preparation for pets comprising inactivated bacterial cells and their metabolites; wherein the bacterial cells are *Lactobacillus plantarum* Ps-8, *Lactobacillus acidophilus* LA-14, *Lactobacillus casei* C-9, and *Bifidobacterium animalis* subsp. *lactospirae* BB-16; the ratio of the number of units of *Lactobacillus plantarum* Ps-8, *Lactobacillus casei* C-9, *Bifidobacterium animalis* subsp. *lactospirae* BB-16, and *Lactobacillus acidophilus* LA-14 is 1–5:1–2:1–52:1–2; preferably 5:1:1:1; the total amount of the bacterial cells is 3 × 10⁻⁶. 10 ~1×10 11 cells / g; preferably 6×10 10 ~1×10 11cells / g; more preferably 1×10 11 cells / g.

[0015] In some embodiments, the metabolites include organic acids.

[0016] In some embodiments, the organic acid includes at least one selected from lactic acid, citric acid, oxalic acid, malic acid, phenyllactic acid, succinic acid, 4-hydroxyphenyllactic acid, 3-phenyllactic acid, salicylic acid, vanillic acid, benzoic acid, phenylalanine, and short-chain fatty acids, wherein the short-chain fatty acid is at least one selected from acetic acid, propionic acid, butyric acid, and valeric acid.

[0017] In some embodiments, the metabolites further include at least one of soybean saponins, soybean isoflavones, γ-aminobutyric acid, active short peptide molecules, genistein glucoside, ursolic acid, allicin, astaxanthin, solanine, 2-hydroxycinnamic acid, and phenylacetaldehyde, wherein the active short peptide molecule is Glu-phe-trp.

[0018] In some embodiments, the raw materials of the pet postbiotic formulation include organic nitrogen sources, organic carbon sources, flavoring agents, pH adjusters, and excipients.

[0019] In some embodiments, the organic nitrogen source is selected from one or more of whole soybean flour, skim milk powder, protein powder, and yeast extract; the organic carbon source is selected from one or more of whole soybean flour, skim milk powder, and protein powder; the pH adjuster is selected from one or more of sodium citrate or potassium citrate; the flavoring agent is selected from one or more of sucrose, xylitol, or erythritol; and the excipient is selected from one or more of maltodextrin and corn syrup, preferably maltodextrin.

[0020] In some embodiments, the raw materials of the pet postbiotic preparation include 4-7 parts by weight of whole soybean powder, 6.5-18 parts by weight of skim milk powder, 5-7 parts by weight of sucrose, 2.5-5 parts by weight of sodium citrate, and 60-78 parts by weight of water.

[0021] In some embodiments, the formulation may be in the form of a liquid formulation or a solid formulation; the formulation may be in the form of an injection, tablet, capsule, powder, granule or ointment.

[0022] In some embodiments, the preparation process of the postbiotic formulation includes the following specific steps:

[0023] S1: Weigh the raw materials, add water, mix, and then perform material processing and homogenization;

[0024] S2: After sterilization and cooling, a liquid solution is obtained;

[0025] S3: Add bacterial cells and lactase to the above liquid, and then carry out constant temperature fermentation to obtain fermentation liquid;

[0026] S4: Sterilize and inactivate, then spray dry to obtain post-biotic powder;

[0027] S5: After mixing postbiotic powder with maltodextrin, a postbiotic preparation is obtained.

[0028] In some embodiments, the conditions for material preparation in step S1 are: 55-60℃ for 10-20 minutes; the conditions for homogenization are: 55-60℃, primary pressure 18-20 MPa, secondary pressure 4-5 MPa; the conditions for sterilization in step S2 are: 90-95℃ for 30-50 minutes; the cooling temperature is 32-37℃; the amount of lactase added in step S3 is 0.1-1.0 mL per kilogram of liquid; the conditions for constant temperature fermentation are: 32-37℃ until pH 4.5-4.6; wherein, after constant temperature fermentation, the total viable cell count is ≥2.0 × 10⁻⁶. 10 CFU / g, preferably ≥3.0×10 10 CFU / g; the amount of maltodextrin added in step S4 is 18-25g per kilogram of fermentation liquid; the inactivation conditions are: inactivation at 75-95℃ for 10-30 minutes.

[0029] In some embodiments, the enzyme activity of the lactase in step S3 is 5000 U / g.

[0030] In some embodiments, the sixth aspect of the present invention provides the use of the above-mentioned pet postbiotic preparation in the preparation of products for improving feline and canine diarrhea.

[0031] In some embodiments, the administration method of the pet postbiotic preparation is direct oral administration, with a dosage of 0.5 g and 1 g of postbiotic preparation administered orally with 2-3 times the amount of water daily.

[0032] In some embodiments, the pet postbiotic preparation is one or more of the following: food ingredients, probiotic powder, food additives, feed additives, or food.

[0033] The seventh aspect of this invention provides the application of the above-mentioned pet postbiotic preparation in the preparation of products to alleviate stress during food change in cats and dogs.

[0034] In some embodiments, the postbiotic preparation for pets can be added to extruded food, baked food, wet food pouches, freeze-dried raw meat and bones, or cat treats. The amount added is 10 grams or 50 grams of postbiotic preparation per 10 kilograms of extruded food, baked food, wet food pouches, freeze-dried raw meat and bones, or cat treats, preferably 10 grams.

[0035] The eighth aspect of the present invention provides the application of the above-mentioned pet postbiotic preparation in the preparation of products for improving diarrhea in cats and dogs.

[0036] The ninth aspect of this invention provides the application of the above-mentioned pet postbiotic preparation in the preparation of products to alleviate stress during food change in cats and dogs.

[0037] The tenth aspect of the present invention provides the use of the above-mentioned pet postbiotic preparation in the preparation of products for treating or improving dental plaque in cats and dogs.

[0038] Technical effect

[0039] The pet postbiotic formulation of this invention is adaptable to various cat / dog food combinations, including but not limited to baked kibble, wet food pouches, extruded kibble, freeze-dried raw meat and bone, and cat treats, covering almost all types of cat / dog food available on the market. Furthermore, this postbiotic formulation is suitable for cats and dogs of different ages (1-10 months) and various common breeds, such as Belgian Shepherds, German Shepherds, Poodles, British Shorthairs, American Shorthairs, Ragdolls, domestic shorthairs, Russian Blues, and Siamese cats, demonstrating a wide range of applications. Moreover, this pet postbiotic formulation offers a variety of benefits, not only improving diarrhea in cats and dogs, alleviating stress during food changes, optimizing their mental and coat condition, and enhancing immunity, but also effectively improving dental plaque and enhancing oral and dental health. Attached Figure Description

[0040] Figure 1 Statistical results of fecal scores in dogs in Experiment 1 (*P<0.05; **P<0.01; ***P<0.001);

[0041] Figure 2 Statistical results of fecal scores in Experiment 2 (*P<0.05; **P<0.01; ***P<0.001);

[0042] Figure 3 : Pulse measurement results of dog in Experiment Example 3;

[0043] Figure 4 Results of respiratory rate measurement in dog in Experiment Example 3;

[0044] Figure 5 The body temperature measurement results of the dog in Experiment Example 3;

[0045] Figure 6 Results of food intake measurement in dog example 3;

[0046] Figure 7 : Results of weight measurement of dogs in Experiment Example 3;

[0047] Figure 8 Results of apparent digestibility of crude protein in dogs in Experiment Example 3 (*P<0.05; **P<0.01; ***P<0.001);

[0048] Figure 9Results of apparent digestibility of crude fat in dogs in Experiment Example 3 (*P<0.05; **P<0.01; ***P<0.001);

[0049] Figure 10 Statistical results of fecal scores in Experimental Example 3 (*P<0.05; **P<0.01; ***P<0.001);

[0050] Figure 11 : Results of food intake measurement in cats in Experiment Example 4;

[0051] Figure 12 : Results of weight measurement of cats in Experiment 4;

[0052] Figure 13 Results of apparent digestibility of crude protein in cats in Experiment 4 (*P<0.05; **P<0.01; ***P<0.001);

[0053] Figure 14 Experimental Example 4: Results of apparent digestibility of crude fat in cats (*P<0.05; **P<0.01; ***P<0.001);

[0054] Figure 15 Statistical results of fecal scores in Experiment 4 (*P<0.05; **P<0.01; ***P<0.001);

[0055] Figure 16 Statistical results of fecal scores in Experimental Example 5 (*P<0.05; **P<0.01; ***P<0.001);

[0056] Figure 17 Results of food intake measurement in dogs in Experiment 5 (*P<0.05; **P<0.01; ***P<0.001);

[0057] Figure 18 Serum cortisol levels in dogs in Experiment 5 (*P<0.05; **P<0.01; ***P<0.001);

[0058] Figure 19 Serum malondialdehyde (MDA) determination results in dogs of Experimental Example 5 (*P<0.05; **P<0.01; ***P<0.001);

[0059] Figure 20 Serum immunoglobulin G assay results of dogs in Experiment Example 5 (*P<0.05; **P<0.01; ***P<0.001);

[0060] Figure 21Serum immunoglobulin A assay results of dogs in Experiment Example 5 (*P<0.05; **P<0.01; ***P<0.001);

[0061] Figure 22 Statistical results of fecal scores in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0062] Figure 23 Results of food intake measurement in cats in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0063] Figure 24 Results of serum malondialdehyde (MDA) determination in cats in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0064] Figure 25 Serum cortisol levels in cats in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0065] Figure 26 Results of serum immunoglobulin G assay in cats in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0066] Figure 27 Results of serum immunoglobulin A assay in cats in Experiment Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0067] Figure 28 Serum D-lactic acid levels in cats in Experiment 6 (*P<0.05; **P<0.01; ***P<0.001);

[0068] Figure 29 Results of serum diamine oxidase assay in cats in Experiment Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0069] Figure 30 Results of weight measurement of cats in Experiment 7 (*P<0.05; **P<0.01; ***P<0.001);

[0070] Figure 31 Results of apparent digestibility of crude protein in cats in Experiment 7 (*P<0.05; **P<0.01; ***P<0.001);

[0071] Figure 32 Results of apparent digestibility of crude fat in cats in Experiment 7 (*P<0.05; **P<0.01; ***P<0.001);

[0072] Figure 33 Statistical results of fecal scores in Experiment 7 (*P<0.05; **P<0.01; ***P<0.001);

[0073] Figure 34 Results of serum immunoglobulin G levels in cats in Experiment Example 7 (*P<0.05; **P<0.01; ***P<0.001);

[0074] Figure 35 Results of serum immunoglobulin A levels in cats in Experiment Example 7 (*P<0.05; **P<0.01; ***P<0.001);

[0075] Figure 36 Experimental Example 7: Results of fecal secretory immunoglobulin A (P<0.05; **P<0.01; ***P<0.001) in cats.

[0076] Figure 37 Statistical results of fecal scores in Experiment 8 (*P<0.05; **P<0.01; ***P<0.001);

[0077] Figure 38 : Results of weight measurement of cats in Experiment 8;

[0078] Figure 39 Experimental Example 8: Results of ammonia content determination in cat feces (*P<0.05; **P<0.01; ***P<0.001);

[0079] Figure 40 Statistical results of fecal scores in Experiment 9 (*P<0.05; **P<0.01; ***P<0.001);

[0080] Figure 41 Statistical results of dental plaque scores in cats in Experiment 9 (*P<0.05; **P<0.01; ***P<0.001).

[0081] Preservation Information

[0082] Name: Ps-8;

[0083] Taxonomy: Lactobacillus plantarum;

[0084] Latin name: Lactobacillus plantarum;

[0085] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0086] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0087] Date of deposit: October 28, 2011;

[0088] Registered with the China National Collection Center (CGMCC) No. 5359.

[0089] Name: LA-14;

[0090] Taxonomy: Lactobacillus acidophilus;

[0091] Latin name: Lactobacillus acidophilus;

[0092] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0093] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0094] Date of deposit: August 6, 2024;

[0095] Registration number at the Collection Center: CGMCC No. 31624.

[0096] Name: C-9;

[0097] Taxonomy: Lactobacillus casei;

[0098] Latin name: Lactobacillus casei;

[0099] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0100] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0101] Date of deposit: October 29, 2012;

[0102] Registered with the Collection Center: CGMCC No. 6734.

[0103] Name: BB-16;

[0104] Taxonomy: Bifidobacterium animalis subsp. lactis;

[0105] Latin name: *Bifidobacterium animalis* subsp. *lactis*;

[0106] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0107] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0108] Date of deposit: August 6, 2024;

[0109] Registration number at the Preservation Center: CGMCC No. 31623. Detailed Implementation

[0110] To facilitate the explanation of the technical solution of this application, the following is a general explanation and definition of the terms and expressions used in this application.

[0111] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0112] The term "chemical dissolution" refers to the process of achieving better dissolution of raw materials.

[0113] The term "homogenization," also known as homogenization, is a process that micronizes and homogenizes the dispersions in a suspension (or emulsion) system. This process simultaneously reduces the size of the dispersions and improves the uniformity of their distribution.

[0114] The term "complex lactic acid bacteria" refers to a combination of Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis subsp. lactis BB-16, and Lactobacillus acidophilus LA-14.

[0115] The strains used in this application are described below:

[0116] Lactobacillus plantarum Ps-8: This strain was isolated from naturally fermented yogurt and has excellent artificial gastric juice, artificial intestinal juice, bile salt tolerance and probiotic properties. It can enhance the body's antioxidant capacity and immunity, promote the maturation of intestinal flora, improve the digestibility of nutrients and relieve stress.

[0117] Lactobacillus acidophilus LA-14: This strain, isolated from naturally fermented yak milk, possesses excellent tolerance to artificial gastric and intestinal fluids, bile salts, and probiotic properties. It can produce high levels of natural active antibacterial substances such as bacteriocins, broadly inhibiting common intestinal pathogens such as Escherichia coli and Salmonella, alleviating problems such as soft stools and watery stools in animals. It also regulates the levels of immune substances in blood and feces, enhancing the body's immunity.

[0118] Lactobacillus casei C-9: This strain was isolated from traditionally fermented mare's milk and has excellent artificial gastric juice, artificial intestinal juice, bile salt tolerance and probiotic properties. It can regulate intestinal flora homeostasis, regulate blood lipid metabolism, protect the liver, enhance the body's immunity and antioxidant capacity, regulate intestinal flora rejuvenation, reduce intestinal bacterial endotoxins, and repair intestinal damage caused by antibiotics.

[0119] Bifidobacterium animalis subsp. lactis BB-16: This strain was isolated from the intestines of healthy pets and has excellent artificial gastric juice, artificial intestinal juice, bile salt tolerance and probiotic properties. It can regulate the intestinal flora, antagonize intestinal pathogens, relieve acute and chronic diarrhea and soft stools, and relieve chronic inflammatory bowel disease.

[0120] The relevant experimental standards and specifications referenced in this application are as follows:

[0121] Temperature measurement: Use an electronic thermometer to measure the dog's temperature while the pet is calm. The measurement site is the cochlea of ​​the dog's ear or the base of the hind leg. The examiner holds the electronic thermometer in place at the measurement site for about 10 seconds, presses the measurement button to perform the measurement, and records the result.

[0122] Pulse measurement: The test is performed when the dog is at rest. During the test, the pet is properly restrained, and the examiner stands behind the pet, holding the hind leg with one hand and reaching into the inner thigh with the other hand to gently press the femoral artery with the fingers to detect and record the dog's pulse.

[0123] Respiratory rate measurement: The test is conducted when the pet is at rest. During the test, the examiner stands in front of the pet's chest and observes the rise and fall of the pet's chest. One rise and fall is counted as one breath, and the number of breaths is recorded.

[0124] Feed intake measurement: The weight of the daily ration before feeding minus the weight of the remaining feed after feeding.

[0125] Weight measurement: During the measurement, the experimenter fixed the pet in the center of the scale and recorded the weight.

[0126] Apparent digestibility of nutrients was determined as follows: Three mixed fecal samples (100g each) were collected from each group on days 0 and 30 of the experiment. These samples were treated with 10% hydrochloric acid for nitrogen fixation, dried to constant weight at 60℃, pulverized, and passed through a 60-mesh sieve before refrigeration. The apparent digestibility of nutrients in the feed diet was determined using the acid-insoluble ash method, following the procedure outlined in GB / T 23742-2009 (Determination of Hydrochloric Acid Insoluble Ash in Feed). Crude ash content was determined using the 550℃ ignition method, following the procedure outlined in GB / T 6438-2007. To determine dry matter content, the moisture content of the sample was first determined according to GB / T 6435-2014, and then the dry matter content was calculated. Crude protein content was determined using an automated Kjeldahl nitrogen analyzer, following the method outlined in GB / T 6433-2006, and crude fat content was determined using Soxhlet extraction. Apparent digestibility of nutrients (%) = [1 - (A / B × C / D)] × 100, where: A: content of a certain nutrient in feces (%); B: content of the corresponding nutrient in the diet (%); C: acid-insoluble ash content in the diet (%); D: acid-insoluble ash content in feces (%).

[0127] Diarrhea disease prevalence statistics: The prevalence of diarrhea in pets was statistically analyzed at different time points according to different implementation methods, and the diarrhea rate was calculated.

[0128] Stool scoring criteria: Watery stool (1 point): Stool is liquid, in clumps rather than a single piece, and may contain mucus or blood. When collected in a container, it can be poured out in a flowing manner, leaving a large amount of stool residue on the ground after collection. This indicates severe diarrhea. Unformed soft stool (2 points): Stool is sticky and does not show any cylindrical shape. It leaves a large amount of stool residue on the ground after collection. This indicates moderate diarrhea. Formed soft stool (3 points): Stool is irregularly columnar and easily breaks. It leaves a small amount of stool residue on the ground after collection. This indicates mild diarrhea. Hard stool (4 points): Hard stool is relatively regular in shape, does not easily break, and leaves a small amount of stool residue on the ground after collection. This indicates a relatively ideal stool condition. Extremely dry stool (5 points): Stool is extremely dry and hard, does not stick to the ground, and may roll on the ground when excreted. This indicates constipation.

[0129] Statistics on the mental state, fur condition, and litter odor of pet cats: The mental state and fur condition of pet cats are based on the results of visual observation by a fixed veterinarian; the odor of litter is counted by the owner, who collects litter with a sampling spoon, fans the litter with their hand to smell it, and then counts the odor.

[0130] Pet Cat Dental Plaque Index Statistics: Teeth are examined through a combination of visual inspection and probing. During the examination, a dental probe is used to lightly scrape the tooth surface, and the amount and thickness of plaque are scored. Four tooth surfaces are examined: mesobob, midbuccal, distobuccal, and lingual. The score for each tooth is the average of the scores for all four surfaces. Scoring criteria are as follows: No plaque: 0 points; Thin plaque, not visible visually but visible when scraped with a probe: 1 point; Moderate amount of plaque visible at the gingival margin or proximal surfaces: 2 points; Abundant soft plaque in the gingival sulcus, gingival margin, or proximal surfaces: 3 points. The dental plaque index is used to evaluate the pet's oral hygiene and the effectiveness of periodontal disease prevention and control.

[0131] Methods for determining serum malondialdehyde (MDA) and cortisol: The content of MDA in serum was detected by the thiobarbituric acid colorimetric method, and the content of cortisol (COR) in serum was detected by the double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA).

[0132] Method for determining serum immunoglobulin G: Whole blood was collected from experimental pets using anticoagulation blood collection tubes containing disodium ethylenediaminetetraacetate (EDTA-2Na). The bottom of the tube was gently tapped to ensure adequate anticoagulation of the blood. Serum was obtained by centrifugation (3000 rpm, 10 min), and the immunoglobulin G content in the serum sample was detected by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies.

[0133] Methods for determining serum diamine oxidase and D-lactic acid: The content of diamine oxidase in serum samples was detected by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using double antibodies. The content of D-lactic acid in serum was detected by enzymatic spectrophotometry.

[0134] Method for determining secretory immunoglobulin A in feces: The content of secretory immunoglobulin A (SIgA) in fecal samples was determined by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies.

[0135] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials are kept consistent to ensure comparability.

[0136] Unless otherwise specified, all reagents and instruments used in the embodiments of this invention can be purchased from the market.

[0137] The following provides a further description of a pet postbiotic preparation, its preparation method, and its application, as provided in this application.

[0138] Example 1: Cultivation and cryopreservation of Lactobacillus plantarum Ps-8

[0139] Lactobacillus plantarum Ps-8 was placed in MRS liquid medium and statically cultured at 37℃ for 18h. Then, sterilized 60% glycerol was mixed with the bacterial culture at a volume ratio of 1:3 to make the final glycerol concentration 15%. The mixture was then pipetted and mixed thoroughly, and the culture was stored at -80℃.

[0140] The MRS liquid culture medium consists of the following ingredients: 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80, 0.2g magnesium sulfate, and 0.05g manganese sulfate. Add these ingredients to 1000mL distilled water, adjust the pH to 6.5, and sterilize at 121℃ for 15min.

[0141] Example 2: Characteristics of Lactobacillus plantarum Ps-8

[0142] 1) Acid and bile salt resistance:

[0143] Frozen Lactobacillus plantarum Ps-8 was inoculated into MRS liquid medium and statically cultured at 37℃ for 18 h. This process was repeated twice to obtain activated fermentation broth.

[0144] Add 3.5 g / L pepsin to sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), filter through a 0.22 μm microporous membrane to sterilize, and prepare simulated gastric juice. Centrifuge the activated strain to collect the cells, add an equal volume of pH 2.5 simulated gastric juice to the culture medium, and incubate at 37°C for 3 h. Determine the viable cell count at 0 h and 3 h using the MRS agar pour method.

[0145] Sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH) was sterilized by adding 0.1% trypsin and 1.8% bovine bile salts and filtering through a 0.22 μm microporous membrane to prepare simulated intestinal fluid. The bacterial culture from the simulated gastric fluid, after 3 hours of treatment, was centrifuged and washed twice to collect the bacteria. Then, an equal volume of simulated intestinal fluid (37℃) was added, and the culture was continued. Viable cell counts were measured using the MRS agar pour method at 4 hours and 8 hours. The experimental results are shown in Table 1.

[0146] Table 1. Survival rates of Ps-8 simulated gastric and intestinal fluids

[0147]

[0148] Survival rate = [N1 / N0] × 100% (N0 - viable bacteria count at 0h; N1 - viable bacteria count after digestion with simulated intestinal and gastric juices), lg CFU / mL represents the logarithmic colony-forming units to the base 10.

[0149] Antibacterial properties:

[0150] The antibacterial effect of *Lactobacillus plantarum* Ps-8 fermentation broth was determined using the well-diffusion agar assay: 20 ml of sterilized MRS agar medium cooled to approximately 50°C was mixed with 200 μL of enteric pathogenic bacteria solution (10 μL of ... 6 Pour the mixture (cfu / ml) into the agar plate and mix well. After the MRS agar medium containing enteropathogenic bacteria has cooled and solidified, use a hole punch to make holes about 8 mm in diameter on the plate.

[0151] Add 100 μL of *Lactobacillus plantarum* Ps-8 fermentation broth to each well, diffuse at 4℃ for 12 h, then incubate at 37℃ for 48 h. Observe the size of the inhibition zone. The diameter of the inhibition zone was measured using vernier calipers (retaining two significant figures). The experimental results are shown in Table 2.

[0152] Table 2. Content and antibacterial properties of phenyllactic acid and 4-hydroxyphenyllactic acid in Ps-8 fermentation broth

[0153]

[0154]

[0155] Note: The diameter of the punch is 8mm; mm is the unit for the diameter of the antibacterial zone.

[0156] As shown in Tables 1 and 2, strain Ps-8 exhibits good acid and bile salt tolerance. After digestion in simulated gastric juice at pH 2.5 for 3 hours, followed by digestion in simulated intestinal juice at pH 8.0 for 8 hours, its survival rate reached as high as 94.60%. Furthermore, strain Ps-8 demonstrates excellent broad-spectrum inhibition of pathogenic bacteria, with a highly significant antibacterial effect.

[0157] Example 3: Cultivation and cryopreservation of Lactobacillus acidophilus LA-14

[0158] Lactobacillus acidophilus LA-14 was placed in MRS liquid medium and statically cultured at 37°C for 18 hours. Then, sterilized 60% glycerol was mixed with the bacterial culture at a volume ratio of 1:3 to make the final glycerol concentration 15%. The mixture was then pipetted and mixed thoroughly, and the culture was stored at -80°C.

[0159] The MRS liquid culture medium consists of the following ingredients: 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80, 0.2g magnesium sulfate, and 0.05g manganese sulfate. Add these ingredients to 1000mL distilled water, adjust the pH to 6.5, and sterilize at 121℃ for 15min.

[0160] Example 4: Characteristics of Lactobacillus acidophilus LA-14

[0161] 1) Acid and bile salt resistance:

[0162] Frozen Lactobacillus acidophilus LA-14 was inoculated into MRS liquid medium and statically cultured at 37℃ for 18 h. This process was repeated twice to obtain activated fermentation broth.

[0163] Add 3.5 g / L pepsin to sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), filter through a 0.22 μm microporous membrane to sterilize, and prepare simulated gastric juice. Centrifuge the activated strain to collect the cells, add an equal volume of pH 2.5 simulated gastric juice to the culture medium, and incubate at 37°C for 3 h. Determine the viable cell count at 0 h and 3 h using the MRS agar pour method.

[0164] Sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH) was sterilized by adding 0.1% trypsin and 1.8% bovine bile salts and filtering through a 0.22 μm microporous membrane to prepare simulated intestinal fluid. The bacterial culture from the simulated gastric fluid, after 3 hours of treatment, was centrifuged and washed twice to collect the bacteria. An equal volume of simulated intestinal fluid was added to the culture, and the mixture was incubated at 37°C. Viable cell counts were determined using the MRS agar pour method at 4 hours and 8 hours. The results are shown in Table 3.

[0165] Table 3 Survival rates of LA-14 artificial simulated gastric and intestinal fluids

[0166]

[0167] Survival rate = [N1 / N0] × 100% (N0 - viable bacteria count at 0h; N1 - viable bacteria count after digestion with simulated intestinal and gastric juices), lg CFU / mL represents the logarithmic colony-forming units to the base 10.

[0168] 2) Antibacterial properties:

[0169] The antibacterial effect of Lactobacillus acidophilus LA-14 fermentation broth was determined using the Well-diffusion Agar Assay: 20 mL of sterilized MRS agar medium cooled to approximately 50°C was mixed with 200 μL of enteric pathogenic bacteria solution (10 μL of ... 6 Pour the mixture (CFU / mL) into the agar plate and mix well. After the MRS agar medium containing enteropathogenic bacteria has cooled and solidified, use a hole punch to make holes about 8 mm in diameter on the plate.

[0170] Add 100 μL of Lactobacillus acidophilus LA-14 fermentation broth to each well, diffuse at 4℃ for 12 h, then incubate at 37℃ for 48 h, and observe the size of the inhibition zone. The diameter of the inhibition zone was measured using vernier calipers (retaining two significant figures). The experimental results are shown in Table 4.

[0171] Table 4. Antibacterial properties of Lactobacillus acidophilus LA-14

[0172]

[0173] Note: The diameter of the punch is 8mm; mm is the unit for the diameter of the antibacterial zone.

[0174] As shown in Tables 1 and 2, strain LA-14 exhibits good acid and bile salt tolerance. After digestion in simulated gastric juice at pH 2.5 for 3 hours, followed by digestion in simulated intestinal juice at pH 8.0 for 8 hours, its survival rate reached as high as 90.30%. Furthermore, strain LA-14 demonstrates excellent broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella, and Clostridium perfringens, with a highly significant antibacterial effect.

[0175] Example 5: Cultivation and cryopreservation of Bifidobacterium animalis subsp. lactis BB-16

[0176] Bifidobacterium animalis subsp. lactis BB-16 was placed in MRS liquid medium and statically cultured at 37°C for 18 hours. Then, sterilized 60% glycerol was mixed with the bacterial culture at a volume ratio of 1:3 to make the final glycerol concentration 15%. The mixture was then pipetted and mixed thoroughly, and the culture was stored at -80°C.

[0177] The MRS liquid culture medium consists of the following ingredients: 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80, 0.2g magnesium sulfate, and 0.05g manganese sulfate. Add these ingredients to 1000mL distilled water, adjust the pH to 6.5, and sterilize at 121℃ for 15min.

[0178] Example 6: Characteristics of Bifidobacterium animalis subsp. lactis BB-16

[0179] 1) Acid and bile salt resistance:

[0180] Frozen Bifidobacterium lactis subsp. BB-16 was inoculated into MRS liquid medium and statically cultured at 37°C for 18 hours. This process was repeated twice to obtain activated fermentation broth.

[0181] Add 3.5 g / L pepsin to sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), filter through a 0.22 μm microporous membrane to sterilize, and prepare simulated gastric juice. Centrifuge the activated strain to collect the cells, add an equal volume of pH 2.5 simulated gastric juice to the culture medium, and incubate at 37°C for 3 h. Determine the viable cell count at 0 h and 3 h using the MRS agar pour method.

[0182] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% bovine bile salt were added and filtered through a 0.22 μm microporous membrane to prepare a simulated intestinal fluid. The bacterial culture from the simulated gastric fluid, after 3 hours of treatment, was centrifuged and washed twice to collect the bacteria. An equal volume of simulated intestinal fluid was added to the culture, and the mixture was incubated at 37°C. Viable cell counts were determined using the MRS agar pour method at 4 hours and 8 hours. The results are shown in Table 5.

[0183] Table 5. Survival rates of BB-16 artificially simulated gastric and intestinal fluids.

[0184]

[0185] Survival rate = [N1 / N0] × 100% (N0 - viable bacteria count at 0h; N1 - viable bacteria count after digestion with simulated intestinal and gastric juices), lg CFU / mL represents the logarithmic colony-forming units to the base 10.

[0186] 2) Antibacterial properties:

[0187] The antibacterial effect of fermentation broth of Bifidobacterium lactis subsp. BB-16 was determined by the Well-diffusion Agar Assay: 20 mL of sterilized MRS agar medium cooled to approximately 50 °C was mixed with 200 μL of enteric pathogenic bacteria solution (10 μL of ... 6 Pour the mixture (CFU / mL) into the agar plate and mix well. After the MRS agar medium containing enteropathogenic bacteria has cooled and solidified, use a hole punch to make holes about 8 mm in diameter on the plate.

[0188] Add 100 μL of Bifidobacterium animalis subsp. lactis BB-16 fermentation broth to each well, diffuse at 4℃ for 12 h, then incubate at 37℃ for 48 h, and observe the size of the inhibition zone. The diameter of the inhibition zone was measured using vernier calipers (retaining two significant figures). The experimental results are shown in Table 6.

[0189] Table 6. Antibacterial properties of Bifidobacterium lactis subsp. BB-16

[0190]

[0191] Note: The diameter of the punch is 8mm; mm is the unit for the diameter of the antibacterial zone.

[0192] As shown in Tables 1 and 2, strain BB-16 exhibits good acid and bile salt tolerance. After digestion in simulated gastric juice at pH 2.5 for 3 hours, followed by digestion in simulated intestinal juice at pH 8.0 for 8 hours, its survival rate reached as high as 91.50%. Furthermore, strain BB-16 demonstrates excellent broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella, and Clostridium perfringens, with highly significant antibacterial effects.

[0193] Example 7: Cultivation and cryopreservation of Lactobacillus casei C-9

[0194] Lactobacillus casei C-9 was placed in MRS liquid medium and statically cultured at 37°C for 18 hours. Then, sterilized 60% glycerol was mixed with the bacterial culture at a volume ratio of 1:3 to make the final glycerol concentration 15%. The mixture was then pipetted and mixed thoroughly, and the culture was stored at -80°C.

[0195] The MRS liquid culture medium consists of the following ingredients: 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80, 0.2g magnesium sulfate, and 0.05g manganese sulfate. Add these ingredients to 1000mL distilled water, adjust the pH to 6.5, and sterilize at 121℃ for 15min.

[0196] Example 8: Characteristics of Lactobacillus casei C-9

[0197] 1) Acid and bile salt resistance:

[0198] Frozen Lactobacillus casei C-9 was inoculated into MRS liquid medium and statically cultured at 37°C for 18 hours. This process was repeated twice to obtain activated fermentation broth.

[0199] Add 3.5 g / L pepsin to sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), filter through a 0.22 μm microporous membrane to sterilize, and prepare simulated gastric juice. Centrifuge the activated strain to collect the cells, add an equal volume of pH 2.5 simulated gastric juice to the culture medium, and incubate at 37°C for 3 h. Determine the viable cell count at 0 h and 3 h using the MRS agar pour method.

[0200] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% bovine bile salt were added and filtered through a 0.22 μm microporous membrane to prepare a simulated intestinal fluid. The bacterial culture from the simulated gastric fluid, after 3 hours of treatment, was centrifuged and washed twice to collect the bacteria. An equal volume of simulated intestinal fluid was added to the culture, and the mixture was incubated at 37°C. Viable cell counts were determined using the MRS agar pour method at 4 hours and 8 hours. The results are shown in Table 7.

[0201] Table 7C-9 Survival rates of artificially simulated gastric and intestinal fluids

[0202]

[0203] Survival rate = [N1 / N0] × 100% (N0 - viable bacteria count at 0h; N1 - viable bacteria count after digestion with simulated intestinal and gastric juices), lg CFU / mL represents the logarithmic colony-forming units to the base 10.

[0204] 2) Antibacterial properties:

[0205] The antibacterial effect of Lactobacillus casei C-9 fermentation broth was determined using the Well-diffusion Agar Assay: 20 mL of sterilized MRS agar medium cooled to approximately 50°C was mixed with 200 μL of enteric pathogenic bacteria solution (10 μL of ... 6 Pour the mixture (CFU / mL) into the agar plate and mix well. After the MRS agar medium containing enteropathogenic bacteria has cooled and solidified, use a hole punch to make holes about 8 mm in diameter on the plate.

[0206] Add 100 μL of Lactobacillus casei C-9 fermentation broth to each well, diffuse at 4℃ for 12 h, then incubate at 37℃ for 48 h, and observe the size of the inhibition zone. The diameter of the inhibition zone was measured using vernier calipers (retaining two significant figures). The experimental results are shown in Table 8.

[0207] Table 8 Antibacterial properties of Lactobacillus casei C-9

[0208]

[0209] Note: The diameter of the punch is 8mm; mm is the unit for the diameter of the antibacterial zone.

[0210] As shown in Tables 1 and 2, strain C-9 exhibits good acid and bile salt tolerance. After digestion in simulated gastric juice at pH 2.5 for 3 hours, followed by digestion in simulated intestinal juice at pH 8.0 for 8 hours, its survival rate reached as high as 74.80%. Furthermore, strain C-9 demonstrates excellent broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella, and Clostridium perfringens, with a highly significant antibacterial effect.

[0211] Example 9: Preparation of inactivated lactic acid bacteria cells and their metabolites

[0212] This embodiment provides an inactivated lactic acid bacteria and its metabolites for improving pet health and stress relief during food change, including inactivated lactic acid bacteria and metabolites of compound lactic acid bacteria.

[0213] The compound lactic acid bacteria include Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis subsp. lactis BB-16, and Lactobacillus acidophilus LA-14.

[0214] The above-mentioned method for preparing postgenes includes the following steps:

[0215] 1) Weighing: Mix 4wt% whole soybean flour, 6.5wt% skim milk powder, 3wt% yeast extract, 5wt% sucrose, 2.5wt% sodium citrate and 75wt% water according to the required proportions;

[0216] 2) Preparing the material: Prepare the material at 55-60℃ for 10-30 minutes;

[0217] 3) Homogenization: 55~60℃, first-stage pressure 18~20Mpa, second-stage pressure 4~5Mpa;

[0218] 4) Sterilization: Sterilize at 90-95℃ for 15-45 minutes;

[0219] 5) Cooling: Cool to 32-37℃ to obtain the liquid material;

[0220] 6) Adding bacterial cells and lactase: Two different compound lactic acid bacteria were added to the feed solution: Compound lactic acid bacteria combination 1 consisted of *Lactobacillus plantarum* Ps-8, *Lactobacillus casei* C-9, *Bifidobacterium animalis* subsp. *lactospirum* BB-16, and *Lactobacillus acidophilus* LA-14, added in a colony-forming unit ratio of 1:1:1:1; Compound lactic acid bacteria combination 2 consisted of *Lactobacillus plantarum* Ps-8, *Lactobacillus casei* C-9, *Bifidobacterium animalis* subsp. *lactospirum* BB-16, and *Lactobacillus acidophilus* LA-14, added in a colony-forming unit ratio of 5:1:1:1; the total inoculum size for both was 6 × 10⁻⁶. 6 CFU / g (solution), and lactase (enzyme activity of 5000U / g) 0.5mL / kg (solution) was added according to production requirements;

[0221] 7) Constant temperature fermentation: Ferment at 32–37℃ until pH reaches 4.5–4.6, approximately 18–19 hours (total viable count of compound lactic acid bacteria ≥ 2.0 × 10⁻⁶). 10 CFU / g);

[0222] 8) Inactivation: Mix 18g / kg of maltodextrin (fermentation liquid) according to production requirements, preheat to 55-60℃, homogenize under first-stage pressure of 18-20Mpa and second-stage pressure of 4-5Mpa, and then sterilize and inactivate (inactivation conditions 75-95℃, 15-30min).

[0223] 9) Spray drying: The inactivated bacterial suspension is spray dried to obtain post-generic powder (i.e., post-generic powder preparation).

[0224] The metabolites in the postbiotic powder prepared by the fermentation agents of the two different strains mentioned above were detected.

[0225] After thoroughly mixing the post-biotic sample with water at a 1:4 (mass ratio), 0.1 mL was pipetted into a 1.5 mL tube, and 0.4 mL of extraction buffer (methanol:acetonitrile = 1:1 (V / V)) was added. The mixture was shaken thoroughly and then placed in an ultrasonic cold water bath for 10 min. The 1.5 mL tube was then incubated at -40℃ for 1 h. After removing the sample, it was centrifuged at 12000×g for 20 min. The supernatant was filtered through a membrane and transferred to a sample vial. An equal volume of supernatant from all samples was used to prepare a quality control sample, which was then analyzed along with the experimental samples. The target compounds were separated using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph with a Waters ACQUITYUPLCBEHAmide (2.1 mm × 100 mm, 1.7 μm) column. Sample tray temperature: 4℃, injection volume: 2 μL. The raw data was converted into mzXML format using Proteo Wizard. Peak detection, extraction, alignment, and integration were then performed using R language and based on the internal program of XCMS to obtain the final metabolite names.

[0226] The test results showed that the final metabolites contained lactic acid, citric acid, oxalic acid, malic acid, phenyllactic acid, succinic acid, 4-hydroxyphenyllactic acid, 3-phenyllactic acid, salicylic acid, vanillic acid, benzoic acid, phenylalanine, acetic acid, propionic acid, butyric acid, valeric acid, soy saponins, soy isoflavones, γ-aminobutyric acid, active short peptide molecules, genistein, gluconic acid, ursolic acid, allicin, astaxanthin, solanine, 2-hydroxycinnamic acid, and phenylacetaldehyde.

[0227] Example 10: Preparation of inactivated lactic acid bacteria cells and their metabolites

[0228] The rest is the same as in Example 9, except that:

[0229] Step 1) Weighing: Mix 7wt% whole soybean flour, 18wt% skim milk powder, 3wt% yeast extract, 7wt% sucrose, 5wt% sodium citrate and 60wt% water according to the required proportions;

[0230] Step 6) The amount of lactase (enzyme activity of 5000 U / g) added is 1 mL / kg (material solution);

[0231] Step 7) After the isothermal fermentation stops, the total viable count of the compound lactic acid bacteria is ≥3.0×10⁻⁶. 10 CFU / g;

[0232] Step 8) The amount of maltodextrin added is 25g / kg of fermentation liquid.

[0233] Example 11: Preparation of post-biotic formulations

[0234] The postbiotic powder prepared from the compound lactic acid bacteria combination 1 in Example 9 was standardized by adding a dilution carrier maltodextrin. The number of inactivated lactic acid bacteria was detected by flow cytometry, and the number of inactivated lactic acid bacteria prepared was 3×10⁻⁶. 10 cells / g, 6×10 10 cells / g and 1×10 11 Three specifications of compound lactic acid bacteria inactivated cells and their metabolites preparations, namely Postbiotic Preparation 1-300, Preparation 1-600 and Preparation 1-1000, are packaged using a powder packaging machine, 1kg / bag.

[0235] Example 12: Preparation of post-biotic formulations

[0236] The postbiotic powder prepared from the compound lactic acid bacteria combination 2 in Example 9 was added to the diluent carrier maltodextrin for standardization treatment, resulting in inactivated lactic acid bacteria counts of 3 × 10⁻⁶. 10 cells / g, 6×10 10 cells / g and 1×10 11 Three specifications of inactivated lactic acid bacteria cells and their metabolites, namely postbiotic formulation 2-300, formulation 2-600 and formulation 2-1000, are packaged using a powder packaging machine, 1kg / bag.

[0237] Example 13: Clinical Trial

[0238] 1. Experimental Example 1: Treatment of diarrhea in dogs using a formulation.

[0239] 1.1 Test Subjects

[0240] Thirty-five Belgian Shepherds aged 2-3 months with diarrhea were selected and divided into experimental group I, experimental group II, experimental group III, experimental group IV and control group, with 7 dogs in each group, including 4 female dogs and 3 male dogs in each group.

[0241] 1.2 Test Methods

[0242] Dogs with diarrhea were evaluated by feeding with inactivated lactic acid bacteria cells and their metabolites preparations 1-300 and 2-300, prepared in Examples 11 and 12, respectively. Experimental group I received 1.5 g / dog / day of preparation 1-300 via gavage; Experimental group II received 3 g / dog / day of preparation 1-300 via gavage; Experimental group III received 1.5 g / dog / day of preparation 2-300 via gavage; and Experimental group IV received 3 g / dog / day of preparation 2-300 via gavage. Specific administration method: The corresponding preparation was dissolved in 2-3 times the volume of water to prepare a solution of inactivated lactic acid bacteria cells and their metabolites, which was then drawn up using a syringe and administered directly. The medication was administered once daily between 10:00 AM and 11:00 AM for 15 consecutive days. The diarrhea rate and fecal scores for each group were recorded at days 0, 7, and 15.

[0243] 1.3 Results

[0244] Before the experiment, all 35 dogs in the 5 groups suffered from diarrhea of ​​varying degrees. After 7 days of the experiment, the diarrhea rates in Experiment I, Experiment II, Experiment III, Experiment IV, and the control group were 71%, 43%, 57%, 29%, and 86%, respectively. After 15 days of the experiment, the diarrhea rates in the five groups were 29%, 14%, 14%, 0%, and 86%, respectively. Fecal scores are as follows: Figure 1 As shown: On day 0 of the experiment, the fecal scores of the five groups were 1.7±0.5, 1.7±0.8, 1.7±0.8, 1.7±0.5, and 1.7±0.8, respectively; on day 7 of the experiment, the fecal scores of the five groups were 2.4±1.3, 3.0±1.3, 2.7±1.3, 3.6±0.8, and 1.9±1.1, respectively. The fecal scores of experimental group II (P<0.05) and experimental group IV (P<0.01) were significantly higher than those of the control group; on day 15 of the experiment, the fecal scores of the five groups were 3.6±0.8, 3.7±0.8, 3.6±1.1, 4.0±0.0, and 2.4±1.0, respectively. The fecal scores of experimental group I (P<0.05), experimental group II (P<0.05), experimental group III (P<0.05), and experimental group IV (P<0.01) were all significantly higher than those of the control group. It is evident that daily oral administration of different doses of inactivated lactic acid bacteria and their metabolites preparations 1-300 and 2-300 significantly alleviated diarrhea in dogs. Experimental group IV showed the best effect; therefore, preparation 2-300 of inactivated lactic acid bacteria and their metabolites is the preferred choice.

[0245] Experimental Example 2: Treatment of diarrhea in cats with a formulation

[0246] 2.1 Test Subjects

[0247] Thirty British Shorthair cats aged 3-5 months with diarrhea were selected and divided into experimental group I, experimental group II, experimental group III, experimental group IV and control group, with 6 cats in each group, including 2 male cats and 4 female cats in each group.

[0248] 2.2 Test Methods

[0249] Cats with diarrhea were evaluated by feeding with inactivated lactic acid bacteria cells and their metabolites preparations 1-300 and 2-300, prepared in Examples 11 and 12, respectively. Experimental group I received 0.5 g / cat / day of preparation 1-300 via gavage; Experimental group II received 1 g / cat / day of preparation 1-300 via gavage; Experimental group III received 0.5 g / cat / day of preparation 2-300 via gavage; and Experimental group IV received 1 g / cat / day of preparation 2-300 via gavage. Specific administration method: The corresponding preparation was dissolved in 2-3 times the volume of water to prepare a solution of inactivated lactic acid bacteria cells and their metabolites, which was then drawn up with a syringe and administered directly. The medication was administered once daily between 10:00 AM and 11:00 AM for 15 consecutive days. The diarrhea rate and fecal scores for each group were recorded at days 0, 7, and 15.

[0250] 2.3 Results

[0251] Before the experiment, all 30 cats in the 5 groups suffered from diarrhea of ​​varying degrees. After 7 days of the experiment, the diarrhea rates in Experiment I, Experiment II, Experiment III, Experiment IV, and the control group were 50%, 33%, 50%, 50%, and 83%, respectively. After 15 days of the experiment, the diarrhea rates in the five groups were 17%, 0%, 17%, 0%, and 67%, respectively. Fecal scores are as follows: Figure 2 As shown, on day 0 of the experiment, the fecal scores of the five groups were 1.7±0.5, 1.5±0.8, 1.8±0.8, 1.5±0.5, and 1.5±0.5, respectively; on day 7 of the experiment, the fecal scores of the seven groups were 3.0±1.3, 3.3±1.0, 2.8±1.3, 3.5±0.5, and 2.2±1.2, respectively. The fecal scores of experimental group II (P<0.05) and experimental group IV (P<0.05) were significantly higher than those of the control group; on day 15 of the experiment, the fecal scores of the five groups were 3.7±0.8, 4.0±0.0, 3.8±0.4, 4.0±0.0, and 2.7±1.2, respectively. The fecal scores of experimental group I (P<0.05), experimental group II (P<0.05), experimental group III (P<0.05), and experimental group IV (P<0.05) were all significantly higher than those of the control group. It is evident that daily oral administration of different doses of inactivated lactic acid bacteria and their metabolites preparations 1-300 and 2-300 significantly alleviated diarrhea in cats. Among them, experimental group IV showed the best effect, therefore, preparation 2-300 of inactivated lactic acid bacteria and their metabolites is preferred.

[0252] Experiment Example 3: A breeding evaluation experiment using extruded food with added additives on dogs.

[0253] 3.1 Test Subjects

[0254] Thirty-six German Shepherds aged 4±1 months were selected and divided into experimental group I, experimental group II, experimental group III and control group, with 9 dogs in each group, including 7 male dogs and 2 female dogs in each group.

[0255] 3.2 Test Methods

[0256] The inactivated lactic acid bacteria cells and their metabolites preparations 2-300, 2-600, and 2-1000 prepared in Example 12 were added to extruded food, with 10 grams of each preparation added per 10 kg of extruded food. Three types of extruded food containing inactivated lactic acid bacteria cells and their metabolites were prepared, corresponding to Experimental Group I, Experimental Group II, Experimental Group III, and the control group, respectively. The experiment lasted for 30 days. Diarrhea rate was recorded and fecal scores were calculated on days 0, 15, and 30. Body temperature, pulse, respiratory rate, feed intake, and body weight of the participating dogs were also measured. The apparent digestibility of crude protein and crude fat was measured on days 0 and 30.

[0257] 3.3 Results

[0258] 3.3.1 Results of pulse, respiratory rate, and body temperature measurements:

[0259] The results are as follows Figure 3-5 The results showed no significant differences in pulse, respiratory rate, and body temperature between the experimental and control groups during the experiment. This indicates that adding the aforementioned inactivated lactic acid bacteria and their metabolites to extruded food had no significant effect on the dogs' basic physiological indicators.

[0260] 3.3.2 Results of feed intake and body weight measurement:

[0261] Feed intake measurement results as follows Figure 6 The results showed that the average feed intake (g / animal / day) for the four groups on day 0 was 518.6±8.2, 518.7±10.2, 518.6±9.1, and 518.8±9.6, respectively; the average feed intake (g / animal / day) on day 15 was 538.3±10.3, 541.6±7.7, 541.9±9.9, and 538.0±10.6, respectively; and the average feed intake (g / animal / day) on day 30 was 567.3±9.9, 572.8±10.2, 573.4±5.4, and 565.4±7.7, respectively. Weight measurement results are as follows... Figure 7 The results showed that the average weight (kg / animal) of the four groups on day 0 of the experiment was 17.2±0.3, 17.3±0.5, 17.3±0.6 and 17.2±0.5, respectively; the average weight (kg / animal) of the four groups on day 15 of the experiment was 18.3±0.6, 18.4±0.8, 18.7±0.4 and 18.1±0.4, respectively; and the average weight (kg / animal) of the four groups on day 30 of the experiment was 19.1±0.8, 19.4±0.3, 19.6±0.5 and 18.8±0.6, respectively.

[0262] At 15 and 30 days of the experiment, the average feed intake and body weight of experimental groups I, II and III were higher than those of the control group. At the same time, experimental groups I, II and III showed an increasing trend, indicating that as the number of inactivated lactic acid bacteria in different preparations increased, the efficacy of promoting feed intake and growth increased.

[0263] 3.3.3 Statistical results of apparent digestibility of major nutrients:

[0264] apparent digestibility of crude protein, such as Figure 8 The results showed that the average apparent digestibility (%) of crude protein in the four groups on day 0 of the experiment were 83.5±1.3, 83.5±1.2, 83.5±1.4, and 83.5±1.2, respectively; and on day 30 of the experiment, the average apparent digestibility of crude protein in the four groups were 84.7±1.4, 86.2±1.4, 86.3±1.1, and 83.8±1.2, respectively. The apparent digestibility of crude protein in groups II (P<0.05) and III (P<0.05) was significantly higher than that in the control group. The apparent digestibility of crude fat was as follows... Figure 9 The results showed that the average apparent digestibility (%) of crude fat in the four groups on day 0 was 93.1±0.9, 92.9±0.7, 93.1±1.1, and 93.5±0.5, respectively; and on day 30, the average apparent digestibility of crude fat in the four groups was 94.2±0.7, 94.6±0.7, 94.8±0.6, and 93.4±0.9, respectively. On day 30, the average apparent digestibility of crude protein and crude fat in experimental groups I, II, and III were all higher than that in the control group. Furthermore, the average apparent digestibility of crude protein and crude fat in experimental groups I, II, and III showed an increasing trend, indicating that the level of nutrient digestibility increased with the increase in the number of inactivated lactic acid bacteria in different formulations.

[0265] 3.3.4 Statistical results of diarrhea rate and stool score:

[0266] Before the experiment, all 36 German Shepherds in the four groups participated in the test suffered from diarrhea of ​​varying degrees. After 15 days of the experiment, the diarrhea rates in Experiment I, Experiment II, Experiment III, and the control group were 56%, 33%, 22%, and 78%, respectively. After 30 days of the experiment, the diarrhea rates in the four groups were 22%, 11%, 11%, and 89%, respectively. Fecal scores are as follows: Figure 10As shown, on day 0 of the experiment, the fecal scores of experimental groups I, II, III, and the control group were 1.6±0.7, 1.8±0.9, 1.6±0.5, and 1.6±0.9, respectively; on day 15 of the experiment, the fecal scores of the five groups were 3.1±0.8, 3.5±0.8, 3.6±0.5, and 2.4±0.7, respectively, with experimental group III (P<0.05) being significantly higher than the control group; on day 30 of the experiment, the fecal scores of the five groups were 3.6±0.5, 3.8±0.5, 3.8±0.5, and 1.9±0.6, respectively, with experimental groups I (P<0.05), II (P<0.05), and III (P<0.01) all being significantly higher than the control group. It is evident that adding inactivated lactic acid bacteria and their metabolite preparations 2-300, 2-600, and 2-1000 to extruded food at a ratio of 1 kg / t can significantly alleviate diarrhea in German Shepherds. Therefore, inactivated lactic acid bacteria and their metabolite preparations 2-600 and 2-1000 are preferred.

[0267] Experiment 4: Feeding evaluation experiment on cats using baked food with added additives.

[0268] 4.1 Test Subjects

[0269] Forty domestic shorthair cats aged 43±5 days were selected and divided into experimental group I (2-300 cats), experimental group II (2-600 cats), experimental group III (2-1000 cats), and control group, with 10 cats in each group.

[0270] 4.2 Test Methods

[0271] The inactivated lactic acid bacteria cells and their metabolites preparations 2-300, 2-600, and 2-1000, prepared in Example 12, were added to baked food, with each preparation added at a dosage of 10 grams per 10 kg of baked food. Three types of baked food containing inactivated lactic acid bacteria cells and their metabolites were prepared, and the experiment lasted for 21 days. Ten cats were included in each of the experimental groups: Experiment I (2-300 group), Experiment II (2-600 group), Experiment III (2-1000 group), and the control group. The diarrhea rate was recorded and fecal scores were calculated on days 0, 14, and 21. Food intake, body weight, and apparent digestibility of crude protein and crude fat were measured on days 0 and 21.

[0272] 4.3 Results

[0273] 4.3.1 Results of feed intake and body weight measurement:

[0274] Feed intake measurement results as follows Figure 11The results showed that the average feed intake (g / animal / day) on day 0 for experimental groups I, II, III, and the control group were 59.4±2.1, 59.7±2.9, 59.3±2.5, and 59.6±2.9, respectively; and the average feed intake (g / animal / day) on day 21 were 65.1±3.6, 68.5±3.3, 68.6±3.2, and 61.8±2.9, respectively. Body weight measurements were as follows... Figure 12 The results showed that the average body weight (kg / animal) of the four groups on day 0 was 1.28±0.16, 1.25±0.10, 1.30±0.19, and 1.28±0.14, respectively; and on day 21, the average body weight (kg / animal) of the four groups was 1.40±0.19, 1.49±0.15, 1.49±0.20, and 1.35±0.16, respectively. On days 14 and 21, the average feed intake and body weight of experimental groups I, II, and III were all higher than those of the control group. This indicates that the addition of three different inactivated lactic acid bacteria cells and their metabolites to the roasted feed all exhibited varying degrees of effects in promoting feed intake and growth.

[0275] 4.3.2 Statistical results of apparent digestibility of major nutrients:

[0276] apparent digestibility of crude protein, such as Figure 13 The results showed that the average apparent digestibility (%) of crude protein in the four groups on day 0 of the experiment were 85.2±1.9, 85.8±1.7, 85.5±1.4, and 86.0±1.3, respectively; and on day 21 of the experiment, the average apparent digestibility of crude protein in the four groups were 88.5±1.4, 90.8±1.5, 91.1±1.8, and 86.1±1.5, respectively. The apparent digestibility of crude protein in groups II (P<0.05) and III (P<0.05) was significantly higher than that in the control group. The apparent digestibility of crude fat was as follows... Figure 14 The results showed that the average apparent digestibility (%) of crude fat in the four groups on day 0 of the experiment were 91.7±1.7, 92.0±0.9, 91.6±1.1, and 91.7±1.5, respectively; and on day 21 of the experiment, the average apparent digestibility of crude fat in the four groups were 92.4±1.4, 92.5±0.7, 92.5±1.0, and 92.2±0.8, respectively. Groups I, II, and III of the experiment were all higher than the control group. Adding three different inactivated lactic acid bacteria and their metabolite preparations to the baked grains promoted the digestibility of the main nutrients, protein and fat.

[0277] 4.3.3 Statistical results of diarrhea rate and stool score:

[0278] Before the experiment, all 40 domestic shorthair cats in the four groups suffered from diarrhea of ​​varying degrees. After 14 days of the experiment, the diarrhea rates in experimental groups I, II, III, and the control group were 30%, 10%, 20%, and 80%, respectively. After 21 days of the experiment, the diarrhea rates in the four groups were 10%, 0%, 0%, and 60%, respectively. Fecal scores are as follows: Figure 15 As shown, on day 0, the fecal scores of experimental groups I, II, III, and the control group were 1.9±0.6, 1.8±0.7, 1.9±0.6, and 1.9±0.6, respectively. On day 14, the fecal scores of the four groups were 3.4±0.7, 3.8±0.5, 3.9±0.4, and 2.4±1.2, respectively, with experimental groups I (P<0.05), II (P<0.01), and III (P<0.01) being significantly higher than the control group. On day 21, the fecal scores of the four groups were 3.8±0.5, 3.9±0.4, 3.9±0.4, and 2.8±0.9, respectively, with experimental groups I (P<0.01), II (P<0.01), and III (P<0.01) all being significantly higher than the control group. It is evident that adding inactivated lactic acid bacteria and their metabolite preparations 2-300, 2-600, and 2-1000 to baked food at a ratio of 1 kg / t can significantly alleviate diarrhea in domestic shorthair cats. Inactivated lactic acid bacteria and their metabolite preparations 2-600 and 2-1000 are preferred.

[0279] Experiment 5: A transition test was conducted on dogs using extruded food with added additives.

[0280] 5.1 Test Subjects

[0281] Forty-two Teddy dogs aged 4±1 months were selected and divided into three groups: Experimental Group I, Experimental Group II, and Experimental Group III, with 14 dogs in each group.

[0282] 5.2 Test Methods

[0283] The inactivated lactic acid bacteria cells and their metabolites preparation 2-1000, prepared in Example 12, were added to two extruded feed formulations, A and B, at a dosage of 10 grams per 10 kg of extruded feed, to prepare two types of extruded feeds containing inactivated lactic acid bacteria cells and their metabolites: A+ and B+. In Experiment I, extruded feed A was fed continuously for 15 days, then directly switched to extruded feed B; in Experiment II, extruded feed A was fed continuously for 15 days, then directly switched to extruded feed B+; in Experiment III, extruded feed A+ was fed continuously for 15 days, then directly switched to extruded feed B+. Diarrhea rate, fecal score, and feed intake were recorded 3 days before and 1, 3, and 7 days after the feed change. Serum cortisol, malondialdehyde, immunoglobulin G, and immunoglobulin A levels were also measured.

[0284] 5.3 Results

[0285] 5.3.1 Statistical results of diarrhea rate and stool score:

[0286] Three days before the food change, the diarrhea rates in Experimental Groups I, II, and III were 43%, 50%, and 14%, respectively; one day after the food change, the diarrhea rates were 93%, 57%, and 29%; three days after the food change, the diarrhea rates were 86%, 29%, and 14%; and seven days after the food change, the diarrhea rates were 57%, 14%, and 7%. Stool scores were as follows: Figure 16 As shown, 3 days before the food change, the fecal scores of Experimental Groups I, II, and III were 2.6±0.9, 2.4±1.0, and 3.4±0.9, respectively, with Experimental Group III significantly higher than Experimental Groups I (P<0.01) and II (P<0.01). One day after the food change, the fecal scores of the three groups were 1.7±0.6, 2.2±1.0, and 3.1±1.2, respectively, with Experimental Group III significantly higher than Experimental Groups I (P<0.01) and II (P<0.05). Experimental Groups II... The fecal scores of the three groups were significantly higher than those of the experimental group I (P < 0.05). Three days before the food change, the fecal scores of the three groups were 1.8 ± 0.9, 2.7 ± 0.9, and 3.4 ± 0.8, respectively, with the scores of experimental group II (P < 0.05) and experimental group III (P < 0.01) significantly higher than those of experimental group I. Seven days before the food change, the fecal scores of the three groups were 2.4 ± 1.2, 3.4 ± 0.7, and 3.5 ± 0.7, respectively, with the scores of experimental group II (P < 0.01) and experimental group III (P < 0.01) significantly higher than those of experimental group I. Therefore, adding 2-1000, a preparation of inactivated lactic acid bacteria and its metabolites, to extruded food at a ratio of 1 kg / t can significantly alleviate diarrhea in Teddy dogs and significantly reduce diarrhea caused by stress due to food intolerance during the food change process.

[0287] 5.3.2 Feed intake measurement results:

[0288] Feed intake measurement results as follows Figure 17The results showed that, in the three days prior to the feed change, the average feed intake (g / bird / day) of Experimental Groups I, II, and III were 231.2±6.8, 228.1±9.6, and 236.8±8.1, respectively, with Experimental Group III showing a higher average feed intake than Experimental Groups I and II. One day after the feed change, the average feed intake (g / bird / day) of the three groups were 187.8±7.9, 216.4±9.1, and 229.3±7.0, respectively, with Experimental Groups II (P<0.05) and III (P<0.01) showing significantly higher average feed intake than Experimental Group I. Three days after the feed change, the average feed intake (g / bird / day) of the three groups were 212.5±11.6, 232.3±9.2, and 241.8±7.4, respectively, with Experimental Groups II and III showing significantly higher average feed intake than Experimental Group I (P<0.05). Seven days after the feed change, the average feed intake (g / animal / day) of the three groups were 239.8±5.6, 258.3±8.2, and 260.2±7.9, respectively. The average feed intake of experimental groups II and III was significantly higher than that of experimental group I (P<0.05). This indicates that adding 2-1000, a preparation of inactivated lactic acid bacteria and its metabolites, at a ratio of 1 kg / t to extruded feeds A and B can reduce anorexia and feed intake decline caused by stress during feed change, and has a certain effect on promoting feed intake. Specifically, in experimental group I, the average feed intake dropped sharply one day after switching from extruded feed A to extruded feed B, but recovered to pre-change levels after seven days. In contrast, experimental groups II and III, which added 2-1000, showed a smaller decrease in average feed intake. In particular, in Experiment III, both the diets before and after the feed change were supplemented with 2-1000 of inactivated lactic acid bacteria and its metabolites. The average feed intake recovered rapidly within 1-3 days after the feed change, showing significant results.

[0289] 5.3.3 Serum cortisol measurement results:

[0290] Serum cortisol test results as follows Figure 18The results showed that, 3 days before the feed change, the serum cortisol levels (ng / mL) in experimental groups I, II, and III were 57.3±3.1, 56.8±3.2, and 56.5±3.6, respectively; 1 day after the feed change, the serum cortisol levels (ng / mL) in the three groups were 181.6±3.7, 98.0±3.5, and 70.8±3.2, respectively. The serum cortisol levels in experimental groups II (P<0.01) and III (P<0.001) were significantly lower than those in experimental group I; 3 days after the feed change, the serum cortisol levels in the three groups... The serum cortisol levels (ng / mL) in the experimental groups were 203.5±5.5, 84.4±2.6, and 65.6±3.3, respectively. The serum cortisol levels in experimental groups II and III were significantly lower than those in experimental group I (P<0.001). Seven days after the feed change, the serum cortisol levels (ng / mL) in the three groups were 120.6±4.0, 65.7±3.8, and 58.4±3.9, respectively. The serum cortisol levels in experimental groups II (P<0.05) and III (P<0.01) were significantly lower than those in experimental group I (P<0.05). Serum cortisol is an adrenal glucocorticoid that reflects the stress level in animals and is commonly used to detect the physiological response level of animals under stress. Comparing the fluctuations in serum cortisol levels in the experimental Teddy dogs throughout the feed change process, the order was: Experimental group III < Experimental group II < Experimental group I, indicating that the stress level during the feed change process was: Experimental group III < Experimental group II < Experimental group I. It is evident that adding 2-1000 inactivated lactic acid bacteria and their metabolite preparations at a ratio of 1 kg / t to extruded grains A and B can effectively reduce stress during grain transition.

[0291] 5.3.4 Serum malondialdehyde (MDA) measurement results:

[0292] Serum malondialdehyde (MDA) test results are as follows Figure 19The results showed that, 3 days before the change of feed, the serum malondialdehyde (mAD) levels (nmol / mL) in experimental groups I, II, and III were 1.91±0.16, 1.89±0.13, and 1.86±0.12, respectively; 1 day after the change of feed, the serum mAD levels (nmol / mL) in the three groups were 3.59±0.40, 2.79±0.19, and 2.20±0.27, respectively. The serum mAD levels in experimental groups II (P<0.05) and III (P<0.01) were significantly lower than those in experimental group I. 3 days after the change of feed... The serum malondialdehyde (MDA) levels (nmol / mL) in the three groups were 3.23±0.31, 2.12±0.22, and 1.93±0.15, respectively. The MDA levels in experimental groups II and III were significantly lower than those in experimental group I (P<0.01). Seven days after the food change, the serum MDA levels (nmol / mL) in the three groups were 2.45±0.26, 1.89±0.19, and 1.88±0.17, respectively. The MDA levels in experimental groups II and III were significantly lower than those in experimental group I (P<0.05). Serum MDA is an important indicator reflecting the level of free radicals and lipid peroxidation metabolism in the body. Comparing the fluctuations in serum MDA levels in the experimental Teddy dogs throughout the food change process, the order was: Experimental group III < Experimental group II < Experimental group I, indicating that the antioxidant capacity of the three groups of dogs was: Experimental group III > Experimental group II > Experimental group I. It is evident that adding 2-1000 inactivated lactic acid bacteria and its metabolites to puffed grains A and B at a ratio of 1 kg / t can enhance the body's ability to scavenge peroxides, rapidly eliminate excess oxygen free radicals generated by stress, and thus reduce adverse reactions caused by stress.

[0293] 5.3.5 Serum Immunoglobulin G Measurement Results:

[0294] Serum immunoglobulin G measurement results are as follows Figure 20The results showed that, three days before the feed change, the immunoglobulin G (mg / mL) levels in experimental groups I, II, and III were 9.7±0.3, 9.5±0.4, and 11.5±0.4, respectively. Among them, the immunoglobulin G level in experimental group III (P<0.05) was significantly higher than that in experimental groups I and II. One day after feed change, the levels of immunoglobulin G (mg / mL) in experimental groups I, II, and III were 9.4±0.6, 10.2±0.5, and 12.0±0.6, respectively, with group III (P<0.05) showing a significantly higher level than group I. Three days after feed change, the levels of serum immunoglobulin G (mg / mL) in the three groups were 9.3±0.6, 10.8±0.5, and 13.1±0.5, respectively, with groups II (P<0.05) and III (P<0.01) showing significantly higher levels of immunoglobulin G than group I. Seven days after feed change, the levels of serum immunoglobulin G (mg / mL) in the three groups were 9.6±0.4, 13.4±0.4, and 14.8±0.2, respectively, with groups II (P<0.01) and III (P<0.01) showing significantly higher levels of immunoglobulin G than group I. It is evident that adding 2-1000 inactivated lactic acid bacteria and its metabolites to extruded food A and B at a ratio of 1 kg / t can significantly increase the content of immunoglobulin G in serum, thereby enhancing immunity, improving the disease resistance of Teddy dogs, and reducing the incidence of diseases such as diarrhea.

[0295] 5.3.6 Serum Immunoglobulin A Measurement Results:

[0296] Serum immunoglobulin A test results are as follows Figure 21 The results showed that, three days before the feed change, the immunoglobulin A (mg / mL) levels in experimental groups I, II, and III were 1.46±0.11, 1.44±0.07, and 1.53±0.08, respectively. One day after the feed change, the levels of immunoglobulin A (mg / mL) in experimental groups I, II, and III were 1.44±0.10, 1.49±0.09, and 1.54±0.07, respectively. Three days after the feed change, the levels of serum immunoglobulin A (mg / mL) in the three groups were 1.42±0.08, 1.55±0.08, and 1.57±0.09, respectively, with group III (P<0.05) showing significantly higher levels of immunoglobulin A than group I. Seven days after the feed change, the levels of serum immunoglobulin A (mg / mL) in the three groups were 1.47±0.08, 1.58±0.09, and 1.61±0.07, respectively. It is evident that adding 2-1000 inactivated lactic acid bacteria and its metabolites to extruded food A and B at a ratio of 1 kg / t can significantly increase the content of immunoglobulin A in serum, thereby enhancing immunity, improving the disease resistance of Teddy dogs, and reducing the incidence of diseases such as diarrhea.

[0297] Experiment 6: A cat food transition test using baked food with added additives.

[0298] 6.1 Test Subjects

[0299] Thirty-three Ragdoll kittens aged 5±1 months were selected and divided into three experimental groups: Experiment I, Experiment II, and Experiment III, with 11 kittens in each group.

[0300] 6.2 Test Methods

[0301] The inactivated lactic acid bacteria cells and their metabolites preparation 2-1000, prepared in Example 12, were added to two baked grain formulations, C and D, respectively, at a dosage of 10 grams per 10 kg of baked grain, to prepare two baked grains, C+ and D+, containing inactivated lactic acid bacteria cells and their metabolites. In Experiment I, baked grain C was fed continuously for 15 days, then directly switched to baked grain D; in Experiment II, baked grain C was fed continuously for 15 days, then directly switched to baked grain D+; in Experiment III, baked grain C+ was fed continuously for 15 days, then directly switched to baked grain D+. Diarrhea rate, fecal score, and feed intake were recorded 1 day before the change and 1, 3, and 7 days after the change. Serum levels of cortisol, malondialdehyde, immunoglobulin G, immunoglobulin A, D-lactic acid, and diamine oxidase were also measured.

[0302] 6.3 Results

[0303] 6.3.1 Statistical results of diarrhea rate and stool score:

[0304] Three days before the food change, the diarrhea rates in Experimental Groups I, II, and III were 64%, 64%, and 21%, respectively. One day after the food change, the diarrhea rates were 79%, 71%, and 21%, respectively. Three days after the food change, the diarrhea rates were 79%, 36%, and 14%, respectively. Seven days after the food change, the diarrhea rates were 57%, 14%, and 14%, respectively. Stool scores were as follows: Figure 22As shown, three days before the food change, the fecal scores of experimental groups I, II, and III were 2.0±0.9, 1.9±0.7, and 2.7±1.0, respectively, with experimental group III significantly higher than experimental groups I (P<0.05) and II (P<0.05). One day before the food change, the fecal scores of the three groups were 1.5±0.5, 1.9±0.5, and 2.7±0.8, respectively, with experimental group III significantly higher than experimental groups I (P<0.01) and II (P<0.05). <0.05); Three days before the food change, the fecal scores of the three groups were 1.7±0.5, 2.5±1.0, and 3.9±0.8, respectively, with experimental group II (P<0.05) and experimental group III (P<0.01) significantly higher than experimental group I; Seven days before the food change, the fecal scores of the three groups were 2.2±1.1, 3.0±0.9, and 3.2±0.8, respectively, with experimental group II (P<0.05) and experimental group III (P<0.05) significantly higher than experimental group I. Therefore, adding inactivated lactic acid bacteria and its metabolite preparation 2-1000 to baked food C and D at a ratio of 1 kg / t can significantly alleviate diarrhea in Ragdoll cats and significantly reduce diarrhea caused by stress due to food intolerance during the food change process.

[0305] 6.3.2 Feed intake measurement results:

[0306] Feed intake measurement results as follows Figure 23The results showed that, in the three days prior to the feed change, the average feed intake (g / bird / day) of Experimental Groups I, II, and III were 77.1±4.0, 76.7±4.5, and 81.5±4.2, respectively, with Experimental Group III showing a higher average feed intake than Experimental Groups I and II. One day after the feed change, the average feed intake (g / bird / day) of the three groups were 64.8±3.8, 74.5±4.1, and 80.6±3.8, respectively, with Experimental Groups II (P<0.05) and III (P<0.01) showing significantly higher average feed intake than Experimental Group I. Three days after the feed change, the average feed intake (g / bird / day) of the three groups were 63.1±4.1, 74.3±3.9, and 81.7±3.4, respectively, with Experimental Groups II (P<0.05) and III (P<0.01) showing significantly higher average feed intake than Experimental Group I. Seven days after the feed change, the average feed intake (g / bird / day) of the three groups were 65.0±4.0, 81.2±3.8, and 86.5±3.3, respectively. The average feed intake of Group II (P<0.01) and Group III (P<0.01) was significantly higher than that of Group I. One day after switching from roasted feed A to roasted feed B, the average feed intake of Group I dropped sharply and did not recover to pre-change levels by the end of the experiment. In Group II, the average feed intake of the supplemented probiotic 2-1000 did not decrease significantly and returned to normal within 7 days. The average feed intake of Group III, the supplemented with 2-1000 of inactivated lactic acid bacteria and its metabolites, continued to increase. Therefore, adding 2-1000 of inactivated lactic acid bacteria and its metabolites at a ratio of 1 kg / t to roasted feeds C and D can reduce anorexia and feed intake decline caused by stress during feed change and has a certain effect on promoting feed intake.

[0307] 6.3.3 Serum malondialdehyde (MDA) measurement results:

[0308] Serum malondialdehyde (MDA) test results are as follows Figure 24The results showed that, 3 days before the change of feed, the serum malondialdehyde (mAD) levels (nmol / mL) in experimental groups I, II, and III were 2.62±0.13, 2.58±0.12, and 2.61±0.13, respectively; 1 day after the change of feed, the serum mAD levels (nmol / mL) in the three groups were 4.24±0.37, 2.76±0.26, and 2.66±0.19, respectively. Among them, the serum mAD levels in experimental groups II (P<0.01) and III (P<0.01) were significantly lower than those in experimental group I. Three days after the food change, the serum malondialdehyde (MDA) levels (nmol / mL) in the three groups were 3.44±0.20, 2.70±0.24, and 2.65±0.17, respectively. The MDA levels in experimental groups II and III were significantly lower than those in experimental group I (P<0.05). Seven days after the food change, the serum MDA levels (nmol / mL) in the three groups were 2.82±0.27, 2.64±0.21, and 2.58±0.18, respectively. The MDA levels in experimental groups II and III were also lower than those in experimental group I. This indicates that adding inactivated lactic acid bacteria and its metabolite preparation 2-1000 to baked food C and D at a ratio of 1 kg / t can significantly reduce the stress caused by food change in Ragdoll cats.

[0309] 6.3.4 Serum cortisol measurement results:

[0310] Serum cortisol test results as follows Figure 25 The results showed that, 3 days before the feed change, the serum cortisol levels (ng / mL) in experimental groups I, II, and III were 44.1±3.0, 43.9±1.4, and 44.2±2.2, respectively; 1 day after the feed change, the serum cortisol levels (ng / mL) in the three groups were 147.2±10.5, 75.8±5.1, and 50.6±6.7, respectively. The serum cortisol levels in experimental groups II (P<0.01) and III (P<0.001) were significantly lower than those in experimental group I; 3 days after the feed change, the serum cortisol levels in the three groups ( The serum cortisol levels (ng / mL) in experimental groups II, III, and D were 149.2±9.6, 62.8±8.7, and 47.2±5.3, respectively. The serum cortisol levels in experimental groups II (P<0.01) and III (P<0.001) were significantly lower than those in experimental group I. Seven days after feed change, the serum cortisol levels (ng / mL) in the three groups were 77.9±8.5, 46.3±4.6, and 44.8±5.2, respectively. The serum cortisol levels in experimental groups II (P<0.05) and III (P<0.05) were significantly lower than those in experimental group I (P<0.05). Further, it was confirmed that adding 2-1000, a preparation of inactivated lactic acid bacteria and its metabolites, at a ratio of 1 kg / t to roasted grains C and D significantly reduced feed change stress.

[0311] 6.3.5 Serum Immunoglobulin G Measurement Results:

[0312] Serum immunoglobulin G measurement results are as follows Figure 26 The results showed that, three days before the feed change, the immunoglobulin G (mg / mL) levels in experimental groups I, II, and III were 6.4±0.4, 6.6±0.5, and 7.7±0.4, respectively. Among them, the immunoglobulin G level in experimental group III (P<0.05) was significantly higher than that in experimental groups I and II. One day after feed change, the levels of immunoglobulin G (mg / mL) in experimental groups I, II, and III were 6.1±0.5, 6.9±0.4, and 7.7±0.3, respectively, with group III (P<0.05) showing a significantly higher level than group I. Three days after feed change, the levels of serum immunoglobulin G (mg / mL) in the three groups were 6.2±0.3, 7.1±0.4, and 7.8±0.4, respectively, with group III (P<0.05) showing a significantly higher level than group I. Seven days after feed change, the levels of serum immunoglobulin G (mg / mL) in the three groups were 6.2±0.5, 7.5±0.4, and 7.8±0.2, respectively, with groups II (P<0.05) and III (P<0.05) showing significantly higher levels of immunoglobulin G than group I. It is evident that adding 2-1000 inactivated lactic acid bacteria and their metabolites to baked food C and D at a ratio of 1 kg / t can significantly increase the content of immunoglobulin G in serum, thereby enhancing immunity, improving the disease resistance of Ragdoll cats, and reducing the incidence of diseases such as diarrhea.

[0313] 6.3.6 Serum Immunoglobulin A Measurement Results:

[0314] Serum immunoglobulin A test results are as follows Figure 27The results showed that, three days before the feed change, the immunoglobulin A (mg / mL) levels in experimental groups I, II, and III were 1.33±0.12, 1.31±0.10, and 1.43±0.07, respectively, with group III (P<0.05) showing a significantly higher level than group II. One day after the feed change, the immunoglobulin A (mg / mL) levels in experimental groups I, II, and III were 1.27±0.09, 1.45±0.08, and 1.47±0.13, respectively, with groups II (P<0.05) and III (P<0.05) showing significantly higher levels than group III. In Experiment I, the serum immunoglobulin A levels (mg / mL) of the three groups three days after the feed change were 1.34±0.13, 1.55±0.11, and 1.49±0.07, respectively. The immunoglobulin A levels in Experiment II (P<0.05) and Group III (P<0.05) were significantly higher than those in Experiment I. Seven days after the feed change, the serum immunoglobulin A levels (mg / mL) of the three groups were 1.32±0.09, 1.53±0.11, and 1.47±0.11, respectively. Again, the immunoglobulin A levels in Experiment II (P<0.05) and Group III (P<0.05) were significantly higher than those in Experiment I. This further indicates that adding inactivated lactic acid bacteria and its metabolite preparation 2-1000 to baked food C and D at a ratio of 1 kg / t can significantly increase the serum immunoglobulin A level, thereby enhancing immunity, improving the disease resistance of Ragdoll cats, and reducing the incidence of diseases such as diarrhea.

[0315] 6.3.7 Serum D-lactic acid assay results:

[0316] Serum D-lactic acid test results are as follows Figure 28The results showed that, three days before the feed change, the D-lactic acid (ng / mL) levels in experimental groups I, II, and III were 0.64±0.10, 0.67±0.12, and 0.46±0.08, respectively, with group III (P<0.05) significantly lower than groups I and II. One day after the feed change, the serum D-lactic acid (mg / mL) levels in the three groups were 0.82±0.10, 0.56±0.09, and 0.47±0.14, respectively, with groups II (P<0.05) and III (P<0.01) significantly lower than groups I and II. In Experiment I, the serum D-lactic acid levels (mg / mL) of the three groups three days after feed change were 0.84±0.09, 0.50±0.11, and 0.47±0.12, respectively. Groups II (P<0.01) and III (P<0.01) showed significantly lower levels than Group I. Seven days after feed change, the serum D-lactic acid levels (mg / mL) of the three groups were 0.72±0.07, 0.43±0.09, and 0.45±0.04, respectively. Groups II (P<0.05) and III (P<0.05) showed significantly lower levels than Group I. D-lactic acid is an important substance reflecting intestinal barrier function. It is produced by the metabolism of certain bacteria in the intestine and is normally excreted through the animal's intestinal barrier. However, in cases of intestinal damage, the intestinal barrier function declines, and D-lactic acid cannot be smoothly excreted, accumulating in the blood and causing an increase in blood D-lactic acid levels. During the food transition, the serum D-lactic acid level in Experiment I showed a significant increase followed by a gradual decrease, indicating a rapid decline in the pets' intestinal barrier function in a short period, which may be closely related to the surge in diarrhea incidence. In Experiment II, the serum D-lactic acid level showed a gradual decrease, indicating that the addition of postbiotic preparation 2-1000 to the baked food at a ratio of 1 kg / t significantly improved intestinal barrier function. In Experiment III, the serum D-lactic acid level remained relatively stable before and after the food transition. This indicates that the addition of C+ and D+ to the baked food at a ratio of 1 kg / t to inactivated lactic acid bacteria and their metabolites preparation 2-1000 both have excellent effects on improving the intestinal barrier.

[0317] 6.3.8 Serum diamine oxidase assay results:

[0318] Serum diamine oxidase test results as follows Figure 29The results showed that, three days before the feed change, the levels of diamine oxidase (μg / dL) in experimental groups I, II, and III were 1.80±0.14, 1.79±0.11, and 1.56±0.10, respectively. Group III (P<0.05) had significantly lower levels than groups I and II. One day after the feed change, the serum diamine oxidase (μg / dL) levels in the three groups were 2.14±0.10, 1.66±0.09, and 1.52±0.04, respectively. Groups II (P<0.05) and III (P<0.01) had significantly higher levels. The serum diamine oxidase (μg / dL) levels in the three groups were lower than those in Experimental Group I on day 3 after the feed change: 1.96±0.07, 1.61±0.10, and 1.53±0.05, respectively. Groups II and III (P<0.05) showed significantly lower levels than Group I. On day 7 after the feed change, the serum diamine oxidase (μg / dL) levels in the three groups were 1.81±0.12, 1.48±0.07, and 1.48±0.04, respectively. Groups II and III (P<0.05) showed significantly lower levels than Group I. Diamine oxidase is another important substance reflecting intestinal barrier function. Normally, histamine-like substances are produced during the metabolic processes of harmful microorganisms in the intestines and during food decomposition. Excessive accumulation of these substances can lead to inflammatory diseases. Diamine oxidase (D+) breaks down these histamine-like substances, reducing their entry into the bloodstream. However, in cases of intestinal damage, the intestinal barrier function declines, leading to a large accumulation of histamine in the blood and an increase in D+ levels. During the food change, the serum D+ level in Experiment I showed a significant increase followed by a gradual decrease, indicating a rapid decline in the pet's intestinal barrier function during the food change, which may be closely related to the surge in diarrhea incidence, consistent with the serum D-lactic acid level test results. In Experiment II, the serum D+ level showed a gradual decrease, further demonstrating that adding 2-1000 kcal of baked food containing inactivated lactic acid bacteria and its metabolites at a ratio of 1 kg / t significantly improved intestinal barrier function. The level of diamine oxidase in the serum of Experiment III group remained relatively stable before and after the diet change, further indicating that the C+ and D+ of the baked grains after adding inactivated lactic acid bacteria cells and their metabolite preparations at a ratio of 1 kg / t had excellent effects on improving the intestinal barrier.

[0319] Experiment 7: A breeding evaluation experiment using wet food meal packs with added supplements for cats.

[0320] 7.1 Test Subjects

[0321] Twenty-four American Shorthair cats aged 8±1 months were selected and divided into three groups: Experimental Group I, Experimental Group II, and Control Group, with six cats in each group.

[0322] 7.2 Test Methods

[0323] The lactic acid bacteria inactivated bacterial cells and their metabolites preparation 2-1000, prepared in Example 12, were added to staple food wet grain meal packs at different amounts: 10 grams and 50 grams per 10 kg of staple food wet grains, respectively, to prepare two types of staple food wet grain meal packs containing lactic acid bacteria inactivated bacterial cells and their metabolites. Experimental group I (10 grams of lactic acid bacteria inactivated bacterial cells and their metabolites preparation 2-1000 per 10 kg of staple food wet grains), experimental group II (50 grams of lactic acid bacteria inactivated bacterial cells and their metabolites preparation 2-1000 per 10 kg of staple food wet grain meal packs), and control group (no lactic acid bacteria inactivated bacterial cells and their metabolites preparation 2-1000 added to the staple food wet grain meal packs) were used for 35 days. At the beginning and end of the experiment, the body weight, apparent digestibility of nutrients, diarrhea rate, and fecal score of the participating cats were measured. The levels of immunoglobulins IgG and IgA in serum and secretory immunoglobulin SIgA in feces were also measured.

[0324] 7.3 Results

[0325] 7.3.1 Weight measurement results:

[0326] Weight measurement results as follows Figure 30 The results showed that the average body weight (kg / animal) of the three groups on day 0 was 3.38±0.12, 3.37±0.12, and 3.38±0.10, respectively; and the average body weight (kg / animal) of the three groups on day 35 was 3.73±0.12, 3.88±0.10, and 3.65±0.14, respectively. The average body weight of groups I and II was higher than that of the control group, with group II being significantly higher than the control group (P<0.05). This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the wet food feed packets has a growth-promoting effect on pet cats, and the promoting effect is significantly enhanced with increasing dosage.

[0327] 7.3.2 Results of apparent digestibility determination of nutrients:

[0328] apparent digestibility of crude protein, such as Figure 31 The results showed that the average apparent digestibility (%) of crude protein in the three groups on day 0 of the experiment was 87.3±0.7, 87.0±0.6, and 87.1±0.7, respectively; and on day 35 of the experiment, the average apparent digestibility of crude protein in the three groups was 89.5±1.5, 90.9±1.1, and 87.5±0.8, respectively. The apparent digestibility of crude protein in groups I (P<0.05) and II (P<0.01) was significantly higher than that in the control group. The apparent digestibility of crude fat was as follows... Figure 32The results showed that the average apparent digestibility (%) of crude fat in the three groups on day 0 of the experiment were 90.5±0.6, 90.8±0.6, and 90.6±0.9, respectively; and on day 35 of the experiment, the average apparent digestibility of crude fat in the three groups were 91.3±0.9, 91.7±0.6, and 90.7±0.8, respectively. Groups I and II of the experiment were higher than the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the staple food wet grain meal packs promoted the digestibility of the main nutrients, protein and fat.

[0329] 7.3.3 Statistical results of diarrhea rate and stool score:

[0330] On day 0 of the experiment, the diarrhea rates in the three groups were 50%, 50%, and 33%, respectively, with all three groups experiencing diarrhea to varying degrees. On day 35 of the experiment, the diarrhea rates in the three groups were 0%, 0%, and 50%, respectively. Groups I and II recovered completely, while the diarrhea in the control group worsened. Stool scores were as follows: Figure 33 As shown, the fecal scores of the three groups on day 0 were 2.7±0.8, 2.5±1.0, and 2.8±1.2, respectively; and on day 35, the fecal scores of the three groups were 3.7±0.5, 3.8±0.4, and 2.7±0.8, respectively. Group I (P<0.01) and Group II (P<0.001) showed significantly higher scores than the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites (2-1000) to wet food packets can significantly alleviate diarrhea in cats, with the effect being more pronounced in Group II.

[0331] 7.3.4 Results of serum immunoglobulin G measurement:

[0332] Serum immunoglobulin G levels (mg / mL) are as follows: Figure 34 The results showed that on day 0, the serum immunoglobulin G levels in the three groups were 9.7±0.4, 9.6±0.5, and 9.7±0.5, respectively; on day 35, the serum immunoglobulin G levels (mg / mL) in the three groups were 11.3±0.7, 11.8±0.5, and 10.5±0.9, respectively. The levels in experimental groups I (P<0.05) and II (P<0.01) were significantly higher than those in the control group. The serum immunoglobulin A levels (mg / mL) were as follows... Figure 35 The results showed that the serum immunoglobulin G levels in the three groups on day 0 were 1.56±0.14, 1.55±0.11, and 1.61±0.12, respectively; and the serum immunoglobulin A levels (mg / mL) in the three groups on day 35 were 1.56±0.12, 1.57±0.10, and 1.53±0.14, respectively, with the levels in experimental groups I and II being higher than those in the control group.

[0333] 7.3.5 Results of fecal secretory immunoglobulin A assay:

[0334] The content of fecal secretory immunoglobulin A (mg / mL) is as follows: Figure 36 The results showed that on day 0, the levels of fecal secretory immunoglobulin A (FIGA) in the three groups were 1.24±0.08, 1.23±0.07, and 1.24±0.07, respectively; and on day 35, the levels were 1.27±0.08, 1.32±0.07, and 1.20±0.07, respectively. Group II (P<0.05) showed a significantly higher level than the control group. Therefore, adding inactivated lactic acid bacteria and their metabolites (2-1000) to the wet food feed as described in Group II significantly increased the fecal IGFA levels, consistent with the results of alleviating diarrhea in cats.

[0335] Experiment Example 8: Feeding Evaluation Experiment of Cats Using Freeze-Dried Raw Meat and Bones with Added Preparations

[0336] 8.1 Test Subjects

[0337] Twenty-seven American Shorthair cats aged 8±1 months were selected and divided into three groups: Experimental Group I, Experimental Group II, and Control Group, with nine cats in each group.

[0338] 8.2 Test Methods

[0339] Using the inactivated lactic acid bacteria cells and their metabolites 2-1000 prepared in Example 12, two types of freeze-dried food containing inactivated lactic acid bacteria cells and their metabolites were prepared by adding 20 grams and 100 grams per 20 kg of freeze-dried raw meat and bones. Before the experiment, the 27 cats were fed a uniform diet consisting of freeze-dried raw meat and bones without the inactivated lactic acid bacteria cells and their metabolites, at a rate of 20 grams per cat per day, with free access to extruded food.

[0340] At the start of the experiment, cats were randomly divided into three groups of nine each. Experimental group I (20g of inactivated lactic acid bacteria and its metabolites 2-1000 per 20kg of freeze-dried raw meat and bones), Experimental group II (100g of inactivated lactic acid bacteria and its metabolites 2-1000 per 20kg of freeze-dried raw meat and bones), and the control group (no inactivated lactic acid bacteria and its metabolites added to the freeze-dried raw meat and bones). All cats were fed 15g of freeze-dried raw meat and bones per day, with free access to extruded food. The experiment lasted 21 days. At the beginning and end of the experiment, the diarrhea rate and fecal scores of the participating cats were recorded, fecal ammonia concentration was measured, and oral odor, litter odor, mental state, and coat condition were also recorded.

[0341] 8.3 Results

[0342] 8.3.1 Statistical results of diarrhea rate and stool score:

[0343] On day 0 of the experiment, four mice in each of the three groups had diarrhea of ​​varying degrees, with a diarrhea rate of 44% in all groups. On day 21 of the experiment, the diarrhea rates in the three groups were 11%, 0%, and 44%, respectively. Group II recovered completely, one mouse in Group I still had diarrhea, and the control group showed slight improvement, but four mice still had diarrhea. Stool scores are as follows: Figure 37 As shown, the fecal scores of the three groups on day 0 were 2.6±0.9, 2.6±0.5, and 2.7±0.7, respectively; and on day 21, the fecal scores of the three groups were 3.3±0.7, 3.7±0.5, and 2.8±0.8, respectively. The scores of groups I (P<0.05) and II (P<0.01) were significantly higher than those of the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites (2-1000) to freeze-dried raw meat and bones can significantly alleviate diarrhea in pet cats, with the effect being more pronounced in group II.

[0344] 8.3.2 Weight measurement results:

[0345] Weight measurement results as follows Figure 38 The results showed that the average body weight (kg / animal) of the three groups on day 0 was 3.42±0.07, 3.43±0.07, and 3.43±0.05, respectively; and the average body weight (kg / animal) of the three groups on day 21 was 3.75±0.07, 3.83±0.05, and 3.67±0.09, respectively. The average body weight of groups I and II was higher than that of the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites (Formula 2-1000) to freeze-dried raw meat and bones promotes growth in pet cats, and the effect increases with increasing dosage.

[0346] 8.3.3 Determination of fecal ammonia content, and statistical analysis of oral odor and cat litter odor:

[0347] Fecal ammonia content test results are as follows Figure 39 The results showed that the average fecal ammonia content (mg / dL) of the three groups on day 0 of the experiment were 25.5±0.1, 25.4±0.3, and 25.5±0.3, respectively; and the average fecal ammonia content (mg / dL) of the three groups on day 21 of the experiment were 17.4±0.3, 15.1±0.2, and 26.3±0.2, respectively. The ammonia content in groups I (P<0.01) and II (P<0.01) was significantly higher than that in the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites to freeze-dried raw meat and bones can significantly reduce the ammonia concentration in pet cat feces, thereby reducing cat litter odor.

[0348] Table 2 shows the statistical results of cat litter odor. Before the experiment, the cat litter in all three groups had a noticeable odor after cleaning feces. After 21 days, the odor in group I was significantly reduced after cleaning feces, group II had no noticeable odor, and the control group had a noticeable odor. This result is consistent with the trend of fecal ammonia concentration detection. Oral odor statistics showed that before the experiment, all three groups of cats had a strong fishy odor and drool stains. After 21 days, group I had a slight fishy odor and a small amount of drool stains, group II had a slight fishy odor and no drool stains, and the control group still had a strong fishy odor and heavy drool stains. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites to freeze-dried raw meat and bones has a certain effect on reducing oral and cat litter odor.

[0349] Table 1. Statistical results of cat litter and oral odor

[0350]

[0351] 8.3.4 Statistics on Mental State and Fur Condition:

[0352] Table 3 shows the statistical results of mental and fur condition. Before the experiment, all three groups of cats had average mental state and poor vitality. In Experiment I, the coat had poor luster and smoothness, poor elasticity, severe shedding, and a high rate of tangling; three cats had mild skin inflammation. In Experiment II, the coat had poor luster and smoothness, poor elasticity, sparse undercoat, and a high rate of tangling; three cats had mild skin inflammation. In the control group, the coat had poor luster and smoothness, poor elasticity, sparse undercoat, and a high rate of tangling; two cats had mild skin inflammation. After 21 days of the experiment, both Experiment I and Experiment II showed increased energy and activity, while the control group remained in the same state as before the experiment. Regarding fur condition, Experiment I had good luster and smoothness, good elasticity, abundant undercoat, reduced shedding, and no obvious tangling; however, one cat still had mild skin inflammation. In Experiment II, the cats exhibited good coat luster and smoothness, good elasticity, abundant underfur, no obvious tangles, and slight hair loss; all three cats with skin inflammation recovered completely. In the control group, the coat luster and smoothness remained poor, elasticity was poor, and hair loss was severe; three cats had mild skin inflammation, with one exhibiting dry, scaly hair loss.

[0353] Table 2. Statistical results of mental and fur condition.

[0354]

[0355] Experiment Example 9: A breeding evaluation experiment using cat treats with added ingredients.

[0356] 9.1 Test Subjects

[0357] Forty-four kittens aged 9±1 months were selected. All participating kittens had varying degrees of oral and diarrhea problems and were divided into experimental group I, experimental group II, experimental group III, and control group, with 11 kittens in each group. Each group had 5 blue cats, 3 Siamese cats, and 3 American Shorthair cats. During the experiment, normal oral treatment and health care were provided.

[0358] 9.2 Test Methods

[0359] Using the preparation of inactivated lactic acid bacteria and its metabolites 2-1000 prepared in Example 12, three types of cat treats containing inactivated lactic acid bacteria and its metabolites were prepared by adding different amounts to cat treats. The experiment lasted for 28 days. The groups were: Experiment I (10 g of inactivated lactic acid bacteria and its metabolites 2-1000 added per 10 kg of cat treat contents), Experiment II (50 g of inactivated lactic acid bacteria and its metabolites 2-1000 added per 10 kg of cat treat contents), Experiment III (150 g of inactivated lactic acid bacteria and its metabolites 2-1000 added per 10 kg of cat treat contents), and Control Group (no inactivated lactic acid bacteria and its metabolites added to the cat treat contents, with the same basic composition as the experimental groups). The cat treat feeding amount was 10 g / cat / day. Diarrhea rate, fecal score, and dental plaque score of the participating cats were recorded on days 0, 5, and 28 of the experiment, and oral odor of the participating cats was also recorded.

[0360] 9.3 Results

[0361] 9.3.1 Statistical results of diarrhea rate and stool score:

[0362] On day 0 of the experiment, all four groups experienced diarrhea of ​​varying degrees: group I (n=7), group II (n=6), group III (n=7), and the control group (n=6), with diarrhea rates of 64%, 55%, 64%, and 55%, respectively. On day 5, the diarrhea rates were 18%, 9%, 0%, and 45%, respectively, indicating that group III had recovered. On day 28, the diarrhea rates were 9%, 0%, 0%, and 45%, respectively, indicating that groups II and III had recovered, group I showed significant improvement, and the control group showed no significant change. Stool scores are as follows: Figure 40As shown, the fecal scores of the four groups on day 0 were 2.3±0.9, 2.4±0.9, 2.2±0.8, and 2.4±0.7, respectively; on day 5, the fecal scores of the four groups were 3.0±0.6, 3.2±0.6, 3.4±0.5, and ±0.7, respectively, with Group II (P<0.05) and Group III (P<0.01) showing significantly higher scores than the control group; on day 28, the fecal scores of the four groups were 3.1±0.8, 3.2±0.4, 3.5±0.5, and 2.6±0.9, respectively, with Group I (P<0.05), Group II (P<0.05), and Group III (P<0.01) showing significantly higher scores than the control group. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites (2-1000) to cat treats can significantly alleviate diarrhea in pet cats, and the effect increases significantly with increasing dosage.

[0363] 9.3.2 Dental plaque scoring results:

[0364] Dental plaque score Figure 41 As shown, the plaque scores of the four groups on day 0 were 2.25±0.40, 2.18±0.28, 2.23±0.44, and 2.20±0.47, respectively; and on day 28, the plaque scores of the four groups were 1.45±0.33, 1.27±0.31, 0.95±0.25, and 1.64±0.38, respectively. Among these, the scores of experimental groups II (P<0.05) and III (P<0.01) were significantly lower than those of the control group, and the score of experimental group I was lower than that of the control group. This indicates that after 28 days, the plaque improvement in experimental group III was the most significant, with most experimental cats showing only a small amount of thin plaque on their teeth. Periodic assessment of oral odor showed that the four groups of cats on day 0 had severe halitosis, with a strong fishy and putrid odor, and most had saliva stains. On day 28 of the experiment, in Experiment III, the cats had a slight fishy odor in their mouths and no drooling. In Experiment II, the cats had a slight fishy odor in their mouths, and one cat had drooling. In Experiment I, one cat had a foul fishy odor in its mouth, but no putrid smell; the rest had a slight fishy odor in their mouths, and two cats had drooling. In the control group, all eight cats still had a foul fishy odor, with six of them having a putrid smell, and all eight cats had drooling. This indicates that adding different amounts of inactivated lactic acid bacteria and their metabolites (2-1000) to the cat treats significantly reduced plaque scores, decreased breath odor, and improved the dental and oral health of the cats. Furthermore, the effect significantly increased with increasing dosage.

[0365] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A postbiotic preparation for pets, characterized in that, The pet-specific postbiotic preparation contains inactivated bacterial cells; wherein the bacterial cells are *Lactobacillus plantarum* Ps-8 (CGMCC No. 5359), *Lactobacillus acidophilus* LA-14 (CGMCC No. 31624), *Lactobacillus casei* C-9 (CGMCC No. 6734), and *Bifidobacterium animalis* subsp. *lactospirae* BB-16 (CGMCC No. 31623); wherein the ratio of the number of units of *Lactobacillus plantarum* Ps-8, *Lactobacillus casei* C-9, *Bifidobacterium animalis* subsp. *lactospirae* BB-16, and *Lactobacillus acidophilus* LA-14 is 5:1:1:1; the total number of bacterial cells is 3 × 10⁻⁶. 10 ~1×10 11 cells / g; The pet postbiotic preparation is applicable to at least one of the following cat and dog foods: baked food, wet food pouches, extruded food, freeze-dried raw meat and bones, and cat treats.

2. The pet postbiotic preparation according to claim 1, characterized in that, The raw materials of the pet postbiotic preparation include an organic nitrogen source, an organic carbon source, a flavoring agent, a pH adjuster, and excipients; the organic nitrogen source is selected from one or more of whole soybean flour, skim milk powder, protein powder, and yeast extract; the organic carbon source is selected from one or more of whole soybean flour, skim milk powder, and protein powder; the pH adjuster is selected from one or more of sodium citrate or potassium citrate; the flavoring agent is selected from one or more of sucrose, xylitol, or erythritol; and the excipients are selected from one or more of maltodextrin and corn syrup.

3. The pet postbiotic preparation according to claim 2, characterized in that, The raw materials of the pet postbiotic preparation include 4-7 parts by weight of whole soybean powder, 6.5-18 parts by weight of skim milk powder, 5-7 parts by weight of sucrose, 2.5-5 parts by weight of sodium citrate, and 60-78 parts by weight of water.

4. The pet postbiotic preparation according to claim 3, characterized in that, The preparation process of the pet postbiotic formulation includes the following specific steps: S1: Weigh the raw materials, add water, mix, and then perform material processing and homogenization; S2: After sterilization and cooling, a liquid solution is obtained; S3: Add bacterial cells and lactase to the above liquid, and then carry out constant temperature fermentation to obtain fermentation liquid; S4: Mix maltodextrin, then sterilize and inactivate it, and spray dry to obtain post-biotic powder; S5: After the post-biotic powder is mixed with maltodextrin again, the post-biotic preparation is obtained.

5. The pet postbiotic preparation according to claim 4, characterized in that, In step S3, the amount of lactase added is 0.1~1.0 mL per kilogram of liquid material; the constant temperature fermentation conditions are: constant temperature fermentation at 32~37℃ until pH 4.5~4.6; wherein, after constant temperature fermentation, the total viable cell count is ≥2.0×10⁻⁶. 10 CFU / g; the amount of maltodextrin added in step S4 is 18-25g per kilogram of fermentation liquid; the sterilization and inactivation conditions are: inactivation at 75-95℃ for 10-30 min.

6. The use of the pet postbiotic preparation according to any one of claims 1-5 in the preparation of products for improving feline and canine diarrhea.

7. The use of the pet postbiotic preparation according to any one of claims 1-5 in the preparation of products to alleviate stress during food change in cats and dogs.

8. The use of the pet postbiotic preparation according to any one of claims 1-5 in the preparation of products for treating or improving dental plaque in cats and dogs.

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