Postbiotic preparation for pets as well as preparation method and application of postbiotic preparation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a postbiotic preparation for pets as well as a preparation method and application of the postbiotic preparation. The metabolite preparation provided by the invention comprises a lactobacillus plantarum Ps-8 inactivated thallus and a metabolite thereof, a lactobacillus casei C-9 inactivated thallus and a metabolite thereof, a bifidobacterium animalis subsp. Lactis BB-16 inactivated thallus and a metabolite thereof, and a lactobacillus acidophilus LA-14 inactivated thallus and a metabolite thereof. The pet food can be applied to various food types of different varieties of cat and dog pets, and has the effects of improving the intestinal barrier function of the pets, enhancing the immunity, reducing the incidence rate of diarrhea, reducing the food change stress, promoting the digestion of nutrient substances, optimizing the state of spirit and fur, reducing the odor of excrement, fading the odor of the oral cavity, improving the health level of the oral cavity and teeth and the like.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a postbiotic preparation for pets, a preparation method and an application thereof. Background Art

[0002] Killed lactic acid bacteria and their metabolites are postbiotics, which are a popular substance for improving the health of the body in recent years. In 2013, Tsilingiri K et al. proposed a definition that postbiotics refer to factors released by the metabolic activities of probiotics and can have beneficial effects on the host in a direct or indirect manner. Since then, different researchers have redefined the concept of postbiotics three times. It was not until 2021 that the International Scientific Society for Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics, proposing for the first time the official concept of postbiotics as "preparations of inanimate microorganisms and / or their components that are beneficial to host health." Compared with probiotics, which have more stringent requirements on the living environment, their effects are greatly affected by storage conditions, their stability is poor, and the drug-resistant genes carried by live probiotics have the risk of transfer. Postbiotics have great application prospects due to their "inanimate" characteristics and their biological activity can be comparable to that of "live" probiotics with very few side effects. They are currently regarded by the industry as the fourth generation of microecological products after probiotics, prebiotics and synbiotic preparations.

[0003] Postbiotics mainly promote animal growth through anti-oxidation, anti-inflammatory, immune and gastrointestinal function regulation, and liver protection. At present, the hot research of postbiotics is mainly concentrated in five aspects: first, based on probiotics (such as lactobacillus and bifidobacterium) to control biofilm formation, regulate gene expression, reduce inflammatory cytokines to regulate immunity; second, exopolysaccharides as a component of postbiotics can reduce inflammation through unclear signal mechanisms to promote barrier function; third, the role of inactivated lactic acid bacteria in the treatment of obesity; fourth, postbiotics improve cell apoptosis, regulate the balance of intestinal flora, promote interleukin production, and improve inflammatory bowel disease; fifth, the health effects of postbiotics on allergic dermatitis.

[0004] Studies have shown that lactobacillus and bifidobacteria in the microbiome are dominant bacteria in the intestines of healthy cats and dogs. They are not only important components of the intestinal barrier, but also can enhance the immune capacity of cats and dogs by stimulating macrophages, inducing interferon production, promoting cell division, and producing antibodies. Data show that after cats and dogs ingested compound lactic acid bacteria preparations for a period of time, the number of Escherichia coli, Clostridium perfringens, and Salmonella in the intestines decreased significantly, while the number of beneficial bacteria increased significantly. At the same time, the apparent digestibility of nutrients in their diets showed a gradual increase. In addition, it has been reported in the literature that feeding lactic acid bacteria and their metabolites to cats and dogs with intestinal disorders and weak constitutions for a period of time can significantly improve their intestinal flora, enhance their physical fitness, and reduce the odor of the mouth and feces. It can be seen that feeding a certain amount of probiotic lactic acid bacteria and their metabolites to cats and dogs can enhance immunity and improve intestinal flora, thereby reducing the use of chemical drugs, which is of great significance to the health of cats and dogs.

[0005] The patents related to postbiotics currently applied for and disclosed for promoting intestinal health of pets are mostly products made of a single pet food product by adding postbiotics and / or probiotics, such as cat strips containing postbiotics and / or probiotics. It is impossible to make postbiotics adapt to diversified pet staple food types at the same time, or to be applicable to cat or dog food at the same time. In addition, finished pet food often contains a wealth of pure meat raw materials, and the unstable meat source is prone to other risks, thus affecting the actual effect of postbiotics. In fact, in reality, drugs or health products used to treat, improve or prevent diseases are often mixed into food, making it easy for cats and dogs to accept them in terms of taste. Due to the complex ingredients of cat and dog food, the staple food of cats and dogs of different ages and breeds also has certain differences. Therefore, whether postbiotic preparation products can adapt to diversified pet staple food types needs to be verified before use. However, the currently disclosed postbiotic preparation patents have relatively few contents that confirm the efficacy of the product through detailed experimental data results or confirm that the product can adapt to diversified pet staple food types, and the product efficacy needs to be verified. In addition, the efficacy of the currently disclosed postbiotic products is also relatively single, and can only be used to treat or improve a certain symptom.

[0006] Therefore, it is necessary to provide a postbiotic preparation that can adapt to cats and dogs of different ages and breeds and a variety of cat and dog food and has rich efficacy for preparing animal food compositions or supplement compositions. Summary of the invention

[0007] Technical issues

[0008] The purpose of the present invention is to provide a houshengyuan preparation that can adapt to diversified pet staple food types and different breeds of cats and dogs of different ages. The preparation also has the effects of promoting the intestinal health of cats / dogs, reducing the stress of food change, and improving the oral and dental health level.

[0009] Technical Solution

[0010] The first aspect of the present invention provides a plant lactobacillus (Lactobacillus plantarum) Ps-8, which has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No.5359, and the deposit date is October 28, 2011.

[0011] The second aspect of the present invention provides a Lactobacillus acidophilus LA-14, which has been deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is GMCC No.31624, and the deposit date is August 6, 2024.

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

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

[0014] The fifth aspect of the present invention provides a postbiotic preparation for pets, which comprises inactivated bacteria and metabolites thereof; wherein the bacteria are Lactobacillus plantarum Ps-8, Lactobacillus acidophilus LA-14, Lactobacillus casei C-9 and Bifidobacterium animalis subspecies lactis BB-16; the ratio of the number of units of Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis subspecies lactis BB-16 and Lactobacillus acidophilus LA-14 is 1-5:1-2:1-S2:1-2; preferably 5:1:1:1; the total amount of the bacteria is 3×10 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 metabolite comprises an organic acid.

[0016] In some embodiments, the organic acid includes at least one of 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 of acetic acid, propionic acid, butyric acid and valeric acid.

[0017] In some embodiments, the metabolites also include at least one of soybean saponins, soybean isoflavones, γ-aminobutyric acid, active short peptide molecules, genistein glucaric acid, 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 postbiotic preparation for pets include an organic nitrogen source, an organic carbon source, a flavoring agent, a pH adjuster, and excipients.

[0019] In some embodiments, the organic nitrogen source is selected from one or more of whole fat soybean powder, skim milk powder, egg white powder and yeast extract powder; the organic carbon source is selected from one or more of whole fat soybean powder, skim milk powder and egg white 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; the auxiliary material is selected from one or more of maltodextrin and corn syrup, preferably maltodextrin.

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

[0021] In some embodiments, the preparation is in the form of a liquid preparation and a solid preparation; the preparation is in the form of an injection, a tablet, a capsule, a powder, a granule or a paste.

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

[0023] S1: Weigh the raw materials, add water and mix them, then dissolve and homogenize them;

[0024] S2: cooling after sterilization to obtain a liquid feed;

[0025] S3: adding bacteria and lactase to the above-mentioned liquid, and then fermenting at a constant temperature to obtain a fermentation liquid;

[0026] S4: sterilization and inactivation, spray drying to obtain postbiotic powder;

[0027] S5: The postbiotic powder is mixed with maltodextrin to obtain a postbiotic preparation.

[0028] In some embodiments, the conditions for the chemical reaction in step S1 are: chemical reaction at 55-60°C for 10-20 min; the conditions for homogenization are: 55-60°C, primary pressure 18-20 MPa, secondary pressure 4-5 MPa; the conditions for sterilization in step S2 are: sterilization at 90-95°C for 30-50 min; the cooling temperature is 32-37°C; 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: constant temperature fermentation at 32-37°C to pH 4.5-4.6; wherein the total viable count of the bacteria after constant temperature fermentation is ≥2.0×10 10 CFU / g, preferably ≥3.0×10 10 CFU / g; the amount of maltodextrin added in step S4 is 18-25 g per kilogram of fermentation liquid; the inactivation conditions are: inactivation at 75-95°C for 10-30 min.

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

[0030] In some embodiments, a sixth aspect of the present invention provides a use of the above-mentioned postbiotic preparation for pets in the preparation of a product for improving diarrhea in cats and dogs.

[0031] In some embodiments, the postbiotic preparation for pets is administered by direct oral administration, and the dosage is: 0.5 grams and 1 gram of the postbiotic preparation are administered orally with 2-3 times the amount of clean water per day.

[0032] In some embodiments, the postbiotic preparation for pets is one or more of food raw materials, probiotic powder, food additives, feed additives or foods.

[0033] A seventh aspect of the present invention provides an application of the above-mentioned postbiotic preparation for pets in the preparation of a product for alleviating the stress of food change for cats and dogs.

[0034] In some embodiments, the postbiotic preparation for pets can be added to puffed food, baked food, staple wet food meal packs, freeze-dried raw bones and meat, or cat strips, and the added amount is 10 grams or 50 grams of the postbiotic preparation per 10 kilograms of puffed food or baked food or staple wet food meal packs or dry raw bones and meat or cat strips, preferably 10 grams.

[0035] An eighth aspect of the present invention provides a use of the above-mentioned postbiotic preparation for pets in the preparation of a product for improving diarrhea in cats and dogs.

[0036] A ninth aspect of the present invention provides a use of the above-mentioned postbiotic preparation for pets in the preparation of a product for alleviating the stress of food change for cats and dogs.

[0037] The tenth aspect of the present invention provides an application of the above-mentioned postbiotic preparation for pets in the preparation of a product for treating or improving dental plaque in cats and dogs.

[0038] Technical Effects

[0039] The postbiotic preparation for pets of the present invention can adapt to the postbiotic combination preparation of a variety of cat / dog food, including but not limited to baked food, wet food meal packs, puffed food, freeze-dried raw bone meat and cat strips, almost covering most types of cat / dog food on the market; in addition, the postbiotic preparation of the present invention can be adapted to cats and dogs of different ages from 1 to 10 months and a variety of common breeds on the market, such as Belgian Shepherd, German Shepherd, Teddy, British Shorthair, American Shorthair, Ragdoll, Rural Cat, Blue Cat, Siamese Cat, with a wide range of applications. Further, the postbiotic preparation for pets of the present invention is rich in efficacy, not only can it improve diarrhea in cats and dogs, relieve food change stress, optimize the spirit and fur state of cats and dogs and enhance immunity, but also can effectively improve dental plaque and improve the oral and dental health of pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Statistical results of feces scores of dogs in Experimental Example 1 (*P<0.05; **P<0.01; ***P<0.001);

[0041] Figure 2 : Statistical results of feces scores of cats in Experimental Example 2 (*P<0.05; **P<0.01; ***P<0.001);

[0042] Figure 3 : Test Example 3: Pulse measurement results of dogs;

[0043] Figure 4 : Test Example 3: Breathing rate measurement results of dogs;

[0044] Figure 5 : Test Example 3 Dog's body temperature measurement results;

[0045] Figure 6 : Test Example 3: Food intake measurement results of dogs;

[0046] Figure 7 : Test Example 3 dog weight measurement results;

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

[0048] Fig. 9: Test Example 3 Results of crude fat apparent digestibility in dogs (*P<0.05; **P<0.01; ***P<0.001);

[0049] Fig.10 : Statistical results of feces scores of dogs in Experimental Example 3 (*P<0.05; **P<0.01; ***P<0.001);

[0050] Fig.11 : Test Example 4 Cat's food intake measurement results;

[0051] Fig.12 : Test Example 4 cat body weight measurement results;

[0052] Fig.13 : Test Example 4 Results of apparent digestibility of crude protein in cats (*P<0.05; **P<0.01; ***P<0.001);

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

[0054] Fig.15 : Statistical results of feces scores of cats in Experimental Example 4 (*P<0.05; **P<0.01; ***P<0.001);

[0055] Fig.16 : Statistical results of feces scores of dogs in Experimental Example 5 (*P<0.05; **P<0.01; ***P<0.001);

[0056] Fig.17 : Test Example 5: Food intake measurement results of dogs (*P<0.05; **P<0.01; ***P<0.001);

[0057] Fig.18 : Test Example 5: Serum cortisol test results of dogs (*P<0.05; **P<0.01; ***P<0.001);

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

[0059] Fig. 20 : Test Example 5: Serum immunoglobulin G test results of dogs (*P<0.05; **P<0.01; ***P<0.001);

[0060] Fig.21: Test Example 5: Serum immunoglobulin A test results of dogs (*P<0.05; **P<0.01; ***P<0.001);

[0061] Fig. 22 : Statistical results of feces scores of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0062] Fig.23 : Test Example 6 Cat food intake measurement results (*P<0.05; **P<0.01; ***P<0.001);

[0063] Fig.24 : Serum malondialdehyde determination results of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0064] Fig.25 : Serum cortisol measurement results of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0065] Fig.26 : Serum immunoglobulin G test results of cats in test example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0066] Fig. 27 : Serum immunoglobulin A determination results of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0067] Fig.28 : Serum D-lactic acid determination results of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0068] Fig.29 : Serum diamine oxidase assay results of cats in Experimental Example 6 (*P<0.05; **P<0.01; ***P<0.001);

[0069] Fig.30 : Test Example 7 Cat body weight measurement results (*P<0.05; **P<0.01; ***P<0.001);

[0070] Fig.31 : Test Example 7: Results of apparent digestibility of crude protein in cats (*P<0.05; **P<0.01; ***P<0.001);

[0071] Fig.32 : Test Example 7 Results of apparent digestibility of crude fat in cats (*P<0.05; **P<0.01; ***P<0.001);

[0072] Fig.33 : Statistical results of feces scores of test example 7 cats (*P<0.05; **P<0.01; ***P<0.001);

[0073] Fig.34 : Test Example 7 Serum immunoglobulin G content determination results of cats (*P<0.05; **P<0.01; ***P<0.001);

[0074] Fig.35 : Test Example 7 Serum immunoglobulin A content determination results of cats (*P<0.05; **P<0.01; ***P<0.001);

[0075] Fig.36 : Test Example 7: Results of determination of secretory immunoglobulin A content in cat feces (*P<0.05; **P<0.01; ***P<0.001);

[0076] Fig.37 : Statistical results of feces scores of cats in Experimental Example 8 (*P<0.05; **P<0.01; ***P<0.001);

[0077] Fig.38 : Test Example 8 cat body weight measurement results;

[0078] Fig.39 : Test Example 8: Fecal ammonia content in cats (*P<0.05; **P<0.01; ***P<0.001);

[0079] Fig.40 : Statistical results of feces scores of test example 9 cats (*P<0.05; **P<0.01; ***P<0.001);

[0080] Fig.41 : Statistical results of dental plaque scores of cats in Experimental Example 9 (*P<0.05; **P<0.01; ***P<0.001).

[0081] Collection Information

[0082] Name: Ps-8;

[0083] Taxonomy: Lactobacillus plantarum;

[0084] Latin name: Lactobacillus plantarum;

[0085] Depository: China General Microbiology Center (CGMCC);

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

[0087] Deposit date: October 28, 2011;

[0088] The registration number of the Collection Center is: CGMCC No.5359.

[0089] Name: LA-14;

[0090] Taxonomy: Lactobacillus acidophilus;

[0091] Latin name: Lactobacillus acidophilus;

[0092] Depository: China General Microbiology Center (CGMCC);

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

[0094] Deposit date: August 6, 2024;

[0095] The registration number of the Collection Center is: CGMCC No.31624.

[0096] Name: C-9;

[0097] Taxonomy: Lactobacillus casei;

[0098] Latin name: Lactobacillus casei;

[0099] Depository: China General Microbiology Center (CGMCC);

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

[0101] Deposit date: October 29, 2012;

[0102] The registration number of the Collection Center is: CGMCC No.6734.

[0103] Name:BB-16;

[0104] Taxonomy: Bifidobacterium animalis subsp. lactis;

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

[0106] Depository: China General Microbiology Center (CGMCC);

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

[0108] Deposit date: August 6, 2024;

[0109] The registration number of the Collection Center is: CGMCC No.31623. DETAILED DESCRIPTION

[0110] To facilitate the technical solution of the application, the following first provides a general description and definition of the terms and expressions involved in the application.

[0111] The terms "comprises", "includes" or any other variation thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a list of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus. In the absence of more restrictions, an element defined by the phrase "comprises a ..." does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0112] The term "dissolving" means to dissolve the raw materials better.

[0113] The term "homogenization" is also called homogenization, which is the process of micronizing and homogenizing the dispersed matter in the suspension (or emulsion) system. This treatment simultaneously reduces the size of the dispersed matter and improves the uniformity of the dispersed matter 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 as follows:

[0116] Lactobacillus plantarum Ps-8: This strain is isolated from naturally fermented yogurt and has excellent artificial gastric juice, artificial intestinal juice, bile salt tolerance and prebiotic 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 is isolated from naturally fermented sour yak milk. It has excellent artificial gastric juice, artificial intestinal juice, bile salt tolerance and probiotic properties. It can produce high amounts of natural active antibacterial substances such as bacteriocins, and can broadly inhibit common intestinal pathogens such as Escherichia coli and Salmonella, and relieve problems such as soft stools and watery stools in animals. It regulates the content of immune substances in blood and feces and enhances the body's immunity.

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

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

[0120] The standards and specifications for the relevant experiments listed in this application are as follows:

[0121] Body temperature measurement: Use an electronic thermometer to detect the dog's body temperature when the pet is quiet. The detection site is the dog's cochlea or the base of the hind limbs. The tester fixes the electronic thermometer on the detection site for about 10 seconds and presses the detection button to detect and record.

[0122] Pulse measurement: The test is carried out when the dog is in a quiet state. During the test, the pet is properly fixed. The tester stands at the back of the pet, holds the hind legs with one hand, and extends the other hand into the inner thigh. The tester uses the fingers to gently press the femoral artery to test and record the dog's pulse.

[0123] Respiratory frequency measurement: The test is carried out when the pet is in a quiet state. During the test, the tester stands in front of the pet's chest and observes the rise and fall of the pet's chest. One rise and fall is one breath, and the number of breaths of the pet is recorded.

[0124] Feed intake was determined by subtracting the weight of the feed left over from the weight of the diet before each feeding.

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

[0126] Determination of apparent digestibility of nutrients: On the 0th and 30th days of the experiment, three mixed feces samples were collected from each group, each weighing 100 g. They were treated with 10% hydrochloric acid for nitrogen fixation, dried to constant weight at 60°C, crushed, sieved with a 60-mesh sieve, and refrigerated for later use. The apparent digestibility of nutrients in the diet was determined by the acid-insoluble ash method, and the method was referred to "Determination of hydrochloric acid insoluble ash in feed" (GB / T 23742-2009); the crude ash content was determined by the 550°C calcination method, and the method was referred to (GB / T 6438-2007); when determining the 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; the crude protein content was determined by a fully automatic Kjeldahl nitrogen analyzer according to the GB / T 6433-2006 method, and the crude fat content was determined by Soxhlet extraction. Apparent digestibility of nutrients (%) = [1-(A / B×C / D)]×100, where: A: content of a certain nutrient in feces (%); B: content of corresponding nutrient in diet (%); C: content of acid-insoluble ash in diet (%); D: content of acid-insoluble ash in feces (%).

[0127] Diarrhea disease status statistics: According to the time points of different embodiments, the diarrhea disease status of pets is counted and the diarrhea rate is calculated.

[0128] Scoring of stool is based on the following criteria: watery stool (scored as 1 point): stool is liquid and in patches rather than a single piece of stool, may contain mucus or blood, is collected in a container and can be poured out in a flowing state, and easily leaves a large amount of stool stains on the ground after collection, which is a severe diarrhea stool state; unformed soft stool (scored as 2 points): stool is viscous and does not show any cylindrical shape, and leaves a large amount of stool stains on the ground after collection, which is a moderate diarrhea stool state; formed soft stool (scored as 3 points): stool is irregularly columnar and easy to crack, and leaves a small amount of stool stains on the ground after collection, which is a mild diarrhea stool state; relatively hard stool (scored as 4 points): hard stool is relatively regular columnar and not easy to crack, and leaves a small amount of stool stains on the ground after collection, which is a relatively ideal stool state; extremely dry stool (scored as 5 points): stool is extremely dry and hard, does not adhere to the ground, and may roll on the ground when discharged, which is a constipation stool state.

[0129] Statistics of pet cats' spirit, fur and cat litter odor: The spirit and fur condition of pet cats are based on the results obtained by a fixed veterinarian through naked eye observation; the odor of cat litter is counted by the breeder, who collects cat litter with a sampling spoon, fans it with his hands, smells it and counts it.

[0130] Statistics of dental plaque index of pet cats: Teeth are examined by visual examination combined with the probe method. During the examination, a dental probe is used to lightly scratch the tooth surface and score according to the amount and thickness of plaque. The four tooth surfaces of the teeth, the mesial buccal surface, the median buccal surface, the distal buccal surface and the lingual surface, are examined. The score for each tooth is the average of the sum of the scores of the four tooth surfaces. The scoring standard is: no plaque on the teeth, 0 points; thin plaque on the teeth, which is not visible during visual examination, but plaque can be seen by scraping the tooth surface with a probe, 1 point; moderate plaque can be seen on the gingival margin or adjacent surface of the teeth, 2 points; a large amount of soft plaque in the gingival sulcus or gingival margin area and adjacent surface of the teeth, 3 points. The oral hygiene status of pets and the prevention and treatment of periodontal disease are evaluated according to the dental plaque index.

[0131] Serum malondialdehyde and cortisol determination method: The malondialdehyde (MDA) content in serum was detected by thiobarbituric acid colorimetry, and the double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the content of cortisol (COR) in serum.

[0132] Serum immunoglobulin G determination method: Use an anticoagulation blood collection tube containing disodium ethylenediaminetetraacetic acid (EDTA-2Na) to collect the whole blood of the experimental pet, flick the bottom of the tube to make the blood fully anticoagulated, and obtain serum by centrifugation (speed 3000rpm, 10min), and use double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) to detect the immunoglobulin G content in the serum sample.

[0133] Serum diamine oxidase and D-lactic acid determination method: Double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the diamine oxidase content in serum samples. Enzyme spectrophotometry was used to detect the D-lactic acid content in serum.

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

[0135] In each group of comparative experiments provided in this application, unless otherwise specified, except for the differences indicated in each group, other experimental conditions, materials, etc. are kept consistent to ensure comparability.

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

[0137] The following is a further description of a postbiotic preparation for pets provided in this application, and its preparation method and application.

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

[0139] Lactobacillus plantarum Ps-8 was placed in MRS liquid culture medium and statically cultured at 37°C for 18 hours; then, sterilized 60% glycerol was mixed with the bacterial solution at a volume of 1:3 to make a final glycerol concentration of 15%, and the mixture was mixed by pipetting and the bacterial strain was stored at -80°C.

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

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

[0142] 1) Acid and bile salt resistance:

[0143] The frozen Lactobacillus plantarum Ps-8 was inoculated into MRS liquid culture medium and statically cultured at 37°C for 18 hours. The culture was subcultured twice to obtain an activated fermentation liquid.

[0144] To a sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), add 3.5 g / L pepsin and filter with a 0.22 μm microporous filter to sterilize to prepare simulated gastric fluid; collect the activated strains by centrifugation, add an equal amount of simulated gastric fluid at pH 2.5 to the culture medium, and culture at 37°C for 3 h. The number of viable bacteria is determined by the MRS agar medium pouring method at 0 h and 3 h.

[0145] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% ox bile salt were added and sterilized by filtering with a 0.22 μm microporous filter membrane to prepare simulated intestinal fluid; the bacterial liquid treated in simulated gastric fluid for 3 hours was centrifuged and washed twice to collect the bacteria, and then the simulated intestinal fluid equal to the previous simulated gastric fluid was added to continue culturing (37°C), and the number of viable bacteria was measured by the MRS agar medium pouring method at 4h and 8h. The experimental results are shown in Table 1:

[0146] Table 1 Survival rate of Ps-8 artificial simulated gastric juice and intestinal juice

[0147]

[0148] Survival rate = [N1 / N0] × 100% (N0 - number of viable bacteria at 0h; N1 - number of viable bacteria after digestion with simulated intestinal and gastric juice), lg CFU / mL represents the colony forming unit in logarithm with base 10.

[0149] Antibacterial properties:

[0150] The antibacterial effect of Lactobacillus plantarum Ps-8 fermentation broth was determined by the well-diffusion agar assay: MRS agar medium (20 ml) cooled to about 50°C after sterilization was mixed with 200 μL of intestinal pathogenic bacteria solution (10 6 After the MRS agar medium with intestinal pathogens is cooled and solidified, a hole with a diameter of about 8 mm is punched on the plate using a hole puncher.

[0151] 100 μL of Lactobacillus plantarum Ps-8 fermentation liquid was added to each well, diffused in a 4°C refrigerator for 12 hours, and then cultured at 37°C for 48 hours, and the size of the inhibition zone was observed. The diameter of the inhibition zone was measured using a vernier caliper (retaining two significant figures), and the experimental results are shown in Table 2:

[0152] Table 2 Contents 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 8 mm; mm is the unit of the diameter of the inhibition zone.

[0156] From the experimental results in Table 1 and Table 2, it can be seen that the Ps-8 strain has good acid and bile resistance. After digestion in artificial simulated gastric juice at pH 2.5 for 3 hours and then in artificial intestinal juice at pH 8.0 for 8 hours, its survival rate is as high as 94.60%. At the same time, the strain Ps-8 has the excellent characteristics of broad-spectrum inhibition of pathogenic bacteria, and the antibacterial effect is very significant.

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

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

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

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

[0161] 1) Acid and bile salt resistance:

[0162] The cryopreserved Lactobacillus acidophilus LA-14 was inoculated into MRS liquid culture medium and statically cultured at 37°C for 18 hours. The culture was subcultured twice to obtain an activated fermentation broth.

[0163] To a sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), add 3.5 g / L pepsin and filter with a 0.22 μm microporous filter to sterilize to prepare simulated gastric fluid; collect the activated strains by centrifugation, add an equal amount of simulated gastric fluid at pH 2.5 to the culture medium, and culture at 37°C for 3 h. The number of viable bacteria is determined by the MRS agar medium pouring method at 0 h and 3 h.

[0164] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% ox bile salt were added and sterilized by filtering with a 0.22 μm microporous filter membrane to prepare simulated intestinal fluid; the bacterial liquid treated in simulated gastric fluid for 3 hours was centrifuged and washed twice to collect the bacteria, and then the simulated intestinal fluid equal to the simulated gastric fluid was added to continue culturing at 37°C, and the number of viable bacteria was measured by the MRS agar medium pouring method at 4 hours and 8 hours. The experimental results are shown in Table 3:

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

[0166]

[0167] Survival rate = [N1 / N0] × 100% (N0 - number of viable bacteria at 0h; N1 - number of viable bacteria after digestion with simulated intestinal and gastric juice), lg CFU / mL represents the colony forming unit in logarithm with base 10.

[0168] 2) Antibacterial properties:

[0169] The antibacterial effect of Lactobacillus acidophilus LA-14 fermentation broth was determined by the well-diffusion agar assay: MRS agar medium (20 mL) cooled to about 50°C after sterilization was mixed with 200 μL of intestinal pathogenic bacteria solution (10 6 CFU / mL) were poured into the plate and mixed. After the MRS agar medium with intestinal pathogens was cooled and solidified, a hole with a diameter of about 8 mm was punched on the plate using a hole puncher.

[0170] 100 μL of Lactobacillus acidophilus LA-14 fermentation liquid was added to each well, diffused in a 4°C refrigerator for 12 hours, and then cultured at 37°C for 48 hours, and the size of the inhibition zone was observed. The diameter of the inhibition zone was measured using a vernier caliper (retaining two significant figures), and 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 8 mm; mm is the unit of the diameter of the inhibition zone.

[0174] From the experimental results in Table 1 and Table 2, it can be seen that the LA-14 strain has good acid and bile resistance. After being digested in artificial simulated gastric juice at pH 2.5 for 3 hours and then continued to be digested in artificial intestinal juice at pH 8.0 for 8 hours, its survival rate is as high as 90.30%. At the same time, the strain LA-14 has excellent characteristics of broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella and Clostridium perfringens, and the antibacterial effect is very significant.

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

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

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

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

[0179] 1) Acid and bile salt resistance:

[0180] The cryopreserved animal Bifidobacterium lactis subsp. BB-16 was inoculated into MRS liquid culture medium and statically cultured at 37° C. for 18 h. The culture was subcultured twice to obtain an activated fermentation liquid.

[0181] To a sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), add 3.5 g / L pepsin and filter with a 0.22 μm microporous filter to sterilize to prepare simulated gastric fluid; collect the activated strains by centrifugation, add an equal amount of simulated gastric fluid at pH 2.5 to the culture medium, and culture at 37°C for 3 h. The number of viable bacteria is determined by the MRS agar medium pouring method at 0 h and 3 h.

[0182] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% ox bile salt were added and sterilized by filtering with a 0.22 μm microporous filter membrane to prepare simulated intestinal fluid; the bacterial liquid treated in simulated gastric fluid for 3 hours was centrifuged and washed twice to collect the bacteria, and then the simulated intestinal fluid equal to the previous simulated gastric fluid was added to continue culturing at 37°C, and the number of viable bacteria was measured by the MRS agar medium pouring method at 4 hours and 8 hours. The experimental results are shown in Table 5:

[0183] Table 5 Survival rate of BB-16 in artificial simulated gastric and intestinal fluids

[0184]

[0185] Survival rate = [N1 / N0] × 100% (N0 - number of viable bacteria at 0h; N1 - number of viable bacteria after digestion with simulated intestinal and gastric juice), lg CFU / mL represents the colony forming unit in logarithm with base 10.

[0186] 2) Antibacterial properties:

[0187] The antibacterial effect of the fermentation liquid of Bifidobacterium lactis subsp. BB-16 was determined by the well-diffusion agar assay: MRS agar medium (20 mL) cooled to about 50°C after sterilization was mixed with 200 μL of intestinal pathogenic bacteria liquid (10 6 CFU / mL) were poured into the plate and mixed. After the MRS agar medium with intestinal pathogens was cooled and solidified, a hole with a diameter of about 8 mm was punched on the plate using a hole puncher.

[0188] 100 μL of animal Bifidobacterium lactis subsp. BB-16 fermentation liquid was added to each well, diffused in a 4°C refrigerator for 12 hours, and then cultured at 37°C for 48 hours, and the size of the inhibition zone was observed. The diameter of the inhibition zone was measured using a vernier caliper (retaining two significant figures), and the experimental results are shown in Table 6:

[0189] Table 6 Antibacterial properties of Bifidobacterium animalis subspecies lactis BB-16

[0190]

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

[0192] From the experimental results in Table 1 and Table 2, it can be seen that the BB-16 strain has good acid and bile resistance. After being digested in artificial simulated gastric juice at pH 2.5 for 3 hours and then continued to be digested in artificial intestinal juice at pH 8.0 for 8 hours, its survival rate is as high as 91.50%. At the same time, the strain BB-16 has excellent characteristics of broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella and Clostridium perfringens, and the antibacterial effect is very significant.

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

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

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

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

[0197] 1) Acid and bile salt resistance:

[0198] The cryopreserved Lactobacillus casei C-9 was inoculated into MRS liquid culture medium and statically cultured at 37°C for 18 hours. The culture was subcultured twice to obtain an activated fermentation broth.

[0199] To a sterile PBS buffer at pH 2.5 (adjusted with 1 mol / L HCl), add 3.5 g / L pepsin and filter with a 0.22 μm microporous filter to sterilize to prepare simulated gastric fluid; collect the activated strains by centrifugation, add an equal amount of simulated gastric fluid at pH 2.5 to the culture medium, and culture at 37°C for 3 h. The number of viable bacteria is determined by the MRS agar medium pouring method at 0 h and 3 h.

[0200] In sterile PBS at pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% ox bile salt were added and sterilized by filtering with a 0.22 μm microporous filter membrane to prepare simulated intestinal fluid; the bacterial liquid treated in simulated gastric fluid for 3 hours was centrifuged and washed twice to collect the bacteria, and then the simulated intestinal fluid equal to the previous simulated gastric fluid was added to continue culturing at 37°C, and the number of viable bacteria was measured by the MRS agar medium pouring method at 4 hours and 8 hours. The experimental results are shown in Table 7:

[0201] Table 7C-9 Survival rate of artificial simulated gastric juice and intestinal juice

[0202]

[0203] Survival rate = [N1 / N0] × 100% (N0 - number of viable bacteria at 0h; N1 - number of viable bacteria after digestion with simulated intestinal and gastric juice), lg CFU / mL represents the colony forming unit in logarithm with base 10.

[0204] 2) Antibacterial properties:

[0205] The antibacterial effect of Lactobacillus casei C-9 fermentation broth was determined by the well-diffusion agar assay: MRS agar medium (20 mL) cooled to about 50°C after sterilization was mixed with 200 μL of intestinal pathogenic bacteria solution (10 6 CFU / mL) were poured into the plate and mixed. After the MRS agar medium with intestinal pathogens was cooled and solidified, a hole with a diameter of about 8 mm was punched on the plate using a hole puncher.

[0206] 100 μL of Lactobacillus casei C-9 fermentation liquid was added to each well, diffused in a 4°C refrigerator for 12 hours, and then cultured at 37°C for 48 hours, and the size of the inhibition zone was observed. The diameter of the inhibition zone was measured using a vernier caliper (retaining two significant figures), and 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 8 mm; mm is the unit of the diameter of the inhibition zone.

[0210] From the experimental results in Table 1 and Table 2, it can be seen that the C-9 strain has good acid and bile resistance. After digestion in artificial simulated gastric juice at pH 2.5 for 3 hours and then in artificial intestinal juice at pH 8.0 for 8 hours, its survival rate is as high as 74.80%. At the same time, the strain C-9 has excellent characteristics of broad-spectrum inhibition against pathogenic bacteria such as Escherichia coli, Salmonella and Clostridium perfringens, and the antibacterial effect is very significant.

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

[0212] This embodiment provides an inactivated lactic acid bacteria body and its metabolites for improving pet health and food change stress, including inactivated lactic acid bacteria body and metabolites of composite lactic acid bacteria;

[0213] The composite 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 preparation method of the above-mentioned postbiotics comprises the following steps:

[0215] 1) Weighing materials: 4wt% of full-fat soybean powder, 6.5wt% of skim milk powder, 3wt% of yeast extract powder, 5wt% of sucrose, 2.5wt% of sodium citrate and 75wt% of water are mixed according to the required proportions;

[0216] 2) Chemical reaction: chemical reaction at 55-60℃ for 10-30min;

[0217] 3) Homogenization: 55-60°C, primary pressure 18-20Mpa, secondary pressure 4-5Mpa;

[0218] 4) Sterilization: 90-95°C for 15-45 minutes;

[0219] 5) Cooling: Cooling to 32-37° C. to obtain a feed liquid;

[0220] 6) Adding bacteria and lactase: Two different composite lactic acid bacteria were added to the liquid: composite lactic acid bacteria combination 1 was Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis lactis subspecies BB-16 and Lactobacillus acidophilus LA-14, which were added at a ratio of 1:1:1:1 in terms of the number of colony forming units; composite lactic acid bacteria combination 2 was Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis lactis subspecies BB-16 and Lactobacillus acidophilus LA-14, which were added at a ratio of 5:1:1:1 in terms of the number of colony forming units; the total inoculation amount was 6×10 6 CFU / g (liquid), and lactase (enzyme activity 5000U / g) 0.5mL / kg (liquid) was added according to production requirements;

[0221] 7) Constant temperature fermentation: Fermentation was carried out at 32-37°C until pH reached 4.5-4.6, which lasted for about 18-19 hours (total viable count of compound lactic acid bacteria ≥ 2.0 × 10 10 CFU / g);

[0222] 8) Inactivation: Mix 18g / kg maltodextrin (fermentation liquid) according to production requirements, preheat to 55-60°C, homogenize at a primary pressure of 18-20Mpa and a secondary pressure of 4-5Mpa, and then sterilize and inactivate (inactivation conditions 75-95°C, 15-30min);

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

[0224] The metabolites in the postbiotic powder prepared by the starter culture of the above two different bacterial strain combinations were detected.

[0225] After the postbiotic sample was mixed with water at a ratio of 1:4 (mass ratio), 0.1 mL was drawn into a 1.5 mL tube, and 0.4 mL of extract (methanol: acetonitrile = 1:1 (V / V)) was added. After sufficient oscillation, the tube was placed in an ultrasonic cold water bath for 10 min; the 1.5 mL tube after the water bath was placed in a -40 °C low-temperature refrigerator for incubation for 1 h; after taking out the sample, it was centrifuged at 12000×g for 20 min; the supernatant was drawn and transferred to the sample bottle after passing through the membrane. In addition, the supernatant of all samples was drawn equally to prepare the quality control sample, which was tested on the machine with the experimental samples. The target compound was chromatographed using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEHAmide (2.1 mm×100 mm, 1.7 μm) liquid chromatography column. Sample plate temperature: 4 °C, injection volume: 2 μL. The raw data were converted into mzXML format by Proteo Wizard, and peak detection, extraction, alignment and integration were completed using R language based on the internal program of XCMS to obtain the final metabolite name.

[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, soybean saponins, soybean isoflavones, γ-aminobutyric acid, active short peptide molecules, genistein, glucaric acid, ursolic acid, allicin, astaxanthin, solanine, 2-hydroxycinnamic acid and phenylacetaldehyde.

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

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

[0229] Step 1) weighing materials: 7wt% of full-fat soybean powder, 18wt% of skim milk powder, 3wt% of yeast extract powder, 7wt% of sucrose, 5wt% of sodium citrate and 60wt% of water are mixed according to the required proportions;

[0230] Step 6) the addition amount of lactase (enzyme activity of 5000U / g) is 1mL / kg (feed liquid);

[0231] Step 7) After the constant temperature fermentation is stopped, the total viable count of the composite lactic acid bacteria is ≥ 3.0 × 10 10 CFU / g;

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

[0233] Example 11: Preparation of postbiotic preparations

[0234] The postbiotic powder prepared by the composite lactic acid bacteria combination 1 of Example 9 was added with the dilution carrier maltodextrin for standardization treatment, and the number of inactivated lactic acid bacteria was detected by flow cytometry. The number of inactivated lactic acid bacteria was 3×10 10 cells / g、6×10 10 cells / g and 1×10 11 There are three specifications of compound lactic acid bacteria inactivated cells and their metabolite preparations, namely postbiotic preparation 1-300, preparation 1-600 and preparation 1-1000, which are packaged in powder packaging machines, 1kg / bag.

[0235] Example 12: Preparation of postbiotic preparations

[0236] The postbiotic powder prepared by the composite lactic acid bacteria combination 2 of Example 9 was added with the dilution carrier maltodextrin for standardization treatment to prepare the inactivated lactic acid bacteria in an amount of 3×10 10 cells / g、6×10 10 cells / g and 1×10 11 cells / g three specifications of inactivated lactic acid bacteria and their metabolites, namely postbiotic preparation 2-300, preparation 2-600 and preparation 2-1000, are packaged using a powder packaging machine, 1kg / bag.

[0237] Example 13: Clinical Trial

[0238] 1. Test Example 1: Treatment of diarrhea in dogs using the preparation

[0239] 1.1 Test subjects

[0240] Thirty-five Belgian Shepherd dogs aged 2-3 months suffering from 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.

[0241] 1.2 Test methods

[0242] The inactivated lactic acid bacteria and their metabolite preparations 1-300 and 2-300 prepared in Example 11 and Example 12 were used to evaluate the feeding of dogs with diarrhea. Among them, the experimental group I was gavaged with preparation 1-300 daily, 1.5 grams / dog / day; the experimental group II was gavaged with preparation 1-300 daily, 3 grams / dog / day; the experimental group III was gavaged with preparation 2-300 daily, 1.5 grams / dog / day; the experimental group IV was gavaged with preparation 2-300 daily, 3 grams / dog / day. Specific gavage method: Use 2-3 times the amount of clean water to dissolve the corresponding preparation to make a solution of inactivated lactic acid bacteria and its metabolites, and use a syringe to absorb it and directly gavage it. Gavage once every day from 10:00 to 11:00 in the morning, and continue for 15 days. The diarrhea rate and feces score of each group were counted on 0 days, 7 days, and 15 days.

[0243] 1.3 Results

[0244] Before the experiment, all 35 dogs in the five groups suffered from diarrhea to varying degrees. Seven days after the experiment, the diarrhea rates of experimental group I, experimental group II, experimental group III, experimental group IV and control group were 71%, 43%, 57%, 29% and 86%, respectively. Fifteen days after the experiment, the diarrhea rates of the five groups were 29%, 14%, 14%, 0% and 86%, respectively. Figure 1 As shown: on the 0th day 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 the 7th day 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, and the experimental group II (P<0.05) and the experimental group IV (P<0.01) were significantly higher than those of the control group; on the 15th day 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, and the experimental group I (P<0.05), the experimental group II (P<0.05), the experimental group III (P<0.05) and the experimental group IV (P<0.01) were significantly higher than those of the control group. It can be seen that daily oral administration of different amounts of inactivated lactic acid bacteria and their metabolites preparations 1-300 and 2-300 can significantly relieve dog diarrhea. Among them, the experimental group IV had the best effect, so the inactivated lactic acid bacteria and their metabolites preparation 2-300 was preferred.

[0245] Test Example 2: Treatment of diarrhea in cats using a preparation

[0246] 2.1 Test subjects

[0247] Thirty British shorthair cats with diarrhea aged 3-5 months 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.

[0248] 2.2 Test methods

[0249] The inactivated lactic acid bacteria and their metabolite preparations 1-300 and 2-300 prepared in Example 11 and Example 12 were used to evaluate the feeding of cats with diarrhea. Among them, the experimental group I was gavaged with preparation 1-300 daily, 0.5 g / cat / day; the experimental group II was gavaged with preparation 1-300 daily, 1 g / cat / day; the experimental group III was gavaged with preparation 2-300 daily, 0.5 g / cat / day; the experimental group IV was gavaged with preparation 2-300 daily, 1 g / cat / day. Specific gavage method: Use 2-3 times the amount of clean water to dissolve the corresponding preparation to prepare a solution of inactivated lactic acid bacteria and its metabolites, and use a syringe to draw it and directly gavage it. Gavage once every day from 10:00 to 11:00 in the morning, and continue for 15 days. The diarrhea rate and fecal score of each group were counted on 0 days, 7 days, and 15 days.

[0250] 2.3 Results

[0251] Before the experiment, all 30 cats in the five groups suffered from diarrhea to varying degrees. Seven days after the experiment, the diarrhea rates of experimental group I, experimental group II, experimental group III, experimental group IV and control group were 50%, 33%, 50%, 50% and 83%, respectively. Fifteen days after the experiment, the diarrhea rates of the five groups were 17%, 0%, 17%, 0% and 67%, respectively. 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, and those in experimental group II (P<0.05) and experimental group IV (P<0.05) were significantly higher than those in 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, and those in 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 significantly higher than those in the control group. It can be seen that daily oral administration of different amounts of inactivated lactic acid bacteria and their metabolites preparations 1-300 and 2-300 can significantly relieve cat diarrhea. Among them, the experimental group IV has the best effect, so the inactivated lactic acid bacteria and their metabolites preparation 2-300 is preferred.

[0252] Test Example 3: Breeding evaluation test on dogs using puffed food with added preparations

[0253] 3.1 Test subjects

[0254] Thirty-six German Shepherd dogs 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 and metabolites preparations 2-300, 2-600 and 2-1000 prepared in Example 12 were added to the puffed grains, and the addition amount of the three preparations was 10 grams per 10 kg of puffed grains to prepare three kinds of puffed grains containing inactivated lactic acid bacteria and metabolites, corresponding to experimental group I, experimental group II, experimental group III and control group, respectively, and the experimental time was 30 days. On the 0th day, 15th day and 30th day of the experiment, the diarrhea rate was counted, the feces were scored, and the body temperature, pulse, respiratory rate, food intake and weight of the participating dogs were measured; on the 0th day and 30th day of the experiment, the apparent digestibility of crude protein and crude fat was measured.

[0257] 3.3 Results

[0258] 3.3.1 Pulse, respiratory rate and body temperature measurement results:

[0259] The results are as follows Figure 3-5 The results showed that during the experiment, there were no significant differences in pulse, respiratory rate and body temperature between the experimental group and the control group, indicating that adding the above-mentioned lactic acid bacteria inactivated bacteria and their metabolites to the puffed food had no significant effect on the basic physiological indicators of dogs.

[0260] 3.3.2 Feed intake and body weight measurement results:

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

[0262] On the 15th and 30th days of the experiment, the average feed intake and body weight of Experimental Group I, Experimental Group II and Experimental Group III were higher than those of the control group; at the same time, Experimental Group I, Experimental Group II and Experimental Group III showed an increasing trend, indicating that with the increase in the number of inactivated lactic acid bacteria in different preparations, the efficacy of promoting feed intake and growth showed an increasing trend.

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

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

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

[0266] Before the experiment, all 36 German Shepherd dogs in the four groups suffered from diarrhea to varying degrees. After 15 days of the experiment, the diarrhea rates of experimental group I, experimental group II, experimental group III and control group were 56%, 33%, 22% and 78% respectively. After 30 days of the experiment, the diarrhea rates of the four groups were 22%, 11%, 11% and 89% respectively. Fig.10As shown, on the 0th day of the experiment, the fecal scores of experimental group I, experimental group II, experimental group III and control group were 1.6±0.7, 1.8±0.9, 1.6±0.5 and 1.6±0.9, respectively; on the 15th day 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, among which the experimental group III (P<0.05) was significantly higher than the control group; on the 30th day 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, and the experimental group I (P<0.05), experimental group II (P<0.05) and experimental group III (P<0.01) were significantly higher than the control group. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparations 2-300, 2-600 and 2-1000 at a ratio of 1 kg / t to the puffed food can significantly alleviate the diarrhea of ​​German Shepherd dogs. Therefore, inactivated lactic acid bacteria and their metabolite preparations 2-600 and 2-1000 are preferred.

[0267] Test Example 4: Cat breeding evaluation test using baked food with added preparations

[0268] 4.1 Test subjects

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

[0270] 4.2 Test methods

[0271] The lactic acid bacteria inactivated thalline and its metabolite preparations 2-300, 2-600 and 2-1000 prepared by Example 12 were added to the baked grains respectively, and the addition amount of the three preparations was 10 grams per 10 kg of baked grains to prepare 3 kinds of baked grains containing lactic acid bacteria inactivated thalline and its metabolites, and the experimental time was 21 days. There were 10 in each of the experimental I group (2-300 group), experimental II group (2-600 group), experimental III group (2-1000 group) and control group. The diarrhea rate of each group was counted on the 0th, 14th and 21st days of the experiment, and the feces were scored. And the feed intake, body weight, and apparent digestibility of crude protein and crude fat of the participating cats were measured on the 0th and 21st days.

[0272] 4.3 Results

[0273] 4.3.1 Feed intake and body weight measurement results:

[0274] The results of feed intake determination were as follows Fig.11The results showed that the average feed intake (g / bird / day) of experimental group I, experimental group II, experimental group III and control group on day 0 of the experiment was 59.4±2.1, 59.7±2.9, 59.3±2.5 and 59.6±2.9 respectively; the average feed intake (g / bird / day) on day 21 of the experiment was 65.1±3.6, 68.5±3.3, 68.6±3.2 and 61.8±2.9 respectively. Fig.12 The results showed that the average body weights (kg / head) of the four groups on day 0 of the experiment were 1.28±0.16, 1.25±0.10, 1.30±0.19 and 1.28±0.14 respectively; the average body weights (kg / head) of the four groups on day 21 of the experiment were 1.40±0.19, 1.49±0.15, 1.49±0.20 and 1.35±0.16 respectively. On days 14 and 21 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. This shows that the addition of three different inactivated lactic acid bacteria and their metabolite preparations to baked food all showed different degrees of effects in promoting feed intake and growth.

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

[0276] The apparent digestibility of crude protein is Fig.13 The results showed that the average crude protein apparent digestibility (%) of the four groups on day 0 of the experiment was 85.2±1.9, 85.8±1.7, 85.5±1.4 and 86.0±1.3 respectively; the average crude protein apparent digestibility of the four groups on day 21 of the experiment was 88.5±1.4, 90.8±1.5, 91.1±1.8 and 86.1±1.5 respectively. The experimental groups II (P<0.05) and III (P<0.05) were significantly higher than those of the control group. Fig.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; the average apparent digestibility of crude fat in the four groups on day 21 of the experiment were 92.4±1.4, 92.5±0.7, 92.5±1.0 and 92.2±0.8 respectively. The digestibility of experimental groups I, II and III was higher than that of the control group. The addition of three different inactivated lactic acid bacteria and their metabolite preparations to baked grains promoted the digestion level 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 cats in the four groups suffered from diarrhea to varying degrees. After 14 days of the experiment, the diarrhea rates of experimental group I, experimental group II, experimental group III and control group were 30%, 10%, 20% and 80%, respectively. After 21 days of the experiment, the diarrhea rates of the four groups were 10%, 0%, 0% and 60%, respectively. Fig.15 As shown, on day 0 of the experiment, the fecal scores of experimental group I, experimental group II, experimental group III and control group were 1.9±0.6, 1.8±0.7, 1.9±0.6 and 1.9±0.6, respectively; on day 14 of the experiment, 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, among which experimental group I (P<0.05), experimental group II (P<0.01) and experimental group III (P<0.01) were significantly higher than those of the control group; on day 21 of the experiment, 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, and experimental group I (P<0.01), experimental group II (P<0.01) and experimental group III (P<0.01) were significantly higher than those of the control group. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparations 2-300, 2-600 and 2-1000 at a ratio of 1 kg / t to baked food can significantly relieve diarrhea in rural cats. Inactivated lactic acid bacteria and their metabolite preparations 2-600 and 2-1000 are preferred.

[0279] Test Example 5: Using puffed food with added preparations to test the diet of dogs

[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 and metabolite preparation 2-1000 prepared in Example 12 were added to the two puffed grain formulas A and B, respectively, and the addition amount was 10 grams per 10 kg of puffed grain to prepare two puffed grains A+ and B+ containing inactivated lactic acid bacteria and their metabolites. In the experimental group I, after continuous feeding of puffed grain A for 15 days, it was directly switched to puffed grain B; in the experimental group II, after continuous feeding of puffed grain A for 15 days, it was directly switched to puffed grain B+; in the experimental group III, after continuous feeding of puffed grain A+ for 15 days, it was directly switched to puffed grain B+. The diarrhea rate, feces score and feed intake were counted 3 days before the change of food and 1, 3 and 7 days after the change of food, and the levels of cortisol, malondialdehyde, immunoglobulin G and immunoglobulin A in serum were measured.

[0284] 5.3 Results

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

[0286] Three days before the diet change, the diarrhea rates of experimental group I, experimental group II and experimental group III were 43%, 50% and 14% respectively; one day after the diet change, the diarrhea rates of the three groups were 93%, 57% and 29% respectively; three days after the diet change, the diarrhea rates of the three groups were 86%, 29% and 14% respectively; seven days after the diet change, the diarrhea rates of the three groups were 57%, 14% and 7% respectively. Fig.16 As shown, 3 days before the diet change, the fecal scores of experimental group I, experimental group II and experimental group III were 2.6±0.9, 2.4±1.0 and 3.4±0.9, respectively, among which experimental group III was significantly higher than experimental group I (P<0.01) and group II (P<0.01); 1 day before the diet change, the fecal scores of the three groups were 1.7±0.6, 2.2±1.0 and 3.1±1.2, respectively, among which experimental group III was significantly higher than experimental group I (P<0.01) and group II (P<0.05), experimental group II was significantly higher than experimental group I (P<0.01) and group II (P<0.05). The fecal scores of the three groups were significantly higher than those of the experimental group I (P < 0.05); 3 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, of which the experimental group II (P < 0.05) and the experimental group III (P < 0.01) were significantly higher than those of the experimental group I; 7 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, of which the experimental group II (P < 0.01) and the experimental group III (P < 0.01) were significantly higher than those of the experimental group I. It can be seen that adding lactic acid bacteria inactivated bacteria and their metabolite preparation 2-1000 at a ratio of 1 kg / t to the extruded food can significantly relieve diarrhea in Teddy dogs and significantly reduce diarrhea caused by stress caused by food intolerance during the food change process.

[0287] 5.3.2 Feed intake measurement results:

[0288] The results of feed intake determination were as follows Fig.17The results showed that the average feed intake (g / bird / day) of experimental group I, experimental group II and experimental group III were 231.2±6.8, 228.1±9.6 and 236.8±8.1 respectively 3 days before the feed change, and the average feed intake of experimental group III was higher than that of experimental group I and group II. The average feed intake (g / bird / day) of the three groups was 187.8±7.9, 216.4±9.1 and 229.3±7.0 respectively 1 day after the feed change, and the average feed intake of experimental group II (P<0.05) and group III (P<0.01) was significantly higher than that of experimental group I. The average feed intake (g / bird / day) of the three groups was 212.5±11.6, 232.3±9.2 and 241.8±7.4 respectively 3 days after the feed change, and the average feed intake of experimental group II and group III was significantly higher than that of experimental group I (P<0.05). Seven days after the feed change, the average feed intake (g / bird / 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). It can be seen that adding inactivated lactic acid bacteria and their metabolite preparations 2-1000 at a ratio of 1kg / t to extruded grains A and B can reduce anorexia and decreased feed intake caused by stress during the feed change, and has a certain effect in promoting feed intake. Among them, one day after the experimental group I was changed from extruded grain A to extruded grain B, the average feed intake dropped sharply, and after 7 days, the average feed intake returned to the level before the feed change. The average feed intake of experimental groups II and III, which were added with inactivated lactic acid bacteria and their metabolite preparations 2-1000, decreased less. In particular, in the experimental group III, inactivated lactic acid bacteria and their metabolite preparation 2-1000 were added to the diet before and after the feed change. The average feed intake was quickly restored within 1-3 days after the feed change, with significant results.

[0289] 5.3.3 Serum cortisol test results:

[0290] Serum cortisol test results Fig.18The results showed that 3 days before the feed change, the serum cortisol levels (ng / mL) of experimental group I, experimental group II and experimental group 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) of the three groups were 181.6±3.7, 98.0±3.5 and 70.8±3.2 respectively, among which the serum cortisol levels of experimental group II (P<0.01) and group III (P<0.001) were significantly lower than those of experimental group I; 3 days after the feed change, the serum cortisol levels of the three groups were The contents (ng / mL) were 203.5±5.5, 84.4±2.6 and 65.6±3.3, respectively. The contents of serum cortisol in experimental group II and group III were significantly lower than those in experimental group I (P<0.001). The contents of serum cortisol in the three groups 7 days after the feed change were 120.6±4.0, 65.7±3.8 and 58.4±3.9, respectively. The contents of serum cortisol in experimental group II (P<0.05) and group III (P<0.01) were significantly lower than those in experimental group I (P<0.05). Serum cortisol is an adrenal glucocorticoid that can reflect the stress level of animals and is often used to detect the physiological response level of animals under stress. The fluctuation of serum cortisol content in experimental Teddy dogs during the whole feed change process showed that experimental group III < experimental group II < experimental group I, indicating that the stress level of the three groups of dogs during the feed change process was experimental group III < experimental group II < experimental group I. It can be seen that adding inactivated lactic acid bacteria and their metabolites preparation 2-1000 at a ratio of 1kg / t to extruded grains A and B can effectively reduce the stress of grain change.

[0291] 5.3.4 Serum malondialdehyde test results:

[0292] Serum malondialdehyde test results Fig.19The results showed that 3 days before the feed change, the serum malondialdehyde levels (nmol / mL) of experimental group I, experimental group II and experimental group III were 1.91±0.16, 1.89±0.13 and 1.86±0.12 respectively; 1 day after the feed change, the serum malondialdehyde levels (nmol / mL) of the three groups were 3.59±0.40, 2.79±0.19 and 2.20±0.27 respectively, among which the serum malondialdehyde levels of experimental group II (P<0.05) and group III (P<0.01) were significantly lower than those of experimental group I; 3 days after the feed change, the serum malondialdehyde levels of experimental group II (P<0.05) and group III (P<0.01) were significantly lower than those of experimental group I. The serum malondialdehyde contents (nmol / mL) of the three groups were 3.23±0.31, 2.12±0.22 and 1.93±0.15, respectively. The serum malondialdehyde contents of experimental groups II and III were significantly lower than those of experimental group I (P<0.01). The serum malondialdehyde contents (nmol / mL) of the three groups 7 days after the feed change were 2.45±0.26, 1.89±0.19 and 1.88±0.17, respectively. The serum malondialdehyde contents of experimental groups II and III were significantly lower than those of experimental group I (P<0.05). Serum malondialdehyde is an important indicator reflecting the level of free radicals and lipid peroxidation metabolism in the body. The fluctuation of serum malondialdehyde content in experimental Teddy dogs during the whole feed change process showed that 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 can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to puffed grains A and B can improve the body's peroxide scavenging ability, quickly remove excess oxygen free radicals produced by stress stimulation in the body, and thus reduce the adverse reactions caused by stress.

[0293] 5.3.5 Serum immunoglobulin G test results:

[0294] Serum immunoglobulin G test results Fig. 20The results showed that 3 days before the feed change, the immunoglobulin G (mg / mL) contents of experimental group I, experimental group II and experimental group III were 9.7±0.3, 9.5±0.4 and 11.5±0.4, respectively. Among them, the immunoglobulin G content of experimental group III (P<0.05) was significantly higher than that of experimental groups I and II. One day after the feed change, the immunoglobulin G contents (mg / mL) of experimental group I, experimental group II and experimental group III were 9.4±0.6, 10.2±0.5 and 12.0±0.6, respectively, among which the immunoglobulin G content of experimental group III (P<0.05) was significantly higher than that of experimental group I; 3 days after the feed change, the serum immunoglobulin G contents (mg / mL) of the three groups were 9.3±0.6, 10.8±0.5 and 13.1±0.5, respectively, among which the immunoglobulin G contents of experimental group II (P<0.05) and group III (P<0.01) were significantly higher than those of experimental group I; 7 days after the feed change, the serum immunoglobulin G contents (mg / mL) of the three groups were 9.6±0.4, 13.4±0.4 and 14.8±0.2, respectively, among which the immunoglobulin G contents of experimental group II (P<0.01) and group III (P<0.01) were significantly higher than those of experimental group I. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to puffed grains A and B 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 test results:

[0296] Serum immunoglobulin A test results Fig.21 It was shown that 3 days before the feed change, the immunoglobulin A (mg / mL) contents of experimental group I, experimental group II and experimental group III were 1.46±0.11, 1.44±0.07 and 1.53±0.08, respectively. One day after the feed change, the immunoglobulin A contents (mg / mL) of experimental group I, experimental group II and experimental group III were 1.44±0.10, 1.49±0.09 and 1.54±0.07, respectively; 3 days after the feed change, the serum immunoglobulin A contents (mg / mL) of the three groups were 1.42±0.08, 1.55±0.08 and 1.57±0.09, respectively, among which the immunoglobulin A contents of group I and group III (P<0.05) were significantly higher than that of experimental group I; 7 days after the feed change, the serum immunoglobulin A contents (mg / mL) of the three groups were 1.47±0.08, 1.58±0.09 and 1.61±0.07, respectively. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to puffed grains A and B 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] Test Example 6: Cat food change test using baked food with added preparations

[0298] 6.1 Test subjects

[0299] Thirty-three ragdoll cats aged 5±1 months were selected and divided into experimental group I, experimental group II, and experimental group III, with 11 cats in each group.

[0300] 6.2 Test methods

[0301] The inactivated lactic acid bacteria and metabolite preparation 2-1000 prepared in Example 12 were added to the two baked grain formulas C and D, respectively, and the addition amount was 10 grams per 10 kg of baked grain to prepare two baked grains C+ and D+ containing inactivated lactic acid bacteria and their metabolites. Experimental group I, after continuous feeding of baked grain C for 15 days, directly switched to baked grain D; experimental group II, after continuous feeding of baked grain C for 15 days, directly switched to baked grain D+; experimental group III, after continuous feeding of baked grain C+ for 15 days, directly switched to baked grain D+. The diarrhea rate, feces score and feed intake were counted 1 day before the change of food and 1, 3 and 7 days after the change of food, and the levels of cortisol, malondialdehyde, immunoglobulin G, immunoglobulin A, D-lactic acid and diamine oxidase in serum were determined.

[0302] 6.3 Results

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

[0304] Three days before the diet change, the diarrhea rates of experimental group I, experimental group II and experimental group III were 64%, 64% and 21% respectively; one day after the diet change, the diarrhea rates of the three groups were 79%, 71% and 21% respectively; three days after the diet change, the diarrhea rates of the three groups were 79%, 36% and 14% respectively; seven days after the diet change, the diarrhea rates of the three groups were 57%, 14% and 14% respectively. Fig. 22As shown in the figure, 3 days before the diet change, the fecal scores of experimental group I, experimental group II and experimental group III were 2.0±0.9, 1.9±0.7 and 2.7±1.0, respectively, among which the experimental group III was significantly higher than that of experimental group I (P<0.05) and group II (P<0.05); 1 day before the diet change, the fecal scores of the three groups were 1.5±0.5, 1.9±0.5 and 2.7±0.8, respectively, among which the experimental group III was significantly higher than that of experimental group I (P<0.01) and group II (P <0.05); 3 days before changing the diet, the stool scores of the three groups were 1.7±0.5, 2.5±1.0 and 3.9±0.8, among which the experimental group II (P<0.05) and the experimental group III (P<0.01) were significantly higher than the experimental group I; 7 days before changing the diet, the stool scores of the three groups were 2.2±1.1, 3.0±0.9 and 3.2±0.8, among which the experimental group II (P<0.05) and the experimental group III (P<0.05) were significantly higher than the experimental group I. It can be seen that adding lactic acid bacteria inactivated bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to baked diets C and D can significantly relieve diarrhea in ragdoll cats and significantly reduce diarrhea caused by stress caused by food intolerance during the diet change process.

[0305] 6.3.2 Feed intake measurement results:

[0306] The results of feed intake determination were as follows Fig.23The results showed that the average feed intake (g / bird / day) of experimental group I, experimental group II and experimental group III were 77.1±4.0, 76.7±4.5 and 81.5±4.2 respectively 3 days before the feed change, and the average feed intake of experimental group III was higher than that of experimental group I and group II. The average feed intake (g / bird / day) of the three groups was 64.8±3.8, 74.5±4.1 and 80.6±3.8 respectively 1 day after the feed change, and the average feed intake of experimental group II (P<0.05) and group III (P<0.01) was significantly higher than that of experimental group I. The average feed intake (g / bird / day) of the three groups was 63.1±4.1, 74.3±3.9 and 81.7±3.4 respectively 3 days after the feed change, and the average feed intake of experimental group II (P<0.05) and group III (P<0.01) was significantly higher than that of experimental group I. Seven days after the feed change, the average feed intake (g / bird / day) of the three groups was 65.0±4.0, 81.2±3.8 and 86.5±3.3, respectively. The average feed intake of experimental groups II (P<0.01) and III (P<0.01) was significantly higher than that of experimental group I. One day after the experimental group I was changed from baked food A to baked food B, the average feed intake dropped sharply, and it had not recovered to the level before the feed change by the end of the experiment. The average feed intake of experimental group II, which was added with postbiotic preparation 2-1000, did not decrease significantly, and the feed intake returned to normal within 7 days. The average feed intake of experimental group III, which was added with inactivated lactic acid bacteria and its metabolite preparation 2-1000, continued to increase. It can be seen that adding inactivated lactic acid bacteria and its metabolite preparation 2-1000 at a ratio of 1kg / t to baked food C and D can reduce anorexia and decreased feed intake caused by stress during the feed change, and has a certain effect of promoting feed intake.

[0307] 6.3.3 Serum malondialdehyde test results:

[0308] Serum malondialdehyde test results Fig.24The results showed that 3 days before the feed change, the serum malondialdehyde levels (nmol / mL) of experimental group I, experimental group II and experimental group III were 2.62±0.13, 2.58±0.12 and 2.61±0.13 respectively; 1 day after the feed change, the serum malondialdehyde levels (nmol / mL) of the three groups were 4.24±0.37, 2.76±0.26 and 2.66±0.19 respectively, among which the serum malondialdehyde levels of experimental group II (P<0.01) and group III (P<0.01) were significantly lower than those of experimental group I; Three days after the feed change, the serum malondialdehyde content (nmol / mL) of the three groups was 3.44±0.20, 2.70±0.24 and 2.65±0.17, respectively. The serum malondialdehyde content of experimental groups II and III was significantly lower than that of experimental group I (P<0.05); 7 days after the feed change, the serum malondialdehyde content (nmol / mL) of the three groups was 2.82±0.27, 2.64±0.21 and 2.58±0.18, respectively. The serum malondialdehyde content of experimental groups II and III was lower than that of experimental group I. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to baked food C and D can significantly reduce the stress of food change on ragdoll cats.

[0309] 6.3.4 Serum cortisol test results:

[0310] Serum cortisol test results Fig.25 The results showed that 3 days before the feed change, the serum cortisol levels (ng / mL) of experimental group I, experimental group II and experimental group 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) of the three groups were 147.2±10.5, 75.8±5.1 and 50.6±6.7 respectively, among which the serum cortisol levels of experimental group II (P<0.01) and group III (P<0.001) were significantly lower than those of experimental group I; 3 days after the feed change, the serum cortisol levels of the three groups ( ng / mL) were 149.2±9.6, 62.8±8.7 and 47.2±5.3, respectively. The serum cortisol content in experimental group II (P<0.01) and group III (P<0.001) was significantly lower than that in experimental group I. The serum cortisol content (ng / mL) of the three groups 7 days after the feed change was 77.9±8.5, 46.3±4.6 and 44.8±5.2, respectively. The serum cortisol content in experimental group II (P<0.05) and group III (P<0.05) was significantly lower than that in experimental group I (P<0.05). It was further confirmed that adding lactic acid bacteria inactivated bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to baked grains C and D can significantly reduce the stress of feed change.

[0311] 6.3.5 Serum immunoglobulin G test results:

[0312] Serum immunoglobulin G test results Fig.26 The results showed that 3 days before the feed change, the immunoglobulin G (mg / mL) contents of experimental group I, experimental group II and experimental group III were 6.4±0.4, 6.6±0.5 and 7.7±0.4, respectively. Among them, the immunoglobulin G content of experimental group III (P<0.05) was significantly higher than that of experimental groups I and II. One day after the feed change, the immunoglobulin G contents (mg / mL) of experimental group I, experimental group II and experimental group III were 6.1±0.5, 6.9±0.4 and 7.7±0.3, respectively, among which the immunoglobulin G content of experimental group III (P<0.05) was significantly higher than that of experimental group I; 3 days after the feed change, the serum immunoglobulin G contents (mg / mL) of the three groups were 6.2±0.3, 7.1±0.4 and 7.8±0.4, respectively, among which the immunoglobulin G content of experimental group III (P<0.05) was significantly higher than that of experimental group I; 7 days after the feed change, the serum immunoglobulin G contents (mg / mL) of the three groups were 6.2±0.5, 7.5±0.4 and 7.8±0.2, respectively, among which the immunoglobulin G contents of experimental group II (P<0.05) and group III (P<0.05) were significantly higher than those of experimental group I. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t to baked grains C and D 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 test results:

[0314] Serum immunoglobulin A test results Fig. 27The results showed that 3 days before the feed change, the immunoglobulin A (mg / mL) contents of experimental group I, experimental group II and experimental group III were 1.33±0.12, 1.31±0.10 and 1.43±0.07, respectively, among which the content of immunoglobulin A in experimental group III (P<0.05) was significantly higher than that in experimental group II; 1 day after the feed change, the content of immunoglobulin A (mg / mL) in experimental group I, experimental group II and experimental group III were 1.27±0.09, 1.45±0.08 and 1.47±0.13, respectively, among which the content of immunoglobulin A in experimental group II (P<0.05) and group III (P<0.05) was significantly higher than that in experimental group II. Experimental group I; 3 days after the feed change, the serum immunoglobulin A content (mg / mL) of the three groups were 1.34±0.13, 1.55±0.11 and 1.49±0.07, among which the immunoglobulin A content of experimental group II (P<0.05) and group III (P<0.05) was significantly higher than that of experimental group I; 7 days after the feed change, the serum immunoglobulin A content (mg / mL) of the three groups were 1.32±0.09, 1.53±0.11 and 1.47±0.11, among which the immunoglobulin A content of experimental group II (P<0.05) and group III (P<0.05) was significantly higher than that of experimental group I. It further shows that adding lactic acid bacteria inactivated bacteria and its metabolite preparation 2-1000 at a ratio of 1kg / t to baked food C and D can significantly increase the content of immunoglobulin A in serum, 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 test results:

[0316] Serum D-lactate test results Fig.28The results showed that 3 days before the feed change, the D-lactic acid (ng / mL) contents of experimental group I, experimental group II and experimental group III were 0.64±0.10, 0.67±0.12 and 0.46±0.08, respectively, among which the experimental group III (P<0.05) was significantly lower than that of experimental group I and experimental group II; 1 day after the feed change, the serum D-lactic acid contents (mg / mL) of the three groups were 0.82±0.10, 0.56±0.09 and 0.47±0.14, respectively, among which the experimental group II (P<0.05) and group III (P<0.01) were significantly lower than that of experimental group I and experimental group II. Experimental group I; 3 days after the feed change, the serum D-lactic acid content (mg / mL) of the three groups were 0.84±0.09, 0.50±0.11 and 0.47±0.12, respectively, among which the experimental group II (P<0.01) and group III (P<0.01) were significantly lower than the experimental group I; 7 days after the feed change, the serum D-lactic acid content (mg / mL) of the three groups were 0.72±0.07, 0.43±0.09 and 0.45±0.04, respectively, among which the experimental group II (P<0.05) and group III (P<0.05) were significantly lower than the experimental group I. D-lactic acid is an important substance reflecting the intestinal barrier function. It is produced by the metabolism of certain specific bacteria in the intestine. Under normal circumstances, it can be discharged through the intestinal barrier of animals. However, in the case of intestinal damage, the intestinal barrier function decreases, and D-lactic acid cannot be discharged smoothly from the body. It will accumulate in the blood through the intestine, causing the D-lactic acid level in the blood to rise. During the food change process, the serum D-lactic acid level in Experimental Group I showed a trend of significant increase and then gradual decrease, indicating that the intestinal barrier function of pets decreased rapidly in the short term during the food change process, which may be closely related to the surge in the incidence of diarrhea. The D-lactic acid level in the serum of Experimental Group II showed a trend of gradual decrease, indicating that the baked grain D+ after adding the postbiotic preparation 2-1000 at a ratio of 1kg / t was effective in improving the intestinal barrier function. The D-lactic acid level in the serum of Experimental Group III was relatively stable before and after the food change. It shows that the baked grains C+ and D+ after adding the inactivated lactic acid bacteria and their metabolite preparation 2-1000 at a ratio of 1kg / t both have excellent intestinal barrier improvement effects.

[0317] 6.3.8 Serum diamine oxidase test results:

[0318] Serum diamine oxidase test results Fig.29The results showed that 3 days before the feed change, the levels of diamine oxidase (μg / dL) in experimental group I, experimental group II and experimental group III were 1.80±0.14, 1.79±0.11 and 1.56±0.10, respectively, among which the experimental group III (P<0.05) was significantly lower than that in experimental group I and experimental group II; 1 day after the feed change, the serum diamine oxidase (μg / dL) levels of the three groups were 2.14±0.10, 1.66±0.09 and 1.52±0.04, respectively, among which the experimental group II (P<0.05) and group III (P<0.01) were significantly lower than that in experimental group I and experimental group II. The serum diamine oxidase (μg / dL) of the three groups 3 days after the feed change was 1.96±0.07, 1.61±0.10 and 1.53±0.05, respectively, of which the experimental groups II (P<0.05) and III (P<0.05) were significantly lower than those of the experimental group I; the serum diamine oxidase (μg / dL) of the three groups 7 days after the feed change was 1.81±0.12, 1.48±0.07 and 1.48±0.04, respectively, of which the experimental groups II (P<0.05) and III (P<0.05) were significantly lower than those of the experimental group I. Diamine oxidase is another important substance that reflects the intestinal barrier function. In general, histamine substances are produced during the metabolic process of harmful microorganisms in the intestine and during the decomposition of food. Excessive accumulation of such substances can lead to inflammatory diseases in the body. The role of diamine oxidase is to decompose such histamine substances and reduce the excessive entry of histamine substances into the blood. However, in the case of intestinal damage, the intestinal barrier function decreases, and a large amount of blood histamine substances accumulate, causing the diamine oxidase level to rise. During the food change process, the diamine oxidase content in the serum of the experimental group I showed a trend of significant increase and then gradually decrease, which also showed that the food change process caused the pet's intestinal barrier function to decline rapidly in a short period of time, which may be closely related to the surge in the incidence of diarrhea, which is consistent with the test results of D-lactic acid levels in serum. The diamine oxidase level in the serum of the experimental group II showed a gradual downward trend, further indicating that the baked grain D+ after adding 2-1000 of lactic acid bacteria and its metabolite preparations at a ratio of 1kg / t was effective in improving the intestinal barrier function. The diamine oxidase level in the serum of Experiment III group was relatively stable before and after the change of diet, which further showed that the baked grains C+ and D+, to which the inactivated lactic acid bacteria and their metabolite preparations 2-1000 were added at a ratio of 1kg / t, had excellent effect in improving the intestinal barrier.

[0319] Test Example 7: Cat breeding evaluation experiment using staple wet food meal packs with added preparations

[0320] 7.1 Test subjects

[0321] Twenty-four American shorthair cats aged 8±1 months were selected and divided into experimental group I, experimental group II and control group, with 6 cats in each group.

[0322] 7.2 Test methods

[0323] The inactivated lactic acid bacteria and its metabolite preparation 2-1000 prepared in Example 12 were added to the staple wet food meal bag in different amounts, and the addition amounts were 10 grams and 50 grams per 10 kilograms of staple wet food, respectively, to prepare two kinds of staple wet food meal bags containing inactivated lactic acid bacteria and its metabolites. Experimental group I (10 grams of inactivated lactic acid bacteria and its metabolite preparation 2-1000 were added to every 10 kilograms of staple wet food), experimental group II (50 grams of inactivated lactic acid bacteria and its metabolite preparation 2-1000 were added to every 10 kilograms of staple wet food meal bags) and control group (staple wet food meal bags were not added with inactivated lactic acid bacteria and its metabolite preparation 2-1000), and the experimental time was 35 days. At the beginning and end of the experiment, the body weight, apparent digestibility of nutrients, diarrhea rate and feces score of the participating cats were measured, and the levels of immunoglobulins IgG and IgA in serum and secretory immunoglobulin SIgA in feces were measured.

[0324] 7.3 Results

[0325] 7.3.1 Body weight measurement results:

[0326] The results of body weight determination are as follows Fig.30 The results showed that the average body weights (kg / cat) of the three groups on day 0 of the experiment were 3.38±0.12, 3.37±0.12 and 3.38±0.10 respectively; the average body weights (kg / cat) of the three groups on day 35 of the experiment were 3.73±0.12, 3.88±0.10 and 3.65±0.14 respectively. The average body weights of the experimental groups I and II were higher than those of the control group, and the experimental group II was significantly higher than the control group (P<0.05). This showed that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the staple wet food meal packs had a growth-promoting effect on pet cats, and the promotion effect was significantly improved with the increase of the added amount.

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

[0328] The apparent digestibility of crude protein is Fig.31 The results showed that the average crude protein apparent digestibility (%) of the three groups on day 0 of the experiment was 87.3±0.7, 87.0±0.6 and 87.1±0.7 respectively; the average crude protein apparent digestibility of the three groups on day 35 of the experiment was 89.5±1.5, 90.9±1.1 and 87.5±0.8 respectively. The experimental group I (P<0.05) and the experimental group II (P<0.01) were significantly higher than the control group. Fig.32The results showed that the average apparent digestibility of crude fat (%) of the three groups on day 0 of the experiment was 90.5±0.6, 90.8±0.6 and 90.6±0.9 respectively; the average apparent digestibility of crude fat of the three groups on day 35 of the experiment was 91.3±0.9, 91.7±0.6 and 90.7±0.8 respectively. The experimental groups I and II were higher than the control group. This showed that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the staple wet food meal packs promoted the digestion level 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 of the three groups were 50%, 50% and 33% respectively, and all three groups had diarrhea to varying degrees; on day 35 of the experiment, the diarrhea rates of the three groups were 0%, 0% and 50% respectively, and the experimental groups I and II completely recovered to normal, while the diarrhea of ​​the control group was aggravated. Fig.33 As shown, the fecal scores of the three groups on day 0 of the experiment were 2.7±0.8, 2.5±1.0 and 2.8±1.2 respectively; the fecal scores of the three groups on day 35 of the experiment were 3.7±0.5, 3.8±0.4 and 2.7±0.8 respectively, among which the scores of experimental group I (P<0.01) and experimental group II (P<0.001) were significantly higher than those of the control group. It can be seen that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the staple wet food meal pack can significantly relieve diarrhea in pet cats, and the effect of adding according to the experimental group II is more significant.

[0331] 7.3.4 Results of serum immunoglobulin G determination:

[0332] Serum immunoglobulin G content (mg / mL) Fig.34 The results showed that the levels of serum immunoglobulin G in the three groups on day 0 were 9.7±0.4, 9.6±0.5 and 9.7±0.5 respectively; the levels of serum immunoglobulin G in the three groups on day 35 were 11.3±0.7, 11.8±0.5 and 10.5±0.9 respectively, and the levels of serum immunoglobulin A in the three groups on day 1 were significantly higher than those in the control group (P<0.05) and group II (P<0.01). Fig.35 The results showed that the serum immunoglobulin G levels of the three groups on day 0 of the experiment were 1.56±0.14, 1.55±0.11 and 1.61±0.12, respectively; the serum immunoglobulin A levels (mg / mL) of the three groups on day 35 of the experiment were 1.56±0.12, 1.57±0.10 and 1.53±0.14, respectively, among which the experimental groups I and II were higher than those of the control group.

[0333] 7.3.5 Results of secretory immunoglobulin A in feces:

[0334] Fecal secretory immunoglobulin A content (mg / mL) Fig.36 The results showed that the contents of secretory immunoglobulin A in the feces of the three groups on day 0 of the experiment were 1.24±0.08, 1.23±0.07 and 1.24±0.07 respectively; the contents of secretory immunoglobulin A in the feces of the three groups on day 35 of the experiment were 1.27±0.08, 1.32±0.07 and 1.20±0.07 respectively, among which the contents of experimental group II (P<0.05) were significantly higher than those of the control group. It can be seen that adding inactivated lactic acid bacteria and their metabolite preparation 2-1000 to the staple wet food meal pack according to the addition method of experimental group II can significantly increase the content of secretory immunoglobulin A in feces, which is consistent with the results of relieving diarrhea in pet cats.

[0335] Test Example 8: Cat breeding evaluation experiment using freeze-dried raw bone meat with added preparations

[0336] 8.1 Test subjects

[0337] Twenty-seven 8±1 month old American Shorthair cats were selected and divided into experimental group I, experimental group II, and control group, with 9 cats in each group.

[0338] 8.2 Test methods

[0339] The inactivated lactic acid bacteria and its metabolites 2-1000 prepared in Example 12 were added to freeze-dried raw bones and meat in different amounts, with the addition amounts being 20 grams and 100 grams per 20 kilograms of freeze-dried raw bones and meat, respectively, to prepare two types of freeze-dried food containing inactivated lactic acid bacteria and its metabolites. Before the experiment, the 27 cats were uniformly fed with freeze-dried raw bones and meat containing no inactivated lactic acid bacteria and its metabolites, with a feeding amount of 20 grams per cat per day, and free access to puffed food.

[0340] At the beginning of the experiment, the cats were randomly divided into 3 groups, with 9 cats in each group. The feeding amount of freeze-dried raw bones and meat was 15 grams per cat per day, and the cats were fed with puffed food freely. The experimental time was 21 days. At the beginning and end of the experiment, the diarrhea rate and fecal score of the participating cats were counted, the fecal ammonia concentration was tested, and the oral odor, cat litter odor, mental state and fur state were recorded.

[0341] 8.3 Results

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

[0343] On day 0 of the experiment, 4 rats in all three groups had diarrhea of ​​varying degrees, with a diarrhea rate of 44%; on day 21 of the experiment, the diarrhea rates in the three groups were 11%, 0%, and 44%, respectively. Group II completely recovered to normal, but one rat in group I still had diarrhea, and the control group showed a slight improvement, but 4 rats still had diarrhea. Fig.37 As shown, the fecal scores of the three groups on day 0 of the experiment were 2.6±0.9, 2.6±0.5 and 2.7±0.7 respectively; the fecal scores of the three groups on day 21 of the experiment were 3.3±0.7, 3.7±0.5 and 2.8±0.8 respectively, among which the scores of experimental group I (P<0.05) and experimental group II (P<0.01) were significantly higher than those of the control group. It can be seen that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to freeze-dried raw bone meat can significantly relieve diarrhea in pet cats, and the effect of adding according to the experimental group II is more significant.

[0344] 8.3.2 Weight measurement results:

[0345] The results of body weight determination are as follows Fig.38 The results showed that the average body weights of the three groups on day 0 of the experiment (kg / cat) were 3.42±0.07, 3.43±0.07 and 3.43±0.05 respectively; the average body weights of the three groups on day 21 of the experiment (kg / cat) were 3.75±0.07, 3.83±0.05 and 3.67±0.09 respectively. The average body weights of the experimental groups I and II were higher than those of the control group. This showed that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to freeze-dried raw bone meat had a growth-promoting effect on pet cats, and the promotion effect increased with the increase in the amount of addition.

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

[0347] The results of fecal ammonia content determination are as follows Fig.39 The results showed that the average fecal ammonia content (mg / dL) of the three groups on day 0 of the experiment was 25.5±0.1, 25.4±0.3 and 25.5±0.3 respectively; the average fecal ammonia content (mg / dL) of the three groups on day 21 of the experiment was 17.4±0.3, 15.1±0.2 and 26.3±0.2 respectively. Among them, the experimental group I (P<0.01) and the experimental group II (P<0.01) were significantly higher than the control group. It shows that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to freeze-dried raw bones and meat can significantly reduce the ammonia concentration in pet cat feces, thereby reducing the odor of cat litter.

[0348] The statistical results of cat litter odor are shown in Table 2. Before the experiment, the cat litters of the three groups had obvious odor after cleaning the feces; 21 days after the experiment, the cat litter odor of the experimental group I was significantly reduced after cleaning the feces, and the cat litter of the experimental group II had no obvious odor after cleaning the feces. The cat litter of the control group had obvious odor after cleaning the feces. This result is consistent with the trend of the fecal ammonia concentration test results. The statistical results of oral odor show that the oral cavity of the three groups of cats before the experiment had a strong fishy smell and saliva marks; 21 days after the experiment, the oral cavity of the experimental group I had a slight fishy smell and a small amount of saliva marks, the oral cavity of the experimental group II had a slight fishy smell and no saliva marks, and the oral cavity of the control group still had a strong fishy smell and heavy saliva marks. It shows that adding different amounts of inactivated lactic acid bacteria and their metabolite preparations 2-1000 to freeze-dried raw bones and meat has a certain effect on reducing the odor of pets' oral cavity and cat litter.

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

[0350]

[0351] 8.3.4 Mental and fur status statistics:

[0352] The statistical results of spirit and fur status are shown in Table 3. Before the experiment, the three groups of pet cats were in general spirit and had poor vitality. In the experimental group I, the fur color gloss and smoothness were poor, the elasticity was poor, the hair loss was serious, the knotting rate was high, and 3 cats had mild inflammation on the skin. In the experimental group II, the fur color gloss and smoothness were poor, the elasticity was poor, the villi were sparse, the knotting rate was high, and 3 cats had mild inflammation on the skin. In the control group, the fur color gloss and smoothness were poor, the elasticity was poor, the villi were sparse, the knotting rate was high, and 2 cats had mild inflammation on the skin. After 21 days of the experiment, both the experimental groups I and II were energetic and lively, and the control group was still in the state before the experiment. In terms of fur status, the fur color gloss and smoothness of the experimental group I was good, the elasticity was good, the villi were abundant, the hair loss was improved, and there was no obvious knotting, but there was still mild inflammation on the skin of 1 cat. In the experimental group II, the fur had good glossiness and smoothness, good elasticity, abundant fluff, no obvious knots, and slight hair loss; the three cats with skin inflammation all recovered to normal. The control group still had poor fur glossiness and smoothness, poor elasticity, severe hair loss, and three cats had slight skin inflammation, one of which had dry scaly hair loss.

[0353] Table 2 Statistical results of mental and fur status

[0354]

[0355] Test Example 9: Cat breeding evaluation experiment using cat strips with added preparations

[0356] 9.1 Test subjects

[0357] Forty-four pet cats aged 9±1 months were selected. All the participating cats had oral and diarrhea problems to varying degrees. They were divided into experimental group I, experimental group II, experimental group III, and control group, with 11 cats in each group. There were 5 blue cats, 3 Siamese cats, and 3 American shorthair cats in each group. Normal oral treatment and health care were performed during the experiment.

[0358] 9.2 Test methods

[0359] The inactivated lactic acid bacteria and metabolite preparation 2-1000 were prepared using Example 12, and added to cat strips in different amounts to prepare three cat strips containing inactivated lactic acid bacteria and metabolites thereof. The experimental time was 28 days, including experimental group I (10 g of inactivated lactic acid bacteria and metabolite preparation 2-1000 was added to every 10 kg of cat strip contents), experimental group II (50 g of inactivated lactic acid bacteria and metabolite preparation 2-1000 was added to every 10 kg of cat strip contents), experimental group III (150 g of inactivated lactic acid bacteria and metabolite preparation 2-1000 was added to every 10 kg of cat strip contents) and a control group (inactivated lactic acid bacteria and metabolite preparation 2-1000 was not added to the cat strip contents, and the basic ingredients were the same as those of the experimental group). The feeding amount of cat strips was 10 g / cat / day. The diarrhea rate, feces score and dental plaque score of the test cats were calculated on the 0th, 5th and 28th days of the experiment, and the oral odor of the test cats was 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 had varying degrees of diarrhea, including 7 animals in group I, 6 animals in group II, 7 animals in group III, and 6 animals in the control group, with diarrhea rates of 64%, 55%, 64%, and 55%, respectively; on day 5 of the experiment, the diarrhea rates of the four groups were 18%, 9%, 0%, and 45%, respectively, indicating that group III had returned to normal; on day 28 of the experiment, the diarrhea rates of the four groups were 9%, 0%, 0%, and 45%, respectively, indicating that groups II and III had returned to normal, diarrhea in group I had significantly improved, and there was no significant change in the control group. Fig.40As shown, the fecal scores of the four groups on day 0 of the experiment were 2.3±0.9, 2.4±0.9, 2.2±0.8 and 2.4±0.7 respectively; the fecal scores of the four groups on day 5 of the experiment were 3.0±0.6, 3.2±0.6, 3.4±0.5 and ±0.7 respectively, among which the experimental group II (P<0.05) and the experimental group III (P<0.01) were significantly higher than the control group; the fecal scores of the four groups on day 28 of the experiment were 3.1±0.8, 3.2±0.4, 3.5±0.5 and 2.6±0.9 respectively, and the experimental group I (P<0.05), the experimental group II (P<0.05) and the experimental group III (P<0.01) were significantly higher than the control group. It can be seen that adding different amounts of inactivated lactic acid bacteria and their metabolite preparation 2-1000 to cat strips can significantly relieve diarrhea in pet cats, and the effect also shows a significant improvement trend with the increase of the added amount.

[0363] 9.3.2 Plaque scoring results:

[0364] Plaque score Fig.41 As shown, the dental plaque scores of the four groups on day 0 of the experiment were 2.25±0.40, 2.18±0.28, 2.23±0.44 and 2.20±0.47, respectively; the dental plaque scores of the four groups on day 28 of the experiment were 1.45±0.33, 1.27±0.31, 0.95±0.25 and 1.64±0.38, respectively. Among them, the dental plaque scores of experimental group II (P<0.05) and experimental group III (P<0.01) were significantly lower than those of the control group, and those of experimental group I were lower than those of the control group. It can be seen that after 28 days of the experiment, the dental plaque of experimental group III improved most significantly, and most of the experimental cats had only a small amount of thin plaque on their teeth. Regular evaluation of oral odor showed that the four groups of cats on day 0 of the experiment had severe bad breath, all with a strong fishy smell mixed with a corrupt smell, and most of them had saliva marks. After 28 days of the experiment, the oral cavity of the experimental group III was slightly fishy, ​​and there were no saliva marks; the oral cavity of the experimental group II was slightly fishy, ​​and one cat had saliva marks; the oral cavity of one cat in the experimental group I was fishy, ​​without corruption, and the rest of the cats had a slight fishy smell, and two cats had saliva marks; the 8 cats in the control group still had a fishy smell, of which 6 cats had corruption, and all 8 cats had saliva marks. It shows that adding different amounts of lactic acid bacteria inactivated bacteria and their metabolite preparations 2-1000 to cat strips can significantly reduce dental plaque scores, reduce oral odor, and improve the dental and oral health of pet cats, and the effect shows a significant improvement trend with the increase of the added amount.

[0365] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above are only specific implementation methods of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent substitutions, improvements, etc. made on the basis of the technical solutions of the present application should be included in the protection scope of the present application.

Claims

1. A Lactobacillus plantarum Ps-8, characterized in that The accession number is CGMCC No.5359.

2. A Lactobacillus acidophilus LA-14, characterized in that The accession number is CGMCC No.31624.

3. A Lactobacillus casei C-9, characterized in that The accession number is CGMCC No.6734.

4. A Bifidobacterium animalis subsp. lactis BB-16, characterized in that The accession number is CGMCC No.31623.

5. A postbiotic preparation for pets, characterized in that: The postbiotic preparation for pets comprises inactivated bacteria and their metabolites; wherein the bacteria are Lactobacillus plantarum Ps-8 according to claim 1, Lactobacillus acidophilus LA-14 according to claim 2, Lactobacillus casei C-9 according to claim 3, and Bifidobacterium animalis subspecies lactis BB-16 according to claim 4; wherein the ratio of the number of units of Lactobacillus plantarum Ps-8, Lactobacillus casei C-9, Bifidobacterium animalis subspecies lactis BB-16, and Lactobacillus acidophilus LA-14 is 1 to 5:1:1:1; the total amount of the bacteria is 3×10 10 ~1×10 11 cells / g.

6. The postbiotic preparation for pets according to claim 5, characterized in that: The raw materials of the postbiotic preparation for pets include an organic nitrogen source, an organic carbon source, a flavoring agent, a pH regulator and auxiliary materials; the organic nitrogen source and the organic carbon source are selected from one or more of whole fat soybean powder, skim milk powder, protein powder and yeast extract powder; the organic carbon source is selected from one or more of soybean powder, milk powder and protein powder; the pH regulator 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; the auxiliary materials are selected from one or more of maltodextrin and corn syrup.

7. The postbiotic preparation for pets according to claim 6, characterized in that: The raw materials of the postbiotic preparation for pets include 4 to 7 parts by weight of whole-fat soybean powder, 6.5 to 18 parts by weight of skim milk powder, 5 to 7 parts by weight of sucrose, 2.5 to 5 parts by weight of sodium citrate and 60 to 78 parts by weight of water.

8. The postbiotic preparation for pets according to claim 7, characterized in that: The preparation process of the postbiotic preparation for pets comprises the following specific steps: S1: Weigh the raw materials, add water and mix them, then dissolve and homogenize them; S2: cooling after sterilization to obtain a liquid feed; S3: adding bacteria and lactase to the above-mentioned liquid, and then fermenting at a constant temperature to obtain a fermentation liquid; S4: sterilization and inactivation, spray drying to obtain postbiotic powder; S5: The postbiotic powder is mixed with maltodextrin to obtain a postbiotic preparation.

9. The postbiotic preparation for pets according to claim 8, characterized in that The amount of lactase added in step S3 is 0.1-1.0 mL per kilogram of feed liquid; the constant temperature fermentation conditions are: constant temperature fermentation at 32-37°C to pH 4.5-4.6; wherein the total viable count of the bacteria after constant temperature fermentation is ≥2.0×10 10 CFU / g; the amount of maltodextrin added in step S4 is 18-25 g per kilogram of fermentation liquid; the sterilization and inactivation conditions are: inactivation at 75-95°C for 10-30 min.

10. Use of the postbiotic preparation for pets according to any one of claims 5 to 9 in the preparation of a product for improving diarrhea in cats and dogs.

11. Use of the postbiotic preparation for pets according to any one of claims 5 to 9 in the preparation of a product for alleviating the stress of food change for cats and dogs.

12. Use of the postbiotic preparation for pets according to any one of claims 5 to 9 in the preparation of a product for treating or improving dental plaque in cats and dogs.

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