A composite probiotic paste and a preparation method and application thereof

Abstract

The present application provides a kind of complex probiotic paste, which combines Enterococcus faecalis, Saccharomyces cerevisiae and vitamin B 12 Producing lactic acid bacteria of canine source, adds prebiotics, flavoring substances and auxiliary substances such as paste matrix preparation.In the paste, the viable count of Enterococcus faecalis, Saccharomyces cerevisiae and lactic acid bacteria is 1-2:1-2:1-8;Paste matrix includes fructooligosaccharide 0.5-3%, psyllium husk powder 0.1-2%, goat milk powder 2-8%, chicken liver powder 1-5%, potassium sorbate 0.5-2%, xanthan gum 1-4%, and the balance is water.The above-mentioned complex probiotic paste can be stably stored for 56 days at 4 DEG C and normal temperature dry place, has good storage stability, good safety, and can adjust intestinal flora and increase the content of intestinal lactic acid bacteria.The above-mentioned complex probiotic paste has good palatability and oral tolerance for pets such as dogs and cats, and has good therapeutic effect on DSS-induced chronic enteritis diarrhea and vitamin B 12 Deficiency.

Description

Technical Field

[0001] This invention relates to the field of probiotic preparation technology, and in particular to a compound probiotic paste, its preparation method, and its application. Background Technology

[0002] Chronic diarrhea in pets is characterized by intermittent or persistent diarrhea lasting 3-4 weeks, and sometimes exceeding two months. It is a common clinical condition in veterinary medicine, characterized by high incidence, long duration, and recurrent episodes. Clinical symptoms include frequent defecation, watery stools, and a strong, foul odor. Treatment for chronic diarrhea primarily involves symptomatic treatment targeting the underlying cause or dietary adjustments. Antibiotics are frequently used in symptomatic treatment; however, antibiotic overuse can lead to bacterial resistance, disrupting the balance of intestinal flora and interfering with the proliferation of beneficial bacteria.

[0003] Vitamin B 12 This typically represents a class of biologically active cobalt-containing coronallinoid compounds, essential trace elements for maintaining normal metabolism and physiological functions. Chronic diarrhea can lead to vitamin B deficiency in pets. 12 Absorption dysfunction leads to vitamin B 12 Deficiency; in addition, inflammatory bowel disease, pancreatic exocrine insufficiency, canine and feline lymphoma, and primary cobalamin malabsorption in dogs and cats can all lead to vitamin B deficiency. 12 Deficiency. Vitamin B 12 A deficiency can cause a variety of diseases, such as the accumulation of homocysteine ​​in the body leading to atherosclerosis, which is associated with methylmalonic acidemia and methylmalonic aciduria. It can also serve as a marker for gastrointestinal diseases and cause neurological disorders, seriously affecting the pet's life and quality of life. Clinically, vitamin B supplementation is often used to treat chronic diarrhea in pets. 12 Supplementing with Vitamin B 12 The traditional method is subcutaneous injection of vitamin B. 12 In addition, vitamin B can also be supplemented orally. 12 .

[0004] Probiotics are microorganisms that improve the balance of gut microbiota, thereby promoting host health. They can regulate gut microbiota balance and have a good effect on treating chronic gastrointestinal diseases in pets. In the treatment of chronic diarrhea in pets, administering probiotics can enhance the digestive function of affected pets, promote recovery, and reduce the likelihood of recurrence. Commonly available pet probiotics on the market include Lactobacillus, Enterococcus, Bifidobacterium, and Saccharomyces cerevisiae, and studies have shown that some probiotics can produce vitamin B. 12It has the ability to [promote / ensure], but its palatability is poor, and it is associated with chronic diarrhea and vitamin B [digestion / allergy]. 12 The effectiveness of treatment for the deficiency is still unclear.

[0005] In recent years, probiotic preparations have been used more and more widely, and pet food companies have begun to add them to pet diets. However, the conditions under which diets are produced can easily inactivate probiotics, resulting in poor efficacy. Currently, most probiotic pet products are available in capsule, tablet, oral liquid, and powder forms. Liquid probiotic preparations are prone to inactivation and mostly require storage at low temperatures; solid probiotic preparations have poor solubility, and freeze-drying requires the addition of antifreeze agents to prevent inactivation due to low temperatures, resulting in high production costs and often poor palatability. This limits the application of probiotic preparations for the prevention and treatment of chronic diarrhea in pets. Summary of the Invention

[0006] To overcome at least one problem in the existing technology, a strain that produces vitamin B has been isolated from dog feces in previous studies. 12 This invention utilizes canine-derived vitamin B1 to produce lactic acid bacteria. 12 Lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae were combined with prebiotics (such as fructooligosaccharides and psyllium husk), flavoring agents (such as goat milk powder and chicken liver powder), and auxiliary food additives (such as xanthan gum and potassium sorbate) to form a compound probiotic paste. The storage stability, palatability, and safety of the compound probiotic paste were evaluated, and its effects on dextran sulfate sodium (DSS)-induced chronic enteritis and diarrhea in mice and vitamin B1 were further verified. 12 Vitamin B deficiency has certain preventive and therapeutic effects, thus providing a basis for clinical application in the prevention and treatment of chronic diarrhea in pets and improving vitamin B levels. 12 The deficiency provides a theoretical basis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a compound probiotic paste comprising a compound probiotic and a paste matrix, wherein the compound probiotic comprises Enterococcus faecalis, Saccharomyces cerevisiae, and canine-derived vitamin B. 12 The ointment matrix contains lactic acid bacteria, and includes prebiotics, flavoring substances, and auxiliary substances.

[0009] Furthermore, the canine-derived vitamin B... 12The lactic acid bacteria is Lactobacillus reuteri, with accession number CGMCC NO.19126, accession date December 13, 2019, and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Furthermore, in the ointment, the live count ratio of Enterococcus faecalis, Saccharomyces cerevisiae, and Lactobacillus is 1–2:1–2:1–8; preferably, the live count ratio of Enterococcus faecalis, Saccharomyces cerevisiae, and Lactobacillus is 1:1:1. The above combination of probiotics has a synergistic effect, which can more effectively prevent and treat chronic diarrhea in pets.

[0011] Furthermore, the number of live bacteria in the compound probiotics in the ointment is 5 × 10⁻⁶. 8 ~5×10 9 CFU / g; preferably, the viable count of the compound probiotics in the ointment is 1×10⁻⁶. 9 CFU / g.

[0012] Furthermore, the prebiotics include fructooligosaccharides and psyllium husk powder, the flavoring substances include goat milk powder and chicken liver powder, and the auxiliary substances include potassium sorbate, xanthan gum, and deionized water.

[0013] The prebiotics used above can selectively stimulate the growth and activation of beneficial bacteria in the host's digestive tract. They mainly exert beneficial effects on the body by promoting the growth and metabolic activity of beneficial bacteria, regulating the intestinal flora, enhancing immunity, and promoting digestion and absorption, thereby improving the host's health. Among them, fructooligosaccharides can increase the number of beneficial bacteria in the pet's intestines and maintain the balance of the flora, while psyllium husk powder can change the number and types of intestinal microorganisms and enhance the body's non-specific immunity.

[0014] This invention combines probiotics and prebiotics to form a synbiotic microecological preparation, which has better effects than single probiotic ingredients, and the addition of prebiotics can extend the shelf life of probiotics. The added flavoring substances—milk powder and chicken liver powder—provide nutrition for pets and alleviate stress, while also increasing aroma appeal, effectively improving the palatability and tolerability of the probiotics. The added auxiliary ingredient, potassium sorbate, has a certain preservative effect, which, together with fructooligosaccharides, extends the shelf life of the ointment. Potassium sorbate, as a stabilizer and thickener, ensures the stability of the ointment's properties and improves the product's texture and taste. The added auxiliary ingredient, xanthan gum, has certain emulsification stability and suspending properties, effectively ensuring that the water and emulsion in the ointment do not separate and that there are minimal particles, making the ointment easier to extrude and more palatable for pets.

[0015] Further, by weight percentage, the ointment matrix comprises 0.5-3% fructooligosaccharides, 0.1-2% psyllium husk powder, 2-8% goat milk powder, 1-5% chicken liver powder, 0.5-2% potassium sorbate, 1-4% xanthan gum, and the balance being water; preferably, the ointment matrix comprises 1% fructooligosaccharides, 0.5% psyllium husk powder, 5% goat milk powder, 3% chicken liver powder, 1% potassium sorbate, 2% xanthan gum, and the balance being water.

[0016] Furthermore, the preparation method of the ointment includes the following steps:

[0017] S1. Preparation of bacterial suspension: Lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae are activated separately, and inoculated and cultured to obtain seed liquids of each bacteria; the seed liquids are inoculated and cultured, the culture liquids are centrifuged, the supernatant is discarded, and bacterial sludge is obtained; the bacterial sludge is resuspended to obtain bacterial suspensions of each bacteria.

[0018] S2. Preparation of ointment matrix: Mix oligofructose, psyllium husk powder, goat milk powder, chicken liver powder, potassium sorbate, xanthan gum, and deionized water in a predetermined ratio, heat and mix evenly, sterilize, and obtain an ointment matrix without probiotics.

[0019] S3. Preparation of probiotic ointment: The bacterial suspensions of lactic acid bacteria, Enterococcus faecalis and Saccharomyces cerevisiae obtained in step S1 are mixed evenly according to a predetermined ratio of live bacteria, and added to the ointment matrix prepared in step S2 for mixing, so that the probiotics are evenly distributed in the ointment matrix to obtain the compound probiotic ointment.

[0020] Furthermore, the above preparation method also includes step S4, packaging of the ointment: vacuum sealing the compound probiotic ointment and storing it in a cool, dry place at low temperature or at room temperature.

[0021] Further, in step S1, the preparation step of the seed liquid specifically includes: inoculating frozen lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae into MRS agar medium, subculturing and activating them twice, then picking single colonies and inoculating them into MRS liquid medium, and culturing them in a constant temperature incubator at 37°C for 24 hours to obtain the seed liquid of each bacterium.

[0022] Further, the MRS agar medium comprises: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 14.0 g / L agar, and 1.0 g / L Tween 80; the MRS liquid medium comprises: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80.

[0023] Further, in step S1, the preparation step of the fungal sludge specifically includes: inoculating the seed liquid into sterilized MRS liquid culture medium at a volume of 2vl%, incubating in a constant temperature shaking incubator for 24h, dispensing the culture medium into 30mL sterile centrifuge tubes, placing them in a low-speed refrigerated centrifuge, centrifuging at 2000r / min and 4℃ for 10min, discarding the supernatant, and obtaining the fungal sludge.

[0024] Furthermore, the preparation steps of the bacterial suspension specifically include: weighing the bacterial mud and adding it to sterilized 0.9% physiological saline at a ratio of 1:1, mixing it quickly and evenly with a shaker to prepare bacterial suspensions of each bacterium.

[0025] Furthermore, the preparation steps of the ointment matrix specifically include: heating and stirring the fructooligosaccharides, psyllium husk powder, goat milk powder, chicken liver powder, potassium sorbate, xanthan gum, and deionized water at 65-75°C until the texture is uniform and thick, and then pasteurizing at 70°C for 30 minutes to obtain the probiotic-free ointment matrix.

[0026] The second aspect of the invention is to provide the use of the compound probiotic paste described in any of the first aspects of the invention, selected from at least one of the following applications: in increasing the content of lactic acid bacteria in the pet's intestines; in improving the palatability and tolerability of pet food; in the preparation of a medicament for the prevention or treatment of chronic enteritis and diarrhea in pets; and in the preparation of a medicament for the prevention or treatment of vitamin B complex in pets. 12 Application in medications for deficiency syndromes.

[0027] Furthermore, the compound probiotic ointment can be used alone or in combination with a drug, including sulfasalazine.

[0028] Furthermore, the pets include dogs, cats, rabbits, rats, sheep, pigs, etc.

[0029] Furthermore, the compound probiotic ointment treats chronic enteritis diarrhea through at least one of the following mechanisms: reducing disease activity index, alleviating colonic damage (length, histopathology), reducing oxidative stress in colonic tissue (MDA, MPO, SOD, T-AOC), and reducing the relative expression of inflammatory factors (IL-6, IL-1β, TNF-α); the compound probiotic ointment treats vitamin B through at least one of the following mechanisms. 12 Deficiency: Increase serum vitamin B levels 12 It reduces the content of homocysteine ​​in serum.

[0030] Furthermore, for rabbits, the feeding amount of the compound probiotic paste is 1.25-2.5g / kg rabbit; for mice, the feeding amount of the compound probiotic paste is 0.1-0.2g / mouse.

[0031] Understandably, the above-mentioned vitamin B 12 Deficiency can be caused by chronic diarrhea or other factors, such as inflammatory bowel disease, pancreatic exocrine insufficiency, pet lymphoma, and primary cobalamin malabsorption in pets. The above-mentioned vitamin B... 12 Deficiencies include vitamin B 12 A deficiency and the various diseases caused by it, such as atherosclerotic diseases, methylmalonic acidemia, and nervous system diseases.

[0032] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0033] This invention involves compounding probiotics and optimizing the composition and ratio of the ointment matrix to produce a high-performance compound probiotic ointment. This enhances palatability for pets, increasing the convenience and compliance of medication administration. The aforementioned compound probiotic ointment exhibits good storage stability, safety, and palatability, and also demonstrates efficacy against chronic enteritis and diarrhea in mice and vitamin B1 deficiency. 12 The deficiency has a good therapeutic effect and can be used as a treatment for chronic diarrhea in dogs and vitamin B deficiency with compound probiotic paste. 12 It provides a theoretical basis for the deficiency syndrome.

[0034] (1) This invention investigated the shelf-life stability of the compound probiotic ointment. Fructooligosaccharides and potassium sorbate were added to the ointment formulation to extend its shelf life. The compound probiotic ointment exhibited good shelf-life stability, maintaining high viable cell levels at both room temperature and 4°C for 56 days. In particular, the compound probiotic ointment stored at 4°C showed strong shelf-life stability, with the viable cell count still reaching 1×10⁻⁶ after 56 days of storage. 8With a CFU / g content, minimal changes in color, odor, texture, and pH value, this compound probiotic ointment is easy to store and use for extended periods, ensuring its effectiveness.

[0035] (2) The safety of the compound probiotic ointment was evaluated in this invention. The test results showed that all indicators of the experimental rabbits in the low-dose and high-dose compound probiotic ointment groups were normal. The compound probiotic ointment had no effect on the liver and kidney function of the experimental rabbits and had good safety. It could also increase the content of lactic acid bacteria in the intestines of the experimental rabbits.

[0036] (3) The present invention evaluated the palatability of compound probiotic paste. The test results showed that the test dogs preferred dog food mixed with compound probiotic paste, and the preference was basically consistent within 5 days, indicating that compound probiotic paste has high palatability and tolerance for dogs; cats also have high palatability and tolerance for compound probiotic paste.

[0037] (4) This invention investigated the efficacy of compound probiotic ointment in treating chronic enteritis and diarrhea in mice. The compound probiotic ointment showed good therapeutic effects on DSS-induced chronic enteritis and diarrhea in mice. Both sulfasalazine and low-dose compound probiotic ointment had therapeutic effects on chronic enteritis and diarrhea in mice, and the combined treatment was more effective than the single treatment. The high-dose compound probiotic ointment group showed significantly better therapeutic effects than the sulfasalazine group and the low-dose compound probiotic ointment group. DSS-induced chronic colitis reduced serum vitamin B in mice. 12 Increase serum HCY levels. Compound probiotic paste is effective in treating vitamin B deficiency in mice. 12 It has a significant effect on blood disorders, and the combined treatment of high-dose compound probiotic ointment, sulfasalazine and low-dose compound probiotic ointment is more effective than using sulfasalazine or low-dose compound probiotic ointment alone. Attached Figure Description

[0038] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0039] Figure 1 This is a sensory score change curve of the compound probiotic ointment in one embodiment of the present invention;

[0040] Figure 2 This is a pH value change curve of the compound probiotic paste in one embodiment of the present invention;

[0041] Figure 3 This is a graph showing the change in the number of live bacteria in the compound probiotic ointment in one embodiment of the present invention;

[0042] Figure 4 This is a schematic diagram showing the results of lactic acid bacteria counting in rabbit feces after oral administration of compound probiotic paste in one embodiment of the present invention; wherein, part a is the MRS plate count of lactic acid bacteria; part b is the effect of oral administration of compound probiotic paste on the number of lactic acid bacteria in rabbit feces;

[0043] Figure 5 This is a schematic diagram showing the results of Escherichia coli count in rabbit feces after oral administration of compound probiotic paste in one embodiment of the present invention; wherein, part a is the Escherichia coli MacConkey plate count; part b is the effect of oral administration of compound probiotic paste on the number of Escherichia coli in rabbit feces;

[0044] Figure 6 This is a schematic diagram showing the effect of oral compound probiotic ointment on the liver and kidney function of healthy rabbits in one embodiment of the present invention; wherein, part a is serum alanine aminotransferase; part b is serum aspartate aminotransferase content; part c is serum urea nitrogen content; and part d is serum creatinine content.

[0045] Figure 7 This is a schematic diagram showing the results of a dog's first choice of a compound probiotic paste in an embodiment of the present invention; wherein, part a is the preferred quantity, representing which food the dog eats first; part b is the first approach quantity, which represents which food the dog sniffs first.

[0046] Figure 8 This is a schematic diagram showing the results of dogs' feeding on the compound probiotic paste in one embodiment of the present invention; wherein, part a is the feeding rate, which is the amount of food fed in the trial diet divided by the sum of the two feeding amounts; part b is the feeding coefficient, which is the feeding rate minus 0.5; and part c is the consumption rate, which is the amount of food fed divided by the amount of food fed in comparison.

[0047] Figure 9 This is a schematic diagram showing the results of cats' consumption of the compound probiotic paste in one embodiment of the present invention; wherein, eating represents the number of cats willing to actively consume the compound probiotic paste, and not eating represents the number of cats unwilling to actively consume the compound probiotic paste;

[0048] Figure 10 This is a schematic diagram showing the changes in mouse body weight after oral administration of a compound probiotic paste in one embodiment of the present invention;

[0049] Figure 11 This is a schematic diagram showing the results of mouse fecal observation and disease activity index after oral administration of compound probiotic paste in one embodiment of the present invention;

[0050] Figure 12 This is a schematic diagram of organ indices in mice after oral administration of a compound probiotic ointment in one embodiment of the present invention; wherein, part a is the spleen index; part b is the liver index; and part c is the kidney index.

[0051] Figure 13 This is a schematic diagram of the colon length in mice after oral administration of a compound probiotic paste according to an embodiment of the present invention; wherein, part a is a representative image of colon length; part b is the average colon length;

[0052] Figure 14 This is a schematic diagram of the small intestine length in mice after oral administration of a compound probiotic paste in one embodiment of the present invention; wherein, part a is a representative image of the small intestine length; part b is the average small intestine length;

[0053] Figure 15 This is a schematic diagram showing the results of pathological changes in mouse colon tissue after oral administration of compound probiotic ointment in one embodiment of the present invention; wherein, part a is a pathological section; part b is the histological score;

[0054] Figure 16 This is a schematic diagram showing the results of a mouse intestinal bacterial translocation experiment after oral administration of a compound probiotic paste in one embodiment of the present invention; wherein, part a shows the colony growth state of a homogenized coating of liver tissue; and part b shows the colony growth state of a homogenized coating of colon tissue.

[0055] Figure 17 This is a schematic diagram showing the results of colonic oxidative stress in mice after oral administration of a compound probiotic paste in one embodiment of the present invention; wherein, part a is the myeloperoxidase content; part b is the malondialdehyde content; part c is the total antioxidant capacity; and part d is the total superoxide dismutase content.

[0056] Figure 18 This is a schematic diagram showing the changes in the expression of pro-inflammatory factor mRNA in the colon of mice after oral administration of a compound probiotic ointment in one embodiment of the present invention; wherein, part a is the relative expression level of IL-6 mRNA in the mouse colon; part b is the relative expression level of IL-1β mRNA in the mouse colon; and part c is the relative expression level of TNF-α mRNA in the mouse colon.

[0057] Figure 19 In one embodiment of the present invention, serum vitamin B in mice after oral administration of a compound probiotic paste is shown to be... 12 Changes in homocysteine; where part a represents mouse serum vitamin B. 12 Content; Part b is the HCY content in mouse serum. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0059] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0060] Example 1 - Optimization of Ointment Matrix and Preparation of Compound Probiotic Ointment

[0061] In this embodiment, the components and their proportions of the ointment matrix were optimized and screened, and a compound probiotic ointment was prepared using the ointment matrix with the optimal proportions.

[0062] (1) Optimization of ointment matrix

[0063] 1) Screening of the main matrix of ointment

[0064] Accurately weigh sodium alginate, gelatin, and xanthan gum, and add equal amounts of fructooligosaccharides, psyllium husk powder, goat milk powder, chicken liver powder, potassium sorbate, and water to each. Heat and stir at 70°C for 5 minutes, mixing until homogeneous and thick. Cool to obtain sodium alginate cream, gelatin cream, and xanthan gum cream. [The mixture is then processed at 4000 rpm.] -1 Centrifuge at 15, 30, and 60 min to observe the layering phenomenon of the cream. Take an appropriate amount of cream and place it on a glass slide, cover it with a coverslip, and gently press it to distribute the particles evenly. Observe it under a 50-100x microscope and calculate the proportion of particles with a diameter greater than 100 μm in the field of view. The results show that xanthan gum cream has the lowest water separation rate at all time points and the lowest proportion of particles with a particle size greater than 100 μm. Therefore, xanthan gum was selected as the main matrix of the cream.

[0065] 2) Screening of prebiotics

[0066] To evaluate the effects of four different prebiotics—inulin, fructooligosaccharides, galactooligosaccharides, and psyllium husk powder—on canine-derived vitamin B12. 12 The effect of lactic acid bacteria will cause canine vitamin B to be produced. 12 After the lactic acid bacteria and the prebiotics sterilized by filtration membrane were prepared into an ointment, viable bacteria were counted after being stored at room temperature for 30 days. A probiotic ointment without prebiotics (Group C1) and a probiotic liquid containing fructooligosaccharides and psyllium husk powder (Group C2, prepared by conventional methods) were set up as controls. The prebiotic groups were further divided into: single prebiotic + lactic acid bacteria ointment groups (Groups A-D), fructooligosaccharides + psyllium husk powder + lactic acid bacteria ointment group (Group E), inulin + psyllium husk powder + lactic acid bacteria ointment group (Group F), and galactooligosaccharides + psyllium husk powder + lactic acid bacteria ointment group (Group G).

[0067] Experimental results showed that after 30 days, the viable bacteria count in the lactic acid bacteria ointment containing prebiotics was significantly higher than that in the control group C1, with the viable bacteria count ranking as follows: Group E > Group G > Group F > Groups A to D. This indicates that the combination of fructooligosaccharides and psyllium husk powder provides superior stability for the preservation of lactic acid bacteria, and the combination of prebiotic fructooligosaccharides, psyllium husk powder, and lactic acid bacteria exhibits a synergistic effect. Compared to the control group C2, the viable bacteria count in the ointment of Group E was significantly higher than that in the bacterial solution of Group C2, indicating that the preservation stability of the lactic acid bacteria ointment formulation is superior to that of the bacterial solution formulation.

[0068] 3) Optimization of the composition ratio of the ointment base

[0069]

[0070]

[0071] Each group of ointment bases in the table above was heated and stirred at 70℃ for 5 minutes until the texture was homogeneous and thick. After cooling, the ointment base was obtained. The appearance and color of each group of ointment bases were observed, and the odor of each group of ointment bases was compared. The particle size distribution, stability, moisturizing properties, heat resistance, low temperature resistance, and freeze resistance were determined according to conventional methods. The results showed that the ointment base of group A was the best formula. Its color was uniform and light yellow. After 3 days, there was almost no water precipitation and the surface was moist with virtually no cracks. It had a rich frankincense and meaty aroma, no sour or fishy smell, a fine and homogeneous texture, and good extensibility.

[0072] (2) Preparation of compound probiotic ointment

[0073] 1) Cultivation of bacterial strains and preparation of bacterial suspension

[0074] Canine-derived vitamin B stored at -80°C 12Lactic acid bacteria (preservation number: CGMCC NO.19126), Enterococcus faecalis (NJFC4003-1), and Saccharomyces cerevisiae (NJNJ4003-5) (all from the Animal Nutrition and Metabolic Disease Laboratory of Nanjing Agricultural University).

[0075] The bacteria were inoculated onto MRS agar medium, subcultured twice for activation, and then single colonies were picked and inoculated onto MRS liquid medium. The cultures were incubated at 37°C for 24 hours to obtain seed cultures of the three bacteria. These seed cultures were then inoculated again at 2% of sterilized MRS liquid medium and incubated at 37°C with shaking for 24 hours. The resulting cultures were then aliquoted into 30mL sterile centrifuge tubes and centrifuged at 4°C for 10 minutes using a low-speed refrigerated centrifuge (2000 rpm). The supernatant was discarded.

[0076] After weighing the bacterial sludge, add sterile physiological saline (0.9% concentration) at a ratio of 1:1. Mix quickly and evenly with a small rapid shaker to prepare a bacterial suspension. Take 2 mL of the bacterial suspension for counting (refer to the "Test Methods for Lactic Acid Bacteria" (GB4789.35-2016) for total colony count determination).

[0077] 2) Preparation of the paste matrix

[0078] Mix 1% fructooligosaccharides, 0.5% psyllium husk powder, 5% goat milk powder, 3% chicken liver powder, 1% potassium sorbate, 2% xanthan gum, and the remainder water (by weight) by heating and stirring (heating at 65-75℃ for 3-5 minutes) until the mixture is homogeneous and thick. Then pasteurize at 70℃ for 30 minutes to obtain a probiotic-free paste matrix.

[0079] 3) Preparation of compound probiotic paste

[0080] The canine-derived vitamin B obtained in step 1) 12 A suspension of lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae was uniformly mixed at a live count ratio of 1:1:1 and added to the emulsified ointment base. The mixture was stirred and mixed under aseptic conditions in a laminar flow hood until the consistency was homogeneous and thick, ensuring even distribution of the probiotics within the ointment base. The initial live count of the resulting compound probiotic ointment was 10. 9 CFU / g. The above-mentioned compound probiotic paste is sealed with a vacuum sealer and can be stored in a refrigerator at 4°C or in a cool, dry place at room temperature.

[0081] Example 2 - Evaluation of the storage stability of compound probiotic paste

[0082] This embodiment describes the compound probiotic paste prepared in Example 1 (with an initial viable count of 10). 9The storage stability of the compound probiotic ointment (CFU / g) was evaluated by storing the vacuum-sealed compound probiotic ointment in a refrigerator at 4°C and in a cool, dry place at room temperature for 56 days. Samples were taken at 0, 7, 14, 21, 28, 35, 42, 49 and 56 days for evaluation.

[0083] (1) Sensory evaluation

[0084] The appearance, color, odor, and texture of the compound probiotic paste were scored to evaluate its acceptability. Scores were assigned according to sensory evaluation standards, and the scores were summed to obtain a final sensory evaluation score. The three sensory evaluation indicators are: 1) Color: Scored based on the overall color of the compound probiotic paste. A uniform, light yellow color is scored as 20 points; a uniform, deep yellow color is scored as 10 points; an uneven color with alternating yellow and white areas is scored as 5 points. 2) Odor: The compound probiotic paste was stirred and smelled, and the odor was scored. A strong, rich milky or meaty aroma with no sour or fishy smell is scored as 20 points; no milky or meaty aroma with no sour or fishy smell is scored as 10 points; no milky or meaty aroma with a sour or fishy smell is scored as 5 points. 3) Texture: The probiotic paste was spread evenly with a glass rod, and the overall texture was observed and scored. A fine, uniform texture with good spreadability is scored as 20 points; an uneven texture with a grainy feel is scored as 10 points; an uneven texture with larger particles is scored as 5 points.

[0085] Samples were taken at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days to evaluate the appearance, color, texture, and odor of the compound probiotic ointment according to sensory evaluation standards. The experimental results are as follows: Figure 1 As shown, from 0 to 7 days, there were no significant changes in color, odor, and texture of the compound probiotic paste stored at both temperatures. After 14 days, the compound probiotic paste stored at room temperature gradually darkened in color, turning deep yellow, and the milky aroma faded, with small particles appearing in the paste. After 21 days, the compound probiotic paste stored at 4℃ showed a weaker aroma but no change in color, with the overall degree of change being less than that of the compound probiotic paste stored at room temperature. At 56 days, the compound probiotic paste stored at 4℃ lost its aroma and color, but the texture remained uniform. The results indicate that the compound probiotic paste stored at 4℃ exhibits better stability in terms of color, odor, and texture.

[0086] (2) pH value measurement

[0087] The pH value was determined in accordance with the National Food Safety Standard for Food (GB 5009.237-2016).

[0088] Take 1g of compound probiotic paste and mix it with an equal volume of distilled water at room temperature by shaking at 180 rpm for 10 minutes. Calibrate the pH meter with a buffer solution of known accurate pH value. Insert the calibrated pH meter into the sample to be tested. After the reading stabilizes, accurately measure the pH value. The same sample should be measured at least three times.

[0089] Samples were taken at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days to determine the pH of the compound probiotic paste. The experimental results are as follows: Figure 2 As shown, during the 56-day storage period, the pH values ​​of the compound probiotic paste stored at 4℃ and room temperature changed from 6.0 to 4.67 and 4.33, respectively, with the change being smaller at 4℃. The results indicate that the compound probiotic paste exhibits good pH stability at both room temperature and 4℃, with better pH stability at 4℃.

[0090] (3) Viable bacteria count

[0091] Take 1g of the compound probiotic paste sample, add 3-5 sterile glass beads and 10mL of sterile water, shake and mix at room temperature for 10min, and then serially dilute to 10. -5 10 -6 10 -7 and 10 -8 Four concentrations. Using a pipette, pipette 100 μL of the diluted sample, shaken and mixed, and place it in the center of an MRS solid agar plate. Sterilize a sterile glass spreader over an alcohol flame, cool for 30 seconds, and then spread the bacterial suspension evenly on the plate, being careful not to touch the edges. Incubate at 37°C for approximately 48 hours. Each sample should be tested on at least three plates. The effective count range is 30-300 colonies. Colonies should be round, white, and surrounded by a clear halo; those not conforming to these morphological characteristics are considered contaminants. Judgment criteria: Each dilution sample must be tested on at least three plates, with uniform colonies on each plate, and an effective count range of 30-300 colonies.

[0092] Lactic acid bacteria count per mL of sample = average colony count per plate × dilution factor × 10 CFU

[0093] Experimental results: Samples were taken at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days to count the number of live bacteria per gram of the compound probiotic paste. The experimental results are as follows: Figure 3 As shown, the number of live bacteria per gram of the compound probiotic paste stored at room temperature decreased slowly from 0 to 14 days, began to decrease rapidly from 14 days, and reached its lowest point at 49 days. By 56 days, the number of live bacteria per gram of the compound probiotic paste had decreased to 1×10⁻⁶. 6 CFU / g. The number of live bacteria per gram of the compound probiotic paste stored at 4℃ showed a slow decline throughout the entire process, but still maintained a high number of live bacteria (1×10⁻⁶) after 56 days. 8 (CFU / g). The results showed that the viable count of the compound probiotic paste was stable at both room temperature and 4℃, with better stability at 4℃.

[0094] The experimental results in this embodiment show that, within a shelf life of 56 days, the viable bacteria count of the compound probiotic paste stored at 4°C and room temperature increased from 1×10⁻⁶. 9 CFU / g decreased to 1×10 8 CFU / g, 1×10 6 The CFU / g and pH values ​​changed from 6.0 to 4.67 and 4.33, respectively. The probiotic paste stored at 4℃ had a uniform texture and a weaker aroma, while the compound probiotic paste stored at room temperature had a darker color and a weaker milky aroma. This indicates that the compound probiotic paste has good stability at both room temperature and 4℃, with better stability at 4℃. The above-mentioned compound probiotic paste exhibits good storage stability, maintaining high levels of viable bacteria at both room temperature and 4℃ for 56 days. In particular, the compound probiotic paste stored at 4℃ showed strong storage stability, with minimal changes in color, odor, texture, and pH value, facilitating long-term storage and use and ensuring the effectiveness of the compound probiotic paste.

[0095] Example 3 - Safety Evaluation of Compound Probiotic Paste

[0096] This embodiment evaluates the safety of oral compound probiotic paste, including the following steps:

[0097] Nine healthy New Zealand rabbits, all from Nanjing Qinglongshan Animal Farm, weighing 2 ± 0.25 kg, were used for 7 days of acclimatization, with free access to water and food. The rabbit hutch was cleaned daily to maintain ventilation and cleanliness. To ensure the rabbits remained healthy for one week, the nine rabbits were randomly divided into three groups: a control group (Control), a low-dose probiotic paste group (Pas-L), and a high-dose probiotic paste group (Pas-H). The control group was fed normal rabbit pellets daily. The low-dose probiotic paste group was fed normal rabbit pellets supplemented with 1.25 g / kg of probiotic paste daily, while the high-dose group was supplemented with 2.5 g / kg of probiotic paste daily for 14 days. The rabbit pellets fed before and after the experiment remained the same.

[0098] Blood and fecal samples were collected on days 0, 7, and 14. One mL of blood was collected from the marginal ear vein of each rabbit. The blood samples were allowed to stand for 2 hours to coagulate, then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant serum was separated and stored at -80°C for later use. Simultaneously, rabbit feces were collected using EP tubes and sent to a clean bench for fecal microbiota counting.

[0099] (1) Effects on the physical condition of experimental rabbits

[0100] During the experimental observation period, compared with the blank control group, the rabbits in the low-dose probiotic paste group and the high-dose probiotic paste group had normal diet and water intake, body condition and no diarrhea or soft stools, indicating that oral probiotic paste does not affect the physical condition of healthy rabbits.

[0101] (2) Fecal microbiota count in experimental rabbits: Accurately weigh 1g of feces, place it in a 10mL EP tube, add 9mL of sterile physiological saline, and shake thoroughly to mix. After mixing the sample thoroughly, perform serial dilution with sterile physiological saline. Dilute to 10... -1 10 -2 10 -3 10 -4 For each dilution, take 100 μL of sample and spread it evenly on agar plates, avoiding the edges. Spread each dilution onto three MRS agar plates (anaerobic for 48 h) and three MacConkey agar plates (aerobic for 24 h), and incubate at 37°C. MRS agar is used for the isolation and identification of lactic acid bacteria; effective colonies should be white, smooth, raised, and round. MacConkey agar is used for the isolation and identification of Escherichia coli and Salmonella; effective colonies should be red, moist, raised, and medium to large. The effective colony count range is 30-300. The average effective colony count is taken as the number of lactic acid bacteria and Escherichia coli in rabbit feces.

[0102] The effects of oral compound probiotic paste on lactic acid bacteria in the rabbit gut, such as Figure 4 As shown, compared with the control group, there was no significant difference in the number of lactic acid bacteria in the feces of rabbits in the low-dose probiotic paste group (Pas-L) and the high-dose probiotic paste group (Pas-H) on day 0 (P>0.05). On day 7, the number of lactic acid bacteria in the feces of rabbits in both the Pas-L and Pas-H groups significantly increased (P<0.01). On day 14, the number of lactic acid bacteria in the feces of rabbits in both the Pas-L and Pas-H groups significantly increased (P<0.01), and the number of lactic acid bacteria in the feces of rabbits in the Pas-L group on day 14 was significantly higher than that on day 7. This indicates that oral administration of compound probiotic paste can increase the content of lactic acid bacteria in the rabbit's intestines, and the high-dose compound probiotic paste is more effective, reaching a relatively high level on day 7, while the low-dose probiotic paste group only reached a relatively high level on day 14.

[0103] The effect of oral compound probiotic paste on the number of Escherichia coli in the rabbit intestine, such as Figure 5 As shown, on days 0, 7, and 14 after oral administration of the compound probiotic paste, there was no significant difference in the number of *E. coli* in the feces of rabbits in the low-dose probiotic paste group (Pas-L) and the high-dose probiotic paste group (Pas-H) compared to the blank control group (Control). (P>0.05) This indicates that oral administration of the compound probiotic paste does not increase the number of *E. coli* in the rabbit intestine.

[0104] (3) Determination of liver and kidney indicators in rabbit serum: The contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are liver function indicators, and blood urea nitrogen (BUN) and creatinine (CREA), which are kidney function indicators, were determined in the serum samples of experimental rabbits using Nanjing Jiancheng test kits to evaluate the effects of compound probiotic ointment on the liver and kidney function of rabbits.

[0105] On days 0, 7, and 14 of the experiment, blood was collected from the marginal ear vein of each rabbit. After the blood clotted, serum was collected, and the biochemical parameters of the rabbit serum were measured. Figure 6 As shown, during days 0, 7, and 14 of the experiment, there were no significant differences (P>0.05) in the serum levels of ALT, AST, BUN, and CREA in the blank control group, the low-dose compound probiotic paste group, and the high-dose compound probiotic paste group, and all of the above indicators were within the normal range. This result indicates that oral administration of compound probiotic paste did not affect the liver and kidney function indicators of healthy rabbits.

[0106] In the experiments of this embodiment, compared with the Control group, the mental state, body condition, and food and water intake of rabbits in the Pas-L and Pas-H groups were normal; serum biochemical indicators such as ALT, AST, BUN, and CREA showed no significant changes; the number of Escherichia coli in rabbit feces also showed no significant changes; on days 7 and 14 of the experiment, compared with the Control group, the number of lactic acid bacteria in the feces of rabbits in the Pas-L and Pas-H groups was significantly increased; on day 14 of the experiment, the number of lactic acid bacteria in the feces of rabbits in the Pas-L group was significantly higher than that on day 7. This indicates that all indicators of rabbits in the low-dose and high-dose compound probiotic paste groups were normal, and the compound probiotic paste had no effect on the liver and kidney function of rabbits, demonstrating good safety. In addition, oral administration of different doses of compound probiotic paste can increase the content of lactic acid bacteria in the rabbit intestine.

[0107] Example 4 - Palatability Evaluation of Compound Probiotic Paste

[0108] This embodiment evaluates the palatability of the compound probiotic paste using a two-basin test and a single-basin test. Palatability refers to the pet's perception when ingesting food, including its perception of appearance, smell, taste, and texture. The experimental steps include:

[0109] (1) Nine healthy dogs were selected, including Chinese rural dogs and beagles; and nine healthy cats were selected, including Chinese rural cats, British Shorthairs, and Ragdolls. All dogs and cats were from the teaching animal hospital of Nanjing Agricultural University. They were allowed to adapt for 7 days, with free access to water and food. The kennels and cat cages were cleaned daily to ensure cleanliness, ventilation, and a suitable temperature.

[0110] (2) Canine palatability test

[0111] Nine healthy dogs were selected and pre-fed for one week to ensure normal appetite and energy levels and no signs of stress before the experiment. The experiment lasted for 5 days, with two food bowls provided each morning. Bowl A contained 100g of dog food, and Bowl B contained 95g of dog food mixed with 5g of compound probiotic paste, thoroughly mixed. The nine dogs were fed sequentially each day, and their eating behavior was observed. The order in which the food was approached and consumed was recorded. The food bowls were left with the dogs for 15 minutes, or until one bowl was completely empty. The amount of food provided in each bowl met the dogs' daily calorie intake. The amount of food consumed from both bowls was recorded, and the total amount consumed was calculated to determine the food intake ratio. During the experimental phase, the left and right bowls were switched to reduce directional bias errors. Animal husbandry was strictly carried out in accordance with the relevant requirements of the "Guidelines for the Care and Use of Laboratory Animals".

[0112] Record relevant indicators, including First choice (FC), First approach (FA), and the amount of food consumed in each of the two bowls and the total amount of food consumed. Calculate the feeding rate (FR), feeding coefficient factor (FCF), and consumption rate (CR).

[0113] Throughout the experiment, the dogs' daily food intake, physical condition, mental state, and appetite remained normal. Figure 7 As shown in Part a, on day 1, 6 dogs chose to consume the compound probiotic paste first; on days 2, 4, and 5, 8 dogs chose to consume the compound probiotic paste first; and on day 3, 7 dogs chose to consume the compound probiotic paste first. Figure 7 As shown in Part b, on days 1, 2, 3, and 4, 7 dogs chose to approach the dog food containing the compound probiotic paste first, and on day 5, 8 dogs chose to approach the dog food containing the compound probiotic paste first. These results indicate that the palatability and tolerability of dog food with the added compound probiotic paste are significantly higher than those of dog food without the added compound probiotic paste.

[0114] The compound probiotic paste was fed to the experimental dogs for 5 consecutive days, and their food intake rate, food intake coefficient, and consumption rate were recorded. Figure 8 As shown, within 5 days of the experiment, the dogs' intake rate, intake coefficient, and consumption rate of the mixed probiotic paste dog food were significantly higher than those of ordinary dog ​​food (P<0.05). These results indicate that the experimental dogs preferred dog food containing the mixed probiotic paste, and this preference remained largely consistent over 5 days, suggesting that the mixed probiotic paste has high palatability and tolerability.

[0115] (3) Cat palatability test

[0116] Nine healthy cats were selected and pre-fed for one week to ensure they had normal appetite and energy levels and showed no signs of stress before the experiment. The experiment lasted 5 days, during which all nine cats were provided with ample food and water to prevent hunger. A compound probiotic paste was administered daily, and the cats' feeding behavior was observed. The probiotic paste was fed to the cats for 5 consecutive days, and feeding behavior and the percentage of cats consuming the probiotic paste were recorded. The percentage of cats actively consuming the probiotic paste was determined based on the number of cats that chose to consume it. Animal husbandry was strictly carried out in accordance with the relevant requirements of the "Guidelines for the Care and Use of Laboratory Animals".

[0117] Throughout the experiment, the cats' physical condition, mental state, and appetite remained normal. To evaluate the cats' preference for the compound probiotic paste, the number of cats that chose to consume the compound probiotic paste daily was recorded, and their proportion was calculated. Figure 9 As shown, on days 1 and 2, 6 cats chose to consume the compound probiotic paste, representing 66.67% of the cats who actively chose to consume it; on days 3, 4, and 5, 7 cats chose to consume the compound probiotic paste, representing 77.78% of the cats who actively chose to consume it. The results indicate that the compound probiotic paste also has high palatability and tolerability for cats.

[0118] In the various experiments of this embodiment, the results of the canine double-basin test showed that, within the 5-day test period, dogs showed significantly higher preferred quantity, closest quantity, consumption rate, consumption coefficient, and consumption rate for the mixed probiotic paste dog food compared to ordinary dog ​​food. The results of the cat single-basin test showed that, within the 5-day test period, 6 cats chose to consume the mixed probiotic paste on 2 days, representing 66.67% of the cats actively choosing to consume it; and 7 cats chose to consume it on 3 days, representing 77.78% of the cats actively choosing to consume it. The experimental dogs showed a greater preference for dog food mixed with the mixed probiotic paste, and this preference remained largely consistent within 5 days, indicating that the mixed probiotic paste has high palatability and tolerability for dogs. Furthermore, cats also showed high palatability and tolerability for the mixed probiotic paste. These results indicate that the mixed probiotic paste has good palatability and tolerability for both dogs and cats, providing conditions for its clinical application in pets.

[0119] Example 5 - Evaluation of the therapeutic effect of compound probiotic ointment on mice with chronic enteritis and diarrhea

[0120] This embodiment uses sodium dextran sulfate (DSS, 36000-50000 Da, Yisheng Biotechnology (Shanghai) Co., Ltd.) to induce and establish a chronic enteritis and diarrhea C57BL / 6 mouse model, and explores the therapeutic effect of compound probiotic ointment on chronic enteritis and diarrhea mice, including the following steps:

[0121] Thirty-six healthy male C57BL / 6 mice, all from the Comparative Medicine Center of Yangzhou University, weighing 19±2g, were used. Mouse food and corn cob bedding were purchased from Nanjing Qinglongshan Animal Farm. The mice were allowed free access to water and food for 7 days of acclimatization. The drinking water was pressurized purified water, which was changed daily. Bedding was changed every two days to maintain cleanliness, ventilation, and a suitable temperature in the cages. The 36 C57BL / 6 mice were randomly divided into 6 groups: a blank control group (CON group), a dextran sulfate sodium model group (DSS group), a sulfasalazine treatment group (SA group, sulfasalazine purchased from Shanghai Yuanye Biotechnology Co., Ltd.), a low-dose compound probiotic ointment treatment group (L group), a combined treatment group of sulfasalazine and low-dose compound probiotic ointment (SA+L group), and a high-dose compound probiotic ointment treatment group (H group).

[0122] Six mice were used in each group. The blank control group was given pressurized purified water throughout the entire treatment. The other five groups (excluding the blank control group) were given 3% DSS solution for 4 consecutive days, followed by a 3-day interval, and then another 4 consecutive days of DSS solution administration. This cycle was repeated for 4 weeks. At the beginning of the third week, the experimental group was given the drug once daily. Specific treatments were as follows: The sulfasalazine treatment group and the sulfasalazine and low-dose compound probiotic ointment combination treatment group received 0.5 g / kg of sulfasalazine by gavage daily. The sulfasalazine and low-dose compound probiotic ointment combination treatment group and the low-dose compound probiotic ointment treatment group were fed 0.1 g of compound probiotic ointment daily, while the high-dose compound probiotic ointment treatment group was fed 0.2 g of compound probiotic ointment daily. The blank control group and the dextran sulfate sodium model group received the same volume of sterilized purified water by gavage daily. Animal husbandry was strictly carried out in accordance with the relevant requirements of the "Guidelines for the Care and Use of Laboratory Animals".

[0123] (1) Observation and scoring of experimental mice

[0124] During the experiment, the mice's condition was observed and recorded every 2 days, including their food and water intake, weight, fecal viscosity, and whether their feces contained blood. The Disease Activity Index (DAI) was calculated based on these indicators (Veenstra JP et al., 2021). The scoring criteria were as follows: (1) Weight: 0 points for a decrease of 1% or less; 1 point for a decrease of 1%–5%; 2 points for a decrease of 6%–10%; 3 points for a decrease of 11%–15%; and 4 points for a decrease of more than 15%. (2) Fecal viscosity: 0 points for normal; 2 points for loose stools; and 4 points for diarrhea. (3) Blood in stool: 0 points for normal; 2 points for occult blood; and 4 points for obvious blood in stool. Occult blood was detected using a fecal and urine occult blood test kit after collecting mouse feces.

[0125] 1) Mouse body weight changes: Starting from day 0, mouse body weight was measured and recorded every 2 days, and a line graph was plotted. The results are as follows: Figure 10 As shown, the body weight of the six groups of mice increased to varying degrees from 0 to 4 days. Starting from day 6, compared to the blank control group (CON group), the body weight of mice in the DSS group, SA group, SA+L group, L group, and H group, which drank 3% DSS solution, began to decrease, reaching its lowest point at day 16. These results indicate that the DSS model was successfully established. On day 18 of the experiment (4 days after treatment), compared to the DSS group, the body weight of mice in the SA group, SA+L group, L group, and H group all showed varying degrees of increase. On day 28 of the experiment, compared to the CON group, the body weight of mice in the DSS group, SA group, SA+L group, L group, and H group was significantly decreased (P<0.01). Compared to the DSS group, the body weight of mice in the SA group, SA+L group, L group, and H group was significantly increased (P<0.01). Compared with group H, the body weight of mice in groups SA, SA+L, and L was significantly reduced (P<0.01), while there was no significant difference in body weight among groups SA, SA+L, and L (P>0.05). These results indicate that feeding mice with sulfasalazine and compound probiotic paste has a therapeutic effect on enteritis and diarrhea. The high-dose probiotic paste showed better therapeutic effects than sulfasalazine, low-dose compound probiotic paste, or the combination of sulfasalazine and low-dose compound probiotic paste.

[0126] 2) Disease Activity Index (DAI) in Mice: The Disease Activity Index (DAI) is an important indicator for evaluating the severity of colitis. DAI is calculated by observing and scoring mice every two days on factors such as weight loss, fecal viscosity, and the presence of blood in the feces, and then summing the scores. Observation of fecal viscosity in mice revealed (see fecal viscosity assessment results)... Figure 11 (Part a) In the CON group, the stool shape of mice remained normal throughout. However, the stool of mice in the DSS group, SA group, SA+L group, L group, and H group, which drank 3% DSS solution, gradually became softer and more viscous from day 2 onwards, eventually becoming unformed. After treatment, the soft stool and diarrhea in all four groups of mice improved. Blood was detected in the mouse stool (see [link to mouse stool blood assessment results]). Figure 11 (Part a) In the CON group, mice never showed any bloody stools. In the DSS, SA, SA+L, L, and H groups, the stools gradually progressed from no bleeding to occult blood, and finally to visible blood in the stool. After treatment, the bloody stools in all four groups improved to varying degrees, and the number of mice with bloody stools and occult blood decreased.

[0127] like Figure 11As shown in section b, the experimental days are plotted on the x-axis, and the DAI score of each group of mice is plotted on the y-axis. Compared with the CON group, the DAI of mice in the DSS, SA, SA+L, L, and H groups increased rapidly, with a brief decrease on day 6, followed by rapid increase from day 6 to day 14. After treatment on day 15, compared with the DSS group, the DAI of mice in the H group showed a stable and slow downward trend, while the DAI growth rate of mice in the SA, SA+L, and L groups also slowed down or even decreased to varying degrees. On day 28, compared with the CON group, the DAI of mice in the DSS, SA, SA+L, L, and H groups was significantly increased (P<0.01). Compared with the DSS group, the DAI of mice in the SA, SA+L, L, and H groups was significantly decreased (P<0.05, P<0.01). There were significant differences in DAI between group H mice and SA mice (P<0.05), and significant differences in DAI between group SA mice and SA+L mice (P<0.05). These results indicate that feeding sulfasalazine and compound probiotic ointment both have therapeutic effects on mice with enteritis and diarrhea. The high-dose compound probiotic ointment was more effective than sulfasalazine. The combined use of sulfasalazine and low-dose compound probiotic ointment was more effective than sulfasalazine alone.

[0128] (2) Collection of blood and tissue samples from experimental mice

[0129] Samples were collected on day 28 of the experiment, including mouse blood and tissue samples. Hemolysis was avoided during blood collection. After collection, the blood samples were left to stand at room temperature for 2 hours to allow coagulation. Then, they were centrifuged at 4°C and 4000 rpm for 20 minutes. The supernatant serum was collected and stored at -80°C for later use. After blood collection, the mice were euthanized by cervical dislocation. The colon and small intestine segments were removed and washed in 0.9% saline solution. The colon and small intestine were placed on a ruler to measure their length. A portion of the colon was fixed in 4% paraformaldehyde fixative for subsequent pathological tissue sectioning. Additionally, the mouse liver, kidney, and spleen were removed, weighed, and their organ indices were calculated. The formula for calculating the organ indices is as follows:

[0130] Organ Index = Organ wet weight (mg) / Mouse body weight (g) × 100%

[0131] like Figure 12 As shown in section a, compared with the CON group, the spleen index of the DSS group, SA group, and L group were significantly increased (P<0.01). Compared with the DSS group, the spleen index of the SA+L group and H group were significantly decreased (P<0.01). This indicates that DSS causes spleen damage, and the combined use of sulfasalazine and low-dose compound probiotic ointment, as well as high-dose compound probiotic ointment, can alleviate this damage. Figure 12 As shown in section b, compared with the CON group, the liver index of the DSS group and SA group was significantly increased (P<0.05, P<0.01), while there was no significant difference between the SA group, L group, and SA+L group. This indicates that DSS causes liver damage, and the compound probiotic ointment can alleviate this damage. Figure 12 As shown in section c, compared with the CON group mice, there were no significant differences in the kidney index among the DSS group, SA group, SA+L group, L group, and H group mice (P>0.05). This indicates that DSS, sulfasalazine, and the compound probiotic ointment have no kidney-damaging effects.

[0132] (3) Preparation of colonic histopathological sections from experimental mice

[0133] 1) Tissue Collection and Fixation: Take 1 cm of colon tissue and preserve it in 5 mL of 4% paraformaldehyde fixative. 2) Dehydration: Replace the water in the tissue with ethanol of different concentrations. 3) Clearing: Clear the tissue twice with xylene, 8 min each time. 4) Paraffin Infiltration and Embedding: Immerse the tissue in molten paraffin for 3 hours, changing the paraffin solution three times to ensure thorough penetration. Then place the tissue in an embedding holder containing molten paraffin. 5) Sectioning and Slide Preparation: Place the paraffin block in a microtome, setting each slide to a thickness of 4 μm. Lay the sectioned tissue slides flat on a glass plate, quickly add 30% ethanol solution to allow the slides to expand, transfer to 40°C warm water, and bake in a 60°C oven for 30-60 min after the slides have fully expanded. 6) HE Staining: Dewax twice with xylene, 10 min each time, then wash with anhydrous ethanol and running water. Stain with hematoxylin for 4 min, then wash with water for 2 min. Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with water for 2 minutes. Reverse blue with 1% dilute ammonia water, then wash with water for 2 minutes. Stain with eosin for 90 seconds, then dehydrate with ethanol for 5 minutes. Wash twice with xylene, 10 minutes each time. 7) Mounting and observation: Mount with neutral resin and observe tissue changes under an optical microscope.

[0134] Colon length: On day 28 of the experiment, colon tissue samples were collected from mice. The colonic segment was removed, and the adipose tissue on the colonic surface was removed, followed by washing in 0.9% saline solution. The colon was placed on a ruler to measure its length and observed. Results are as follows: Figure 13 As shown in section a, compared to the CON group, the DSS group mice had shorter colons, with visible redness and swelling. To more clearly illustrate the differences in colon length between the groups, a graph was plotted with the group number on the x-axis and the colon length of each group on the y-axis. Figure 13(Part b). The results showed that, compared with the CON group, the colons of mice in the DSS, SA, SA+L, L, and H groups were significantly shortened (P<0.05, P<0.01). Compared with the DSS group, the colon length of mice in the SA+L and H groups was significantly increased (P<0.01), while there was no significant difference in colon length between the SA and L groups and the DSS group (P>0.05). These results indicate that drinking DSS solution causes colonic damage and shortening of colon length, and that sulfasalazine and compound probiotic ointment are both effective in treating DSS-induced colonic shortening in mice. Furthermore, the combined use of sulfasalazine or low-dose compound probiotic ointment, and high-dose compound probiotic ointment, resulted in better therapeutic effects.

[0135] Small intestine length: On day 28 of the experiment, mice were euthanized by cervical dislocation, and small intestine tissue samples were collected. The small intestine segment was removed and washed in 0.9% saline solution. The small intestine was placed on a ruler to measure its length. Results are as follows: Figure 14 As shown in section a, compared to the CON group, the small intestine length of mice in the five groups that drank 3% DSS solution was shortened. To more clearly illustrate the differences in small intestine length among the groups, a graph was plotted with the group number on the x-axis and the small intestine length of each group on the y-axis. Figure 14 As shown in section b, compared with the CON group, the DSS group showed a significant reduction in small intestinal length, while there were no significant differences among the SA, SA+L, L, and H groups. These results indicate that drinking DSS solution causes small intestinal damage and shortening of the small intestine, and that sulfasalazine and the compound probiotic ointment both have therapeutic effects on DSS-induced small intestinal shortening in mice.

[0136] (4) Histopathological scoring of colon tissue in experimental mice

[0137] Examine the slides under a microscope, and randomly select three fields of view for each slide. Score each field of view, and calculate the colonic histopathological score by multiplying the four scores (Cooper HS et al., 1993).

[0138] Colonic histopathological score = Inflammation score × Depth of injury score × Recess injury score × Extent of inflammation score

[0139] The scoring criteria for colonic histopathology are as follows: 1) Inflammation severity score: 0 points for no inflammation, 1 point for mild inflammation, 2 points for moderate inflammation, and 3 points for severe inflammation. 2) Lesion depth score: 0 points for no lesion, 1 point for lesion involving the colonic mucosa, 2 points for lesion involving the submucosa, 3 points for lesion involving the muscularis mucosa, and 4 points for lesion involving the serosa. 3) Cun destruction score: 0 points for no cun damage, 1 point for damage to 1 / 3 of the cun, 2 points for damage to 2 / 3 of the cun, 3 points for damage to all cun, and 4 points for damage to all cun and epithelium. 4) Inflammation extent score: 0 points for no lesion, 1%–25% of the lesion area is 1 point, 26%–50% is 2 points, 51%–75% is 3 points, and 76%–100% is 4 points.

[0140] Colonic tissue sections were prepared from each group of mice. Hematoxylin-eosin staining was performed, and the sections were observed using a tissue scanner. The results are as follows: Figure 15 As shown in section a, the colonic tissue of the CON group mice was intact, the crypt structure was complete, the thickness of the mucosa and muscularis propria was normal, there was no loss of goblet cells, and no inflammatory cell infiltration was observed. Compared with the CON group mice, the DSS, SA, SA+L, L, and H groups mice had more severe colonic inflammation, with a large number of inflammatory cell infiltrations, reduced goblet cells, damaged crypt structure, and thinning of the mucosa and muscularis propria. Compared with the DSS group mice, the pathological changes in the colonic tissue of the four treatment groups were alleviated, with reduced inflammation, fewer inflammatory cells, crypt structure closer to normal, and intact intestinal wall tissue. To better compare the pathological damage of the colonic tissue in the experimental mice, a score was calculated, with the group as the x-axis and the histopathological score as the y-axis. The results are as follows: Figure 15 As shown in section b, compared with the CON group, the histopathological scores of colon tissue in mice in the DSS group, SA group, SA+L group, L group, and H group were significantly increased (P<0.01). Compared with the DSS group, the histopathological scores of colon tissue in mice in the SA group, SA+L group, L group, and H group were significantly decreased (P<0.05, P<0.01), indicating that both sulfasalazine and compound probiotic ointment can improve the histopathological damage of colon tissue in mice, and the effect of compound probiotics is better than that of sulfasalazine.

[0141] (5) Experimental mouse liver and colon bacterial translocation test

[0142] To investigate bacterial translocation in the mouse intestine, the liver and colon of mice were aseptically removed. An appropriate amount of sterile physiological saline was added at a ratio of 1:1 (m / v) of tissue to physiological saline. After homogenization with a tissue homogenizer, 100 μL of the homogenate from each tissue was taken and spread on the surface of MacConkey agar using a sterile spreader. The mixture was incubated at 37°C for 24 h to observe whether there was bacterial growth.

[0143] The liver and colon were aseptically removed, and an appropriate amount of sterile saline was added at a tissue-to-saline ratio of 1:1 (m / v). After homogenization using a tissue homogenizer, 100 μL of the homogenate was taken from each tissue and spread onto MacConkey agar using a sterile spreader. The mixture was incubated at 37°C for 24 hours. The liver homogenate results are shown below. Figure 16 As shown in section a, colonies grew on MacConkey agar in the DSS and L groups, but no colonies grew in the CON, SA+L, and H groups. The colon smear results are as follows: Figure 16 As shown in section b, the CON group, DSS group, SA group, SA+L group, L group, and H group all showed a large number of colonies growing on MacConkey agar, while the DSS group had relatively fewer colonies. These results indicate that drinking 3% DSS solution leads to bacterial translocation, while low and high doses of the compound probiotic paste do not cause bacterial translocation; the bacteria only colonize the intestines.

[0144] (6) Determination of oxidative stress status in colon tissue of experimental mice

[0145] Take 10 mg of colon tissue, add an appropriate amount of sterile saline at a ratio of 1:1 (m / v) of tissue to physiological saline, grind using a tissue homogenizer, and take the supernatant of each tissue homogenate separately. Use a kit from Nanjing Jiancheng Biotechnology Co., Ltd. to determine the contents of MDA, MPO, T-AOC and SOD in mouse colon tissue.

[0146] The levels of myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant capacity (T-AOC), and total superoxide dismutase (SOD) in mouse colon tissue were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd. The results are as follows: Figure 17 As shown, compared with the CON group, the DSS group showed significantly increased MPO and MDA levels (P<0.01) and significantly decreased T-AOC and SOD levels (P<0.05), indicating that the oxidative stress of the mice in the DSS group was aggravated. Compared with the DSS group, the MPO and MDA levels of mice in the SA, SA+L, L, and H groups were significantly decreased (P<0.05, P<0.01), while the T-AOC and SOD levels of the SA+L and H groups were significantly increased (P<0.05, P<0.01), and the SOD level of the SA and L groups was significantly increased (P<0.05, P<0.01). These results indicate that both sulfasalazine and compound probiotic ointment can alleviate colonic oxidative stress, with high-dose compound probiotic ointment showing better effects than either sulfasalazine or low-dose compound probiotic ointment.

[0147] (7) Effects of colonic tissue inflammation in experimental mice

[0148] The mRNA levels of colonic inflammatory factors IL-6, IL-1β, and TNF-α in mice were detected using quantitative real-time PCR: 1) Colonic tissue homogenization: 5 mg of colonic tissue was minced and placed in a 2 mL EP tube. 1 mL of pre-chilled Trizol was added, and the tissue was homogenized using a homogenizer in either intestinal or vascular mode to obtain a colonic tissue homogenate. 2) RNA extraction and purification: 200 μL of chloroform (frozen at -20°C) was added to the tissue homogenate. The mixture was vortexed for 15 seconds, allowed to stand for 5 minutes, and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The clear liquid at the top was transferred to a new 1.5 mL EP tube, and an equal volume of pre-chilled isopropanol (pre-chilled at -20°C) was added. The mixture was vortexed and allowed to stand for 2 hours. The mixture was then centrifuged at 4°C for 10 minutes at 12,000 rpm and the supernatant was discarded. Add 1 mL of 75% ethanol pre-cooled overnight at -20°C, then centrifuge at 7000 rpm for 10 min at 4°C. Remove the supernatant and repeat the washing process twice. Place the mixture on a clean bench and allow it to air dry at room temperature. Add an appropriate amount of DEPC water to dissolve the RNA precipitate, and then use an ultra-micro UV spectrophotometer to detect its concentration. If the purity meets the requirements, proceed to the next step of synthesis. 3) cDNA synthesis: Perform the synthesis according to the Takara reverse transcription kit instructions. 4) Real-time PCR: Follow the steps in the Takara instructions for the specific operation of the synthesized cDNA.

[0149] The results are as follows Figure 18 As shown, compared with the CON group, the relative mRNA expression levels of IL-6, IL-1β, and TNF-α were significantly increased in the DSS and SA groups (P<0.05, P<0.01), and the relative mRNA expression level of TNF-α was significantly increased in the SA+L, L, and H groups (P<0.05). Compared with the DSS group, the relative mRNA expression levels of IL-6 were significantly decreased in the SA+L, L, and H groups (P<0.05), the relative mRNA expression levels of IL-1β were significantly decreased in the SA+L and H groups (P<0.05), and the relative mRNA expression level of TNF-α was significantly decreased in the H group (P<0.05). These results indicate that drinking 3% DSS solution increases the expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) in the colonic tissue of mice, and that the use of sulfasalazine and compound probiotic ointment can reduce the expression of inflammatory factors (IL-6, IL-1β, and TNF-α) to some extent and alleviate inflammation. Furthermore, the combined use of sulfasalazine and low-dose compound probiotic ointment, and the high-dose compound probiotic ointment showed better therapeutic effects than the low-dose compound probiotic ointment group and the group using sulfasalazine alone.

[0150] The experiments in this embodiment demonstrate that the compound probiotic ointment has a good therapeutic effect on DSS-induced chronic enteritis and diarrhea in mice. Compared with the DSS group, mice in the SA group, SA+L group, L group, and H group showed significant increases in body weight, significant decreases in disease activity index, significant relief of colonic damage (length, histopathological score), significant reduction in colonic tissue oxidative stress (MDA, MPO, SOD, T-AOC), and significant decreases in the relative expression levels of inflammatory factors (IL-6, IL-1β, TNF-α). These results suggest that both sulfasalazine and low-dose compound probiotic ointment have therapeutic effects on chronic enteritis and diarrhea in mice, and the combined use is more effective than using either alone. The high-dose compound probiotic ointment group showed significantly better therapeutic effects than the sulfasalazine-only treatment group and the low-dose compound probiotic ointment treatment group.

[0151] Example 6 - Compound probiotic paste on vitamin B 12 Evaluation of the treatment effect of deficiency

[0152] This embodiment verifies that the compound probiotic paste improves vitamin B12 deficiency caused by chronic diarrhea. 12 The study investigated the effects of vitamin B deficiency, specifically whether drinking a 3% DSS solution would cause vitamin B deficiency in mice. 12 Can blood disorders and compound probiotic paste alleviate vitamin B deficiency in mice? 12 Blood deficiency. Serum vitamin B in mice was detected using ELISA. 12 Homocysteine ​​(HCY) content (sample: mouse serum sample collected in Example 5):

[0153] Vitamin B1 in the serum of experimental mice was determined using a double-antibody sandwich method. 12 And homocysteine ​​content. Measured serum vitamin B in experimental mice. 12 The specific steps are as follows: purify mouse vitamin B... 12 Antibodies were coated onto microplates to prepare solid-phase antibodies, and then vitamin B was added. 12 Add sequentially to the coated monoclonal antibody microwells, and HRP-labeled vitamin B. 12 Antibody complexes form an antigen-antibody-enzyme-labeled antibody complex. After thorough washing, TMB is added for color development. TMB is catalyzed by HRP alcohol to turn blue, and then, under the catalysis of the enzyme, finally turns yellow. The intensity of the yellow color is related to the vitamin B content in the sample. 12 The content was positively correlated. The absorbance of the samples at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve was finally plotted to statistically analyze the levels of vitamin B in mouse serum. 12 content.

[0154] 1) Sample Addition: Set up blank wells and sample wells. Do not add sample or enzyme-labeled reagent to the blank wells. The other steps are the same. Add 40 μL of sample dilution to the sample wells of the enzyme-labeled plate, and then add 10 μL of sample. Try to add the sample to the bottom of the wells of the enzyme-labeled plate, and do not touch the plate wall. 2) Incubation: Apply the sealing film to the enzyme-labeled plate and incubate at 37°C for 30 min. 3) Solution Preparation: Dilute the concentrated washing buffer 30 times with distilled water and set aside. 4) Thorough Washing: Carefully remove the sealing film, discard the liquid, shake dry, add washing buffer to each well, let stand for 30 s and discard, repeat several times. 5) Enzyme Addition: Add 50 μL of enzyme-labeled reagent to each well except the blank wells. 6) Incubation: Same as above. 7) Thorough Washing: Same as above. 8) Color development: Add 50 μL of colorimetric reagent A to each well, then add 50 μL of colorimetric reagent B, gently shake to mix, and incubate at 37°C in the dark for 15 min. 9) Termination: Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). 10) Measurement: Zero the instrument using a blank well and measure the OD value at 450 nm using a microplate reader. The measurement should be completed within 15 min.

[0155] The procedure for detecting homocysteine ​​in mouse serum is the same as above.

[0156] Experimental results are as follows Figure 19 As shown. Figure 19 As shown in section a, compared with the CON group, the serum vitamin B in the DSS group and the L group was lower. 12 A significant decrease (P<0.01) was observed in serum vitamin B in mice in the SA+L and H groups compared to the DSS group. 12 The content increased significantly (P<0.05, P<0.01). For example... Figure 19 As shown in section b, compared with the CON group, serum HCY levels in the DSS, SA, and L groups were significantly increased (P<0.05, P<0.01). Compared with the DSS group, serum HCY levels in the SA, SA+L, L, and H groups were significantly decreased (P<0.05, P<0.01). These results indicate that DSS-induced chronic colitis reduces serum vitamin B in mice. 12 The increased HCY levels in mice led to low vitamin B content. 12 Blood disorders and hyperhCY. In addition, sulfasalazine and a compound probiotic ointment were effective in treating vitamin B deficiency in mice. 12 It has significant effects on blood disorders and hyperHCY blood disorders, and the combined effect of sulfasalazine and low-dose compound probiotic ointment, as well as the high-dose compound probiotic ointment, is better than the use of sulfasalazine or low-dose compound probiotic ointment alone.

[0157] The experimental results in this embodiment indicate that DSS-induced chronic colitis reduces serum vitamin B in mice. 12Increase serum HCY levels. The compound probiotic paste is effective in treating low vitamin B levels caused by colitis in mice. 12 It has a significant effect on blood disorders, and the combined treatment of high-dose compound probiotic ointment, sulfasalazine and low-dose compound probiotic ointment is more effective than using sulfasalazine or low-dose compound probiotic ointment alone.

[0158] As demonstrated by the above embodiments, this invention successfully developed a compound probiotic ointment. Its unique ointment formulation enhances palatability for pets, increasing the convenience and compliance of medication administration. After being sealed using a vacuum sealer, the compound probiotic ointment can be stably stored for 56 days at 4°C and in a dry place at room temperature, exhibiting good storage stability. The aforementioned compound probiotic ointment demonstrates good safety in healthy rabbits. Furthermore, the compound probiotic ointment can regulate the intestinal flora of healthy rabbits, increasing the content of lactic acid bacteria in the intestines. The aforementioned compound probiotic ointment exhibits good storage stability, safety, palatability, and tolerability, and it also shows good therapeutic effects on DSS-induced chronic enteritis and diarrhea in mice, while simultaneously increasing serum vitamin B in mice. 12 Levels, improve vitamin B in mice 12 Deficiency, which is treated with a compound probiotic paste for chronic diarrhea in pets and vitamin B deficiency. 12 The deficiency syndrome provides a theoretical basis.

[0159] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A compound probiotic paste, characterized in that, The ointment is made of a compound probiotic and an ointment base, wherein the compound probiotic consists of Enterococcus faecalis, Saccharomyces cerevisiae, and canine-derived vitamin B. 12 The ointment base is composed of lactic acid bacteria and is made of prebiotics, flavoring substances and auxiliary substances. Among them, the canine-derived vitamin B 12 The lactic acid bacteria is Lactobacillus reuteri ( Lactobacillus reuteri Its accession number is: CGMCC NO.19126; In the ointment, the ratio of viable counts of Enterococcus faecalis, Saccharomyces cerevisiae, and Lactobacillus is 1-2:1-2:1-8, and the viable count of the compound probiotics in the ointment is 5 × 10⁻⁶. 8 ~ 5×10 9 CFU / g; The prebiotic is made from fructooligosaccharides and psyllium husk powder, the flavoring agent is goat milk powder and chicken liver powder, and the auxiliary agent is potassium sorbate, xanthan gum, and deionized water. By weight percentage, the ointment matrix is ​​made from 0.5-3% fructooligosaccharides, 0.1-2% psyllium husk powder, 2-8% goat milk powder, 1-5% chicken liver powder, 0.5-2% potassium sorbate, 1-4% xanthan gum, and the balance being water.

2. The compound probiotic ointment according to claim 1, characterized in that, The ratio of viable counts of Enterococcus faecalis, Saccharomyces cerevisiae, and Lactobacillus is 1:1:1, and the viable count of the compound probiotics in the ointment is 1×10⁻⁶. 9 CFU / g; by weight percentage, the ointment matrix is ​​made of 1% fructooligosaccharides, 0.5% psyllium husk powder, 5% goat milk powder, 3% chicken liver powder, 1% potassium sorbate, 2% xanthan gum and the balance water.

3. The compound probiotic ointment according to claim 1, characterized in that, The preparation method of the ointment includes the following steps: S1. Preparation of bacterial suspension: Lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae are activated separately, inoculated and cultured to obtain seed culture of each bacteria; each seed culture is inoculated and cultured, the culture solution is centrifuged, the supernatant is discarded, and bacterial sludge is obtained; the bacterial sludge is resuspended to obtain bacterial suspension of each bacteria. S2. Preparation of ointment matrix: Mix oligofructose, psyllium husk powder, goat milk powder, chicken liver powder, potassium sorbate, xanthan gum, and deionized water in a predetermined ratio, heat and mix evenly, sterilize, and obtain an ointment matrix without probiotics. S3. Preparation of probiotic ointment: The bacterial suspensions of lactic acid bacteria, Enterococcus faecalis and Saccharomyces cerevisiae obtained in step S1 are mixed evenly according to a predetermined ratio of live bacteria, and added to the ointment matrix prepared in step S2 for mixing, so that the probiotics are evenly distributed in the ointment matrix to obtain the compound probiotic ointment.

4. The compound probiotic ointment according to claim 3, characterized in that, It also includes step S4, packaging of the ointment: vacuum sealing the compound probiotic ointment and storing it in a cool, dry place at low temperature or at room temperature.

5. The compound probiotic ointment according to claim 3, characterized in that, In step S1, the seed culture preparation step specifically includes: inoculating frozen lactic acid bacteria, Enterococcus faecalis, and Saccharomyces cerevisiae into MRS agar medium, subculturing and activating them twice, then picking single colonies and inoculating them into MRS liquid medium, and culturing them in a 37°C constant temperature incubator for 24 h to obtain the seed culture of each bacterium. In step S1, the preparation of the mycelial sludge specifically includes: inoculating the seed liquid into sterilized MRS liquid culture medium at a volume of 2 vol%, incubating in a constant temperature shaking incubator at 37℃ for 24 h, dispensing the culture medium into 30 mL sterile centrifuge tubes, placing them in a low-speed refrigerated centrifuge, centrifuging at 2000 r / min at 4℃ for 10 min, discarding the supernatant, and obtaining the mycelial sludge; In step S1, the preparation of the bacterial suspension specifically includes: weighing the bacterial mud and adding it to sterile 0.9% physiological saline at a ratio of 1:1, mixing it quickly and evenly with a shaker to prepare bacterial suspensions of each bacterium; In step S2, the preparation step of the ointment matrix specifically includes: heating and stirring the fructooligosaccharides, psyllium husk powder, goat milk powder, chicken liver powder, potassium sorbate, xanthan gum, and deionized water at 65-75 ℃ until the texture is uniform and thick, and then pasteurizing at 70 ℃ for 30 min to obtain the ointment matrix that does not contain probiotics.

6. The compound probiotic ointment according to claim 5, characterized in that, The MRS agar medium composition is: 10.0 g / L peptone, beef extract powder. 8.0 g / L yeast extract The MRS liquid culture medium consists of the following ingredients: 4.0 g / L glucose, 20.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 2.0 g / L sodium acetate, 5.0 g / L magnesium sulfate, 0.2 g / L agar, 14.0 g / L Tween 80, and 1.0 g / L Tween 80. 8.0 g / L yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L.

7. The application of a compound probiotic paste as described in any one of claims 1 to 6, characterized in that, The application is selected from at least one of the following applications: application in the preparation of products that improve the palatability and endurance of pet food; application in the preparation of medicines for the prevention or treatment of chronic enteritis and diarrhea in pets; and application in the preparation of vitamin B for the prevention or treatment of vitamin B in pets. 12 Application in medications for deficiency syndromes.

8. The application according to claim 7, characterized in that, The compound probiotic ointment can be used alone or in combination with a drug, wherein the drug is sulfasalazine.

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