A probiotic strain and its application, a composition for preventing and treating infantile diarrhea

Abstract

The present application provides a probiotic strain and its application, a composition for preventing and treating infantile diarrhea, and belongs to the technical field of probiotics. The probiotic strain is Lactobacillus reuteri FPH2951, which is classified and named as Lactobacillus reuteri Limosilactobacillus reuteri, the preservation number is GDMCC NO.62946, the preservation date is November 3, 2022, the preservation unit is Guangdong Microbial Culture Collection Center, the address is No. 59, Building 5, Guangzhou Xianlie Middle Road 100, Guangdong Academy of Microbiology, and the postcode is 510070. The probiotic strain can prevent, relieve and treat infantile diarrhea, has the advantages of good curative effect, quick effect and short treatment cycle, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a probiotic strain and its application, and to compositions for the prevention and treatment of childhood diarrhea. Background Technology

[0002] According to data released by the National Health and Family Planning Commission, the exclusive breastfeeding rate for infants aged 0-6 months is 27.8% (the international average is 38%), with 30.3% in rural areas and 15.8% in urban areas. In 1998, China's breastfeeding rate was as high as 67%. In recent years, with the widespread dissemination of education promoting the benefits of breastfeeding, the breastfeeding rate among mothers born in the 1990s has been increasing, but it is still far below the 50% breastfeeding rate target set in China's Child Development Program. Numerous studies have shown a significant difference in the risk of diarrheal diseases between breastfed and non-breastfed infants; that is, non-breastfed infants are more prone to diarrheal diseases.

[0003] Diarrhea in children is a group of diseases characterized by diarrhea, caused by multiple pathogens and factors. Its main features are increased frequency and altered stool consistency. Pathogens can include viruses (primarily human rotavirus and other enteroviruses), bacteria (pathogenic Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, invasive Escherichia coli, as well as Salmonella typhimurium, Campylobacter jejuni, Yersinia, Staphylococcus aureus, etc.), parasites, and fungi. Extraintestinal infections, intestinal flora imbalance caused by antibiotic abuse, allergies, improper feeding, and climatic factors can also contribute to the disease. Because children's immune systems are not fully developed and they are in a period of physiological immune insufficiency, they are more susceptible to functional intestinal disorders such as diarrhea. Diarrhea is extremely common in childhood, with diarrhea and vomiting being its main clinical manifestations. If children do not receive timely and effective treatment, electrolyte imbalances usually occur, and in severe cases, it can be life-threatening. Summary of the Invention

[0004] The purpose of this invention is to propose a probiotic strain and its application, as well as a composition for the prevention and treatment of childhood diarrhea. This composition can effectively prevent, alleviate, and treat childhood diarrhea, and has the advantages of good efficacy, rapid effect, and short treatment cycle, and has broad application prospects.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides the use of a strain in the preparation of compositions for the prevention and treatment of colic, diarrhea, and intestinal disorders caused by pathogens such as *Escherichia coli*, *Salmonella enteritidis*, *Clostridium difficile*, or *Campylobacter jejuni*. The strain exhibits antibacterial activity against these pathogens. The strain includes *Lactobacillus reuteri*, specifically *Lactobacillus reuteri* FPH2951, classified as *Limosilactobacillus reuteri*, with accession number GDMCC NO.62946, deposited on November 3, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, 510070, China.

[0007] As a further improvement of the present invention, the 16S rRNA sequence of the Lactobacillus reuteri FPH2951 is shown in SEQ ID No. 1.

[0008] As a further improvement of the present invention, the Lactobacillus reuteri FPH2951 is a Gram-positive rod-shaped bacterium. On MRS medium, the colonies are round, with neat edges, milky white, raised, smooth, and do not produce spores.

[0009] As a further improvement of the present invention, the diarrhea is infantile diarrhea.

[0010] This invention further protects a method for preparing a composition for preventing and treating infantile diarrhea, comprising the following steps:

[0011] S1. Enzymatic hydrolysis: Kudzu root and licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and a compound enzyme was added for enzymatic hydrolysis. The enzyme was inactivated, filtered, and the solid was kept for use. The filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0012] S2. Water extraction: Add the solid from step S1 to water, heat to boiling and extract, filter, retain the solid, dry the filtrate to obtain the water extract;

[0013] S3. Strain activation: Inoculate probiotics into Gao's medium, activate and culture them to obtain strain seed solution;

[0014] S4. Fermentation: Add the solid from step S2 to water, sterilize, inoculate with the probiotic seed liquid prepared in step S3, ferment and culture, filter, retain the solid, freeze dry, and obtain the fermentation product;

[0015] S5. Ultrafine powder mixing: The solid from step S4 is dried, pulverized and sieved to obtain fine powder, which is then mixed evenly with montmorillonite powder to obtain ultrafine powder;

[0016] S6. Preparation of prebiotics: Mix fructooligosaccharides and xylooligosaccharides evenly to obtain prebiotics;

[0017] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: The fermentation product obtained in step S4 is added to water, sodium alginate, sodium carboxymethyl cellulose, and emulsifier are added, and the mixture is stirred and mixed evenly. The mixture is then added to fish oil, rapidly emulsified, and calcium chloride solution is added dropwise. The mixture is solidified at room temperature and freeze-dried to obtain fermentation product microspheres. These microspheres are then mixed evenly with the enzymatic hydrolysate obtained in step S1, the aqueous extract obtained in step S2, the ultrafine powder obtained in step S5, and the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0018] As a further improvement of the present invention, in step S1, the mass ratio of kudzu root and licorice is 5-7:3-5, the solid-liquid ratio of the herbal powder and water is 1:5-10 g / mL, the compound enzyme is cellulase and pectin methyl esterase in a mass ratio of 7-10:5, the amount of the compound enzyme added is 2-3% of the total mass of the system, and the enzymatic hydrolysis temperature is 45-50℃ for 2-4 hours; in step S2, the mass ratio of solid to water is 10-15:50-70, the heating and boiling extraction time is 1-3 hours, and the operation is repeated 1-2 times; in step S3, the activation culture temperature is 36-38℃ for 18-24 hours, and the bacterial count of the inoculum seed solution is 10. 8 -10 9 cfu / mL.

[0019] As a further improvement of the present invention, in step S4, the mass ratio of solid to water is 20-30:100-120, and the fermentation conditions are 36-38℃, 50-70 r / min, and fermentation for 48-56 h; in step S5, the mesh size of the sieve is 500-1000 mesh, and the mass ratio of the solid powder to montmorillonite powder is 10-12:15-20; in step S6, the mass ratio of fructooligosaccharide to xylooligosaccharide is 7-10:3-5; in step S7, the fermentation product, sodium alginate, and... The mass ratio of sodium carboxymethyl cellulose and emulsifier is 10-12:7-10:3-5:0.5-1. The emulsifier is selected from at least one of lecithin, sodium stearoyl lactylate, calcium stearoyl lactylate, diacetyl tartrate monoglyceride, sucrose fatty acid ester, and distilled monoglyceride. The pore size of the rapid membrane is 1-5 micrometers. The curing time at room temperature is 20-30 minutes. The mass ratio of fermentation product microspheres, enzymatic hydrolysis products, water extracts, ultrafine powder, and prebiotics is 15-20:2-3:3-5:12-15:2-3.

[0020] As a further improvement of the present invention, the probiotic is Lactobacillus reuteri FPH2951, and the inoculation amount of the probiotic seed solution is 2-3%.

[0021] As a further improvement of the present invention, the probiotic is a composition of Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16, and the inoculation amount of the probiotic seed solution is: 2-3% for Lactobacillus reuteri FPH2951, 0.5-1% for Saccharomyces boulardii, and 1-2% for Lactobacillus acidophilus LA16.

[0022] The present invention further protects a composition for the prevention and treatment of childhood diarrhea prepared by the above-described preparation method.

[0023] The present invention has the following beneficial effects: The present invention provides a Lactobacillus reuteri FPH2951 with high gastric acid resistance and survival rate, which has a good preventive, relieving and therapeutic effect on infantile diarrhea, colic, diarrhea and intestinal disorders. It can secrete beneficial substances to promote the proliferation and colonization of probiotics in the body, inhibit the colonization of harmful bacteria, build a biological barrier on the surface of the gastrointestinal mucosa of the child to prevent the invasion of harmful bacteria, improve the intestinal environment of the child, avoid the continuous reproduction and growth of harmful bacteria, and ultimately improve the state of the gastrointestinal flora of the child, relieve diarrhea symptoms, and has the advantages of good efficacy, rapid effect and short treatment cycle.

[0024] The present invention also provides a composition for the prevention and treatment of childhood diarrhea. Wherein:

[0025] Kudzu root contains puerarin, xyloside, soy isoflavones, β-sitosterol, arachidic acid, and other components, which have antipyretic, antispasmodic, intestinal-constricting, and mildly blood-circulating effects. Licorice contains glycyrrhizic acid, glycyrrhizin, isoglycyrrhizin, and other components, which have detoxifying, anti-inflammatory, anti-allergic, expectorant, antitussive, gastric mucosal-protective, gastric ulcer-treating, antispasmodic, and liver-damaging effects. The combination of these two ingredients effectively reduces fever, improves blood circulation, and protects the gastric mucosa. Enzymatic hydrolysis using compound enzymes (cellulase and pectin methyl esterase) promotes cell wall rupture and the dissolution of active medicinal components, significantly enhancing efficacy. Water extraction further extracts most of the water-soluble components.

[0026] The probiotic composition of this invention can not only break down the cell walls of traditional Chinese medicine through fermentation, promoting the dissolution of active components in kudzu root and licorice and improving efficacy, but also improve the patient's digestive and immune capabilities. During drug use, it can maintain the balance of gastrointestinal flora, improve the intestinal environment, repair the intestinal mucosa, improve clinical diarrhea symptoms, optimize the ability to resist pathogens, control the regeneration of pathogens in the patient's body, eliminate residual bacteria in the body, and shorten the patient's recovery time and disease course.

[0027] Among them, Lactobacillus reuteri FPH2951 has a high survival rate against gastric acid, can secrete beneficial substances to promote the proliferation and colonization of probiotics in the body, inhibit the colonization of harmful bacteria, build a biological barrier on the surface of the gastrointestinal mucosa of the child to prevent the invasion of harmful bacteria, improve the intestinal environment of the child, avoid the continuous reproduction and growth of harmful bacteria, and ultimately improve the status of the gastrointestinal flora of the child and relieve diarrhea symptoms.

[0028] Saccharomyces boulardii powder is a non-pathogenic fungal probiotic that can release large amounts of polyamines in the body, increasing the activity of metabolic enzymes such as α-glucosidase and alkaline phosphatase, delaying the growth and reproduction of pathogenic microorganisms, preventing pathogenic microorganisms from invading and damaging mucosal epithelial cells, generating short peptide proteins to degrade bacterial toxins, playing a role in resisting pathogenic microorganisms, improving the intestinal microecological environment, and relieving symptoms such as diarrhea in children.

[0029] The addition of Lactobacillus acidophilus LA16 can inhibit the adhesion of harmful bacteria to the intestinal mucosa, while having high resistance to gastric acid, improving the gastrointestinal microecological environment of children, maintaining the stability of the number of gastrointestinal flora, inhibiting the occurrence of inflammation, reducing diarrhea, and improving the immune function disorder of children.

[0030] Lactobacillus reuteri FPH2951 alone has already shown good preventive, alleviating, and therapeutic effects. The synergistic combination of the three probiotics can effectively prevent and reduce infections in patients, improve the gastrointestinal microecological environment of children, inhibit inflammation, reduce diarrhea, and improve immune dysfunction in children.

[0031] Montmorillonite powder can enhance the mucosal coverage of the digestive tract and effectively inhibit viruses, bacteria, and toxins in the digestive tract, thereby increasing the mucosal barrier defense function of the digestive tract in children and promoting the recovery of gastrointestinal function. However, it can easily cause constipation. Therefore, this invention dries and ultrafinely pulverizes the solid residue after fermentation and extraction of traditional Chinese medicine to obtain fine powder, mixes it evenly with montmorillonite powder, and adds it to the composition. This mixture can work synergistically with montmorillonite powder to enhance the mucosal coverage of the digestive tract. At the same time, the fiber of the traditional Chinese medicine can promote intestinal peristalsis, relieve constipation caused by montmorillonite powder, and expel harmful substances fixed by montmorillonite powder from the intestine, thus making the composition safer and more effective.

[0032] Prebiotics are a compound mixture of fructooligosaccharides and xylooligosaccharides. By selectively stimulating the growth and proliferation of probiotic colonies added in this invention, they can reduce the production of inflammatory factors, promote antioxidant responses, improve the gastrointestinal microecological environment of children, maintain a stable number of gastrointestinal flora, reduce diarrhea, and improve the immune function disorders of children.

[0033] After encapsulation, the present invention can resist the gastric acid environment to a certain extent and further increase the content of probiotics directly delivered to the intestines, thereby achieving better prevention, relief and treatment effects. The resulting composition for the prevention and treatment of childhood diarrhea has the advantages of good efficacy, rapid effect and short treatment cycle, and has broad application prospects. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a colony morphology diagram of Lactobacillus reuteri FPH2951.

[0036] Figure 2 This is a morphological diagram of Lactobacillus reuteri FPH2951. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0038] This invention provides a Lactobacillus reuteri FPH2951, classified as Lactobacillus reuteri, with accession number GDMCC NO.62946, accession date November 3, 2022, and deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, postcode: 510070.

[0039] Lactobacillus acidophilus LA16, classified as Lactobacillus acidophilus, with accession number CGMCCNO.22771, deposited on June 24, 2021, is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0040] MRS liquid culture medium, with the following components: peptone 10 g / L, beef extract 5 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, Tween 80 1 mL / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0041] MRS solid culture medium, with the following components: peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14 g / L, and Tween 80 1 mL / L, purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0042] Example 1: Screening and identification of Lactobacillus reuteri FPH2951

[0043] 1. Screening of Lactobacillus reuteri FPH2951

[0044] 1.1 Sample Source

[0045] The strains used in this invention were all collected from fermented tofu products from Sichuan—fermented tofu, that is, the present invention is based on fermented tofu from Sichuan as a sample.

[0046] 1.2 Isolation of strains

[0047] Add 1g of sample to 10mL of MRS liquid culture medium, mix well, and incubate at 36℃. Then, in a clean bench, take 1mL of enrichment solution and perform a tenfold serial dilution. Select 10... -5 10 -6 10 -7 Three dilution gradients of bacterial suspension, 100 μL each, were spread onto petri dishes containing sterile MRS solid medium and incubated statically at 36°C for 24-48 h under aerobic conditions until clear single colonies were formed. Then, plates with 70-100 single colonies were selected from the medium, and typical colonies were picked and streaked multiple times on MRS solid medium for purification until the colonies on the entire plate had a uniform morphology. The culture was then used for strain identification.

[0048] 1.3 Preservation of strains

[0049] Select single colonies and culture them in MRS liquid medium at 37°C for 36 hours. Add 500 μL of the bacterial culture to a 0.8 mL tube of 60% (v / v) glycerol and store at -80°C.

[0050] 2. Identification of Lactobacillus reuteri FPH2951

[0051] 2.1 Colony characteristics

[0052] After culturing Lactobacillus reuteri FPH2951 in MRS solid medium for 48 hours, the colonies were milky white, round, with regular, raised edges, and a smooth surface. Figure 1 .

[0053] 2.2 Microscopic morphology

[0054] Lactobacillus reuteri FPH2951 colony smear: Gram-positive, bacteria are rod-shaped, arranged singly or in clusters, see Figure 2 .

[0055] 2.3 Identification of 16S rRNA

[0056] Testing and Authentication Agency: Guangdong Provincial Center for Microbiology Analysis and Testing

[0057] Identification sequence:

[0058]

[0059] Identification results: Based on the sequence alignment results and physiological and biochemical results of the strain, the selected strain was identified as Limosilactobacillus reuteri.

[0060] Example 2: Preparation of Lactobacillus reuteri FPH2951 powder

[0061] The glycerol tube containing *Lactobacillus reuteri* FPH2951 was removed from -80°C and activated for culture, yielding a bacterial count of 10. 8 A 3 wt% inoculum of *Lactobacillus reuteri* FPH2951 was obtained by diluting the inoculum 100 times with cfu / g of the bacterial seed solution and inoculating it into MRS liquid medium. The culture was then anaerobic at 36°C for 24 hours (7% carbon dioxide and 85% nitrogen, by volume). After centrifugation and freeze-drying, the *Lactobacillus reuteri* FPH2951 powder was obtained. The viable count of *Lactobacillus reuteri* FPH2951 strain was determined to be 10⁻⁶. 11 cfu / g.

[0062] In the following examples, the activation and fermentation cultures were conducted in an anaerobic environment with a carbon dioxide concentration of 5 v / v% and a nitrogen concentration of 95 v / v%.

[0063] Saccharomyces boulardii, 20 billion CFU / g, purchased from Xi'an Hetaiyuan Biotechnology Co., Ltd.; cellulase, 10,000 U / g, pectin methyl esterase, 6,000 U / g, purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.; montmorillonite powder, content >99%, purchased from Inner Mongolia Mengchuang Jiamu Biotechnology Co., Ltd.

[0064] Test Example 1: Antibiotic Resistance Detection

[0065] The sensitivity of the Lactobacillus reuteri FPH2951 strain of this invention to antimicrobial drugs was tested in accordance with the "Guidelines for Identification and Safety Evaluation of Strains for Direct Feeding Microorganisms and Fermentation Products" (Agricultural Office Animal Husbandry

[2021] No. 43) to obtain the minimum inhibitory concentration (MIC) value.

[0066] The results are shown in Table 1.

[0067] Table 1

[0068] Antibacterial drugs MIC (mg / L) Cut-off (mg / L) Conclusion Gentamicin 256 8 Resistant Kanamycin >2048 64 Resistant Streptomycin 1024 64 Resistant Tetracycline 64 32 Resistant Erythromycin 8 1 Resistant Clindamycin 16 4 Resistant Chloramphenicol 16 4 Resistant Ampicillin 8 2 Resistant

[0069] As shown in the table above, Lactobacillus reuteri FPH2951 in this invention exhibits excellent resistance to antibacterial drugs.

[0070] Example 3

[0071] This embodiment provides a method for preparing a composition for preventing and treating childhood diarrhea, comprising the following steps:

[0072] S1. Enzymatic hydrolysis: 5 parts by weight of kudzu root and 3 parts by weight of licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and the solid-liquid ratio of the Chinese medicine powder to water was 1:5 g / mL. A compound enzyme was added, and the amount of the compound enzyme added was 2% of the total mass of the system. The mixture was enzymatically hydrolyzed at 45°C for 2 hours, the enzyme was inactivated, the mixture was filtered, the solid was retained, and the filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0073] The composite enzyme is cellulase and pectin methyl esterase in a mass ratio of 7:5.

[0074] S2. Water extraction: Add 10 parts by weight of the solid from step S1 to 50 parts by weight of water, heat to boiling and extract for 1 hour, repeat the operation once, filter, keep the solid, dry the filtrate to obtain the water extract.

[0075] S3. Strain activation: The glycerol tube containing *Lactobacillus reuteri* FPH2951 was removed from -80℃ and inoculated into Gao's medium. Activation culture was carried out at 36℃ for 18 hours, yielding a bacterial count of 10... 8 CFU / mL bacterial seed solution;

[0076] S4. Fermentation: Add 20 parts by weight of the solid from step S2 to 100 parts by weight of water, sterilize, inoculate with the Lactobacillus reuteri FPH2951 seed culture obtained in step S3, wherein the inoculation amount of the Lactobacillus reuteri FPH2951 seed culture is 2.5%, ferment at 36℃ and 50 r / min for 48 h, filter, retain the solid, freeze dry to obtain the fermentation product;

[0077] S5. Ultrafine powder mixing: Dry 10 parts by weight of the solid from step S4, pulverize it through a 500-mesh sieve to obtain fine powder, and mix it evenly with 15 parts by weight of montmorillonite powder to obtain ultrafine powder.

[0078] S6. Preparation of prebiotics: Mix 7 parts by weight of fructooligosaccharide and 3 parts by weight of xylooligosaccharide evenly to obtain prebiotics;

[0079] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 10 parts by weight of the fermentation product obtained in step S4 are added to 100 parts by weight of water, 7 parts by weight of sodium alginate, 3 parts by weight of sodium carboxymethyl cellulose, and 0.5 parts by weight of lecithin are added and stirred for 20 minutes. Then, 200 parts by weight of fish oil are added and rapidly emulsified using a membrane with a pore size of 1 micrometer. 20 parts by weight of 5 wt% calcium chloride solution are added dropwise, and the mixture is solidified at room temperature for 20 minutes. The mixture is then freeze-dried to obtain fermentation product microspheres. 15 parts by weight of the fermentation product microspheres are mixed evenly with 2 parts by weight of the enzymatic hydrolysate obtained in step S1, 3 parts by weight of the aqueous extract obtained in step S2, 12 parts by weight of the ultrafine powder obtained in step S5, and 2 parts by weight of the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0080] Example 4

[0081] This embodiment provides a method for preparing a composition for preventing and treating childhood diarrhea, comprising the following steps:

[0082] S1. Enzymatic hydrolysis: 7 parts by weight of kudzu root and 5 parts by weight of licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and the solid-liquid ratio of the Chinese medicine powder to water was 1:10 g / mL. A compound enzyme was added, and the amount of the compound enzyme added was 3% of the total mass of the system. The enzyme was hydrolyzed at 50°C for 4 hours, the enzyme was inactivated, filtered, the solid was retained, and the filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0083] The composite enzyme is cellulase and pectin methyl esterase in a mass ratio of 10:5.

[0084] S2. Water extraction: Add 15 parts by weight of the solid from step S1 to 70 parts by weight of water, heat to boiling and extract for 3 hours. Repeat the operation twice, filter, keep the solid, dry the filtrate, and obtain the water extract.

[0085] S3. Strain Activation: The glycerol tube containing *Lactobacillus reuteri* FPH2951 was removed from -80℃ and inoculated into Gao's medium along with *Saccharomyces boulardii* and *Lactobacillus acidophilus* LA16. The culture was activated at 38℃ for 24 hours, yielding a bacterial count of 10... 9 CFU / mL bacterial seed solution;

[0086] S4. Fermentation: Add 30 parts by weight of the solid from step S2 to 120 parts by weight of water, sterilize, and inoculate with the Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16 seed culture prepared in step S3. The inoculation amounts of Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16 seed culture are 2%, 0.5%, and 1%, respectively. Ferment and culture at 38°C and 70 r / min for 56 h, filter, retain the solid, freeze-dry, and obtain the fermentation product.

[0087] S5. Ultrafine powder mixing: Dry 12 parts by weight of the solid from step S4, pulverize it through a 1000-mesh sieve to obtain fine powder, and mix it evenly with 20 parts by weight of montmorillonite powder to obtain ultrafine powder;

[0088] S6. Preparation of prebiotics: Mix 10 parts by weight of fructooligosaccharide and 5 parts by weight of xylooligosaccharide evenly to obtain prebiotics;

[0089] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 12 parts by weight of the fermentation product obtained in step S4 were added to 100 parts by weight of water, 10 parts by weight of sodium alginate, 5 parts by weight of sodium carboxymethyl cellulose, and 1 part by weight of diacetyl tartrate monoglyceride. The mixture was stirred and mixed for 20 minutes. Then, 200 parts by weight of fish oil were added and rapidly emulsified using a membrane with a pore size of 5 micrometers. 20 parts by weight of 5 wt% calcium chloride solution were added dropwise, and the mixture was solidified at room temperature for 30 minutes. The mixture was then freeze-dried to obtain fermentation product microspheres. 20 parts by weight of the fermentation product microspheres were mixed evenly with 3 parts by weight of the enzymatic hydrolysate obtained in step S1, 5 parts by weight of the aqueous extract obtained in step S2, 15 parts by weight of the ultrafine powder obtained in step S5, and 3 parts by weight of the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0090] Example 5

[0091] This embodiment provides a method for preparing a composition for preventing and treating childhood diarrhea, comprising the following steps:

[0092] S1. Enzymatic hydrolysis: 6 parts by weight of kudzu root and 4 parts by weight of licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and the solid-liquid ratio of the Chinese medicine powder to water was 1:7 g / mL. A compound enzyme was added, and the amount of the compound enzyme added was 2.5% of the total mass of the system. The mixture was enzymatically hydrolyzed at 47°C for 3 hours. The enzyme was then inactivated, filtered, and the solid was retained. The filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0093] The composite enzyme is cellulase and pectin methyl esterase in a mass ratio of 8.5:5;

[0094] S2. Water extraction: Add 12 parts by weight of the solid from step S1 to 60 parts by weight of water, heat to boiling and extract for 2 hours. Repeat the operation twice, filter, keep the solid, dry the filtrate to obtain the water extract.

[0095] S3. Strain Activation: The glycerol tube containing *Lactobacillus reuteri* FPH2951 was removed from -80℃ and inoculated into Gao's medium along with *Saccharomyces boulardii* and *Lactobacillus acidophilus* LA16. The culture was activated at 37℃ for 21 hours, yielding a strain with a bacterial count of 10... 9 CFU / mL bacterial seed solution;

[0096] S4. Fermentation: 25 parts by weight of the solid from step S2 are added to 110 parts by weight of water, sterilized, and inoculated with the Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16 seed culture prepared in step S3. The inoculation amounts of Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16 seed culture are 2.5%, 0.7%, and 1.5%, respectively. Fermentation is carried out at 37°C and 60 r / min for 52 h. After filtration, the solid is retained and freeze-dried to obtain the fermentation product.

[0097] S5. Ultrafine powder mixing: 11 parts by weight of the solid from step S4 are dried, pulverized and passed through a 700-mesh sieve to obtain fine powder, which is then mixed evenly with 17 parts by weight of montmorillonite powder to obtain ultrafine powder.

[0098] S6. Preparation of prebiotics: 8.5 parts by weight of fructooligosaccharide and 4 parts by weight of xylooligosaccharide are mixed evenly to obtain prebiotics;

[0099] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 11 parts by weight of the fermentation product obtained in step S4 were added to 100 parts by weight of water, along with 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethyl cellulose, and 0.7 parts by weight of sodium stearoyl lactylate. The mixture was stirred and mixed for 20 minutes. Then, 200 parts by weight of fish oil were added, and the mixture was rapidly emulsified using a membrane with a pore size of 3 micrometers. 20 parts by weight of 5 wt% calcium chloride solution were added dropwise, and the mixture was solidified at room temperature for 25 minutes. The mixture was then freeze-dried to obtain fermentation product microspheres. 17 parts by weight of the fermentation product microspheres were mixed evenly with 2.5 parts by weight of the enzymatic hydrolysate obtained in step S1, 4 parts by weight of the aqueous extract obtained in step S2, 13.5 parts by weight of the ultrafine powder obtained in step S5, and 2.5 parts by weight of the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0100] Example 6

[0101] The difference from Example 5 is that the complex enzyme is a single cellulase.

[0102] Example 7

[0103] The difference from Example 5 is that the complex enzyme is a single pectin methyl esterase.

[0104] Comparative Example 1

[0105] The difference from Example 5 is that kudzu root was not added in step S1.

[0106] Specifically as follows:

[0107] S1. Enzymatic hydrolysis: 10 parts by weight of licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and the solid-liquid ratio of the Chinese medicine powder to water was 1:7 g / mL. A compound enzyme was added, and the amount of the compound enzyme added was 2.5% of the total mass of the system. The mixture was enzymatically hydrolyzed at 47°C for 3 hours. The enzyme was then inactivated, filtered, and the solid was retained. The filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0108] Comparative Example 2

[0109] The difference from Example 5 is that licorice was not added in step S1.

[0110] Specifically as follows:

[0111] S1. Enzymatic hydrolysis: 10 parts by weight of kudzu root were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and the solid-liquid ratio of the Chinese medicine powder to water was 1:7 g / mL. A compound enzyme was added, and the amount of the compound enzyme added was 2.5% of the total mass of the system. The mixture was enzymatically hydrolyzed at 47°C for 3 hours. The enzyme was then inactivated, filtered, and the solid was retained. The filtrate was freeze-dried to obtain the enzymatic hydrolysis product.

[0112] Comparative Example 3

[0113] The difference from Example 5 is that enzymatic hydrolysis is performed in step S1.

[0114] Specifically as follows:

[0115] S1. Wash 6 parts by weight of kudzu root and 4 parts by weight of licorice root, dry them, and grind them into powder to obtain Chinese medicine powder.

[0116] Comparative Example 4

[0117] Compared with Example 5, the difference is that step S2 was not performed and water extract was not added in step S7.

[0118] Specifically as follows:

[0119] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 11 parts by weight of the fermentation product obtained in step S4 were added to 100 parts by weight of water, along with 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethyl cellulose, and 0.7 parts by weight of sodium stearoyl lactylate. The mixture was stirred and mixed for 20 minutes. Then, 200 parts by weight of fish oil were added, and the mixture was rapidly emulsified using a membrane with a pore size of 3 micrometers. 20 parts by weight of 5 wt% calcium chloride solution were added dropwise, and the mixture was solidified at room temperature for 25 minutes. The mixture was then freeze-dried to obtain fermentation product microspheres. 17 parts by weight of the fermentation product microspheres were mixed evenly with 2.5 parts by weight of the enzymatic hydrolysis product obtained in step S1, 13.5 parts by weight of the ultrafine powder obtained in step S5, and 2.5 parts by weight of the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0120] Comparative Example 5

[0121] The difference from Example 5 is that Lactobacillus reuteri FPH2951 was not inoculated in step S4.

[0122] Specifically as follows:

[0123] S4. Fermentation: Add 25 parts by weight of the solid from step S2 to 110 parts by weight of water, sterilize, and inoculate with the Saccharomyces boulardii and Lactobacillus acidophilus LA16 seed culture prepared in step S3, wherein the inoculation amounts of Saccharomyces boulardii and Lactobacillus acidophilus LA16 seed culture are 0.7% and 4%, respectively. Ferment at 37°C and 60 r / min for 52 h, filter, retain the solid, freeze dry, and obtain the fermentation product.

[0124] Comparative Example 6

[0125] The difference from Example 5 is that Lactobacillus acidophilus LA16 was not inoculated in step S4.

[0126] Specifically as follows:

[0127] S4. Fermentation: Add 25 parts by weight of the solid from step S2 to 110 parts by weight of water, sterilize, and inoculate with the Lactobacillus reuteri FPH2951 and Saccharomyces boulardii seed culture prepared in step S3. The inoculation amounts of Lactobacillus reuteri FPH2951 and Saccharomyces boulardii seed culture are 4% and 0.7%, respectively. Fermentation is carried out at 37°C and 60 r / min for 52 h. After filtration, the solid is retained and freeze-dried to obtain the fermentation product.

[0128] Comparative Example 7

[0129] Compared with Example 5, the difference is that step S4 fermentation was not performed, and fermentation product microspheres were not added in step S7.

[0130] Specifically as follows:

[0131] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 2.5 parts by weight of the enzymatic hydrolysate obtained in step S1, 4 parts by weight of the aqueous extract obtained in step S2, 13.5 parts by weight of the ultrafine powder obtained in step S5, and 2.5 parts by weight of the prebiotic obtained in step S6 are mixed evenly to obtain a composition for the prevention and treatment of childhood diarrhea.

[0132] Comparative Example 8

[0133] The difference from Example 5 is that no solid fine powder was added in step S5.

[0134] Specifically as follows:

[0135] S5. Ultrafine powder mixing: Montmorillonite powder is used as ultrafine powder.

[0136] Comparative Example 9

[0137] The difference from Example 5 is that montmorillonite powder was not added in step S5.

[0138] Specifically as follows:

[0139] S5. Ultrafine powder mixing: Dry 11 parts by weight of the solid from step S4, pulverize it through a 700-mesh sieve to obtain a fine powder, which is used as ultrafine powder.

[0140] Comparative Example 10

[0141] The difference from Example 5 is that no ultrafine powder was added in step S7.

[0142] Specifically as follows:

[0143] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 11 parts by weight of the fermentation product obtained in step S4 were added to 100 parts by weight of water, along with 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethyl cellulose, and 0.7 parts by weight of sodium stearoyl lactylate. The mixture was stirred and mixed for 20 minutes. Then, 200 parts by weight of fish oil were added, and the mixture was rapidly emulsified using a membrane with a pore size of 3 micrometers. 20 parts by weight of 5 wt% calcium chloride solution were added dropwise, and the mixture was solidified at room temperature for 25 minutes. The mixture was then freeze-dried to obtain fermentation product microspheres. 17 parts by weight of the fermentation product microspheres were mixed evenly with 2.5 parts by weight of the enzymatic hydrolysate obtained in step S1, 4 parts by weight of the aqueous extract obtained in step S2, and 2.5 parts by weight of the prebiotic obtained in step S6 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0144] Comparative Example 11

[0145] The difference from Example 5 is that no prebiotics were added in step S7.

[0146] Specifically as follows:

[0147] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 11 parts by weight of the fermentation product obtained in step S4 were added to 100 parts by weight of water, along with 8.5 parts by weight of sodium alginate, 4 parts by weight of sodium carboxymethyl cellulose, and 0.7 parts by weight of sodium stearoyl lactylate. The mixture was stirred and mixed for 20 minutes. Then, 200 parts by weight of fish oil were added, and the mixture was rapidly emulsified using a membrane with a pore size of 3 micrometers. 20 parts by weight of 5 wt% calcium chloride solution were added dropwise, and the mixture was solidified at room temperature for 25 minutes. The mixture was then freeze-dried to obtain fermentation product microspheres. 17 parts by weight of the fermentation product microspheres were mixed evenly with 2.5 parts by weight of the enzymatic hydrolysis product obtained in step S1, 4 parts by weight of the aqueous extract obtained in step S2, and 13.5 parts by weight of the ultrafine powder obtained in step S5 to obtain a composition for the prevention and treatment of childhood diarrhea.

[0148] Comparative Example 12

[0149] Compared with Example 5, the difference is that step S7 did not involve embedding, and the components were simply mixed.

[0150] Specifically as follows:

[0151] S7. Preparation of a composition for the prevention and treatment of childhood diarrhea: 17 parts by weight of fermentation product, 2.5 parts by weight of enzymatic hydrolysate obtained in step S1, 4 parts by weight of aqueous extract obtained in step S2, 13.5 parts by weight of ultrafine powder obtained in step S5, and 2.5 parts by weight of prebiotic obtained in step S6 are mixed evenly to obtain a composition for the prevention and treatment of childhood diarrhea.

[0152] The therapeutic effect of test example 2 on mice with diarrhea

[0153] 1. Grouping of animals:

[0154] KM mice (20-25g) were randomly divided into a normal group, a model group, Example 2-7 groups, and Comparative Examples 1-12 groups after 3 days of acclimatization in the laboratory. Each group consisted of 10 mice, half male and half female, and they were housed separately.

[0155] 2. Model making:

[0156] (1) Preparation of antibiotic mixture: Prepare an antibiotic mixture with a concentration of 65g / L by mixing gentamicin sulfate injection (produced by Yichang Renfu Pharmaceutical Co., Ltd.) and cefadroxil capsules (produced by Suzhou Zhonghua Pharmaceutical Co., Ltd.). Prepare and use immediately.

[0157] (2) Except for the normal group, the other groups were given 25 mL / (kg·d) of antibiotic mixture by gavage, and the normal group was given an equal amount of sterile water twice a day for 5 days. The model was successful when the mice showed lethargy, loose and wet feces and unclean perianal area.

[0158] 3. Treatment methods:

[0159] After successful modeling, the oral administration of antibiotic mixture was stopped. Based on the effective dose per surface area of ​​mice, mice in Examples 2-7 and Comparative Examples 1-12 were administered the corresponding prepared bacterial powder or composition by oral administration at 1 g / (mouse·day). Mice in the normal group and model group were given an equal amount of sterile water, twice a day, for 7 days.

[0160] 4. Stool consistency (dryness / wetness)

[0161] The stains formed on the filter paper by fecal contamination are classified into four levels: Level 1 is a stain with a diameter < 1.0 cm, indicated by "+"; Level 2 is a stain with a diameter of 1.0 to 1.9 cm, indicated by "++"; Level 3 is a stain with a diameter of 2.0 to 3.0 cm, indicated by "+++"; and Level 4 is a stain with a diameter > 3.0 cm, indicated by "++++".

[0162] The results are shown in Table 2.

[0163] Table 2

[0164]

[0165]

[0166] Note: "+" indicates the degree of dryness of the stool; the more "+", the higher the moisture content.

[0167] As shown in the table above, the moisture content of mouse feces was significantly reduced after treatment with the bacterial powder prepared in Example 2 of this invention or the composition for preventing and treating infantile diarrhea prepared in Examples 3-5.

[0168] 5. Diarrhea rate

[0169] According to *Pharmacological Experimental Methodology* (4th Edition), the diarrhea rate of mice was determined on days 0, 3, and 7 of the experiment. The calculation formula is as follows:

[0170] Diarrhea rate = (Number of animals with diarrhea / Total number of animals in the group) × 100%

[0171] The results are shown in Table 3.

[0172] Table 3

[0173]

[0174]

[0175] As shown in the table above, the diarrhea rate in mice was significantly reduced after treatment with the bacterial powder prepared in Example 2 of this invention or the compositions for preventing and treating childhood diarrhea prepared in Examples 3-5.

[0176] 6. Inflammatory factor markers

[0177] Twenty-four hours after the last administration, blood was collected from the eyeballs of mice in each group and serum was separated. After the mice were sacrificed, a portion of the small intestine was removed, and serum from each group was collected and small intestinal homogenate was prepared. The levels of inflammatory cytokines TNF-α and IL-6 in the serum and small intestinal tissue homogenate supernatant were measured according to the ELISA kit instructions.

[0178] The results are shown in Table 4.

[0179] Table 4

[0180]

[0181]

[0182] Note: * indicates P < 0.05 compared to the normal group; # indicates P < 0.05 compared to the model group.

[0183] As shown in the table above, after treatment with the bacterial powder prepared in Example 2 of this invention or the composition for preventing and treating infantile diarrhea prepared in Examples 3-5, the inflammatory factors in the serum and intestinal tissue of mice decreased significantly.

[0184] Compared to Example 5, Examples 6 and 7 use a single cellulase or pectinase as the complex enzyme. Comparative Example 3, compared to Example 5, involves enzymatic hydrolysis in step S1. The diarrhea rate decreased, stool moisture increased after treatment, and inflammatory factor indicators improved. This invention, through enzymatic hydrolysis using a complex enzyme (cellulase and pectinase), promotes cell wall rupture and the dissolution of active components of traditional Chinese medicine, thereby significantly improving efficacy. After water extraction, most of the water-soluble components are extracted.

[0185] Compared with Example 5, Comparative Examples 1 and 2 did not include kudzu root or licorice in step S1. The diarrhea rate decreased, but stool moisture increased and inflammatory factor indicators improved after treatment. Kudzu root contains puerarin, xyloside, soy isoflavones, β-sitosterol, arachidic acid, and other components, possessing antipyretic, antispasmodic, intestinal-constricting, and mildly blood circulation-improving effects. Licorice contains glycyrrhizic acid, glycyrrhizin, isoglycyrrhizin, and other components, possessing detoxifying, anti-inflammatory, anti-allergic, expectorant, antitussive, gastric mucosal-protective, gastric ulcer-treating, antispasmodic, and liver-damaging effects. The combination of these two ingredients effectively reduces fever, improves blood circulation, reduces inflammation, and protects the gastric mucosa.

[0186] Compared to Example 5, Comparative Example 4 did not perform step S2, and no water extract was added in step S7. The diarrhea rate decreased, but stool moisture increased after treatment, and inflammatory factor indicators improved. The water extracts of kudzu root and licorice contain abundant active substances that effectively reduce fever, improve blood circulation, reduce inflammation, and protect the gastric mucosa.

[0187] Compared with Example 5, Comparative Examples 5 and 6 did not involve inoculation with *Lactobacillus reuteri* FPH2951 or *Lactobacillus acidophilus* LA16 in step S4. Comparative Example 7, compared with Example 5, did not undergo fermentation in step S4, and did not add fermentation product microspheres in step S7. The diarrhea rate decreased, but stool moisture increased after treatment, and inflammatory factor indicators improved. The probiotic composition of this invention not only breaks down the cell walls of traditional Chinese medicines through fermentation, promoting the dissolution of active components in kudzu root and licorice, thus improving efficacy, but also enhances the patient's digestive and immune capabilities. During drug use, it can maintain the balance of intestinal flora, improve the intestinal environment, repair the intestinal mucosa, improve clinical diarrhea symptoms, optimize the ability to resist pathogens, control the regeneration of pathogens in the patient's body, eliminate residual bacteria, and shorten the patient's recovery time and disease duration. Among them, *Lactobacillus reuteri* FPH2951 has a high survival rate against gastric acid, can secrete beneficial substances to promote the proliferation and colonization of probiotics in the body, inhibit the colonization of harmful bacteria, build a biological barrier on the surface of the gastrointestinal mucosa of the child to prevent the invasion of harmful bacteria, improve the intestinal environment of the child, avoid the continuous reproduction and growth of harmful bacteria, and ultimately improve the state of the intestinal flora of the child and relieve diarrhea symptoms. *Saccharomyces boulardii* powder is a non-pathogenic fungal microecological probiotic that can release a large amount of polyamines in the body, increase the activity of metabolic enzymes such as α-glucosidase and alkaline phosphatase, delay the growth and reproduction of pathogenic microorganisms, prevent pathogenic microorganisms from invading and damaging mucosal epithelial cells, generate short peptide proteins to degrade bacterial toxins, play a role in resisting pathogenic microorganisms, improve the intestinal microecological environment, and relieve symptoms such as diarrhea in the child. The addition of Lactobacillus acidophilus LA16 can inhibit the adhesion of harmful bacteria to the intestinal mucosa, while also exhibiting high resistance to gastric acid, improving the gastrointestinal microecological environment of children, maintaining a stable number of intestinal flora, inhibiting inflammation, reducing diarrhea, and improving immune dysfunction in children. Lactobacillus reuteri FPH2951 alone has already shown good preventive, alleviating, and therapeutic effects. The synergistic combination of the three probiotics can effectively prevent and reduce infections in patients, improve the gastrointestinal microecological environment of children, inhibit inflammation, reduce diarrhea, and improve immune dysfunction in children.

[0188] Compared with Example 5, Comparative Examples 8 and 9 did not add solid fine powder or montmorillonite powder in step S5. Compared with Example 5, Comparative Example 10 did not add ultrafine powder in step S7. The diarrhea rate decreased, and the stool moisture increased after treatment. Montmorillonite powder has the effect of strengthening the mucosal coverage of the digestive tract and can effectively inhibit viruses, bacteria and toxins in the digestive tract, thereby increasing the mucosal barrier defense function of the digestive tract in children and promoting the recovery of gastrointestinal function. However, it can easily cause constipation and other problems. Therefore, this invention dries the solid residue after fermentation and extraction of traditional Chinese medicine, ultrafinely pulverizes it to obtain fine powder of traditional Chinese medicine, mixes it evenly with montmorillonite powder, and adds it to the composition. It can work synergistically with montmorillonite powder to strengthen the mucosal coverage of the digestive tract. At the same time, the fiber of traditional Chinese medicine can promote intestinal peristalsis, relieve constipation caused by montmorillonite powder, and expel harmful substances fixed by montmorillonite powder from the intestine, thereby making the composition safer and more effective.

[0189] Compared to Example 5, Comparative Example 11 did not include prebiotics in step S7. The diarrhea rate decreased, but stool moisture increased after treatment, and inflammatory factor indicators rose. The prebiotics are a mixture of fructooligosaccharides and xylooligosaccharides. By selectively stimulating the growth and proliferation of the probiotic colonies added in this invention, they reduce the production of inflammatory factors, promote antioxidant responses, improve the gastrointestinal microecological environment of the children, maintain a stable number of intestinal flora, reduce diarrhea, and improve the children's immune dysfunction.

[0190] Compared to Example 5, Comparative Example 12 did not involve encapsulation in step S7; the components were simply mixed. The diarrhea rate decreased, but stool moisture increased after treatment, and inflammatory factor indicators improved. The fermentation product of this invention, after encapsulation, can resist the gastric acid environment to a certain extent, further increasing the content of probiotics directly delivered to the intestines, thereby achieving better preventative, alleviating, and therapeutic effects. The resulting composition for preventing and treating childhood diarrhea has advantages such as good efficacy, rapid onset of action, and short treatment cycle, and has broad application prospects.

[0191] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of Lactobacillus reuteri FPH2951 in the preparation of a composition for treating diarrhea caused by gentamicin sulfate and cephalexin, wherein the Lactobacillus reuteri FPH2951 is classified as Lactobacillus reuteri. Limosilactobacillus reuteri, The accession number is GDMCC NO.62946, the accession date is November 3, 2022, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology, postcode: 510070.

2. The use according to claim 1, characterized in that, The 16S rRNA sequence of *Lactobacillus reuteri* FPH2951 is shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that, The Lactobacillus reuteri FPH2951 is a Gram-positive rod-shaped bacterium. On MRS medium, its colonies are round with neat edges, milky white, raised, smooth, and do not produce spores.

4. A method for preparing a composition for treating diarrhea caused by gentamicin sulfate and cefadroxil, characterized in that, Includes the following steps: S1. Enzymatic hydrolysis: Kudzu root and licorice were washed, dried, and pulverized to obtain Chinese medicine powder. The powder was added to water, and a compound enzyme was added for enzymatic hydrolysis. The enzyme was inactivated, filtered, and the solid was retained. The filtrate was freeze-dried to obtain the enzymatic hydrolysis product. The compound enzyme was cellulase and pectin methyl esterase. S2. Water extraction: Add the solid from step S1 to water, heat to boiling and extract, filter, retain the solid, dry the filtrate to obtain the water extract; S3. Strain activation: The probiotics are inoculated into Gao's medium and activated to obtain a strain seed solution; the probiotics are Lactobacillus reuteri FPH2951 or a combination of Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16; S4. Fermentation: Add the solid from step S2 to water, sterilize, inoculate with the probiotic seed liquid prepared in step S3, ferment and culture, filter, retain the solid, freeze dry, and obtain the fermentation product; S5. Ultrafine powder mixing: The solid from step S4 is dried, pulverized and sieved to obtain fine powder, which is then mixed evenly with montmorillonite powder to obtain ultrafine powder; S6. Preparation of prebiotics: Mix fructooligosaccharides and xylooligosaccharides evenly to obtain prebiotics; S7. Preparation of the composition: The fermentation product obtained in step S4 is added to water, sodium alginate, sodium carboxymethyl cellulose, and emulsifier are added, and the mixture is stirred and mixed evenly. The mixture is then added to fish oil, rapidly emulsified with a membrane, and calcium chloride solution is added dropwise. The mixture is then solidified at room temperature and freeze-dried to obtain fermentation product microspheres. These microspheres are then mixed evenly with the enzymatic hydrolysate obtained in step S1, the aqueous extract obtained in step S2, the ultrafine powder obtained in step S5, and the prebiotic obtained in step S6 to obtain a composition for treating diarrhea caused by gentamicin sulfate and cephalexin.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass ratio of kudzu root and licorice is 5-7:3-5; the solid-liquid ratio of the herbal powder and water is 1:5-10 g / mL; the mass ratio of cellulase and pectin methyl esterase in the compound enzyme is 7-10:5; the amount of the compound enzyme added is 2-3% of the total mass of the system; the enzymatic hydrolysis temperature is 45-50℃; and the time is 2-4 h. In step S2, the mass ratio of solid to water is 10-15:50-70; the heating and boiling extraction time is 1-3 h; and the operation is repeated 1-2 times. In step S3, the activation culture temperature is 36-38℃; the time is 18-24 h; and the bacterial count of the inoculum is 10. 8 -10 9 cfu / mL.

6. The preparation method according to claim 4, characterized in that, In step S4, the mass ratio of solid to water is 20-30:100-120, and the fermentation conditions are 36-38℃, 50-70 r / min, and fermentation time of 48-56 h; in step S5, the sieve mesh size is 500-1000 mesh, and the mass ratio of the solid powder to montmorillonite powder is 10-12:15-20; in step S6, the mass ratio of fructooligosaccharide to xylooligosaccharide is 7-10:3-5; in step S7, the fermentation product, sodium alginate, and carboxymethyl cellulose... The mass ratio of sodium to emulsifier is 10-12:7-10:3-5:0.5-1. The emulsifier is selected from at least one of lecithin, sodium stearoyl lactylate, calcium stearoyl lactylate, diacetyl tartaric acid monoglyceride, sucrose fatty acid ester, and distilled monoglyceride. The pore size of the rapid membrane is 1-5 micrometers. The curing time at room temperature is 20-30 minutes. The mass ratio of fermentation product microspheres, enzymatic hydrolysis products, water extracts, ultrafine powder, and prebiotics is 15-20:2-3:3-5:12-15:2-3.

7. The preparation method according to claim 4, characterized in that, When the probiotic is Lactobacillus reuteri FPH2951, the inoculation amount of the probiotic seed solution is 2-3%.

8. The preparation method according to claim 4, characterized in that, When the probiotic is a combination of Lactobacillus reuteri FPH2951, Saccharomyces boulardii, and Lactobacillus acidophilus LA16, the inoculation amount of the probiotic seed solution is as follows: 2-3% for Lactobacillus reuteri FPH2951, 0.5-1% for Saccharomyces boulardii, and 1-2% for Lactobacillus acidophilus LA16.

9. A composition for treating diarrhea caused by gentamicin sulfate and cefadroxil, prepared by any one of claims 4-8.

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