WO2019227417A1 - 一种组合物及其应用 - Google Patents

一种组合物及其应用 Download PDF

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WO2019227417A1
WO2019227417A1 PCT/CN2018/089317 CN2018089317W WO2019227417A1 WO 2019227417 A1 WO2019227417 A1 WO 2019227417A1 CN 2018089317 W CN2018089317 W CN 2018089317W WO 2019227417 A1 WO2019227417 A1 WO 2019227417A1
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composition
composition according
bacteria
food
lactobacillus
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PCT/CN2018/089317
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English (en)
French (fr)
Inventor
邹远强
薛文斌
肖亮
李晓平
余靖宏
刘传
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深圳华大生命科学研究院
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Priority to CN201880093363.0A priority Critical patent/CN112236155A/zh
Priority to PCT/CN2018/089317 priority patent/WO2019227417A1/zh
Publication of WO2019227417A1 publication Critical patent/WO2019227417A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system

Definitions

  • the present application relates to the field of microbial preparations, and in particular, to a microorganism or a microorganism-related composition and application thereof.
  • IBD Inflammatory bowel disease
  • UC ulcerative enteritis
  • CD Crohn's disease
  • Ulcerative colitis is an important type of inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the cause of the disease is unknown.
  • the main lesion is in the submucosa of the colonic mucosa, which is a chronic disease.
  • Intestinal diseases Based on current research, the main causes of ulcerative enteritis are host genetic susceptibility, intestinal flora, and intestinal mucosal immune response.
  • the clinical and pathological manifestations are persistent abdominal pain, diarrhea, and mucus and bloody stools, and the condition is repeated.
  • UC in China The number of patients has a significant upward trend.
  • the clinical medications for UC include salicylic acid drugs, adrenal glucocorticoid drugs and immunosuppressants. These three types of drugs can alleviate UC to a certain extent, but they also have shortcomings.
  • Salicylic acid drugs can better inhibit prostaglandin synthesis and scavenge oxygen free radicals in order to alleviate the inflammatory response, but they can only be relieved in a short period of time and cannot be cured.
  • Salicylic acid western medicines commonly used in the clinical treatment of UC are mainly Sulfasalazine (SASP), mainly for patients with mild, moderate and chronic UC.
  • SASP Sulfasalazine
  • salicylic acid drugs also have many side effects, such as triggering gastrointestinal reactions, headaches, increased reticulocytes, decreased sperm, and rashes, liver toxicity, leukopenia, and anemia caused by allergic reactions. These drugs also have Antibacterial effect, easy to cause flora disorder and enhanced drug resistance.
  • Adrenal glucocorticoids are the first choice for patients with severe or explosive UC. Typical drugs such as betamethasone; however, adrenal glucocorticoids can cause side effects such as metabolic disorders and water retention. They can only be used as emergency medication. Cannot be taken for a long time.
  • Immunosuppressive agents such as cyclosporine, can suppress UC by inhibiting the production of IL-2 in T cells, which affects the progress of the immune response; however, immunosuppressive therapy is highly drug-dependent, has a long treatment cycle, and is easy Causes nephrotoxicity and secondary infections, and can only be used as a means of adjuvant therapy.
  • the purpose of this application is to provide a composition and its use.
  • composition comprising Lactobacillus gasseri and / or a metabolite thereof, and Lactobacillus acidophilus and / or a metabolite thereof.
  • Lactobacillus gasseri and Lactobacillus acidophilus can prevent and treat inflammation and inflammation-related diseases, especially it can effectively prevent and treat ulcerative enteritis; research shows that, Its preventive or therapeutic preventive effect is mainly composed of two aspects.
  • Lactobacillus gasseri and Lactobacillus acidophilus improve the intestinal microecology of the body and form an ecological protective barrier composed of beneficial bacteria, thereby playing inflammation or inflammation.
  • Prevention and treatment effects of related diseases on the other hand, the metabolites of Lactobacillus gasseri and Lactobacillus acidophilus are used as probiotic materials to prevent and treat inflammation or inflammation-related diseases. Therefore, the composition of the present application is mainly used for preventing or treating inflammation or inflammation-related diseases, especially for preventing or treating ulcerative enteritis or related diseases.
  • the composition of Lactobacillus gasseri and Lactobacillus acidophilus is used to improve the intestinal microecology of the body through two bacteria to form an ecological protection composed of beneficial bacteria. Barrier to prevent and treat ulcerative enteritis. It can be understood that this micro-ecological improvement not only has prevention and treatment effects on ulcerative enteritis, but also has effects on other micro-ecological-related diseases, such as common enteritis or gastritis; therefore, the composition of the present application can be used for Prevent or treat inflammation or inflammation-related diseases.
  • Lactobacillus gasseri is Lactobacillus gasseri TF08-1 with deposit number GDMCC 60092
  • the Lactobacillus acidophilus is Lactobacillus acidophilus AM13-1 with deposit number GDMCC 60091.
  • Lactobacillus gasseri and Lactobacillus acidophilus can prevent and treat inflammation and inflammation-related diseases
  • These two strains are the two strains with better combined use effect found in the research process of this application, so the two strains have been deposited separately.
  • Lactobacillus gasseri and Lactobacillus acidophilus from other strains can be used to achieve or even exceed the effects of the two strains of the present application under the inventive idea of the present application; Or if the prevention or treatment of inflammation-related diseases is less demanding, other strains of Lactobacillus gasseri and Lactobacillus acidophilus can also be used.
  • composition of the present application further comprises Collins shenzhenii and / or its metabolites.
  • the Collinsella Shenzhen is the Collinsella Shenzhen TF06-26 with the deposit number GDMCC 60090.
  • composition of the present application further includes Roseburia inulinivorans and / or its metabolites.
  • Roseburia inulinivorans is Roseburia inulinivorans with accession number DSM 16841.
  • composition of the present application further includes butyric acid and / or a metabolite thereof.
  • the butyric acid bacterium is butyric acid TF01-11 having the accession number CGMCC 10984.
  • composition of the present application further includes Bifidobacterium pseudosmallii and / or a metabolite thereof.
  • the Bifidobacterium pseudominis is Bifidobacterium pseudominis TM12-14 with the accession number GDMCC 60089.
  • the composition of the present application further comprises Monomonas monomorpha and / or its metabolites, and human fecal anaerobic coryneform bacteria and / or its metabolites.
  • the Monomorpha monomorpha is Monomonas monomorpha AF24-28AC of deposit number GDMCC 60093
  • the human fecal anaerobic coryneform bacteria is human fecal anaerobic coryneform AM25-6 of deposit number GDMCC 60087.
  • Lactobacillus garneri and Lactobacillus acidophilus can prevent and treat inflammation and inflammation-related diseases, without affecting the effect of the combined use of Lactobacillus garneri and Lactobacillus acidophilus.
  • composition of the present application further contains other probiotics and / or prebiotics.
  • the key to the composition of the present application is that the combined use of both Lactobacillus gasseri and Lactobacillus acidophilus can prevent and treat inflammation and inflammation-related diseases. It is understood that without affecting Lactobacillus gasseri and acidophilus In the case of the combined effect of the two Lactobacillus, other probiotics or prebiotics can also be added to make the composition of the present application have more functions or to enhance its original efficacy. These probiotics or prebiotics can be used The probiotics or prebiotics reported in the existing research are not specifically limited here.
  • the prebiotics of the composition of the present application are selected from the group consisting of fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), and lactulose oligosaccharide (LACT). ), Soy oligosaccharides (SOS), inulin and oligosaccharides.
  • FOS fructooligosaccharide
  • GOS galactooligosaccharide
  • XOS xylooligosaccharide
  • LACT lactulose oligosaccharide
  • SOS Soy oligosaccharides
  • inulin and oligosaccharides are selected from the group consisting of fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), and lactulose oligosaccharide (LACT).
  • the composition of the present application further contains a substance that helps maintain the viability of at least one of each strain in the composition.
  • the substance that helps maintain the viability of the strain is selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, dibutylmethyltoluene, tocopherol, and bamboo leaf At least one of an oxide, D-isoascorbic acid or its sodium salt, sodium ascorbate, calcium ascorbate, a phospholipid, vitamin C, and vitamin E.
  • composition of the present application further comprises a pharmaceutically or food-acceptable carrier or excipient.
  • composition of the present application has the effect of preventing and treating inflammation and inflammation-related diseases.
  • the composition of the present application is mainly used to achieve the prevention and treatment effect. Therefore, a pharmaceutically or food-acceptable carrier or excipient may also be included in the composition for ease of use.
  • a pharmaceutically or food-acceptable carrier or excipient is selected from glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, poly At least one of oxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, and physiological saline.
  • the other side of the present application discloses the application of the composition of the present application in the preparation of food, health products, food additives or medicines for treating or preventing inflammation or inflammation-related diseases.
  • composition of the present application has the effects of preventing and treating inflammation and inflammation-related diseases.
  • the composition of the present application can be made into various foods, health products, food additives or medicines.
  • composition of the present application can be used in particular for preparing food, health products, food additives or drugs for treating or preventing ulcerative enteritis or related diseases.
  • composition of the present application discloses the use of the composition of the present application in the preparation of a food, a health product, a food additive, or a medicine for controlling weight loss in a mammal.
  • the weight loss of mammals especially the weight loss of mammals due to inflammation.
  • the inflammation is ulcerative enteritis, that is, controlling weight loss caused by ulcerative enteritis in a mammal.
  • composition of the present application discloses the use of the composition of the present application in the preparation of a food, a health product, a food additive, or a medicine for reducing the disease activity index of a mammal.
  • Another aspect of the present application discloses the application of the composition of the present application in the preparation of foods, health products, food additives, or medicines for improving intestinal lesions in mammals.
  • composition of the present application can prevent and treat inflammation and inflammation-related diseases, and the key lies in being able to control the weight loss caused by inflammation and inflammation-related diseases and reduce the disease activity index caused by inflammation and inflammation-related diseases And improve mammalian intestinal pathology; therefore, the composition of the present application can also be used alone to prepare foods, health products, and food additives that control mammalian weight loss, reduce mammalian disease activity index, or improve mammalian intestinal pathology. Or medicine.
  • Another aspect of the present application discloses a method for treating or preventing inflammation or inflammation-related diseases using the composition of the present application.
  • Another aspect of the present application discloses a method for controlling weight loss in a mammal using the composition of the present application.
  • Another aspect of the present application discloses a method for reducing the disease activity index of a mammal using the composition of the present application.
  • Another aspect of the present application discloses a method for improving intestinal pathological changes in mammals by using the composition of the present application.
  • composition of the present application is mainly used to treat or prevent inflammation or inflammation-related diseases, control the weight loss of mammals, reduce the mammalian disease activity index, or improve the mammalian intestine.
  • Another aspect of the present application discloses a food, which contains the composition of the present application.
  • the food is a lactic acid drink or a soy milk drink.
  • the food in the present application is an edible article existing in any form in a broad sense, and is not limited to a lactic acid drink or a soy milk drink.
  • the food may be a fermented food or an animal feed.
  • the food of the present application contains the composition of the present application, it also has the effects of treating or preventing inflammation or inflammation-related diseases, controlling weight loss of mammals, reducing mammalian disease activity index, and improving mammalian intestinal diseases. .
  • the key to the food of the present application is that it contains the composition of the present application.
  • the specific form of the food such as solid, liquid, etc., it can be determined according to different food products or use requirements, and is not specifically limited here.
  • the composition of the present application is mainly made into a common lactic acid drink or soy milk drink for easy drinking; of course, it can also be made into solid foods such as milk slices and cheese bars, etc. Be specific.
  • the amount of active bacteria or ingestion of Lactobacillus gasseri and Lactobacillus acidophilus in the food of the present application is not specifically limited, and in practical applications, it can be flexibly selected according to actual conditions.
  • Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 as examples, the research in this application shows that the daily intake of Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 is 10 9 cfu
  • the composition of 0.2 mL / mL has a good therapeutic effect on ulcerative enteritis.
  • the dose of the active bacteria can be used as a reference amount or a reference intake of food, health products, food additives, or medicines.
  • Another aspect of the present application discloses a health product, which contains the composition of the present application.
  • the health product of the present application contains the composition of the present application, it also has the advantages of treating or preventing inflammation or inflammation-related diseases, controlling weight loss of mammals, reducing mammalian disease activity index, and improving mammalian intestinal diseases. effect.
  • Another aspect of the present application discloses a food additive containing the composition of the present application.
  • composition of the present application can be used in combination with common food materials.
  • cereals and potatoes include rice, noodles, miscellaneous grains, potatoes including potatoes, sweet potatoes, etc .; animal foods, including meat, poultry, fish, milk, eggs, etc .; beans and their products, including soybeans and other dried Beans; vegetables and fruits, including fresh beans, rhizomes, leafy vegetables, eggplants, etc .; pure thermal energy foods, including animal and vegetable oils, starch, edible sugar, and alcohol; therefore, the composition of the present application can be used alone as a food additive Or the modulator is added to various foods and eaten directly to play a therapeutic or preventive effect on inflammation and related diseases.
  • Another aspect of the present application discloses a medicine containing the composition of the present application.
  • the medicine is a tablet, granule, powder, enteric solvent, solution or suspension.
  • the medicine of the present application has the composition of the present application, so it has the effect of treating or preventing inflammation and related diseases; the medicine of the present application may be the composition of the present application alone, or it may be combined with other inflammations.
  • the drugs can be used in combination as long as they do not affect the activity of each other. It can be understood that, as long as the medicine of the present application does not affect the activity of each strain in the composition, the medicine can adopt various existing dosage forms.
  • the medicines in the present application may further include auxiliary materials commonly used in medicines or dosage forms, such as stabilizers, wetting agents, emulsifiers, adhesives, isotonic agents, and the like.
  • the medicine of the present application can be administered in any form of oral solution, tablet, injection, orally disintegrating tablet, lyophilized powder preparation or enteric solvent type.
  • An enteric solvent type such as a capsule or an enteric tablet, is preferred so that the active ingredient of the medicine, that is, the microorganism can pass through the stomach smoothly without being destroyed by gastric acid. More preferably, the medicine of the present application can be made into enteric tablets for oral use.
  • the enteric solvent type in the present application refers to a pharmaceutical dosage form that does not disintegrate in gastric juice and can be disintegrated and absorbed in intestinal fluid.
  • the enteric solvent type includes capsules and enteric tablets. Among them, capsules are formed by powdered medicines enclosed in capsule shells that are allowed for conventional medicines; enteric-coated tablets are formed by wrapping an enteric coating on the outside of ordinary tablet medicines.
  • the "enteric coating” referred to in the present application is referred to as "enteric coating”, and includes all the coatings allowed for conventional drugs. These coatings are not degraded by gastric acid, but can be fully decomposed in the small intestine and quickly release the drug of the present application.
  • the thickness of the casing is 5-100 ⁇ m, and the ideal thickness is 20-80 ⁇ m.
  • the casing components are selected from conventional materials which are publicly known.
  • the active bacteria content or the dosage of each strain of the composition in the medicine of the present application is not particularly limited. In practical applications, it can be flexibly selected according to the health status of the subject to be administered. However, the study of this application shows that daily intake of 0.2 mL of a composition in which the concentrations of Lactobacillus garnerii TF08-1 and Lactobacillus acidophilus AM13-1 are both 10 9 cfu / mL has a good therapeutic effect on ulcerative enteritis This amount can be used as a reference for the content of active bacteria in the drug or the dosage.
  • composition of the present application through the combined use of both Lactobacillus gasseri and Lactobacillus acidophilus, can have a good therapeutic and preventive effect on inflammation, especially ulcerative enteritis, and its related diseases. Treatment and prevention provide a new composition that is safe, effective, has little toxic and side effects, and does not easily develop resistance.
  • FIG. 1 is a control group, a model group, a VSL # 3 group, a probiotic composition treatment group, a combination of bacteria 1, a combination of bacteria 2, a combination of bacteria 3, a combination of bacteria 4, and a combination of bacteria 5 Change curve of body weight;
  • FIG. 2 is a control group, a model group, a VSL # 3 group, a probiotic composition treatment group, a combination of bacteria 1, a combination of bacteria 2, a combination of bacteria 3, a combination of bacteria 4, and a combination of bacteria 5 Change curve of DAI index.
  • the Lactobacillus gasseri TF08-1 of the present application was deposited at the Guangdong Provincial Center for Microbial Strains on October 13, 2016.
  • the address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong province, China.
  • the number is GDMCC 60092.
  • Lactobacillus acidophilus AM13-1 was deposited in the Guangdong Provincial Center for Microbial Strains on October 13, 2016.
  • the address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong province, China, and the deposit number is GDMCC. 60091.
  • Collinsella Shenzhen TF06-26 was deposited at the Guangdong Provincial Center for Microbial Strains on October 13, 2016.
  • the location of the depository is the fifth floor of the Provincial Microbiology Institute Laboratory Building, 100 Xianlie Middle Road, Guangzhou, Guangdong province, China, and the deposit number is GDMCC 60090. .
  • Roseburia inulinivorans DSM 16841 was purchased from the German Microbial Collection.
  • Butyric acid TF01-11 was deposited on June 16, 2015 at the General Microbiology Center of the China Microbial Strain Collection Management Committee. The deposit location is Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The number is CGMCC 10984.
  • Pseudomonas aeruginosa TM12-14 was deposited in the Guangdong Provincial Center for Microbial Strains on October 13, 2016.
  • the address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong, China.
  • the number is GDMCC 60089.
  • Monomorpha monomorpha AF24-28AC was deposited at the Guangdong Provincial Center for Microbial Strains on October 13, 2016.
  • the address of the deposit unit is the fifth floor of the Experimental Building of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong, China. It is GDMCC 60093.
  • ulcerative enteritis is closely related to the composition of intestinal microorganisms.
  • the imbalance of intestinal bacteria is closely related to the intestinal inflammatory response.
  • the excessive proliferation of harmful bacteria can trigger an inflammatory response.
  • beneficial bacteria constitute the first biological barrier of the intestine.
  • the composition includes Lactobacillus gasseri and / or its metabolites, and Lactobacillus acidophilus and / or its metabolites.
  • the composition of the present application not only has the function of treating and preventing ulcerative enteritis, but also has effects on other microecological-related diseases, such as common enteritis or gastritis; therefore, the composition of the present application can be used to prevent or treat inflammation Or inflammation-related diseases.
  • a composition consisting of Lactobacillus gasseri TF08-1 with deposit number GDMCC 60092 and Lactobacillus acidophilus AM13-1 with deposit number GDMCC 60091 is superior to the treatment effect of ulcerative enteritis VSL # 3 composite probiotics produced by the American company Alfasigma can be used to prepare foods, health products, food additives or drugs for the treatment or prevention of inflammation and related diseases.
  • a mouse model of ulcerative enteritis induced by sodium sulfate dextran sulfate (abbreviated DSS) with a molecular weight of 36,000-50000 was used as the research object.
  • Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus were studied.
  • the therapeutic effect of AM13-1 composition on ulcerative enteritis was studied.
  • Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 are added probiotics to the composition of Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1, and use them in combination, these added probiotics include: Shenzhen Collins (Collinsella shenzhenensis) TF06-26, Roseburia inulinivorans DSM 16841, Butyribacter intestini TF01-11, Bifidobacterium pseudocatenulatum TM12-14, Monomonas monomorpha (Megamonas funiformis) AF24-28AC and Anaerofustis bacteriacorihominis AM25-6. details as follows:
  • Lactobacillus gasseri TF08-1 was isolated using PYG medium, and the isolation condition was 37 ° C anaerobic condition.
  • TF08-1 was cultured in PYG medium for 2 days. The colonies were white, low raised, nearly round, and wavy. The diameter of the colonies was about 1-2 mm. The microscopic morphology of the bacteria was rod-shaped. Gram-positive and non-producing. Spores and flagella. The strains were deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60092.
  • Lactobacillus gasseri TF08-1 The specific isolation and identification steps of Lactobacillus gasseri TF08-1 are as follows:
  • the isolated sample was from a fecal sample of a 16-year-old healthy female volunteer living in Shenzhen, Guangdong. The diet and physical conditions of the volunteer were recorded in detail.
  • the medium is PYG medium purchased from Huankai Microbial Technology Co., Ltd.
  • the specific ingredients are: peptone 5g, tryptic casein 5g, yeast powder 10g, beef paste 5g, glucose 5g, K 2 HPO 4 2g, Tween 80 1mL, Cysteine-HCl ⁇ H 2 O 0.5g, sodium sulfide 0.25g, heme 5mg, vitamin K 1 1 ⁇ L, inorganic salt solution 40mL, resazurin 1mg, distilled water 950mL, pH 6.8 ⁇ 7.0, 115 Sterilize at °C for 25min.
  • the solid medium was added with 1.5% agar and poured in an anaerobic operation box.
  • Each 1 L of the inorganic salt solution contains 0.25 g of CaCl 2 ⁇ 2H 2 O, 0.5 g of MgSO 4 ⁇ 7H 2 O, 1 g of K 2 HPO 4, 1 g of KH 2 PO 4 , 10 g of NaHCO 3 , and 2 g of NaCl.
  • the collected fresh stool samples were transferred to an anaerobic box, 0.2 g of stool was suspended in 1 mL of sterile phosphate buffered saline (abbreviated PBS), thoroughly mixed, and then subjected to gradient dilution, and 100 ⁇ L of the diluted solution was plate coated, 37 ° C
  • the isolated strain was identified by 16S rDNA to determine the species classification information of the strain.
  • the obtained isolated strain was cultured in liquid PYG medium for 24 hours, and 1 mL of the bacterial solution was centrifuged at 10,000 r / min for 5 min. The bacterial cells were collected, the genomic DNA of the strain was extracted, and 16S rDNA was amplified using the genomic DNA as a template. 16S rDNA was used. Universal primers.
  • the 16S rDNA PCR amplification system is: 10 ⁇ PCR buffer 3 ⁇ L, dNTP 2.5 ⁇ L, upstream primer 27F 0.5 ⁇ L, downstream primer 1492R 0.5 ⁇ L, Taq enzyme 0.3 ⁇ L, template 1 ⁇ L, ddH 2 O 18.2 ⁇ L.
  • the 16S rDNA amplification conditions are: pre-denaturation at 95 ° C for 4 min, and then enters 30 cycles: denaturation at 95 ° C for 30s, annealing at 57 ° C for 40s, and extension at 72 ° C for 1min to 30s.
  • the 16S rDNA PCR product was purified, sequenced at 3730 to obtain the 16S rDNA sequence of the strain, and then compared with the NCBI database.
  • the upstream and downstream primers of the 16S rDNA universal primer in this experiment are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.
  • the 16SrDNA sequence of the isolated strain TF08-1 showed the sequence shown in SEQ ID NO.3.
  • the NCBI blast comparison showed that the TF08-1 strain isolated in this example had the highest homology with Lactobacillus gasseri, with a similarity of 99.9%. Therefore, TF08-1 was judged as Lactobacillus gasseri and was named Lactobacillus gasseri TF08-1 And preserve it.
  • SEQ ID NO. 2 5'-TAGGGTTACCTTGTTACGACTT-3 '.
  • Lactobacillus acidophilus AM13-1 was isolated using PYG medium under 37 ° C anaerobic conditions.
  • the colonies of AM13-1 cultured in PYG medium for 2 days were white, raised, thick, opaque, round, with neat edges, about 2-3 mm in diameter.
  • the microscopic morphology of the cells was rod-shaped and Gram-positive. No spores and flagella.
  • the strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60091.
  • Lactobacillus acidophilus AM13-1 The specific isolation and identification steps of Lactobacillus acidophilus AM13-1 are as follows:
  • the isolated sample was from a fecal sample from a healthy male in Shenzhen.
  • the isolation process of Lactobacillus acidophilus AM13-1 was as follows:
  • Genomic DNA was extracted, and 16S rDNA was amplified using DNA as a template.
  • 16S rDNA universal primers were used for PCR amplification.
  • the amplified PCR product was purified and sequenced at 3730 to obtain the full length 16S rDNA sequence of AM13-1.
  • AF13- The 16S rDNA sequence of 1 is aligned in the NCBI database.
  • the 16S rDNA universal primers, PCR amplification system and conditions of this experiment are the same as "16. rDNA identification of strain 1.1.3".
  • the 16SrDNA sequence of the isolated strain AM13-1 showed the sequence shown in SEQ ID NO.4. NCBI blast comparison results showed that the AM13-1 strain isolated in this example had the highest homology with Lactobacillus acidophilus, with a similarity of 100%. Therefore, AM13-1 was judged as Lactobacillus acidophilus and named Lactobacillus acidophilus AM13-1 And preserve it.
  • the Collinella TF06-26 of Shenzhen was cultured in PYG medium, and the culture condition was 37 ° C anaerobic condition.
  • the colonies of Shenzhen Collins TF06-26 cultured for 2 days were white and raised, the colonies were thicker, and the diameter of the colonies was about 1-2 mm.
  • Bacterial cells are short rod-shaped under the microscope, Gram-negative, and free of spores and flagella. The strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60090.
  • the Collinsella TF06-26 of this test was isolated from a stool sample of a healthy young female volunteer in Shenzhen.
  • the culture medium used for this experiment is an anaerobic PYG medium purchased from Huankai Microbial Technology Co., Ltd.
  • the specific composition is 1L medium containing: peptone 5g, tryptic casein 5g, yeast powder 10g, beef extract 5g , Glucose 5g, K 2 HPO 4 2g, Tween 80 1mL, Cysteine-HCl ⁇ H 2 O 0.5g, heme 5mg, vitamin K 1 1 ⁇ L, inorganic salt solution 40mL, resazurin 1mg, distilled water 950mL, adjust pH to 6.8 ⁇ 7.0.
  • the sterilization conditions were autoclaved at 115 ° C for 25 minutes.
  • each liter of the inorganic salt solution contained 0.25 g of CaCl 2 ⁇ 2H 2 O, 0.5 g of MgSO 4 ⁇ 7H 2 O, 1 g of K 2 HPO 4, 1 g of KH 2 PO 4 , 10 g of NaHCO 3 , and 2 g of NaCl.
  • Preparation of PBS Weigh Nag 8g, KCl 0.2g, Na 2 HPO 4 ⁇ 12H 2 O 3.63g, KH 2 PO 4 0.24g, cysteine hydrochloride 0.5g, dissolve in 900mL double distilled water, and use hydrochloric acid Adjust the pH value to 7.4 with NaOH, add water to make up to 1L, pass in N 2 for deaeration for 30s, seal the anaerobic bottle, and then autoclave at 115 ° C for 25min before using.
  • the collected fresh stool samples were immediately transferred to the anaerobic box.
  • 16S rDNA sequencing was performed on the isolated purely cultured strains to obtain classification information of each strain.
  • the strain was cultured in liquid PYG medium for 24 hours to reach a concentration of about 10 8 cfu / ml.
  • Genomic extraction was performed on the bacterial solution, 16S rDNA PCR amplification was performed using the extracted genomic DNA as a template, and the obtained 16S rDNA was amplified.
  • the products were detected by electrophoresis, purified, and sequenced at 3730 to obtain the 16S rDNA sequence of the strains, and then compared with the EZBio Cloud database to identify the isolated strains.
  • the primers, PCR amplification system, and conditions for 16S rDNA PCR amplification in this test were the same as "16S rDNA identification of 1.1.3 strain".
  • the sequencing results showed that the 16SrDNA sequence of the TF06-26 strain isolated in this example was the sequence shown in SEQ ID NO.5.
  • the EZBio Cloud database comparison results show that the TF06-26 strain isolated in this example has the highest homology with Collinsella aerofaciens JCM 10188 purchased from the Japan Microbial Species Collection Center.
  • TF06-26 is catalase-negative, oxidase-negative, non-motile, growth temperature range is 25-45 ° C, growth pH range is 5.0-8.0, NaCl tolerance concentration is 2%, bile salt tolerance concentration is 0.3%.
  • TF06-26 and related reference bacteria were purchased from the Collinsella aerofaciens and JCM 10188 substrates of Japan Microbial Collection Center, API 20A and API 50CHL. See Table 1 for details. "+” In Table 1 indicates a positive reaction, "- "” Indicates a negative reaction, and "w” indicates a weak positive reaction.
  • TF06-26 and JCM 10188 are in lactose, sucrose, sucrose, galactose, fructose, mannose, arbutin, cellobiose, maltose, honey
  • disaccharides, trehalose, and 2-keto-gluconate are significantly different, which shows that TF06-26 and JCM 10188 are not the same species.
  • TF06-26 had 51% homology with JCM 10188.
  • the DNA hybridization value of TF06-26 and JCM 10188 is less than 70%, so TF06 -26 is a new strain that is different from known bacteria.
  • IBSP International Bacterial Classification Commission
  • this new strain was named Collinsella shenzhenzhenensis sp.nov, and TF06-26 was used as a model strain of this species. That is, the Collinella shenzhenensis TF06-26 in this example, and it is preserved
  • Roseburia inulinivorans DSM 16841 was cultured in a PYG anaerobic medium at 37 ° C.
  • the colonies cultured in PYG medium for 2 days were light yellow and about 1 mm in diameter.
  • the microscopic morphology of the fungus is short rod-shaped, Gram-positive, and does not produce spores and flagella.
  • the strain was purchased from the German Microbial Collection Center (DSMZ) and the deposit number was DSM 16841.
  • the butyric acid TF01-11 of this example was cultured in a PYG medium, and the culture conditions were 37 ° C anaerobic conditions. After 48 hours of culture of butyric acid TF01-11 on anaerobic PYG medium, the colonies appeared off-white, opaque, smooth, irregularly shaped like pseudoroots, and the diameter of the colonies was about 2mm. According to Gram staining and microscopic observation, TF01-11 is a Gram-positive bacterium, long rod-shaped, spore-free, with flagella, movable, the diameter of the bacterium is about 0.5-1.0mm, and the length is about 2.0-8.0mm. The strains were provided and deposited by the General Microbial Center of the China Microbial Species Collection Management Committee (CGMCC), and the deposit number was CGMCC 10984.
  • CGMCC General Microbial Center of the China Microbial Species Collection Management Committee
  • TM12-14 Pseudo-small chain Bifidobacterium TM12-14 was isolated using PYG medium under 37 ° C anaerobic conditions. The colonies of TM12-14 cultured in PYG medium for 2 days were white, raised, round, and the edges were neat. The diameter of the colonies was about 1-2 mm. The microscopic morphology of the bacteria showed a divergent rod shape. The Gram stain was positive and no spore And flagella are produced. The strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60089.
  • the isolated sample was from the feces of a 14-year-old healthy male.
  • the feces were collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.
  • the collected fresh samples were immediately transferred to the anaerobic operation box, 0.2g of the sample was taken in 1mL of sterile PBS, thoroughly shaken and mixed, and then gradient dilution coating was performed.
  • the obtained pure culture strain was cultured to a concentration of about 10 9 cfu / mL, and 400 ⁇ L of the bacterial solution was added to 400 ⁇ L of 40% glycerol to make the glycerol concentration reach 20%, and then stored at -80 ° C. at ultra-low temperature.
  • the obtained isolated strain was cultured in liquid PYG medium for 24 hours, and 1 mL of the bacterial solution was centrifuged at 10,000 r / min for 5 minutes. The bacterial cells were collected and genomic DNA was extracted. Using genomic DNA as a template, 16S rDNA universal primers were used for PCR amplification. The PCR amplification primers, systems and conditions were the same as "16. rDNA identification of strain 1.1.3".
  • the 16S rDNA amplification products obtained were subjected to electrophoretic detection, purification, and 3730 sequencing to obtain the 16S rDNA sequences of the strains, and then the comparison of the NCBI database was performed.
  • the macromonas monomorpha AF24-28AC in this example was cultured in a PYG medium under 37 ° C anaerobic conditions.
  • the colonies of AF24-28AC cultured in PYG medium for 2 days were light yellow, with irregular wavy edges, flat, low water content, and about 2-3 mm in diameter.
  • the microscopic morphology of the fungus is rod-shaped, Gram-negative, and does not produce spores and flagella.
  • the strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60093.
  • Women stool sample isolated from a healthy, were plated using serial dilution method isolates, using PYG medium were purchased from Central Kay Microbial Technology Company, anaerobic culture, the anaerobic gas component is N 2 : CO 2 : H 2 90: 5: 5, cultured for 48 h, single colonies were picked and purified by streaking to obtain a pure culture of each single strain.
  • the genomic DNA of the isolated strain was extracted, and the 16S rDNA universal primer was used for PCR amplification.
  • the obtained 16S rDNA amplified product was subjected to electrophoresis detection, purification, and 3730 sequencing to obtain the 16S rDNA sequence of the strain, and then performed in the EZBio Cloud database Compare and identify the isolates.
  • the 16S rDNA PCR amplification system is: 10 ⁇ PCR buffer 3 ⁇ L, dNTPs 2.5 ⁇ L, upstream primer 27F 0.5 ⁇ L, downstream primer 1492R 0.5 ⁇ L, Taq enzyme 0.3 ⁇ L, template 1 ⁇ L, ddH 2 O 18.2 ⁇ L.
  • the amplification conditions of 16S rDNA are as follows:
  • 65 °C -57 °C 40s means that the temperature decreases proportionally after each cycle, that is, the annealing temperature in the first cycle is 65 °C, and finally decreases to 57 °C in the 20th cycle.
  • the upstream primer of the 16S rDNA universal primer in this experiment is 27f, the upstream primer sequence is the sequence shown in SEQ ID NO. 1, the downstream primer is 1492r, and the downstream primer sequence is the sequence shown in SEQ ID NO. 2.
  • the 16SrDNA sequence of the isolated strain AF24-28AC was the sequence shown in SEQ ID NO.7.
  • EZBio Cloud database comparison results show that the AF24-28AC strain isolated in this example has the highest homology with Megamonas fungiformis DSM 19343 purchased from the German Breed Collection Center, with a similarity of 99.09%.
  • Monomonas named Monomonas monomorpha AF24-28AC, and deposited them.
  • the human fecal anaerobic coryneform AM25-6 in this example was cultured in PYG medium, and the culture conditions were 37 ° C anaerobic conditions.
  • the colonies of AM25-6 cultured in PYG medium for 2 days were light yellow, the colonies were small, needle-shaped, and the diameter was about 0.5mm.
  • the microscopic morphology of the fungus is short rod-shaped, Gram-positive, and does not produce spores and flagella.
  • the strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC60087.
  • the isolated sample was from a healthy male feces.
  • the feces were collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.
  • the collected fresh samples were immediately transferred to the anaerobic operation box, and 0.2 g of the sample was suspended in 1 mL of sterile phosphate buffered saline (abbreviated PBS), and thoroughly shaken and mixed.
  • Strain isolation was performed using a gradient dilution plate method, and the culture medium was PYG medium purchased from Huankai Microbial Technology Company.
  • the obtained pure culture strain was cultured to a concentration of about 10 9 cfu / mL, and 400 ⁇ L of the bacterial solution was added to 400 ⁇ L of 40% glycerol to make the glycerol concentration reach 20%, and then stored at -80 ° C. at ultra-low temperature.
  • the genomic DNA of the isolated strain was extracted, and the 16S rDNA universal primer was used for PCR amplification.
  • the obtained 16S rDNA amplified product was subjected to electrophoretic detection, purification, and 3730 sequencing to obtain the 16S rDNA sequence of the strain, and then compared with the EZBio Cloud database. Yes, the isolated strains are identified.
  • the primers, PCR amplification system, and conditions for 16S rDNA PCR amplification in this test were the same as "16S rDNA identification of 1.1.3 strain".
  • the sequencing results showed that the 16S rDNA sequence of the AM25-6 strain isolated in this example was the sequence shown in SEQ ID NO.8.
  • EZBio Cloud database comparison results show that the AM25-6 strain isolated in this example has the highest homology with Anaerofustis stercorihominis DSM 17244 purchased from the German Bacteria Collection Center. Corynebacterium, named human fecal anaerobic coryneform AM25-6, and preserved it.
  • the mouse model selected in this example is: DSS (dextran sodium sulfate Dextran Sulfate, Na, molecular weight 36000-50000) induced ulcerative enteritis mouse model.
  • mice 84 mouse strains of C57bl / 6 mice purchased from Hubei Medical Experimental Animal Center were used. All mice were 8 weeks old and weighed 20g ⁇ 2g. They were reared in a SPF-grade mouse room environment. 96 mice were randomly divided into 8 groups, and 12 mice in each group were followed up.
  • mice were continued to drink 0.15% DSS for seven days to obtain a mouse model of ulcerative enteritis.
  • mice were randomly divided into 8 groups of 12 mice each, and the 8 groups were the normal group (i.e. the control group), the model group, the probiotic composition treatment group, the combined bacteria group 1, the combined bacteria group 2, and the combined bacteria group 3 Group, combined bacteria group 4, combined bacteria group 5 and VSL # 3 treatment group, the specific treatment of each group is as follows:
  • Normal group they were fed with ordinary feed, and each mouse was fed with 0.2 mL of PBS buffer.
  • Model group fed with the same feed, and subjected to DSS modeling: DSS was added to the drinking water of the mice, the DSS was added at a final concentration of 0.15%, and fed for seven days. Each mouse was fed with 0.2 mL of PBS buffer daily. liquid.
  • Probiotic composition treatment group fed with the same feed, 3 days before DSS modeling, each mouse was administrated with 0.2 mL of probiotic composition liquid daily, and then DSS modeling: DSS modeling It was added to the drinking water of mice, and the DSS was added at a final concentration of 0.15%. After being fed for seven days, each mouse was fed with 0.2 mL of the probiotic composition bacterial solution daily.
  • Combined bacteria group 1 Feed the same feed, and 3 days before DSS modeling, each mouse was given 0.2 mL of combined bacteria 1 bacteria solution daily, and then DSS modeling was performed: DSS was added to the mice. Rats drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of the combined bacteria 1 bacterial solution daily.
  • Combined bacteria 2 group fed with the same feed, 3 days before DSS modeling, each mouse was given 0.2 mL of combined bacteria 2 bacteria solution daily, and then DSS modeling: DSS was added to Rats drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of the combined bacteria 2 bacterial solution daily.
  • Combined bacteria group 3 Feed the same feed, and 3 days before DSS modeling, each mouse was given 0.2 mL of combined bacteria 3 bacteria solution daily, and then DSS modeling was performed: DSS was added to Rats drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of the combined bacteria 3 bacterial solution daily.
  • Combined bacteria group 4 fed with the same feed, 3 days before DSS modeling, each mouse was fed with 0.2 mL of combined bacteria 4 bacteria solution daily, and then DSS modeling was performed: DSS was added to Rats drank water, DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of the combined bacteria 4 bacteria solution daily.
  • Combined bacteria group 5 fed with the same feed, 3 days before DSS modeling, each mouse was fed with 0.2 mL of combined bacteria 5 bacteria solution daily, and then DSS modeling: DSS was added to Rats drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of the combined bacteria 5 bacterial solution daily.
  • VSL # 3 treatment group fed with the same feed, 3 days before DSS modeling, each mouse was given 0.2 mL of VSL # 3 bacteria solution daily, and then DSS modeling was performed: DSS was added to Mice drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of VSL # 3 bacteria solution daily.
  • the bacterial liquid of the probiotic composition and the bacterial liquid of the combined bacteria 1 to 5 are prepared by the following method:
  • Lactobacillus gasseri TF08-1 Lactobacillus acidophilus AM13-1, Shenzhen Collins TF06-26, Roseburia inulinivorans DSM 16841, Butyric acid TF01-11, Bifidobacterium pseudominis TM12- 14.
  • C. monomorpha AF24-28AC and human fecal anaerobic coryneform AM25-6 were cultured for 24 hours, the cells were collected by centrifugation, each cell was suspended with PBS, and the bacteria concentration was adjusted to 10 9 cfu / mL of bacterial suspension, used to prepare each bacterial solution.
  • Probiotic composition bacterial solution Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 are mixed in equal volumes to obtain a probiotic composition bacterial solution;
  • Combined bacteria 1 bacterial solution Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Shenzhen Collins TF06-26 were mixed in equal volumes to obtain a combined bacteria 1 bacterial liquid;
  • Combination bacteria 2 bacterial solution Lactobacillus garnerii TF08-1, Lactobacillus acidophilus AM13-1, and a bacterial suspension of Roseburia inulinivorans DSM 16841 are mixed in equal volumes to obtain a combination bacterial liquid;
  • Combined bacteria 3 bacterial solution Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and suspensions of butyric acid TF01-11 were mixed in equal volumes to obtain a combined bacteria 3 bacterial liquid;
  • Combined bacteria 4 bacterial solution Lactobacillus garnerii TF08-1, Lactobacillus acidophilus AM13-1, and a suspension of Bifidobacterium pseudomonas TM12-14 are mixed in equal volumes to obtain a combined bacteria 3 bacterial liquid;
  • Combination bacteria 5 bacterial liquid equal volume of bacterial suspension of Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, Monomonas monomorpha AF24-28AC and human fecal anaerobic coryneform AM25-6, That is, a combined bacteria 5 bacteria liquid was obtained.
  • VSL # 3 bacteria solution is prepared by the following method:
  • VSL # 3 was purchased from the American company Alfasigma, and is a kind of strains containing Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus germani subsp. Thermophilus, Bifidobacterium longum, Bifidobacterium short, A composite probiotic of 8 beneficial bacteria such as Bifidobacterium infantis; similarly, VSL # 3 was suspended in PBS and adjusted to a concentration of 10 9 cfu / mL to obtain a VSL # 3 bacterial solution.
  • the DSS model was used to record the body weight, diet and drinking water of the mice, and observe the fecal characteristics and occult blood of the mice.
  • the disease activity of the mice was calculated on the 1st, 3rd, 5th, and 7th days, respectively. Index (abbreviation DAI), DAI scoring standards are shown in Table 2.
  • Mice were sacrificed at the end of the experiment. All mice were bled, neck removed, colon removed, photographed, weighed, and colon length measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde. Among them, the recording time of the normal group was the same as that of DSS modeling.
  • mice The weights of the mice on the 1st, 3rd, 5th, and 7th days were counted, and the average weights of the mice in each group are shown in Table 3 and FIG. 1.
  • Control refers to the normal group.
  • Probiotics is the probiotic composition treatment group
  • combination bacteria 1 is the combination bacteria 1 treatment group
  • combination bacteria 2 is the combination bacteria 2 treatment group
  • combination bacteria 3 is the combination bacteria.
  • combination bacteria 4 is the combination bacteria 4 treatment group
  • combination bacteria 5 is the combination bacteria 5 treatment group
  • VSL # 3 is the VSL # 3 treatment group.
  • This example shows that the probiotic group consisting of Lactobacillus garneri TF08-1 and Lactobacillus acidophilus AM13-1, and the combined bacteria 1, 2, 3, 4, 5 and VSL # 3 can control the weight loss caused by UC .
  • the mice of this example Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1, constituted a probiotic composition group and mice with combined bacteria 1, 2, 3, 4, and 5 had slightly higher body weights.
  • VSL # 3 it is shown that the probiotic composition composed of Lactobacillus gabriensis TF08-1 and Lactobacillus acidophilus AM13-1, and the combination of other probiotics in addition to this in controlling the weight loss of UC mice Slightly better than VSL # 3.
  • DSA-induced ulcerative enteritis in mice caused changes in DAI index due to changes in body weight, stool traits, and blood in the stool.
  • the statistical values of the DAI index on mice on days 1, 3, 5, and 7 were as follows: Table 4 and Figure 2.
  • Table 4 the DAI of each group of mice is taken as the average value of each group of mice.
  • Control refers to the normal group.
  • Probiotics is the probiotic composition treatment group
  • combination bacteria 1 is the combination bacteria 1 treatment group
  • combination bacteria 2 is the combination bacteria 2 treatment group
  • combination bacteria 3 is the combination bacteria.
  • combination bacteria 4 is the combination bacteria 4 treatment group
  • combination bacteria 5 is the combination bacteria 5 treatment group
  • VSL # 3 is the VSL # 3 treatment group.
  • the data in Table 4 and Figure 2 show that the DAI of the mice in the Control group is basically the same, and with the induction of DSS, the model group, the probiotic group, and the combination of other probiotics based on the probiotic group 1, 2, 3, 4
  • the DAI of mice in the 5 and VSL # 3 groups gradually increased.
  • the DAI of the model group mice began to be significant on the 3rd day (* P ⁇ 0.05), and the DAI of the model group mice reached the highest on the 7th day. Level (ie relative to control group ** P ⁇ 0.01).
  • Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 combined with probiotics, and the combination of probiotics combined with other probiotics to form a combination of bacteria 1, 2, 3, 4, and 5 can control the DAI.
  • the colon tissue of UC model mice will be changed, mainly due to the shortening of colon tissue due to the occurrence of ulcers and inflammation. After treatment, the colon length of the mice measured by anatomy is shown in Table 5.
  • Control refers to the normal group.
  • Probiotics is the probiotic composition treatment group
  • combination bacteria 1 is the combination bacteria 1 treatment group
  • combination bacteria 2 is the combination bacteria 2 treatment group
  • combination bacteria 3 is the combination bacteria.
  • 3 treatment groups combination bacteria 4 is the combination bacteria 4 treatment group
  • combination bacteria 5 is the combination bacteria 5 treatment group
  • VSL # 3 is the VSL # 3 treatment group.
  • ** refers to the significant difference in colon length between the model group and the normal group.
  • ⁇ refers to “probiotics”, “combined bacteria 1", “combined bacteria 2", “combined bacteria 3” Significant differences in colon length between mice in the ", combined bacteria 4", “combined bacteria 5", and “VSL # 3" groups compared to the model group mice P ⁇ 0.05.
  • composition of Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 which has the effect of treating and preventing ulcerative enteritis in Example 1, is made into a common food, as follows:
  • Milk, vitamin C, white sugar, and other auxiliary materials are mixed with the cultured Lactobacillus garnerii TF08-1 and Lactobacillus acidophilus AM13-1 according to the formula in Table 6 to prepare foods with ulcerative enteritis treatment and prevention functions.
  • the cow's milk product of this example was added to the feed of mice modeled by DSS, and fed and tested according to the probiotic composition treatment group of Example 1. The difference was that only the cow's milk of this example was added to the feed. The product does not additionally administer the probiotic composition bacterial liquid.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 1 proved to have ulcerative enteritis treatment and prevention effects in Example 1 is made into a common food, as follows:
  • Milk, vitamin C, sugar and other auxiliary materials are mixed with the cultured Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and Collins Shenzhen TF06-26 according to the formula in Table 7 to prepare the treatment and prevention of ulcerative enteritis Functional food.
  • the cow's milk product of this example was added to the DSS modeled mouse feed, and fed and tested according to the combination bacteria 1 treatment group of Example 1. The difference is that this example only added the cow's milk product of this example to the feed. , No additional intragastric combination bacteria 1 bacterial solution.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index DAI of the mice, improve the intestinal lesions, and have ulcerative enteritis treatment and prevention effects.
  • composition of the combined bacterium 2 which has the therapeutic and preventive effects of ulcerative enteritis in Example 1 is made into a common food, as follows:
  • the milk product of this example was added to the DSS modeled mouse feed, and fed and tested according to the combination bacteria 2 treatment group of Example 1. The difference is that this example only added the milk product of this example to the feed. , No additional intragastric combination 2 bacteria liquid.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 3, which has the therapeutic and preventive effects of ulcerative enteritis in Example 1, is made into a common food, as follows:
  • Milk, vitamin C, sugar and other auxiliary materials are mixed with the cultured Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Butyric acid TF01-11 according to the formula in Table 9 to prepare ulcerative enteritis treatment and prevention Functional food.
  • the milk product of this example was added to the feed of mice modeled by DSS, and fed and tested according to the combined bacteria 3 treatment group of Example 1. The difference was that only the milk product of this example was added to the feed. , No additional intragastric combination of 3 bacteria liquid.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 3, which has the therapeutic and preventive effects of ulcerative enteritis in Example 1, is made into a common food, as follows:
  • the milk product of this example was added to the DSS-modeled mouse feed, and fed and tested according to the combined bacteria 4 treatment group of Example 1. The difference was that this example only added the milk product of this example to the feed. , No additional intragastric combination of 4 bacteria liquid.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 5 having the therapeutic and preventive effects of ulcerative enteritis in Example 1 is made into a common food, as follows:
  • the cow's milk product of this example was added to the DSS-modeled mouse feed, and fed and tested according to the combined bacteria 5 treatment group of Example 1. The difference was that only the cow's milk product of this example was added to the feed. , No additional intragastric combination of 5 bacteria liquid.
  • the test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index DAI of the mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • composition of Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus AM13-1 which has the therapeutic and preventive effects of ulcerative enteritis in Example 1, is prepared into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 12. :
  • the capsule pharmaceutical composition of this example was used to replace the bacterial solution of the probiotic composition in the treatment group of the probiotic composition of Example 1, and the capsule pharmaceutical composition of this example was orally administered in the same manner as in Example 1.
  • One capsule was tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 1 which has the therapeutic and preventive effects of ulcerative enteritis in Example 1 is made into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 13:
  • the capsule pharmaceutical composition of this example was used to replace the combined bacteria 1 bacterial solution in the combined bacteria 1 treatment group of Example 1, and the capsule pharmaceutical composition of this example was orally administered in the same manner as in Example 1, and one capsule was orally administered daily.
  • the capsules were tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 2 proved to have ulcerative enteritis treatment and prevention effects in Example 1 is made into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 14:
  • the capsule pharmaceutical composition of this example was used to replace the combined bacteria 2 bacterial solution in the combined bacteria 2 treatment group of Example 1.
  • the capsule pharmaceutical composition of this example was orally administered one capsule per day.
  • the capsules were tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 3 having the therapeutic and preventive effects of ulcerative enteritis in Example 1 is made into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 15:
  • a capsule which is made up of Lactobacillus gastroseri TF08-1, Lactobacillus acidophilus AM13-1 and Butyribacter intestini TF01-11 capsule pharmaceutical composition of three strains of probiotics.
  • the capsule pharmaceutical composition of this example was used to replace the combined bacteria 3 bacterial solution in the combined bacteria 3 treatment group of Example 1.
  • the capsule pharmaceutical composition of this example was orally administered one capsule per day.
  • the capsules were tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 4 which has been proved to have the effect of treating and preventing ulcerative enteritis in Example 1 is made into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 16:
  • the capsule pharmaceutical composition of this example was used to replace the combined bacteria 4 bacteria solution in the combined bacteria 4 treatment group of Example 1, and the capsule pharmaceutical composition of this example was orally administered in the same manner as in Example 1, and one capsule was orally administered daily.
  • the capsules were tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • DAI disease activity index
  • composition of the combined bacterium 5 having the therapeutic and preventive effects of ulcerative enteritis in Example 1 is made into a medicament for treating ulcerative enteritis.
  • the formula is shown in Table 17:
  • the capsule pharmaceutical composition of this example was used to replace the combined bacteria 5 bacterial solution in the combined bacteria 5 treatment group of Example 1, and the capsule pharmaceutical composition of this example was orally administered in the same manner as in Example 1, and one capsule was orally administered daily.
  • the capsules were tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index DAI of mice, improve intestinal lesions, and has ulcerative enteritis treatment and prevention effects.
  • Lactobacillus gasseri and Lactobacillus acidophilus can treat and prevent ulcerative enteritis; and in addition to Lactobacillus gasseri and Lactobacillus acidophilus, it is also possible to add Shenzhen Collins and Rose
  • the combination of Roseburia inulinivorans, butyric acid or Pseudomonas aeruginosa, or the addition of Monomonas morphogenes and human anaerobic coryneform bacteria can all treat and prevent ulcerative enteritis.
  • the above combined composition can be made into various foods or medicines; of course, it can be understood that since the above combined composition can be made into various foods and medicines, it can also be made into various health products or food additives.

Abstract

一种组合物及其应用。该组合物，包括加氏乳杆菌和/或其代谢产物，以及嗜酸乳杆菌和/或其代谢产物。该组合物，通过加氏乳杆菌和嗜酸乳杆菌两者联合使用，能够对炎症，尤其是溃疡性肠炎，及其相关疾病有良好的治疗和预防效果，为炎症及其相关疾病的治疗和预防提供了一种安全、有效、毒副作用小，且不易产生抗性的组合物。

Description

一种组合物及其应用 技术领域
本申请涉及微生物制剂领域，特别是涉及一种微生物或微生物相关的组合物及其应用。
背景技术
炎症性肠道疾病(inflammatory bowel disease，IBD)是一种病因不明的慢性炎症性肠道疾病，该疾病易反复，严重影响患者的生活质量。现代医学认为，引发炎症性肠道疾病(IBD)的因素有遗传、饮食、感染、自身免疫、心理因素以及环境等。炎症性肠道疾病包括溃疡性肠炎(UC)和克罗恩病(CD),均属于炎症相关疾病。
溃疡性肠炎(ulcerative colitis，UC)是炎症性肠道疾病(inflammatory bowel disease，IBD)一种重要的疾病类型，其发病原因不明，主要病变部位是在结肠粘膜的粘膜下层，属于一种慢性的肠道疾病。基于目前的研究，主要认为引发溃疡性肠炎的病因有宿主遗传易感性、肠道菌群以及肠粘膜的免疫反应，临床病理表现为持续腹痛、腹泻和黏液血便，且病情反复，近年来我国UC的患病人数呈明显上升趋势。
目前，临床上针对UC并的用药主要有水杨酸类药物、肾上腺糖皮质激素类药物和免疫抑制剂。这三类药物均可以一定程度上对UC进行缓解，但是也都存在不足。
水杨酸类药物可以比较好的抑制前列腺素合成，清除氧自由基从而达到缓解炎症反应的目的，但也只能短期缓解，无法实现根治，临床上治疗UC常见的水杨酸类西药主要是柳氮磺胺吡啶(SASP)，主要针对轻度、中度以及慢性UC患者。并且，水杨酸类药物还具有很多副作用，例如引发消化道反应、头痛、网织红细胞增多、精子减少，以及过敏反应引起的皮疹、肝毒性、白细胞减少、贫血等，这类药物同时也具有抗菌作用，容易引起菌群紊乱以及耐药性增强。
肾上腺糖皮质激素类药物是重症或者爆发性UC患者的首选用药，典型的药物比如倍他米松；但是，肾上腺糖皮质激素类药物会导致机体代谢紊乱，水潴留等副作用，仅可作为应急用药，不能长期服用。
免疫抑制剂，例如环孢素，可以通过抑制T细胞IL-2的产生，影响免疫反应的进展，从而对UC进行抑制；但是，免疫抑制剂治疗对药物依赖性较大，治 疗周期长，容易引起肾毒性及二次感染，只能作为一种辅助治疗的手段。
因此，目前来说尚没有安全有效的炎症相关疾病尤其是溃疡性肠炎的治疗药物。
发明内容
本申请的目的是提供一种组合物及其应用。
本申请采用了以下技术方案：
本申请的一方面公开了一种组合物，该组合物包括加氏乳杆菌和/或其代谢产物，以及嗜酸乳杆菌和/或其代谢产物。
需要说明的是，本申请的关键在于，研究发现加氏乳杆菌和嗜酸乳杆菌两者联合使用能够预防和治疗炎症及炎症相关疾病，尤其是能够有效预防和治疗溃疡性肠炎；研究显示，其预防或治疗预防作用主要由两方面组成，一方面，是加氏乳杆菌和嗜酸乳杆菌对体内肠道微生态进行改善，形成由有益菌构成的生态保护屏障，从而起到炎症或炎症相关疾病的预防和治疗效果；另一方面，是加氏乳杆菌和嗜酸乳杆菌的代谢产物作为益生材料，起到炎症或炎症相关疾病的预防和治疗作用。因此，本申请的组合物，其重要的用途就是用于预防或治疗炎症或炎症相关疾病，尤其是用于预防或治疗溃疡性肠炎或其相关疾病。
还需要说明的是，本申请的一种实现方式中，由加氏乳杆菌和嗜酸乳杆菌的组合物，通过两个菌对体内肠道微生态进行改善，形成由有益菌构成的生态保护屏障，起到溃疡性肠炎预防和治疗效果。可以理解，这种微生态的改善，不仅对溃疡性肠炎有预防和治疗效果，对于其它与微生态相关疾病，例如普通肠炎或胃炎等，同样具有效果；因此，本申请的组合物可以用于预防或治疗炎症或炎症相关疾病。
优选的，加氏乳杆菌为保藏号GDMCC 60092的加氏乳杆菌TF08-1，所述嗜酸乳杆菌为保藏号GDMCC 60091的嗜酸乳杆菌AM13-1。
需要说明的是，本申请的关键在于研究发现加氏乳杆菌和嗜酸乳杆菌两者联合使用能够预防和治疗炎症及炎症相关疾病，而加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1，这两个菌株是本申请研究过程中发现的联合使用效果较好的两个菌株，因此分别对两个菌株进行了保藏。可以理解，一方面，不排除在本申请的发明思路下，还可以采用其它菌株的加氏乳杆菌和嗜酸乳杆菌，达到甚至超过本申请两个菌株的效果；另一方面，在对炎症或炎症相关疾病预防或治疗效果要求较低的情况下，也可以采用其它菌株的加氏乳杆菌和嗜酸乳杆菌。
优选的，本申请的组合物还包括深圳柯林斯菌和/或其代谢产物。
优选的，深圳柯林斯菌为保藏号GDMCC 60090的深圳柯林斯菌TF06-26。
优选的，本申请的组合物还包括罗斯氏菌(Roseburia inulinivorans)和/或其代谢产物。
优选的，罗斯氏菌(Roseburia inulinivorans)为保藏号DSM 16841的罗斯氏菌(Roseburia inulinivorans)。
优选的，本申请的组合物还包括丁酸杆菌和/或其代谢产物。
优选的，丁酸杆菌为保藏号CGMCC 10984的丁酸杆菌TF01-11。
优选的，本申请的组合物还包括假小链状双歧杆菌和/或其代谢产物。
优选的，假小链状双歧杆菌为保藏号GDMCC 60089的假小链状双歧杆菌TM12-14。
优选的，本申请的组合物还包括单形巨单胞菌和/或其代谢产物，以及人粪厌氧棒形菌和/或其代谢产物。
优选的，单形巨单胞菌为保藏号GDMCC 60093的单形巨单胞菌AF24-28AC，人粪厌氧棒形菌为保藏号GDMCC 60087的人粪厌氧棒形菌AM25-6。
需要说明的是，本申请研究证实加氏乳杆菌和嗜酸乳杆菌两者联合使用能够预防和治疗炎症及炎症相关疾病，在不影响加氏乳杆菌和嗜酸乳杆菌联合使用效果的情况下，还可以加入深圳柯林斯菌、罗斯氏菌(Roseburia inulinivorans)、丁酸杆菌或假小链状双歧杆菌，或者还可以在加氏乳杆菌和嗜酸乳杆菌的基础上加入单形巨单胞菌和人粪厌氧棒形菌。以上组合方案都够预防和治疗炎症及炎症相关疾病。
优选的，本申请的组合物还含有其它益生菌和/或益生元。
需要说明的是，本申请的组合物，其关键在于加氏乳杆菌和嗜酸乳杆菌两者联合使用能够预防和治疗炎症及炎症相关疾病，可以理解，在不影响加氏乳杆菌和嗜酸乳杆菌两者联合作用效果的情况下，还可以添加其它益生菌或者益生元，使得本申请的组合物具备更多的功能，或者对其原本的功效进行加强，这些益生菌或益生元可以采用现有研究报道过的益生菌或益生元，在此不做具体限定。
优选的，本申请的一种实现方式中，本申请组合物的益生元选自低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)和寡聚糖中的至少一种。
优选的，本申请的组合物还含有有助于保持组合物中各菌株的至少一种的活力的物质。
可以理解，为了保持组合物中组合物中各菌株的活力，保障其功效，还可以在组合物中添加各种维持菌株活力的物质，这些活力物质可以是现有研究报道过的活力物质，在此不做具体限定。
优选的，本申请的一种实现方式中，有助于保持菌株活力的物质，选自半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸或其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C和维生素E中的至少一种。
优选的，本申请的组合物还包括药学上或食品上可接受的载体或辅料。
需要说明的是，本申请的组合物具有预防和治疗炎症及炎症相关疾病的效果，本申请的一种实现方式中，主要通过食用本申请的组合物，达到预防和治疗效果。因此，组合物中还可以包括药学上或食品上可接受的载体或辅料，以方便使用。
优选的，药学上或食品上可接受的载体或辅料，选自葡萄糖、乳糖、蔗糖、淀粉、甘露醇、糊精、脂肪酸甘油酯、聚乙二醇、羟乙基淀粉、乙二醇、聚氧乙烯山梨糖醇酐脂肪酸酯、氨基酸、明胶、白蛋白、水和生理食盐水中的至少一种。
本申请的另一面公开了本申请的组合物在制备用于治疗或预防炎症或炎症相关疾病的食品、保健品、食品添加剂或药品中的应用。
可以理解，本申请的组合物具有预防和治疗炎症及炎症相关疾病的效果，为了方便使用，可以将本申请的组合物制成各种食品、保健品、食品添加剂或药品。
优选的，本申请的组合物尤其可以用于制备治疗或预防溃疡性肠炎或其相关疾病的食品、保健品、食品添加剂或药品。
本申请的再一面公开了本申请的组合物在制备用于控制哺乳动物体重下降的食品、保健品、食品添加剂或药品中的应用。
其中，哺乳动物体重下降，尤其指哺乳动物因炎症导致的体重下降。
优选的，炎症为溃疡性肠炎，即控制哺乳动物因溃疡性肠炎导致的体重下降。
本申请的再一面公开了本申请的组合物在制备用于降低哺乳动物的疾病活动指数的食品、保健品、食品添加剂或药品中的应用。
本申请的再一面公开了本申请的组合物在制备用于改善哺乳动物肠道病变的食品、保健品、食品添加剂或药品中的应用。
需要说明的是，本申请的组合物能够预防和治疗炎症及炎症相关疾病，其 关键就体现在能够对炎症及炎症相关疾病引起的体重下降进行控制、降低炎症及炎症相关疾病引起的疾病活动指数，并改善哺乳动物肠道病变；因此，本申请的组合物也可以单独的用于制备控制哺乳动物体重下降、降低哺乳动物疾病活动指数或改善哺乳动物肠道病变的食品、保健品、食品添加剂或药品。
本申请的再一面公开了采用本申请的组合物进行炎症或炎症相关疾病的治疗或预防的方法。
本申请的再一面公开了采用本申请的组合物控制哺乳动物体重下降的方法。
本申请的再一面公开了采用本申请的组合物降低哺乳动物的疾病活动指数的方法。
本申请的再一面公开了采用本申请的组合物改善哺乳动物肠道病变的方法。
需要说明的是，以上各种方法中，主要是通过食用本申请的组合物，以起到治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数或改善哺乳动物肠道病变的效果。
本申请的再一面公开了一种食品，该食品中含有本申请的组合物。
优选的，食品为乳酸饮品或豆乳饮品。本申请的食品，是广义上的以任何形态存在的可食用物品，并不仅限于乳酸饮品或豆乳饮品，例如还可以是发酵食品，也可以是动物饲料等。
需要说明的是，本申请的食品由于含有本申请的组合物，因此同样具有治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数和改善哺乳动物肠道病变的效果。可以理解，本申请的食品，其关键在于其中含有本申请的组合物，至于食品的具体形态，如固体、液体等，可以根据不同的食品产品或使用需求而定，在此不做具体限定。本申请的一种实现方式中，主要是将本申请的组合物制成常见的乳酸饮品或豆乳饮品，以方便饮用；当然，也可以制成例如奶片、奶酪条等固体食品，在此不做具体限定。
还需要说明的是，本申请的食品中加氏乳杆菌和嗜酸乳杆菌的活性菌量或者摄取量不做具体限定，实际应用中，可以根据实际状况灵活选择。以加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1为例，本申请的研究显示，每天摄入加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1的浓度都是10 9cfu/mL的组合物0.2mL，对溃疡性肠炎有良好的治疗效果，该活性菌剂量可以作为食品、保健品、食品添加剂或药品的参考用量或参考摄取量。
本申请的再一面公开了一种保健品，该保健品中含有本申请的组合物。
需要说明的是，本申请的保健品由于含有本申请的组合物，因此同样具有治疗或预防炎症或炎症相关疾病、控制哺乳动物体重下降、降低哺乳动物疾病活动指数和改善哺乳动物肠道病变的效果。
本申请的再一面公开了一种食品添加剂，该食品添加剂中含有本申请的组合物。
需要说明的是，本申请的组合物可以与通常的食品材料配合食用。例如，谷类及薯类，谷类包括米、面、杂粮，薯类包括马铃薯、红薯等；动物性食物，包括肉、禽、鱼、奶、蛋等；豆类及其制品，包括大豆及其它干豆类；蔬菜水果类，包括鲜豆、根茎、叶菜、茄果等；纯热能食物，包括动植物油、淀粉、食用糖和酒类等；因此，本申请的组合物可以单独的作为食品添加剂或者调制剂加入各种食材中直接食用，起到炎症及其相关疾病的治疗或预防效果。
本申请的再一面公开了一种药品，该药品中含有本申请的组合物。
优选的，药品为片剂、颗粒剂、散剂、肠溶剂、溶液剂或悬浮剂。
需要说明的是，本申请的药品由于含有本申请的组合物，因此具有炎症及其相关疾病的治疗或预防效果；本申请的药品，可以是单独的本申请的组合物，也可以与其它炎症药物配合使用，只要相互之间不影响活性即可。可以理解，本申请的药品，只要不影响组合物中各菌株的活性，药品可以采用现有的各种剂型。而本申请的药品中，进一步的还可以包括药品或剂型中常用的辅料，例如稳定剂、湿润剂、乳化剂、粘合剂、等渗剂等。
本申请的药品可以以口服液、片剂、针剂、口崩片、冻干粉制剂或肠溶剂型的任一种形式给药。优选肠溶剂型，如胶囊或肠溶片，以便于药品的活性成分即微生物能顺利通过胃而不被胃酸所破坏。更优选的，本申请的药品可制成肠溶片供口服使用。
本申请的肠溶剂型是指在胃液中不崩解，而在肠液中能够崩解和吸收的一种药品剂型，肠溶剂型包括胶囊和肠溶片。其中，胶囊由粉末状的药品封装在的常规药物允许使用的胶囊壳中形成；肠溶片则是在普通的片剂药品外面包裹一层肠溶包衣而成。本申请的“肠溶包衣”简称“肠衣”，包括所有常规药物允许使用的包衣，这些包衣不被胃酸降解，但在小肠中能充分分解并快速释放出本申请的药品。例如，本申请的肠衣能在合成胃酸如pH＝1的HCl溶液中在36-38℃维持2小时以上，并优选在合成肠液如pH＝7.0的缓冲液中在1.0小时内分解。
优选的，本申请的肠溶片中，其肠衣厚度为5-100μm，理想的厚度为20-80μm。肠衣成分选自已公开知晓的常规材料。
本申请的药品中组合物的各菌株的活性菌含量，或者药剂量不受特别限制，实际应用中，可以根据给药对象的健康状况灵活选择。但是，本申请的研究显示，每天摄入加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1的浓度都是10 9cfu/mL的组合物0.2mL，对溃疡性肠炎有良好的治疗效果，此用量可以作为药品中活性菌含量或者给药剂量参考。
本申请的有益效果在于：
本申请的组合物，通过加氏乳杆菌和嗜酸乳杆菌两者联合使用，能够对炎症，尤其是溃疡性肠炎，及其相关疾病有良好的治疗和预防效果，为炎症及其相关疾病的治疗和预防提供了一种新的安全、有效、毒副作用小，且不易产生抗性的组合物。
附图说明
图1是本申请实施例中Control组、模型组、VSL #3组、益生菌组合物治疗组、组合菌1、组合菌2、组合菌3、组合菌4、组合菌5治疗组小鼠的体重的变化曲线；
图2是本申请实施例中Control组、模型组、VSL #3组、益生菌组合物治疗组、组合菌1、组合菌2、组合菌3、组合菌4、组合菌5治疗组小鼠的DAI指数的变化曲线。
本申请的加氏乳杆菌TF08-1于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60092。
嗜酸乳杆菌AM13-1于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60091。
深圳柯林斯菌TF06-26于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60090。
罗斯氏菌(Roseburia inulinivorans)DSM 16841于购自德国微生物菌种保藏中心。
丁酸杆菌TF01-11于2015年06月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏单位的地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏号为CGMCC 10984。
假小链状双歧杆菌TM12-14于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60089。
单形巨单胞菌AF24-28AC于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60093。
人粪厌氧棒形菌AM25-6于2016年10月13日保藏于广东省微生物菌种保藏中心，保藏单位的地址为中国广东省广州市先烈中路100号省微生物所实验楼五楼，保藏号为GDMCC 60087。
具体实施方式
随着肠道微生态的深入研究，发现溃疡性肠炎的发病与肠道微生物的组成密切相关，肠道细菌的失衡与肠粘膜炎症反应有着密切的关系，其中有害菌的过度增殖可以引发炎症反应，进而诱发溃疡性肠炎的发病。健康的人体肠道中存在着大量的有益菌，这些有益菌构成肠道的第一层生物屏障。
基于以上研究和认识，本申请研发并提出了一种新的组合物，该组合物包括加氏乳杆菌和/或其代谢产物，以及嗜酸乳杆菌和/或其代谢产物。本申请的组合物不仅具有溃疡性肠炎治疗和预防功能，而且，对于其它与微生态相关疾病，例如普通肠炎或胃炎等，同样具有效果；因此，本申请的组合物能够用于预防或治疗炎症或炎症相关疾病。
本申请的一种实现方式中，由保藏号GDMCC 60092的加氏乳杆菌TF08-1和保藏号为GDMCC 60091的嗜酸乳杆菌AM13-1组成的组合物，对溃疡性肠炎的治疗效果优于美国Alfasigma公司生产的VSL #3复合益生菌剂，能够用于制备治疗或预防炎症及其相关疾病的食品、保健品、食品添加剂或药品。
下面通过具体实施例对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例一
本例以分子量36000-50000的硫酸葡聚糖钠(缩写DSS)诱导的溃疡性肠炎小鼠模型为研究对象，研究加氏乳杆菌(Lactobacillus gasseri)TF08-1和嗜酸乳杆菌(Lactobacillus acidophilus)AM13-1组合物对溃疡性肠炎的治疗效果。并分别在加氏乳杆菌(Lactobacillus gasseri)TF08-1和嗜酸乳杆菌(Lactobacillus  acidophilus)AM13-1组合物中添加不同的益生菌，与之联合使用，这些添加的益生菌包括：深圳柯林斯菌(Collinsella shenzhenensis)TF06-26、罗斯氏菌(Roseburia inulinivorans)DSM 16841、丁酸杆菌(Butyribacter intestini)TF01-11、假小链状双歧杆菌(Bifidobacterium pseudocatenulatum)TM12-14、单形巨单胞菌(Megamonas funiformis)AF24-28AC和人粪厌氧棒形菌(Anaerofustis stercorihominis)AM25-6。具体如下：
一、材料和方法
1.菌株培养和鉴定
1.1 加氏乳杆菌TF08-1
本例的加氏乳杆菌TF08-1采用PYG培养基进行分离，分离条件为37℃厌氧条件。TF08-1在PYG培养基培养2天的菌落为白色、低凸起、近圆形、边缘波状，菌落直径约1-2mm，菌体的显微形态为杆状，革兰氏阳性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60092。
加氏乳杆菌TF08-1的具体分离鉴定步骤如下：
1.1.1 样品收集
分离的样品来自于一位16岁健康女性志愿者的粪便样品，志愿者居住于广东省深圳市。并详细记录该志愿者的饮食情况和身体情况。
1.1.2 菌株的分离培养
提前制备好分离培养基，培养基选用购自环凯微生物科技公司的PYG培养基，具体成分为：蛋白胨5g，胰化酪蛋白5g，酵母粉10g，牛肉膏5g，葡萄糖5g，K 2HPO 4 2g，Tween 80 1mL，Cysteine-HCl·H 2O 0.5g，硫化钠0.25g，血红素5mg，维生素K 11μL，无机盐溶液40mL，刃天青1mg，蒸馏水950mL，pH6.8～7.0，115℃灭菌25min。固体培养基加入1.5％的琼脂，在厌氧操作箱中倾倒。每1L无机盐溶液含有CaCl 2·2H 2O 0.25g，MgSO 4·7H 2O 0.5g，K 2HPO 4 1g，KH 2PO 4 1g，NaHCO 3 10g，NaCl 2g。
收集的新鲜粪便样品转移至厌氧箱，取0.2g粪便悬浮于1mL无菌磷酸盐缓冲液(缩写PBS)中，充分混匀，然后进行梯度稀释，取100μL稀释液进行平板涂布，37℃厌氧培养3-4天，厌氧的气体组分为N 2：CO 2：H 2＝90：5：5。待平板长出菌落选取单个菌落进行划线分纯，获得纯培养菌株，然后进行鉴定和功能验证。
1.1.3 菌株的16S rDNA鉴定
分离得到的菌株进行16S rDNA鉴定，以确定菌株的物种分类信息。将获得的分离菌株在液体PYG培养基中培养24h，取1mL菌液进行10000r/min离心 5min，收集菌体，提取菌株的基因组DNA，以基因组DNA作为模板进行16S rDNA的扩增，使用16S rDNA的通用引物。
16S rDNA的PCR扩增体系为：10×PCR缓冲液3μL、dNTP 2.5μL、上游引物27F 0.5μL、下游引物1492R 0.5μL、Taq酶0.3μL、模板1μL、ddH 2O 18.2μL。
16S rDNA的扩增条件为：95℃预变性4min，然后进入30个循环：95℃变性30s、57℃退火40s、72℃延伸1min 30s。
对16S rDNA的PCR产物进行纯化，3730测序，获得菌株的16S rDNA序列，然后进行NCBI的数据库的比对。
本试验的16S rDNA通用引物的上下游引物分别为SEQ ID NO.1和SEQ ID NO.2所示序列。分离获得的菌株TF08-1的16S rDNA序列结果为SEQ ID NO.3所示序列。NCBI blast比对结果显示，本例分离的TF08-1菌株与Lactobacillus gasseri同源性最高，相似度为99.9％，因此，判断TF08-1为加氏乳杆菌，命名为加氏乳杆菌TF08-1，并对其进行保藏。
SEQ ID NO.1：5’-AGAGTTTGATCATGGCTCAG-3’
SEQ ID NO.2：5’-TAGGGTTACCTTGTTACGACTT-3’。
1.2 嗜酸乳杆菌AM13-1
嗜酸乳杆菌AM13-1采用PYG培养基进行分离，分离条件为37℃厌氧条件。AM13-1在PYG培养基培养2天的菌落为白色，凸起，较粘稠，不透明，圆形，边缘整齐，直径约2-3mm，菌体的显微形态为杆状，革兰氏阳性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60091。
嗜酸乳杆菌AM13-1的具体分离鉴定步骤如下：
1.2.1 分离培养
分离的样品来自于深圳市一位健康男性的粪便样品，嗜酸乳杆菌AM13-1的分离过程如下：
(1)将样品转移至厌氧箱中，取约0.2g样品悬浮于1mL无菌PBS中，充分混匀，然后进行梯度稀释；
(2)取100μL稀释液于PYG培养基平板上，然后进行涂布，涂布均匀后放置在37℃厌氧环境中进行培养，厌氧的气体组成为：氮气：氢气：二氧化碳＝90:5:5；PYG培养基与“1.1.2菌株的分离培养”相同；
(3)培养4天，待平板上长出菌落之后，挑选单菌落进行划线分纯，37℃厌氧培养；
(4)对分纯的单菌进行甘油保藏和真空冷冻干燥保藏。
1.2.2 AM13-1的16S rDNA鉴定
提取基因组DNA，以DNA作为模板进行16S rDNA扩增，采用16S rDNA的通用引物进行PCR扩增，扩增的PCR产物进行纯化，3730测序，获得AM13-1的16S rDNA全长序列，将AF13-1的16S rDNA序列在NCBI的数据库比对。
本试验的16S rDNA通用引物、PCR扩增体系和条件都与“1.1.3菌株的16S rDNA鉴定”相同。分离获得的菌株AM13-1的16S rDNA序列结果为SEQ ID NO.4所示序列。NCBI blast比对结果显示，本例分离的AM13-1菌株与Lactobacillus acidophilus同源性最高，相似度为100％，因此，判断AM13-1为嗜酸乳杆菌，命名为嗜酸乳杆菌AM13-1，并对其进行保藏。
1.3 深圳柯林斯菌TF06-26
本例的深圳柯林斯菌TF06-26采用PYG培养基进行培养，培养条件为37℃厌氧条件。在厌氧的PYG培养基上深圳柯林斯菌TF06-26培养2天的菌落为白色，凸起，菌落较粘稠，菌落直径约1-2mm。菌体细胞在显微镜下呈短杆状，革兰氏阴性，没有芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60090。
深圳柯林斯菌TF06-26的具体分离鉴定步骤如下：
1.3.1 样品采集
本试验的深圳柯林斯菌TF06-26分离自深圳市的一位健康青少年女性志愿者的粪便样品。
1.3.2 准备培养基和磷酸盐缓冲液PBS
本试验所采用菌株分离的培养基是购自环凯微生物科技公司的厌氧的PYG培养基，具体成分是1L培养基中包含：蛋白胨5g，胰化酪蛋白5g，酵母粉10g，牛肉膏5g，葡萄糖5g，K 2HPO 4 2g，Tween 80 1mL，Cysteine-HCl·H 2O 0.5g，血红素5mg，维生素K 1 1μL，无机盐溶液40mL，刃天青1mg，蒸馏水950mL，调节pH至6.8～7.0。灭菌条件为115℃高压灭菌25min。固体培养基需在厌氧操作箱中倾倒平板。其中，每升1L无机盐溶液含有CaCl 2·2H 2O 0.25g，MgSO 4·7H 2O 0.5g，K 2HPO 4 1g，KH 2PO 4 1g，NaHCO 3 10g，NaCl 2g。
PBS的准备：称取NaCl 8g，KCl 0.2g，Na 2HPO 4·12H 2O 3.63g，KH 2PO 4 0.24g，半胱氨酸盐酸盐0.5g，溶于900mL双蒸水中，用盐酸和NaOH调pH值至7.4，加水定容至1L，通入N 2除氧30s，厌氧瓶密封，然后115℃高压灭菌25min，备用。
1.3.3 菌株分离
收集的新鲜粪便样品立即转移至厌氧箱，厌氧箱的气体组成为：氮气： 氢气：二氧化碳＝90：5：5，v/v，取约0.2g粪便于PBS中进行悬浮，充分混匀，进行10倍为单位的梯度稀释，然后涂平板，37℃厌氧条件下培养2天，选择单菌落进行划线纯化，获得纯培养菌株，并进行-80℃甘油冷冻保藏和真空冷冻干燥保藏。
1.3.4 16S rDNA鉴定
对分离得到的纯培养菌株进行16S rDNA测序，获得每株菌的分类信息。菌株在液体的PYG培养基中培养24小时，达到菌浓约10 8cfu/ml，对菌液进行基因组提取，以提取的基因组DNA作为模板进行16S rDNA PCR扩增，对获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得菌株的16S rDNA序列，然后进行EZBio Cloud数据库的比对，对分离菌株进行鉴定。
本试验16S rDNA PCR扩增的引物、PCR扩增体系和条件都与“1.1.3菌株的16S rDNA鉴定”相同。
测序结果显示，本例分离的TF06-26菌株的16S rDNA序列为SEQ ID NO.5所示序列。EZBio Cloud数据库比对结果显示，本例分离的TF06-26菌株与购自日本微生物菌种保藏中心的Collinsella aerofaciens JCM 10188同源性最高。
1.3.5 TF06-26的生理生化特征
TF06-26为过氧化氢酶阴性，氧化酶阴性，不具运动性，生长温度范围是25-45℃，生长pH值范围是5.0-8.0，NaCl耐受浓度为2％，胆盐耐受浓度是0.3％。TF06-26和近缘参考菌购自日本微生物菌种保藏中心的Collinsella aerofaciens JCM 10188的底物利用情况，API 20A和API 50CHL，详见表1，表1中“+”表示阳性反应，“-”表示阴性反应，“w”表示弱阳性反应。
表1 TF06-26和JCM 10188的底物利用情况
Figure PCTCN2018089317-appb-000001
Figure PCTCN2018089317-appb-000002
表1中TF06-26与JCM 10188的碳源利用情况比较显示，TF06-26同JCM 10188在乳糖、蔗糖、柳醇、半乳糖、果糖、甘露糖、熊果甙、纤维二糖、 麦芽糖、蜜二糖、海藻糖以及2-酮基-葡萄糖酸盐的利用有着明显不同，由此可见TF06-26跟JCM 10188不是同一个物种。
1.3.6 新物种TF06-26同亲缘菌株JCM 10188的基因组杂交试验
参考TF06-26的16S rDNA比对结果，亲缘关系最近的菌为Collinsella aerofaciens JCM 10188，16S rDNA相似度为99.9％，从16S rDNA序列来看，无法将TF06-26和JCM 10188进行种水平的区分，因此需要进行DNA杂交进一步的确认。
DNA杂交结果显示，TF06-26同JCM 10188的同源性为51％。根据《伯杰氏细菌鉴定手册》，两株菌的DNA杂交值高于70％，可以判定这两株菌属于同一个物种，TF06-26和JCM 10188的DNA杂交值低于70％，所以TF06-26是一株不同于已知菌的新菌种。按照国际细菌分类委员会(IBSP)细菌命名规则，将这株新菌命名为Collinsella shenzhenensis sp.nov，TF06-26作为这个种的模式菌株。即本例的深圳柯林斯菌TF06-26，并对其进行保藏
1.4 罗斯氏菌DSM 16841
本例的罗斯氏菌(Roseburia inulinivorans)DSM 16841，培养基采用PYG厌氧培养基，37℃厌氧培养。在PYG培养基培养2天的菌落为浅黄色，直径约1mm。菌体的显微形态为短杆状，革兰氏阳性、不产芽孢和鞭毛。菌株购自德国微生物菌种保藏中心(DSMZ)，保藏编号为DSM 16841。
1.5 丁酸杆菌TF01-11
本例的丁酸杆菌TF01-11采用PYG培养基进行培养，培养条件为37℃厌氧条件。在厌氧的PYG培养基上丁酸杆菌TF01-11培养48h之后菌落呈现灰白色，不透明，平滑，边缘不规则似假根状，菌落直径约2mm。通过革兰氏染色、显微观察，TF01-11为革兰氏阳性菌，长杆状，无芽孢，具有鞭毛，可运动，菌体直径约0.5-1.0mm，长度约2.0-8.0mm。菌株由中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)提供和保藏，保藏编号为CGMCC 10984。
1.6 假小链状双歧杆菌TM12-14
假小链状双歧杆菌TM12-14采用PYG培养基进行分离，分离条件为37℃厌氧条件。TM12-14在PYG培养基培养2天的菌落为白色、凸起、圆形、边缘整齐，菌落直径约1-2mm，菌体显微形态呈现分歧杆状，革兰氏染色为阳性，没有芽孢和鞭毛产生。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60089。
假小链状双歧杆菌TM12-14的具体分离鉴定步骤如下：
1.6.1 样品收集
分离样品来自于一位14岁健康的男性粪便，将粪便采集至无菌的样品管中，1h内带回实验室进行分选。
1.6.2 假小链状双歧杆菌的分离纯化
收集的新鲜样品立刻转移至厌氧操作箱中，取0.2g样品于1mL无菌的PBS中，充分震荡混匀，然后进行梯度稀释涂布，培养基采用PYG培养基平板，于37℃厌氧培养，厌氧的气体组分为N 2：CO 2：H 2＝90：5：5。培养3天后，挑取单菌落进行划线分纯，获得每株单菌的纯培养。其中，PYG培养基与“1.1.2菌株的分离培养”相同。
1.6.3 菌种保藏
对获得的纯培养菌株进行培养，至浓度约为10 9cfu/mL，取400μL菌液添加40％甘油400μL，使其甘油浓度达到20％，然后进行-80℃超低温保藏。
1.6.4 16S rDNA鉴定
将获得的分离菌株在液体PYG培养基中培养24h，取1mL菌液进行10000r/min离心5min，收集菌体，提取基因组DNA。以基因组DNA作为模板，使用16S rDNA通用引物进行PCR扩增，PCR扩增引物、体系和条件与“1.1.3菌株的16S rDNA鉴定”相同。
将获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得菌株的16S rDNA序列，然后进行NCBI的数据库的比对。
测序结果显示，菌株TM12-14的16S rDNA长度为1400bp，序列如SEQ ID NO.6所示。NCBI blast比对结果显示，菌株TM12-14与Bifidobacterium pseudocatenulatum同源性最高，鉴定其为假小链状双歧杆菌Bifidobacterium pseudocatenulatum，命名为假小链状双歧杆菌TM12-14，并对其进行保藏。
1.7 单形巨单胞菌AF24-28AC
本例的单形巨单胞菌AF24-28AC采用PYG培养基进行培养，培养条件为37℃厌氧条件。AF24-28AC在PYG培养基培养2天的菌落为浅黄色，边缘不规则波状，扁平，含水量低，直径约2-3mm。菌体的显微形态为杆状，革兰氏阴性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60093。
单形巨单胞菌AF24-28AC的具体分离鉴定步骤如下：
1.7.1 菌株的分离培养
分离样品来自于一位健康的女性粪便，采用梯度稀释涂平板方法进行菌株分离，培养基采用购自环凯微生物科技公司的PYG培养基，进行厌氧培养，厌氧的气体组分为N 2：CO 2：H 2＝90：5：5，培养48h，挑取单菌落进行划线 分纯，获得每株单菌的纯培养。
1.7.2 菌株的16S rDNA鉴定
对分离菌株的基因组DNA进行提取，采用16S rDNA通用引物进行PCR扩增，对获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得菌株的16S rDNA序列，然后在EZBio Cloud数据库中进行比对，对分离菌株进行鉴定。
16S rDNA的PCR扩增体系为：10×PCR缓冲液3μL、dNTPs 2.5μL、上游引物27F 0.5μL、下游引物1492R 0.5μL、Taq酶0.3μL、模板1μL、ddH 2O 18.2μL。
16S rDNA的扩增条件如下：
Figure PCTCN2018089317-appb-000003
其中，65℃-57℃40s是指每个循环后温度等比递减，即第1个循环时的退火温度为65℃，最终在第20个循环降低到57℃。
本试验的16S rDNA通用引物的上游引物为27f，上游引物序列为SEQ ID NO.1所示序列，下游引物为1492r，下游引物序列为SEQ ID NO.2所示序列。
分离获得的菌株AF24-28AC的16S rDNA序列结果为SEQ ID NO.7所示序列。EZBio Cloud数据库比对结果显示，本例分离的AF24-28AC菌株与购自德国菌种保藏中心的Megamonas funiformis DSM 19343同源性最高，相似度为99.09％，因此，判断AF24-28AC为单形巨单胞菌，命名为单形巨单胞菌AF24-28AC，并对其进行保藏。
1.8 人粪厌氧棒形菌AM25-6
本例的人粪厌氧棒形菌AM25-6采用PYG培养基进行培养，培养条件为37℃厌氧条件。AM25-6在PYG培养基培养2天的菌落为浅黄色，菌落较小，针尖状，直径约0.5mm。菌体的显微形态为短杆状，革兰氏阳性、不产芽孢和鞭毛。菌株保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC 60087。
人粪厌氧棒形菌AM25-6的具体分离鉴定步骤如下：
1.8.1 菌株的分离培养
分离样品来自于一位健康的男性粪便，将粪便采集至无菌的样品管中，1h内带回实验室进行分选。收集的新鲜样品立刻转移至厌氧操作箱中，取0.2g样品悬浮在1mL无菌的磷酸缓冲液(缩写PBS)中，充分震荡混匀。采用梯度稀释涂平板方法进行菌株分离，培养基采用购自环凯微生物科技公司的PYG培养基。涂布的平板置于37℃厌氧培养，厌氧的气体组分为N 2：CO 2：H 2＝90：5：5。培养3天后，挑取单菌落进行划线分纯，获得每株单菌的纯培养。对获得的纯培养菌株进行培养，至浓度约为10 9cfu/mL，取400μL菌液添加40％甘油400μL，使其甘油浓度达到20％，然后进行-80℃超低温保藏。
1.8.2 菌株16S rDNA鉴定
对分离菌株的基因组DNA进行提取，采用16S rDNA通用引物进行PCR扩增，对获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得菌株的16S rDNA序列，然后进行EZBio Cloud数据库的比对，对分离菌株进行鉴定。
本试验16S rDNA PCR扩增的引物、PCR扩增体系和条件都与“1.1.3菌株的16S rDNA鉴定”相同。
测序结果显示，本例分离的AM25-6菌株的16S rDNA序列为SEQ ID NO.8所示序列。EZBio Cloud数据库比对结果显示，本例分离的AM25-6菌株与购自德国菌种保藏中心的Anaerofustis stercorihominis DSM 17244同源性最高，同源性99.86％，因此，判断AM25-6为人粪厌氧棒形菌，命名为人粪厌氧棒形菌AM25-6，并对其进行保藏。
2.小鼠模型
本例所选取的小鼠模型为：DSS(硫酸葡聚糖钠Dextran Sulfate,Na，分子量36000-50000)诱导的溃疡性肠炎小鼠模型。
具体的，采用购自湖北医学实验动物中心的小鼠品系为C57bl/6小鼠84只，所有小鼠8周龄，体重20g±2g，在SPF级别的鼠房环境中饲养。96只小鼠随机分为8组，每组12只进行后续试验。
DSS造模：续给小鼠饮用0.15％的DSS七天，即获得溃疡性肠炎小鼠模型。
3.试验方法
84只小鼠随机分为8组，每组12只，8组分别为正常组(即control组)、模型组、益生菌组合物治疗组、组合菌1组、组合菌2组、组合菌3组、组合菌4组、组合菌5组和VSL #3治疗组，各组的具体处理方式如下：
正常组：采用普通饲料进行饲喂，每只小鼠每天灌胃0.2mL的PBS缓冲液。
模型组：采用相同的饲料饲喂，并进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的PBS缓冲液。
益生菌组合物治疗组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的益生菌组合物菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的益生菌组合物菌液。
组合菌1组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的组合菌1菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的组合菌1菌液。
组合菌2组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的组合菌2菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的组合菌2菌液。
组合菌3组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的组合菌3菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的组合菌3菌液。
组合菌4组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的组合菌4菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的组合菌4菌液。
组合菌5组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的组合菌5菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的组合菌5菌液。
VSL #3治疗组：采用相同的饲料饲喂，在进行DSS造模前3天，每天给每只小鼠灌胃0.2mL的VSL #3菌液，然后再进行DSS造模：将DSS添加到小鼠饮水中，DSS添加量为终浓度0.15％，饲喂七天，每只小鼠每天灌胃0.2mL的VSL #3菌液。
其中，益生菌组合物菌液、组合菌1至组合菌5菌液采用如下方法配制：
将加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1、深圳柯林斯菌TF06-26、罗斯 氏菌(Roseburia inulinivorans)DSM 16841、丁酸杆菌TF01-11、假小链状双歧杆菌TM12-14、单形巨单胞菌AF24-28AC和人粪厌氧棒形菌AM25-6分别培养24h，离心分别收集菌体，用PBS对各菌体进行悬浮，分别调整菌浓度至10 9cfu/mL的菌悬液，用于配制各菌液。
益生菌组合物菌液：加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1的菌悬液等体积混合，即获得益生菌组合物菌液；
组合菌1菌液：加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和深圳柯林斯菌TF06-26的菌悬液等体积混合，即获得组合菌1菌液；
组合菌2菌液：加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和罗斯氏菌(Roseburia inulinivorans)DSM 16841的菌悬液等体积混合，即获得组合菌2菌液；
组合菌3菌液：加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和丁酸杆菌TF01-11的菌悬液等体积混合，即获得组合菌3菌液；
组合菌4菌液：加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14的菌悬液等体积混合，即获得组合菌3菌液；
组合菌5菌液：加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1、单形巨单胞菌AF24-28AC和人粪厌氧棒形菌AM25-6的菌悬液等体积混合，即获得组合菌5菌液。
VSL #3菌液采用如下方法配制：
VSL #3购自美国Alfasigma公司，是一种，包含干酪乳杆菌、植物乳杆菌、嗜酸乳杆菌、德式乳杆菌保加利亚亚种、嗜热链球菌、长双歧杆菌、短双歧杆菌、婴儿双岐杆菌等8种有益菌的复合益生菌剂；同样，采用PBS对VSL #3进行悬浮，调节浓度至10 9cfu/mL，即获得VSL #3菌液。
DSS造模后每天记录小鼠体重、饮食和饮水情况，同时观察小鼠的粪便性状及粪便隐血情况，分别在第1天、第3天、第5天和第7天计算小鼠的疾病活动指数(缩写DAI)，DAI评分标准详见表2。实验结束后处死小鼠，所有小鼠取血、脱颈、取结肠、拍照、称重、量取结肠长度。结肠组织保存于-80℃冰箱和多聚甲醛中。其中，正常组的记录时间与DSS造模相同。
表2 DAI指数评分表
Figure PCTCN2018089317-appb-000004
Figure PCTCN2018089317-appb-000005
表2中的“大便性状”，“正常”大便是指成形大便，“松散”大便是指不粘附于肛门的糊状、半成型大便，“稀便”是指可粘附于肛门的稀样水便。“大便隐血/弱眼血便”中，“正常”是指小鼠便血为阴性；“肉眼血便”是指肉眼可以直接观察到红色或褐色便血；“隐血阳性”是指不明显的肉眼血便，使用四甲基联苯胺进行检测为便血阳性。DAI指数等于体重、“大便性状”以及“大便隐血/弱眼血便”三个积分之和。
二、结果及分析
1.体重变化
统计小鼠分别在第1天、第3天、第5天和第7天的体重，各组小鼠的平均体重如表3和图1所示。
表3 各自小鼠平均体重
分组 第1天(g) 第3天(g) 第5天(g) 第7天(g)
Control 22.32±0.45 23.73±0.64 24.98±0.96 25.52±1.26
模型组 22.41±0.52 21.65±0.71* 20.02±1.26* 18.21±1.57**
益生菌 22.29±0.50 21.85±0.79 21.41±1.27 ▲ 21.40±1.46 ▲
组合菌1 22.31±0.48 21.89±0.72 21.43±1.09 ▲ 21.51±1.50 ▲
组合菌2 22.22±0.62 21.90±0.81 21.48±1.14 ▲ 21.48±1.61 ▲
组合菌3 22.50±0.56 21.94±0.78 21.47±1.19 ▲ 21.54±1.59 ▲
组合菌4 22.38±0.61 21.97±0.85 21.47±1.35 21.32±1.49 ▲
组合菌5 22.09±0.47 21.59±0.81 21.42±1.37 ▲ 21.21±1.75 ▲
VSL #3 22.54±0.71 21.87±0.92 21.32±1.48 20.01±1.66 ▲
表3中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，组合菌1即组合菌1治疗组，组合菌2即组合菌2治疗组，组合菌3即组合菌3治疗组，组合菌4即组合菌4治疗组，组合菌5即组合菌5治疗组，“VSL #3” 即VSL #3治疗组。“*”是指模型组相对于正常组小鼠体重的差异显著水平P<0.05，“**”是指模型组相对于正常组小鼠体重的差异显著水平P<0.01，“▲”是指“益生菌”、“组合菌1”、“组合菌2”、“组合菌3”、“组合菌4”、“组合菌5”和“VSL #3”组小鼠相对于模型组的差异显著水平P<0.05。
表3和图1的结果显示，Control组小鼠的体重呈缓慢升高的趋势，DSS诱导的8组小鼠的体重均持续下降，模型组与对照组相比，在第3天体重下降开始显著(*P<0.05)，第7天，二者之间的差异显著程度更加明显(**P<0.01)。而益生菌组、组合菌1，2，3，4，5和VSL#3的干预都可以减缓UC小鼠体重的下降，在第7天，这7组小鼠的体重下降的控制相对于模型组比较显著(▲P<0.05)。说明本例由加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组成的益生菌组，以及组合菌1，2，3，4，5和VSL#3都可以控制UC引起的体重下降情况。并且，在第7天本例的加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组成益生菌组合物组以及组合菌1，2，3，4，5的小鼠的体重都略高于VSL#3，说明加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组成益生菌组合物，以及在此基础上添加其他益生菌的组合菌在控制UC小鼠体重降低的效果上都略优于VSL#3。
2.DAI的变化
DSS诱导的溃疡性肠炎的小鼠由于体重下降、大便性状和便血情况的变化引起DAI指数的变化，小鼠DAI指数在第1天、第3天、第5天和第7天的统计值如表4和图2。表4中，各组小鼠的DAI取各组小鼠的平均值。
表4 小鼠DAI值
分组 第1天 第3天 第5天 第7天
Control 1.1±0.5 1.1±0.7 1.2±0.8 1.3±0.8
模型组 1.1±0.5 3.6±1.1* 7.2±1.6** 9.4±2.0**
益生菌 1.2±0.4 3.5±1.1 6.1±1.5 ▲ 6.5±1.9 ▲
组合菌1 1.1±0.4 3.4±1.2 6.0±1.5 ▲ 6.4±1.8 ▲
组合菌2 1.2±0.4 3.3±1.3 5.9±1.4 ▲ 6.4±1.7 ▲
组合菌3 1.1±0.5 3.5±1.2 6.0±1.6 ▲ 6.6±1.9 ▲
组合菌4 1.2±0.4 3.3±1.2 6.0±1.5 ▲ 6.5±1.8 ▲
组合菌5 1.1±0.4 3.4±1.2 6.0±1.7 ▲ 6.4±2.1 ▲
VSL #3 1.1±0.4 3.4±1.3 6.6±1.6 7.8±1.9 ▲
表4中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，组合菌1即组合菌1治疗组，组合菌2即组合菌2治疗组，组合菌3即组合菌3治疗组，组合菌4即组合菌4治疗组，组合菌5即组合菌5治疗组，“VSL #3”即VSL #3治疗组。“*”是指模型组相对于正常组小鼠DAI指数的差异显著水平P<0.05，“**”是指模型组相对于正常组小鼠DAI指数的差异显著水平P<0.01，“▲”是指“益生菌”、“组合菌1”、“组合菌2”、“组合菌3”、“组合菌4”、“组合菌5”和“VSL #3”组小鼠相对于模型组的差异显著水平P<0.05。
表4和图2数据显示，Control组小鼠的DAI基本持平，而随着DSS诱导，模型组、益生菌组和基于益生菌组基础上添加其他益生菌的组合菌1，2，3，4，5和VSL#3组小鼠的DAI逐渐升高，与对照组相比，第3天模型组小鼠DAI变得开始显著(*P<0.05)，第7天模型组小鼠DAI达到最高水平(即相对于control组**P<0.01)。加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组合益生菌组合、在益生菌组合基础上添加其他益生菌形成的组合菌1，2，3，4，5的干预可以控制DAI的升高，这几组小鼠在第5天和第7天的DAI值相对于模型组得到了显著的控制(▲P<0.05)，并且，在第7天益生菌组合和组合菌1，2，3，4，5干预的小鼠的DAI略低于VSL#3，说明加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组成益生菌组合物，以及在此基础上添加其他益生菌的组合菌在控制UC小鼠DAI升高的效果都优于VSL#3。
3.结肠长度的变化
UC模型小鼠的结肠组织会发生改变，主要是因为溃疡和炎症的发生导致结肠组织缩短，治疗结束后，通过解剖测量的小鼠结肠长度如表5所示。
表5 小鼠结肠长度
分组 结肠长度(cm)
Control 8.38±0.49
模型组 5.02±0.87**
VSL #3 6.10±0.67 ▲
益生菌 6.62±0.69 ▲
组合菌1 6.64±0.58 ▲
组合菌2 6.66±0.71 ▲
组合菌3 6.65±0.79 ▲
组合菌4 6.63±0.71 ▲
组合菌5 6.63±0.62 ▲
表5中，“Control”是指正常组，“益生菌”即益生菌组合物治疗组，组合菌1即组合菌1治疗组，组合菌2即组合菌2治疗组，组合菌3即组合菌3治疗组，组合菌4即组合菌4治疗组，组合菌5即组合菌5治疗组，“VSL #3”即VSL #3治疗组。“**”是指模型组相对于正常组小鼠结肠长度差异显著水平P<0.01，“▲”是指“益生菌”、“组合菌1”、“组合菌2”、“组合菌3”、“组合菌4”、“组合菌5”和“VSL #3”组小鼠相对于模型组小鼠结肠长度差异显著水平P<0.05。
表5的结果显示，在进行DSS诱导7天后的模型组小鼠的结肠组织缩短情况比较严重，与control组相比较非常显著(**P<0.01)。而益生菌组合物、组合菌1，2，3，4，5和VSL#3的干预可以显著控制小鼠结肠的缩短，相对于模型组得到了显著的控制(*P<0.05)。通过表中数据可以发现益生菌组合物和组合菌1，2，3，4，5干预的小鼠的结肠长度比VSL#3组小鼠结肠长度长，可以说明加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组成益生菌组合物，以及在此基础上添加其他益生菌的组合菌在控制UC小鼠结肠缩短的能力强于VSL#3。
表3至表5以及图1和图2的结果显示，本例的益生菌组合物，以及在此基础上的组合菌1，2，3，4，5对溃疡性肠炎具有治疗和预防作用，并且治疗效果都略优于现有的VSL #3产品。
实施例二
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1按照表6的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表6 含益生菌组合物的食品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.2
Lactobacillus acidophilus AM13-1 0.2
牛奶 90.0
白糖 9.0
维生素C 0.6
按照表6的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(即维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1和嗜酸乳杆菌Lactobacillus acidophilus AM13-1两种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的益生菌组合物治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃益生菌组合物菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例三
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌1的组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和深圳柯林斯菌TF06-26按照表7的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表7 含益生菌组合物的食品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.2
Lactobacillus acidophilus AM13-1 0.2
Collinsella shenzhenensis TF06-26 0.2
牛奶 90.0
白糖 8.8
维生素C 0.6
按照表7的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和深圳柯林斯菌Collinsella shenzhenensis TF06-26三种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的组合菌1治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃组合菌1菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠 炎治疗和预防效果。
实施例四
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌2的组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和罗斯氏菌(Roseburia inulinivorans)DSM 16841按照表8的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表8 含益生菌组合物的食品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.2
Lactobacillus acidophilus AM13-1 0.2
Roseburia inulinivorans DSM 16841 0.2
牛奶 90.0
白糖 8.8
维生素C 0.6
按照表8的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和罗斯氏菌Roseburia inulinivorans DSM 16841三种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的组合菌2治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃组合菌2菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例五
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌3的组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和丁酸杆菌TF01-11按照表9的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表9 含益生菌组合物的食品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.2
Lactobacillus acidophilus AM13-1 0.2
Butyribacter intestini TF01-11 0.2
牛奶 90.0
白糖 8.8
维生素C 0.6
按照表9的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和丁酸杆菌Butyribacter intestini TF01-11三种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的组合菌3治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃组合菌3菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例六
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌3的组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1和假小链状双歧杆菌TM12-14按照表10的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表10 含益生菌组合物的食品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.15
Lactobacillus acidophilus AM13-1 0.15
Bifidobacterium pseudocatenulatum TM12-14 0.15
牛奶 90.0
白糖 9.0
维生素C 0.55
按照表10的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压 力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14三种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的组合菌4治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃组合菌4菌液。检测结果显示，本例的牛乳产品同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例七
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌5的组合物制成常见的食品，具体如下：
将牛奶、维生素C和白糖等辅料，与培养的加氏乳杆菌TF08-1、嗜酸乳杆菌AM13-1、单形巨单胞菌AF24-28AC和人粪厌氧棒形菌AM25-6、按照表11的配方混合，制备具有溃疡性肠炎治疗和预防功能的食品。
表11 含益生菌组合物的食品配方
原料 质量百分比(％)
Megamonas funiformis AF24-28AC 0.15
Anaerofustis stercorihominis AM25-6 0.15
Lactobacillus gasseri TF08-1 0.15
Lactobacillus acidophilus AM13-1 0.15
牛奶 90.0
白糖 8.8
维生素C 0.6
按照表11的配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入保护剂(维生素C)，并接种1-100×10 6cfu/g的加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1、单形巨单胞菌Megamonas funiformis AF24-28AC和人粪厌氧棒形菌Anaerofustis stercorihominis AM25-6四种混合益生菌的食品组合物。
将本例的牛乳产品加入DSS造模的小鼠饲料中，按照实施例一的组合菌5治疗组进行饲喂和检测，所不同的是，本例仅仅在饲料中添加了本例的牛乳产品，不额外灌胃组合菌5菌液。检测结果显示，本例的牛乳产品同样能够控制 UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例八
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的加氏乳杆菌TF08-1和嗜酸乳杆菌AM13-1组合物制成用于治疗溃疡性肠炎的药物，配方如表12所示：
表12 含益生菌组合物的药品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.75％
Lactobacillus acidophilus AM13-1 0.75％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 93％
维生素C 0.5％
按照表12的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1和嗜酸乳杆菌Lactobacillus acidophilus AM13-1活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1和嗜酸乳杆菌Lactobacillus acidophilus AM13-1两株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一益生菌组合物治疗组中的益生菌组合物菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例九
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌1的组合物制成用于治疗溃疡性肠炎的药物，配方如表13所示：
表13 含益生菌组合物的药品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.75％
Lactobacillus acidophilus AM13-1 0.75％
Collinsella shenzhenensis TF06-26 0.75％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 92.25％
维生素C 0.5％
按照表13的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和深圳柯林斯菌Collinsella shenzhenensis TF06-26活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和深圳柯林斯菌Collinsella shenzhenensis TF06-26三株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一组合菌1治疗组中的组合菌1菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例十
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌2的组合物制成用于治疗溃疡性肠炎的药物，配方如表14所示：
表14 含益生菌组合物的药品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.75％
Lactobacillus acidophilus AM13-1 075％
Roseburia inulinivorans DSM 16841 0.75％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 92.25％
维生素C 0.5％
按照表14的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和罗斯氏菌Roseburia inulinivorans DSM 16841活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和罗斯氏菌Roseburia inulinivorans DSM 16841三株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一组合菌2治疗组中的组合菌2菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例十一
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌3的组合物制成用于治疗溃疡性肠炎的药物，配方如表15所示：
表15 含益生菌组合物的药品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.75％
Lactobacillus acidophilus AM13-1 0.75％
Butyribacter intestini TF01-11 0.75％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 92.25％
维生素C 0.5％
按照表15的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和丁酸杆菌Butyribacter  intestini TF01-11活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和丁酸杆菌Butyribacter intestini TF01-11三株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一组合菌3治疗组中的组合菌3菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例十二
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌4的组合物制成用于治疗溃疡性肠炎的药物，配方如表16所示：
表16 含益生菌组合物的药品配方
原料 质量百分比(％)
Lactobacillus gasseri TF08-1 0.75％
Lactobacillus acidophilus AM13-1 0.75％
Bifidobacterium pseudocatenulatum TM12-14 0.75％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 92.25％
维生素C 0.5％
按照表16的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1和假小链状双歧杆菌Bifidobacterium pseudocatenulatum TM12-14三株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一组合菌4治疗组中的组合菌4菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
实施例十三
本例将实施例一证实具有溃疡性肠炎治疗和预防效果的组合菌5的组合物制成用于治疗溃疡性肠炎的药物，配方如表17所示：
表17 含益生菌组合物的药品配方
原料 质量百分比(％)
Megamonas funiformis AF24-28AC 0.75％
Anaerofustis stercorihominis AM25-6 0.75％
Lactobacillus gasseri TF08-1 0.5％
Lactobacillus acidophilus AM13-1 0.5％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 92％
维生素C 0.5％
按照表17的比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入保护剂(维生素C)，分别接入1-50×10 6cfu/mL加氏乳杆菌Lactobacillus gasseri TF08-1、嗜酸乳杆菌Lactobacillus acidophilus AM13-1、单形巨单胞菌Megamonas funiformis AF24-28AC和人粪厌氧棒形菌Anaerofustis stercorihominis AM25-6活菌，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备组合益生菌冷冻干燥物。称取0.5克冷冻干燥物与等量的麦芽糊精混合后装入胶囊中，即制成含单形巨单胞菌Megamonas funiformis AF24-28AC、人粪厌氧棒形菌Anaerofustis stercorihominis AM25-6、加氏乳杆菌Lactobacillus gasseri TF08-1和嗜酸乳杆菌Lactobacillus acidophilus AM13-1四株组合益生菌的胶囊剂药物组合物。
采用本例的胶囊剂药物组合物替换实施例一组合菌5治疗组中的组合菌5菌液，按照实施例一相同的方式，灌胃本例的胶囊剂药物组合物，每天灌胃一粒胶囊，按照实施例一相同的方法进行检测。结果显示，本例的胶囊剂药物组 合物同样能够控制UC小鼠体重降低、降低小鼠疾病活动指数DAI、改善肠道病变，具有溃疡性肠炎治疗和预防效果。
以上各实施例证明加氏乳杆菌和嗜酸乳杆菌两者联合使用能够治疗和预防溃疡性肠炎；并且，在加氏乳杆菌和嗜酸乳杆菌的基础上，还可以添加深圳柯林斯菌、罗斯氏菌(Roseburia inulinivorans)、丁酸杆菌或假小链状双歧杆菌，或者添加单形巨单胞菌和人粪厌氧棒形菌，这些组合都能够治疗和预防溃疡性肠炎。以上组合的组合物可以制成各种食品或药品使用；当然，可以理解，以上组合的组合物既然可以制成各种食品和药品，同样也可以制成各种保健品或食品添加剂。
另外，研究显示，加氏乳杆菌和嗜酸乳杆菌组合的治疗效果很大程度上是基于微生态的改善，而这种微生态的改善不仅对溃疡性肠炎有治疗和预防效果，对其它与微生态相关疾病，例如普通肠炎、胃炎等，同样具有效果；因此，本申请的组合物可以用于预防或治疗炎症或炎症相关疾病，特别是各种肠炎、胃炎。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。

Claims (28)

  1. 一种组合物，其特征在于：所述组合物包括加氏乳杆菌和/或其代谢产物，以及嗜酸乳杆菌和/或其代谢产物。
  2. 根据权利要求1所述的组合物，其特征在于：所述加氏乳杆菌为保藏号GDMCC 60092的加氏乳杆菌TF08-1，所述嗜酸乳杆菌为保藏号GDMCC 60091的嗜酸乳杆菌AM13-1。
  3. 根据权利要求1所述的组合物，其特征在于：所述组合物还包括深圳柯林斯菌和/或其代谢产物；优选的，所述深圳柯林斯菌为保藏号GDMCC 60090的深圳柯林斯菌TF06-26。
  4. 根据权利要求1所述的组合物，其特征在于：所述组合物还包括罗斯氏菌和/或其代谢产物；优选的，所述罗斯氏菌为保藏号DSM 16841的罗斯氏菌。
  5. 根据权利要求1所述的组合物，其特征在于：所述组合物还包括丁酸杆菌和/或其代谢产物；优选的，所述丁酸杆菌为保藏号CGMCC 10984的丁酸杆菌TF01-11。
  6. 根据权利要求1-5任一项所述的组合物，其特征在于：所述组合物还含有其它益生菌和/或益生元；优选的，所述益生元选自低聚果糖、低聚半乳糖、低聚木糖、低聚乳果糖、大豆低聚糖、菊粉和寡聚糖中的至少一种。
  7. 根据权利要求1-5任一项所述的组合物，其特征在于：所述组合物还含有有助于保持组合物中各菌株的至少一种的活力的物质。
  8. 根据权利要求7所述的组合物，其特征在于：所述有助于保持组合物中各菌株的至少一种的活力的物质，选自半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸或其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C和维生素E中的至少一种。
  9. 根据权利要求1-5任一项所述的组合物，其特征在于：所述组合物还包括药学上或食品上可接受的载体或辅料。
  10. 根据权利要求9所述的组合物，其特征在于：所述药学上或食品上可接受的载体或辅料，选自葡萄糖、乳糖、蔗糖、淀粉、甘露醇、糊精、脂肪酸甘油酯、聚乙二醇、羟乙基淀粉、乙二醇、聚氧乙烯山梨糖醇酐脂肪酸酯、氨基酸、明胶、白蛋白、水和生理食盐水中的至少一种。
  11. 根据权利要求1-10任一项所述的组合物在制备用于治疗或预防炎症或炎症相关疾病的食品、保健品、食品添加剂或药品中的应用。
  12. 根据权利要求11所述的应用，其特征在于：所述炎症为溃疡性肠炎。
  13. 根据权利要求1-10任一项所述的组合物在制备用于控制哺乳动物体重下降的食品、保健品、食品添加剂或药品中的应用。
  14. 根据权利要求13所述的应用，其特征在于：所述哺乳动物体重下降，是指哺乳动物因炎症导致的体重下降；优选的，所述炎症为溃疡性肠炎。
  15. 根据权利要求1-10任一项所述的组合物在制备用于降低哺乳动物的疾病活动指数的食品、保健品、食品添加剂或药品中的应用。
  16. 根据权利要求1-10任一项所述的组合物在制备用于改善哺乳动物肠道病变的食品、保健品、食品添加剂或药品中的应用。
  17. 一种采用权利要求1-10任一项所述的组合物进行炎症或炎症相关疾病的治疗或预防的方法。
  18. 一种采用权利要求1-10任一项所述的组合物控制哺乳动物体重下降的方法。
  19. 一种采用权利要求1-10任一项所述的组合物降低哺乳动物的疾病活动指数的方法。
  20. 一种采用权利要求1-10任一项所述的组合物改善哺乳动物肠道病变的方法。
  21. 一种食品，其特征在于：所述食品中含有权利要求1-10任一项所述的组合物。
  22. 根据权利要求21所述的食品，其特征在于：所述食品为乳酸饮品或豆乳饮品。
  23. 一种保健品，其特征在于：所述保健品中含有权利要求1-10任一项所述的组合物。
  24. 一种食品添加剂，其特征在于：所述食品添加剂中含有权利要求1-10任一项所述的组合物。
  25. 一种药品，其特征在于：所述药品中含有权利要求1-10任一项所述的组合物。
  26. 根据权利要求25所述的药品，其特征在于：所述药品为片剂、颗粒剂、散剂、肠溶剂、溶液剂或悬浮剂。
  27. 根据权利要求25所述的药品，其特征在于：所述药品为肠溶剂，所述肠溶剂为胶囊或肠溶片；优选的，所述药品为肠溶片。
  28. 根据权利要求27所述的药品，其特征在于：所述肠溶片的肠溶包衣厚度为5-100μm，优选的，厚度为20-80μm。
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