WO2018112740A1 - 一种加氏乳杆菌及其培养方法和应用 - Google Patents

一种加氏乳杆菌及其培养方法和应用 Download PDF

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WO2018112740A1
WO2018112740A1 PCT/CN2016/111027 CN2016111027W WO2018112740A1 WO 2018112740 A1 WO2018112740 A1 WO 2018112740A1 CN 2016111027 W CN2016111027 W CN 2016111027W WO 2018112740 A1 WO2018112740 A1 WO 2018112740A1
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lactobacillus gasseri
pharmaceutical composition
gasseri
lactobacillus
preparation
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PCT/CN2016/111027
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English (en)
French (fr)
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邹远强
薛文斌
肖亮
李晓平
余靖宏
刘传
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深圳华大基因研究院
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Priority to CN201680091073.3A priority Critical patent/CN109983115B/zh
Priority to PCT/CN2016/111027 priority patent/WO2018112740A1/zh
Publication of WO2018112740A1 publication Critical patent/WO2018112740A1/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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a Lactobacillus gasseri and a culture method and application thereof.
  • Ulcerative colitis is a type of Inflammatory bowel disease (IBD), a chronic intestinal inflammatory disease with unknown pathogenesis.
  • IBD Inflammatory bowel disease
  • the pathological study of UC is mainly related to its pathogenesis and susceptibility genes, mucosal immunity and intestinal microbes. Its clinical pathological manifestations include persistent abdominal pain, diarrhea and mucous bloody stools, and the condition is repeated.
  • the site of inflammation of ulcerative enteritis is mainly in the colon and rectum, and the main lesions are in the colonic mucosa and submucosa.
  • the main western salicylate drugs for the treatment of UC are sulfasalazine (SASP), mainly for light Patients with moderate, moderate, and chronic UC; glucocorticoids are the first choice for patients with severe or explosive UC, such as betamethasone; immunosuppressive agents such as cyclosporine can inhibit immune response by inhibiting IL-2 production by T cells. Progress, thereby inhibiting UC.
  • SASP sulfasalazine
  • glucocorticoids are the first choice for patients with severe or explosive UC, such as betamethasone
  • immunosuppressive agents such as cyclosporine can inhibit immune response by inhibiting IL-2 production by T cells. Progress, thereby inhibiting UC.
  • All of the above three drugs can alleviate UC to a certain extent, but there are certain side effects.
  • the side effects of salicylic acid are gastrointestinal reactions, headache, reticulocyte increase, sperm reduction and rash caused by allergic reactions. Hepatotoxicity, leukopenia, anemia, etc.
  • Glucocorticoids can cause side effects such as metabolic disorders and water retention, and can only be used as an emergency medication and cannot be taken for a long time.
  • Immunosuppressive therapy is highly dependent on drugs, has a long treatment period, and is prone to cause nephrotoxicity and secondary infection. As a means of adjuvant therapy.
  • the present invention provides a novel species of Lactobacillus gasseri, which has the effect of preventing and/or treating ulcerative enteritis.
  • the present invention further provides a method for cultivating the new gut bacterial species, a product thereof, and an application thereof.
  • the present invention provides a Lactobacillus gasseri TF08-1 deposited with the Guangdong Provincial Collection of Microorganisms and Cultures under the accession number GDMCC 60092.
  • the present invention provides a method for culturing the Lactobacillus gasseri TF08-1 of the first aspect, wherein the Lactobacillus gasseri TF08-1 is inoculated into a PYG medium for anaerobic culture.
  • the present invention provides a microecological preparation comprising Lactobacillus gasseri TF08-1 of the first aspect and/or a metabolite thereof.
  • microecological preparations As is generally understood in the art, all formulations which promote the growth and reproduction of normal microbial populations and inhibit the growth and reproduction of pathogenic bacteria are referred to as "microecological preparations".
  • microecological preparation refers to a preparation made by using Lactobacillus gasseri TF08-1 and/or its metabolite, which has the effect of regulating the intestinal tract and rapidly constructing the intestinal microecological balance.
  • a typical microecological preparation may be a probiotic preparation for the prevention/treatment of ulcerative enteritis.
  • Lactobacillus gasseri TF08-1 has the effect of treating ulcerative enteritis, and can further change the type of probiotic preparation, using different packaging and processing methods, such as using embedding technology to maintain the activity of the strain to reach the corresponding The therapeutic effect, or the addition of prebiotics (bacterial powder, oligosaccharides, etc.) combined with Lactobacillus gargle TF08-1 to treat ulcerative enteritis can achieve the same therapeutic effect.
  • the probiotic lactic acid bacteria TF08-1 of the present invention can alleviate ulcerative enteritis, and may also exert its therapeutic effect in other inflammation-related diseases (common enteritis, gastritis, etc.).
  • the present invention provides a food composition, a nutraceutical or an auxiliary additive comprising Lactobacillus gasseri TF08-1 and/or a metabolite thereof of the first aspect.
  • the food composition of the present invention may contain various food materials, food additives, and the like, such as milk, white sugar, and vitamins, in addition to Lactobacillus gasseri TF08-1 and/or its metabolites.
  • Excipient additives in the present invention such as various edible additives.
  • the present invention provides a pharmaceutical composition comprising Lactobacillus garerii TF08-1 of the first aspect and/or a metabolite thereof.
  • the pharmaceutical composition of the present invention may contain, in addition to Lactobacillus gasseri TF08-1 and/or its metabolites, various pharmaceutically acceptable carriers and/or adjuvants including, but not limited to, lactose and yeast powder. , peptone, purified water, starch, vitamins, etc., may also contain various excipients, and can be formulated into tablets or capsules. Further, the pharmaceutical composition of the present invention may further contain a substance which contributes to the maintenance of the activity of L. gasseri TF08-1, such as a protective agent, and a typical, but non-limiting protective agent is vitamin C.
  • the content of Lactobacillus gasseri TF08-1 may be, based on the total volume or total weight of the pharmaceutical composition, for example, typically but not limited to 1 x 10 -1 to 1 x 10 20 cfu.
  • Lactobacillus gasseri TF08-1 of /mL or cfu/g preferably contains 1 x 10 4 to 1 x 10 15 cfu/mL or cfu/g of L. gasseri TF08-1.
  • the present invention provides the use of the Lactobacillus gasseri TF08-1 of the first aspect for the preparation of a medicament for preventing and/or treating ulcerative colitis.
  • the present invention provides the use of the Lactobacillus gasseri TF08-1 of the first aspect for the preparation of a cholesterol-lowering drug.
  • the invention provides the use of the Lactobacillus gasseri TF08-1 of the first aspect for the preparation of a microecological preparation.
  • the invention provides the use of the Lactobacillus gasseri TF08-1 of the first aspect for the preparation of a food composition, a health supplement or an adjuvant additive.
  • the present invention provides the use of the Lactobacillus gasseri TF08-1 of the first aspect for producing an exopolysaccharide.
  • the present invention provides a method for preventing and/or treating ulcerative colitis
  • the method comprises administering to a subject a Lactobacillus gasseri TF08-1 or a pharmaceutical composition of the fifth aspect of the first aspect.
  • the invention provides a method of lowering blood lipids, controlling a decrease in body weight of a mammal, and/or reducing a disease activity index (DAI) of a mammal, comprising administering to the subject a first aspect Lactobacillus gasseri TF08-1 or a pharmaceutical composition of the fifth aspect.
  • DAI disease activity index
  • the subject may be a human or other mammal.
  • the Lactobacillus gasseri TF08-1 of the present invention belongs to a new strain discovered by the inventors, and it has been found that the new strain has obvious remission effect on ulcerative enteritis, and the specific expression can significantly improve the appearance of mice with ulcerative colitis.
  • the state reduces the disease activity index of mice and reduces the inflammatory response in mice.
  • the probiotic function it also has the production of extracellular polysaccharides, and can effectively lower cholesterol.
  • Figure 1 shows a picture of colonies cultured for 48 hours of Lactobacillus gasseri TF08-1.
  • the colonies are white, low convex, nearly circular, edge wavy, and the colony diameter is about 1-2 mm.
  • Fig. 2 shows a Gram-stained picture (1000 times) of the Lactobacillus gasseri TF08-1 of the present invention under a microscope.
  • the microscopic morphology of the cells is rod-shaped, Gram-positive, and does not produce spores and flagella.
  • Figure 3 shows the cholesterol standard curve, using phthalaldehyde colorimetric method (OPA method) for cholesterol detection by using different concentrations (20ug/mL, 40ug/mL, 60ug/mL, 80ug/mL) of cholesterol and OPA The reaction was developed to obtain a standard curve.
  • OPA method phthalaldehyde colorimetric method
  • Figure 4 shows changes in body weight of control, model group, VSL # 3 and TF08-1 treated mice.
  • Figure 5 shows the changes in the DAI index of the control, model group, VSL # 3 and TF08-1 treated mice.
  • Example 1 Isolation and Identification of Lactobacillus gasseri TF08-1
  • the isolated samples were from a stool sample of a 16-year-old healthy female volunteer living in Shenzhen, Guangdong province. The volunteer's diet and physical condition were recorded in detail.
  • the separation medium was prepared in advance, and the medium was selected from PYG medium (purchased from Huanqi Microbiology Technology Co., Ltd.).
  • the specific ingredients were: peptone 5g, trypticin 5g, yeast powder 10g, beef extract 5g, glucose 5g, K 2 HPO.
  • sterile PBS phosphate buffer
  • the isolated strain was subjected to 16S rDNA identification to determine the species classification information of the strain.
  • the obtained isolates were cultured in liquid PYG medium for 24 hours, and 1 ml of the bacterial solution was centrifuged at 10000 r/min for 5 min, the cells were collected, the genomic DNA of the strain was extracted, and 16S rDNA was amplified using genomic DNA as a template, using 16S rDNA.
  • the colonies cultured in TF08-1 for 48 hours were white, low convex, nearly round, and edge wavy.
  • the colony diameter was about 1-2 mm (Fig. 1).
  • the microscopic morphology of the cells was rod-shaped and Gram-positive (Fig. 2). ), no spores and flagella.
  • the catalase reaction of TF08-1 was negative, oxidase was negative, and facultative anaerobic, and the utilization of carbon source was detected using API 20A (purchased from Mérieux, France) kit.
  • the results are shown in Table 1 (+, indicating a positive reaction; -, indicating a negative reaction; w indicating a weak positive reaction).
  • Numbering reaction result Numbering reaction result 1 ⁇ produce - 11 Gelatin hydrolysis - 2 Urea (urease) - 12 Qiyeling + 3 glucose + 13 glycerin w 4 Mannitol w 14 Cellobiose + 5 lactose w 15 Mannose + 6 sucrose + 16 Pine syrup w
  • Example 2 Biologically active substance of Lactobacillus gasseri TF08-1
  • the biologically active substance of TF08-1 mainly investigates the production of organic acids and short-chain fatty acids in the fermentation products. Take 1 ml of the fermentation broth of TF08-1 cultured for 48 hours, centrifuge at 10000r/min, and take the supernatant for the detection of organic acids and short-chain fatty acids.
  • the main active substances detected by organic acids are: 3-methylbutyric acid, valeric acid.
  • quinic acid lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, anti-fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L- Ascorbic acid; active substances mainly detected by short-chain fatty acids are: acetic acid, propionic acid, butyric acid, valeric acid. Detection was performed using an Agilent gas chromatograph (GC-7890B, Agilent).
  • the detection conditions of the organic acid are: the column is 122-5532G DB-5ms (40m ⁇ 0.25mm ⁇ 0.25um), the column temperature: 270 ⁇ 290°C; the inlet temperature: 250°C; the gas flow rate: 0.86ml/min;
  • the detection conditions of short-chain fatty acids are: HP-INNOWax (Cross-Linked PEG), 30m ⁇ 0.25mm ⁇ 0.25um capillary column for analysis, the detector is a hydrogen flame ion detector, and the GC parameter is set to the column temperature: 180 ⁇ 200°C; gasification chamber temperature: 240°C; detection temperature: 210°C; injection volume: 2 ⁇ L; carrier gas flow rate: N 2 , 50mL/min; hydrogen flow rate: 50mL/min; air flow rate: 600 ⁇ 700ml/ Min.
  • Table 2 The results of the measurement of organic acids and short-chain fatty acids are shown in Table 2.
  • TF08-1 was sensitive to antibiotics other than oxacillin and ceftriaxone sodium.
  • Example 4 Survival rate of Lactobacillus gasseri TF08-1 in the gastrointestinal tract
  • TF08-1 maintained a high survival rate (90% and 87%) under the conditions of pH 3 and 0.3% bile salt, respectively, indicating that TF08-1 has strong acid and bile salt tolerance.
  • the bile salt hydrolase was detected by TDCA method, and the TDAC plate was prepared. 4% of TDAC (sodium taurodeoxycholate) and 0.37 g/L of CaCl 2 were added to the PYG solid medium, and TF08-1 was cultured to a concentration of about 5%. 10 8 cfu/ml, take 10ul of bacteria droplets on a 0.6mm diameter filter paper, place the filter paper on the surface of the TDAC plate, and incubate at 37 °C for 2 days to observe the white precipitate produced around the filter paper. The diameter of the white precipitate represents the gallbladder. Salt hydrolase activity.
  • the white precipitate of TF08-1 had a diameter of 10 mm, indicating that TF08-1 has a bile salt hydrolase activity.
  • the method for determining the content of cholesterol is determined by the o-phthalaldehyde colorimetric method (OPA method), and the degradation ability of cholesterol is examined by the change of the cholesterol content of the strain in a certain concentration of cholesterol medium for a period of time.
  • OPA method o-phthalaldehyde colorimetric method
  • a certain amount of cholesterol was weighed and dissolved in ethanol at a concentration of 10 mg/mL, and sterilized by filtration. Add the prepared PYG medium to 10 mg/mL bile salt (autoclave), 10% mass concentration of sodium thioglycolate (filter sterilization) and cholesterol, mix well, and then wait for 3% of the inoculum. The test strain was inoculated into the medium. In addition to TF08-1, another strain of commercial cholesterol-lowering probiotic Lactobacillus plantarum Lp299v (purchased from Probi, Sweden) was used for comparison. Both strains were anaerobic at 37 °C. Incubate for 72 h under the conditions.
  • the culture solution containing the cholesterol-containing PYG medium was centrifuged at 10,000 r/min, and the supernatant was collected for cholesterol detection, and the uninoculated cholesterol PYG medium was used as a blank control group.
  • Take 1ml of the sample to be tested in a clean test tube add 6ml of 95% ethanol and 4ml of 50% KOH, shake and mix, then saponify in a 60 °C water bath for 10min, rapidly cool, add 10ml of n-hexane for extraction, fully Mix well, let stand for 20 min at room temperature, measure 8 ml organic phase (n-hexane layer) into another clean test tube, then dry it in nitrogen in a 60 ° C water bath, add 4 ml 0.5 g / L phthalaldehyde acetic acid solution, room temperature After standing for 10 min, 2 ml of concentrated H 2 SO 4 was added for 10 min, and finally, the absorbance at 550 nm was measured.
  • the cholesterol content in the medium before and after culture was calculated according to the standard curve.
  • the degradation rate of cholesterol was calculated according to the following formula:
  • L cholesterol degradation rate
  • A cholesterol content in the cholesterol medium not inoculated
  • B cholesterol content in the culture medium for 48 hours after the test strain is cultured.
  • the cholesterol degradation rate of TF08-1 was 84.4%, and the degradation rate of Lp299v was 70%, which indicated that TF08-1 had stronger cholesterol degradation ability than Lp299v.
  • the detection of extracellular polysaccharides is carried out by the phenolic phenol method.
  • the phenolic phenol can react with the free monosaccharides, oligosaccharides and hexoses in the polysaccharides, and the color produced is proportional to the absorbance, and the absorption wavelength is 490 nm.
  • the specific experimental process is as follows:
  • the experimental strain was cultured in PYG medium (formulation as in Example 1) for 2 days, treated with 10 ml of bacterial liquid in boiling water bath for 30 min, then centrifuged at 10000 r/min, and the supernatant was taken, and 80% trichloroacetic acid was added to a final concentration of 8%.
  • the protein was precipitated by treatment at 4 ° C overnight. Centrifuge at 10,000 r/min for 30 min, and adjust the pH of the supernatant to 6.0 with NaOH.
  • the content of extracellular polysaccharide in the TF08-1 fermentation broth cultured for 2 days was 380.29 mg/L.
  • Example 7 Effect of Lactobacillus delbrueckii TF08-1 on ulcerative enteritis in mice
  • the mouse model was used to treat ulcerative enteritis (UC) with Lactobacillus gasseri TF08-1, and a probiotic VSL # 3 (purchased from Sigma Tau, USA) was used as a reference to observe the body weight of the mice.
  • Pathological indicators such as mortality, colon length, DAI index, and intestinal mucosal pathology were used to evaluate the treatment of UC with TF08-1.
  • mice were cultured with C57bl/6 strain (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20g ⁇ 2g, and the feeding environment of mice was SPF grade, and the feeding was performed for 1 week.
  • the modeling method was induced by dextran sulfate sodium (DSS).
  • DSS dextran sulfate sodium
  • the mice were free to drink 0.2% DSS for 7 days.
  • the experimental group was divided into 4 groups, 12 mice in each group. The details are as follows:
  • the first group the control group (blank control group) - normal mice, did not undergo DSS induction
  • the second group UC model group - DSS modeling, using sterile PBS for intragastric administration
  • VSL # 3 treatment group - DSS modeling VSL # 3 treatment group (bacteria concentration 10 9 cfu / ml)
  • the fourth group TF08-1 treatment group - DSS modeling, TF08-1 treatment group (bacteria concentration 10 9 cfu / ml)
  • mice weight loss score + blood test score + stool trait score, the specific indicators are shown in Table 4.
  • Blood in the stool normal mice have positive blood in the stool; the blood of the naked eye is red or brown; the occult blood is positive for the naked blood, which is detected by tetramethylbenzidine.
  • the DAI index is an important indicator for judging the severity of UC mice.
  • DSS-induced UC model mice cause weight loss in mice, inflammation and ulceration in the colon, causing bleeding, and affecting stool traits, resulting in an increase in DAI index. .
  • the DAI values of the mice in each group during the experiment are shown in Table 6 and Figure 5.
  • Example 8 Food composition containing Lactobacillus gasseri TF08-1
  • Lactobacillus gasseri TF08-1 bacteria Mix milk and sugar according to the above formula ratio, stir until completely mixed, preheat, homogenize at 20Mpa, sterilize for 5-10 minutes at 90 °C, cool to 40-43 °C, mix with protective agent vitamin C, inoculate 1-100 ⁇ 10 6 cfu/g of Lactobacillus gasseri TF08-1 bacteria, that is, a food composition containing Lactobacillus gasseri TF08-1 bacteria.
  • Example 8 Pharmaceutical composition containing Lactobacillus gasseri TF08-1
  • the raw material ratio is shown in Table 9.
  • lactose, yeast powder and peptone are mixed uniformly with purified water, preheated to 60-65 ° C, homogenized at 20 Mpa, sterilized at 90 ° C for 20-30 minutes, cooled to 36-38 ° C, mixed with protective agent vitamin C, Access to L. gasseri TF08-1 live bacteria (1-500 ⁇ 10 6 cfu/mL), ferment at pH 36-38 ° C to pH 6.0, centrifuge, freeze-dry to a moisture content of less than 3%, ie prepare Garcinia Lactobacillus TF08-1 bacteria freeze-dried.
  • 0.5 g of the lyophilized product of Lactobacillus gasseri TF08-1 was weighed and mixed with maltodextrin in an equal amount, and then placed in a capsule to prepare a pharmaceutical composition containing Lactobacillus gasseri TF08-1.
  • Example 9 Preparation method of medicine for treating ulcerative enteritis (UC)
  • Lactobacillus gasseri TF08-1 (1 ⁇ 10 9 cfu/ml) was subjected to anaerobic culture, and anaerobic medium was cultured in PYG medium, and subjected to anaerobic fermentation at 37 ° C for 2-3 days.
  • Preparation of pharmaceutical dosage form Add 5 volumes of growth factor and 1 volume of protective agent vitamin C to 100 volumes of TF08-1 fermented bacterial solution, mix well and mix, then add starch adjuvant (such as maltodextrin) to prepare.
  • starch adjuvant such as maltodextrin

Abstract

提供了一种加氏乳杆菌及其培养方法和应用，该加氏乳杆菌(Lactobacillus gasseri) TF08-1，保藏于广东省微生物菌种保藏中心，其保藏编号为GDMCC 60092。所述菌株对溃疡性肠炎有明显的缓解作用，并具有产胞外多糖及降胆固醇的功能。

Description

一种加氏乳杆菌及其培养方法和应用 技术领域
本发明涉及微生物技术领域，尤其涉及一种加氏乳杆菌(Lactobacillus gasseri)及其培养方法和应用。
背景技术
溃疡性肠炎(Ulcerative colitis，UC)是炎症性肠病(Inflammatory bowel disease，IBD)的一种，是一种发病机制不明的慢性肠道炎症性疾病。目前临床上对UC的病理研究主要认为与其发病与易感基因、粘膜免疫和肠道微生物有关，其临床病理表现为持续腹痛、腹泻和黏液血便，且病情反复。其中溃疡性肠炎的炎症发生部位主要在结肠和直肠，主要病变是在结肠粘膜和粘膜下层。
由于病理机制不明确，临床的治疗也缺乏特异性和针对性，临床上主要有营养治疗、手术治疗和药物治疗，其中药物治疗是最主要的治疗方式，临床上针对UC并的用药主要有水杨酸类、糖皮质激素、免疫制剂。水杨酸类药物可以比较好的抑制前列腺素合成，清除氧自由基从而到达缓解炎症反应的目的，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；糖皮质激素是重症或者爆发性UC患者的首选用药，比如倍他米松；免疫抑制剂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。
上述三类药物均可以一定程度上对UC进行缓解，但是也都存在一定的副作用，水杨酸类的副作用是引发消化道反应、头痛、网织红细胞增多、精子减少及过敏反应引起的皮疹、肝毒性、白细胞减少、贫血等。糖皮质激素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。
发明内容
本发明提供一种加氏乳杆菌新种，具有预防和/或治疗溃疡性肠炎的作用。本发明进一步提供该肠道细菌新种的培养方法以及其制成的产品和其应用。
本发明包括如下技术方案：
根据本发明的第一方面，本发明提供一种加氏乳杆菌(Lactobacillus gasseri)TF08-1，其保藏于广东省微生物菌种保藏中心，其保藏编号为GDMCC 60092。
根据本发明的第二方面，本发明提供一种第一方面的加氏乳杆菌TF08-1的培养方法，将所述加氏乳杆菌TF08-1接种于PYG培养基中进行厌氧培养。
根据本发明的第三方面，本发明提供一种含有第一方面的加氏乳杆菌TF08-1和/或其代谢产物的微生态制剂。
按照本领域的通常理解，一切能促进正常微生物群生长繁殖及抑制致病菌生长繁殖的制剂都称为“微生态制剂”。本发明中，所称的“微生态制剂”，是指利用加氏乳杆菌TF08-1和/或其代谢产物制成的制剂，其具有调节肠道之功效，快速构建肠道微生态平衡。典型的微生态制剂可以是益生菌制剂，用于预防/治疗溃疡性肠炎。作为本发明的益生菌，加氏乳杆菌TF08-1具有治疗溃疡性肠炎的效果，可通过进一步改变益生菌制剂类型，采用不同包装及加工方法，比如采用包埋技术保持菌种活性从而到达相应的治疗效果，或者通过额外添加益生元(菌粉、寡聚糖等)联用加氏乳杆菌TF08-1来治疗溃疡性肠炎都可实现同等的治疗效果。另外本发明的益生菌加氏乳杆菌TF08-1可以缓解溃疡性肠炎，还可能会在其他的一些炎症相关的疾病(普通肠炎、胃炎等)中发挥其治疗作用。
根据本发明的第四方面，本发明提供一种含有第一方面的加氏乳杆菌TF08-1和/或其代谢产物的食品组合物、保健品或辅料添加剂。
本发明中的食品组合物，除含有加氏乳杆菌TF08-1和/或其代谢产物以外，还可以含有各种食品原料或食品添加剂等，例如牛奶、白糖和维生素等。本发明中的辅料添加剂，例如各种食用性添加剂。
根据本发明的第五方面，本发明提供一种含有第一方面的加氏乳杆菌TF08-1和/或其代谢产物的药物组合物。
本发明中的药物组合物，除含有加氏乳杆菌TF08-1和/或其代谢产物以外，还可以含有各种药学上可接受的载体和/或辅料，包括但不限于：乳糖、酵母粉、蛋白胨、纯净水、淀粉和维生素等，还可以含有各种赋形剂，可以制成片剂或胶囊制剂等。此外，本发明中的药物组合物还可以含有有助于保持加氏乳杆菌TF08-1活力的物质，如保护剂，典型但非限定性的保护剂是维生素C。
本发明的药物组合物中，加氏乳杆菌TF08-1的含量可以按药物组合物的总体积或总重量计，例如，典型但非限定性地含有1×10-1至1×1020cfu/mL或cfu/g的加氏乳杆菌TF08-1，较佳地含有1×104至1×1015cfu/mL或cfu/g的加氏乳杆菌TF08-1。
根据本发明的第六方面，本发明提供第一方面的加氏乳杆菌TF08-1在制备预防和/或治疗溃疡性肠炎的药物中的应用。
根据本发明的第七方面，本发明提供第一方面的加氏乳杆菌TF08-1在制备降胆固醇的药物中的应用。
根据本发明的第八方面，本发明提供第一方面的加氏乳杆菌TF08-1在制备微生态制剂中的应用。
根据本发明的第九方面，本发明提供第一方面的加氏乳杆菌TF08-1在制备食品组合物、保健品或辅料添加剂中的应用。
根据本发明的第十方面，本发明提供第一方面的加氏乳杆菌TF08-1在生产胞外多糖中的应用。
根据本发明的第十一方面，本发明提供一种预防和/或治疗溃疡性肠炎的方 法，包括给受试对象施用第一方面的加氏乳杆菌TF08-1或第五方面的药物组合物。
根据本发明的第十二方面，本发明提供一种降低血脂、控制哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法，包括给受试对象施用第一方面的加氏乳杆菌TF08-1或第五方面的药物组合物。
本发明中，受试对象可以是人或其它哺乳动物。
本发明的加氏乳杆菌TF08-1属于发明人发现的新菌株，经过研究发现这株新菌株对溃疡性肠炎有明显的缓解作用，具体表现在能够显著改善溃疡性结肠炎小鼠的表观状态，降低小鼠疾病活动指数，并降低小鼠炎症性反应。同时在益生功能方面还体现在具有产胞外多糖，并可以有效的降胆固醇。
保藏信息
菌株名称：Lactobacillus gasseri TF08-1
保藏日期：2016年10月13日
保藏单位：广东省微生物菌种保藏中心(GDMCC)
保藏地址：广东省广州市先烈中路100号大院59号楼5楼
保藏编号：GDMCC 60092
附图说明
图1显示了加氏乳杆菌TF08-1培养48h菌落的图片，菌落为白色、低凸起、近圆形、边缘波状，菌落直径约1-2mm。
图2显示了本发明加氏乳杆菌Lactobacillus gasseri TF08-1在显微镜下的革兰氏染色图片(1000倍)，菌体的显微形态为杆状，革兰氏阳性，不产芽孢和鞭毛。
图3显示了胆固醇标准曲线，采用邻苯二甲醛比色法(OPA法)进行胆固 醇的检测，通过使用不同浓度(20ug/mL，40ug/mL，60ug/mL，80ug/mL)的胆固醇与OPA进行反应显色，得到标准曲线，线性回归的方程式为：y＝0.0085x+0.0072；相关系数R2为0.9992。
图4显示了对照组、模型组、VSL#3和TF08-1治疗组小鼠的体重的变化。
图5显示了对照组、模型组、VSL#3和TF08-1治疗组小鼠的DAI指数的变化。
具体实施方式
下面结合实施例对本发明做进一步的说明。
实施例1：加氏乳杆菌TF08-1的分离鉴定
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。
2、菌株的分离培养
提前制备好分离培养基，培养基选用PYG培养基(购自环凯微生物科技公司)，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，K2HPO4 2g，Tween 80 1mL，Cysteine-HCl·H2O 0.5g，硫化钠0.25g，血红素5mg，维生素K11μL，无机盐溶液(每L无机盐溶液含有CaCl2·2H2O 0.25g，MgSO4·7H2O 0.5g，K2HPO4 1g，KH2PO4 1g，NaHCO3 10g，NaCl 2g)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。
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PBS(磷酸盐缓冲液)中，充分混匀，然后进行梯度稀释，取100ul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N2：CO2：H2＝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。
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(8F-AGAGTTTGATCATGGCTCAG(SEQ ID NO：1)和1492R-TAGGGTTACCTTGTTACGACTT(SEQ ID NO：2))，16S rDNA的扩增条件为：95℃预变性4min，然后95℃变性30s，57℃退火40s，72℃延伸1min30s，30个循环。对产生的16S rDNA产物进行纯化，3730测序，获得菌株的16S rDNA序列(SEQ ID NO：3)，然后进行NCBI的数据库的比对。TF08-1同数据库中同源性最高的菌为Lactobacillus gasseri，相似度为99.9％。可以判定TF08-1属于加氏乳杆菌。
4、TF08-1的生理生化特征
TF08-1培养48小时的菌落为白色、低凸起、近圆形、边缘波状，菌落直径约1-2mm(图1)，菌体的显微形态为杆状，革兰氏阳性(图2)，不产芽孢和鞭毛。TF08-1的过氧化氢酶反应为阴性，氧化酶阴性，兼性厌氧，碳源利用情况使用API 20A(购自法国梅里埃)试剂盒进行检测。结果如表1(+，表示阳性反应；-，表示阴性反应；w表示弱阳性反应)。
表1
编号 反应 结果 编号 反应 结果
1 吲哚产生 - 11 明胶水解 -
2 脲素(脲酶) - 12 七叶灵 +
3 葡萄糖 + 13 甘油 w
4 甘露醇 w 14 纤维二糖 +
5 乳糖 w 15 甘露糖 +
6 蔗糖 + 16 松叁糖 w
7 麦芽糖 + 17 棉籽糖 w
8 柳醇 + 18 山梨醇 w
9 木糖 + 19 鼠李糖 w
10 阿拉伯糖 w 20 海藻糖 +
实施例2：加氏乳杆菌TF08-1的生物活性物质
TF08-1的生物活性物质主要考察发酵产物中的有机酸和短链脂肪酸的产生情况。取培养48h的TF08-1的发酵液1ml，进行10000r/min离心处理，取上清进行有机酸和短链脂肪酸的检测，有机酸主要检测的活性物质有：3-甲基丁酸，戊酸，奎宁酸，乳酸，草酸，丙二酸，苯甲酸，马来酸，丁二酸，反富马酸，苹果酸，己二酸，酒石酸，莽草酸，柠檬酸，异柠檬酸和L-抗坏血酸；短链脂肪酸主要检测的活性物质有：乙酸、丙酸、丁酸、戊酸。使用安捷伦气相色谱(GC-7890B,Agilent)进行检测。有机酸的检测条件是：色谱柱为122-5532G DB-5ms(40m×0.25mm×0.25um)，柱温：270～290℃；进样口温度：250℃；气体流量:0.86ml/min；短链脂肪酸的检测条件是：色谱柱是HP-INNOWax(Cross-Linked PEG)，30m×0.25mm×0.25um的毛细柱进行分析，检测器为氢火焰离子检测器，GC参数设置为柱温：180～200℃；气化室温度：240℃；检测温度：210℃；进样量：2μL；载气流量：N2，50mL/min；氢气流量：50mL/min；空气流量：600～700ml/min。测得有机酸和短链脂肪酸的结果详见表2。
表2
Figure PCTCN2016111027-appb-000001
Figure PCTCN2016111027-appb-000002
实施例3：加氏乳杆菌TF08-1的抗生素敏感情况
考察TF08-1对常见20种抗生素的敏感情况，采用药敏纸片法进行实验，取培养至对数期的TF08-1的菌液100ul进行平板涂布，将抗生素药敏片贴在平板表面，37℃培养48h，测量抑菌圈大小，其结果如表3。
表3
Figure PCTCN2016111027-appb-000003
Figure PCTCN2016111027-appb-000004
药敏试验表明，TF08-1对除了苯唑西林和头孢曲松钠之外的抗生素均比较敏感。
实施例4：加氏乳杆菌TF08-1的在胃肠道中的存活率
益生菌的功能研究需要考察其在胃肠道中(酸和胆盐的环境)的耐受的能力，将浓度为109cfu/ml的TF08-1接入到pH3.0的人工胃液中，37℃处理2h，然后进行平板菌落计数，通过计算，人工胃液处理后TF08-1的存活率为90％。
同时将109cfu/ml的TF08-1接种至含0.3％的胆盐MRS培养基中，37℃处理2h，然后进行平板菌落计数，通过计算，0.3％的胆盐处理后的TF08-1的存活率为87％。
通过以上耐受实验，TF08-1分别在pH3和0.3％的胆盐的条件下维持一个很高的存活率(90％和87％)，表明TF08-1具有很强的酸和胆盐耐受能力，绝大多数活菌能够通过人体的胃液和小肠到达大肠发挥其功能。
实施例5：加氏乳杆菌TF08-1的降胆固醇功能
1、TF08-1的胆盐水解酶活性
胆盐水解酶采用TDCA法进行检测，配制TDAC平板，PYG固体培养基中添加4％的TDAC(牛磺去氧胆酸钠)和0.37g/L的CaCl2，将TF08-1培养至浓度约108cfu/ml，取10ul菌液滴在直径为0.6mm的滤纸片上，滤纸片置于TDAC平板表面，37℃培养2天，观察滤纸片周边产生的白色沉淀情况，白色沉淀的直径代表胆盐水解酶的活性。
通过测量，TF08-1的白色沉淀的直径为10mm，表明TF08-1具有胆盐水解酶的活性。
2、TF08-1的体外降胆固醇情况
胆固醇的含量测定方法采用邻苯二甲醛比色法(OPA法)，通过菌株在含一定浓度的胆固醇培养基中培养一段时间的胆固醇含量前后的变化来考察对胆固醇的降解能力。具体方法如下：
(1)胆固醇培养基的配制和实验菌株的培养
称取一定质量的胆固醇溶解于乙醇中，浓度为10mg/mL，过滤除菌。将配置好的PYG培养基分别加入10mg/mL的胆盐(高压灭菌)，10％质量浓度的巯基乙酸钠(过滤除菌)和胆固醇，充分混匀，然后按照3％的接种量将待测菌株接种至该培养基中，待测菌株除了TF08-1，还选用另外一株商业降胆固醇益生菌植物乳杆菌Lp299v(购自瑞典Probi公司)作比较，两种菌都在37℃厌氧条件下培养72h。
(2)标准曲线的制作
精确量取0.5mg/mL的胆固醇标准溶液40uL，80uL，120uL，160uL，200uL于干净试管中，加入无水乙醇定容至1mL，每个试管中加入OPA 4mL(0.5mg邻苯二甲醛加入到1mL冰醋酸)，震荡混匀，室温静置10min，然后加入2mL的浓硫酸混匀，静置反应10min，于550nm处测定吸光度。以浓度作为横坐标，吸光度作为纵坐标绘制标准曲线(图3)，通过计算，线性回归的方程式为：y＝0.0085x+0.0072；相关系数R2为0.9992。
(3)培养基中胆固醇的测定
将含有胆固醇的PYG培养基培养好的菌液进行10000r/min的离心，收集上清，进行胆固醇检测，同时以未接种的胆固醇PYG培养基作为空白对照组。取1ml待测样品于干净的试管中，加入95％乙醇6ml和50％的KOH 4ml，震荡混匀，然后在60℃水浴中进行皂化反应10min，迅速进行冷却，加入10ml正己烷进行萃取，充分混匀，室温静置20min，量取8ml有机相(正己烷层)到另一洁 净试管中，然后在60℃水浴中进行氮气吹干，加入4ml 0.5g/L邻苯二甲醛乙酸溶液，室温静置10min，添加2ml浓H2SO4反应10min，最后测量在550nm处的吸光值。
(4)胆固醇降解率的计算
根据标准曲线计算培养前后培养基中胆固醇的含量，胆固醇的降解率按以下公式进行计算：
L＝(A-B)/A×100％
L：胆固醇降解率；A：未接种菌的胆固醇培养基中胆固醇的含量；B：待测菌株培养48h培养液中胆固醇的含量。
(5)胆固醇降解结果
通过计算，得到TF08-1的胆固醇降解率为84.4％，而Lp299v降解率为70％，由此说明TF08-1比Lp299v具有更强的胆固醇降解能力。
实施例6：加氏乳杆菌TF08-1的胞外多糖(EPS)产生情况
胞外多糖的检测采用硫酸苯酚法，硫酸苯酚可以与游离的单糖、寡糖和多糖中的己糖等发生显色反应，产生的颜色同吸光度成正比，其吸收波长为490nm。具体实验过程如下：
(1)多糖的提取
实验菌株在PYG培养基(配方同实施例1)中培养2天，取10ml菌液沸水浴处理30min，然后10000r/min离心，取上清，加入80％三氯乙酸至终浓度为8％，4℃过夜处理，沉淀蛋白。取出10000r/min离心30min，用NaOH调节上清液的pH至6.0。加入2倍体积的无水乙醇进行多糖沉淀，4℃过夜处理，取出进行10000r/min离心30min，弃掉上清，沉淀用预热的蒸馏水进行溶解，然后转移至超滤管(3000Da滤径)中进行超滤，5000r/min离心30min，内管中截留的多糖转移至容量瓶中蒸馏水定容至10ml，备用。
(2)葡萄糖标准曲线的制作
精密称取标准葡萄糖20mg至100ml容量瓶中，加水至刻度线，然后分别配置20、40、60、80、100μg/ml的葡萄糖标准液。每组标准液取400ul，三个平行，依次加入400ul 5％的苯酚和1ml浓硫酸进行反应，沸水浴15min后冷却至室温，测量在490nm的吸光度。然后以吸光度作为纵坐标，葡萄糖标准液浓度为横坐标绘制标准曲线。
(3)检测提取的多糖的浓度
量取多糖溶液400ul，依次加入400ul 5％的苯酚和1ml浓硫酸进行反应，沸水浴15min后冷却至室温，测量在490nm的吸光度。根据葡萄糖标准曲线计算多糖的浓度。
(4)结果
通过计算，培养2天的TF08-1发酵液中胞外多糖的含量为380.29mg/L。
实施例7：加氏乳杆菌TF08-1治疗小鼠溃疡性肠炎的效果
对加氏乳杆菌TF08-1治疗溃疡性肠炎(UC)采用小鼠模型进行验证，同时选取一种治疗UC的益生菌VSL#3(购自美国Sigma Tau)作为参照，通过观察小鼠的体重、死亡率、结肠长度、DAI指数以及肠粘膜病理改变等病理学指标来评价TF08-1对UC的治疗情况。
实验小鼠采用C57bl/6品系(购自湖北医学实验动物中心)，8周龄，体重20g±2g，小鼠饲养环境为SPF级，适应性喂养1周进行造模。造模方法采用葡聚糖硫酸钠(DSS)诱导，通过小鼠自由饮用含0.2％的DSS，持续7天，实验组共分为4个组，每组12只小鼠，详细情况如下：
第一组：对照组(空白对照组)——正常小鼠，未进行DSS诱导
第二组：UC模型组——DSS造模，使用无菌PBS进行灌胃
第三组：VSL#3治疗组——DSS造模，VSL#3治疗组(菌浓109cfu/ml)
第四组：TF08-1治疗组——DSS造模，TF08-1治疗组(菌浓109cfu/ml)
每日观察记录小鼠的饮食、饮水、活动等一般情况，并且进行称重，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天计算小鼠疾病活动指数(DAI指数，表4)，实验干预治疗为期7天，益生菌的日灌胃量为200ul。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。
小鼠的DAI＝体重降低分数+便血评分+大便性状评分，具体各项指标详见表4。
表4
Figure PCTCN2016111027-appb-000005
表中的大便性状：正常大便—成形大便；松散大便—不粘附于肛门的糊状、半成型大便；稀便—可粘附于肛门的稀样水便。
便血情况：正常小鼠便血为阳性；肉眼血便为红色或褐色；隐血阳性为不明显的肉眼血便，使用四甲基联苯胺进行检测。
TF08-1对UC小鼠的干预结果：
1、体重变化(表5和图4)
表5
Figure PCTCN2016111027-appb-000006
Figure PCTCN2016111027-appb-000007
表5和图4中数据表明，随着DSS的诱导(模型组)，小鼠的体重呈现下降的趋势，自第3天开始下降比较显著(*P<0.05)，第5天开始，下降变得极显著(**P<0.01)。但是益生菌(包括TF08-1和VSL#3)的干预可以控制UC小鼠体重的下降，在第7天，TF08-1和VSL#3对DSS小鼠的体重下降的控制比较显著(相对于模型组，▲P<0.05)，同时TF08-1组小鼠第7天的体重高于VSL#3组，说明TF08-1对UC小鼠体重下降的控制略好于VSL#3。
2、DAI指数的变化
DAI指数是评判UC小鼠严重程度的一个重要的指标，DSS诱导的UC模型小鼠会引起小鼠体重下降，结肠出现炎症和溃疡，引起出血，同时影响大便的性状，造成DAI指数会升高。实验过程中各组小鼠的DAI数值详见表6和图5。
表6
Figure PCTCN2016111027-appb-000008
如表6和图5所示，随着DSS诱导的UC模型，小鼠的DAI指数逐渐升高，从第3天开始DSS诱导的小鼠(模型组)相对于对照组的小鼠DAI升高差异极显著(*P<0.01)。但是益生菌(包括TF08-1和VSL#3)的干预可以控制UC小鼠DAI的上升。在第7天，TF08-1和VSL#3对DSS小鼠的DAI升高的控制比较显著(相对于模型组，▲P<0.05)，同时TF08-1组小鼠第7天的DAI值低于 VSL#3组的，说明TF08-1对UC小鼠疾病缓解优于VSL#3。
3、TF08-1对DSS诱导的UC小鼠结肠长度缩短的控制
DSS诱导的UC小鼠结肠组织发生溃疡，会造成结肠长度的缩短，治疗结束后，通过解剖测量的小鼠结肠长度见表7。
表7
Figure PCTCN2016111027-appb-000009
结果显示，模型组小鼠的结肠缩短比较显著(相对于对照组**P<0.01)，同时益生菌(包括TF08-1和VSL#3)的干预可以一定程度上控制结肠缩短(相对于模型组，▲P<0.05)。TF08-1组小鼠结肠要长于VSL#3组，说明TF08-1在控制小鼠结肠缩短的能力强于VSL#3。
综合以上数据，TF08-1在控制UC小鼠的体重下降、DAI指数升高以及小鼠结肠长度的效果均优于VSL#3，说明TF08-1对UC的缓解能力强于VSL#3，在对UC的治疗和应用有着更大的价值。
实施例8：含加氏乳杆菌TF08-1的食品组合物
原料配比如表8。
表8
原料 质量百分比(％)
加氏乳杆菌TF08-1 0.5
牛奶 90.0
白糖 9.0
维生素C 0.5
按照上述配方比例混合牛奶、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，接种1-100×106cfu/g的加氏乳杆菌TF08-1菌，即制成含加氏乳杆菌TF08-1菌的食品组合物。
实施例8：含加氏乳杆菌TF08-1的药物组合物
原料配比见表9。
表9
原料 质量百分比(％)
加氏乳杆菌TF08-1 1.0
乳糖 2.0
酵母粉 2.0
蛋白胨 1.0
纯净水 93.5
维生素C 0.5
按照比例将乳糖、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，接入加氏乳杆菌TF08-1活菌(1-500×106cfu/mL)，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备加氏乳杆菌TF08-1菌冷冻干燥物。称取0.5克加氏乳杆菌TF08-1冷冻干燥物与麦芽糊精等量混合后装入胶囊中，即制成含加氏乳杆菌TF08-1的药物组合物。
实施例9：用于治疗溃疡性肠炎(UC)的药物的制备方法
1、菌液准备：将加氏乳杆菌TF08-1(1×109cfu/ml)进行厌氧培养，厌氧培养基采用PYG培养基，经过37℃厌氧发酵2-3天。
2、生长因子制备：将脱脂牛奶、酪蛋白进行混合、离心、超滤获得牛奶生长因子粗提物(含有维生素类物质、嘌呤类物质、嘧啶类物质的营养物质)。
3、药物剂型制作：将5体积的生长因子和1体积的保护剂维生素C加入到100体积的TF08-1发酵的菌液中，充分搅拌混匀，然后加入淀粉辅料(如麦芽糊精)制备药物剂型。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (15)

  1. 一种加氏乳杆菌(Lactobacillus gasseri)TF08-1，其保藏于广东省微生物菌种保藏中心，其保藏编号为GDMCC 60092。
  2. 一种权利要求1所述的加氏乳杆菌TF08-1的培养方法，其特征在于，将所述加氏乳杆菌TF08-1接种于PYG培养基中进行厌氧培养。
  3. 一种含有权利要求1所述的加氏乳杆菌TF08-1和/或其代谢产物的微生态制剂。
  4. 一种含有权利要求1所述的加氏乳杆菌TF08-1和/或其代谢产物的食品组合物、保健品或辅料添加剂。
  5. 一种含有权利要求1所述的加氏乳杆菌TF08-1和/或其代谢产物的药物组合物。
  6. 如权利要求5所述的药物组合物，其特征在于，按所述药物组合物的总体积或总重量计，所述药物组合物含有1×10-1至1×1020cfu/mL或cfu/g的加氏乳杆菌TF08-1，较佳地含有1×104至1×1015cfu/mL或cfu/g的加氏乳杆菌TF08-1。
  7. 如权利要求5所述的药物组合物，其特征在于，所述药物组合物还含有药学上可接受的载体和/或辅料。
  8. 如权利要求5所述的药物组合物，其特征在于，所述药物组合物还含有有助于保持加氏乳杆菌TF08-1活力的物质。
  9. 权利要求1所述的加氏乳杆菌TF08-1在制备预防和/或治疗溃疡性肠炎的药物中的应用。
  10. 权利要求1所述的加氏乳杆菌TF08-1在制备降胆固醇的药物中的应用。
  11. 权利要求1所述的加氏乳杆菌TF08-1在制备微生态制剂中的应用。
  12. 权利要求1所述的加氏乳杆菌TF08-1在制备食品组合物、保健品或辅料添加剂中的应用。
  13. 权利要求1所述的加氏乳杆菌TF08-1在生产胞外多糖中的应用。
  14. 一种预防和/或治疗溃疡性肠炎的方法，其特征在于，给受试对象施用权利要求1所述的加氏乳杆菌TF08-1或权利要求5所述的药物组合物。
  15. 一种降低血脂、控制哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数的方法，其特征在于，给受试对象施用权利要求1所述的加氏乳杆菌TF08-1或权利要求5所述的药物组合物。
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