WO2018112741A1 - Lactobacillus acidophilus, sa méthode de culture et son application - Google Patents

Lactobacillus acidophilus, sa méthode de culture et son application Download PDF

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WO2018112741A1
WO2018112741A1 PCT/CN2016/111028 CN2016111028W WO2018112741A1 WO 2018112741 A1 WO2018112741 A1 WO 2018112741A1 CN 2016111028 W CN2016111028 W CN 2016111028W WO 2018112741 A1 WO2018112741 A1 WO 2018112741A1
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lactobacillus acidophilus
pharmaceutical composition
acid
preparation
cfu
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PCT/CN2016/111028
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English (en)
Chinese (zh)
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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 PCT/CN2016/111028 priority Critical patent/WO2018112741A1/fr
Priority to CN201680091076.7A priority patent/CN110023486B/zh
Publication of WO2018112741A1 publication Critical patent/WO2018112741A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • 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
    • C12R2001/23—Lactobacillus acidophilus

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a Lactobacillus acidophilus and a culture method and application thereof.
  • IBD Inflammatory bowel disease
  • 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 pathological study of ulcerative enteritis mainly believes that its pathogenesis is related to 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. With the improvement of living standards and changes in dietary structure, the incidence of UC is on the rise.
  • 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 cause the machine Side effects such as body metabolism disorder and water retention 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. It can only be used as a means of adjuvant therapy.
  • the present invention provides a novel species of Lactobacillus acidophilus 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.
  • a Lactobacillus acidophilus AM13-1 deposited at the Guangdong Provincial Collection of Microorganisms and Cultures under the accession number GDMCC 60091.
  • the present invention provides a method for culturing the Lactobacillus acidophilus AM13-1 of the first aspect, wherein the Lactobacillus acidophilus AM13-1 is inoculated into a PYG medium for anaerobic culture.
  • a microecological preparation comprising Lactobacillus acidophilus AM13-1 and/or a metabolite thereof of the first aspect.
  • 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 prepared by using Lactobacillus acidophilus AM13-1 and/or its metabolite, which has an effect of regulating the intestinal tract and rapidly constructs an intestinal microecological balance.
  • a typical microecological preparation may be a probiotic preparation for the prevention/treatment of ulcerative enteritis.
  • Lactobacillus acidophilus AM13-1 has the effect of treating ulcerative enteritis, and can be further modified by different types of probiotic preparations, using different packaging and processing methods, such as embedding technology to maintain the activity of the strain to reach the corresponding The therapeutic effect, or the addition of prebiotics (bacteria powder, oligosaccharide, etc.) combined with Lactobacillus acidophilus AM13-1 to treat ulcerative enteritis can achieve the same therapeutic effect.
  • the probiotic Lactobacillus acidophilus AM13-1 of the present invention can alleviate ulcerative enteritis, and may also exert its treatment in other inflammation-related diseases (common enteritis, gastritis, etc.). Therapeutic effect.
  • the present invention provides a food composition, a health supplement or an auxiliary additive comprising the Lactobacillus acidophilus AM13-1 of the first aspect and/or a metabolite thereof.
  • the food composition of the present invention may contain, in addition to Lactobacillus acidophilus AM13-1 and/or its metabolites, various food materials, food additives, and the like, such as milk, white sugar, and vitamins. Excipient additives in the present invention, such as various edible additives.
  • the present invention provides a pharmaceutical composition comprising the Lactobacillus acidophilus AM13-1 of the first aspect and/or a metabolite thereof.
  • the pharmaceutical composition of the present invention may contain, in addition to Lactobacillus acidophilus AM13-1 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 Lactobacillus acidophilus AM13-1, such as a protective agent, and a typical, but non-limiting protective agent is vitamin C.
  • the content of Lactobacillus acidophilus AM13-1 may be, for example, but not limited to, typically 1 ⁇ 10 -1 to 1 ⁇ 10 20 cfu, based on the total volume or total weight of the pharmaceutical composition. /mL or cfu/g of Lactobacillus acidophilus AM13-1, preferably containing 1 x 10 4 to 1 x 10 15 cfu/mL or cfu/g of Lactobacillus acidophilus AM13-1.
  • the present invention provides the use of the Lactobacillus acidophilus AM13-1 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 acidophilus AM13-1 of the first aspect in the preparation of a cholesterol-lowering drug.
  • the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect in the preparation of a microecological preparation.
  • the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect in the preparation of a food composition, a health care product or an auxiliary additive.
  • the present invention provides the use of the Lactobacillus acidophilus AM13-1 of the first aspect for producing a short-chain fatty acid or an organic acid.
  • the organic acid in the present invention such as formic acid, acetic acid, butyric acid, etc., the organic acid may further include 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, One or more of succinic acid, fufuic acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid, and L-ascorbic acid.
  • the present invention provides a method for preventing and/or treating ulcerative colitis comprising administering a subject of the first aspect of the Lactobacillus acidophilus AM13-1 or the pharmaceutical composition of the fifth 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 acidophilus AM13-1 or a pharmaceutical composition of the fifth aspect.
  • DAI disease activity index
  • the subject may be a human or other mammal.
  • the Lactobacillus acidophilus AM13-1 of the present invention belongs to a new strain discovered by the inventors. It has been found that Lactobacillus acidophilus AM13-1 can effectively lower cholesterol and has obvious relief effect on ulcerative enteritis, which can be significantly improved. The apparent state of mice with ulcerative colitis reduces the index of disease activity in mice and improves the changes in the colon of mice.
  • Figure 1 shows a picture of a colony of Lactobacillus acidophilus AM13-1 cultured for 48 hours.
  • the colonies are white, convex, viscous, opaque, round, with neat edges and a diameter of about 2-3 mm.
  • Figure 2 shows the Gram stain image (1000 times) of Lactobacillus acidophilus AM13-1 under the microscope.
  • the microscopically amplified 1000-fold AM13-1 cells are rod-shaped, Gram-positive, non-spore 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 AM13-1 treated mice.
  • Figure 5 shows changes in the DAI index of the control, model group, VSL # 3 and AM13-1 treated mice.
  • the separated sample was from a stool sample of a healthy male in Shenzhen.
  • the separation process of Lactobacillus acidophilus AM13-1 was as follows:
  • Inorganic salt component Content (1L) CaCl 2 ⁇ 2H 2 O 0.25g MgSO 4 ⁇ 7H 2 O 0.5g K 2 HPO 4 1g KH 2 PO 4 1g NaHCO 3 10g
  • AM13-1 has the following microbiological characteristics:
  • the colonies of AM13-1 cultured on a PYG plate at 37 ° C for 2 days were white, convex, viscous, opaque, round, with neat edges and a diameter of about 2-3 mm (Fig. 1).
  • AM13-1 is negative for catalase, negative for oxidase, and facultative anaerobic.
  • Substrate utilization of AM13-1 API 20A (purchased from Mérieux, France) The results of the experiment are shown in Table 2 (+, indicating a positive reaction; -, indicating a negative reaction; w indicating a weak positive reaction).
  • Genomic DNA was extracted and 16S rDNA amplification was performed using DNA as a template, and 16S rDNA universal primers (8F-AGAGTTTGATCATGGCTCAG (SEQ ID NO: 1) and 1492R-TAGGGTTACCTTGTTACGACTT (SEQ ID NO: 2)) were used, and the amplification conditions were 95 °C. Pre-denaturation for 4 min, then denaturation at 95 ° C for 30 s, annealing at 57 ° C for 40 s, extension at 72 ° C for 1 min 30 s, 30 cycles. The amplified PCR product was purified and sequenced at 3730 to obtain the full length 16S rDNA sequence of AM13-1 (SEQ ID NO: 3).
  • the bioactive substances of AM13-1 mainly investigate the production of short-chain fatty acids (SCFA) and organic acids.
  • the AM13-1 was cultured for 48 hours, and 1 ml of the bacterial solution was centrifuged at 10,000 r/min for 5 minutes, and the supernatant was taken to prepare for detection of short-chain fatty acids (SCFA) and organic acids.
  • SCFA short-chain fatty acids
  • the short-chain fatty acid was determined by an external standard method, and acetic acid, propionic acid, butyric acid, and valeric acid were used to prepare a standard curve.
  • acetic acid, propionic acid, butyric acid, and valeric acid were used to prepare a standard curve.
  • HP-INNOWax Cross-Linked PEG
  • 30m ⁇ 0.25mm ⁇ 0.25um capillary column was used for analysis.
  • the detector was a hydrogen flame ion detector, and the GC parameter was set to 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.
  • the selection criteria for organic acids are: 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, and propylene Acid, benzoic acid, maleic acid, succinic acid, fufuic acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-ascorbic acid.
  • GC-7890B Agilent Meteorological Chromatograph
  • the column is selected from 122-5532G DB-5ms (40m ⁇ 0.25mm ⁇ 0.25um), column temperature: 270 ⁇ 290 ° C; inlet temperature: 250 ° C; gas Flow rate: 0.86 ml/min.
  • AM13-1 was sensitive to antibiotics other than oxacillin and cefazolin.
  • PYG medium with pH 2.5 was prepared, and AM13-1 cultured to log phase was inoculated into PYG medium of pH 2.5 according to 10% inoculation amount. After incubation for 2 hours at 37 °C, AM13 cultured in the same amount of normal PYG medium was taken. -1 Bacterial solution and AM13-1 culture solution cultured in PYG medium of pH 2.5 were plated and counted, and the survival rate of AM13-1 bacteria treated under the condition of pH 2.5 was calculated according to the following formula:
  • pH2.5 treatment survival rate (number of bacterial liquid plate coated colonies in PYG medium cultured at pH 2.5 / number of bacterial liquid plate coated colonies in normal PYG medium culture) ⁇ 100%
  • 0.3% bile salt medium was prepared. By adding 0.3% bile salt to PYG, AM13-1 cultured to log phase was inoculated to 0.3% PYG bile salt medium at 10% inoculation, and cultured at 37 °C. After 2 h, the AM13-1 broth cultured in an equal amount of normal PYG medium and the AM13-1 broth cultured in 0.3% PYG bile salt medium were plated and counted, and the condition of 0.3% bile salt was calculated according to the following formula. Survival rate of the treated AM13-1 strain:
  • AM13-1 maintained a high survival rate (92% and 85%) under the conditions of pH 2.5 and 0.3% bile salt, respectively, indicating that AM13-1 has a strong acid and Bile salt tolerance, the vast majority of live bacteria can reach their function through the stomach juice and small intestine of the human body.
  • 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 AM13-1 was cultured to a concentration of about 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 AM13-1 had a diameter of 12 mm, indicating that AM13-1 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 AM13-1, another strain of commercial cholesterol-lowering probiotic Lactobacillus plantarum Lp299v (purchased from Probi, Sweden) was used for comparison. Both bacteria 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 AM13-1 was 78%, and the degradation rate of Lp299v was 70%, which indicated that AM13-1 had stronger cholesterol degradation ability than Lp299v.
  • mice were C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20g ⁇ 2g, and the mouse breeding environment was SPF grade, and the feeding was performed for 1 week.
  • the modeling method was performed using a dextran sulfate sodium (DSS) method, and the mice were self-drinking 0.2% DSS for 7 days.
  • the experiment was divided into 4 groups of 12 mice each, as follows:
  • Control group Normally reared mice were not subjected to DSS induction;
  • VSL#3 treatment group DSS-induced UC model, each mouse was intragastrically administered with 200 ul of VSL#3 bacterial agent (purchased from Sigma Tau, USA);
  • AM13-1 treatment group DSS-induced UC model, each mouse was intragastrically administered with 200 ul of AM13-1 bacteria per day.
  • VSL#3 Take a certain amount of VSL#3 powder dissolved in PBS, mix well, adjust the bacteria to 10 9 cfu/ml;
  • AM13-1 was cultured to logarithmic growth phase, and the bacterial solution was centrifuged at 8000 r/min, and the cells were suspended in PBS to adjust the concentration to 10 9 cfu/ml.
  • DSS induction and intragastric intervention were performed according to the grouping of mice.
  • the intervention method was performed by side-molding.
  • the body weight, diet and drinking water were recorded every day.
  • the fecal traits and fecal occult blood were observed on the first day.
  • the disease activity index (DAI) of the mice was calculated on the 3rd, 5th, and 7th days.
  • the DAI scores are shown in Table 5.
  • the experiment lasted for 7 days, and the daily gavage amount of probiotics and PBS was 200 ul/mouse.
  • the mice were sacrificed. All mice were bled, necked, colonic, photographed, weighed, and the length of the colon was measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde.
  • 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 equal to the sum of the three points of weight, stool traits, and fecal occult blood/weak blood.
  • DSS-induced ulcerative enteritis model mice caused weight loss.
  • the mice in the model group showed a significant decrease in body weight (*P ⁇ 0.05).
  • the body weight of the model group became extremely different from that of the control group.
  • the intervention of probiotic AM13-1 could effectively control the decrease of body weight in mice.
  • the weight loss of AM13-1 and VSL # 3 mice was effectively controlled compared with the model group ( ⁇ P ⁇ 0.05). This indicates that the two groups of probiotics can control the weight loss caused by UC.
  • the DAI index is an important indicator for judging UC mice. It characterizes the severity of the disease in this mouse. DSS-induced UC model mice cause weight loss in mice, inflammation and ulceration in the colon, bleeding, and affecting stool traits. , causing the DAI index to rise. The DAI values of the mice in each group during the experiment are shown in Table 7 and Figure 5.
  • the colon of UC mice induced by DSS caused the colon to shorten due to inflammation and ulceration. Therefore, the shortening of the length of the colon can be used as an important indicator of the severity of UC.
  • the end of the experiment the colon length of each group of mice is shown in Table 8. .
  • Example 7 Food composition containing Lactobacillus acidophilus AM13-1
  • Example 8 Pharmaceutical composition containing Lactobacillus acidophilus AM13-1
  • the ratio of raw materials is shown in Table 10.
  • 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, Connected to Lactobacillus acidophilus AM13-1 live bacteria (1-500 ⁇ 10 6 cfu/mL), fermented to pH 6.0 at 36-38 ° C, centrifuged, freeze-dried to a moisture content of less than 3%, ie prepare acidophilus Lactobacillus AM13-1 bacteria freeze-dried. 0.5 g of Lactobacillus acidophilus AM13-1 lyophilized product was weighed in an equal amount with maltodextrin, and then filled into a capsule to prepare a pharmaceutical composition containing Lactobacillus acidophilus AM13-1.
  • Example 9 Preparation method of medicine for treating ulcerative enteritis (UC)
  • Lactobacillus acidophilus AM13-1 (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 microorganism C to 100 volumes of AM13-1 fermented bacterial solution, mix well and mix, then add starch adjuvant (such as maltodextrin) to prepare.
  • starch adjuvant such as maltodextrin

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Abstract

La présente invention concerne un Lactobacillus acidophilus, sa méthode de culture et son application. Le Lactobacillus acidophilus AM13-1 est conservé dans un Centre de cultures microbiologiques de Guangdong, et le numéro de conservation est GDMCC No : 60091. Le Lactobacillus acidophilus AM13-1 peut réduire efficacement le cholestérol et atténuer significativement l'entérite ulcéreuse.
PCT/CN2016/111028 2016-12-20 2016-12-20 Lactobacillus acidophilus, sa méthode de culture et son application WO2018112741A1 (fr)

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CN201680091076.7A CN110023486B (zh) 2016-12-20 2016-12-20 一种嗜酸乳杆菌及其培养方法和应用

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN113755370A (zh) * 2021-08-23 2021-12-07 微康益生菌(苏州)股份有限公司 嗜酸乳杆菌la85在制备降血脂药物或保健食品上的应用
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