WO2008101428A1 - Milieu de culture pour lactobacillus acidophilus et streptococcus faecalis, composition le comprenant et son procédé de préparation - Google Patents
Milieu de culture pour lactobacillus acidophilus et streptococcus faecalis, composition le comprenant et son procédé de préparation Download PDFInfo
- Publication number
- WO2008101428A1 WO2008101428A1 PCT/CN2008/070255 CN2008070255W WO2008101428A1 WO 2008101428 A1 WO2008101428 A1 WO 2008101428A1 CN 2008070255 W CN2008070255 W CN 2008070255W WO 2008101428 A1 WO2008101428 A1 WO 2008101428A1
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- WIPO (PCT)
- Prior art keywords
- powder
- weight
- lactobacillus acidophilus
- protective agent
- bifidobacterium longum
- Prior art date
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- 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
- A61P1/10—Laxatives
-
- 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
- A61P1/12—Antidiarrhoeals
-
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/225—Faecalis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/533—Longum
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/853—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/853—Lactobacillus
- Y10S435/854—Lactobacillus acidophilus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/885—Streptococcus
Definitions
- Lactobacillus acidophilus / Enterococcus faecalis culture medium preparation and process thereof
- the present invention relates to a triple live bacterial preparation and a preparation method thereof, and more particularly to a triple live bacterial preparation comprising Bifidobacterium longum, Lactobacillus acidophilus and Streptococcus faecalis and a preparation method thereof. Background technique
- Bifidobacteria, Lactobacillus acidophilus and Streptococcus faecalis are important members of the normal flora in humans and animals. They are concentrated in the lower intestinal mucosa and exert beneficial effects on the body. They can inhibit the invasion of foreign pathogens and provide metabolites. Host essential vitamins to participate in and assist in the digestion and absorption of nutrients. The above bacteria can stimulate the body's immune function. Reduce the absorption of intestinal toxicity. Bifidobacteria in the body are gradually reduced due to digestive diseases or extensive use of antibiotics and with age. In order to maintain the normal levels of the above beneficial bacteria, preparations containing these beneficial bacteria have been studied. For example, Chinese Patent No.
- CN1103584 discloses three oral liquids and a preparation method thereof, which contain Bifidobacterium, Lactobacillus acidophilus and Streptococcus faecalis.
- the oral liquid has poor stability and the survival rate of the bacteria is low, and it is not suitable for long-term preservation.
- Chinese patent CN1119154C discloses a bifid triple live bacterial preparation and a preparation method thereof.
- This document suggests that specific protective agents can be used to increase the stability of Bifidobacterium, Lactobacillus acidophilus and Streptococcus faecalis.
- specific protective agents can be used to increase the stability of Bifidobacterium, Lactobacillus acidophilus and Streptococcus faecalis.
- the use of the above protective agent can improve the stability of Bifidobacterium, Lactobacillus acidophilus and Streptococcus faecalis to a certain extent, the effect of further improving the stability is not very satisfactory.
- Another object of the present invention is to provide a process for preparing the above-mentioned triple live bacterial preparation which can conveniently prepare the triple live bacterial preparation.
- the invention provides a triple live bacterial preparation comprising:
- the Bifidobacterium longum powder comprises Bifidobacterium longum and a first protective agent, the first protective agent comprising 8-50% of skim milk powder, 0.01-10% of sodium glutamate, and 8-50% of isolactose. , Vc-Na 0.01-5% starch 5-30%, based on the total weight of the first protective agent,
- the Lactobacillus acidophilus powder comprises Lactobacillus acidophilus powder comprising Lactobacillus acidophilus and a second protective agent, the Streptococcus faecalis powder comprising Streptococcus faecalis and a second protective agent, the second protective agent comprising Skimmed milk powder 6-70%, sodium glutamate 0.01-10%, isolose 8-50%, Vc-Na0.01-5% starch 4-30%, based on the total weight of the second protective agent
- a protective agent is provided, the protective agent comprising 6-70% of skim milk powder, 0.01-10% of sodium glutamate, 8-50% of isolactose, Vc-Na0.01-5% starch 4-30 %, based on the total weight of the protective agent.
- the invention also provides a method of manufacturing the triple live bacterial preparation, the method comprising:
- step (d) fermenting the Bifidobacterium longum inoculated product obtained in the step (a) in a first fermentation medium to obtain a fermentation product of Bifidobacterium longum;
- step (e) fermenting the Lactobacillus acidophilus inoculated product obtained in the step (b) in a second fermentation medium to obtain a fermentation product of Lactobacillus acidophilus;
- step (f) fermenting the S. faecalis inoculated product obtained in the step (c) in a second fermentation medium to obtain a fermentation product of Streptococcus faecalis;
- the first seed medium comprises 1.1 to 6.5% by weight of a nitrogen source, 0.5 to 3% by weight of a carbon source, 0.6 to 1.5% by weight of a nutrient, and 0.15 to 5% by weight.
- the second seed culture medium contains 1.1 to 6.5 wt% of nitrogen.
- Source 0.2-3 wt% carbon source, 0.5-25 wt% nutrient and 65.5-98.2 wt% solvent.
- the first fermentation medium comprises 1.1 to 6.5% by weight of a nitrogen source, 0.5 to 3% by weight of a carbon source, 0.6 to 1.5% by weight of a nutrient, and 0.15 to 5% by weight.
- the second fermentation medium comprises 1.1 to 6.5% by weight of a nitrogen source, 0.2 to 3% by weight of a carbon source, 0.5 to 25% by weight of nutrients, and 65.5 to 98.8% by weight. Solvent.
- the invention also provides a seed culture medium comprising 1.1-6.5 wt% nitrogen source, 0.2-3 wt% carbon source, 0.5-25 wt% nutrient and 65.5-98.2 wt% Solvent.
- the invention further provides a fermentation medium comprising 1. 1-6.5 wt% nitrogen source, 0.2-3 wt% carbon source, 0.5-25 wt% nutrient and 65.5-98.2 wt. % solvent.
- the invention further provides the use of the triple live bacterial preparation for the treatment of constipation.
- the preparation process of the fermentation liquid of the invention improves the recovery rate of the living bacteria; the protective agent used in freeze-drying of the bacteria obviously improves the survival rate of the bacteria; the preparation prepared by using the live dried lyophilized powder prolongs the survival period of the bacteria and improves Formulation stability.
- the invention provides a triple live bacterial preparation comprising:
- Lactobacillus acidophilus powder Lactobacillus acidophilus powder
- the Bifidobacterium longum powder comprises Bifidobacterium longum and a first protective agent, the first protective agent comprising 8-50% of skim milk powder, 0.01-10% of sodium glutamate, and 8-50% of isolactose. Vc-Na 0.01-5% starch 5-30%, based on the total weight of the first protective agent.
- the Lactobacillus acidophilus powder comprises Lactobacillus acidophilus powder comprising Lactobacillus acidophilus and a second protective agent, the Streptococcus faecalis powder comprising Streptococcus faecalis and a second protective agent, the second protective agent comprising Skim milk powder 6-70%, sodium glutamate 0.01-10%, isolose 8-50%, Vc-Na0.01-5% starch 4-30%, based on the total weight of the second protective agent
- Said Bifidobacterium longum, Lactobacillus acidophilus and Streptococcus faecalis are conventional in the art, it has It is disclosed in the authorized US6368591, wherein the Bifidobacterium longum is deposited under the number CCTCC M98003, the Lactobacillus acidophilus is deposited under the number CCTCC M98004, and the deposited strain of Streptococcus faecalis is CCTCC M9800
- Bifidobacterium longum is an anaerobic bacterium, Gram-positive, uneven coloration, no spores, no capsule, no flagella, the cells are straight or curved, and can appear "Y" or "V” The bifurcation, the rod shape, and the like.
- the Bifidobacterium longum is Bifidobacterium longum (6-1) (CCTCC M98003)
- Lactobacillus acidophilus is a facultative anaerobic bacterium, Gram-positive, non-spore, non-capsulated, flagella-free, short rod-shaped rods at both ends, single or double.
- the Lactobacillus acidophilus is Lactobacillus acidophilus YIT2004 (CCTCC M98004).
- Streptococcus faecalis is a facultative anaerobic bacterium, Gram-positive, non-spore, non-capsulated, flagella-free, oval-shaped, mostly double-arranged, also short-chain.
- the Streptococcus faecalis is Streptococcus faecalis YIT0027 (CCTCC M98005).
- the first protecting agent is conventional, and one of ordinary skill in the art can know which specific protecting agents are useful in the present invention based on the prior art, for example, see CN1119154C.
- the first protective agent comprises skim milk powder 8-50%, sodium glutamate 0.01-10%, isolose 8-50%, Vc-NaO.01-5%, starch 5 -30%, based on the total weight of the first protective agent.
- the skim milk powder is contained in an amount of 10 to 40%, preferably 15 to 35%, more preferably 20 to 30%, most preferably 22 to 28%, in the The total weight of a protective agent.
- the sodium glutamate is contained in an amount of 0.1 to 8%, preferably 0.5 to 5%, more preferably 1-3%, most preferably 1.5 to 2.5%.
- the total weight of the first protective agent is stated.
- the content of the isolose is 10-45%, preferably 15-35%, more preferably 20-30%, most preferably 22-25%, The total weight of a protective agent.
- the Vc-Na content is 0.1 to 4%, preferably 0.5 to 3%, more preferably 1 to 2.5%, most preferably 1.5 to 2%, The total weight of the first protective agent.
- the starch is contained in an amount of 8 to 25%, preferably 10 to 20%, more preferably 12 to 18%, most preferably 13 to 17%, in the first The total weight of the protective agent.
- the second protective agent comprises 6-70% of skim milk powder, 0.01-10% of sodium glutamate, 8-50% of isolactose, Vc-NaO.01-5%, and starch of 4-30%. Based on the total weight of the second protective agent.
- the skim milk powder is contained in an amount of 10 to 60%, preferably 20 to 50%, more preferably 30 to 40%, most preferably It is 33-37 %, based on the total weight of the second protective agent.
- the content of the sodium glutamate is from 0.1 to 8%, preferably from 0.5 to 5%, more preferably from 1 to 3%, most preferably from 1.5 to 2.5%.
- the content of the isolactose is from 10 to 45%, preferably from 15 to 35 %, more preferably from 20 to 30%, most preferably from 22 to 25%, The total weight of a protective agent.
- the Vc-Na content is 0.1 to 4%, preferably 0.5 to 3%, more preferably 1 to 2.5%, most preferably 1.5 to 2%, The total weight of the second protective agent.
- the starch is contained in an amount of 5 to 25%, preferably 10 to 20%, more preferably 12 to 18%, most preferably 13 to 17%, in the second The total weight of the protective agent.
- the second protective agent consists of the following materials:
- the skim milk powder is conventional, and those of ordinary skill in the art can directly obtain which skim milk powder can be used in the present invention according to the prior art.
- the skimmed milk powder is selected from the group consisting of commercially available skim milk powder.
- the sodium glutamate is conventional, and one of ordinary skill in the art can directly obtain which sodium glutamate can be used in the present invention according to the prior art.
- the sodium glutamate is selected from the group consisting of commercially available sodium glutamate.
- the isolose is conventional, and those of ordinary skill in the art can directly obtain which isolose can be used in the present invention according to the prior art.
- the isolactose is selected from the group consisting of commercially available isolactose.
- the VcNa is conventional, and one of ordinary skill in the art can directly obtain which VcNa can be used in the present invention according to the prior art.
- the VcNa is selected from the group consisting of commercially available VcNa.
- the starch is conventional, and those skilled in the art can directly obtain which starch can be used in the present invention according to the prior art.
- the starch is selected from the group consisting of commercially available starches.
- step (d) fermenting the Bifidobacterium longum inoculated product obtained in the step (a) in a first fermentation medium to obtain a fermentation product of Bifidobacterium longum;
- step (e) fermenting the Lactobacillus acidophilus inoculated product obtained in the step (b) in a second fermentation medium to obtain a fermentation product of Lactobacillus acidophilus;
- step (f) fermenting the S. faecalis inoculated product obtained in the step (c) in a second fermentation medium to obtain a fermentation product of Streptococcus faecalis;
- the inoculation method comprises a flame inoculation method, that is, clamping the ethanol cotton with tweezers, igniting, burning up and down the seed can inoculation head, and sterilizing the flame. Then unscrew the seed canister to inoculate the head cover and quickly insert the rubber tube containing the seed inoculating bottle into the inoculation head under flame. Slowly open the inoculation valve and pour the seeds into the can.
- the method of inoculating the Lactobacillus acidophilus is conventional, and those skilled in the art can directly know how to inoculate the Lactobacillus acidophilus according to the prior art.
- a specific inoculation method can be found in CN1119145C.
- the inoculation method comprises a flame inoculation method in which ethanol cotton is clamped with tweezers, and after ignition, the seed can inoculation head is burned up and down, and the flame is sterilized. Unscrew the seed canister inoculation cap and quickly insert the rubber tube containing the seed inoculating bottle into the inoculating head under flame. Slowly open the inoculum valve and pour the seeds into the can.
- the method of inoculating the Streptococcus faecalis is conventional, and those skilled in the art can directly know how to inoculate the Streptococcus faecalis according to the prior art. Specific inoculation methods can be found CN1119145C.
- the inoculation method comprises a flame inoculation method, that is, clamping the ethanol cotton with tweezers, igniting, burning up and down the seed can inoculation head, and sterilizing the flame. Unscrew the seed can to inoculate the head cover and quickly insert the rubber tube containing the seed inoculating bottle into the inoculation head under flame.
- the method of fermenting the Bifidobacterium longum inoculation product is conventional, and those skilled in the art can know how to carry out the Bifidobacterium longum inoculation product according to the description of the present invention in combination with the prior art. Fermentation. Specific fermentation methods can be found in CN1119145C.
- the fermentation method comprises a liquid culture method (that is, the medium used is one or more aqueous solutions such as: peptone water, nutrient broth, etc.), solid state culture method (the medium contains solid matter) Such as: agar, gelatin and other media, solidified or semi-solidified).
- the method of fermenting the product of the Lactobacillus acidophilus inoculum is conventional, and those skilled in the art can know how to ferment the Lactobacillus acidophilus inoculum product according to the description of the present invention in combination with the prior art.
- a specific fermentation method can be found in CN1119145C.
- the fermentation method includes a liquid culture method (the medium is one or more aqueous solutions such as: peptone water, nutrient broth, etc.), and a solid state culture method (the medium contains solid substances of different concentrations) Such as: agar, gelatin and other media, solidified or semi-solidified).
- the method of inoculating the product of S. faecalis is conventional, and those skilled in the art can know how to ferment the S. faecalis inoculation product according to the description of the present invention in combination with the prior art.
- a specific fermentation method can be found in CN1119145C.
- the fermentation method includes a liquid culture method (the medium is one or more aqueous solutions such as: peptone water, nutrient broth, etc.), and a solid state culture method (the medium contains solid substances of different concentrations) Such as: agar, gelatin and other media, solidified or semi-solidified).
- the lyophilization method is conventional, and a person skilled in the art can know the specific method of lyophilization according to the description of the present invention and the prior art. Specific lyophilization methods can be found in CN1119145C.
- the lyophilization process comprises a low temperature freeze drying process, which refers to the process of removing moisture or other solvent from the frozen biological product by sublimation. Sublimation refers to the process by which solvents, such as water, like dry ice, change from a solid to a gaseous state without going through a liquid state.
- the first seed medium is conventional in the art, and one of ordinary skill in the art can directly obtain which seed medium can be used for inoculation of Bifidobacterium longum according to the prior art.
- the specific first seed culture medium can be found in “Optimization of Bifidobacterium longum enrichment medium” (Food Industry Science and Technology, 2004, No. 04).
- the first seed culture medium comprises 1.1 to 6.5% by weight of a nitrogen source, 0.5 to 3% by weight of a carbon source, 0.6 to 1.5% by weight of a nutrient and 0. 15-5 by weight. % mineral salt and 84.00-97.65 weight
- the first seed medium comprises the following components:
- the nitrogen source is conventional, and one of ordinary skill in the art can directly know which specific nitrogen sources are useful in the present invention based on the prior art.
- An example of a specific nitrogen source can be found in CN1119154C.
- the nitrogen source is selected from one or more of skim milk powder, peptone, tryptone, and ammonium salts.
- the nitrogen source is selected from one or both of skimmed milk powder and ammonium sulfate.
- the nitrogen source is present in an amount of from 1.1 to 6.5% by weight, preferably from 1.5 to 5.5% by weight, more preferably from 2.0 to 5.0% by weight, most preferably from 3.0, based on the total weight of the first seed medium. - 4.0% by weight.
- the carbon source is conventional, and one of ordinary skill in the art can directly know which carbon sources are useful in the present invention in accordance with the description of the present invention in conjunction with the prior art.
- An example of a specific carbon source can be found in CN1119145C.
- the carbon source is selected from one or more of the group consisting of glucose, lactose, lactose, galactose, glycerin, sorbitol, trehalose, and maltose.
- the carbon source is selected from one or both of glucose and isoglucan.
- the carbon source is contained in an amount of from 0.8 to 2.8% by weight, preferably from 1.0 to 2.5% by weight, more preferably from 1.5 to 2.0% by weight.
- the nutrient is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which nutrients are useful in the present invention.
- An example of a specific nutrient can be found in CN1119145C.
- the nutritive substance is yeast powder.
- the nutrient content is from 0.6 to 1.5% by weight, preferably from 0.7 to 1.2% by weight, more preferably from 0.8 to 1.0% by weight, based on the total weight of the first seed medium.
- the mineral salts are conventional, and those skilled in the art can directly know which mineral salts can be used in the present invention in accordance with the description of the present invention in conjunction with the prior art.
- An example of a specific mineral salt can be found in CN1119145C.
- the mineral salt is selected from one or more of the phosphates.
- the mineral salt is selected from one or more of potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
- the mineral salt is present in an amount of from 0.15 to 5% by weight, preferably from 0.5 to 4.5% by weight, more preferably from 1.0 to 3.0%, most preferably from 1.5% by weight based on the total weight of the first seed medium. 2.5 wt%.
- the solvent is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which solvents are useful in the present invention.
- An example of a specific solvent can be found in CN1119145C.
- the solvent is a solvent commonly used in biological media, such as water (pure water) and the like.
- the second seed medium is conventional in the art, and one of ordinary skill in the art can directly obtain which seed medium can be used for inoculating the sour milk according to the prior art.
- Bacillus for the specific second seed culture medium, please refer to “Optimization of Lactobacillus acidophilus enrichment medium” ( ⁇ Food Industry Science and Technology ⁇ 2002 ⁇ 06 ⁇ ) and "Study on Lactobacillus acidophilus, Streptococcus faecalis health yoghurt starter” (Journal of Anhui Agricultural and Technical Teachers College, 2000, No. 01)
- the second seed medium contains 1.1 to 6.5% by weight of a nitrogen source, 0.2 to 3% by weight of a carbon source, 0.5 to 25% by weight of nutrients, and 65.5 to 98.8% by weight. Solvent.
- the second seed medium comprises: 4000g of preparation
- the nitrogen source is conventional, and one of ordinary skill in the art can directly know which specific nitrogen sources are useful in the present invention based on the prior art.
- An example of a specific nitrogen source can be found in CN1119154C.
- the nitrogen source is selected from one or more of skim milk powder, peptone, tryptone, and ammonium salts.
- the nitrogen source is selected from one or both of pancreatic fistula and peptone.
- the nitrogen source is contained in an amount of from 1.1 to 6.5 % by weight, preferably from 1.5 to 5.5% by weight, more preferably from 2.0 to 5.0% by weight, most preferably from 3.0, based on the total weight of the second sub-culture medium. - 4.0% by weight.
- the carbon source is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which carbon sources are available for use in the present invention.
- An example of a specific carbon source can be found in CN1119145C.
- the carbon source is selected from one or more of the group consisting of glucose, lactose, isodolacose, galactose, glycerin, sorbitol, trehalose, and maltose.
- the carbon source is selected from one or both of glucose and lactose.
- the carbon source is contained in an amount of from 0.5 to 2.8% by weight, preferably from 1.0 to 2.5% by weight, more preferably from 1.5 to 2.0% by weight, based on the total mass of the second sub-culture medium.
- the nutrient is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which nutrients are useful in the present invention.
- An example of a specific nutrient can be found in CN1119145C.
- the nutritive substance is selected from one or more of the group consisting of succulent, kimchi, and yeast powder.
- the nutritive substance is selected from one or more of the group consisting of tomato and yeast powder.
- the nutrient is present in an amount of from 0.5 to 25% by weight, preferably from 5 to 20% by weight, more preferably from 10 to 15% by weight, based on the total weight of the second seed medium.
- the solvent is conventional, and one of ordinary skill in the art according to the present invention The description of the invention can be directly known in the prior art to know which solvents are useful in the present invention.
- An example of a specific solvent can be found in CN1119145C.
- the solvent is a solvent commonly used in biological media, such as water (pure water) and the like. Fermentation medium
- the first fermentation medium is conventional, and those skilled in the art can know which fermentation medium can be used for fermenting Bifidobacterium longum according to the description of the present invention in combination with the prior art.
- a specific fermentation medium can be found in CN1119145C.
- the first fermentation medium comprises 1.1 to 6.5 wt% of a nitrogen source, 0.5 to 3 wt% of a carbon source, 0.6 to 1.5 wt% of a nutrient, and 0.15 to 5 wt% of Mineral salt and 84.00-97.65 wt% solvent.
- the first fermentation medium comprises:
- the nitrogen source is conventional, and one of ordinary skill in the art can directly know which specific nitrogen sources are useful in the present invention based on the prior art.
- An example of a specific nitrogen source can be found in CN1119154C.
- the nitrogen source is selected from one or more of skim milk powder, peptone, tryptone, and ammonium salts.
- the nitrogen source is selected from one or both of skimmed milk powder and ammonium sulfate.
- the nitrogen source is present in an amount of from 1.1 to 6.5% by weight, based on the total weight of the first fermentation medium, It is preferably from 1.5 to 5.5% by weight, more preferably from 2.0 to 5.0% by weight, most preferably from 3.0 to 4.0% by weight.
- the carbon source is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which carbon sources are available for use in the present invention.
- An example of a specific carbon source can be found in CN1119145C.
- the carbon source is selected from one or more of the group consisting of glucose, lactose, isodolacose, galactose, glycerin, sorbitol, trehalose, and maltose.
- the carbon source is selected from one or both of glucose and isoglucan.
- the carbon source is contained in an amount of from 0.8 to 2.8% by weight, preferably from 1.0 to 2.5% by weight, more preferably from 1.5 to 2.0% by weight, based on the total mass of the first fermentation medium.
- the nutrient is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which nutrients are useful in the present invention.
- An example of a specific nutrient can be found in CN1119145C.
- the nutritive substance is yeast powder.
- the nutrient content is from 0.6 to 1.5% by weight, preferably from 0.7 to 1.2% by weight, more preferably from 0.8 to 1.0% by weight, based on the total weight of the first fermentation medium.
- the mineral salt (mineral) is conventional, and one of ordinary skill in the art, based on the description of the present invention and in conjunction with the prior art, can directly know which mineral salts are useful in the present invention.
- An example of a specific mineral salt can be found in CN1119145C.
- the mineral salt is selected from one or more of the phosphates.
- the mineral salt is selected from one or more of potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
- the mineral salt is present in an amount of from 0.15 to 5% by weight, preferably from 0.5 to 4.5% by weight, more preferably from 1.0 to 3.0%, most preferably from 1.5% by weight based on the total weight of the first fermentation medium. 2.5 wt%.
- the solvent is conventional, and one of ordinary skill in the art, based on the description of the present invention and in conjunction with the prior art, can directly know which solvents are useful in the present invention.
- An example of a specific solvent can be found in CN1119145C.
- the solvent is a solvent commonly used in biological media, such as water (pure water) and the like.
- the second fermentation medium is conventional, and those skilled in the art can know which fermentation medium can be used in the present invention according to the description of the present invention and the prior art. Fermentation of Lactobacillus acidophilus and Streptococcus faecalis.
- the second fermentation medium contains 1.1-6.5 wt% of a nitrogen source, 0.2-3 wt% of a carbon source, 0.5-25 wt% of nutrients, and 65.5-98.2 wt% of a solvent, the second fermentation medium comprising: 400kg preparation
- the nitrogen source is conventional, and one of ordinary skill in the art can directly know which specific nitrogen sources are useful in the present invention based on the prior art.
- An example of a specific nitrogen source can be found in CN1119154C.
- the nitrogen source is selected from one or more of skim milk powder, peptone, tryptone, and ammonium salts.
- the nitrogen source is selected from one or both of pancreatic fistula and peptone.
- the nitrogen source is present in an amount of from 1.1 to 6.5 wt%, preferably from 1.5 to 5.5 wt%, more preferably from 2.0 to 5.0 wt%, most preferably from 3.0, based on the total weight of the second fermentation medium. - 4.0% by weight.
- the carbon source is conventional, and one of ordinary skill in the art, in light of the description of the present invention, in conjunction with the prior art, can directly know which carbon sources are available for use in the present invention.
- An example of a specific carbon source can be found in CN1119145C.
- the carbon source is selected from one or more of the group consisting of glucose, lactose, isodolacose, galactose, glycerin, sorbitol, trehalose, and maltose.
- the carbon source is selected from one or both of glucose and lactose.
- the carbon source is contained in an amount of from 0.5 to 2.8% by weight, preferably from 1.0 to 2.5% by weight, more preferably from 1.5 to 2.0% by weight, based on the total mass of the second fermentation medium.
- the nutrient is conventional, and one of ordinary skill in the art, based on the description of the present invention and in conjunction with the prior art, can directly know which nutrients are useful in the present invention.
- An example of a specific nutrient can be found in CN1119145C.
- the nutritive substance is selected from one or more of the group consisting of succulent, kimchi, and yeast powder.
- the nutritive substance is selected from one or more of the group consisting of tomato and yeast powder.
- the nutrient is present in an amount of from 0.5 to 25% by weight, preferably from 5 to 20% by weight, more preferably from 10 to 15% by weight, based on the total weight of the second fermentation medium.
- the solvent is conventional, and one of ordinary skill in the art according to the present invention The description of the invention can be directly known in the prior art to know which solvents are useful in the present invention.
- An example of a specific solvent can be found in CN1119145C.
- the solvent is a solvent commonly used in biological media, such as water (pure water) and the like.
- the triple live bacterial preparation can be prepared into various suitable dosage forms such as oral liquid, tablets, capsules, orally disintegrating tablets and the like.
- the dosage form is a capsule.
- the dosage form is a tablet.
- the triple live bacterial preparation can be prepared into various dosage forms by a method known in the art, for example, a tablet, a capsule, a granule, or the like can be prepared according to the method described in CN1119154C.
- a tablet, a capsule, a granule, or the like can be prepared according to the method described in CN1119154C.
- Capsule manufacturing method pretreatment of raw materials, baking powder, mixing of raw and auxiliary materials, filling of capsules, bottling, packing;
- Granule manufacturing method raw material pretreatment, granulation, baking powder, raw and auxiliary material mixing and sieving, powder filling, blistering, packing and packing, packing;
- Tablet manufacturing method raw material pretreatment, granulation, baking powder, raw and auxiliary material mixing sieving, tableting, inner packaging, cartoning, packing
- Tomato juice was prepared by centrifugation of the tomato juice after heat sterilization.
- Example 1 Preparation of seed medium inoculated with Bifidobacterium longum
- the above solution and suspension were ground by a colloid mill (Wenzhou Lucheng Emulsifying Machinery Co., Ltd., JM-L80) and placed in a seed bottle. Then, the drinking medium was added to 4000 g to prepare a seed culture medium of Bifidobacterium breve, and then added. A small amount of dilute sodium hydroxide solution adjusts the pH of the medium.
- the above seed bottle mouth was sealed, and the seed bottle was sterilized in a sterilizing pot at 121 ° C for 10-15 minutes.
- Example 2 Preparation of seed medium inoculated with Lactobacillus acidophilus and Streptococcus faecalis
- the above solution and suspension are uniformly ground by a colloid mill and placed in a seed bottle, and then added to the drinking water.
- Example 3 The above inoculated products A1-A5 were separately placed in the fermentation medium prepared in Example 3 (Prescription 1-5) In the 600L fermenter (Zhangjiagang Jiangnan Chemical Machinery Factory), the fermentation product Bl-B5 was obtained by fermentation at 37.0 ⁇ 2.0°C and tank pressure 0.05 ⁇ 0.03MPa.
- the above fermentation products B 1-B5 were respectively introduced into a centrifuge tube of a GQ142 tube centrifuge manufactured by Shanghai Institute of Centrifugal Machinery for centrifugation at room temperature.
- the flow rate of the fermentation liquid to be introduced is controlled at 4000 ⁇ 9000 ml/min. After centrifugation for 90-150 minutes, the centrifuge tube is taken out, and the slime C1-C5 is excavated from the centrifuge tube and placed in a stainless steel barrel.
- Example 6 Preparation of Lactobacillus acidophilus
- the above fermentation products E1-E5 were respectively introduced into a centrifuge tube of a GQ142 tube centrifuge manufactured by Shanghai Institute of Centrifugal Machinery for centrifugation at room temperature.
- the flow rate of the introduced fermentation liquid was controlled at 4000 9000 ml/min. After centrifugation for 40-80 minutes, the centrifuge tube was taken out, and the fungus F1-F5 was excavated from the centrifuge tube and placed in a stainless steel barrel.
- Example 7 Preparation of Streptococcus faecalis
- the inoculum products F1-F5 were placed in a 600 L fermentor (Zhangjiagang Jiangnan Chemical Machinery Factory) containing the fermentation medium (Prescription 1-5) obtained in Example 4, and the fermentation was carried out at a pressure of 0.05 ⁇ 0.03 MPa. , the fermentation product Hl-H5 was obtained.
- the above-mentioned fermentation products H1-H5 were respectively introduced into a centrifuge tube of a GQ142 tubular centrifuge manufactured by Shanghai Institute of Centrifugal Machinery for centrifugation at room temperature.
- the flow rate of the fermented liquid to be introduced is controlled at 4000 ⁇ 9000 ml/min. After centrifugation for 40 to 80 minutes, the centrifuge tube is taken out, and the bacterial sludge 11-15 is excavated from the centrifuge tube and placed in a stainless steel drum.
- Example 8 Preparation of long Bifidobacterium powder
- Example 5 In the bacterial sludge C1-C5 obtained in Example 5, a protective agent having the composition shown in the following table was added (in which the slime was added) Add the prescription 1 - Prescription 5 protective agent to C1-C5, and grind it evenly with a colloid mill. The resulting homogeneous mixture was placed in a freeze dryer (Beijing Tianli Technology, GLZ-6) for lyophilization. During the lyophilization process, the inlet temperature of the laminar layer is cooled and pre-frozen, and the product is pre-frozen to about -40 °C. After pre-freezing, open the condensation to lower the cold trap to -40 ° C, then open the vacuum pump to evacuate the tank. After the vacuum of the cabinet reaches the vacuum set value (15pa), the product is warmed up. When the color of the product is the same, the lyophilization is finished, that is, the powder Jl-J5 is obtained.
- a freeze dryer Beijing Tianli Technology, GL
- F1-F5 is added to the prescription 1 - prescription 5 protective agent), and is evenly ground by a colloid mill.
- the resulting homogeneous mixture was placed in a freeze dryer (Beijing Tianli Technology, GLZ-6) for lyophilization.
- a freeze dryer Beijing Tianli Technology, GLZ-6
- the inlet temperature of the laminar layer is cooled and pre-frozen, so that the product is pre-frozen to about -40 °C.
- open the condensation to reduce the cold trap to -40 ° C then open the vacuum pump to evacuate the tank.
- the vacuum of the cabinet reaches the vacuum set point (15pa), the product is warmed up.
- the lyophilization is finished, that is, the bacterial powder Kl-K5 is obtained.
- Example 10 Preparation of Streptococcus faecalis powder
- Example 7 To the bacterial sludge 11-15 obtained in Example 7, a protective agent having the composition shown in the following table was added (preparation of the protective agent of Formulation 1 - Prescription 5 in the slime 11-15), and the mixture was uniformly ground by a colloid mill. The resulting homogeneous mixture was placed in a freeze dryer (Beijing Tianli Technology, GLZ-6) for lyophilization. During the lyophilization process, the inlet temperature of the laminar layer is cooled and pre-frozen, so that the product is pre-frozen to about -40 °C. After pre-freezing, open the condensation to reduce the cold trap to -40 ° C, then open the vacuum pump to evacuate the tank. After the vacuum of the cabinet reaches the vacuum set value (15pa), the product is warmed up. When the color of the product is the same, the lyophilization is completed, that is, the powder Ll-L5 is obtained. Table 7
- Lactobacillus acidophilus powder K1-K5 (>5.0x l0 8 CPU/g) 6.0kg
- Streptococcus faecalis powder L1-L5 (>5.0x l0 8 CPU/g) 3.0kg
- the components shown in the table below were put into a multi-directional motion mixer (HDA-600, Wenzhou Pharmaceutical Equipment Factory) and mixed 30-40 to obtain a homogeneous mixture. Open the powder filling machine (Shanghai Songchuan Packaging Machinery Factory multi-row packaging machine, SL-8) The uniform mixture is made into 100,000 pieces of powder N1-N5, and the powder Ni contains long Bifidobacterium powder Ji, soluophilic milk Bacillus powder Ki and Streptococcus faecalis powder Li, i are integers from 1 to 5.
- the blank particles were prepared as follows: The components shown in Table 10 below were spray granulated and dried in an oven at 100 °C. Table 9
- Microbial preparations i.e., Comparative Examples 1-3, were prepared according to Examples 1-3 of CN1119154C. Use and embodiment
- Example 14 Application of triple live bacterial preparation
- Capsules Ml prepared in Example 1 were administered to a patient with functional constipation three times a day, each time The results are as follows:
- This example describes the effect of the triple live bacterial preparation of the present invention in treating diarrhea.
- the patient deactivates other drugs (antibiotics, hormones, other antidiarrheal drugs or emollients) before treatment, and the capsule M2 obtained in Example 12 is administered to the patient at the following dosage: 5 capsules per adult, 2-3 times per day; 0 + -1 year old children 1 capsule, 3 times / day; 1-6 years old children 2 capsules, 3 times / day; 6-13 years old children 3 capsules, 3 times / day.
- Statistical processing was performed according to chi-square test and Ridit analysis.
- Capsule M2 obtained in Example 12 was used to treat 401 cases of mild or moderate acute diarrhea and unexplained chronic diarrhea in adults or children. The total effect was 87.5 %. In addition, the effect of capsule M2 in the treatment of acute diarrhea is better than the treatment of chronic diarrhea (there is a significant difference between the two treatments 1 3 ⁇ 0.05). The total effective rate of treatment for acute diarrhea in adults and children was 92.3 % and 90.7 %, respectively, while the total effective rate for chronic diarrhea was 80.0 % and 76.3%, respectively.
- Capsules M2 is effective in the treatment of infectious diarrhea (pathogens including: dysentery bacilli, salmonella, pathogenic coliform, Campylobacter jejuni, Aeromonas and rotavirus, etc.) in addition to diarrhea of unknown cause.
- infectious diarrhea pathogens including: dysentery bacilli, salmonella, pathogenic coliform, Campylobacter jejuni, Aeromonas and rotavirus, etc.
- pathogens including: dysentery bacilli, salmonella, pathogenic coliform, Campylobacter jejuni, Aeromonas and rotavirus, etc.
- Capsule M2 is less effective than chronic diarrhea for chronic diarrhea, but it does have some effect.
- Chronic diarrhea patients have different levels of other drugs before the test, no significant effect, or long-term diagnosis of "intestinal irritation syndrome", after taking capsule M2 80.0% of adults and 76.3 % of children recovered.
- Capsule M2 has the function of adjusting the intestinal flora. From the results of the detection of the main flora before and after treatment of 19 adults and 16 children with diarrhea, the patients had different degrees of intestinal flora disorder before treatment, mainly in: Enterobacter aerobic or facultative anaerobic bacteria The anaerobic bacteria such as bifidobacteria are low, and the above conditions are improved with the disappearance of clinical symptoms after treatment, and the intestinal flora returns to normal.
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Description
嗜酸乳杆菌 /粪肠球菌培养基、 制剂及其工艺 技术领域
本发明涉及三联活菌制剂及其制备方法,更具体涉及包含长型双歧杆菌、嗜酸乳 杆菌和粪链球菌的三联活菌制剂及其制备方法。 背景技术
双歧杆菌、嗜酸乳杆菌和粪链球菌是人和动物体内参见的正常菌群重要成员,集 中粘附在下肠道粘膜, 对机体发挥有益作用, 能抑制外籍病原菌的入侵, 其代谢产物 提供宿主必需的维生素, 参与和协助营养的消化和吸收。 上述细菌能激发机体的免疫 功能。减少肠源性毒性的吸收。 由于消化***疾病或大量使用抗生素及随着年龄的增 长, 体内双歧杆菌逐渐减少。 为保持上述有益菌的正常水平, 人们开始研究含有这些 有益菌的制剂。 如中国专利 CN1103584公开了三株口服液及其制法, 其含有双歧杆 菌、 嗜酸乳杆菌和粪链球菌。 但是, 该口服液稳定性差, 菌的存活率低, 不宜长期保 存。
中国专利 CN1119154C 公开了一种双歧三联活菌制剂及其制备方法。 该文献提 出可以使用特定保护剂来提高双歧杆菌、 嗜酸乳杆菌和粪链球菌的稳定性。 虽然使用 上述保护剂可在一定程度上提高双歧杆菌、 嗜酸乳杆菌和粪链球菌的稳定性, 但是进 一步提高稳定性的效果并不十分令人满意。
另外, 目前并没有文献报道改进双歧杆菌、嗜酸乳杆菌和粪链球菌的种子液培养 基和发酵培养基来提高双歧杆菌、 嗜酸乳杆菌和粪链球菌稳定性的方法。
因此, 本领域中需一种具有较高的稳定性新的三联活菌制剂。 发明内容
本发明的目的在于提供一种新的三联活菌制剂, 它具有较高的稳定性。
本发明的另一个目的在于提供一种制备上述三联活菌制剂的方法,所述方法能方 便地制备所述三联活菌制剂。 本发明一方面提供了一种三联活菌制剂, 它包括:
长型双歧杆菌菌粉;
嗜酸乳杆菌菌粉;
粪链球菌菌粉,
所述长型双歧杆菌菌粉包含长型双歧杆菌和第一保护剂,所述第一保护剂包含脱 脂奶粉 8-50%、 谷氨酸钠 0.01-10%、 异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 5-30%, 以所 述第一保护剂的总重量计,
所述嗜酸乳杆菌菌粉包含嗜酸乳杆菌菌粉包含嗜酸乳杆菌和第二保护剂,所述粪 链球菌菌粉包含粪链球菌和第二保护剂, 所述第二保护剂包含脱脂奶粉 6-70%、 谷氨 酸钠 0.01-10%、异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 4-30%, 以所述第二保护剂的总重 本发明另一方面提供了一种保护剂, 所述保护剂包含包含脱脂奶粉 6-70%、 谷氨 酸钠 0.01-10%、异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 4-30%, 以所述保护剂的总重量计。
本发明还提供了一种制造所述三联活菌制剂的方法, 所述方法包括:
(a) 将长型双歧杆菌在第一种子培养基中接种, 得到长型双歧杆菌接种产物;
(b) 将嗜酸乳杆菌在第二种子培养基中接种, 得到嗜酸乳杆菌接种产物; (c) 将粪链球菌在第二种子培养基中接种, 得到粪链球菌接种产物;
(d)将步骤(a)所得长型双歧杆菌接种产物置于第一发酵培养基中发酵, 得到 长型双歧杆菌发酵产物;
(e)将步骤(b)所得嗜酸乳杆菌接种产物置于第二发酵培养基中发酵, 得到嗜 酸乳杆菌发酵产物;
(f)将步骤 (c)所得粪链球菌接种产物置于第二发酵培养基中发酵, 得到粪链 球菌发酵产物;
(g) 混合长型双歧杆菌与第一保护剂, 冻干得到长型双歧杆菌菌粉;
(h) 将嗜酸乳杆菌和粪链球菌分别与第二保护剂混合, 分别冻干得到嗜酸乳杆 菌菌粉和粪链球菌菌粉;
(i) 混合上述长型双歧杆菌菌粉、 嗜酸乳杆菌菌粉和粪链球菌菌粉得到三联活 菌制剂。
在本发明的一个优选实例中, 所述第一种子培养基包含 1.1-6.5重量%的氮源、 0.5-3 重量%的碳源、 0.6-1.5 重量%的营养物质和 0.15-5 重量%的矿物盐和 84.00-97.65重量%的溶剂。
在本发明的另一个优选实例中, 所述第二种子培养基包含 1.1-6.5 重量%的氮
源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。 在本发明的另一个优选实例中,所述第一发酵培养基包含 1.1-6.5重量%的氮 源、 0.5-3 重量%的碳源、 0.6- 1.5重量%的营养物质和 0.15-5 重量%的矿物盐和 84.00-97.65重量%的溶剂。
在本发明的另一个 优选实例中, 所述第二发酵培养基包含 1.1-6.5重量%的 氮源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。
本发明还提供了一种种子培养基, 所述种子培养基含 1.1-6.5 重量%的氮源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。
本发明又提供了一种发酵培养基, 所述发酵培养基包含 1. 1-6.5 重量%的氮 源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。
本发明进一步提供了所述三联活菌制剂在治疗便秘中的用途。 本发明发酵液配制生产工艺使活菌收得率提高; 细菌冻干时所用的保护剂, 明显 提高细菌的存活率; 使用该活菌冻干粉制备成的制剂, 延长了细菌存活期, 提高制剂 稳定性。 具体实施方式
三联活菌制剂
本发明一方面提供了一种三联活菌制剂, 它包括:
长型双歧杆菌菌粉;
嗜酸乳杆菌菌粉;
粪链球菌菌粉,
所述长型双歧杆菌菌粉包含长型双歧杆菌和第一保护剂,所述第一保护剂包含脱 脂奶粉 8-50%、 谷氨酸钠 0.01-10%、 异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 5-30%, 以所 述第一保护剂的总重量计。
所述嗜酸乳杆菌菌粉包含嗜酸乳杆菌菌粉包含嗜酸乳杆菌和第二保护剂,所述粪 链球菌菌粉包含粪链球菌和第二保护剂, 所述第二保护剂包含脱脂奶粉 6-70%、 谷氨 酸钠 0.01-10%、异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 4-30%, 以所述第二保护剂的总重 本发明所述的长型双歧杆菌、 嗜酸乳杆菌和粪链球菌在本领域中是常规的, 它已
经公开在已授权的 US6368591中, 其中长型双歧杆菌的保藏号为 CCTCC M98003, 嗜酸乳杆菌的保藏号为 CCTCC M98004, 粪链球菌的保藏号为 CCTCC M98005。
长型双歧杆菌为厌氧性细菌, 革兰氏染色阳性, 着色不均匀, 无芽孢, 无荚膜, 无鞭毛, 菌体呈直形或弯曲状, 可出现 "Y "或" V"型的分叉状, 棒状等多多形态。在本 发明的一个优选实例中, 所述长型双歧杆菌为长型双歧杆菌 (6— 1) ( CCTCC M98003
嗜酸乳杆菌为兼性厌氧菌, 革兰氏染色阳性、 无芽孢, 无荚膜, 无鞭毛, 两端圆 的短杆球杆状, 单个或成双排列。 在本发明的一个优选实例中, 所述嗜酸乳杆菌为嗜 酸乳杆菌 YIT2004 (CCTCC M98004)。
粪链球菌为兼性厌氧菌, 革兰氏染色阳性、 无芽孢, 无荚膜, 无鞭毛, 卵圆 形, 多成双排列, 也呈短链。 在本发明的一个优选实例中, 所述粪链球菌为粪链 球菌 YIT0027 (CCTCC M98005) 。 保护剂
在本发明中,所述第一保护剂是常规的,本领域的普通技术人员根据现有技术可 以知道哪些具体的保护剂可用于本发明中, 例如参见 CN1119154C。 在本发明的一个 优选实例中,所述第一保护剂包含脱脂奶粉 8-50%、谷氨酸钠 0.01-10%、异乳糖 8-50%、 Vc-NaO.01-5%, 淀粉 5-30%, 以所述第一保护剂的总重量计。 在本发明的另一个优选实 例中, 所述脱脂奶粉的含量为 10-40%、较好为 15-35%、 更好为 20-30%、 最好为 22-28 %, 以所述第一保护剂的总重量计。 在本发明的另一个优选实例中, 所述谷氨酸钠的含 量为 0.1-8%、 较好为 0.5-5%、 更好为 1-3%、 最好为 1.5-2.5%, 以所述第一保护剂 的总重量计。 在本发明的另一个优选实例中, 所述异乳糖的含量为 10-45%、 较好为 15-35%、 更好为 20-30%、 最好为 22-25%, 以所述第一保护剂的总重量计。 在本发 明的另一个优选实例中, 所述 Vc-Na的含量为 0.1-4%、较好为 0.5-3%、更好为 1-2.5 %、 最好为 1.5-2%, 以所述第一保护剂的总重量计。 在本发明的另一个优选实例中, 所述淀粉的含量为 8-25%、 较好为 10-20%、 更好为 12-18%、 最好为 13-17%, 以所 述第一保护剂的总重量计。
在本发明中, 所述第二保护剂包含脱脂奶粉 6-70%、 谷氨酸钠 0.01-10%、 异乳糖 8-50%、 Vc-NaO.01-5%, 淀粉 4-30%, 以所述第二保护剂的总重量计。 在本发明的一个 优选实例中, 所述脱脂奶粉的含量为 10-60%、 较好为 20-50%、 更好为 30-40%、 最好
为 33-37 %, 以所述第二保护剂的总重量计。 在本发明的另一个优选实例中, 所述谷氨 酸钠的含量为 0.1-8 %、 较好为 0.5-5 %、 更好为 1-3 %、 最好为 1.5-2.5 %, 以所述第 二保护剂的总重量计。在本发明的另一个优选实例中, 所述异乳糖的含量为 10-45 %、 较好为 15-35 %、 更好为 20-30%、 最好为 22-25 %, 以所述第一保护剂的总重量计。 在本发明的另一个优选实例中, 所述 Vc-Na的含量为 0.1-4%、 较好为 0.5-3 %、 更好 为 1-2.5 %、 最好为 1.5-2%, 以所述第二保护剂的总重量计。 在本发明的另一个优选 实例中, 所述淀粉的含量为 5-25 %、 较好为 10-20%、 更好为 12-18 %、 最好为 13-17 % , 以所述第二保护剂的总重量计。
在本发明的一个优选实例中, 所述第二保护剂由下列物质组成:
在本发明中,所述谷氨酸钠是常规的,本领域的普通技术人员根据现有技术可以 直接得到哪些谷氨酸钠可用于本发明中。在本发明的一个优选实例中, 所述谷氨酸钠 选自市售谷氨酸钠。
在本发明中,所述异乳糖是常规的,本领域的普通技术人员根据现有技术可以直 接得到哪些异乳糖可用于本发明中。在本发明的一个优选实例中, 所述异乳糖选自市 售异乳糖。
在本发明中, 所述 VcNa是常规的, 本领域的普通技术人员根据现有技术可以直 接得到哪些 VcNa可用于本发明中。 在本发明的一个优选实例中, 所述 VcNa选自市 售 VcNa。
在本发明中,所述淀粉是常规的,本领域的普通技术人员根据现有技术可以直接 得到哪些淀粉可用于本发明中。在本发明的一个优选实例中,所述淀粉选自市售淀粉。 制造三联活菌制剂的方法
本发明另一方面提供了一种制造上述三联活菌制剂的方法, 所述方法包括:
(a) 将长型双歧杆菌在第一种子培养基中接种, 得到长型双歧杆菌接种产物;
(b) 将嗜酸乳杆菌在第二种子培养基中接种, 得到嗜酸乳杆菌接种产物; (C) 将粪链球菌在第二种子培养基中接种, 得到粪链球菌接种产物;
(d)将步骤(a)所得长型双歧杆菌接种产物置于第一发酵培养基中发酵, 得到 长型双歧杆菌发酵产物;
(e)将步骤(b)所得嗜酸乳杆菌接种产物置于第二发酵培养基中发酵, 得到嗜 酸乳杆菌发酵产物;
(f)将步骤 (c)所得粪链球菌接种产物置于第二发酵培养基中发酵, 得到粪链 球菌发酵产物;
(g) 混合长型双歧杆菌与第一保护剂, 冻干得到长型双歧杆菌菌粉;
(h) 将嗜酸乳杆菌和粪链球菌分别与第二保护剂混合, 分别冻干得到嗜酸乳杆 菌菌粉和粪链球菌菌粉;
(i) 混合上述长型双歧杆菌菌粉、 嗜酸乳杆菌菌粉和粪链球菌菌粉得到三联活 菌制剂。
在上述步骤 (a) 中, 所述接种长型双歧杆菌的方法是常规的, 本领域的普通技术 人员根据现有技术可直接知道怎样对所述长型双歧杆菌进行接种。具体的接种方法可 参见 CN1119145C。 在本发明的一个优选实例中, 所述接种方法包括火焰接种法, 即 用镊子夹住乙醇棉花, 点燃后, 在种子罐接种头上下燃烧, 火焰消毒。 然后旋开种子 罐接种头盖, 在火焰下快速将装有种子接种瓶的橡皮管***接种头上。 缓慢打开接种 口阀门, 将种子倒入罐内。
在上述步骤(b) 中, 接种所述嗜酸乳杆菌的方法是常规的, 本领域的普通技术人 员根据现有技术可直接知道怎样对所述嗜酸乳杆菌进行接种。具体的接种方法可参见 CN1119145C。 在本发明的一个优选实例中, 所述接种方法包括火焰接种法, 即用镊 子夹住乙醇棉花, 点燃后, 在种子罐接种头上下燃烧, 火焰消毒。 旋开种子罐接种头 盖, 在火焰下快速将装有种子接种瓶的橡皮管***接种头上。 缓慢打开接种口阀门, 将种子倒入罐内。
在上述步骤 (c) 中, 接种所述粪链球菌的方法是常规的, 本领域的普通技术人员 根据现有技术可直接知道怎样对所述粪链球菌进行接种。 具体的接种方法可参见
CN1119145C。 在本发明的一个优选实例中, 所述接种方法包括火焰接种法, 即用镊 子夹住乙醇棉花, 点燃后, 在种子罐接种头上下燃烧, 火焰消毒。 旋开种子罐接种头 盖, 在火焰下快速将装有种子接种瓶的橡皮管***接种头上。 缓慢打开接种口阀门, 将种子倒入罐内。 在上述步骤 (d) 中, 发酵长型双歧杆菌接种产物的方法是常规的, 本领域的普 通技术人员根据本发明的描述再结合现有技术可知道怎样对长型双歧杆菌接种产物 进行发酵。 具体的发酵方法可参见 CN1119145C。 在本发明的一个优选实例中, 所述 发酵方法包括液态培养方法(即所用培养基是一种或多种水溶液如: 蛋白胨水、 营养 肉汤等) 、 固态培养方法 (培养基是含有固态物如: 琼脂、 明胶等的培养基, 呈凝固 状或者半凝固状) 。
在上述步骤 (e) 中, 发酵嗜酸乳杆菌接种产物的方法是常规的, 本领域的普通 技术人员根据本发明的描述再结合现有技术可知道怎样对嗜酸乳杆菌接种产物进行 发酵。 具体的发酵方法可参见 CN1119145C。 在本发明的一个优选实例中, 所述发酵 方法包括液态培养方法 (培养基是一种或多种水溶液如: 蛋白胨水、 营养肉汤等) 、 固态培养方法(培养基是含有不同浓度固态物如: 琼脂、 明胶等的培养基, 呈凝固状 或者半凝固状) 。
在上述步骤 (f) 中, 发酵粪链球菌接种产物的方法是常规的, 本领域的普通技 术人员根据本发明的描述再结合现有技术可知道怎样对粪链球菌接种产物进行发酵。 具体的发酵方法可参见 CN1119145C。 在本发明的一个优选实例中, 所述发酵方法包 括液态培养方法 (培养基是一种或多种水溶液如: 蛋白胨水、 营养肉汤等) 、 固态培 养方法(培养基是含有不同浓度固态物如: 琼脂、 明胶等的培养基, 呈凝固状或者半 凝固状) 。
在上述步骤(g)和 (h) 中, 所述冻干方法是常规的, 本领域的普通技术人员根 据本发明的描述再结合现有技术可以知道进行冻干的具体方法。具体的冻干方法可参 见 CN1119145C。 在本发明的一个优选实例中, 所述冻干方法包括低温冷冻干燥法, 所述冷冻干燥是指通过升华从冻结的生物产品中去掉水份或其他溶剂的过程。升华指 的是溶剂, 比如水, 像干冰一样, 不经过液态, 从固态直接变为气态的过程。 第一种子培养基
在上述步骤 (a) 中, 所述第一种子培养基在本领域中是常规的, 本领域的普通 技术人员根据现有技术可直接得到哪些种子培养基可用于接种长型双歧杆菌。具体的 第一种子培养基可参见 《长双歧杆菌增菌培养基的优化》 ( 《食品工业科技》 2004 年第 04期) 。
但是, 为了得到更加稳定的长型双歧杆菌, 优选使用特定的种子培养基。在本发 明的一个优选实例中, 所述第一种子培养基包含 1.1-6.5重量%的氮源、 0.5-3重量 %的碳源、0.6- 1.5重量%的营养物质和 0. 15-5重量%的矿物盐和 84.00-97.65重量
%的溶剂。 在本发明的另一个优选实例中, 所述第一种子培养基包含如下组分:
在所述第一种子培养基中,所述氮源是常规的,本领域的普通技术人员根据现有 技术可直接知道哪些具体氮源可用于本发明中。具体氮源的例子可参见 CN1119154C。 在本发明的一个优选例子中, 所述氮源选自脱脂奶粉、 蛋白胨、 胰胨、 铵盐中的一种 或多种。 在本发明的另一个优选实例中, 所述氮源选自脱脂奶粉、 硫酸铵中的一种或 两种。 通常, 以所述第一种子培养基的总重量计, 所述氮源的含量为 1.1-6.5重量%, 较好为 1.5-5.5重量%, 更好为 2.0-5.0重量%, 最好为 3.0-4.0重量%。
在所述第一种子培养基中,所述碳源是常规的,本领域的普通技术人员根据本发 明的描述再结合现有技术可以直接知道哪些碳源可用于本发明中。具体碳源的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述碳源选自葡萄糖、 乳糖、 异乳 糖、 半乳糖、 甘油、 山梨醇、 海藻糖、 麦芽糖中的一种或多种。 在本发明的另一个实 例中, 所述碳源选自葡萄糖、 异乳糖中的一种或两种。 通常, 以所述第一种子培养基
的总重量计,所述碳源的含量为 0.8-2.8重量%,较好为 1.0-2.5重量%,更好为 1.5-2.0 重量%。
在所述第一种子培养基中,所述营养物质是常规的,本领域的普通技术人员根据 本发明的描述再结合现有技术可以直接知道哪些营养物质可用于本发明中。具体营养 物质的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述营养物质是酵母 粉。 通常, 以所述第一种子培养基的总重量计, 所述营养物质的含量为 0.6-1.5 重量 % , 较好为 0.7-1.2重量%, 更好为 0.8-1.0重量%。
在所述第一种子培养基中, 所述矿物盐(矿物质)是常规的, 本领域的普通技术 人员根据本发明的描述再结合现有技术可以直接知道哪些矿物盐可用于本发明中。具 体的矿物盐的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述矿物盐选 自磷酸盐中的一种或多种。在本发明的另一个优选实例中, 所述矿物盐选自磷酸二氢 钾、 磷酸氢二钾中的一种或多种。 通常, 以所述第一种子培养基的总重量计, 所述矿 物盐的含量为 0.15-5重量%,较好为 0.5-4.5重量%,更好为 1.0-3.0%,最好为 1.5-2.5 重量%。
在所述第一种子培养基中,所述溶剂是常规的,本领域的普通技术人员根据本发 明的描述再结合现有技术可以直接知道哪些溶剂可用于本发明中。具体溶剂的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述溶剂是生物学培养基中常用的 溶剂, 例如水 (纯水) 等。 第二种子培养基
在上述步骤(b )和 (c) 中, 所述第二种子培养基在本领域中是常规的, 本领域 的普通技术人员根据现有技术可直接得到哪些种子培养基可用于接种嗜酸乳杆菌。具 体的第二种子培养基可参见《嗜酸乳杆菌增菌培养基的优化》 (《食品工业科技》 2002 年第 06期) 和 《嗜酸乳杆菌、 粪链球菌保健酸奶发酵剂的研究》 (安徽农业技术师 范学院学报 2000年第 01期)
但是, 为了得到更加稳定的嗜酸乳杆菌, 优选使用特定的第二种子培养基。在本 发明的一个优选实例中, 所述第二种子培养基含 1.1-6.5重量%的氮源、 0.2-3重量 %的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。 所述第二种子培 养基包含:
4000g的配制量
西红柿汁 营养物质 500g
葡萄糖 碳源 60g
酵母粉 营养物质 43g
胰胨 氮源 160g
蛋白胨 氮源 36g
乳糖 碳源 13g
水 溶剂 余量 在所述第二种子培养基中,所述氮源是常规的,本领域的普通技术人员根据现有 技术可直接知道哪些具体氮源可用于本发明中。具体氮源的例子可参见 CN1119154C。 在本发明的一个优选例子中, 所述氮源选自脱脂奶粉、 蛋白胨、 胰胨、 铵盐中的一种 或多种。 在本发明的另一个优选实例中, 所述氮源选自胰胨、 蛋白胨中的一种或两 种。 通常, 以所述第二子培养基的总重量计, 所述氮源的含量为 1.1-6.5 重量%, 较 好为 1.5-5.5重量%, 更好为 2.0-5.0重量%, 最好为 3.0-4.0重量%。
在所述第二种子培养基中,所述碳源是常规的,本领域的普通技术人员根据本发 明的描述再结合现有技术可以直接知道哪些碳源可用于本发明中。具体碳源的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述碳源选自葡萄糖、 乳糖、 异乳 糖、 半乳糖、 甘油、 山梨醇、 海藻糖、 麦芽糖中的一种或多种。 在本发明的另一个 实例中, 所述碳源选自葡萄糖、 乳糖中的一种或两种。 通常, 以所述第二子培养基 的总重量计,所述碳源的含量为 0.5-2.8重量%,较好为 1.0-2.5重量%,更好为 1.5-2.0 重量%。
在所述第二种子培养基中,所述营养物质是常规的,本领域的普通技术人员根据 本发明的描述再结合现有技术可以直接知道哪些营养物质可用于本发明中。具体营养 物质的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述营养物质选自西 红柿、 泡菜汁、 酵母粉中的一种或多种。 在本发明的另一个优选实例中, 所述营养 物质选自西红柿、 酵母粉中的一种或多种。 通常, 以所述第二种子培养基的总重量 计, 所述营养物质的含量为 0.5-25重量%, 较好为 5-20重量%, 更好为 10-15重量 %。
在所述第二种子培养基中,所述溶剂是常规的,本领域的普通技术人员根据本发
明的描述再结合现有技术可以直接知道哪些溶剂可用于本发明中。具体溶剂的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述溶剂是生物学培养基中常用的 溶剂, 例如水 (纯水) 等。 发酵培养基
在上述步骤 (d) 中, 所述第一发酵培养基是常规的, 本领域的普通技术人员根 据本发明的描述再结合现有技术可知道哪些发酵培养基可用于发酵长型双歧杆菌。具 体的发酵培养基可参见 CN1119145C。
但是, 为了得到更加稳定的长型双歧杆菌, 优选使用特定的第一发酵培养基。在 本发明的一个优选实例中, 所述第一发酵培养基包含 1.1-6.5 重量%的氮源、 0.5-3 重量%的碳源、 0.6-1.5重量%的营养物质和 0.15-5重量%的矿物盐和 84.00-97.65 重量%的溶剂。 在本发明的一个 优选实例中, 所述第一发酵培养基包含:
在所述第一发酵培养基中,所述氮源是常规的,本领域的普通技术人员根据现有 技术可直接知道哪些具体氮源可用于本发明中。具体氮源的例子可参见 CN1119154C。 在本发明的一个优选例子中, 所述氮源选自脱脂奶粉、 蛋白胨、 胰胨、 铵盐中的一种 或多种。 在本发明的另一个优选实例中, 所述氮源选自脱脂奶粉、 硫酸铵中的一种或 两种。 通常, 以所述第一发酵培养基的总重量计, 所述氮源的含量为 1.1-6.5重量%,
较好为 1.5-5.5重量%, 更好为 2.0-5.0重量%, 最好为 3.0-4.0重量%。
在所述第一发酵培养基中,所述碳源是常规的,本领域的普通技术人员根据本发 明的描述再结合现有技术可以直接知道哪些碳源可用于本发明中。具体碳源的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述碳源选自葡萄糖、 乳糖、 异乳 糖、 半乳糖、 甘油、 山梨醇、 海藻糖、 麦芽糖中的一种或多种。 在本发明的另一个实 例中, 所述碳源选自葡萄糖、 异乳糖中的一种或两种。 通常, 以所述第一发酵培养基 的总重量计,所述碳源的含量为 0.8-2.8重量%,较好为 1.0-2.5重量%,更好为 1.5-2.0 重量%。
在所述第一发酵培养基中,所述营养物质是常规的,本领域的普通技术人员根据 本发明的描述再结合现有技术可以直接知道哪些营养物质可用于本发明中。具体营养 物质的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述营养物质是酵母 粉。 通常, 以所述第一发酵培养基的总重量计, 所述营养物质的含量为 0.6-1.5 重量 % , 较好为 0.7-1.2重量%, 更好为 0.8-1.0重量%。
在所述第一发酵培养基中, 所述矿物盐(矿物质)是常规的, 本领域的普通技术 人员根据本发明的描述再结合现有技术可以直接知道哪些矿物盐可用于本发明中。具 体的矿物盐的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述矿物盐选 自磷酸盐中的一种或多种。在本发明的另一个优选实例中, 所述矿物盐选自磷酸二氢 钾、 磷酸氢二钾中的一种或多种。 通常, 以所述第一发酵培养基的总重量计, 所述矿 物盐的含量为 0.15-5重量%,较好为 0.5-4.5重量%,更好为 1.0-3.0%,最好为 1.5-2.5 重量%。
在所述第一发酵培养基中, 所述溶剂是常规的, 本领域的普通技术人员根据本发明的 描述再结合现有技术可以直接知道哪些溶剂可用于本发明中。具体溶剂的例子可参见 CN1119145C。在本发明的一个优选实例中, 所述溶剂是生物学培养基中常用的溶剂, 例如水 (纯水) 等。
在上述步骤 (e) 和 (f) 中, 所述第二发酵培养基是常规的, 本领域的普通技术 人员根据本发明的描述再结合现有技术可知道哪些发酵培养基可用于本发明中发酵 嗜酸乳杆菌和粪链球菌。在本发明的一个优选实例中,所述第二发酵培养基含 1.1-6.5 重量%的氮源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量% 的溶剂, 所述第二发酵培养基包含:
400kg的配制量
西红柿汁 营养物质 50 kg
葡萄糖 碳源 6 kg
酵母粉 营养物质 4.3 kg
胰胨 氮源 16 kg
蛋白胨 氮源 3.6 kg
乳糖 碳源 1.3 kg
水 溶剂 余量 在所述第二发酵培养基中,所述氮源是常规的,本领域的普通技术人员根据现有 技术可直接知道哪些具体氮源可用于本发明中。具体氮源的例子可参见 CN1119154C。 在本发明的一个优选例子中, 所述氮源选自脱脂奶粉、 蛋白胨、 胰胨、 铵盐中的一种 或多种。 在本发明的另一个优选实例中, 所述氮源选自胰胨、 蛋白胨中的一种或两 种。 通常, 以所述第二发酵培养基的总重量计, 所述氮源的含量为 1.1-6.5 重量%, 较好为 1.5-5.5重量%, 更好为 2.0-5.0重量%, 最好为 3.0-4.0重量%。
在所述第二发酵培养基中,所述碳源是常规的,本领域的普通技术人员根据本发 明的描述再结合现有技术可以直接知道哪些碳源可用于本发明中。具体碳源的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述碳源选自葡萄糖、 乳糖、 异乳 糖、 半乳糖、 甘油、 山梨醇、 海藻糖、 麦芽糖中的一种或多种。 在本发明的另一个 实例中, 所述碳源选自葡萄糖、 乳糖中的一种或两种。 通常, 以所述第二发酵培养 基的总重量计,所述碳源的含量为 0.5-2.8重量%,较好为 1.0-2.5重量%,更好为 1.5-2.0 重量%。
在所述第二发酵培养基中,所述营养物质是常规的,本领域的普通技术人员根据 本发明的描述再结合现有技术可以直接知道哪些营养物质可用于本发明中。具体营养 物质的例子可参见 CN1119145C。 在本发明的一个优选实例中, 所述营养物质选自西 红柿、 泡菜汁、 酵母粉中的一种或多种。 在本发明的另一个优选实例中, 所述营养 物质选自西红柿、 酵母粉中的一种或多种。 通常, 以所述第二发酵培养基的总重量 计, 所述营养物质的含量为 0.5-25重量%, 较好为 5-20重量%, 更好为 10-15重量 %。
在所述第二发酵培养基中,所述溶剂是常规的,本领域的普通技术人员根据本发
明的描述再结合现有技术可以直接知道哪些溶剂可用于本发明中。具体溶剂的例子可 参见 CN1119145C。 在本发明的一个优选实例中, 所述溶剂是生物学培养基中常用的 溶剂, 例如水 (纯水) 等。
在上述步骤 (g) - (h) 中所述的第一保护剂和第二保护剂的定义如上述。 其他
所述三联活菌制剂可制成各种合适的剂型, 例如口服液、 片剂、 胶囊、 口崩片等 等。 在本发明的一个优选实例中, 所述剂型为胶囊。 在本发明的另一个优选实例中, 所述剂型为片剂。
在本发明中,所述三联活菌制剂可通过本领域中已知的方法制成各种剂型,例如 可根据 CN1119154C所述方法制成片剂、 胶囊、 颗粒剂等等。 例如:
1. 胶囊制造方法: 原辅料预处理、 烘粉、 原辅料混合过筛、 胶囊填充、 装瓶、 装箱;
2. 颗粒制造方法: 原辅料预处理、 制粒、 烘粉、 原辅料混合过筛、 散剂填充、 装 泡罩、 装单中盒、 装箱;
3. 片剂制造方法: 原辅料预处理、 制粒、 烘粉、 原辅料混合过筛、 压片、 内包装、 装盒、 装箱 实施例
西红柿汁的制备
准确称取果色艳丽, 果皮光亮、 肉质厚、 无柄、 无斑点、 无裂痕的西红柿(可根 据实际需要量决定) 。将上述经过初步筛选后的西红柿放入水池中洗干净后放置于干 净的筐中。 将洗净的西红柿依次放入粉碎机中粉碎后倒入加热灭菌锅中灭菌。
加热灭菌后的西红柿汁离心后即制备成西红柿汁。 实施例 1: 制备接种长型双歧杆菌的种子培养基
用 500ml 的纯水溶解下表 1 所示数量的葡萄糖 (购自河北圣雪葡萄糖有限公 司) 、 酵母粉 (德国 MERCK KGAA. ) 、 硫酸铵 (江苏永华精细化学品有限公司, AR级) 、 磷酸二氢钾 (汕头金砂化工厂有限公司, AR级) 、 磷酸氢二钾 (汕头金 砂化工厂有限公司, AR级) 和异乳糖 (意大利 INALCO S.P.A. ) , 得到溶液。 并 用 1500ml纯水将下表 1所示数量的脱脂奶粉(新西兰 FONTERRA LTD. )用匀浆机
(上海标本模型厂, DS-1 ) 打匀, 得到混悬液。
将上述溶液和混悬液经过胶体磨(温州鹿城乳化机械公司, JM-L80 )研磨均匀 后放至种子瓶中, 然后用饮用水加至 4000g配制成长型双歧杆菌的种子培养基, 再加 入少许稀氢氧化钠溶液调节培养基的 pH。
封好上述种子瓶口, 并将所述种子瓶在消毒锅中于 121 °C消毒 10-15分钟。
表 1
4000g的配制量
用 500ml的纯水溶解下表 2所示数量的葡萄糖 (河北圣雪葡萄糖有限公司) 、 酵母粉 (德国 MERCK KGAA. ) 、 乳糖 (上海申美医药开发科技有限公司) 、 西 红柿汁 (上海浦东新区金杨街道鑫鑫副食品经营部) , 得到溶液。 并用 1500ml纯 水将下表 2所示数量的胰胨(德国 MERCK KGAA. )和蛋白胨(日本制药株式会社) 用匀浆机 (上海标本模型厂, DS-1 ) 打匀, 得到混悬液。
将上述溶液和混悬液经过胶体磨研磨均匀后放至种子瓶中, 然后用饮用水加至
4000g配制成长型双歧杆菌的种子培养基, 再加入少许稀氢氧化钠溶液调节培养基的 pH。
封好上述种子瓶口, 并将所述种子瓶在消毒锅中于 121 °C消毒 10-15分钟。
表 2
4000g的配制:
将下表 3所示除碳酸钙以外的组分倒入不锈钢桶中, 再将 30千克饮用水倒入不 锈钢桶内, 同时用不锈钢棒搅拌, 得到培养基的混悬液。 将所得混悬液倒入胶体磨中 碾磨均匀。 将碾磨均匀的混悬液倒入装有搅拌机的发酵罐中, 用水补充至 400kg。 开 启发酵罐搅拌机,同时用 NaOH调节培养基的 pH 值,再加入下表 3所示的加碳酸钙, 同时用水加至 400kg, 得到发酵培养基。 将所述发酵培养基于 121 °C进行 10-15分钟 的消毒。 表 3
400kg的配制量
组分 处方 1 处方 2 处方 3 处方 4 处方 5
脱脂奶粉 4.4 kg 4.4 kg 20 kg 26 kg 26 kg 葡萄糖 2 kg 2 kg 9 kg 12 kg 12 kg
酵母粉 2.4 kg 6.2 kg 6.2 kg 6.2 kg 6 kg
硫酸铵 0.2 kg 2.3 kg 2.3 kg 2.3 kg 4 kg
磷酸二氢钾 0.1 kg 1.1 kg 1.1 kg 0.1 kg 2 kg 磷酸氢二钾 0.1 kg 4 kg 2.1 kg 0.1 kg 4 kg 碳酸钙 0.2 kg 10 kg 5.3 kg 0.2 kg 10kg 实施例 4: 制备嗜酸乳杆菌和 /或粪肠杆菌发酵培养基
将下表 4所示除西红柿汁以外的组分倒入不锈钢桶中, 再将 30千克饮用水倒入 不锈钢桶内, 同时用不锈钢棒搅拌, 得到培养基的混悬液。 将所得悬浮液倒入胶体磨 中碾磨均匀。将碾磨均匀的悬浮液倒入装有搅拌机的发酵罐中, 用水补充至总体积为 40升。 开启发酵罐搅拌机, 同时用 NaOH调节培养基的 pH 值, 再加入下表 4所示 的西红柿汁, 同时用水加至 400kg, 得到发酵培养基。 将所述发酵培养基于 121°C进 行 10-15分钟的消毒。 表 4
400kg的配制量
将长型双歧杆菌种子(购自中国生物制品检定所) 40ml分别置于装有实施例 1 中所得种子培养基 (处方 1-5 ) 的 5000ml玻璃血清瓶中, 于 37.0±2.0°C接种, 分别得到接种产物 Al-A5。
将上述接种产物 A1-A5分别置于装有实施例 3所得发酵培养基 (处方 1-5 )
的 600L发酵罐 (张家港江南化工机械厂) 中 37.0±2.0°C、 罐压 0.05±0.03MPa发 酵, 得到发酵产物 Bl-B5。
将上述发酵产物 B 1-B5分别通入上海离心机械研究所生产的 GQ142管式离 心机的离心管进行常温离心。 通入的发酵液体流量控制在 4000〜9000 毫升 /分钟, 离心 90-150分钟结束后取出离心管, 从离心管中挖出菌泥 C1-C5放入不锈钢桶。 实施例 6: 制备嗜酸乳杆菌菌泥
将嗜酸乳杆菌(购自中国生物制品检定所) 40ml分别置于装有实施例 2中所 得种子培养基 (处方 1-5 ) 的 5000ml玻璃血清瓶中 37.0±2.0°C接种, 分别得到接 种产物 Dl-D5。
将上述接种产物 D1-D5分别置于实施例 4所得发酵培养基 (处方 1-5 ) 600L 发酵罐 (张家港江南化工机械厂) 中 37.0±2.0°C、 罐压 0.05±0.03MPa发酵, 得到 发酵产物 El-E5。
将上述发酵产物 E1-E5分别通入上海离心机械研究所生产的 GQ142管式离心 机的离心管进行常温离心。 通入的发酵液体流量控制在 4000 9000 ml/min, 离心 40-80分钟结束后取出离心管, 从离心管中挖出菌泥 F1-F5放入不锈钢桶。 实施例 7: 制备粪链球菌菌泥
将粪链球菌(购自中国生物制品检定所) 40ml分别置于装有实施例 2中所得 种子培养基 (处方 1-5 ) 的 5000ml玻璃血清瓶中 37.0±2.0°C接种, 分别得到接种 产物 Gl-G5。
将上述接种产物 F1-F5分别置于装有实施例 4所得发酵培养基 (处方 1-5 ) 的 600L发酵罐 (张家港江南化工机械厂) 中 37.0±2.0°C、 罐压 0.05±0.03MPa发 酵, 得到发酵产物 Hl-H5。
将上述发酵产物 H1-H5分别通入上海离心机械研究所生产的 GQ142管式离 心机的离心管进行常温离心。 通入的发酵液体流量控制在 4000~9000 ml/min, 离 心 40〜80分钟结束后取出离心管, 从离心管中挖出菌泥 11-15放入不锈钢桶。 实施例 8: 制备长型双歧杆菌菌粉
在实施例 5所得菌泥 C1-C5中加入分别下表所示组成的保护剂 (其中菌泥
C1-C5中分别加入处方 1-处方 5的保护剂) , 同时用胶体磨循环研磨均匀。 将所 得均匀混合物放入冷冻干燥机 (北京天利科技, GLZ-6) 中进行冻干。 在冻干过 程中, 对板层进口温度进行降温预冻, 使产品预冻到 -40°C左右。 预冻后, 开冷凝 使冷阱降至 -40°C, 然后开真空泵对箱体进行抽空。 箱体真空达到真空设定值 ( 15pa)后,开始对产品进行升温。当产品颜色一致时,冻干结束,即得菌粉 Jl-J5。
在实施例 6所得菌泥 F1-F5中加入分别下表所示组成的保护剂 (其中菌泥
F1-F5中分别加入处方 1-处方 5的保护剂) , 同时用胶体磨循环研磨均匀。 将所 得均匀混合物放入冷冻干燥机 (北京天利科技, GLZ-6) 中进行冻干。 在冻干过 程中, 对板层进口温度进行降温预冻, 使产品预冻到 -40°C左右。 预冻后, 开冷凝 使冷阱降至 -40°C, 然后开真空泵对箱体进行抽空。 箱体真空达到真空设定值 ( 15pa)后,开始对产品进行升温。当产品颜色一致时,冻干结束,即得菌粉 Kl-K5。 表 6
组分 处方 1 处方 2 处方 3 处方 4 处方 5
奶粉 1.6kg 2kg 2.4kg 3.2kg 4kg 谷氨酸钠 400g 480g 560g 640g 720g 异乳糖 3.2kg 4kg 2.8kg 2.4kg 1.44kg
Vc-Na 400g 320g 240g 160g 80g 淀粉 2.4kg 1.2kg 2kg 1.6kg 1.76kg
实施例 10: 制备粪链球菌菌粉
在实施例 7所得菌泥 11-15中加入分别下表所示组成的保护剂 (在菌泥 11-15 中分别加入处方 1-处方 5的保护剂) , 同时用胶体磨循环研磨均匀。 将所得均匀 混合物放入冷冻干燥机 (北京天利科技, GLZ-6) 中进行冻干。 在冻干过程中, 对板层进口温度进行降温预冻, 使产品预冻到 -40°C左右。 预冻后, 开冷凝使冷阱 降至 -40°C, 然后开真空泵对箱体进行抽空。箱体真空达到真空设定值(15pa)后, 开始对产品进行升温。 当产品颜色一致时, 冻干结束, 即得菌粉 Ll-L5。 表 7
按下表所示数量的组分投入到多向运动混合机 (HDA-600, 温州市制药设备 厂) 中混合 30分钟, 得到均匀混合物。 使用胶囊填充机(浙江富昌机械有限公司 全自动胶囊充填机, NJP-2000B ) 制得胶囊 M1-M5各 60万粒, 其中胶囊 Mi包含 长型双歧杆菌菌粉 Ji、嗜酸乳杆菌菌粉 Ki和粪链球菌菌粉 Li, i为 1到 5的整数。 表 8
公斤数
长型双歧杆菌菌粉 J1-J5 (>10x l08CPU/g) 12.0kg
嗜酸乳杆菌菌粉 K1-K5 (>5.0x l08CPU/g ) 6.0kg
粪链球菌菌粉 L1-L5 (>5.0x l08CPU/g) 3.0kg
预胶化淀粉 52.5 kg
乳糖 52.5kg
实施例 12: 制备三联活菌散剂
将下表所示的组分投入多向运动混合机(HDA-600, 温州市制药设备厂) 中混 合 30-40, 得到均匀混合物。 开启散剂填充机 (上海松川包装机械厂多列包装机, SL-8 ) 将所述均匀混合物制成散剂 N1-N5各 10万支, 散剂 Ni包含长型双歧杆菌 菌粉 Ji、 嗜酸乳杆菌菌粉 Ki和粪链球菌菌粉 Li, i为 1到 5的整数。
其中空白颗粒如下制备: 将下表 10所示的组分喷雾制粒后, 经 100°C烘箱干 燥。 表 9
表 10
散剂空白制粒处方
用 量
脱脂奶粉 70kg
麦芽糊精 21kg
异乳糖 14kg
HPMC (2910 ) 0.3% 150g
95%乙醇 25kg
水 25kg
实施例 13: 稳定性试验
将下述菌粉分别用铝塑袋抽真空封口包藏,置于相对应的温度中,每月取出 保存温度: 2-8 °C
保存时间 Jl Kl L1
0 5.7χ109 7.8χ109 7.1χ109
3月 6.2χ109 4.5χ109 6.3χ109
6月 4.1χ109 3.0χ109 5.0χ109
12月 2.1χ109 3.7χ109 Ι.ΟχΙΟ9
24月 8.9χ107 2.4χ108 1.9χ108 保存温度: 2-8 °C
保存时间 J2 K2 L2
0 6.1x109 7.1x109 5.9x109
3月 3.1x109 7.7x109 6.7x109
6月 2.0x109 6.7x109 3.8x109
12月 9.8x108 5.0x109 8.8x108
24月 3.0x108 1.7x108 7.6x107
保存温度: 2-8 °C
保存时间 J5 L5 L5
0 3.8χ109 8.2χ109 7.9χ109
3月 4.4χ109 7.6χ109 8.0χ109
6月 3.0χ109 3.8χ109 6.7χ109
12月 1.3χ109 8.7χ108 7.8χ108
24月 1.2χ108 3.7χ108 2.0χ108
保存温度: 25 °C 保存时间 Jl Kl L1
0 5.7χ109 7.8χ109 7.1χ109
3月 6.2χ109 4.5χ109 6.3χ109
6月 8.2χ108 Ι.ΟχΙΟ9 4.7χ108
12月 2.3χ108 3.2χ108 1.8χ108
18月 7.7χ107 8.3χ107 7.8χ107 保存温度: 25 °C 保存时间 J2 K2 L2
0 6.1χ109 7.1χ109 5.9χ109
3月 3.1χ109 7.7χ109 6.7χ109
6月 7.7χ108 8.3χ108 5.0χ108
12月 3.5χ108 1.9χ108 2.0χ108
18月 8.0χ107 5.6χ107 4.7χ107 保存温度: 25 °C 保存时间 J5 L5 L5
0 3.8χ109 8.2χ109 7.9χ109
3月 4.4χ109 7.6χ109 8.0χ109
6月 3.9χ108 5.0χ108 8.1χ108
12月 Ι.ΟχΙΟ8 8.8χ107 1.3χ108
18月 5.7χ107 7.8χ107 6.7χ107
保存温度: 37°C 保存时间 Jl Kl LI
0 5.7χ109 7.8χ109 7.1χ109
1月 1.7χ109 3.1χ109 30χ109
2月 2.3χ108 7.8χ108 6.7χ108
3月 1.8χ107 1.5χ108 9.7χ107
保存温度: 37°C
保存时间 J2 K2 L2
0 6.1χ109 7.1χ109 5.9χ109
1月 8.3χ108 7.8χ108 4.6χ108
2月 3.0χ108 4.7χ108 3.9χ108
3月 5.6χ107 3.7χ107 4.5χ107 保存温度: 37°C
保存时间 J5 L5 L5
0 3.8χ109 8.2χ109 7.9χ109
1月 8.9χ108 Ι.ΙχΙΟ9 9.7χ108
2月 1.7χ108 5.1χ108 9.8χ107
3月 Ι.ΙχΙΟ8 5.8χ107 3.4χ107 对比例 1-3
根据 CN1119154C的实施例 1-3分别制备微生物制剂, 即对比例 1-3。用与实施例
13相同的方法测试对比例 1-3所得微生物制剂中的长型双歧杆菌、嗜酸乳杆菌和粪肠 杆菌的稳定性, 结果如下:
+ 保存温度: 2-8°C (对比例 1)
保存时间 B L S
0 3.3χ109 3.0χ109 3.0χ109
3月 2.0χ109 1.7χ109 2.4χ109
6月 5.7x10s 8.0x10s Ι.ΟχΙΟ9
12月 Ι.ΟχΙΟ8 4.1χ108 7.0χ109
24月 Ι.όχΙΟ7 6.0χ107 Ι.ΙχΙΟ7
保存温度: 2-8°C (对比例 2) 保存时间 B L S
0 1.3χ109 8.0χ109 2.0χ109
3月 Ι.ΟχΙΟ9 6.4χ109 Ι.ΟχΙΟ9
6月 7.9χ108 3.7χ109 9.2χ108
12月 3.0χ108 Ι.ΟχΙΟ9 6.5χ108
24月 8.8χ106 1.7χ107 7.1χ107 保存温度: 2-8°C (对比例 3) 保存时间 B L S
0 2.0χ1010 Ι.ΟχΙΟ10 3.1χ109
3月 Ι.ΟχΙΟ10 8.3χ109 Ι.ΟχΙΟ9
6月 8.0χ109 5.4χ109 7.4χ108
12月 8.0χ108 2.0χ109 4.0χ108
24月 3.6χ107 4.5χ107 9.0χ106 保存温度: 25 °C (对比例 1) 保存时间 B L S
0 3.3χ109 3.0χ109 3.0χ109
6月 5.6χ108 4.1χ108 7.0χ108
12月 7.2χ107 8.1χ107 Ι.ΟχΙΟ8
18月 Ι.ΟχΙΟ7 2.6χ107 5.1χ107
保存温度: 25 °C (对比例 2) 保存时间 B L S
0 1.3χ109 8.0χ109 2.0χ109
6月 3.0x10s Ι.ΟχΙΟ9 7.6χ108
12月 5.0χ107 4.6χ107 2.3χ108
18月 8.6χ106 Ι.ΟχΙΟ7 7.1χ107
保存温度: 25 °C (对比例 3)
保存时间 B L S
0 2.0χ1010 Ι.ΟχΙΟ10 3.1χ109
6月 7.0χ109 2.8χ109 5.7χ108
12月 2.1χ108 8.7χ108 Ι.ΟχΙΟ8
18月 5.6χ107 4.0χ108 4.0χ107 保存温度: 37°C (对比例 1)
保存时间 B L S
0 3.3χ109 3.0χ109 3.0χ109
1月 5.7χ108 8.0χ108 7.0χ109
2月 Ι.ΟχΙΟ7 2.3χ107 1.7χ107
3月 6.3χ106 5.2χ106 4.7χ106 保存温度: 37°C (对比例 2)
保存时间 B L S
0 1.3χ109 8.0χ109 2.0χ109
1月 6.6χ108 7.3χ108 3.7χ108
2月 4.5χ107 6.0χ107 4.8χ107
3月 6.4χ106 7.0χ106 5.8χ106 保存温度: 37°C (对比例 3)
保存时间 B L S
0 2.0χ1010 Ι.ΟχΙΟ10 3.1χ109
1月 8.0χ109 8.4χ109 6.0x10s
2月 7.1χ108 6.1χ108 5.1χ107
3月 6.2χ107 5.9χ107 7.0χ106
其中, Β表示长型双歧杆菌, L表示嗜酸乳杆菌, S表示粪链球菌。 实施例 14: 三联活菌制剂的应用
使功能性便秘患者口服实施例 1所制得的胶囊 Ml, 每天三次, 每次
得结果如下:
1 ) 有效性结果:
本研究共入组病例 141例。 结果显示: 功能性便秘患者服用胶囊 Ml持续一个 月后, 日均排便次数较治疗前 (0.30, 0.35 ) 显著增多至 0.63次 /日, P<0.0001, 2 周治疗后, 周 Bristol分型均值显著改善 (0.61, 0.63至 1.76) , P<0.0001。
便秘的伴随症状疗效评价: 在治疗 1个月后便秘伴随症状改善有效率为 46.2 % , 在治疗 3个月后伴随症状改善有效率为 81.5 %。
对单个症状改善情况的分析显示: 服用胶囊 Ml持续一个月后单个症状积分较 治疗前显著降低 (PO.05 ) 。
对便秘常见症状的总积分改善情况分析显示: 功能性便秘患者服用胶囊 Ml持 续一个月后总积分较治疗前显著下降 (PO.001 ) 。
2) 安全性结果: 无不良反应发生。 实施例 15: 三联活菌制剂的应用
本实施例描述了本发明三联活菌制剂治疗腹泻的效果。
A. 入选病例
1. 成人急性腹泻:
( 1 ) 轻型或者普通型急性菌痢:
*轻型一体温 38°C以下, 临床症状不明显, 腹泻次数不超过 4次 -5次 /日, 大便 稀烂, 肉眼不见脓血, 培养阳性。
*普通型一体温 38-39°C, 白血球 <20000/ml, 有轻度里急后重, 大便 5-15次 / 日, 带粘液或脓血, 神志清醒, 无嗜睡、 惊厥, 心血管功能良好, 呼吸平稳。
(2 ) 原因不明急性腹泻 (包括细菌性和病毒性等) : 起病急, 有不洁饮食史, 腹泻水便> 3次 /日, 大便外观无脓血, 镜检高倍视野白细胞 < 15个。
2. 成人慢性腹泻: 大便 >2-3次 /日, 大便性状不正常, 持续 2-3个月以上, 反复发作以及近期又犯者。
3. 成人便秘: 大便 >2-3次 /天, ***干结, 且有排便困难, 常需依靠开塞露 或润肠剂, 症状持续 1周以上者。
4. 小儿各种病因的急、 慢性腹泻 (感染性或非感染性) 或便秘之 0+-13周岁 门诊或住院病儿。
B. 研究方法
病人于治疗前停用其它药物 (抗生素、 激素、 其它止泻药或润肠剂) , 将实施 例 12所得的胶囊 M2让患者服用, 剂量如下: 成人每次 5粒, 2-3次 /日; 0+-1周 岁的儿童每次 1粒, 3次 /日; 1-6周岁的儿童每次 2粒, 3次 /日; 6-13周岁的儿 童每次 3粒, 3次 /日。 根据卡方检验和 Ridit分析进行统计学处理。
C. 结论:
1. 用实施例 12所得胶囊 M2治疗成人或儿童轻、 中型的急性腹泻和原因不明 的慢性腹泻共 401例。 总疗效为 87.5 %。 另外, 胶囊 M2治疗急性腹泻的效果优 于治疗慢性腹泻(二者疗效相比有显著性差异13< 0.05 ) 。 治疗成人和儿童急性腹 泻的总有效率分别为 92.3 %和 90.7 % ; 而对慢性腹泻的总有效率则分别为 80.0 % 和 76.3 %。
2. 胶囊 M2除了可用于治疗原因不明的腹泻外, 尚对感染性腹泻 (病原包括: 痢疾杆菌、 沙门氏菌、 致病大肠菌、 空肠弯曲菌、 气单胞菌及轮状病毒等) 有疗 效。 其中 401例病例中有 52例治疗前检査出病原菌 (细菌类) , 有 8例检査出轮 状病毒, 治疗有 52例中有 50例 (96.2 % ) 转阴, 8例轮状病毒性肠炎患者治疗后 全部显效。
3. 胶囊 M2对慢性腹泻的疗效虽不及急性腹泻, 但确有一定效果。 慢性腹泻 患者试验前均有不同程度的服过其它药物而无明显效果, 或长期被诊断为 "肠激 惹综合症" , 服用胶囊 M2后 80.0 %的成人和 76.3 %的儿童患者痊愈。
4. 胶囊 M2有调整肠道菌群的功能。 从对 19例成人和 16例儿童腹泻患者治 疗前后主要菌群的检测结果看, 治疗前病人都不同程度的存在肠菌群紊乱, 主要 表现在: 肠杆菌需氧或兼性厌氧菌偏高, 双歧杆菌等厌氧菌偏低, 治疗后随着临 床症状的消失上述状况得到改善, 肠菌群恢复正常。
5. 未发现服用胶囊 M2后有明显副作用。
Claims
1. 一种三联活菌制剂, 它包括:
长型双歧杆菌菌粉;
嗜酸乳杆菌菌粉;
粪链球菌菌粉,
所述长型双歧杆菌菌粉包含长型双歧杆菌和第一保护剂,所述第一保护剂包含脱 脂奶粉 8-50%、 谷氨酸钠 0.01-10%、 异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 5-30%, 以所 述第一保护剂的总重量计,
所述嗜酸乳杆菌菌粉包含嗜酸乳杆菌菌粉包含嗜酸乳杆菌和第二保护剂,所述粪 链球菌菌粉包含粪链球菌和第二保护剂, 所述第二保护剂包含脱脂奶粉 6-70%、 谷氨 酸钠 0.01-10%、异乳糖 8-50%、 Vc-Na0.01-5% 淀粉 4-30%, 以所述第二保护剂的总重
2. 一种保护剂, 所述保护剂包含包含脱脂奶粉 6-70%、 谷氨酸钠 0.01-10%、 异 乳糖 8-50%、 Vc-Na0.01-5% 淀粉 4-30%, 以所述保护剂的总重量计。
3. 一种制造权利要求 1所述三联活菌制剂的方法, 所述方法包括:
(a) 将长型双歧杆菌在第一种子培养基中接种, 得到长型双歧杆菌接种产物;
(b) 将嗜酸乳杆菌在第二种子培养基中接种, 得到嗜酸乳杆菌接种产物;
(c) 将粪链球菌在第二种子培养基中接种, 得到粪链球菌接种产物;
(d)将步骤(a)所得长型双歧杆菌接种产物置于第一发酵培养基中发酵, 得到 长型双歧杆菌发酵产物;
(e)将步骤(b)所得嗜酸乳杆菌接种产物置于第二发酵培养基中发酵, 得到嗜 酸乳杆菌发酵产物;
(f)将步骤 (c)所得粪链球菌接种产物置于第二发酵培养基中发酵, 得到粪链 球菌发酵产物;
(g) 混合长型双歧杆菌与第一保护剂, 冻干得到长型双歧杆菌菌粉;
(h) 将嗜酸乳杆菌和粪链球菌分别与第二保护剂混合, 分别冻干得到嗜酸乳杆 菌菌粉和粪链球菌菌粉;
(i) 混合上述长型双歧杆菌菌粉、 嗜酸乳杆菌菌粉和粪链球菌菌粉得到三联活 菌制剂。
4. 如权利要求 3 所述的方法, 其特征在于, 所述第一种子培养基包含 1.1-6.5 重量%的氮源、 0.5-3重量%的碳源、 0.6-1.5重量%的营养物质和 0.15-5重量%的 矿物盐和 84.00-97.65重量%的溶剂。
5. 如权利要求 3 所述的方法, 其特征在于, 所述第二种子培养基包含 1.1-6.5 重量%的氮源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量% 的溶剂。
6. 如权利要求 3所述的方法, 其特征在于, 所述第一发酵培养基包含 1.1-6.5 重量%的氮源、 0.5-3重量%的碳源、 0.6-1.5重量%的营养物质和 0.15-5重量%的 矿物盐和 84.00-97.65重量%的溶剂。
7. 如权利要求 4所述的方法, 其特征在于, 所述第二发酵培养基包含 1.1-6.5 重量%的氮源、 0.2-3重量%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量% 的溶剂。
8. 一种种子培养基, 所述种子培养基含 1.1-6.5重量%的氮源、 0.2-3重量%的 碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。
9. 一种发酵培养基, 所述发酵培养基包含 1.1-6.5重量%的氮源、 0.2-3重量
%的碳源、 0.5-25重量%的营养物质和 65.5-98.2重量%的溶剂。
10. 权利要求 1所述的三联活菌制剂在治疗便秘中的用途。
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CN103540550A (zh) * | 2013-10-26 | 2014-01-29 | 兰亚朝 | 乳酸粪链球菌的发酵方法 |
CN114891685A (zh) * | 2022-05-30 | 2022-08-12 | 微康益生菌(苏州)股份有限公司 | 一种提高嗜酸乳杆菌抗逆性的组合物及其制备方法和应用 |
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CN101244089A (zh) | 2008-08-20 |
US9040037B2 (en) | 2015-05-26 |
CN101244089B (zh) | 2013-09-25 |
US20100119487A1 (en) | 2010-05-13 |
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