CN111528478A - Compound beneficial microbial preparation for treating constipation and preparation method thereof - Google Patents
Compound beneficial microbial preparation for treating constipation and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 73
- 230000000813 microbial effect Effects 0.000 title claims abstract description 53
- 206010010774 Constipation Diseases 0.000 title claims abstract description 38
- 230000009286 beneficial effect Effects 0.000 title claims abstract description 29
- 150000001875 compounds Chemical class 0.000 title claims abstract description 29
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 40
- 241000186660 Lactobacillus Species 0.000 claims abstract description 39
- 229940039696 lactobacillus Drugs 0.000 claims abstract description 39
- 238000001694 spray drying Methods 0.000 claims abstract description 14
- 240000005373 Panax quinquefolius Species 0.000 claims abstract description 11
- 235000003140 Panax quinquefolius Nutrition 0.000 claims abstract description 11
- 229940076742 senna leaves Drugs 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 6
- 244000144725 Amygdalus communis Species 0.000 claims abstract description 4
- 235000003893 Prunus dulcis var amara Nutrition 0.000 claims abstract description 4
- 239000000706 filtrate Substances 0.000 claims description 98
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 49
- 239000000284 extract Substances 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- 238000000855 fermentation Methods 0.000 claims description 29
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- 238000001704 evaporation Methods 0.000 claims description 28
- 238000001914 filtration Methods 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 28
- 239000000843 powder Substances 0.000 claims description 28
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- 238000000605 extraction Methods 0.000 claims description 19
- 238000001035 drying Methods 0.000 claims description 15
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- 240000003183 Manihot esculenta Species 0.000 claims description 14
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 238000012807 shake-flask culturing Methods 0.000 claims description 14
- 230000009965 odorless effect Effects 0.000 claims description 9
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- 239000001116 FEMA 4028 Substances 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 241000170793 Phalaris canariensis Species 0.000 claims description 7
- 244000018633 Prunus armeniaca Species 0.000 claims description 7
- 235000009827 Prunus armeniaca Nutrition 0.000 claims description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 230000003698 anagen phase Effects 0.000 claims description 7
- 235000015278 beef Nutrition 0.000 claims description 7
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 7
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 7
- 229960004853 betadex Drugs 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 235000013312 flour Nutrition 0.000 claims description 7
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 7
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 238000007873 sieving Methods 0.000 claims description 7
- 235000020183 skimmed milk Nutrition 0.000 claims description 7
- 239000002002 slurry Substances 0.000 claims description 7
- 235000017281 sodium acetate Nutrition 0.000 claims description 7
- 239000001632 sodium acetate Substances 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 239000007921 spray Substances 0.000 claims 1
- 210000001124 body fluid Anatomy 0.000 abstract description 2
- 239000010839 body fluid Substances 0.000 abstract description 2
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- 230000009967 tasteless effect Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000013872 defecation Effects 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 239000002775 capsule Substances 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
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- 208000009531 Fissure in Ano Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 241000522641 Senna Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229960002983 loperamide hydrochloride Drugs 0.000 description 1
- PGYPOBZJRVSMDS-UHFFFAOYSA-N loperamide hydrochloride Chemical compound Cl.C=1C=CC=CC=1C(C=1C=CC=CC=1)(C(=O)N(C)C)CCN(CC1)CCC1(O)C1=CC=C(Cl)C=C1 PGYPOBZJRVSMDS-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
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- 229940124513 senna glycoside Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
-
- 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
Abstract
The invention relates to a microbial preparation, and particularly discloses a compound beneficial microbial preparation for treating constipation and a preparation method thereof. According to the mass parts, 5-7 parts of senna leaves, 10-20 parts of bitter almonds, 10-20 parts of American ginseng and 8 parts of I type microbial preparation are prepared, wherein the I type microbial preparation is prepared by performing strain culture on 5 parts of bacillus and 4 parts of anaerobic lactobacillus and then performing spray drying. The invention has the functions of relaxing bowels, tonifying qi and promoting the secretion of saliva or body fluid, establishes a basic process by combining the related technology of modern microbial preparations, has reasonable, feasible, stable and reliable method, can effectively control the quality of the preparation, and lays a foundation for developing the preparation into health-care food.
Description
Technical Field
The invention relates to a microbial preparation, and particularly discloses a compound beneficial microbial preparation for treating constipation and a preparation method thereof.
Background
Constipation is a common clinical multiple disorder that is generally considered a clinical symptom rather than a disease and has developed into a global public health problem. The constipation patients mainly have the symptoms of difficult defecation, dry feces, water shortage, prolonged defecation period and the like. According to the study, the general patients with constipation are 12-19%. And with the increase of age, the prevalence rate of constipation is also improved correspondingly; generally, women have a higher prevalence than men. Constipation persists for a long time, and a series of complications can occur, such as: anal fissure, rectal prolapse, hemorrhoids, autointoxication and the like can cause a series of problems including physiological, psychological and social aspects of patients and have great influence on the life of the patients.
The intestinal tract is not only an important system for human digestion and absorption, but also the largest immune barrier and nervous system. Unlike the brain center, the gut must rely on gut microbes to perform normal physiological functions. As the largest and most complex microecosystem of human body, the intestinal microorganisms and metabolites thereof can regulate the health of the intestinal tract, so that the invention of a compound beneficial microbial preparation for treating constipation is necessary.
Disclosure of Invention
The invention aims to select proper strains and a processing technology to provide a compound beneficial microbial preparation for treating constipation, the preparation takes bacillus and anaerobic lactobacillus as active components, and the total bacterial number of the product is not less than 1 × 1010cfu/g, average particle diameter 10 μm.
The invention aims to provide a compound beneficial microbial preparation for treating constipation, which comprises the following raw materials in parts by weight: 5-7 parts of senna leaves, 10-20 parts of bitter apricot seeds, 10-20 parts of American ginseng and 8 parts of type I microbial preparation.
Another objective of the present invention is to provide a method for preparing a compound beneficial microbial preparation for treating constipation, comprising the following steps:
step one, culture and preparation of strains
1. Seed culture medium: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus, 4 parts of anaerobic lactobacillus and a slant culture medium are taken for culture for 2d at 37 ℃;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into a 500mL shake flask, placing the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm and maintaining the culture temperature at 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a seed culture medium under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, crushing the dried senna leaves, adding 90 percent ethanol by mass for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the concentrated filtrate in an evaporating dish until no alcohol smell exists in a water bath kettle, and drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, crushing the dried bitter almond, adding water at 80 ℃ for 3h, filtering by double-layer gauze, combining the filtrates, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is odorless in a water bath, then concentrating the filtrate at 60 ℃ and 0.01MPa under reduced pressure to a certain volume, and putting the concentrated filtrate into the evaporation dish to obtain dry extract;
step four, crushing dried American ginseng, adding 90% by mass of ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.2 MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the concentrated filtrate at 60 ℃ and-0.2 MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, and preparing 90% by mass of ethanol into particles;
and step five, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding the I-type microbial preparation to prepare the compound beneficial microbial preparation for treating constipation.
Compared with the prior art, the health-care product has the functions of relaxing the bowels, tonifying qi and promoting the secretion of saliva or body fluid and has the health-care function for constipation patients. According to the physicochemical properties and the design process requirements, the invention adds I type microbial preparation of bacillus and anaerobic lactobacillus into three traditional Chinese medicines of senna leaf, bitter apricot seed and American ginseng; the experimental result shows that the bacillus can produce various digestive enzymes to help constipation patients to digest and absorb nutrient substances, is high-temperature resistant, is suitable for being added into foods with higher temperature, and establishes a basic process by combining the related technology of modern microbial preparations.
Detailed Description
The invention is further described with reference to specific examples, but the scope of the invention is not limited thereby.
The materials and equipment used in the following examples are commercially available.
Example 1
A preparation method of a compound beneficial microbial preparation for treating constipation comprises the following steps:
step one, culture and preparation of strains
1. Seed culture medium: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into 500mL sterilized shake flask, placing the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm and maintaining the culture temperature at 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a seed culture medium under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, taking 7 parts of dried senna leaves according to the mass part, crushing, adding 90 percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and then drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, crushing 10 parts of dried bitter apricot kernels according to the mass part, adding 80 ℃ water for extraction for 3h, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.02 MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is tasteless, then concentrating the filtrate at 60 ℃ and-0.01 MPa under reduced pressure to a certain volume, and putting the concentrated filtrate into the evaporation dish to obtain dry extract;
step four, crushing 10 parts of dried American ginseng according to the mass part, adding 90% ethanol for extraction for 3 times, 2 hours each time, filtering by using double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.2MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the filtrate at 60 ℃ and 0.2MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, and preparing the ethanol with the mass percent of 90% into particles;
and step five, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding 8 parts of the I-type microbial preparation according to the mass part to prepare the compound beneficial microbial preparation for treating constipation.
Example 2
A preparation method of a compound beneficial microbial preparation for treating constipation comprises the following steps:
step one, culture and preparation of strains
1. Culture medium for seed culture: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into 500mL sterilized shake flask, placing the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm and maintaining the culture temperature at 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a seed culture medium under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, taking 7 parts of dried senna leaves according to the mass part, crushing, adding 90 percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and then drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, taking 20 parts of dried bitter apricot kernels according to the mass part, crushing, adding 80 ℃ water for extraction for 3h, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.02 MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is tasteless, then concentrating the filtrate at 60 ℃ and-0.01 MPa under reduced pressure to a certain volume, and putting the filtrate into the evaporation dish to obtain dry extract;
step four, taking 20 parts of dried American ginseng according to the mass part, adding 90% ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.2MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the filtrate at 60 ℃ and 0.2MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, granulating by 90% ethanol by mass, and preparing into capsules;
and step five, crushing the capsules by using an ultrafine crusher, sieving the crushed capsules by using a 300-mesh sieve, and adding 8 parts of the I-type microbial preparation according to the mass part to prepare the compound beneficial microbial preparation for treating constipation.
Example 3
A preparation method of a compound beneficial microbial preparation for treating constipation comprises the following steps:
step one, culture and preparation of strains
1. Culture medium for seed culture: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into a 500mL sterilized shake flask, putting the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm and maintaining the culture temperature at 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a culture medium for seed culture under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, crushing 5 parts of dried senna leaves according to the mass part, adding 90 percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and then drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, taking 15 parts of dried bitter apricot kernels according to the mass part, crushing, adding 80 ℃ water for extraction for 3h, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is tasteless, then concentrating the filtrate at 60 ℃ and 0.01MPa under reduced pressure to a certain volume, and putting the concentrated filtrate into the evaporation dish to obtain dry extract;
step four, taking 15 parts of dried American ginseng according to the mass part, adding 90% ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.2MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the filtrate at 60 ℃ and 0.2MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, and preparing the ethanol with the mass percent of 90% into particles;
and step five, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding 8 parts of the I-type microbial preparation according to the mass part to prepare the compound beneficial microbial preparation for treating constipation.
Example 4
A preparation method of a compound beneficial microbial preparation for treating constipation comprises the following steps:
step one, culture and preparation of strains
1. Culture medium for seed culture: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into 500mL sterilized shake flask, placing the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm at a culture temperature of 37 ℃; (ii) a
c. Seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a culture medium for seed culture under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, taking 6 parts of dried senna leaves according to the mass part, crushing, adding 90 percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and then drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, taking 20 parts of dried bitter apricot kernels according to the mass part, crushing, adding 80 ℃ water for extraction for 3h, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.02 MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is tasteless, then concentrating the filtrate at 60 ℃ and-0.01 MPa under reduced pressure to a certain volume, and putting the filtrate into the evaporation dish to obtain dry extract;
step four, crushing 12 parts of dried American ginseng according to the mass part, adding 90% ethanol for extraction for 3 times, 2 hours each time, filtering by using double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.2MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the filtrate at 60 ℃ and 0.2MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, and preparing the ethanol with the mass percent of 90% into particles;
and step five, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding 8 parts of the I-type microbial preparation according to the mass part to prepare the compound beneficial microbial preparation for treating constipation.
Example 5
A preparation method of a compound beneficial microbial preparation for treating constipation comprises the following steps:
step one, culture and preparation of strains
1. Culture medium for seed culture: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 g of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 1 part of water;
2. fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
3. strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into 500mL sterilized shake flask, placing the shake flask on a shaking table, starting a switch, and culturing for 2d under a shaking environment of 200rpm and maintaining the culture temperature at 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a culture medium for seed culture under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: spray drying at inlet temperature of 120-150 ℃ and outlet temperature of 65-75 ℃ to generate the powdery I type microbial preparation containing bacillus and anaerobic lactobacillus with average grain diameter of 10 mu m.
Step two, taking 7 parts of dried senna leaves according to the mass part, crushing, adding 90 percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and then drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
step three, crushing 14 parts of dried bitter almond according to the mass parts, adding 80 ℃ water for extraction for 3h, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is tasteless, then concentrating the filtrate at 60 ℃ and 0.01MPa under reduced pressure to a certain volume, and putting the concentrated filtrate into the evaporation dish to obtain dry extract;
step four, taking 20 parts of dried American ginseng according to the mass part, adding 90% ethanol for extraction for 3 times, 2 hours each time, filtering by using double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and 0.2MPa under reduced pressure, putting the filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the filtrate at 60 ℃ and 0.2MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, mixing the dry extract with the two dry extracts uniformly, adding beta-cyclodextrin, and preparing the ethanol with the mass percent of 90% into particles;
and step five, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding 8 parts of the I-type microbial preparation according to the mass part to prepare the compound beneficial microbial preparation for treating constipation.
Drug effect test of compound beneficial microbial preparation for treating constipation
1. Design of animal experiments
40 male SPF-grade BALB/c mice at 7 weeks of age were divided into blank and example groups, each group consisting of 10 mice. After 7 days of adaptation, the blank group is filled with sterile normal saline every day, the mice in the example group are filled with loperamide hydrochloride to make the mice constipation, after 1 hour, the blank group is filled with sterile normal saline, and the example group is filled with a compound beneficial microbial preparation normal saline suspension for treating the constipation, wherein the volume of each gastric administration is 0.3 mL.
The preparation method of the physiological saline suspension of the compound beneficial microbial preparation comprises the following steps: the compound beneficial microbial preparation for treating constipation prepared in each example is diluted by taking 1 part of the compound beneficial microbial preparation in 100 parts of normal saline.
2. Results
The results of the effects of the compound beneficial microbial preparation for treating constipation of the invention on the number of the mouse excrement particles, the weight and the water content are shown in table 1, and the effects of the example groups on the number of the constipation particles, the weight of the excrement and the water content of the excrement of the constipation-treated mice tend to be enhanced along with the prolonged administration time, so that the formula selected by the invention can be used for relieving constipation.
TABLE 1 number of particles for defecation in 1 st, 7 th and 14d mice in each group
TABLE 2 3h defecation weight of groups 0, 7, 14d mice
TABLE 3 moisture content of feces from groups 0, 7 and 14d mice
Note: the letters a-c in the same row indicate significant differences between groups (p <0.05)
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (3)
1. The compound beneficial microbial preparation for treating constipation is characterized by comprising, by mass, 5-7 parts of folium sennae, 10-20 parts of bitter apricot seeds, 10-20 parts of American ginseng and 8 parts of type I microbial preparation.
2. A preparation method of a compound beneficial microbial preparation for treating constipation is characterized by comprising the following steps:
step one, crushing dried senna leaves, adding 90 mass percent ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, concentrating the filtrate at 60 ℃ and 0.02MPa under reduced pressure, placing the filtrate in an evaporating dish until no alcohol smell exists in a water bath, and drying at 60 ℃ and 0.02MPa under reduced pressure to obtain dry extract;
crushing dried bitter almonds, adding water at 80 ℃ for 3h, filtering by using double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.02 MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is odorless in a water bath, then concentrating the concentrated filtrate at 60 ℃ and-0.01 MPa under reduced pressure to a certain volume, and putting the concentrated filtrate into the evaporation dish to obtain dry extract;
step three, crushing dried American ginseng, adding 90% by mass of ethanol for extraction for 3 times, 2 hours each time, filtering by double-layer gauze, combining filtrates, concentrating the filtrate at 60 ℃ and-0.2 MPa under reduced pressure, putting the concentrated filtrate into an evaporation dish until the filtrate is odorless in a water bath, drying the concentrated filtrate at 60 ℃ and-0.2 MPa under reduced pressure until the filtrate is constant weight to obtain dry extract, uniformly mixing the dry extract with the two dry extracts prepared in the step one and the step two, and adding beta-cyclodextrin and 90% by mass of ethanol to prepare particles;
and step four, crushing the particles by using an ultrafine crusher, sieving the particles by using a 300-mesh sieve, and adding the I-type microbial preparation to prepare the compound beneficial microbial preparation for treating constipation.
3. The method for preparing a compound beneficial microbial preparation for treating constipation according to claim 2, wherein the method for preparing the type I microbial preparation comprises the following steps:
step one, a seed culture medium: according to the mass parts, 10 parts of peptone, 3 parts of yeast extract, 2 parts of beef extract, 5 parts of glucose, 3 parts of sodium acetate, 2 parts of ammonium citrate, 1 part of magnesium sulfate heptahydrate, 3 parts of potassium hydrogen phosphate, 20 parts of agar powder and 100 parts of water;
step two, fermentation medium: according to the mass parts, 6 parts of porous starch, 6 parts of skimmed milk powder, 5 parts of skimmed soybean powder, 7 parts of cassava powder, 1 part of salt, 4 parts of glucose and 100 parts of distilled water; wherein the cassava flour is obtained after being crushed;
step three, strain culture
a. Slant culture: according to the mass parts, 5 parts of bacillus and 4 parts of anaerobic lactobacillus are taken to be cultured for 2d at 37 ℃ in a slant culture medium;
b. and (3) shake flask culture: inoculating bacillus and anaerobic lactobacillus obtained by slant culture into a sterilized shake flask of a 500mL triangular flask, putting the shake flask on a shaker, starting a switch, and culturing for 2d under a shaking environment of 200rpm and a culture temperature of 37 ℃;
c. seed culture: inoculating bacillus and anaerobic lactobacillus obtained by shake flask culture into a seed culture medium under an aseptic condition, and culturing until the logarithmic growth phase of thalli is reached;
d. culturing in a fermentation tank: inoculating a culture solution obtained in seed culture to a fermentation culture medium, uniformly mixing, stirring for 5 minutes/3 hours under the culture condition of 37 ℃ and the pressure of 0.02-0.04 MPa, and continuously fermenting for 48 hours; the bacillus microscopicus and the anaerobic lactobacillus have normal shapes and no mixed bacteria, and the fermentation is stopped; making into slurry with a stirrer, and filtering;
e. spray drying: and (3) drying the bacillus and the anaerobic lactobacillus cultured in the fermentation tank by using a spray dryer with the inlet temperature of 120-150 ℃ and the outlet temperature of 65-75 ℃ to generate a powdery I type microbial preparation containing the bacillus and the anaerobic lactobacillus and with the average particle size of 10 mu m.
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