CN112375703A - Therapeutic milk-derived composite probiotics and preparation method and application thereof - Google Patents
Therapeutic milk-derived composite probiotics and preparation method and application thereof Download PDFInfo
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- CN112375703A CN112375703A CN202011260970.7A CN202011260970A CN112375703A CN 112375703 A CN112375703 A CN 112375703A CN 202011260970 A CN202011260970 A CN 202011260970A CN 112375703 A CN112375703 A CN 112375703A
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
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- 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
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
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- 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
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- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
Abstract
The invention discloses a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof, and is characterized in that the preparation method comprises the following steps: inoculating strain, culturing in seed tank, culturing, adding carrier, drying, sieving, inspecting quality, and culturing probiotic bacteria in MRS culture dish in the concentration of 1% for anaerobic culture. The treatment type milk-derived composite probiotics and the preparation method and application thereof are characterized in that the seed tank is used for fully activating lactobacillus acidophilus and bifidobacterium to provide certain nutrients for the lactobacillus acidophilus and the bifidobacterium, the lactobacillus acidophilus and the bifidobacterium are centrifuged, cleaned and frozen to ensure the good effect of mixing the two thalli and ensure the reproduction effect of the thalli after being frozen and stored for reuse, calcium alginate is used for embedding the thalli to form composite probiotic particles, and the acid resistance of the composite probiotics is improved under the condition that the tolerance of bile salts of the probiotics is not influenced.
Description
Technical Field
The invention relates to the technical field of probiotic preparation, in particular to a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof.
Background
The probiotics can regulate normal flora of gastrointestinal tract of organism, keep microecological balance, improve food digestibility and biological value, reduce serum cholesterol, control endotoxin, inhibit growth and propagation of putrefying bacteria and generation of putrefying products in intestinal tract, produce nutrient substances, and stimulate tissue development, thereby acting on nutritional state, physiological function, cell infection, drug effect, toxic reaction, immunoreaction, tumorigenesis, aging process, sudden emergency reaction, etc. of organism.
Commonly used probiotics are usually prepared from a single bacterium, and the acid resistance of such probiotics needs to be improved, so that the probiotics are propagated and stored in vivo.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof, and solves the problems.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof are characterized in that the preparation method comprises the following steps: inoculating and culturing strains, culturing in a seeding tank, culturing in a production way, adding a carrier, drying, sieving, and inspecting quality to finish the probiotics.
Preferably, the method comprises the following steps:
s1, selecting freeze-dried lactobacillus acidophilus and bifidobacterium, and placing lactobacillus acidophilus and bifidobacterium with the concentration of 1% in two MRS culture dishes respectively for anaerobic culture for 24 hours;
s2, extracting the acidophilic lactobacillus species and the bifidobacteria species after partial culture, putting the acidophilic lactobacillus species and the bifidobacteria species into a seed tank for activation, keeping the oxygen concentration inside the seed tank at 10-15%, and keeping the culture time for 12-24 h;
s3, mixing 3% of inoculum size of the lactobacillus acidophilus and bifidobacterium after primary culture with skim milk sterilized at 100-115 ℃ for 15 minutes, placing the mixed raw materials in a constant temperature box at 35-37 ℃ for culturing for 20 hours, and repeating twice;
s4, placing the cultured raw materials under the action of a centrifugal machine at 4000r/min-6000r/min, and performing centrifugal operation for 20 min;
s5, taking a phosphoric acid buffer with the pH value of 7.0 to wash the centrifuged raw material to obtain thalli;
s6, freeze-drying the strains in the skim milk culture medium at-30 to-40 deg.C for 2-5 h;
s7, embedding the strains in the calcium alginate carrier to form probiotic particles, and screening and quality testing the probiotic particles to obtain the finished product of the probiotic.
Preferably, the skim milk comprises 10-15% of skim milk powder, 5-8% of inulin and 77-85% of water.
Preferably, a liquid level detection sensor and an online cell density instrument are arranged inside the seeding tank, and a gas cylinder and a control cabinet connected with the seeding tank are arranged outside the seeding tank.
Preferably, the phosphate buffer is prepared by mixing potassium dihydrogen phosphate, sodium hydroxide solution and water.
(III) advantageous effects
The invention provides a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof, and the therapeutic milk-derived composite probiotic has the following beneficial effects: the treatment type milk-derived composite probiotics and the preparation method and application thereof are characterized in that the seed tank is used for fully activating lactobacillus acidophilus and bifidobacterium to provide certain nutrients for the lactobacillus acidophilus and the bifidobacterium, the lactobacillus acidophilus and the bifidobacterium are centrifuged, cleaned and frozen to ensure the good effect of mixing the two thalli and ensure the reproduction effect of the thalli after being frozen and stored for reuse, calcium alginate is used for embedding the thalli to form composite probiotic particles, and the acid resistance of the composite probiotics is improved under the condition that the tolerance of bile salts of the probiotics is not influenced.
Detailed Description
All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The invention provides a technical scheme that: a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof are characterized in that the preparation method comprises the following steps: the method comprises the following steps of strain inoculation culture, seeding tank culture, production culture, carrier addition, drying, sieving, quality inspection and probiotic completion, and comprises the following steps:
selecting freeze-dried lactobacillus acidophilus and bifidobacterium, and placing lactobacillus acidophilus and bifidobacterium with the concentration of 1% in two MRS culture dishes respectively for anaerobic culture for 24 h; extracting the cultured Lactobacillus acidophilus and Bifidobacterium, activating in seed tank with oxygen concentration of 10%, and culturing for 12 hr; mixing lactobacillus acidophilus and bifidobacterium after primary culture with 3% of inoculum size in skim milk sterilized at 100 ℃ for 15 minutes, placing the mixed raw materials in a constant temperature box at 35 ℃ for culturing for 20 hours, and repeating twice; placing the cultured raw materials in a centrifuge at 4000r/min, and centrifuging for 20 min; washing the centrifuged raw material by using a phosphate buffer with a pH value of 7.0 to obtain thalli; freeze drying the strain in skim milk culture medium at-30 deg.C for 2 hr; embedding the strains in the calcium alginate carrier to form probiotic particles, and screening and quality testing the probiotic particles to obtain the finished product of the probiotic.
The skimmed milk comprises skimmed milk powder 10%, inulin 5%, and water 85%; a liquid level detection sensor and an online cell density instrument are arranged inside the seed tank, and a gas cylinder and a control cabinet connected with the seed tank are arranged outside the seed tank; the phosphate buffer solution is prepared by mixing potassium dihydrogen phosphate, sodium hydroxide solution and water.
Example two
The invention provides a technical scheme that: a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof are characterized in that the preparation method comprises the following steps: the method comprises the following steps of strain inoculation culture, seeding tank culture, production culture, carrier addition, drying, sieving, quality inspection and probiotic completion, and comprises the following steps:
selecting freeze-dried lactobacillus acidophilus and bifidobacterium, and placing lactobacillus acidophilus and bifidobacterium with the concentration of 1% in two MRS culture dishes respectively for anaerobic culture for 24 h; extracting the cultured Lactobacillus acidophilus and Bifidobacterium, activating in seed tank with oxygen concentration of 13%, and culturing for 18 hr; mixing lactobacillus acidophilus and bifidobacterium after primary culture with 3% of inoculum size in skim milk sterilized at 110 ℃ for 15 minutes, placing the mixed raw materials in a constant temperature box at 36 ℃ for culturing for 20 hours, and repeating twice; placing the cultured raw materials in a centrifuge of 5000r/min, and centrifuging for 20 min; washing the centrifuged raw material by using a phosphate buffer with a pH value of 7.0 to obtain thalli; freeze drying the strain in skim milk culture medium at-35 deg.C for 3 hr; embedding the strains in the calcium alginate carrier to form probiotic particles, and screening and quality testing the probiotic particles to obtain the finished product of the probiotic.
The skimmed milk comprises skimmed milk powder 12%, inulin 6%, and water 82%; a liquid level detection sensor and an online cell density instrument are arranged inside the seed tank, and a gas cylinder and a control cabinet connected with the seed tank are arranged outside the seed tank; the phosphate buffer solution is prepared by mixing potassium dihydrogen phosphate, sodium hydroxide solution and water.
EXAMPLE III
The invention provides a technical scheme that: a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof are characterized in that the preparation method comprises the following steps: the method comprises the following steps of strain inoculation culture, seeding tank culture, production culture, carrier addition, drying, sieving, quality inspection and probiotic completion, and comprises the following steps:
selecting freeze-dried lactobacillus acidophilus and bifidobacterium, and placing lactobacillus acidophilus and bifidobacterium with the concentration of 1% in two MRS culture dishes respectively for anaerobic culture for 24 h; extracting the cultured Lactobacillus acidophilus and Bifidobacterium, activating in seed tank with oxygen concentration of 15%, and culturing for 24 hr; mixing lactobacillus acidophilus and bifidobacterium after primary culture with 3% of inoculum size in skim milk sterilized at 115 ℃ for 15 minutes, placing the mixed raw materials in a constant temperature box at 37 ℃ for culturing for 20 hours, and repeating twice; placing the cultured raw materials in a centrifuge of 6000r/min, and centrifuging for 20 min; washing the centrifuged raw material by using a phosphate buffer with a pH value of 7.0 to obtain thalli; freeze drying the strain in skim milk culture medium at-40 deg.C for 5 hr; embedding the strains in the calcium alginate carrier to form probiotic particles, and screening and quality testing the probiotic particles to obtain the finished product of the probiotic.
The skimmed milk comprises skimmed milk powder 15%, inulin 8%, and water 77%; a liquid level detection sensor and an online cell density instrument are arranged inside the seed tank, and a gas cylinder and a control cabinet connected with the seed tank are arranged outside the seed tank; the phosphate buffer solution is prepared by mixing potassium dihydrogen phosphate, sodium hydroxide solution and water.
And (4) experimental conclusion: the invention provides a therapeutic milk-derived composite probiotic as well as a preparation method and application thereof, which have the characteristic of obviously improving the acid resistance of the probiotic and can well meet the survival efficiency of the probiotic in vivo.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A therapeutic milk-derived composite probiotic as well as a preparation method and application thereof are characterized in that the preparation method comprises the following steps: inoculating and culturing strains, culturing in a seeding tank, culturing in a production way, adding a carrier, drying, sieving, and inspecting quality to finish the probiotics.
2. The therapeutic milk-derived composite probiotic as well as the preparation method and the application thereof according to claim 1 are characterized by comprising the following steps:
s1, selecting freeze-dried lactobacillus acidophilus and bifidobacterium, and placing lactobacillus acidophilus and bifidobacterium with the concentration of 1% in two MRS culture dishes respectively for anaerobic culture for 24 hours;
s2, extracting the acidophilic lactobacillus species and the bifidobacteria species after partial culture, putting the acidophilic lactobacillus species and the bifidobacteria species into a seed tank for activation, keeping the oxygen concentration inside the seed tank at 10-15%, and keeping the culture time for 12-24 h;
s3, mixing 3% of inoculum size of the lactobacillus acidophilus and bifidobacterium after primary culture with skim milk sterilized at 100-115 ℃ for 15 minutes, placing the mixed raw materials in a constant temperature box at 35-37 ℃ for culturing for 20 hours, and repeating twice;
s4, placing the cultured raw materials under the action of a centrifugal machine at 4000r/min-6000r/min, and performing centrifugal operation for 20 min;
s5, taking a phosphoric acid buffer with the pH value of 7.0 to wash the centrifuged raw material to obtain thalli;
s6, freeze-drying the strains in the skim milk culture medium at-30 to-40 deg.C for 2-5 h;
s7, embedding the strains in the calcium alginate carrier to form probiotic particles, and screening and quality testing the probiotic particles to obtain the finished product of the probiotic.
3. The therapeutic milk-derived composite probiotic as well as the preparation method and the application thereof according to claim 2 are characterized in that: the components of the skim milk comprise 10-15% of skim milk powder, 5-8% of inulin and 77-85% of water.
4. The therapeutic milk-derived composite probiotic as well as the preparation method and the application thereof according to claim 2 are characterized in that: the seeding tank is characterized in that a liquid level detection sensor and an online cell density meter are arranged inside the seeding tank, and a gas cylinder and a control cabinet which are connected with the seeding tank are arranged outside the seeding tank.
5. The therapeutic milk-derived composite probiotic as well as the preparation method and the application thereof according to claim 2 are characterized in that: the phosphate buffer solution is formed by mixing potassium dihydrogen phosphate, sodium hydroxide solution and water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011260970.7A CN112375703A (en) | 2020-11-12 | 2020-11-12 | Therapeutic milk-derived composite probiotics and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011260970.7A CN112375703A (en) | 2020-11-12 | 2020-11-12 | Therapeutic milk-derived composite probiotics and preparation method and application thereof |
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| CN112375703A true CN112375703A (en) | 2021-02-19 |
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| CN202011260970.7A Pending CN112375703A (en) | 2020-11-12 | 2020-11-12 | Therapeutic milk-derived composite probiotics and preparation method and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012098239A1 (en) * | 2011-01-21 | 2012-07-26 | Vesale Pharma Sa | Microencapsulated probiotic substance and process of manufacture |
| CN104095148A (en) * | 2014-07-21 | 2014-10-15 | 九江礼涞生物科技有限公司 | Preparation method of products containing animal intestinal beneficial bacteria |
| CN106860483A (en) * | 2017-01-06 | 2017-06-20 | 家家乐购（北京）科技有限公司 | Probiotics complex composition and application containing Bifidobacterium and lactobacillus acidophilus |
| CN112956698A (en) * | 2021-03-26 | 2021-06-15 | 四川农业大学 | Probiotics microcapsule embedded blasting bead and preparation method thereof |
-
2020
- 2020-11-12 CN CN202011260970.7A patent/CN112375703A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012098239A1 (en) * | 2011-01-21 | 2012-07-26 | Vesale Pharma Sa | Microencapsulated probiotic substance and process of manufacture |
| CN104095148A (en) * | 2014-07-21 | 2014-10-15 | 九江礼涞生物科技有限公司 | Preparation method of products containing animal intestinal beneficial bacteria |
| CN106860483A (en) * | 2017-01-06 | 2017-06-20 | 家家乐购（北京）科技有限公司 | Probiotics complex composition and application containing Bifidobacterium and lactobacillus acidophilus |
| CN112956698A (en) * | 2021-03-26 | 2021-06-15 | 四川农业大学 | Probiotics microcapsule embedded blasting bead and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| MINTO MICHAEL 等: ""Plant extract enhances the viability of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus in probiotic nonfat yogurt"", 《FOOD SCIENCE & NUTRITION》 * |
| 孙昕 等: ""乳源性复合益生菌对2型糖尿病大鼠GLP-1的影响及调控机制"", 《中国微生态学杂志》 * |
| 宋茂清: "《益生菌及其功效》", 30 November 2018, 科学技术文献出版社 * |
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Application publication date: 20210219 |