CN105838637A - Method for culturing high biomass mycobacterium phlei - Google Patents
Method for culturing high biomass mycobacterium phlei Download PDFInfo
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- CN105838637A CN105838637A CN201511013469.XA CN201511013469A CN105838637A CN 105838637 A CN105838637 A CN 105838637A CN 201511013469 A CN201511013469 A CN 201511013469A CN 105838637 A CN105838637 A CN 105838637A
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- Prior art keywords
- mycobacterium phlei
- radix ginseng
- fermentation
- biomass
- medicine residues
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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
Abstract
The invention relates to the field of microbial fermentation and discloses a medium raw material for improving mycobacterium phlei biomass and a use thereof. Ginseng residue fine powder subjected to cellulase-based enzymatic hydrolysis, drying and crushing or to bacillus subtilis fermentation, sterilization, drying and crushing is used as a medium raw material for mycobacterium phlei fermentation so that fermentation biomass is observably improved and a practical scheme is provided for mycobacterium phlei industrial production, development and application. The method provides an effective approach for herb residue use.
Description
Technical field
The present invention relates to field of microbial fermentation, a kind of side cultivating the Mycobacterium phlei obtaining high-biomass
Method.
Background technology
Along with Chinese herbal medicine industry development, the discarded amount of medicinal residues produced during Chinese patent medicine production etc. increases the most day by day.
There are nearly 1500 Chinese medicine enterprises in China at present, annual discharge Chinese medicine slag about 13,000,000 t.These medicinal residues usually owing to dealing with improperly,
Cause serious environmental pollution and the wasting of resources.When some Chinese patent medicines containing Radix Ginseng produce, Radix Ginseng mainly extracts ginsenoside
Active component, still contains a large amount of polysaccharide in residue medicine residues of Radix Ginseng, is a kind of well fermentable raw material.
Mycobacterium phlei (Mycobacterium phlei) is to be distributed widely in the one of nature in antiacid Mycobacterium
Plant people and animal no pathogenicity, the saprophytic bacteria of fast growth, be the natural pharmaceutical resources of a kind of preciousness.Mycobacterium phlei is 1889
Year is found, and has biological affinity with tubercule bacillus, is widely present in soil, plant and drinking-water.At present, Mycobacterium phlei
Being widely used as humans and animals multifunction immunity reinforcing agent to use, it is many that it is mainly composed of muramyldipeptide (MDP), mycobacteria
Sugar (MPS), CpG immune modulatory sequence etc..
At present, the research to Mycobacterium phlei is concentrated mainly on preparation research, application process and effect disquisition etc..But giving birth to
The Mycobacterium phlei obtaining more multi-biomass during product is the most significant.The application to solve the technical problem that and i.e. to exist
On the basis of prior art, obtain the Mycobacterium phlei of more multi-biomass by adding medicine residues of Radix Ginseng fine powder in the medium,
Industrialized production and the exploitation application of Mycobacterium phlei provide the most practical scheme；Meanwhile, also for the utilization offer of Chinese medicine slag
A kind of effective way.
Notification number is patent of invention " a kind of Mycobacterium phlei Sq-1 and the system of compound micro-ecological preparation of CN102206590A
Preparation Method " (application number: CN201010572928.9) to this Mycobacterium phlei Sq-1 and the preparation method of compound micro-ecological preparation
Announced.Described Mycobacterium phlei Sq-1 (Mycobacterium plei Sq-1), preserving number: CGMCC NO0916
With bacillus subtilis (Bacillus subtilis) preserving number: ACCC10634.
The preservation information of biological material specimens is: Mycobacterium phlei Mycobacterium phlei, Sq-1 depositary institution:
China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, preservation date: on April 1st, 2003, deposit number: CGMCC NO.0916.Hay spore
Bacillus (Bacillus subtilis), purchased from Chinese agriculture Microbiological Culture Collection administrative center, is purchased from October 12nd, 2013,
Preserving number: ACCC10634.
This preparation method fermentation biomass is limited, it would be highly desirable to improve.
Summary of the invention
A kind of method that it is an object of the invention to provide Mycobacterium phlei cultivated and obtain high-biomass.
It is an object of the invention to be realized by following approach: a kind of side cultivating the Mycobacterium phlei obtaining high-biomass
Method, its step is as follows:
A. seed activation, the composition of seed culture medium: the 20-25g/L Han sucrose in pure water, peptone 2.5-3g/L, sodium citrate
20-25g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, agar 15-20g/L, pH7.0-7.2g/L；
Inoculation, cultivates 48 hours for 30 ± 1 DEG C, and microscopy is without miscellaneous bacteria and robust growth；
B. seed tank fermentation, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 2.0-2.5g/L, Fructus Citri Limoniae
Acid sodium 15-22g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-7.2；Inoculation, adds automatically
Adding defoamer, ventilation 1:0.6-0.8, cultivate 28-32 hour for 30 ± 1 DEG C, microscopy is without miscellaneous bacteria and robust growth；
C. ferment tank, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 1.5-2.0g/L, Fructus Citri Limoniae
Acid sodium 15-20g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-7.2；Inoculation, adds automatically
Adding defoamer, ventilation 1:0.8, cultivate 46-54 hour for 30 ± 1 DEG C, bacterium number reaches 1010Individual/mL stops fermentation, obtains grass branch
Bacillus body.
As the further optimization of this programme, the described preferred addition of medicine residues of Radix Ginseng fine powder is containing Radix Ginseng in every liter of pure water
Medicinal residues 40g/L.
As the further optimization of this programme, described medicine residues of Radix Ginseng fine powder is processed by cellulase degradation or by withered
Grass fermentation of bacillus processes.
As the further optimization of this programme, described medicine residues of Radix Ginseng fine powder fine powder fineness is 80-200 mesh.
A kind of method cultivating the Mycobacterium phlei obtaining high-biomass of the present invention, contains by adding in the fermentation medium
Medicine residues of Radix Ginseng fine powder, it is thus achieved that Mycobacterium phlei fermentation medium and the fermentation process of fermentation biomass can be significantly improved, for
Industrialized production and the exploitation application of Mycobacterium phlei provide the most practical scheme；Meanwhile, also for the utilization offer of Chinese medicine slag
A kind of effective way.
Detailed description of the invention
A kind of method cultivating the Mycobacterium phlei obtaining high-biomass of the present invention, its step is as follows:
A. seed activation, the composition of seed culture medium: the 20-25g/L Han sucrose in pure water, peptone 2.5-3g/L, sodium citrate
20-25g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, agar 15-20g/L, pH7.0-7.2；Connect
Kind, to cultivate 48 hours for 30 ± 1 DEG C, microscopy is without miscellaneous bacteria and robust growth；
B. seed tank fermentation, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 2.0-2.5g/L, Fructus Citri Limoniae
Acid sodium 15-22g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-7.2；Inoculation, adds automatically
Adding defoamer, ventilation 1:0.6-0.8, cultivate 28-32 hour for 30 ± 1 DEG C, microscopy is without miscellaneous bacteria and robust growth；
C. ferment tank, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 1.5-2.0g/L, Fructus Citri Limoniae
Acid sodium 15-20g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-7.2；Inoculation, adds automatically
Adding defoamer, ventilation 1:0.8, cultivate 46-54 hour for 30 ± 1 DEG C, bacterium number reaches 1010Individual/mL stops fermentation, obtains grass branch
Bacillus body.
The described preferred addition of medicine residues of Radix Ginseng fine powder is the 40g/L Han medicine residues of Radix Ginseng in pure water, and medicine residues of Radix Ginseng fine powder passes through
Cellulase degradation is processed or is processed by fermentation of bacillus subtilis, and medicine residues of Radix Ginseng fine powder fine powder fineness is 80-200 mesh.
Following example employing Mycobacterium phlei sq-1(preserving number: CGMCC NO.0916) carry out concrete bidding documents, it is possible to adopt
With the Mycobacterium phlei bacterial strain can being commercially available on market.
One, produce medicine residues of Radix Ginseng powder with the medicinal residues preparing SHENMAI ZHUSHEYE, be further used as culture medium raw material, carry out grass branch
Bacillus fermentation.
Experimental example one takes the medicine residues of Radix Ginseng during SHENMAI ZHUSHEYE produces, and adds cellulase, enzymolysis, dries, pulverize to obtain fineness
The fine powder of 80-200 mesh.Join in Mycobacterium phlei fermentation medium by 4% addition, ferment, result count.
Experimental example two takes the medicine residues of Radix Ginseng during SHENMAI ZHUSHEYE produces, and adds bacillus subtilis, fermentation, and sterilizing is dried,
Pulverize to obtain the fine powder of fineness 80-200 mesh.Join in Mycobacterium phlei fermentation medium by 4% addition, ferment, result
Counting.
Two, produce medicine residues of Radix Ginseng powder with the medicinal residues preparing Shenqi oral liquid, make culture medium raw material, carry out Mycobacterium phlei
Fermentation.
Experimental example three takes the medicine residues of Radix Ginseng during stilbene ginseng oral liquid produces, and adds cellulase, enzymolysis, dries, pulverize to obtain fineness
The fine powder of 80-200 mesh.Join in Mycobacterium phlei fermentation medium by 4% addition, ferment, result count.
Experimental example four takes the medicine residues of Radix Ginseng during stilbene ginseng oral liquid produces, and adds bacillus subtilis, fermentation, and sterilizing is dried,
Pulverize to obtain the fine powder of fineness 80-200 mesh.Join in Mycobacterium phlei fermentation medium by 4% addition, ferment, result
Counting.
Three, contrast method is by patent of invention that notification number is CN102206590A " a kind of Mycobacterium phlei Sq-1 and multiple
Closing the preparation method of microbial ecological agent " Mycobacterium phlei fermentation process described in (application number: CN201010572928.9) carries out
Fermentation, result count.
Note: SHENMAI ZHUSHEYE is extracted formed by Radix Ginseng Rubra, Radix Ophiopogonis, and the ratio of Radix Ginseng Rubra Radix Ophiopogonis is 11.
Stilbene ginseng oral liquid is extracted made by the Radix Astragali, Radix Ginseng, Radix Glycyrrhizae, and the ratio of Radix Astragali Radix Ginseng Radix Glycyrrhizae is 322.
Four, Comparative result
By upper table, processed by cellulase degradation or the medicine residues of Radix Ginseng powder of fermentation of bacillus subtilis process by adding
Culture medium carry out mycobacterium graminis fermentation, compared with art methods, its fermentation biomass be respectively increased 7.22 times,
5.28 times, 10.28 times, 6.11 times.
Claims (4)
1. the method cultivating the Mycobacterium phlei obtaining high-biomass, it is characterised in that: its step is as follows:
A. seed activation, the composition of seed culture medium: the 20-25g/L Han sucrose in pure water, peptone 2.5-3g/L, sodium citrate
20-25g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, agar 15-20g/L, pH7.0-7.2；Connect
Kind, to cultivate 48 hours for 30 ± 1 DEG C, microscopy is without miscellaneous bacteria and robust growth；
B. seed tank fermentation, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 2.0-2.5g/L, Fructus Citri Limoniae
Acid sodium 15-22g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-7.2；Inoculation, adds automatically
Adding defoamer, ventilation 1:0.6-0.8, cultivate 28-32 hour for 30 ± 1 DEG C, microscopy is without miscellaneous bacteria and robust growth；
C. ferment tank, medium component is: the 20-60g/L Han medicine residues of Radix Ginseng in pure water, peptone 1.5-2.0g/L, the Radix Astragali
Extractum 15g/L, sodium citrate 15-20g/L, potassium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.5-2.0g/L, PH6.5-
7.2；Inoculation, automatically adds defoamer, ventilation 1:0.8, cultivates 46-54 hour for 30 ± 1 DEG C, and bacterium number reaches 1010Individual/mL stops
Fermentation, obtains Mycobacterium phlei body.
A kind of method cultivating the Mycobacterium phlei obtaining high-biomass the most as claimed in claim 1, it is characterised in that: described
The preferred addition of medicine residues of Radix Ginseng fine powder be the 40g/L Han medicine residues of Radix Ginseng in pure water.
A kind of method cultivating the Mycobacterium phlei obtaining high-biomass the most as claimed in claim 1, it is characterised in that: described
Medicine residues of Radix Ginseng fine powder processed by cellulase degradation or processed by fermentation of bacillus subtilis.
A kind of method cultivating the Mycobacterium phlei obtaining high-biomass the most as claimed in claim 1, it is characterised in that: described
Medicine residues of Radix Ginseng fine powder fine powder fineness be 80-200 mesh.
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| CN201511013469.XA CN105838637A (en) | 2015-12-31 | 2015-12-31 | Method for culturing high biomass mycobacterium phlei |
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| CN201511013469.XA CN105838637A (en) | 2015-12-31 | 2015-12-31 | Method for culturing high biomass mycobacterium phlei |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058796A (en) * | 2007-04-25 | 2007-10-24 | 中国人民解放军第三军医大学第一附属医院 | Composite culture medium for bifidobacterium fermentation and preparation method thereof |
| CN102206590A (en) * | 2010-12-03 | 2011-10-05 | 张素琴 | Preparation methods of Mycobacterium plei Sq-1 and composite microecological preparation thereof |
-
2015
- 2015-12-31 CN CN201511013469.XA patent/CN105838637A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058796A (en) * | 2007-04-25 | 2007-10-24 | 中国人民解放军第三军医大学第一附属医院 | Composite culture medium for bifidobacterium fermentation and preparation method thereof |
| CN102206590A (en) * | 2010-12-03 | 2011-10-05 | 张素琴 | Preparation methods of Mycobacterium plei Sq-1 and composite microecological preparation thereof |
Non-Patent Citations (5)
| Title |
|---|
| JINGHONG Z等: "Inhaled inactivated-Mycobacterium phlei modulates", 《ASIAN PAC J ALLERGY IMMUNOL》 * |
| 刘彦亮: "黄芪及其它中草药对双歧杆菌增菌的影响研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 孙永欣: "黄芪多糖对刺参肠道褐藻酸降解菌生物量及产酶活性的影响", 《中国饲料》 * |
| 封晔: "高产纤维素酶菌株的筛选及药渣发酵工艺研究", 《食品安全导刊》 * |
| 金美玲等: "草分支杆菌疫苗治疗哮喘模型小鼠的实验研究", 《复旦学报(医学版)》 * |
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