CN1626648A - CZ-81 strain of 'Baibachi' bacterium and artificial culture method - Google Patents
CZ-81 strain of 'Baibachi' bacterium and artificial culture method Download PDFInfo
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Abstract
A Baipichi strain CZ-81 (CGMCC No.1016) is disclosed, which features high stability, metablitic power and safety. Its artificial culture method includes slant culture, shake culture, seeding-tank culture, fermenting and extracting. Its advantages are short time dicreased by 20%, and high content of active components.
Description
Technical field:
The present invention relates to the high-performance bacterial classification that a kind of fungal organism engineering submerged fermentation is cultivated, i.e. the cultural method of white rake teeth bacterium " CZ-81 " bacterial strain.
Background technology:
White rake teeth bacterium Irpex lacteus Fr. belongs to hedgehog fungus section, and a kind of fungi of rake teeth Pseudomonas, white rake teeth bacterial classification is called white bag hole.Different name: Hirschioporus lacteus (Fr.) Teng; Deng Shuqun, the fungi of China, 484.1963.Its sporophore is born on the dry wood or dead and dying trees of deciduous tree.Morphological specificity: basidioma (sporophore) is calm, expansion, white, soft leather matter; Or calm and warp, warp partly is 8～15 * 10～20mm.Sometimes fall to give birth to, it is storied, fan-shaped to be multiple watt shape, narrow and small base portion is arranged, transverse diameter 2～4cm, former 2～3mm; The capping oyster white has down, and spun silk shape gloss is arranged, and ring grain is arranged, and thin edge is curved down, and is wavy to shallow splitting.Bacterial context white is about former 1mm.Thorn is irpiciform, and is short, irregular, white to yellow-white, long 1～2cm, and the spore ellipse, level and smooth, colourless, 4～5 * 2～3mm.Cystidium clavate or fusiform, 50～150 * 5～10 μ m have draped over one's shoulders crystallization sometimes, mainly are distributed in provinces and regions [1] such as Forest in Changbai Mountain Forest Region and Heilungkiang, Hebei, Shanxi, Gansu, Jiangxi, Fujian, Yunnan and Tibet.
Become the fermented product extract of bacterial strain that the treatment chronic glomerulonephritis is had the good clinical effect by the separation and purification of white rake teeth bacterium, existing The stability of strain is poor, the deficiency that metabolic function is weak, how artificial culture goes out high-quality white rake teeth bacteria strain, the cultural method that never obtains.
Summary of the invention:
The technical issues that need to address of the present invention provide a kind of stable performance, and resistance of aging is strong, the white rake teeth bacteria strain that the biological metabolism performance is high.
The present invention discloses the cultural method of this bacterial strain.
Technical scheme of the present invention is as follows:
White its bacterial classification of rake teeth bacterium Irpex lacteus Fr. provided by the present invention is the CZ-81 bacterial strain, be deciduous tree dry wood or the long sporophore that white rake teeth bacterium is arranged of dead and dying trees, obtain through separation, purifying, screening and long-term breeding from Chinese Forest in Changbai Mountain Forest Region theropencedrymion band.
The syntrophism law of development of organism be by juvenile stage to growing animated period, Zhongdao senescence phase is until decline.The Mycophyta biology also is that so Mycophyta biological fermentation engineering principle is exactly the application of fermentation method, promotes the continuous breeding of mycelia and the accumulation of meta-bolites, extracts the effective active composition, for pharmacy provides reliable raw material.Because long-term production expands numerous bacterial classification, certainly will cause the degeneration of bacterial classification.Therefore, provide a kind of good stability, energetic, growth rate is fast, metabolism performance height, and the bacterial classification that ageing resistance is strong is important key technical problem to fermentative production.
This bacterial classification is on September 25th, 2003, at the preservation of the microbial strains preservation council of the Chinese Academy of Sciences, preserving number: CGMCC No.1016.
The bacterial classification application standard:
1, bacterial classification source explanation: the CZ-81 bacterial strain be from Forest in Changbai Mountain Forest Region rotten wood, through separation, purifying, screening, and examines the bacterial strain of the stable performance that obtains by systematic breeding in the long sporophore that white rake teeth bacterium arranged.CZ-is meant the abbreviation prefix of Changchun Traditional Chinese Medical College.
2, the form standard of bacterial classification
(1) mode of appearance
Slant strains: bacterium colony is white in color, and mycelia is that white cotton is cotton-shaped, and along test tube wall extend upward, dense.The mycelial growth development rate is fast, just can cover with full inclined-plane in 5 days.
Shake a bottle bacterial classification: clear and bright, the tawny of bacterium liquid, it is spherical that mycelia is radioactivity.Sense of taste has dense fruit aroma.
The seeding tank bacterial classification: with the shake-flask seed form, but the mycelia densification in the bacterium ball, dry weight increases, and sense of taste is the same.
(2) microscopy form
By the inspection of 16 * 100 times of biomicroscopes, mycelia color depth (U.S. blue single stain), and evenly, branch is many, is netted, and is sturdy, clamp connexion obviously as seen, no living contaminants.
3, the general standard of bacterial classification application
Bacterial classification is being produced when using and must carried out by following standard:
(1) uses the new numerous fresh bacterial classification (refer to newly expand numerous bacterial classification and preserve, its slant strains kind age not above 30 days) that expands at 2～4 ℃ of refrigerators
(2) the standby bacterial classification that uses refrigerator to preserve will activate more than 12 hours in advance, can use.
(3) put the slant strains of 2～4 ℃ of refrigerator prolonged preservation, must be once 120 days left and right sides tubes reasons.Expand through tube and numerously to reach bacterial classification required standard (referring to outward appearance, microscopy form and vigor) time and re-use.
(4) to use the source clear, detail record be arranged, dependable performance, the conformance with standard specified requirement, the excellent species of CZ-81 bacterial strain.
(5) thin as the bacterium colony that slant strains occurs, very thin, the variable color of white cotton-shaped mycelia seldom has again along the upwardly extending mycelia of tube wall, illustrates that bacterial classification is aging, vigor reduces, can not re-use.
(6) CZ-81 bacterial strain stable performance certainly will cause the bacterium potential drop low but go down to posterity for a long time, and this bacterial strain was difficult for degenerating in 20 generations.It is then obviously aging gradually to surpass for 20 generations, can not re-use, and must change performance " CZ-81 " novel bacterial reliably, guarantees quality product.
4, the classification technique administrative standard of bacterial classification
(1) primary inclined plane bacterial classification:
Substratum: comprehensive PDA culture medium: glucose 2%, potato 20%, agar 2%, KH
2PO
40.3%, MgSO
40.2%, VB1 0.05%, PH6.5 (before disappearing).
If special messenger, special case (refrigerator), special record management.To bacterial classification performance and aging conditions examine or check at any time, record.Carry out tube go down to posterity mark and record, produce the bacterial classification that uses belong to which in generation (passage number) must be clear.Note abnormalities and the aged bacterial classification will in time be eliminated, not can be applicable to produce.
26 ± 1 ℃ of slant strains culture temperature, mycelia was covered with the inclined-plane in 110-130 hour, put refrigerator (2～4 ℃) and preserved standby.
(2) secondary shakes a bottle bacterial classification:
Substratum: glucose 3%, peptone 1.5%, yeast extract 0.5%, KH
2PO
40.2%, MgSO
40.15%, PH6.5 (before disappearing).It is standby to sterilize.
Get that cell age is short, the sturdy dense slant strains of mycelia, under aseptic condition, insert in the shake-flask culture base of having sterilized (in 1: 6 ratio, promptly 1 inclined-plane connects 6 and shakes bottle), 26 ± 1 ℃ of temperature controls are cultivated, and shaking bottle revolution is 160-260r/min (rotary), when the bacterium sphere volume be weight in wet base account for 30% or more (get the 100ml nutrient solution high-volume tin in, static 15 minutes, the settled percentage of bacterium ball); Microscopy reaches standard under the bacterial classification item, and sense of taste has dense fruit aroma, and no living contaminants, incubation time are 90-96 hour, can change the seeding tank fermentation culture over to.
(3) three grades of seeding tank bacterial classifications
Substratum: glucose 2%, peptone 1%, yeast powder 0.5%, soybean cake powder soak juice 5%, MgSO
40.15%, KH
2PO
40.2%, PH6.5 (before disappearing).
Get fresh (refrigerator is preserved standby person, does not surpass a week) and shake a bottle bacterial classification, in 10% ratio, insert under aseptic condition in the seed tank culture base of having sterilized, 26 ± 1 ℃ of temperature controls are cultivated, logical sterile filtration pneumatic blending, and its flow is 1: 0.4vvm.When bacterium ball weight in wet base accounts for more than 60%; The microscopy mycelia color depth, and evenly, sturdy, branch is many, is netted, and clamp connexion obviously as seen; Fermented liquid is tawny, clear and bright, and dense fruit aroma is arranged; PH reduces to 5.5-6; Amino nitrogen (N) is reduced to 30-40%, and reducing sugar (C) is reduced to 3-5%; Incubation time is 68-76 hour; During no living contaminants, can change the fermentor tank inoculation culture over to.(the fermentor cultivation inoculum size is inoculated in 10% ratio)
5, the technological process of production and metabolic capacity appraisal standards
Use this bacterial strain and adopt following technical process:
Qualified
(extraction)---→ raw material work in-process---→ detect---→ finished product bulk drug, standby.
1) to put behind the jar fermented liquid after testing " white rake teeth granulose " (Crude polysaccharides) content should not be less than 1.5～2mg/ml.
2) extract dry product " polysaccharide " content of making through aftertreatment processing and must not be less than 150～200mg/g (detection of 3.5-dinitrosalicylic acid method)
The production application effect:
[fermentor cultivation and survey requirement :]
1, the bacterial classification that sets out: " CZ-81 bacterial strain "
2, substratum: with the seeding tank substratum
3, pH value 6.5 (before disappearing)
4, inoculum size: 10%
5, fermentor cultivation condition:
Temperature: 26 ± 1 ℃
Air flow quantity: 1: 0.4vvm
Tank pressure: 0.5kg/cm
2
6, put a jar standard
(1) outward appearance: bacterium liquid is clear and bright, tawny, mycelia weight in wet base (bacterium ball) reach more than 70%.
(2) sense of taste: little fruit aroma is arranged.
(3) microscopy: painted shallow, thin, the branch minimizing of mycelia, clamp connexion rare (or nothing), accidental empty spore.
(4) biochemical indicator:
PH value is reduced to about 4.5～5
C source (reducing sugar) reduces to 2～2.5%.
N source (amino nitrogen) reduces to 20%.
(5) polysaccharide content:
The liquid that turns sour detects that (put jar time)---content is more than 1.5mg/ml.
Dry product---content is more than 200mg/g to extract the processing back.
(6) incubation time: 36～40h.
White rake teeth bacterium CZ-81 bacterial strain provided by the invention is cultivate through seed selection for many years stable strong, metabolic capacity height, safe bacterial classification.This bacterial classification does not jeopardize human health or animal and plant cause of disease or contaminate environment in the enforcement of regulations process.Through system endurance, this strain passage difficult degeneration of 20 generations, and metabolic index is higher than more than 1 times of former primary dcreening operation bacterial strain.Control science, reasonable more at submerged fermentation culture process and each production link thereof simultaneously, whole fermented incubation time has shortened for nearly 20% time.Promptly improve the content of effective active components, shortened fermented incubation time again, reduced production cost.
And detect through security, pharmacology and to compare no abnormal finding with the fermentating metabolism product of former primary dcreening operation bacterial classification.Have higher using value, utilize the tunning of the CZ-81 bacterial strain of new seed selection, the rake teeth granulose of extraction for making treatment chronic nephritis medicine, provides long-range, science, a reliable medicine source approach.
Embodiment
From the long sporophore that white rake teeth bacterium arranged of the deciduous tree dry wood of Chinese Forest in Changbai Mountain Forest Region theropencedrymion band or dead and dying trees through separation, purifying, screening, and the bacterial strain of the stable performance that obtains by the systematic breeding examination, cultivate management process and be:
(1) primary inclined plane bacterial classification:
Substratum: comprehensive PDA culture medium: glucose 2%, potato 20%, agar 2%, KH
2PO
40.3%, MgSO
40.2%, VB1 0.05%, PH6.5 (before disappearing).
If special messenger, special case (refrigerator), special record management.To bacterial classification performance and aging conditions examine or check at any time, record.Carry out tube go down to posterity mark and record, produce the bacterial classification that uses belong to which in generation (passage number) must be clear.Note abnormalities and the aged bacterial classification will in time be eliminated, not can be applicable to produce.
26 ℃ of slant strains culture temperature, mycelia was covered with the inclined-plane in 120 hours, put refrigerator (3 ℃) and preserved standby.
(2) secondary shakes a bottle bacterial classification:
Substratum: glucose 3%, peptone 1.5%, yeast extract 0.5%, KH
2PO
40.2%, MgSO
40.15%, PH6.5 (before disappearing).It is standby to sterilize.
Get that cell age is short, the sturdy dense slant strains of mycelia, under aseptic condition, insert in the shake-flask culture base of having sterilized (in 1: 6 ratio, promptly 1 inclined-plane connects 6 and shakes bottle), 26 ℃ of temperature controls are cultivated, and shaking bottle revolution is 160-260r/min (rotary), when the bacterium sphere volume be weight in wet base account for 30% or more (get the 100ml nutrient solution high-volume tin in, static 15 minutes, the settled percentage of bacterium ball); Microscopy reaches standard under the bacterial classification item, and sense of taste has dense fruit aroma, no living contaminants, and incubation time is 93 hours, changes the seeding tank fermentation culture over to.
(3) three grades of seeding tank bacterial classifications
Substratum: glucose 2%, peptone 1%, yeast powder 0.5%, soybean cake powder soak juice 5%, MgSO
40.15%, KH
2PO
40.2%, PH6.5 (before disappearing).
Get fresh (refrigerator is preserved standby person, does not surpass a week) and shake a bottle bacterial classification, in 10% ratio, insert under aseptic condition in the seed tank culture base of having sterilized, 26 ℃ of temperature controls are cultivated, logical sterile filtration pneumatic blending, and its flow is 1: 0.4vvm.When bacterium ball weight in wet base accounts for more than 60%; The microscopy mycelia color depth, and evenly, sturdy, branch is many, is netted, and clamp connexion obviously as seen; Fermented liquid is tawny, clear and bright, and dense fruit aroma is arranged; PH reduces to 5.5; Amino nitrogen (N) reduces to 35%, and reducing sugar (C) reduces to 4%; Incubation time is 70 hours; During no living contaminants, can change the fermentor tank inoculation culture over to.(the fermentor cultivation inoculum size is inoculated in 10% ratio)
To put behind the jar fermented liquid after testing " white rake teeth granulose " (Crude polysaccharides) content be 2mg/ml.
Extracting dry product " polysaccharide " content of making through aftertreatment processing is 200mg/g (detection of 3.5-dinitrosalicylic acid method).
Claims (9)
1, white rake teeth bacterium CZ-81 bacterial strain (lrpes lacteus Fr.) CZ-81 bacterial strain CGMCC No.1016.
2, white rake teeth bacterium CZ-81 bacterial strain is characterized in that: metabolic capacity, when carrying out, put a jar secondary fermentation meta-bolites by requirement under this normal term---and " white rake teeth granulose " content should be more than 1.5mg/ml; Extraction is made the dry product polysaccharide content more than 200mg/g, and the hydrolysate of gained polysaccharide is a seminose.The molecular weight that gel filtration method records is about 80000.
3, according to claim 1,2 described white rake teeth bacterium CZ-81 bacterial strain artificial culture methods, its artificial culture method is to comprise spawn culture, and promptly slant strains is cultivated; Shake a bottle spawn culture; The seeding tank spawn culture; Cultivate and put the extraction processing behind the jar with fermentative production.
4, according to claim 3 described cultural methods, it is characterized in that it is comprehensive PDA culture medium that the slant strains addressed is cultivated used, its culture condition is PH6-6.5 (before disappearing), and culture temperature is 26 ± 1 ℃, and incubation time is 110-130h.
5, according to claim 3 described cultural methods, it is characterized in that the used substratum of being addressed of bottle spawn culture that shakes is a glucose peptone synthetic medium, PH6-6.5 (before disappearing), culture temperature is 26 ± 1 ℃, the shake-flask culture revolution is 160-260r/min, and the cultivation fate is 90-96h.
6,, it is characterized in that the used substratum of being addressed of seeding tank spawn culture soaks the juice synthetic medium for the glucose soybean cake powder according to claim 3 described cultural methods; PH value is 6-6.5 (before disappearing), and the same shake-flask culture of temperature, sterile filtration pneumatic blending are cultivated, and its air flow quantity is 1: 0.4vvm.Tank pressure 0.5kg/cm
2Incubation time is 68-76h.
7, according to the described white rake teeth bacterium CZ-81 bacterial strain of claim 1-2, it is characterized in that the slant strains of being addressed should be the dense cotton-shaped bacterium colony of white cotton; Shake a bottle bacterial classification bacterium ball weight in wet base and account for more than 30%, sense of taste has dense fruit aroma; Microscopy: color depth in U.S. blue simple stain, mycelia is sturdy, branch is many, is netted, clamp connexion obviously as seen.PH value is reduced to 5.5-6; Seed tank culture, its bacterium ball weight in wet base should account for more than 60%, and fermented liquid is clear and bright, does not have muddiness, and dense fruit aroma is arranged.Microscopy: with shaking a bottle bacterial standard; Change inspection: amino nitrogen is reduced to 30-40%, and reducing sugar reduces to 5%, and PH reduces to 5.5-6.
8, according to claim 3 described fermentation process, the substratum that it is characterized in that the fermentor cultivation of addressing is that glucose (industrial) soybean cake powder soaks the juice synthetic medium, and pH value 6-6.5 (before disappearing) is through 1.3-1.5kg/cm
2Pressure behind the sterilization 1h, is cultivated, and jar temperature: 26 ± 1 ℃, the sterile filtration air stirs the companion, air flow quantity 1: 0.4vvm.Tank pressure 0.5kg/cm
2Inoculum size 10% when bacterium ball weight in wet base accounts for more than 70%, has little fruit aroma; Microscopy: mycelia is painted shallow, thinner, and branch is gradually few, and rare clamp connexion, or accidental empty spore do not take place but there is the mycelia autolysis.This moment, amino nitrogen can reduce to 20%, and reducing sugar is reduced to 2-2.5%, and pH value is reduced to 4.5-5, and incubation time is 36-40h.
9, according to claim 3,8 described methods, it is characterized in that end fermented liquid when putting jar of fermentor cultivation, white after testing rake teeth granulose (Crude polysaccharides) content should be more than 1.5mg/ml; Through being processed into dry product, polysaccharide content should be more than 200m/g.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690147A (en) * | 2012-06-06 | 2012-09-26 | 吉林大学 | Additive for liquid nutrient medium for Irpex lacteus fermentation |
| CN106399132A (en) * | 2016-12-16 | 2017-02-15 | 泰安市煜达生物科技有限公司 | Irpex lacteus and application thereof |
| CN104403950B (en) * | 2014-11-04 | 2017-03-29 | 贵州大学 | A kind of white rake teeth bacterium and application thereof |
| CN106635821A (en) * | 2016-08-13 | 2017-05-10 | 辽宁省农业科学院微生物工程中心 | Mycorrhiza fungi for promoting nutrient absorption of blueberry and preparation method and application of mycorrhiza fungi |
| CN110791438A (en) * | 2016-08-31 | 2020-02-14 | 郭清子 | Rapex cacteus extract and preparation method and application thereof |
| CN111961596A (en) * | 2020-08-25 | 2020-11-20 | 中南林业科技大学 | Rapex baichii capable of efficiently degrading lignin |
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2003
- 2003-12-12 CN CN 200310115915 patent/CN1279159C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690147A (en) * | 2012-06-06 | 2012-09-26 | 吉林大学 | Additive for liquid nutrient medium for Irpex lacteus fermentation |
| CN104403950B (en) * | 2014-11-04 | 2017-03-29 | 贵州大学 | A kind of white rake teeth bacterium and application thereof |
| CN106635821A (en) * | 2016-08-13 | 2017-05-10 | 辽宁省农业科学院微生物工程中心 | Mycorrhiza fungi for promoting nutrient absorption of blueberry and preparation method and application of mycorrhiza fungi |
| CN106635821B (en) * | 2016-08-13 | 2019-10-25 | 辽宁省农业科学院微生物工程中心 | A kind of mycorrhizal fungi and the preparation method and application thereof for promoting blueberry nutrient to absorb |
| CN110791438A (en) * | 2016-08-31 | 2020-02-14 | 郭清子 | Rapex cacteus extract and preparation method and application thereof |
| CN110804557A (en) * | 2016-08-31 | 2020-02-18 | 郭清子 | Culture method and culture medium of irpex cacteus |
| CN106399132A (en) * | 2016-12-16 | 2017-02-15 | 泰安市煜达生物科技有限公司 | Irpex lacteus and application thereof |
| CN111961596A (en) * | 2020-08-25 | 2020-11-20 | 中南林业科技大学 | Rapex baichii capable of efficiently degrading lignin |
| CN111961596B (en) * | 2020-08-25 | 2022-05-06 | 中南林业科技大学 | Rapex baichii capable of efficiently degrading lignin |
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