CN1259570A - Mortierella Diding, its isolation cultivation method and fermentation technology - Google Patents
Mortierella Diding, its isolation cultivation method and fermentation technology Download PDFInfo
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Abstract
The present invention relates to a new variety fungus sporum Diding, its isolation and culture method and fermentation technology. It is cultured on MA culture medium under 25 deg.C for 10 days or is cultured on cha medium under 25 deg.C for 10 days. The isolating culture method is characterized in that: inoculate small piece corpse from freshly died caterpillar fungus on SDAY culture medium, culture in illuminated culture box at 18 deg.C, transfer and inoculate the grown bacterial colony until pure bacterial colony is grown. The fermentation technology is three stage culture, that is slant culture, liquid culture and fermentation tank culture. Said invention can be used in the development of food and medicine.
Description
The present invention relates to a kind of fungi and cultural method, exactly is a kind of Chinese new record cordyceps species mortierella Diding (Acremonium terricola, CGMCC0346) and isolation cultivation method and fermentation technology.
In the strategy of sustainable development of 21 century, the rational exploitation and utilization of species diversity is to supporting socio-economic development very important.At present, the protection of species diversity is subjected to unprecedented attention, and lot of manpower and material resources has been dropped in countries in the world, is devoted to the research and the protection work of species diversity.In the research and utilization of species diversity, the species diversity of microorganism is still being studied inadequately at present, but but very important research field.Wherein, the species diversity of fungi it " weight " in " weight " again especially.Because fungi is a lot of pharmaceutical industries, foodstuffs industry " raw material storage ", " gene pool ".In recent years, farsighted medicine scholar of developed countries such as American and Britain, day and drugmaker all transfer to fungi with attention from actinomycetes etc. comes up, wherein " Chinese caterpillar fungus " because of its exclusive pharmacology is worth, " attracting attention " more induces one again.
The development of resources of Chinese caterpillar fungus class not only has important social value, and considerable economic is arranged.Because different Chinese caterpillar funguses contains different biologically active substances, and the pharmacological action difference, thereby a kind of discovery of new cordyceps species, often mean a kind of generation of new biological product.
The objective of the invention is to: a kind of Chinese new record microorganism pure growth---mortierella Diding and isolation cultivation method thereof and fermentation technology with food, drug development purposes is provided.
Described goal of the invention realizes by following proposal.
CGMCC0346 goes up the hyphomycete that an isolated strain produces coremium from the infected insect polypide of interior sclerotium of picking up from the little spore mutation of Cordyceps gunnii (Berk.) Berk. [Cordyceps gunnii var.minor] that the wilderness area falls in Qimen, Anhui bull, belongs to first in China and finding.
1, CGMCC0346 is Chinese new record kind, it is characterized in that:
A, on the MA substratum, the cultivation form of CGMCC0346 is 25 ℃ cultivated 10 days, the bacterium colony circle, diameter 13～21mm, white, the middle part powdery, the edge is velvet-like, forms coremium sometimes, the back side is fallow;
B, on czapek's solution, the cultivation form of CGMCC0346 is 25 ℃ cultivated 10 days, bacterium colony garden shape, diameter 21～22mm forms coremium, white, the edge incarnadine, back side cream color is to ochre yellow.Its microscopic morphology is: hyphae colorless, smooth, tool barrier film, wide 1.5 μ m; Upright the connecing from mycelia of bottle grows taper, many Dan Sheng, 10.8～19.8 * 1.8～2.9 μ m; Conidium is colourless, smooth, forms the exsiccant chain, long fusiform, two ends point, 3.6～5.8 * 0.9～1.4 μ m, length-to-diameter ratio (L/D) 3～3.5.
Above-mentioned CGMCC0346 is characterized in that:
Described MA substratum is: wort 30g, agar 15g, water 1000ml.
Above-mentioned CGMCC0346 is characterized in that:
Above-mentioned czapek's solution is: NaNO
32g, K
2HPO
32g, KCL 0.5g, MgSO
44g, FeSO
40.01g, sucrose 20g, agar 20g, water 1000ml.
2, the method for a kind of separation and Culture CGMCC0346 is characterized in that:
2.1, in Bechtop, under aseptic condition, with sterilized water with the long fresh worm corpse flushing that the Chinese caterpillar fungus stroma arranged three times;
2.2, have the fresh worm corpse of Chinese caterpillar fungus stroma carefully to cut with the aseptic operation blade with long, in this operating process, keep off brokenly the insect dorsal vessel;
2.3, in incision site, take out the worm corpse piece of small rice grain size, be inoculated on the SDAY substratum plate of sterilization;
2.4, place the natural light of 18 ℃ of illumination boxs to cultivate 14～16 days down postvaccinal substratum plate;
2.5, wait to grow bacterium colony after, transfer in another SDAY substratum plate with the inoculating needle mycelia that takes a morsel;
2.6, the substratum plate after will transferring places the natural light of 18 ℃ of illumination boxs to cultivate down, until the bacterium colony that grows single purifying;
2.7, with the bacterial classification point of purifying plant in sterilization, diameter is that 9cm, every ware have on the plate of 15ml MA liquid medium, cultivated 10 days for 25 ℃;
Or with the bacterial classification point of purifying plant in sterilization, have on the plate of czapek's solution liquid, cultivated 10 days for 25 ℃.
The method of a kind of separation and Culture CGMCC0346 is characterized in that: described SDAY substratum is glucose 40g, peptone 10g, yeast extract powder 10g, agar 20g, water 1000ml.
The method of a kind of separation and Culture CGMCC0346 is characterized in that: described MA substratum is bud juice 30g, agar 15g, water 1000ml.
The method of a kind of separation and Culture CGMCC0346 is characterized in that: described czapek's solution is NaNO
32g, K
2HPO
32g, KCL 0.5g, MgSO
44g, FeSO
40.01g, sucrose 20g, agar 20g, water 1000ml.
3, a kind of fermentation technology of mortierella Diding is characterized in that:
3.1, with the bacterial classification inoculation of purifying in the SDAY medium slant, cultivated 7 days down, after waiting to produce a large amount of coremiums, promptly can be used as the liquid shaking bottle bacterial classification for 24～26 ℃;
3.2, above-mentioned two slant strains are inoculated in one in the 500ml triangular flask of dress liquid nutrient medium 200ml, inoculum size 5～10%, under 24～26 ℃, placed on the rotary shaking table 150～160r/m shaking culture 24～26 hours, promptly obtain the liquid seeds of first class seed pot;
3.3, in 50 liters of column plate type fermentor tanks, add 30 liters of liquid nutrient mediums, and add 0.03% defoamer bubble enemy, 14.71 * 10
4Under the Pa pressure, pressurize 30 minutes after the sterilization cooling, inserts cultivation at 24～26 ℃ with shake-flask seed, air flow 1: 0.5, and 4.903～7.0845 * 10
4Pressurize under the Pa, and through 48～60 hours aerated culture.
The fermentation technology of a kind of CGMCC0346 is characterized in that: described SDAY substratum is glucose 40g, yeast extract powder 10g, peptone 10g, agar 20g, water 1000ml.
The fermentation technology of a kind of CGMCC0346 is characterized in that: described liquid nutrient medium is a white sugar 1.5%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0%, water 95%, and PH6.0-7.0.
The fermentation technology of a kind of CGMCC0346 is characterized in that: the liquid nutrient medium in the fermentor tank is: white sugar 2.0%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0%, water 94.5%.
Bacterial classification of the present invention has been defined in specified depositary institution of Patent Office of State Intellectual Property Office---China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation by the 25 of patent law detailed rules for the implementation, preservation date: on April 22nd, 1998, preserving number: CGMCC0346, and attach the viability report.
This bacterial classification has been used for industrial fermentation production.The mycelium capsule that utilizes this bacterial classification to produce through the functional examination that Jiangsu Sanitation and Antiepidemic Station did, has tangible immunologic function (cellular immunization, humoral immunization, mononuclear-macrophage phagocytic function), meets the immunologic function judgement criteria of protective foods.Measure through Inst., of Clinical Pharmacology, Anhui Medical Univ., its mycelium also has anti rheumatism action.By the exocellular polysaccharide that extracts in its fermented liquid, measure through Inst., of Clinical Pharmacology, Anhui Medical Univ., tangible antihepatitic activity is arranged.In with the anti-ageing test that fruit bat did, can obviously prolong life span of drosophila melanogaster, show fabulous anti-aging effects.The possibility that is developed as protective foods and medicine is arranged.
Below the present invention is further described.
This bacterium is separated from soil in nineteen fifty-seven first by Miller, and it is belonged in the Fusidium genus, names the Miller into Fusidium terricola, Giddens Foster.Onions in 1967 etc. are renamed as Paecilomyces terricola (Miller et al.) Onions Barron with it.Gams general's in 1971 commentaries on classics belongs in the mould genus of top spore (Acremonium), and names the Gams into Acremonium terricola (Miller et al.).
The original description of Miller when nineteen fifty-seven is delivered novel species is:
On the MA substratum, cultivated 10 days colony diameter 12～22mm, positive floury, white, back side tawny for 25 ℃.It is coremium that mycelia assembles, hyphae colorless, the most Dan Sheng of bottle stalk, very thin, taper, 12～20～(25) * 1.2～2.0 μ m, top 0.5～1.0 μ m, conidium forms the exsiccant long-chain, and is colourless, smooth, long fusiform, the two ends point, 3.8～5.4～(6.0) * 0.9～1.5 μ m, L/D 2.8～5.0.
The CGMCC0346 of this discovery belongs to Chinese new record kind.Determining of the evaluation of bacterial classification and new record kind mainly determined with reference to relevant classified documents with microscopic morphology according to its cultural characters.
The cultural characters of this China new record kind CGMCC0346: the bacterial classification point of purifying is planted in sterilization (1.5Kg/cm
230 minutes) MA substratum plate on, cultivated 10 days for 25 ℃; The bacterium colony circle, diameter 13～21mm, white, the middle part powdery, the edge is velvet-like, forms coremium sometimes, and the back side is fallow.Described MA substratum is: wort 30g, agar 15g, water 1000ml, described plate is: the culture dish of diameter 9cm, every ware 15ml substratum.The bacterial classification point of purifying is planted on the czapek's solution of sterilization, cultivated 10 days for 25 ℃: the bacterium colony circle, diameter 21～22mm forms coremium, white, the edge incarnadine, back side cream color is to ochre yellow.Hyphae colorless, smooth, tool barrier film, wide 1.5 μ m.Upright the connecing from mycelia of bottle grows taper, many Dan Sheng, 10.8～19.8 * 1.8～2.9 μ m.Conidium is colourless, smooth, forms the exsiccant chain, long fusiform, two ends point, 3.6～5.8 * 0.9～1.4 μ m, length-to-diameter ratio (L/D) 3～3.5.
The separation and Culture condition of CGMCC0346 is: in Bechtop, to there be the fresh worm corpse of Chinese caterpillar fungus stroma carefully to cut with the length of aseptic water washing three times with the aseptic operation blade, in this operating process, note keeping off brokenly the insect dorsal vessel, take out the worm corpse piece of small rice grain size, be inoculated on the SDAY substratum plate of sterilization (1.5Kg/cm30 minute), place 18 ℃ of illumination boxs (under the natural light) to cultivate 15 days, wait to grow behind the bacterium colony with a small amount of mycelia of inoculating needle picking (SDAY substratum) the same method of transferring in another plate and cultivate, till the bacterium colony that grows single purifying.Described SDAY substratum is: glucose 40g, peptone 10g, yeast extract powder 10g, agar 20g, water 1000ml; Described plate is: diameter 9cm, every ware 15ml substratum.
The fermentation technology of CGMCC0346 was divided into for three steps, and the one, the primary inclined plane spawn culture; The 2nd, liquid seeds is cultivated; The 3rd, fermentor cultivation.Following proceed step by step explanation:
1, primary inclined plane spawn culture
The preservation bacterial classification inoculation in the SDAY medium slant, was cultivated 7 days down for 24～26 ℃, wait to produce a large amount of coremiums after, promptly can be used as the liquid shaking bottle bacterial classification.Described SDAY substratum is: glucose 40g, yeast extract powder 10g, peptone 10g, agar 20g, water 1000ml.
2, liquid seeds is cultivated
Connect in one the 500ml triangular flask of dress liquid nutrient medium 200ml with two slant strains, inoculum size 5～10% under 24～26 ℃, places rotary shaking table (160r/m) to go up shaking culture 24～36 hours, promptly can be used as the liquid seeds of first class seed pot.Described liquid nutrient medium is: white sugar 1.5%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0%, water 95%; Its pH value is 6.0～7.0.
3, fermentor cultivation
50 liters of column plate type fermentor tanks, calculate to cultivate 30 liters of liquid spawns, in fermentor tank, add white sugar 2.%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0% then adds entry 95.5%, water temperature is more than or equal to 95 ℃, and adding defoamer bubble enemy as polyoxyethylene oxypropylene glycerine, consumption is 0.03%, stirs.14.71 * 10
4Under the Pa pressure, pressurize 30min, sterilization is cooled to 24～26 ℃, shake-flask seed is inserted cultivate, and air flow 1: 0.5 is 4.903～7.0845 * 10
4Pressurize under the Pa, through 48～60 hours aerated culture, the mycelia raised growth also began to produce spore and can put jar.
Claims (9)
1, mortierella Diding (Acremonium terricola, CGMCC0346) is Chinese new record kind, it is characterized in that:
A, on the MA substratum, the microscopic morphology of CGMCC0346 is the bacterium colony circle, diameter 13～21mm, white, the middle part powdery, the edge is velvet-like, forms coremium sometimes, the back side is fallow;
B, on czapek's solution, the microscopic morphology of CGMCC0346 is diameter 21～22mm, forms coremium, white, the edge incarnadine, back side cream color is to ochre yellow; Hyphae colorless, smooth, tool barrier film, wide 1.5 μ m; Upright the connecing from mycelia of bottle grows taper, many Dan Sheng, 10.8～19.8 * 1.8～2.9 μ m; Conidium is colourless, smooth, forms the exsiccant chain, long fusiform, two ends point, 3.6～5.8 * 0.9～1.4 μ m, length-to-diameter ratio (L/D) 3～3.5.
2, a kind of method of separation and Culture mortierella Diding is characterized in that:
2.1, in Bechtop, under aseptic condition, with sterilized water with the long fresh worm corpse flushing that the Chinese caterpillar fungus stroma arranged three times;
2.2, have the fresh worm corpse of Chinese caterpillar fungus stroma carefully to cut with the aseptic operation blade with long, in this operating process, keep off brokenly the insect dorsal vessel;
2.3, in incision site, take out the worm corpse piece of small rice grain size, be inoculated on the SDAY substratum plate of sterilization;
2.4, place the natural light of 18 ℃ of illumination boxs to cultivate 14～16 days down postvaccinal substratum plate;
2.5, wait to grow bacterium colony after, transfer in another SDAY substratum plate with the inoculating needle mycelia that takes a morsel;
2.6, the substratum plate after will transferring places the natural light of 18 ℃ of illumination boxs to cultivate down, until the bacterium colony that grows single purifying;
2.7, with the bacterial classification point of purifying plant in sterilization, diameter is that 9cm, every ware have on the plate of 15ml MA substratum, cultivated 10 days for 25 ℃; Or with the bacterial classification point of purifying plant in sterilization, on the plate with czapek's solution, cultivated 10 days for 25 ℃.
3, the method for a kind of separation and Culture mortierella Diding according to claim 2 is characterized in that: described SDAY substratum is: glucose 40g, peptone 10g, yeast extract powder 10g, agar 20g, water 1000ml.
4, the method for a kind of separation and Culture mortierella Diding according to claim 2 is characterized in that: described MA substratum is: wort 30g, agar 15g, water 1000ml.
5, the method for a kind of separation and Culture mortierella Diding according to claim 2 is characterized in that: described czapek's solution is NaNO
32g, K
2HPO
32g, KCL 0.5g, MgSO
44g, FeSO
40.01g, sucrose 20g, agar 20g, water 1000ml.
6, a kind of fermentation technology of mortierella Diding is characterized in that:
6.1, with the bacterial classification inoculation of purifying in the SDAY medium slant, cultivated 7 days down at 24～26 ℃, after waiting to produce a large amount of coremiums, promptly can be used as the liquid shaking bottle bacterial classification;
6.2, above-mentioned two slant strains are inoculated in one in the 500ml triangular flask of dress liquid nutrient medium 200ml, inoculum size 5～10%, under 24～26 ℃, placed on the rotary shaking table 150～160r/m shaking culture 24～26 hours, promptly obtain the liquid seeds of first class seed pot;
6.3, in 50 liters of column plate type fermentor tanks, add 30 liters of liquid nutrient mediums, and add 0.03% defoamer bubble enemy, 14.71 * 10
4Under the Pa pressure, pressurize 30 minutes after the sterilization cooling, inserts cultivation at 24～26 ℃ with shake-flask seed, air flow 1: 0.5, and 4.903～7.0845 * 10
4Pressurize under the Pa, and through 48～60 hours aerated culture.
7, the fermentation technology of a kind of mortierella Diding according to claim 6 is characterized in that: described SDAY substratum is: glucose 40g, yeast extract powder 10g, peptone 10g, agar 20g, water 1000ml.
8, the fermentation technology of a kind of mortierella Diding according to claim 6 is characterized in that: the liquid nutrient medium in the described triangular flask is: white sugar 1.5%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0%, water 95%, and pH value 6.0-7.0.
9, the fermentation technology of a kind of mortierella Diding according to claim 6 is characterized in that: the liquid nutrient medium in the fermentor tank is: white sugar 2.0%, dried silkworm chrysalis meal 2.5%, yeast powder 1.0%, water 94.5%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195804B (en) * | 2006-12-05 | 2011-02-09 | 上海中医药大学 | Huperzia serrata endogenetic epiphyte and uses thereof |
| CN102168108A (en) * | 2010-12-17 | 2011-08-31 | 广东省微生物研究所 | Cordyceps cardinalis, method for preparing oosporin by using same and application of Cordyceps cardinalis |
| CN101797014B (en) * | 2009-02-10 | 2013-08-07 | 李晓祥 | Production method and products of nutritive eggs with low cholesterol |
| CN103734482A (en) * | 2014-01-20 | 2014-04-23 | 安徽康智生物科技有限责任公司 | Production method of feed additive 'Acremonium terricola culture' |
| CN103756916A (en) * | 2014-01-14 | 2014-04-30 | 合肥迈可罗生物工程有限公司 | Acremonium terricola mutant strain and application thereof |
| CN107475132A (en) * | 2017-09-20 | 2017-12-15 | 广东容大生物股份有限公司 | A kind of mortierella Diding culture and its application |
| CN109182135A (en) * | 2018-08-30 | 2019-01-11 | 广东容大生物股份有限公司 | A kind of mortierella Diding Shake flask medium and its application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1095103A (en) * | 1993-05-07 | 1994-11-16 | 中国中医研究院 | Producing process of Chinese caterpillar fungus hypha fermentation |
| JPH0853387A (en) * | 1994-08-12 | 1996-02-27 | Sankyo Co Ltd | New compound f-11263 |
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1999
- 1999-10-27 CN CN99114522A patent/CN1091150C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195804B (en) * | 2006-12-05 | 2011-02-09 | 上海中医药大学 | Huperzia serrata endogenetic epiphyte and uses thereof |
| CN101797014B (en) * | 2009-02-10 | 2013-08-07 | 李晓祥 | Production method and products of nutritive eggs with low cholesterol |
| CN102168108A (en) * | 2010-12-17 | 2011-08-31 | 广东省微生物研究所 | Cordyceps cardinalis, method for preparing oosporin by using same and application of Cordyceps cardinalis |
| CN102168108B (en) * | 2010-12-17 | 2013-04-24 | 广东省微生物研究所 | Cordyceps cardinalis, method for preparing oosporin by using same and application of Cordyceps cardinalis |
| CN103756916A (en) * | 2014-01-14 | 2014-04-30 | 合肥迈可罗生物工程有限公司 | Acremonium terricola mutant strain and application thereof |
| CN103734482A (en) * | 2014-01-20 | 2014-04-23 | 安徽康智生物科技有限责任公司 | Production method of feed additive 'Acremonium terricola culture' |
| CN103734482B (en) * | 2014-01-20 | 2016-05-11 | 安徽康智生物科技有限责任公司 | The production method of a kind of feed addictive " mortierella Diding culture " |
| CN107475132A (en) * | 2017-09-20 | 2017-12-15 | 广东容大生物股份有限公司 | A kind of mortierella Diding culture and its application |
| CN107475132B (en) * | 2017-09-20 | 2020-10-13 | 广东容大生物股份有限公司 | Acremonium terricola culture and application thereof |
| CN109182135A (en) * | 2018-08-30 | 2019-01-11 | 广东容大生物股份有限公司 | A kind of mortierella Diding Shake flask medium and its application |
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