CN102533567B - Paecilomyces tenuipes mutagenesis bacterial strain and culturing method thereof - Google Patents
Paecilomyces tenuipes mutagenesis bacterial strain and culturing method thereof Download PDFInfo
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Abstract
A mutagenic new paecilomyces tenuipes bacterial strain subcultured for more than 10 times can not retrograde, and has genetic stability, and the contents of intracellular polysaccharide, adenosine and mannitol are many times more than those of original bacteria.
Description
Technical field
The present invention discloses a kind of paecilomyces tenuipes mutagenic strain, is a kind of new bacterial strain kind, and the present invention also provides the method for cultivation of this bacterial strain, belongs to microbial technology field.
Background technology
Paecilomyces tenuipes
Paecilomyces tenuipes(Peck) Samson is a kind of common insect pathogenic fungus, is under the jurisdiction of the lepidopteran Deuteromycotina in the classification, hyphomycetes, and paecilomyces is mountain, Kaohsiung anamorph of Cordyceps.Doing check analysis with natural cordyceps shows, the Main chemical component of paecilomyces tenuipes such as sterols, glycitols, alkaloid, organic acid and trace element and the former are quite similar, amino acid contained kind is also identical, but multiple amino acids content is higher than Cordyceps sinensis.The pharmacological action aspect studies show that paecilomyces tenuipes has antitumor, antibiotic, anti-oxidant and immunoloregulation function, has good prospect in medicine and exploitation value.
In the present invention, seed selection one strain paecilomyces tenuipes strain excellent, polysaccharide, N.F,USP MANNITOL and adenosine content all are significantly increased than the wild-type paecilomyces tenuipes in its mycelium, and be significant to its further investigation and widespread use.
Summary of the invention
The object of the present invention is to provide a kind of paecilomyces tenuipes mutagenic strain, be a kind of new bacterial strain, polysaccharide, adenosine, mannitol content are compared with wild mushroom and are significantly increased in its mycelium.
The present invention also provides the method for cultivation of this bacterial strain, is applicable to suitability for industrialized production.
Paecilomyces tenuipes mutagenic strain of the present invention: in the center preservation of Chinese Typical Representative culture collection, preservation address: Wuhan Wuhan University, preservation date: on April 28th, 2011, specific name: paecilomyces tenuipes N45 Paecilomyces tenuipes Samson N45; Preservation registration number is CCTCC NO.M 2011145.
Paecilomyces tenuipes bacterial strain method of cultivation of the present invention may further comprise the steps:
1) with the paecilomyces tenuipes original strain
Paecilomyces tenuipesSamson Pt196 is inoculated in the PDA inclined-plane, cultivates 3-7 days in the 24-26 ℃ of thermostat container, injects stroke-physiological saline solution in slant tube, filters with aseptic absorbent cotton after the vibration, and being diluted to concentration is 3 * 10
7The spore suspension of individual/mL;
2) get the spore suspension 5mL for preparing, change over to and carry out autoclaving in the sterilization packaging bag.After processing 30min under 26 ℃ of temperature, the pressure 200M Pa condition.Get spore suspension stepwise dilution in sterile distilled water after the autoclaving, the PDA culture medium flat plate was cultivated after 3-4 days, and bacterial strain is preserved in 96 orifice plates;
3) utilize the dull and stereotyped preliminary screening rapidly bacterial strain of growing;
4) will screen the bacterial strain that obtains and in shaking flask, recover, go down to posterity rear 26 ℃, cultivate 4 days in the 150rpm constant-temperature table, get mycelium, measure mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, the paecilomyces tenuipes bacterial strain of screening high yield.
5) the high yield paecilomyces tenuipes mutagenic strain that obtains is through continuous passage, culture condition is 26 ℃, cultivates 96h in the 150rpm constant-temperature table, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, investigate the genetic stability of mutagenic strain, thereby finally determine the purpose bacterial strain.
The present invention cultivates the substratum of paecilomyces tenuipes bacterial strain, it is characterized in that being made by ratio of weight and the number of copies by following medicine:
Glucose 40 g/L, extractum carnis 10 g/L, soy peptone 10 g/L, KH
2PO
40.688 g/L, MgSO
47H
2O 1 g/L, NaCl 0.5 g/L, VB
10.201 g/L, VB
120.130 g/L;
Being characterized as of paecilomyces tenuipes mutagenic fungi of the present invention:
1) content of paecilomyces tenuipes mutagenic strain mycelium dry weight and adenosine, four kinds of effective constituents of Crude polysaccharides and N.F,USP MANNITOL is compared all with original strain and is significantly improved, wherein, the thalline soma 15.83g/L that weighs, the more former bacterium that sets out has improved 3.735%; Adenosine content can reach 0.054 g/L, and the more former bacterium that sets out has improved 80%; Polysaccharide content can reach 0.542 g/L, and the more former bacterium that sets out has improved 44.15%; Mannitol content can reach 2.019 g/L, and the more former bacterium that sets out has improved 78.989%.
2) the paecilomyces tenuipes mutagenic strain is cultivated through the continuous passage more than 20 times and is not degenerated, and has genetic stability.
Positively effect of the present invention is: the new bacterial strain of the paecilomyces tenuipes of mutagenesis is cultivated through going down to posterity more than 10 times and is not degenerated, and have genetic stability, and intracellular polyse, adenosine and mannitol content is far away than high times of original bacterium.
Description of drawings
Fig. 1, carbon source are on the impact of expected value;
Fig. 2, nitrogenous source are on the impact of expected value;
Fig. 3, inorganic salt are on the impact of expected value;
Response surface and isogram between Fig. 4, each factor;
Fig. 5, hidden layer node number with
DThe a correlogram;
Fig. 6, genetic algebra and match value correlogram.
Specific implementation method
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood to be to explaination of the present invention but not to any type of restriction of the present invention.
Embodiment 1: the mutagenesis of paecilomyces tenuipes mutant strain and selection
(1) paecilomyces tenuipes mutagenesis
With the paecilomyces tenuipes original strain
Paecilomyces tenuipesSamson Pt196 is inoculated in the PDA inclined-plane, cultivates 5 days in 26 ℃ of thermostat containers, injects stroke-physiological saline solution 1.5mL in slant tube, filters with aseptic absorbent cotton after the vibration, and being diluted to concentration is 3 * 10
7The spore suspension of individual/mL.Under aseptic condition, pack in the aluminium foil Polypropylene Bag, seal.After processing 30min under 26 ℃ of temperature, the pressure 200M Pa condition, get spore suspension stepwise dilution in sterile distilled water, the PDA culture medium flat plate was cultivated after 4 days, and bacterial strain is preserved in 96 orifice plates.Toothpick with sterilization is chosen bacterial strain in 96 orifice plates on the flat board of PDA substratum, and 20 bacterium colonies of every plate were cultivated 3 days in 26 ℃ of constant incubators; The more rapidly bacterium colony of will growing is chosen in the PDA substratum again, about every plate 10 strains, cultivates 3 days in 26 ℃ of constant incubators; With growth more rapidly bacterium colony choose in the shaking flask that fills the PDA substratum recovery 5 days; Strain passage is cultivated, and in the 250mL Erlenmeyer flask that fills 100mL PDA substratum, inoculation paecilomyces tenuipes mutagenic strain bacteria suspension 2mL cultivates 4d in 26 ℃ of constant-temperature tables.The mutant strain that mutagenesis is obtained is stored in 48 orifice plates, centrifugal mycelium, and 5000rpm, centrifugal 10min washes once, and mycelium is laid on dull and stereotyped freeze-drying, and lyophilized powder is weighed, and pulverizes, and crosses 60 mesh sieves.
The extraction of (2) four kinds of effective constituents and the mensuration of content
Take by weighing paecilomyces tenuipes mycelium dry powder 0.1g, add 5mL distilled water, 80 ℃ of water-bath lixiviate 3h, centrifugal 10 min of 8000 g/min get supernatant and measure mycelium polysaccharides content.Take by weighing paecilomyces tenuipes mycelium dry powder 0.1g, add 5mL distilled water, 45 ℃ of water-bath lixiviate 3h, centrifugal 10 min of 8000 g/min get supernatant and measure mycelium adenosine and mannitol content.Adopt anthracene copper-sulfuric acid process to measure total sugar content; The DNS method is measured reducing sugar content, polysaccharide content=total sugar content-reducing sugar content; Adopt the HPLC method to measure adenosine content in the paecilomyces tenuipes mycelium; Utilize the spectrophotometry mannitol content.
(3) screening of high yield mutagenic fungi
From the paecilomyces tenuipes mutant of mutagenesis, filter out mycelium dry weight and four kinds of strain excellents (seeing Table 1) that active constituent content obviously improves, then the cultivation of going down to posterity, measure bacterial strain dry weight and three kinds of active constituent contents every a generation, the same step of measuring method (2), cultivated altogether for 10 generations, investigate the genetic stability feature that screening obtains the high yield mutagenic fungi.
Table 1 mutagenic strain four indices improvement value
| Index | Dry weight (g/L) | Intracellular polyse (g/L) | Adenosine (g/L) | N.F,USP MANNITOL (g/L) |
| Original strain | 15.260 | 0.376 | 0.030 | 1.128 |
| N-45 | 15.830 | 0.542 | 0.054 | 2.019 |
| Improvement value (%) | 3.735 | 44.150 | 80.000 | 78.989 |
Embodiment 2:
The optimization of paecilomyces tenuipes mutagenic strain fermention medium
(1) sets up expectation function
Take the output of intracellular polyse, dry cell weight, adenosine and cordycepic acid as investigating index, adopt expectation function with a plurality of investigation index comprehensives be one investigate the index expected value (
DV), paecilomyces tenuipes mutagenic strain fermention medium is optimized, each is investigated index (response value) and all converts nondimensional expected value to by formula (1), the balance yardstick d scope of expectation function is 0 ~ 1, explanation response value substantial deviation target value during d=0, d=1 explanation response value and target value approach.D increases and increases along with desired response value.
（1）
wI is the weighted value of i index.
The investigation index of table 2 paecilomyces tenuipes N45 fermentation and the minimum response value of expectation, the highest response value and weighted value
| Index | Intracellular polyse/g.L -1 | Dry weight/g.L -1 | Adenosine/g.L -1 | Cordycepic acid/g.L -1 |
| L i | 0.20 | 10.00 | 0.01 | 0.60 |
| E i | 1.50 | 35.00 | 0.15 | 5.00 |
| w i | 0.30 | 0.10 | 0.35 | 0.25 |
(2) experiment of single factor
In the process of medium optimization, utilize experiment of single factor that the carbon source, nitrogenous source and the Inorganic Salts that affect expected value are investigated.
1. carbon source kind screening
Take the PDA substratum as basic medium, adopt respectively 40 g/L sucrose, fructose, glycerine, maltose, lactose and glucose as carbon source, the preparation substratum, the substratum liquid amount is 100mL/250mL, respectively with 5% inoculum size access seed liquor, 150rpm, 26 ℃ cultivate 4d.Measure each index components content under every kind of carbon source culture medium condition, calculate
DThe v value is to select suitable carbon source kind.The result as shown in Figure 1, when adopting glucose as carbon source, its expected value
DV is the highest, and therefore selecting glucose is optimum carbon source.
2. nitrogenous source kind screening
Take the PDA substratum as basic medium, adopt respectively 20 g/L soy peptones, yeast extract paste, extractum carnis, peptone and yeast to soak powder as nitrogenous source, the preparation substratum, the substratum liquid amount is 100mL/250mL, inoculum size access seed liquor with 5%, 150rpm, 26 ℃ of cultivation 4d.Measure each index components content under every kind of nitrogenous source culture medium condition, calculate
DThe v value.The result as shown in Figure 2, when with the extractum carnis of 20g/L during as nitrogenous source, expected value is high than the expected value of other several nitrogenous source substratum, therefore selects extractum carnis as optimum nitrogenous source.On the basis of nitrogenous source kind screening, consider the content of each effective constituent, select compound nitrogen source to carry out subsequent experimental, and to utilize experiment of single factor screening compound nitrogen source be the optimum proportion of extractum carnis and soy peptone, the optimum proportion of compound nitrogen source is 1:1.
3. the screening of inorganic ion kind
On the basis of the suitable carbon and nitrogen sources kind of screening, in substratum, add respectively following inorganic salt: CuSO
45H
2O, ZnCl
2, MnCl
24H
2O, FeCl
36H
2O, KH
2PO
4, MgSO
47H
2O, CaCl
2And NaCl, every kind of inorganic salt are made as 0.001%, 0.05% and 0.1% 3 concentration.Measure each index components content in the mycelium, calculate
DThe v value is with the suitableeest inorganic ion kind of screening.The result as shown in Figure 3, the optimal inorganic salts of paecilomyces tenuipes N45 fermention medium is 0.05% KH
2PO
44H
2O.
(3) partial factors experiment
According to the experiment of single factor result, adopt 9 factors, 2 horizontal component factorial experiments, in the screening culture medium expected value is affected significant factor.Experimental data is carried out simple linear regression analysis, obtains a linear model,
FCheck checks the linear model significance, thus near the peak response zone.The result shows, KH
2PO
4, VB
1And VB
12That expected value on paecilomyces tenuipes N45 shake flask fermentation affects significant factor.Therefore, select KH
2PO
44H
2O, VB
1And VB
123 factors are further optimized.
(4) Box-Behnken contrived experiment
On above-mentioned experimental result basis, adopt the Box-Behnken contrived experiment of 3 factors, 3 levels further to optimize KH
2PO
4, VB
1And VB
12The optimum concn of 3 factors obtains the best medium prescription.Adopt respectively even experiment design (result as shown in Figure 4) and neural network model (result such as Fig. 5, shown in Figure 6) to come the match experimental data, obtain the best medium prescription through software analysis and be (g/L): glucose 40, extractum carnis 10, soy peptone 10, KH
2PO
40.688, MgSO
47H
2O 1, NaCl 0.5, VB
10.201, VB
120.130.
Claims (1)
1. paecilomyces tenuipes mutagenic strain, (
Paecilomyces tenuipesSamson), be preserved on May 5th, 2011 that " Chinese Typical Representative culture collection " center ", its preserving number is: CCTCC NO:M 2011145.
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