CN105039170A - High-yield Tricholoma matsutake sing mutagenesis strain and breeding method thereof - Google Patents
High-yield Tricholoma matsutake sing mutagenesis strain and breeding method thereof Download PDFInfo
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- 238000002703 mutagenesis Methods 0.000 title claims abstract description 18
- 238000009395 breeding Methods 0.000 title abstract 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 30
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a Tricholoma matsutake sing mutagenesis strain and a breeding method thereof. According to the present invention, a new high-yield Tricholoma matsutake sing strain is obtained through chemical mutagenesis screening separation, wherein the preservation number is CCTCC N0:2014671, the mycelium dry weight is increased by 69.68% compared with the original strain, the adenosine content is increased by 33.96%, the polysaccharide content is increased by 75.72%, the protein content is increased by 35.17%, and the mannitol content is increased by 52.8%; and after subculturing is performed on the new Tricholoma matsutake sing strain more than or equal to 10 times, no degeneration is generated, and the genetic stability is provided. The present invention further discloses an optimal fermentation culture medium of the Tricholoma matsutake sing mutagenesis strain.
Description
Technical field
The present invention relates to a kind of high yield matsutake mutagenic strain, be a kind of new strain variety, present invention also offers the method for cultivation of this bacterial strain, belong to bio-fermentation engineering field.
Background technology
Matsutake (TricholomamatsutakeSing.) belongs to basidiomycetes, Basidiomycetes, Agaricales, Tricholomataceae, Tricholoma, has another name called China fir gill fungus, loose mushroom, closes bacterium, platform bacterium, loose bacterium.Matsutake uses with a long history, and distribution on global is extensive, and the delicate plumpness of meat, is of close texture, and have strong special aroma, it is nutritious, containing effective constituents such as protein, amino acid, multivitamin, carbohydrate and mineral substance, is famous and precious wild mushrooms.The primary bioactivity material of matsutake is polysaccharide, polypeptide and sterol etc., and the research of pharmacological action aspect shows that matsutake has antitumor, immunomodulatory, anti-oxidant, the function such as radioprotective and treatment diabetes, has good prospect in medicine and Development volue.
Current matsutake is mainly derived from wild harvesting, and artificial culture is very difficult, and a large amount of market requirements causes matsutake resource exhaustion, ecotope suffers to destroy and causes price high simultaneously, and this just brings very large obstacle to industry chain of Tricholoma spp development.Adopt modern biotechnology, by the artificial modified of fermentation strain and optimization two kinds of approach of fermentation technology process, obtain the effective product of matsutake fast and effectively and develop barrier at present to break, for industry chain of Tricholoma spp exploitation lays the foundation.
Summary of the invention
The object of the present invention is to provide a kind of matsutake mutagenic strain, be a kind of new bacterial strain, its mycelium dry weight, polysaccharide, adenosine, mannitol content are compared with wild mushroom and are significantly increased.
Present invention also offers the method for cultivation of this bacterial strain, be applicable to suitability for industrialized production.
Matsutake mutagenic strain provided by the present invention: in China typical culture collection center preservation, preservation address: Wuhan University, preservation date: on December 25th, 2014, specific name: matsutake T35TricholomamatsutakeT35; Preservation registration number is CCTCCNO:M2014671.
Matsutake bacterial strain method of cultivation of the present invention, comprises the following steps:
(1) be CGMCC5.793 matsutake bacterial strain by preserving number, be inoculated in PDA inclined-plane after activation, in 23-27oC thermostat container, cultivate 4-8 days, pH6.0 phosphoric acid buffer is injected in slant medium, then use 4 layers of filtered through gauze, dilute spore concentration to 106-107/mL, for selection by mutation;
(2) 10mg nitrosoguanidine is taken in the culture dish of diameter 7cm, add cosolvent acetone a little after, add sterile phosphate buffer 9mL (pH=6.0), open magnetic stirring apparatus slowly to stir, after NTG fully dissolves, draw 1mL bacteria suspension put into plate and start mutagenesis (nitrosoguanidine final concentration is lmg/mL).Respectively after mutagenesis 2min, 5min, 10min, 15min and 20min, get 0.3mL bacteria suspension in 2.7mL sterile distilled water and stepwise dilution;
(3) to the spore suspension after mutagenesis, carry out gradient dilution, PDA culture medium flat plate was cultivated after 3-7 days, is equipped with by the single bacterium colony grown in 96 orifice plates of PDA solid medium cultivates with the point-to-point implantation of sterilized toothpick.Be inoculated in one by one in 250mL Erlenmeyer flask by cultured mutant strain inoculating needle and cultivate, 26oC150rpm cultivates and obtains tricholoma matsutake mycelium in 4-6 days, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, the matsutake bacterial strain of screening high yield;
(4) the high yield matsutake mutagenic strain obtained is through continuous passage, culture condition is cultivate 90-120h in 26oC, 150rpm constant-temperature table, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, investigate the genetic stability of mutagenic strain, thus finally determine object bacterial strain.
This matsutake mutagenic strain T35 mycelial growth is quick, and mycelium is white, and can carry out quick liquid deep drainpipe, the liquid submerged fermentation feature of this bacterial strain is as follows:
(1) primary inclined plane bacterial classification
Slant medium: glucose 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-1g/L, KH2PO40.1-0.7g/L, MgSO7.H2O0.1-0.6g/L, vitaminB10 .05-0.5g/L, agar 25g/L;
Slant strains culture temperature 26 ± 1oC, within 4-7 days, mycelia covers with inclined-plane, puts refrigerator (2-4oC) and saves backup;
(2) second-level shake flask bacterial classification
Seed culture medium: glucose 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, KH2PO40.1-0.7g/L, MgSO7.H2O0.1-0.6g/L, vitaminB10 .05-0.5g/L;
Seed culture method: inclined-plane mother is planted access liquid seed culture medium, at the shaking table of 26 ± 10C, 120-160r/min, cultivates 5d;
(3) shake flask fermentation is cultivated
By seed liquor by 5% inoculum size access liquid fermentation medium (glucose 18-25g/L, yeast leaching powder 15-25g/L, KH2PO40.3-0.6g/L, MgSO7.H2O0.3-0.6g/L, vitaminB10 .05-0.3g/L) in carry out liquid fermenting, fermention medium liquid amount is shaking flask dress 100mL, the culture temperature 260C shaking speed of 250mL is 150rpm, gathers in the crops mycelium after cultivating 5d;
(4) 100L fermentor tank amplification culture
By seed liquor by 7% inoculum size access 60L liquid fermentation medium, in 100L fermentor tank, carry out liquid submerged fermentation cultivation, 26oC, 150r/min, air flow 3-5L/min, initial pH is 5.0, cultivates 48h.
the feature of matsutake mutagenic fungi of the present invention is:
(1) content of matsutake mutagenic strain mycelium dry weight, adenosine, Crude polysaccharides and N.F,USP MANNITOL four kinds of effective constituents is all significantly improved compared with original strain, and wherein thalline soma weighs 15.178/L, and the more former bacterium that sets out improves 69.68%; Adenosine content can reach 0.0359g/L, and the more former bacterium that sets out improves 33.96%; Polysaccharide content can reach 1.780g/L, and the more former bacterium that sets out improves 75.72%; Mannitol content can reach 0.327g/L, and the more former bacterium that sets out improves 52.8%.
(2) matsutake mutagenic strain through more than 20 times continuous passage cultivate do not degenerate, there is genetic stability.
Positively effect of the present invention is: the matsutake new strains of mutagenesis is not degenerated through more than 10 times Secondary Culture, has genetic stability, and intracellular polyse, adenosine and mannitol content more high than original bacteria times far away.
Accompanying drawing explanation
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 between each factor of Fig. 4, Fig. 5, Fig. 6 and isogram.
Embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood to be explaination of the present invention but not to any type of restriction of the present invention.
embodiment 1:
the mutagenesis of matsutake mutant strain and selection
(1) matsutake mutagenesis
Be the bacterial classification that sets out of CGMCC5.793 matsutake bacterial strain by preserving number, after activation, be inoculated in PDA inclined-plane, cultivate 6 days in 26oC thermostat container, pH6.0 phosphoric acid buffer is injected in slant medium, then use 4 layers of filtered through gauze, dilution spore concentration to 107/mL, for selection by mutation.Take 10mg nitrosoguanidine in the culture dish of diameter 7cm, add cosolvent acetone a little after, add sterile phosphate buffer 9mL (pH=6.0), open magnetic stirring apparatus slowly to stir, after NTG fully dissolves, draw 1mL bacteria suspension put into plate and start mutagenesis (nitrosoguanidine final concentration is lmg/mL).Respectively after mutagenesis 2min, 5min, 10min, 15min and 20min, get 0.3mL bacteria suspension in 2.7mL sterile distilled water and stepwise dilution.To the spore suspension after mutagenesis, carry out gradient dilution, after PDA culture medium flat plate cultivates 5 days, the single bacterium colony grown is equipped with the point-to-point implantation of sterilized toothpick in 96 orifice plates of PDA solid medium and cultivates.Be inoculated in one by one in 250mL Erlenmeyer flask by cultured mutant strain inoculating needle and cultivate, 26oC150rpm cultivates and obtains tricholoma matsutake mycelium in 5 days, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, the matsutake bacterial strain of screening high yield.The high yield matsutake mutagenic strain obtained is through continuous passage, culture condition is cultivate 120h in 26oC, 150rpm constant-temperature table, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, investigate the genetic stability of mutagenic strain, thus finally determine object bacterial strain.
(2) four kinds of extractions of effective constituent and the mensuration of content
Mutant strain mutagenesis obtained is stored in 48 orifice plates, centrifugal mycelium, 5000rpm, centrifugal 10min, and once, mycelium is laid on dull and stereotyped freeze-drying, and lyophilized powder is weighed in washing, pulverizes, and crosses 60 mesh sieves.Take tricholoma matsutake mycelium dry powder 0.1g, add 5mL distilled water, 80oC water-bath lixiviate 3h, the centrifugal 10min of 8000g/min, get supernatant and measure mycelium polysaccharides content.Take tricholoma matsutake mycelium dry powder 0.1g, add 5mL distilled water, 45oC water-bath lixiviate 3h, the centrifugal 10min of 8000g/min, get supernatant and measure mycelium adenosine and mannitol content.Green onion copper-sulfuric acid process is adopted to measure total sugar content; DNS method measures reducing sugar content, polysaccharide content two total sugar content one reducing sugar content.
(3) screening of high yield mutagenic fungi
From the matsutake mutant of mutagenesis, filter out the strain excellent (see table 1) that mycelium dry weight and four kinds of active constituent contents significantly improve, then Secondary Culture is carried out, bacterial strain dry weight and three kinds of active constituent contents are measured every a generation, the same step of measuring method (2), in Dual culture 10 generation, investigate the genetic stability feature that screening obtains high yield mutagenic fungi.
Table 1 mutagenic strain four indices improvement value
Table 2 mutagenic strain T35 genetic stability is investigated
| Index | Dry weight (g/L) | Intracellular polyse (g/L) | Adenosine (g/L) | N.F,USP MANNITOL (g/L) |
| 1st generation | 14.985 | 1.744 | 0.0378 | 0.351 |
| 2nd generation | 15.011 | 1.787 | 0.0345 | 0.342 |
| 3rd generation | 14.877 | 1.712 | 0.0367 | 0.356 |
| 4th generation | 14.967 | 1.765 | 0.0328 | 0.371 |
| 5th generation | 15.321 | 1.786 | 0.0389 | 0.387 |
| 6th generation | 15.228 | 1.793 | 0.0396 | 0.353 |
| 7th generation | 15.153 | 1.749 | 0.0357 | 0.361 |
| 8th generation | 14.841 | 1.728 | 0.0378 | 0.373 |
| 9th generation | 14.979 | 1.766 | 0.0364 | 0.351 |
| 10th generation | 15.013 | 1.735 | 0.0351 | 0.365 |
embodiment 2:
the optimization of matsutake mutagenic strain fermention medium
1, expectation function is set up
Respectively with dry cell weight, intracellular polyse, adenosine, N.F,USP MANNITOL is inspection target, is optimized matsutake zymotechnique, adopts expectation function to be comprehensively an inspection target expected value (D) by multiple inspection target, each inspection target (response value) all converts nondimensional expected value to by formula (1), when the balance yardstick d scope of expectation function is 0 ~ 1, d=0, response value substantial deviation target value is described, d=1 illustrate response value and target value close.D increases along with desired response value and increases.
（1）
be the response value of i-th inspection target, Li is that i-th inspection target expectation can not lower than this response value; The highest response value that Ei i-th inspection target is expected.
（2）
Wi is the weighted value of i-th index, and the inspection target of Tricholoma matsutake (lto et lmai) Singer fermentation and the minimum response value of expectation, expect that the highest response value and weighted value are as table 3.
The inspection target that table 3 matsutake T35 ferments and the minimum response value of expectation, the highest response value and weighted value
| Parameter | Dry weight/gL-1 | Adenosine/gL-1 | N.F,USP MANNITOL/gL-1 | Intracellular polyse/gL-1 |
| yil | 2.00 | 0.01 | 0.00 | 0.10 |
| yih | 15.00 | 0.15 | 0.80 | 2.50 |
| wi | 0.25 | 0.30 | 0.15 | 0.30 |
2, experiment of single factor
In the process of medium optimization, experiment of single factor is utilized to investigate affecting the carbon source of expected value, nitrogenous source and Inorganic Salts.
(1) carbon source kind screening
Substratum based on PDA substratum, adopt 20g/L glucose, sucrose, lactose, fructose, N.F,USP MANNITOL, Zulkovsky starch and maltose as carbon source respectively, prepare substratum, substratum liquid amount 100mL/250mL, access seed liquor with the inoculum size of 5% respectively, 150rpm, 26oC cultivate 4d.Each index components content under measuring often kind of carbon source culture medium condition, calculates Dv value, to select suitable carbon source kind.As shown in Figure 1, when adopting glucose as carbon source, its expected value Dv is the highest for result, therefore selects glucose to be optimum carbon source.
(2) nitrogenous source kind screening
Substratum based on PDA substratum, adopt the leaching of 10g/L peptone, yeast powder, extractum carnis, soy peptone, Tryptones and ammonium sulfate as nitrogenous source respectively, prepare substratum, substratum dress liquid 100mL/250mL, with the inoculum size access seed liquor of 5%, 150rpm, 26oC cultivate 4d.Each index components content under measuring often kind of nitrogen source medium condition, calculates Dv value.As shown in Figure 2, when using the yeast of 10g/L leaching powder as nitrogenous source, expected value is high compared with the expected value of other several nitrogen source medium for result, therefore selects yeast leaching powder 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 utilize experiment of single factor to screen the old optimum proportion of compound nitrogen source and yeast leaching powder and soybean protein, the optimum proportion of compound nitrogen source is 1:1.
(3) screening of inorganic ion kind
On the basis of the suitable carbon and nitrogen sources kind of screening, add following inorganic salt in the medium respectively: CuSO4.5H2O, ZnCl2, FeCl3.6H2O, KH2PO4, NaCl, CaCl2 and MgSO4.7H2O, often kind of inorganic salt are set to two concentration 0.05% and 0.1%, measure each index components content in mycelium, calculate Dv value, to screen the suitableeest inorganic ion kind.As shown in Figure 3, the optimal inorganic salts of matsutake T35 fermention medium is KH2PO4 to result.H2O。
3, significant composition is affected in PB experiment sieving matsutake mutagenic strain T35 fermention medium
On the basis of above test, carry out BP experiment sieving glucose, yeast leaching powder and inorganic salt to the significant composition of mutagenic strain T35 fermentation impact.Result glucose sugar (X1) wherein, yeast leaching powder (X2), KH2PO4 (X3) are remarkable item.Therefore, glucose, yeast leaching powder and KH2PO4 is selected to optimize further.
4, hill climbing test approaches the optimal concentration affecting remarkable composition
Carry out hill climbing test design on the basis of BP test, make the concentration of the remarkable composition of impact close to optimal region.
5, matsutake mutagenic fungi T35 fermentation center combination design test
Center combination test is carried out on the basis of above test, glucose, yeast leaching powder and KH2PO4 are to accurate optimization matsutake fermention medium, adopt even experiment design (RSM) respectively, analyze accordingly test-results and search best medium scheme, result as shown in Figure 4,5, 6.Carry out statistical study to the model of RSM matching to obtain optimal medium formula and be: glucose 25.62g/L, yeast leaching powder 10.74g/L, soy peptone 10g/L, KH2PO40.57g/L, MgSO47H2O0.5g/L and VB10.25g/L.
Claims (4)
1. a Tricholoma matsutake (lto et lmai) Singer mutagenic strain, on December 25th, 2014 in China typical culture collection center preservation, preservation registration number is CCTCCNO.M2014671, specific name: matsutake T35TricholomamatsutakeT35.
2. obtain the method for matsutake mutagenic strain described in claim 1, comprised the following steps:
1) be CGMCC5.793 matsutake bacterial strain by preserving number, be inoculated in PDA inclined-plane after activation, at 23-27
ocultivate 4-8 days in C thermostat container, in slant medium, inject pH6.0 phosphoric acid buffer, then use 4 layers of filtered through gauze, dilution spore concentration to 10
6-10
7individual/mL, for selection by mutation;
2) 10mg nitrosoguanidine is taken in the culture dish of diameter 7cm, add cosolvent acetone a little after, add sterile phosphate buffer 9mL (pH=6.0), open magnetic stirring apparatus slowly to stir, after NTG fully dissolves, draw 1mL bacteria suspension put into plate and start mutagenesis (nitrosoguanidine final concentration is lmg/mL);
Respectively after mutagenesis 2min, 5min, 10min, 15min and 20min, get 0.3mL bacteria suspension in 2.7mL sterile distilled water and stepwise dilution;
3) to the spore suspension after mutagenesis, carry out gradient dilution, PDA culture medium flat plate was cultivated after 3-7 days, is equipped with by the single bacterium colony grown in 96 orifice plates of PDA solid medium cultivates with the point-to-point implantation of sterilized toothpick;
Cultured mutant strain inoculating needle is inoculated in 250mL Erlenmeyer flask one by one and cultivates, 26
oc150rpm cultivates and obtains tricholoma matsutake mycelium in 4-6 days, measures mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, the matsutake bacterial strain of screening high yield;
4) the high yield matsutake mutagenic strain obtained is through continuous passage, and culture condition is 26
ocultivate 96h in C, 150rpm constant-temperature table, measure mycelium dry weight, polysaccharide, N.F,USP MANNITOL and adenosine content, investigate the genetic stability of mutagenic strain, thus finally determine object bacterial strain.
3. obtain the cultural method of matsutake mutagenic strain described in claim 1, comprised the following steps:
1) primary inclined plane bacterial classification
Slant medium: glucose 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-1g/L, KH
2pO
40.1-0.7g/L, MgSO
7.H
2o0.1-0.6g/L, vitamins B
10.05-0.5g/L, agar 25g/L;
Slant strains culture temperature 26 ± 1
owithin C, 4-7 days, mycelia covers with inclined-plane, puts refrigerator (2-4
oc) save backup;
2) second-level shake flask bacterial classification
Seed culture medium: glucose 15-25g/L, peptone 5-15g/L, yeast leaching powder 5-15g/L, KH
2pO
40.1-0.7g/L, MgSO
7.H
2o0.1-0.6g/L, vitamins B
10.05-0.5g/L;
Seed culture method: inclined-plane mother is planted access liquid seed culture medium, 26 ± 1
0the shaking table of C, 120-160r/min, cultivates 5d;
3) shake flask fermentation is cultivated
By seed liquor by 5% inoculum size access liquid fermentation medium (glucose 18-25g/L, yeast leaching powder 15-25g/L, KH
2pO
40.3-0.6g/L, MgSO
7.H
2o0.3-0.6g/L, vitamins B
1carry out liquid fermenting 0.05-0.3g/L), fermention medium liquid amount is the shaking flask dress 100mL of 250mL, culture temperature 26
0c shaking speed is 150rpm, gathers in the crops mycelium after cultivating 5d;
4) 100L fermentor tank amplification culture
By seed liquor by 7% inoculum size access 60L liquid fermentation medium, in 100L fermentor tank, carry out liquid submerged fermentation cultivation, 26
oc, 150r/min, air flow 3-5L/min, initial pH are 5.0, cultivate 48h, to obtain final product.
4. cultivating the substratum of matsutake bacterial strain described in claim 1, it is characterized in that by following raw materials by weight portion than making:
Glucose 25.62g/L, yeast leaching powder 10.74g/L, soy peptone 10g/L, KH
2pO
40.57g/L, MgSO
47H
2o0.5g/L and VB
10.25g/L.
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