CN104911109A - High-yield paecilomyces hepiali mutant strain and cultivation method - Google Patents

High-yield paecilomyces hepiali mutant strain and cultivation method Download PDF

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CN104911109A
CN104911109A CN201510280881.1A CN201510280881A CN104911109A CN 104911109 A CN104911109 A CN 104911109A CN 201510280881 A CN201510280881 A CN 201510280881A CN 104911109 A CN104911109 A CN 104911109A
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滕利荣
王娟
孟庆繁
王迪
刘燕隔
逯家辉
王春月
蔡广胜
权宇彤
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Jilin University
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Abstract

The invention discloses a high-yield paecilomyces hepiali mutant strain and a cultivation method thereof. The strain (PH40) is obtained through a chemical mutagenesis method in a screening mode. Compared with an original strain, the fermentation mycelium dry weight yield of the strain PH40 is improved by 44.06%; the mycelium intracellular adenosine content is improved by 3.59%; the mycelium intracellular mannitol content is improved by 104.7%; the mycelium intracellular polysaccharide content is improved by 139.83%. The paecilomyces hepiali mutant strain does not degenerate after subculture of 10 generations, and hereditary stability is achieved. The invention further discloses a most proper fermentation medium of the paecilomyces hepiali mutant strain (PH40).

Description

A kind of high yield peacilomyce hepiahi mutagenic strain and method of cultivation
technical field:
The present invention relates to a kind of high yield peacilomyce hepiahi mutagenic strain, be a kind of new strain variety, present invention also offers the method for cultivation of this bacterial strain, belong to fermentation engineering field.
background technology:
Peacilomyce hepiahi is the endophyte be separated in natural cordyceps, the anamorph of ubiquitous a kind of Chinese caterpillar fungus in natural cordyceps, this bacterium shows through the mycelium of submerged fermentation gained and the comparative study of natural cordyceps chemical composition, both main component, pharmacological action basic simlarity are the substitute of natural cordyceps effective constituent and effect.In addition, this bacterium is put into " the fungi strain list that can be used for healthcare products ".Economical and practical with it, best in quality, the easy to use new situation opening Chinese caterpillar fungus application of peacilomyce hepiahi bacterium filament.
summary of the invention:
The object of this invention is to provide a strain peacilomyce hepiahi mutagenic strain, is a kind of new bacterial strain, and the output of its mycelium dry weight and effective constituent adenosine, polysaccharide and cordycepic acid is compared to have with original strain and significantly improved.
Peacilomyce hepiahi mutagenic strain disclosed by the invention, called after: peacilomyce hepiahi PH 40(called after: paecilomyces hepidl chenpH40), this mutagenic strain is on December 26th, 2014 preservation" Chinese Typical Representative culture preservationcenter ", preservationnumber be: CCTCC NO:M 2014670, show according to the systematics of fungi and the evolutionary tree of qualification, bacterial strain belongs to peacilomyce hepiahi.
Peacilomyce hepiahi mutagenic strain of the present invention, it has following methods mutagenic and breeding and obtains, with peacilomyce hepiahi (buy and preserve center RCEF1429 from Agricultural University Of Anhui microorganism) for starting strain, adopt chemomorphosis treatment technology, the peacilomyce hepiahi selecting high yield is high produce prominentbecome bacterial strain PH 40.
The feature of peacilomyce hepiahi mutagenic strain of the present invention is: the content of mycelium dry weight, adenosine, N.F,USP MANNITOL and polysaccharide is all significantly improved, wherein the more former starting strain of mycelium dry weight improves 44.06 %, adenosine content improves 3.59%, mannitol content improves 104.7%, and polysaccharide content improves 139.83%.
Adopt peacilomyce hepiahi mutagenic strain of the present invention to carry out the method for liquid submerged fermentation, comprised the following steps:
(1) primary inclined plane bacterial classification:
Slant medium: potato 200 g/L, glucose 18-22 g/L, KH 2pO 40.1-0.5 g/L, MgSO 47H 2o 0.1-0.5 g/L, agar 18-25 g/L.
Slant strains culture temperature 26 ± 1 DEG C, within 4-5 days, mycelia covers with inclined-plane, puts refrigerator (2-4 DEG C) and saves backup.
(2) second-level shake flask bacterial classification
Seed culture medium: glucose 18-22 g/L, peptone 8-12 g/L, yeast leaching powder 8-12 g/L, KH 2pO 40.1-0.5 g/L, MgSO 47H 2o 0.1-0.5 g/L, vitamins B 10.05-0.1 g/L.
Seed culture method: inclined-plane mother is planted access liquid seed culture medium, at 26 ± 1 DEG C, 4d cultivated by the shaking table of 120-160 r/min.
(3) shake flask fermentation is cultivated
By seed liquor by 5% inoculum size access liquid fermentation medium (sucrose: 26-32 g/L, yeast leaching powder: 15-20g/L, peptone: 8-12g/L, (NH 4) 2sO 4: 8-12 g/L, KH 2pO 4: 2-4 g/L, MgSO 4: 2-4 g/L, VB 1: 0.1-0.3g/L, zinc chloride: 0.01-0.02g/L) in carry out liquid fermenting, fermention medium liquid amount is the shaking flask dress 100mL of 250mL, and culture temperature 26 DEG C, shaking speed is 150rpm, cultivates after 4d and gathers in the crops mycelium.
(4) 100L fermentor tank amplification culture
By seed liquor by 5 % inoculum size access 60L liquid fermentation mediums, in 100L fermentor tank, carry out liquid submerged fermentation cultivation, 26 DEG C, 200 r/min, air flow 4 L/min, initial pH is 5.0, cultivates 72 h.
positively effect of the present invention is:peacilomyce hepiahi mutagenic strain (the PH provided 40) be a kind of new strains, the content of mycelium dry weight, adenosine, N.F,USP MANNITOL and polysaccharide is all significantly improved, and wherein the more former starting strain of mycelium dry weight improves 44.06 %, and adenosine content improves 3.59%, mannitol content improves 104.7%, and polysaccharide content improves 139.83%.This peacilomyce hepiahi mutagenic fungi PH 40mycelial growth is quick, and the mycelium initial stage is brown, and the later stage gradually becomes brown color, can carry out quick liquid deep drainpipe.
Embodiment
embodiment 1
Peacilomyce hepiahi PH 40mutagenic breeding method
The mutafacient system of peacilomyce hepiahi mutagenic strain of the present invention is nitrosoguanidine mutagenesis.
1) by peacilomyce hepiahi bacterium strain, streak inoculation is in slant medium, and 26 DEG C of incubators cultivate 5 days, and mycelium is white, and in inclined-plane, inject stroke-physiological saline solution, after concussion, adopt 9 layers of filtered through gauze, being diluted to concentration is 10 6the spore suspension of individual/ml, after adopting the aseptic nitroso guanidine solution process 30min of 10ug/ml, gets spore suspension and adopts stroke-physiological saline solution stepwise dilution, be applied in seed culture medium flat board and cultivate after 7 days, choose single bacterium colony and preserve in 96 orifice plates.
2) adopt aseptic inoculation pin to choose one by one in mutagenic strain to seed culture medium to activate, in 26 DEG C of constant-temperature tables, 150rpm cultivates 5 days, measures mycelium dry weight, adenosine, polysaccharide and cordycepic acid content, high to screen produce prominentmutant.HPLC method is adopted to measure adenosine content in mycelium; Anthracene copper-sulfuric acid process is adopted to measure total sugar content; DNS method is adopted to measure contents of monosaccharides; Utilize spectrophotometry cordycepic acid content.
Described peacilomyce hepiahi mutagenic strain, called after: peacilomyce hepiahi PH40(paecilomyces hepiali chen & dai, PH 40), this mutagenic strain is on December 26th, 2014 preservation" Chinese Typical Representative culture preservationcenter ", preservationnumber be: CCTCC M 2014670, show according to the systematics of fungi and the evolutionary tree of qualification, bacterial strain belongs to peacilomyce hepiahi.
embodiment 2
Peacilomyce hepiahi mutagenic fungi (PH 40) to compare with starting strain and genetic stability is investigated
1) by original strain and mutagenic strain PH 40be inoculated in seed culture medium (sucrose: 26-32 g/L, yeast leaching powder: 15-20g/L, peptone: 8-12g/L, (NH respectively 4) 2sO 4: 8-12 g/L, KH 2pO 4: 2-4 g/L, MgSO 4: 2-4 g/L, VB 1: 0.1-0.3g/L, zinc chloride: 0.01-0.02g/L) in, in 26 DEG C, 150rpm, cultivate 4d and carry out fermentation culture, the content of peacilomyce hepiahi mutagenic strain mycelium dry weight, adenosine, N.F,USP MANNITOL and polysaccharide is all significantly improved, wherein, thalline soma weighs 14.40 g/L, and more former starting strain improves 44.06 %; Adenosine content can reach 0.1038g/l, and the more former bacterium that sets out improves 3.59%; Cordycepic acid content can reach 1.275 g/L, and comparatively original strain improves 104.7%; Polysaccharide content can reach 1.364 g/L, and the more former bacterium that sets out improves 139.83%.
table 1former starting strain and mutagenic strain PH 40active constituent content compares
Dry weight (gL -1） Adenosine (mgL -1） Cordycepic acid (gL -1） Polysaccharide (gL -1）
Former starting strain 9.996 0.1002 0.6228 0.5687
Mutagenic strain PH of the present invention 40 14.40 0.1038 1.275 1.364
Improve ratio (%) 44.06 3.593 104.7 139.83
2) whether the high productivity energy hereditary property in order to study mutagenic strain is stablized, and adopts the side that colony goes down to posterity method is investigatedmutagenic strain PH 40genetic stability, bacterial strain passes continuously and connect for ten generations, and every generation is inoculated in seed culture medium respectively, in 26 DEG C, 150rpm, cultivate 4d carry out fermentation culture, survey the output of effective constituent after fermentation ends.
table 2mutagenic strain PH 40genetic stability is investigated
Passage number Dry weight (gL -1） Adenosine (mgL -1） Cordycepic acid (gL -1） Polysaccharide (gL -1）
1st generation 14.12 0.1045 1.315 1.414
2nd generation 14.05 0.1080 1.214 1.488
3rd generation 14.44 0.1078 1.222 1.589
4th generation 14.21 0.1032 1.172 1.345
5th generation 14.36 0.1089 1.123 1.574
6th generation 14.42 0.1109 1.065 1.569
7th generation 14.21 0.1104 1.395 1.384
8th generation 14.70 0.1094 1.215 1.552
9th generation 14.39 0.1012 1.375 1.553
10th generation 14.38 0.1037 1.203 1.492
embodiment 3,
Peacilomyce hepiahi superior strain (PH 40) screening of fermention medium
1) foundation of expectation function
Respectively with dry cell weight, intracellular polyse, adenosine, cordycepic acid is inspection target, to mutagenic strain PH 40zymotechnique is optimized, expectation function is adopted 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), the balance yardstick d scope of expectation function is 0 ~ 1, during 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 expect 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 peacilomyce hepiahi fermentation and the minimum response value of expectation, expect the highest response value and weighted value as table 3
table 3inspection target and the minimum response value of expectation, the highest response value and weighted value
Index Dry weight/gL -1 Adenosine/gL -1 Cordycepic acid/gL -1 Intracellular polyse/gL -1
L i 10.00 0.08 1.00 1.00
E i 20.00 0.15 1.50 2.00
w i 0.25 0.30 0.15 0.30
2) peacilomyce hepiahi high yield mutagenic strain PH 40fermenting carbon source screens
Take seed culture medium as initial medium.Respectively the kind of glucose, maltose, fructose, sucrose, lactose, N.F,USP MANNITOL is screened, single factor test is used on acquired results basis method is investigatedthe concentration of optimum carbon source, often kind of parallel twice experiment of carbon source substratum, the sucrose filtering out 2.5% is optimum carbon source.Fermentation condition is: planting age is 3.5 d, and inoculum size is 5 %, and culture temperature is 26 DEG C, and shaking speed is 150 rpm, and incubation time is 4d.
3) peacilomyce hepiahi high yield mutagenic strain PH 40fermentation nitrogen source screens
Sucrose with 2.5% is optimum carbon source, investigates peptone, extractum carnis, multivalent protein peptone, urea, yeast powder and yeast extract paste respectively to the impact of fermentation mycelium active constituent content.Two kinds of nitrogenous sources that screening expected value D is the highest are compound nitrogen source, carry out the response surface optimum experimental nitrogen concentration of 2 factor 5 levels.Often kind of parallel twice experiment of nitrogen source medium, filters out the yeast leaching powder that compound nitrogen source ratio is 1% peptone and 1.8%.
4) PB experiment sieving peacilomyce hepiahi high yield mutagenic strain PH 40significant composition is affected in fermention medium
On the basis of above test, carry out BP experiment sieving sucrose, yeast leaching powder, peptone and inorganic salt to mutagenic strain PH 40the significant composition of fermentation impact.The leaching of result sucrose wherein, yeast powder, VB 1for remarkable item.Therefore, sucrose, yeast is selected to soak powder, VB 1further optimization.
table 4.1 Plackett-Burman experimental factor level and encoded radio
5) peacilomyce hepiahi high yield mutagenic strain PH 40the test of fermentation center combination design
Center combination test is carried out on the basis of above test, sucrose, yeast leaching powder and VB 1to accurate optimization peacilomyce hepiahi PH 40fermention medium, adopts even experiment design (RSM) respectively, analyzes accordingly and search best medium scheme to test-results.Statistical study is carried out to the model of RSM matching, SAS work box give can obtain maximum response ( y) x 1, x 2, x 3corresponding encoded radio x 1=0.397, x 2=-0.115, x 3=0.940, be yeast leaching powder concentration 18.970 g/L, sucrose concentration 18.850 g/L, VB 1consumption 0.235 g/L.The optimal medium formula that statistical analysis method obtains is: 30 g/L sucrose, 10 g/L peptones, 18 g/L yeast leaching powder, 3 g/L KH 2pO 43H 2o, 3 g/L MgSO 47H 2o, 0.235 g/L VB 1, 0.011 g/L ZnCl 2, 10 g/L (NH 4) 2sO 4.Be: 150 rpm, 26 DEG C that cultivate 4.5d, the D value now obtained is 0.5195 at culture condition.
7) peacilomyce hepiahi high yield mutagenic strain PH 40fermentation proof test
3 parallel fermentation tests are carried out, with the reliability of Confirming model and optimization method and accuracy by the substratum scheme of optimum.
table 4peacilomyce hepiahi mutagenic strain PH 40fermentation proof test result
Test number Dry weight/gL -1 Adenosine/gL -1 Cordycepic acid/gL -1 Polysaccharide/gL -1
1 14.89 0.1102 1.350 1.596
2 15.01 0.1123 1.386 1.654
3 15.11 0.1098 1.401 1.569

Claims (2)

1. a high yield peacilomyce hepiahi mutagenic strain, in preservation on December 26 " China typical culture collection center " in 2014, called after: paecilomyces hepidl chenpH40; Peacilomyce hepiahi bacterium strain PH40, preserving number is: CCTCC NO:M 2014670.
2. adopt high yield peacilomyce hepiahi mutagenic strain described in claim 1 to carry out the method for liquid submerged fermentation, comprised the following steps:
(1) primary inclined plane bacterial classification:
Slant medium: potato 200 g/L, glucose 18-22 g/L, KH 2pO 40.1-0.5 g/L, MgSO 47H 2o 0.1-0.5 g/L, agar 18-25 g/L;
Slant strains culture temperature 26 ± 1 DEG C, within 4-5 days, mycelia covers with inclined-plane, puts refrigerator (2-4 DEG C) and saves backup;
(2) second-level shake flask bacterial classification
Seed culture medium: glucose 18-22 g/L, peptone 8-12 g/L, yeast leaching powder 8-12 g/L, KH 2pO 40.1-0.5 g/L, MgSO 47H 2o 0.1-0.5 g/L, vitamins B 10.05-0.1 g/L;
Seed culture method: inclined-plane mother is planted access liquid seed culture medium, and at 26 ± 1 DEG C, 4 d cultivated by the shaking table of 120-160 r/min;
(3) shake flask fermentation is cultivated
By seed liquor by 5% inoculum size access liquid fermentation medium (sucrose: 26-32 g/L, yeast leaching powder: 15-20g/L, peptone: 8-12g/L, (NH 4) 2sO 4: 8-12 g/L, KH 2pO 4: 2-4 g/L, MgSO 4: 2-4 g/L, VB 1: 0.1-0.3g/L, zinc chloride: 0.01-0.02g/L) in carry out liquid fermenting, fermention medium liquid amount be 250mL shaking flask dress 100mL, culture temperature 26 DEG C, shaking speed is 150rpm, gathers in the crops mycelium after cultivating 4 d;
(4) 100L fermentor tank amplification culture
By seed liquor by 5 % inoculum size access 60L liquid fermentation mediums, in 100L fermentor tank, carry out liquid submerged fermentation cultivation, 26 DEG C, 200 r/min, air flow 4 L/min, initial pH is 5.0, cultivates 72 h.
CN201510280881.1A 2015-05-28 2015-05-28 High-yield paecilomyces hepiali mutant strain and cultivation method Pending CN104911109A (en)

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CN105886411A (en) * 2016-05-13 2016-08-24 青海省畜牧兽医科学院 Culture medium and culture method for inducing morphological transformation of Paecilomyces hepiali strain SH-1, and strain
CN108676730A (en) * 2018-05-25 2018-10-19 江西国药有限责任公司 A kind of fermentation manufacturing technique of Paecilomyces hepiali chen Cs-4
CN108865895A (en) * 2018-06-15 2018-11-23 浙江工业大学 Paecilomyces hepiali chen ZJB18001 and its application
CN109673393A (en) * 2019-02-18 2019-04-26 浙江方格药业有限公司 A kind of production method of peacilomyce hepiahi bacterium filament
CN110592161A (en) * 2019-09-24 2019-12-20 长春工业大学 Preparation method of polysaccharide, health-care oral liquid and preparation method
CN111139277A (en) * 2020-03-03 2020-05-12 长春工业大学 Crude polysaccharide and preparation process thereof, health-care oral liquid and preparation method thereof
CN111671136A (en) * 2020-07-24 2020-09-18 四川中烟工业有限责任公司 Method for solid state fermentation of tobacco stems and cut stems and application thereof
CN111685364A (en) * 2020-07-24 2020-09-22 四川中烟工业有限责任公司 Method for solid-state fermentation of tobacco stems by using paecilomyces liquid filtrate and application thereof
CN117187076A (en) * 2023-07-24 2023-12-08 江苏神华药业有限公司 Paecilomyces hepialid mutagenesis strain with high adenosine yield and preparation method and application thereof

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
CN105779299A (en) * 2016-01-07 2016-07-20 江苏苏中药业集团股份有限公司 Paecilomyces hepialid strain capable of realizing high yield of adenosine and mannite type substances and application
CN105779299B (en) * 2016-01-07 2022-01-25 江苏苏中药业集团股份有限公司 Paecilomyces hepiali strain for producing adenosine and mannitol substances and application thereof
CN105886411A (en) * 2016-05-13 2016-08-24 青海省畜牧兽医科学院 Culture medium and culture method for inducing morphological transformation of Paecilomyces hepiali strain SH-1, and strain
CN108676730B (en) * 2018-05-25 2021-02-19 江西国药有限责任公司 Fermentation production process of paecilomyces hepiali Cs-4
CN108676730A (en) * 2018-05-25 2018-10-19 江西国药有限责任公司 A kind of fermentation manufacturing technique of Paecilomyces hepiali chen Cs-4
CN108865895A (en) * 2018-06-15 2018-11-23 浙江工业大学 Paecilomyces hepiali chen ZJB18001 and its application
CN109673393A (en) * 2019-02-18 2019-04-26 浙江方格药业有限公司 A kind of production method of peacilomyce hepiahi bacterium filament
CN110592161A (en) * 2019-09-24 2019-12-20 长春工业大学 Preparation method of polysaccharide, health-care oral liquid and preparation method
CN111139277A (en) * 2020-03-03 2020-05-12 长春工业大学 Crude polysaccharide and preparation process thereof, health-care oral liquid and preparation method thereof
CN111671136A (en) * 2020-07-24 2020-09-18 四川中烟工业有限责任公司 Method for solid state fermentation of tobacco stems and cut stems and application thereof
CN111685364A (en) * 2020-07-24 2020-09-22 四川中烟工业有限责任公司 Method for solid-state fermentation of tobacco stems by using paecilomyces liquid filtrate and application thereof
CN111685364B (en) * 2020-07-24 2022-03-22 四川中烟工业有限责任公司 Method for solid-state fermentation of tobacco stems by using paecilomyces liquid filtrate and application thereof
CN111671136B (en) * 2020-07-24 2022-04-15 四川中烟工业有限责任公司 Method for solid state fermentation of tobacco stems and cut stems and application thereof
CN117187076A (en) * 2023-07-24 2023-12-08 江苏神华药业有限公司 Paecilomyces hepialid mutagenesis strain with high adenosine yield and preparation method and application thereof

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