CN101693881B - High-yield strain streptomyces lydicus, breeding and fermentation thereof - Google Patents
High-yield strain streptomyces lydicus, breeding and fermentation thereof Download PDFInfo
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- CN101693881B CN101693881B CN2009100708298A CN200910070829A CN101693881B CN 101693881 B CN101693881 B CN 101693881B CN 2009100708298 A CN2009100708298 A CN 2009100708298A CN 200910070829 A CN200910070829 A CN 200910070829A CN 101693881 B CN101693881 B CN 101693881B
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Abstract
The invention discloses a high-yield strain streptomyces lydicus, as well as breeding and fermentation thereof. The fermentation of the high-wield stain streptomyces lydicus E9 CGMCC No.3075 comprises the following steps: (1) preparation of a shake flask, seeds and a fermentation medium; (2) shake flask culture; (2) seed culture; (4) fermentation pot culture: leading a seed culture liquid in a fermentation pot filled with the fermentation medium and culturing for 24-96 hours to obtain a high-wield stain streptomyces lydicus E9 fermentation liquor under the condition of ventilating, stirring, temperature of 25-35 DEG C, and pH of 4-9; and carrying out ultrasonication for 20-40 min. The strain of the invention can generate large quantity of substances with antimycotic activity in the fermentation liquor, and the fermentation liquor has evident inhibiting effect on rhizoctonia solani; and the bacterial inhibition rate of the fermentation liquor which is diluted by 50 times reaches above 97.8% after 48 hour fermentation.
Description
Technical field
The invention belongs to the industrial microorganism field, relate to a kind of new microbe high-yield bacterial strain streptomyces lydicus and the seed selection and the fermentation of this bacterial strain.
Background technology
In China, fungal diseases of plants occupies first of the plant three big diseases (bacterium, fungi, virus), brings about great losses to eagroforestry like untimely control meeting.And at present fungal diseases of plants adopts chemical pesticide to prevent and treat more, though the chemical pesticide instant effect, its action time is short, seriously polluted to environment, and pathogenic bacteria is prone to chemical pesticide is developed immunity to drugs, and medicine efficient by other at last, environmental protection substitutes.And microbial pesticide is exactly a kind of agricultural chemicals of efficient, environmental protection.
My laboratory is separated from soil and is obtained a strain streptomyces lydicus bacterial strain (Streptomyces lydicus AS 4.2501) CGMCC NO.1692; Its fermented liquid has the antifungic action of wide spectrum; But the throughput of starting strain is too low, can not satisfy requirements of actual production.Therefore need antimycotic bacterial strain that throughput improves badly.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of superior strain streptomyces lydicus is provided.
Second purpose of the present invention provides a kind of seed selection of superior strain streptomyces lydicus.
The 3rd purpose of the present invention provides a kind of fermentation of superior strain streptomyces lydicus.
Technical scheme of the present invention is summarized as follows:
Superior strain streptomyces lydicus (Streptomyces lydicus) E9 CGMCC NO.3075.
The seed selection of superior strain streptomyces lydicus (Streptomyces lydicus) E9 CGMCC NO.3075, form by following steps:
(1) the natural seed selection of starting strain: under room temperature, be 10 with the concentration of 0.1mL
2～10
8The streptomyces lydicus of individual/mL (Streptomyces lydicus AS 4.2501) CGMCC NO.1692 monospore suspension-s is added on the isolation medium flat board, smoothens each flat board with spreading rod 26 ℃～30 ℃; Cultivate; When treating to grow on the flat board white colony of needle point size, with carrying out shake-flask culture behind single bacterium colony agar block picking, after again the shake flask fermentation liquid of each bacterial strain being filtered and diluting 10 times; Adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, select the bacterial strain of good antimicrobial effect;
(2) complex mutation and self-product resistance screening: under room temperature, the concentration that said step (1) is prepared is 10
4～10
8Individual/mL streptomyces lydicus bacterial strain monospore suspension-s is that 254nm, irradiation distance are UV irradiation 20s～60s under the 30cm condition at wavelength; Use 0.001～0.0001M nitrous acid aqueous solution mutagenic treatment, 2～5min again; With after handling monospore suspension-s carry out flat board and be coated with, be applied to the selection culture medium flat plate of the shake flask fermentation liquid that contains 0.5ml, 1ml, 1.5ml, 2ml, 2.5ml, 3ml streptomyces lydicus respectively, in 24 ℃～28 ℃; Constant temperature culture 7～10 days; Picking selects the enlightening streptomycete spore on the culture medium flat plate profit to carry out shake-flask culture, with the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, compare with starting strain; Filter out superior strain and the cultivation of going down to posterity, obtain stable superior strain streptomyces lydicus (Streptomyces lydicus) E9.
Said streptomyces lydicus monospore suspension-s is to process with following method: the saline water with 45mL～55mL washs cultured streptomyces lydicus (Streptomyces lydicus AS 4.2501) thalline inclined-plane; And gently spore is scraped with transfering loop; Washings is poured in the aseptic triangular flask of the 250mL that granulated glass sphere is housed, and 29 ℃, the 210rpm 30～45min that vibrates breaks up agglomerating spore on shaking table; And with the absorbent cotton filtration, dilution monospore suspension-s to 10
5～10
6Individual/mL, then with spore suspension in 28 ℃ of held 15min, make the spore thermal activation.
Said step (2) is preferably: under room temperature, be 10 with the concentration of said step (1) preparation
7Individual/mL streptomyces lydicus bacterial strain monospore suspension-s is that 254nm, irradiation distance are UV irradiation 40s under the 30cm condition at wavelength; Use 0.001～0.0001M nitrous acid aqueous solution mutagenic treatment 4min again; With after handling monospore suspension-s carry out flat board and be coated with, be applied to the selection culture medium flat plate of the shake flask fermentation liquid that contains the 1ml streptomyces lydicus respectively, in 27 ℃; Constant temperature culture 9 days; Picking selects the enlightening streptomycete spore on the culture medium flat plate profit to carry out shake-flask culture, with the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, compare with starting strain; Filter out superior strain and the cultivation of going down to posterity, obtain stable superior strain streptomyces lydicus (Streptomyces lydicus) E9.
The fermentation of superior strain streptomyces lydicus E9, form by following steps:
(1) preparation of shake-flask culture base, seed culture medium and fermention medium:
The preparation of shake-flask culture base: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g adds water and is settled to 1 liter, transferring pH is 5～9, sterilization;
The preparation of seed culture medium: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g adds water and is settled to 1 liter, transferring pH is 5～9, sterilization;
The preparation of fermention medium: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g, bubble enemy 1～8g adds water and is settled to 1 liter, and transferring pH is 5～9, sterilization;
(2) shake-flask culture: superior strain streptomyces lydicus (Streptomyces lydicus) E9 CGMCC NO.3075 is inserted the 250mL～1000mL that contains 50mL shake-flask culture base shake in the bottle; At 26～30 ℃; Under the condition of shaking speed 250～280rpm; Cultivated 20～80 hours, and obtained fermented liquid;
(3) seed culture: 10mL～40mL fermented liquid that step (2) is obtained inserts in the seed culture medium of 90mL～360mL; Shake greatly in the bottle at 500mL～2L, at 25～35 ℃, shaking speed is under 160～300rpm condition; Cultivated 20～80 hours, and made seed culture fluid;
(4) fermentor cultivation, carry out with a kind of of following method:
Method one:
1. said seed culture fluid is inserted and be equipped with in the 5L fermentor tank of 1～4L fermention medium; At air flow is 0.5～3vvm; Mixing speed is 300～600rpm, and temperature is 25～35 ℃, and using acid-alkali accommodation pH is under 4～9 the condition; Cultivated 24～96 hours, and obtained superior strain streptomyces lydicus E9 fermented liquid; 2. at 40～70 ℃, ultrasonication 20～40min;
Method two:
1. insert with said seed culture fluid or through the superior strain streptomyces lydicus E9 fermented liquid that 1. step (4) method one obtains and be equipped with in the 30L fermentor tank of 10～28L fermention medium; At air flow is 0.1～2.5vvm; Mixing speed is 300～600rpm, and temperature is 25～35 ℃, under the condition with acid-alkali accommodation pH4～9; The normal hexane of interpolation 120mL～260mL or n-dodecane were cultivated 24～96 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid; 2. the superior strain streptomyces lydicus E9 fermented liquid that obtains is at 40～70 ℃, ultrasonication 20～40min.
The preparation of shake-flask culture base is preferably: with starch 70～90g, and glucose 7～10g, steeping water 4～5g, soybean cake powder 7～9g, NaCl 0.3～0.5g, K
2HPO
40.2～0.3g, MgSO
47H
2O 0.2～0.4g, CaCO
30.3～0.5g adds water and is settled to 1 liter, transferring pH is 5～7, sterilization.
The preparation of fermention medium is preferably: with starch 50～80g, and glucose 8～11g, steeping water 5～8g, soybean cake powder 9～12g, NaCl 0.4～0.7g, K
2HPO
40.2～0.4g, MgSO
47H
2O 0.3～0.6g, CaCO
30.5～0.8g, bubble enemy 2～5g adds water and is settled to 1 liter, and transferring pH is 5～9, sterilization.
Said step (2) shake-flask culture is preferably: superior strain streptomyces lydicus E9 is inserted the 250mL～1000mL that contains 50mL shake-flask culture base shake in the bottle, at 29 ℃, shaking speed is under the 160rpm condition, cultivates the acquisition fermented liquid 48 hours;
Said step (3) seed culture is preferably: the 10mL fermented liquid that step (2) is obtained inserts in the 90mL seed culture medium, shakes greatly in the bottle at 500mL～2L, and at 27 ℃, shaking speed is under the condition of 230rpm, cultivates 72 hours, makes seed culture fluid.
Said step (4) fermentor cultivation, method one be preferably:
1. said seed culture fluid being inserted and be equipped with in the 5L fermentor tank of 3L fermention medium, is 2.5vvm at air flow, and mixing speed is 500rpm, and temperature is 28 ℃, with NaOH and H
2SO
4Regulate pH and be under 5 the condition, cultivated 48 hours, obtain superior strain streptomyces lydicus E9 fermented liquid; 2. at 60 ℃, ultrasonication 30min.
Said step (4) fermentor cultivation, method two be preferably:
2L is equipped with the 30L fermentor tank of 18L fermention medium from the superior strain streptomyces lydicus E9 fermented liquid access that 1. step (4) method one obtains, and is 1vvm at air flow, and mixing speed is 400rpm, and temperature is 29 ℃, with NaOH and H
2SO
4Regulate pH and be under 6 the condition, the normal hexane or the n-dodecane that add 120mL～260mL were cultivated 72 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid.
Advantage of the present invention:
Superior strain streptomyces lydicus (Streptomyces lydicus) E9 can produce a large amount of biologically active substances with anti-mycotic activity in fermented liquid (comprising ferment filtrate and mycelium); This material is present in mycelium and removes in the mycelial ferment filtrate, and is wherein higher with mycelial bacteriostatic activity.Its fermented liquid also has the obvious suppression effect to dry thread Pyrenomycetes, and the 50 times of bacteriostasis rates of secondary fermentation liquid dilution in 48 hours that ferment reach 97.8%.
The present invention adopts nature seed selection and complex mutation and the two steps screening of self-product resistance.Antimycotic biologically active substance is produced self product resistance seed selection of bacterium, and this resistant mutant strain can guarantee in the presence of high density self microbiotic, and not only thalli growth is bred unaffectedly, and antimycotic biologically active substance output also can constantly be accumulated.
The present invention has simultaneously confirmed that a kind of suitable streptomyces lydicus high productive mutant E9 produces the zymotechnique of antifungus active substance, and what comprise seed and fermentation culture based component and proportioning confirms 5L fermentor tank and the new fermentating controling process of 30L fermentor tank.The enforcement of novel process has improved the fermentation level of streptomyces lydicus high productive mutant E9.
Description of drawings
Fig. 1 is 40 times of fungistatic effect figure of 5L jar fermented liquid dilution, and 1-1 is a blank; 1-2 is 40 times of 5L jar fermented liquid dilutions.
Fig. 2 is 50 times of fungistatic effect figure of 30L jar fermented liquid dilution.2-1 is a blank, and 2-2 is 50 times of 30L jar fermented liquid dilutions.
Embodiment
Superior strain streptomyces lydicus of the present invention (Streptomyces lydicus) E9CGMCC NO.3075; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 21st, 2009; It abbreviates CGMCC as, and deposit number is CGMCC NO.3075.
The morphological specificity of this bacterial strain, cultural characteristic, the result that molecular biological characteristic is analyzed is accredited as streptomyces lydicus.Concrete qualification result is following:
One. form and physio-biochemical characteristics
1. growth characteristics on different substratum
| Substratum | Aerial hyphae | Substrate mycelium | Soluble pigment |
| Czapek's agar glucose aspartic acid agar glycerine aspartic acid agar inorganic salt Starch Agar ISP-2 agar oat agar Gause I agar Sang Tasi agar | The no white oyster white pearl pearl cuttlefish grey white of white | Yellow oyster white dirt grey shallow khaki color light brown khaki color of the brown shallow millet of cloves and grey black cassia bark light brown | Do not have |
2. microscopic morphology characteristic
The incomplete volution of fibrillae of spores, hook-shaped, ring-type; Spore is oval.
3, physio-biochemical characteristics
Two .16SrRNA gene sequencing results:
CCACAAGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAA
CGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCG
AACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGC
AGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAG
TCCCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC
GACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGACCCTGTCTCCAGGGTTTTCCGGTGTA
TGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAAT
TCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACGGACGACGTGGA
ATGTCGCCCACACCTAGTTCCCAACGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTC
GCTCCTCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCT
ACACCAGGAATTCCGATCTCCCCTACCGAACTCTAGCCTGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGGGCT
TTCACATCCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTA
CCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTCTCGCTTCTTCCCTGCTGAAAGAGG
TTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTG
CTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCG
CCTTGGTAGGCCATCACCCCACCAACAAGCTGATAGGCCGCGGGCTCATCCTTCACCGCCGGAGCTTTCCACACGGAG
GTCATGCGACCCCGTGTCGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGAAGGGCAGATTGCCCAC
GTGTTACTCACCCGTTCGCCACTAATCCCCTCCCGAAGGAGGTTCATCGTTC
Below in conjunction with specific embodiment the present invention is described further:
Embodiment 1
The seed selection of superior strain streptomyces lydicus E9, form by following steps:
(1) the natural seed selection of starting strain: under room temperature, be 10 with the concentration of 0.1mL
7The streptomyces lydicus of individual/mL (Streptomyces lydicus AS 4.2501) CGMCC NO.1692 monospore suspension-s is added on the isolation medium flat board; Smoothen with spreading rod then; 30 ℃ of cultivations; When treating to grow the white colony of needle point size on the flat board, use the punch tool punching of internal diameter, with carrying out shake-flask culture behind single bacterium colony agar block picking as 8mm; Then the shake flask fermentation liquid of each bacterial strain is filtered and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, select the starting strain of the bacterial strain of good antimicrobial effect as the next stage;
(2) complex mutation and self-product resistance screening: under room temperature, be 10 with the concentration for preparing
7The optimization bacterial strain monospore suspension-s that the step of individual/mL (1) obtains is that 254nm, irradiation distance are UV irradiation 40s under the 30cm condition at wavelength; Use 0.005M nitrous acid aqueous solution mutagenic treatment 4min again; Monospore suspension-s after handling is carried out the flat board coating; Be applied to the selection culture medium flat plate of the shake flask fermentation liquid of the streptomyces lydicus that contains 1mL respectively, in 27 ℃ of constant temperature culture 9 days, picking selected the enlightening streptomycete spore on the culture medium flat plate profit to carry out shake-flask culture; With the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity; Compare with starting strain, filter out superior strain and the cultivation of going down to posterity, obtain stable superior strain streptomyces lydicus (Streptomyces lydicus) E9 and be passaged to stable.
Embodiment 2
The seed selection of superior strain streptomyces lydicus E9, form by following steps:
(1) the natural seed selection of starting strain: under room temperature, be 10 with the concentration of 0.1mL
2The streptomyces lydicus of individual/mL (Streptomyces lydicus AS 4.2501) CGMCC NO.1692 monospore suspension-s is added on the isolation medium flat board; Smoothen with spreading rod then; 26 ℃ of cultivations; When treating to grow the white colony of needle point size on the flat board, use the punch tool punching of internal diameter, with carrying out shake-flask culture behind single bacterium colony agar block picking as 8mm; Then the shake flask fermentation liquid of each bacterial strain is filtered and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, select the starting strain of the bacterial strain of good antimicrobial effect as the next stage; (2) complex mutation and self-product resistance screening: under room temperature, be 10 with the concentration for preparing
4The optimization bacterial strain monospore suspension-s that the step of individual/mL (1) obtains is that 254nm, irradiation distance are UV irradiation 20s under the 30cm condition at wavelength; Use 0.0001M nitrous acid aqueous solution mutagenic treatment 5min again; Monospore suspension-s after handling is carried out the flat board coating; Be applied to the selection culture medium flat plate of the shake flask fermentation liquid of the streptomyces lydicus that contains 0.5ml, 1ml, 1.5ml, 2ml, 2.5ml, 3ml respectively, in 26 ℃ of constant temperature culture 10 days, picking selected the enlightening streptomycete spore on the culture medium flat plate profit to carry out shake-flask culture; With the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity; Compare with starting strain, filter out superior strain and the cultivation of going down to posterity, obtain stable superior strain streptomyces lydicus (Streptomyceslydicus) E9 and be passaged to stable.
Embodiment 3
The seed selection of superior strain streptomyces lydicus E9, form by following steps:
(1) the natural seed selection of starting strain: under room temperature, be 10 with the concentration of 0.1mL
8The streptomyces lydicus of individual/mL (Streptomyces lydicus AS 4.2501) CGMCC NO.1692 monospore suspension-s is added on the isolation medium flat board; Smoothen with spreading rod then; 28 ℃ of cultivations; When treating to grow the white colony of needle point size on the flat board, use the punch tool punching of internal diameter, with carrying out shake-flask culture behind single bacterium colony agar block picking as 8mm; Then the shake flask fermentation liquid of each bacterial strain is filtered and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity, select the starting strain of the bacterial strain of good antimicrobial effect as the next stage;
(2) complex mutation and self-product resistance screening: under room temperature, be 10 with the concentration for preparing
8The optimization bacterial strain monospore suspension-s that the step of individual/mL (1) obtains is that 254nm, irradiation distance are UV irradiation 60s under the 30cm condition at wavelength; Use 0.001M nitrous acid aqueous solution mutagenic treatment 2min again; Monospore suspension-s after handling is carried out the flat board coating; Be applied to the selection culture medium flat plate of the shake flask fermentation liquid of the streptomyces lydicus that contains 1ml, 1.5ml, 2ml, 3ml respectively, in 28 ℃ of constant temperature culture 7 days, picking selected the enlightening streptomycete spore on the culture medium flat plate profit to carry out shake-flask culture; With the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after adopt the double-layer plate cylinder plate method to measure bacteriostatic activity; Compare with starting strain, filter out superior strain and the cultivation of going down to posterity, obtain stable superior strain streptomyces lydicus (Streptomyces lydicus) E9 and be passaged to stable.
Embodiment 4
The preparation of double-layer plate in the nature seed selection: bottom shop 15mL lower floor's substratum (slant medium), it is 10 that dry thread Pyrenomycetes is made into concentration
9The bacteria suspension of individual/mL joins in the upper strata substratum that is cooled to about 50 ℃ (PDA) by the amount that adds 1mL in every 100mL substratum, gets 5mL behind the mixing and is laid on rapidly on the bottom substratum that has prepared, makes double-layer plate;
Cylinder plate method: 3 Oxford cups of equidistant placement (high 10mm, internal diameter 6mm) on flat board, with the shake flask fermentation liquid of each bacterial strain filter and dilute 10 times after be added drop-wise in the cup of Oxford; Cultivated 30 hours in 27 ℃; Measure antibacterial circle diameter with ruler, three repetitions are averaged.
Embodiment 5
The preparation of streptomyces lydicus monospore suspension-s: the saline water with 45mL washs cultured streptomyces lydicus (Streptomyces lydicus AS 4.2501) thalline inclined-plane; And gently spore is scraped with transfering loop; Washings is poured in the aseptic triangular flask of the 250mL that granulated glass sphere is housed, and 29 ℃, the 210rpm 45min that vibrates breaks up agglomerating spore on shaking table; And with the absorbent cotton filtration, dilution monospore suspension-s to 10
6Individual/mL, then with spore suspension in 28 ℃ of held 15min, make the spore thermal activation.
Embodiment 6
The preparation of streptomyces lydicus monospore suspension-s: the saline water with 55mL washs cultured streptomyces lydicus (Streptomyces lydicus AS 4.2501) thalline inclined-plane; And gently spore is scraped with transfering loop; Washings is poured in the aseptic triangular flask of the 250mL that granulated glass sphere is housed, and 29 ℃, the 210rpm 30min that vibrates breaks up agglomerating spore on shaking table; And with the absorbent cotton filtration, dilution monospore suspension-s to 10
5Individual/mL, then with spore suspension in 28 ℃ of held 15min, make the spore thermal activation.
Embodiment 7
Shake flask fermentation:
1. the preparation of shake-flask culture base: contain starch 70g in one liter of substratum, glucose 7g, steeping water 4g, soybean cake powder 7g, NaCl 0.3g, K
2HPO
40.2g, MgSO
47H
2O 0.2g, CaCO
30.3g the preparation of substratum water also is settled to 1 liter, transferring pH is 6, sterilization.
2. shake-flask culture: the high yield streptomyces lydicus E9 of embodiment 1 preparation is inserted the 500mL that contains 50mL shake-flask culture base shake in the bottle, 29 ℃, cultivated 48 hours, shaking speed 160rpm must shake flask fermentation liquid.
Embodiment 8
Shake flask fermentation:
1. the preparation of shake-flask culture base: contain starch 90g in one liter of substratum, glucose 10g, steeping water 10g, soybean cake powder 9g, NaCl 0.5g, K
2HPO
40.3g, MgSO
47H
2O 0.4g, CaCO
30.5g the preparation of substratum water also is settled to 1 liter, transferring pH is 6, sterilization.
2. shake-flask culture: the high yield streptomyces lydicus E9 of embodiment 2 preparations is inserted the 250mL that contains 50mL shake-flask culture base shake in the bottle, 26 ℃, to cultivate 80 hours, shaking speed is 250rpm, must shake flask fermentation liquid.
Embodiment 9
Shake flask fermentation:
1. the preparation of shake-flask culture base: contain starch 30g in one liter of substratum, glucose 5g, steeping water 3g, soybean cake powder 6g, NaCl 0.2g, K
2HPO
40.1g, MgSO
47H
2O 0.1g, CaCO
30.2g the preparation of substratum water also is settled to 1 liter, transferring pH is 5, sterilization.
2. shake-flask culture: the high yield streptomyces lydicus E9 of embodiment 1 preparation is inserted the 1000mL that contains 50mL shake-flask culture base shake in the bottle, 30 ℃, to cultivate 20 hours, shaking speed is 280rpm, must shake flask fermentation liquid.
Embodiment 10
Shake flask fermentation:
1. the preparation of shake-flask culture base: contain starch 100g in one liter of substratum, glucose 12g, steeping water 5g, soybean cake powder 13g, NaCl 0.9g, K
2HPO
40.8g, MgSO
47H
2O 0.8g, CaCO
30.9g the preparation of substratum water also is settled to 1 liter, transferring pH is 7, sterilization.
2. shake-flask culture: the high yield streptomyces lydicus E9 of embodiment 3 preparations is inserted the 500mL that contains 50mL shake-flask culture base shake in the bottle, 29 ℃, to cultivate 48 hours, shaking speed is 160rpm, must shake flask fermentation liquid.
Embodiment 11
The preparation of seed culture medium and cultivation
1. the preparation of seed culture medium: contain starch 60g in one liter of substratum, glucose 9g, steeping water 6g, soybean cake powder 11g, NaCl 0.2g, K
2HPO
40.4g, MgSO
47H
2O 0.7g, CaCO
30.4g pH is 8, the preparation of substratum water also is settled to 1 liter, sterilization;
2. seed culture: the 10mL shake flask fermentation liquid that makes is inserted in the 90mL seed culture medium, shake greatly in the bottle at 1L, 27 ℃, to cultivate 72 hours, shaking speed is 230rpm, makes seed culture fluid; (The Scarlet Letter increases according to claims)
Embodiment 12
The preparation of seed culture medium and cultivation
1. the preparation of seed culture medium: contain starch 30g in one liter of substratum, glucose 12g, steeping water 3g, soybean cake powder 13g, NaCl 0.3g, K
2HPO
40.1g, MgSO
47H
2O 0.8g, CaCO
30.2g pH is 8, the preparation of substratum water also is settled to 1 liter, sterilization;
2. seed culture: the 30mL shake flask fermentation liquid that makes is inserted in the 120mL seed culture medium, shake greatly in the bottle at 500mL, 25 ℃, to cultivate 80 hours, shaking speed is 160rpm, makes seed culture fluid; (The Scarlet Letter increases according to claims)
Embodiment 13
The preparation of seed culture medium and cultivation
1. the preparation of seed culture medium: contain starch 100g in one liter of substratum, glucose 5g, steeping water 10g, soybean cake powder 6g, NaCl 0.9g, K
2HPO
40.8g, MgSO
47H
2O 0.1g, CaCO
30.9g pH is 8, the preparation of substratum water also is settled to 1 liter, sterilization;
2. seed culture: the 40mL shake flask fermentation liquid that makes is inserted in the 300mL seed culture medium, and 2L shakes greatly in the bottle, and 35 ℃, to cultivate 20 hours, shaking speed is 300rpm, makes seed culture fluid; (The Scarlet Letter increases according to claims)
Embodiment 14
Fermentor cultivation
1. the preparation of fermention medium: contain starch 50g in one liter of substratum, glucose 8g, steeping water 5g, soybean cake powder 9g, NaCl 0.4g, K
2HPO
40.2g, MgSO
47H
2O 0.3g, CaCO
30.5g, bubble enemy 2g, pH is 6, the preparation of substratum water also is settled to 1 liter, sterilization;
2. cultivate: the seed culture fluid access that embodiment 11 makes is equipped with in the 5L fermentor tank of 3L fermention medium, is 2.5vvm at air flow, and mixing speed is 500rpm, and temperature is 28 ℃, with NaOH and H
2SO
4Regulate pH and be under 5 the condition, cultivated 48 hours, obtain superior strain streptomyces lydicus E9 fermented liquid.
Embodiment 15
Fermentor cultivation
1. the preparation of fermention medium: contain starch 80g in one liter of substratum, glucose 11g, steeping water 8g, soybean cake powder 12g, NaCl 0.7g, K
2HPO
40.4g, MgSO
47H
2O 0.6g, CaCO
30.8g, bubble enemy 5g, pH is 7, the preparation of substratum water also is settled to 1 liter, sterilization;
2. cultivate: the seed culture fluid access that embodiment 12 makes is equipped with in the 5L fermentor tank of 1L fermention medium, is 0.5vvm at air flow, and mixing speed is 600rpm, and temperature is 35 ℃, with NaOH and H
2SO
4Regulate pH and be under 9 the condition, cultivated 24 hours, obtain superior strain streptomyces lydicus E9 fermented liquid.
Embodiment 16
Fermentor cultivation
1. the preparation of fermention medium: contain starch 30g in one liter of substratum, glucose 5g, steeping water 3g, soybean cake powder 6g, NaCl 0.9g, K
2HPO
40.8g, MgSO
47H
2O 0.1g, CaCO
30.6g, bubble enemy 1g, pH is 5, the preparation of substratum water also is settled to 1 liter, sterilization;
2. cultivate: the seed culture fluid access that embodiment 13 makes is equipped with in the 5L fermentor tank of 4L fermention medium, is 3vvm at air flow, and mixing speed is 300rpm, and temperature is 25 ℃, with NaOH and H
2SO
4Regulate pH and be under 4 the condition, cultivated 96 hours, obtain superior strain streptomyces lydicus E9 fermented liquid.
Embodiment 17
Fermentor cultivation
1. the preparation of fermention medium: contain starch 100g in one liter of substratum, glucose 12g, steeping water 10g, soybean cake powder 13g, NaCl 0.4g, K
2HPO
40.1g, MgSO
47H
2O 0.8g, CaCO
30.2g, bubble enemy 8g, pH is 9, the preparation of substratum water also is settled to 1 liter, sterilization;
2. the superior strain streptomyces lydicus E9 fermented liquid that 2L is obtained from embodiment 14 inserts and is equipped with the 30L fermentor tank of 18L fermention medium, is 1vvm at air flow, and mixing speed is 400rpm, and temperature is 29 ℃, with NaOH and H
2SO
4Regulate pH and be under 6 the condition, the normal hexane that adds 200ml was cultivated 72 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 40 ℃, ultrasonication 40min.
Embodiment 18
Fermentor cultivation
1. the preparation of fermention medium: with embodiment 14
2. the superior strain streptomyces lydicus E9 fermented liquid that 2L is obtained from embodiment 14 inserts and is equipped with the 30L fermentor tank of 10L fermention medium, is 0.1vvm at air flow, and mixing speed is 600rpm, and temperature is 35 ℃, with NaOH and H
2SO
4Regulate pH and be under 4 the condition, the normal hexane that adds 120mL was cultivated 96 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 70 ℃, ultrasonication 20min.
Embodiment 19
Fermentor cultivation
1. the preparation of fermention medium: with embodiment 14
2. the superior strain streptomyces lydicus E9 fermented liquid that 2L is obtained from embodiment 14 inserts and is equipped with the 30L fermentor tank of 28L fermention medium, is 2.5vvm at air flow, and mixing speed is 300rpm, and temperature is 25 ℃, with NaOH and H
2SO
4Regulate pH and be under 9 the condition, the n-dodecane that adds 260mL was cultivated 24 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 40 ℃, ultrasonication 30min.
Embodiment 20
Fermentor cultivation
1. the preparation of fermention medium: with embodiment 14
2. the seed culture fluid that embodiment 11 is made inserts and is equipped with in the 30L fermentor tank of 10L fermention medium, is 1.5vvm at air flow, and mixing speed is 500rpm, and temperature is 28 ℃, with NaOH and H
2SO
4Regulate pH and be under 5 the condition, the n-dodecane that adds 160mL was cultivated 48 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 60 ℃, ultrasonication 30min.
Embodiment 21
Fermentor cultivation
1. the preparation of fermention medium: with embodiment 14
2. the seed culture fluid that embodiment 11 is made inserts and is equipped with in the 30L fermentor tank of 28L fermention medium, is 2.5vvm at air flow, and mixing speed is 300rpm, and temperature is 25 ℃, with NaOH and H
2SO
4Regulate pH and be under 9 the condition, the n-dodecane that adds 260mL was cultivated 24 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 40 ℃, ultrasonication 40min.
Embodiment 22
Fermentor cultivation
1. the preparation of fermention medium: with embodiment 14
2. the seed culture fluid that embodiment 11 is made inserts to be equipped with to insert and is equipped with in the 30L fermentor tank of 20L fermention medium, is 0.1vvm at air flow, and mixing speed is 600rpm, and temperature is 35 ℃, with NaOH and H
2SO
4Regulate pH and be under 4 the condition, the normal hexane that adds 120mL was cultivated 96 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid;
3. at 70 ℃, ultrasonication 20min.
Embodiment 23
Anti-mycotic activity is measured
Mycelia piece method: with the petridish of sterilized pipette, extract 15mLPDA substratum injection diameter 12cm, the 0.3mL fermented liquid mixing with putting into wherein is cooled to and solidifies.28 ℃ of activity check indicator bacterium colonies of having cultivated 48h on the PDA substratum are prepared in advance; Use sterilize, internal diameter is the punch tool of 6mm, beats the circular bacterium piece that diameter is 6mm at this colony edge.Pathogenic bacteria bacterium piece is positioned over above-mentioned having solidified in the good PDA substratum, and every ware is placed 1～3 pathogenic bacteria bacterium piece, cultivates 64h for 29 ℃, measures colony diameter (mm), and repeatedly experiment is averaged, and calculates bacteriostasis rate with following formula.Sterilized water with same volume replaces sample as blank;
Bacteriostasis rate (%)=[(blank group bacterium colony mean diameter
2-bacterium colony mean diameter
2) ÷ (blank group bacterium colony mean diameter
2-6
2)] * 100
The fermented liquid anti-mycotic efficiency that embodiment 14-22 is obtained
| Embodiment | 14 | 15 | 16 | 17 | 18 |
| Bacteriostasis rate % | 98.9±0.97 | 71.4±1.4 | 96.3±3.5 | 98.2±0.9 | 85.2±1.3 |
| Embodiment | 19 | 20 | 21 | 22 | |
| Bacteriostasis rate % | 87.9±3.5 | 98.5±3.0 | 87.3±2.7 | 88.9±1.7 |
Claims (5)
1. superior strain streptomyces lydicus (Streptomyces lydicus) E9 CGMCC NO.3075.
2. the fermentation of the superior strain streptomyces lydicus E9 of claim 1 is characterized in that being made up of following steps:
(1) preparation of shake-flask culture base, seed culture medium and fermention medium:
The preparation of shake-flask culture base: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g adds water and is settled to 1 liter, transferring pH is 5～9, sterilization;
The preparation of seed culture medium: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g adds water and is settled to 1 liter, transferring pH is 5～9, sterilization;
The preparation of fermention medium: with starch 30～100g, glucose 5～12g, steeping water 3～10g, soybean cake powder 6～13g, NaCl 0.2～0.9g, K
2HPO
40.1～0.8g, MgSO
47H
2O 0.1～0.8g, CaCO
30.2～0.9g, bubble enemy 1～8g adds water and is settled to 1 liter, and transferring pH is 5～9, sterilization;
(2) shake-flask culture: the superior strain streptomyces lydicus E9 of claim 1 is inserted the 250mL～1000mL that contains 50mL shake-flask culture base shake in the bottle,, under the condition of shaking speed 160～280rpm, cultivated 20～80 hours, the acquisition fermented liquid at 26～30 ℃;
(3) seed culture: 10mL～40mL fermented liquid that step (2) is obtained inserts in the seed culture medium of 90mL～360mL; Shake greatly in the bottle at 500mL～2L, at 25～35 ℃, shaking speed is under 160～300rpm condition; Cultivated 20～80 hours, and made seed culture fluid;
(4) fermentor cultivation, carry out with a kind of of following method:
Method one:
1. said seed culture fluid is inserted and be equipped with in the 5L fermentor tank of 1～4L fermention medium; At air flow is 0.5～3vvm; Mixing speed is 300～600rpm, and temperature is 25～35 ℃, and using acid-alkali accommodation pH is under 4～9 the condition; Cultivated 24～96 hours, and obtained superior strain streptomyces lydicus E9 fermented liquid; 2. at 40～70 ℃, ultrasonication 20～40min;
Method two:
1. insert with said seed culture fluid or through the superior strain streptomyces lydicus E9 fermented liquid that 1. step (4) method one obtains and be equipped with in the 30L fermentor tank of 10～28L fermention medium; At air flow is 0.1～2.5vvm; Mixing speed is 300～600rpm, and temperature is 25～35 ℃, under the condition with acid-alkali accommodation pH4～9; The normal hexane of interpolation 120ml～260ml or n-dodecane were cultivated 24～96 hours as oxygen carrier; Obtain superior strain streptomyces lydicus E9 fermented liquid; 2. the superior strain streptomyces lydicus E9 fermented liquid that obtains is at 40～70 ℃, ultrasonication 20～40min.
3. the fermentation of superior strain streptomyces lydicus E9 according to claim 2 is characterized in that being formulated as of said shake-flask culture base: with starch 70～90g, and glucose 7～10g, steeping water 4～5g, soybean cake powder 7～9g, NaCl 0.3～0.5g, K
2HPO
40.2～0.3g, MgSO
47H
2O 0.2～0.4g, CaCO
30.3～0.5g adds water and is settled to 1 liter, transferring pH is 5～7, sterilization.
4. the fermentation of superior strain streptomyces lydicus E9 according to claim 2 is characterized in that the preparation of said fermention medium: with starch 50～80g, and glucose 8～11g, steeping water 5～8g, soybean cake powder 9～12g, NaCl 0.4～0.7g, K
2HPO
40.2～0.4g, MgSO
47H
2O 0.3～0.6g, CaCO
30.5～0.8g, bubble enemy 2～5g adds water and is settled to 1 liter, and transferring pH is 5～9, sterilization.
5. the fermentation of superior strain streptomyces lydicus E9 according to claim 2; It is characterized in that said step (2) shake-flask culture is: the superior strain streptomyces lydicus E9 of claim 1 is inserted the 250mL～1000mL that contains 50mL shake-flask culture base shake in the bottle; At 29 ℃; Shaking speed is under the 250rpm condition, cultivates 48 hours, obtains fermented liquid; Said step (3) seed culture is: the 10mL fermented liquid that step (2) is obtained inserts in the 90mL seed culture medium, shakes greatly in the bottle at 500mL～2L, and at 27 ℃, shaking speed is under the condition of 230rpm, cultivates 72 hours, makes seed culture fluid.
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