WO2022110088A1 - Culture medium for screening high-yield gibberellin strain - Google Patents

Culture medium for screening high-yield gibberellin strain Download PDF

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Publication number
WO2022110088A1
WO2022110088A1 PCT/CN2020/132546 CN2020132546W WO2022110088A1 WO 2022110088 A1 WO2022110088 A1 WO 2022110088A1 CN 2020132546 W CN2020132546 W CN 2020132546W WO 2022110088 A1 WO2022110088 A1 WO 2022110088A1
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Prior art keywords
gibberellin
medium
screening high
cyanobacterial
vitamin
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PCT/CN2020/132546
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French (fr)
Chinese (zh)
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张恒
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南京溧水高新创业投资管理有限公司
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Priority to PCT/CN2020/132546 priority Critical patent/WO2022110088A1/en
Publication of WO2022110088A1 publication Critical patent/WO2022110088A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
    • A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • the invention belongs to the technical field of microorganism culture, in particular to a medium for screening high-yielding gibberellin strains.
  • Gibberellin is a tetracyclic diterpenoid compound, its chemical structure is relatively complex, the basic structure is gibberellin. More than 100 kinds of gibberellins have been found, and different gibberellins have different biological activities, and gibberellin GA3 has the highest activity.
  • Gibberellin GA3 is a natural, broad-spectrum, high-efficiency plant growth regulator, which can promote the growth and development of crops, make them mature earlier, increase yield, and improve quality; Germination; reduce the shedding of buds, flowers, bolls and fruits, improve the fruiting rate or form seedless fruits; widely used in agricultural production, with good results.
  • the main process used for domestic production of gibberellin GA3 is microbial fermentation. Since the activity of microorganisms grows to a certain stage or decreases, resulting in a decrease in gibberellin production, selecting an appropriate medium can delay the time when the number of bacteria is reduced to a certain extent and improve gibberellin production.
  • MCs microcystins
  • MCs microcystins
  • LR and RR microcystins LR and RR have the highest relative content, more existence and the greatest toxicity. Due to the large amount of toxins contained in cyanobacterial cells, most of the cyanobacterial blooms are discarded or dehydrated into algal sludge and used as organic fertilizer after being collected. However, the large amount of organic matter contained in cyanobacteria has not been fully utilized.
  • a medium with cyanobacterial extract as the main nutritional component is expected, which can be used in the culture of strains, and can screen high gibberellin-producing strains, thereby increasing the yield of gibberellin.
  • the main purpose of the present invention is to overcome the deficiencies in the prior art and provide a medium for screening high-yielding gibberellin strains.
  • the medium of the invention has sufficient sources of raw materials and can be used for screening high-gibberellin-producing strains.
  • the invention provides a medium for screening high-yielding gibberellin strains, characterized in that the medium is composed of: cyanobacteria extract 40-60 g/L, complex vitamins 0.01-0.05 g/L, minerals 0.01 ⁇ 0.1g/L, potassium dihydrogen phosphate 1 ⁇ 5g/L, alanine 0.01 ⁇ 0.1g/L, methionine 0.01 ⁇ 0.1g/L, threonine 0.01 ⁇ 0.1g/L, trace inorganic Salt 0.01 ⁇ 0.02g/L.
  • the medium is composed of: cyanobacteria extract 50g/L, multivitamin 0.03g/L, minerals 0.05g/L, potassium dihydrogen phosphate 3g/L, alanine 0.04g/L, methionine Acid 0.06g/L, threonine 0.05g/L, trace inorganic salt 0.01g/L.
  • the cyanobacterial extract is prepared according to the following method: inoculating the mutagenized Rhodococcus ruber into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of bacteria and algae ratio of 1:1000 ⁇ 1:2000 In the light incubator, bacteria and algae are co-cultivated for 7 to 10 days, and then the cultured mixture is centrifuged and filtered to obtain a precipitate. Spirulina Extract.
  • the mutagenized Rhodococcus ruber is obtained according to the following method: take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 7 to 1 ⁇ 10 8 CFU/mL and spread it on the carrier The slides were then placed in the ARTP mutagenesis breeder for mutagenesis.
  • the mutagenesis conditions are as follows: the mutagenesis power is set to 260-320W, the airflow rate is set to 8-12 SLM, and the mutagenesis treatment time is set to 50-60s.
  • the cyanobacteria slurry obtained by preliminary dehydration is obtained according to the following method: the cyanobacterial slurry salvaged from the water with a water content greater than 99% is processed in a centrifugal dehydrator to obtain a concentrated water content of about 97% cyanobacteria slurry.
  • the percentage composition of the multivitamins is: 30% vitamin B1, 25% vitamin B12, 30% vitamin C, and 25% vitamin E.
  • the minerals are selected from one or a mixture of two or more of magnesium sulfate, zinc sulfate, sodium sulfate, and potassium sulfate.
  • the trace inorganic salts are composed of: CoCl 2 ⁇ 6H 2 O 150 mg/L, CuCl 2 ⁇ 5H 2 O 200 mg/L, H 3 BO 3 100 mg/L, MnCl 2 ⁇ 4H 2 O 150 mg/L, Na 2 MoO 4 ⁇ 2H 2 O 150mg/L, ferric citrate 100mg/L.
  • the pH of the medium is 7.0 ⁇ 0.5.
  • the present invention at least has the following advantages: the present invention takes cyanobacteria as the main material source, and uses the treated cyanobacteria as the main components of the culture medium for screening high-yielding nystatin strains, on the one hand, it can make full use of the cyanobacteria contained.
  • Organic and inorganic substances on the other hand, cyanobacteria are not only rich in sources, but also fully utilized to reduce environmental pollution.
  • the medium of the invention has simple components, and can be used for screening high-yielding gibberellin strains in the culture process, and can increase the density of the strains, thereby increasing the gibberellin yield.
  • the special medium of the present invention has rich sources of components, can effectively screen out high gibberellin-producing strains, and the method is simple. It has many of the above advantages and practical value, and there is no similar design published or used in similar products and methods, but it is indeed an innovation. It has great improvements in both methods and functions. It has made great progress, and has produced easy-to-use and practical effects. Compared with the existing products, it has improved many functions, so it is more suitable for practical use, and has the value of extensive use in the industry. It is a novel, progressive, and Practical new design.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 290W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 55s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained after preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1500, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured
  • the resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 250° C. for 12 min, and then cooled to room temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.03g/L, magnesium sulfate 0.05g/L, diphosphate Potassium hydrogen 3g/L, alanine 0.04g/L, methionine 0.06g/L, threonine 0.05g/L, trace inorganic salt 0.01g/L (composition: CoCl 2 ⁇ 6H 2 O 150mg/L L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L), pH 7.0 ⁇ 0.5.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 7 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 260W, the air flow was set to 12SLM, and the mutagenesis treatment time was set to 60s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacteria slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the culture was carried out.
  • the resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.02g/L, potassium sulfate 0.08g/L, phosphate diphosphate Potassium hydrogen 3g/L, alanine 0.06g/L, methionine 0.06g/L, threonine 0.06g/L, trace inorganic salts (composition: CoCl 2 ⁇ 6H 2 O 150mg/L, CuCl 2 ⁇ 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.01g/L, pH 7.0 ⁇ 0.5.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 280W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 60s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 200° C. for 10 min, and then cooled to room temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.02g/L, potassium sulfate 0.08g/L, phosphate diphosphate Potassium hydrogen 3g/L, alanine 0.06g/L, methionine 0.06g/L, threonine 0.06g/L, trace inorganic salts (composition: CoCl 2 ⁇ 6H 2 O 150mg/L, CuCl 2 ⁇ 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.01g/L, pH 7.0 ⁇ 0.5.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power is set to 300W, the airflow is set to 8SLM, and the mutagenesis processing time is set to 50s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured
  • the resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.03g/L, zinc sulfate 0.1g/L, phosphate diphosphate Potassium hydrogen 2g/L, alanine 0.01g/L, methionine 0.01g/L, threonine 0.01g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150 mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L , pH 7.0 ⁇ 0.5.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power is set to 300W, the airflow is set to 8SLM, and the mutagenesis processing time is set to 50s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured
  • the resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.05g/L, sodium sulfate 0.01g/L, diphosphate Potassium hydrogen 1g/L, alanine 0.1g/L, methionine 0.1g/L, threonine 0.01g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L, pH 7.0 ⁇ 0.5.
  • Mutagenesis treatment of Rhodococcus ruber take 20 ⁇ L of Rhodococcus ruber spore suspension with a concentration of 1 ⁇ 10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 320W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 50s.
  • cyanobacterial extract The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%.
  • the mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained after preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1500, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured
  • the resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 250° C. for 15 min, and then cooled to room temperature to obtain a cyanobacterial extract.
  • composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.01g/L, magnesium sulfate 0.01g/L, phosphate diphosphate Potassium hydrogen 5g/L, alanine 0.01g/L, methionine 0.01g/L, threonine 0.1g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L, pH 7.0 ⁇ 0.5.

Abstract

Disclosed is a culture medium for screening a high-yield gibberellin strain, characterised in that the culture medium is composed of: 40-60 g/L of a blue-green algae extract, 0.01-0.05 g/L of decavitamin, 0.01-0.1 g/L of minerals, 1-5 g/L of potassium dihydrogen phosphate, 0.01-0.1 g/L of alanine, 0.01-0.1 g/L of methionine, 0.01-0.1 g/L of threonine and 0.01-0.02 g/L of a trace inorganic salt. By means of taking blue-green algae as the main substance source, treated blue-green algae are used as the main ingredient of a culture medium for screening a high-yield nysfungin strain. Organic and inorganic substances contained in blue-green algae can be fully utilized, and also blue-green algae are abundant and can be fully utilized, thereby reducing environmental pollution. The culture medium has a simple composition and during the culture process for screening a high-yield gibberellin strain, the density of the strain can be increased, thereby increasing the yield of gibberellin.

Description

用于筛选高产赤霉素菌种的培养基Medium for screening high gibberellin-producing strains 技术领域technical field
本发明属于微生物培养相关技术领域，具体涉及一种用于筛选高产赤霉素菌种的培养基。The invention belongs to the technical field of microorganism culture, in particular to a medium for screening high-yielding gibberellin strains.
背景技术Background technique
赤霉素，为四环二萜类化合物，其化学结构比较复杂，基本结构是赤霉素烷，在赤霉素烷上由于双键、羟基数目和位置不同，形成了各种赤霉素，现已发现100多种，不同的赤霉素生物活性不同，而赤霉素GA3的活性最高。赤霉素GA3是一种天然、广谱、高效的植物生长调节剂，可促进作物生长发育，使之提早成熟、提高产量、改进品质；能迅速打破种子、块茎和鳞茎等器官的休眠，促进发芽；减少蕾、花、铃、果实的脱落，提高果实结果率或形成无籽果实；普遍应用于农业生产上，具有良好的使用效果。Gibberellin is a tetracyclic diterpenoid compound, its chemical structure is relatively complex, the basic structure is gibberellin. More than 100 kinds of gibberellins have been found, and different gibberellins have different biological activities, and gibberellin GA3 has the highest activity. Gibberellin GA3 is a natural, broad-spectrum, high-efficiency plant growth regulator, which can promote the growth and development of crops, make them mature earlier, increase yield, and improve quality; Germination; reduce the shedding of buds, flowers, bolls and fruits, improve the fruiting rate or form seedless fruits; widely used in agricultural production, with good results.
目前国内生产赤霉素GA3主要采取的工艺是微生物发酵法。由于微生物生长到一定阶段后活性或降低，从而导致赤霉素产量下降，因此选择合适的培养基能够在一定程度上延缓菌群数量降低的时间，提高赤霉素产量。At present, the main process used for domestic production of gibberellin GA3 is microbial fermentation. Since the activity of microorganisms grows to a certain stage or decreases, resulting in a decrease in gibberellin production, selecting an appropriate medium can delay the time when the number of bacteria is reduced to a certain extent and improve gibberellin production.
世界各地25％-70％的蓝藻水华可产生毒素，其中微囊藻毒素(以下简称MC)是一类分布最广泛且与人类关系最密切的七肽单环肝毒素。MC通常大部分存在于藻细胞内，当细胞破裂或衰老时毒素释放进入水中。MC的组成十分复杂，其中相对含量较高、存在较多、毒性最大的是微囊藻毒素LR、RR。由于蓝藻细胞内含有大量毒素，因此蓝藻水华被收集后大多被废弃或脱水处理为藻泥后用做有机肥，然而蓝藻内还含有的大量有机物质没有得到充分利用。25%-70% of cyanobacterial blooms around the world can produce toxins, among which microcystins (hereinafter referred to as MCs) are the most widely distributed and most closely related to humans. MCs are usually found mostly within algal cells, and toxins are released into the water when cells rupture or age. The composition of MC is very complex, among which the microcystins LR and RR have the highest relative content, more existence and the greatest toxicity. Due to the large amount of toxins contained in cyanobacterial cells, most of the cyanobacterial blooms are discarded or dehydrated into algal sludge and used as organic fertilizer after being collected. However, the large amount of organic matter contained in cyanobacteria has not been fully utilized.
基于以上，期待一种以蓝藻提取物为主要营养成分的培养基，该培养基用于菌种培养中，能够筛选高产赤霉素菌种，从而提高赤霉素产量。Based on the above, a medium with cyanobacterial extract as the main nutritional component is expected, which can be used in the culture of strains, and can screen high gibberellin-producing strains, thereby increasing the yield of gibberellin.
发明内容SUMMARY OF THE INVENTION
本发明的主要目的在于克服现有技术中的不足，提供一种用于筛选高产赤霉素菌种的培养基。本发明的培养基原料来源充足，可用于筛选高产赤霉素的菌种。The main purpose of the present invention is to overcome the deficiencies in the prior art and provide a medium for screening high-yielding gibberellin strains. The medium of the invention has sufficient sources of raw materials and can be used for screening high-gibberellin-producing strains.
本发明的目的及解决其技术问题是采用以下技术方案来实现的。The purpose of the present invention and the solution to its technical problems are achieved by adopting the following technical solutions.
本发明提供了一种用于筛选高产赤霉素菌种的培养基，其特征在于，所述培养基组成为：蓝藻提取物40～60g/L，复合维生素0.01～0.05g/L，矿物质0.01～0.1g/L，磷酸二氢钾1～5g/L，丙氨酸0.01～0.1g/L，甲硫氨酸0.01～0.1g/L，苏氨酸0.01～0.1g/L，微量无机盐0.01～0.02g/L。The invention provides a medium for screening high-yielding gibberellin strains, characterized in that the medium is composed of: cyanobacteria extract 40-60 g/L, complex vitamins 0.01-0.05 g/L, minerals 0.01～0.1g/L, potassium dihydrogen phosphate 1～5g/L, alanine 0.01～0.1g/L, methionine 0.01～0.1g/L, threonine 0.01～0.1g/L, trace inorganic Salt 0.01～0.02g/L.
优选地，所述培养基组成为：蓝藻提取物50g/L，复合维生素0.03g/L，矿物质0.05g/L，磷酸二氢钾3g/L，丙氨酸0.04g/L，甲硫氨酸0.06g/L，苏氨酸0.05g/L，微量无机盐0.01g/L。Preferably, the medium is composed of: cyanobacteria extract 50g/L, multivitamin 0.03g/L, minerals 0.05g/L, potassium dihydrogen phosphate 3g/L, alanine 0.04g/L, methionine Acid 0.06g/L, threonine 0.05g/L, trace inorganic salt 0.01g/L.
优选地，所述蓝藻提取物按照以下方法制备得到：将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:1000～1:2000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在200～300℃热处理10～15min后，冷却至常温，得到蓝藻提取物。Preferably, the cyanobacterial extract is prepared according to the following method: inoculating the mutagenized Rhodococcus ruber into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of bacteria and algae ratio of 1:1000～1:2000 In the light incubator, bacteria and algae are co-cultivated for 7 to 10 days, and then the cultured mixture is centrifuged and filtered to obtain a precipitate. Spirulina Extract.
优选地，所述所述经过诱变处理的赤红球菌(Rhodococcus ruber)是按照以下方法得到：取20μL浓度为1×10 7～1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理。 Preferably, the mutagenized Rhodococcus ruber is obtained according to the following method: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 7 to 1×10 8 CFU/mL and spread it on the carrier The slides were then placed in the ARTP mutagenesis breeder for mutagenesis.
优选地，所述所述诱变条件为：诱变功率设定为260～320W，气流量设定为8～12SLM，诱变处理时间设定为50～60s。Preferably, the mutagenesis conditions are as follows: the mutagenesis power is set to 260-320W, the airflow rate is set to 8-12 SLM, and the mutagenesis treatment time is set to 50-60s.
优选地，所述所述经过初步脱水得到的蓝藻浆泥是按照以下方法得到：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。Preferably, the cyanobacteria slurry obtained by preliminary dehydration is obtained according to the following method: the cyanobacterial slurry salvaged from the water with a water content greater than 99% is processed in a centrifugal dehydrator to obtain a concentrated water content of about 97% cyanobacteria slurry.
优选地，所述复合维生素的百分比组成为：30％维生素B1、25％维生素B12，30％维生素C，25％维生素E。Preferably, the percentage composition of the multivitamins is: 30% vitamin B1, 25% vitamin B12, 30% vitamin C, and 25% vitamin E.
优选地，所述矿物质选自硫酸镁、硫酸锌、硫酸钠、硫酸钾中的一种或者两种以上的混合物。Preferably, the minerals are selected from one or a mixture of two or more of magnesium sulfate, zinc sulfate, sodium sulfate, and potassium sulfate.
优选地，所述微量无机盐组成为：CoCl 2·6H 2O 150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L。 Preferably, the trace inorganic salts are composed of: CoCl 2 ·6H 2 O 150 mg/L, CuCl 2 ·5H 2 O 200 mg/L, H 3 BO 3 100 mg/L, MnCl 2 ·4H 2 O 150 mg/L, Na 2 MoO 4 ·2H 2 O 150mg/L, ferric citrate 100mg/L.
优选地，所述培养基的pH为7.0±0.5。Preferably, the pH of the medium is 7.0 ± 0.5.
借由上述技术方案，本发明至少具有下列优点：本发明以蓝藻为主要物质来源，将经过处理的蓝藻作为筛选高产制霉素菌种的培养基主要成分，一方面能够充分利用蓝藻所含有的有机和无机物，另一方面，蓝藻不仅来源丰富，还能得到充分利用，降低的对环境的污染。本发明的培养基成分简单，用于筛选高产赤霉素菌种的培养过程中，能够提高菌种的密度，从而提高赤霉素产量。By the above-mentioned technical scheme, the present invention at least has the following advantages: the present invention takes cyanobacteria as the main material source, and uses the treated cyanobacteria as the main components of the culture medium for screening high-yielding nystatin strains, on the one hand, it can make full use of the cyanobacteria contained. Organic and inorganic substances, on the other hand, cyanobacteria are not only rich in sources, but also fully utilized to reduce environmental pollution. The medium of the invention has simple components, and can be used for screening high-yielding gibberellin strains in the culture process, and can increase the density of the strains, thereby increasing the gibberellin yield.
综上所述，本发明特殊的培养基成分来源丰富，能有效筛选出高产赤霉素的菌种，且方法简单。其具有上述诸多的优点及实用价值，并在同类产品和方法中未见有类似的设计公开发表或使用而确属创新，其不论在方法上或功能上皆有较大的改进，在技术上有较大的进步，并产生了好用及实用的效果，且较现有的产品具有增进的多项功效，从而更加适于实用，而具有产业的广泛利用价值，诚为一新颖、进步、实用的新设计。To sum up, the special medium of the present invention has rich sources of components, can effectively screen out high gibberellin-producing strains, and the method is simple. It has many of the above advantages and practical value, and there is no similar design published or used in similar products and methods, but it is indeed an innovation. It has great improvements in both methods and functions. It has made great progress, and has produced easy-to-use and practical effects. Compared with the existing products, it has improved many functions, so it is more suitable for practical use, and has the value of extensive use in the industry. It is a novel, progressive, and Practical new design.
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例详细说明如后。The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, and to implement according to the content of the description, the preferred embodiments of the present invention are described in detail below.
具体实施方式Detailed ways
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。In order to make it easy to understand the technical means, creative features, goals and effects realized by the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments It is only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
实施例1Example 1
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 8CFU/mL的赤 红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为290W，气流量设定为10SLM，诱变处理时间设定为55s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 290W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 55s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:1500的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在250℃热处理12min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained after preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1500, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 250° C. for 12 min, and then cooled to room temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素B12，30％维生素C，25％维生素E)0.03g/L，硫酸镁0.05g/L，磷酸二氢钾3g/L，丙氨酸0.04g/L，甲硫氨酸0.06g/L，苏氨酸0.05g/L，微量无机盐0.01g/L(组成为：CoCl 2·6H 2O 150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.03g/L, magnesium sulfate 0.05g/L, diphosphate Potassium hydrogen 3g/L, alanine 0.04g/L, methionine 0.06g/L, threonine 0.05g/L, trace inorganic salt 0.01g/L (composition: CoCl 2 ·6H 2 O 150mg/L L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L), pH 7.0 ± 0.5.
实施例2Example 2
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 7CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为260W，气流量设定为12SLM，诱变处理时间设定为60s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 7 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 260W, the air flow was set to 12SLM, and the mutagenesis treatment time was set to 60s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:1000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在300℃热处理15min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacteria slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the culture was carried out. The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素B12，30％维生素C，25％维生素E)0.02g/L，硫酸钾0.08g/L，磷酸二氢钾3g/L，丙氨酸0.06g/L，甲硫氨酸0.06g/L，苏氨酸0.06g/L，微量无机盐(组成为：CoCl 2·6H 2O150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)0.01g/L，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.02g/L, potassium sulfate 0.08g/L, phosphate diphosphate Potassium hydrogen 3g/L, alanine 0.06g/L, methionine 0.06g/L, threonine 0.06g/L, trace inorganic salts (composition: CoCl 2 ·6H 2 O 150mg/L, CuCl 2 · 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.01g/L, pH 7.0±0.5.
实施例3Example 3
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为280W，气流量设定为10SLM，诱变处理时间设定为60s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 280W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 60s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:2000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在200℃热处理10min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 200° C. for 10 min, and then cooled to room temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素B12，30％维生素C，25％维生素E)0.02g/L，硫酸钾0.08g/L，磷酸二氢钾3g/L，丙氨酸0.06g/L，甲硫氨酸0.06g/L，苏氨酸0.06g/L，微量无机盐(组成为：CoCl 2·6H 2O150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)0.01g/L，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.02g/L, potassium sulfate 0.08g/L, phosphate diphosphate Potassium hydrogen 3g/L, alanine 0.06g/L, methionine 0.06g/L, threonine 0.06g/L, trace inorganic salts (composition: CoCl 2 ·6H 2 O 150mg/L, CuCl 2 · 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.01g/L, pH 7.0±0.5.
实施例4Example 4
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为300W，气流量设定为8SLM，诱变处理时间设定为50s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power is set to 300W, the airflow is set to 8SLM, and the mutagenesis processing time is set to 50s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:2000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在300℃热处理15min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素B12，30％维生素C，25％维生素E)0.03g/L，硫酸锌0.1g/L，磷酸二氢钾2g/L，丙氨酸0.01g/L，甲硫氨酸0.01g/L，苏氨酸0.01g/L，微量无机盐(组成为：CoCl 2·6H 2O 150 mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)0.02g/L，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.03g/L, zinc sulfate 0.1g/L, phosphate diphosphate Potassium hydrogen 2g/L, alanine 0.01g/L, methionine 0.01g/L, threonine 0.01g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150 mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L , pH 7.0 ± 0.5.
实施例5Example 5
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为300W，气流量设定为8SLM，诱变处理时间设定为50s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power is set to 300W, the airflow is set to 8SLM, and the mutagenesis processing time is set to 50s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:2000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在300℃热处理15min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained by preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:2000, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 300° C. for 15 min, and then cooled to normal temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素B12，30％维生素C，25％维生素E)0.05g/L，硫酸钠0.01g/L，磷酸二氢钾1g/L，丙氨酸0.1g/L，甲硫氨酸0.1g/L，苏氨酸0.01g/L，微量无机盐(组成为：CoCl 2·6H 2O 150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)0.02g/L，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.05g/L, sodium sulfate 0.01g/L, diphosphate Potassium hydrogen 1g/L, alanine 0.1g/L, methionine 0.1g/L, threonine 0.01g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L, pH 7.0 ± 0.5.
实施例6Example 6
赤红球菌(Rhodococcus ruber)的诱变处理：取20μL浓度为1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理，诱变功率设定为320W，气流量设定为10SLM，诱变处理时间设定为50s。 Mutagenesis treatment of Rhodococcus ruber: take 20 μL of Rhodococcus ruber spore suspension with a concentration of 1×10 8 CFU/mL and spread it on the slide, then put the slide into the ARTP mutagenesis breeder for mutagenesis treatment , the mutagenesis power was set to 320W, the air flow was set to 10SLM, and the mutagenesis treatment time was set to 50s.
制备蓝藻提取物：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:1500的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在250℃热处理15min后，冷却至常温，得到蓝藻提取物。Preparation of cyanobacterial extract: The cyanobacterial slurry salvaged from the water with a moisture content of more than 99% is processed in a centrifugal dehydrator to obtain a concentrated cyanobacterial slurry with a moisture content of about 97%. The mutagenized Rhodococcus ruber was inoculated into the cyanobacterial slurry obtained after preliminary dehydration according to the volume ratio of the bacteria and algae ratio of 1:1500, and the bacteria and algae were co-cultivated in a light incubator for 7 to 10 days, and then the cultured The resulting mixture was centrifuged and filtered to obtain a precipitate, and then the precipitate was heat-treated at 250° C. for 15 min, and then cooled to room temperature to obtain a cyanobacterial extract.
配制培养基组成为：蓝藻提取物50g/L，复合维生素(30％维生素B1、25％维生素 B12，30％维生素C，25％维生素E)0.01g/L，硫酸镁0.01g/L，磷酸二氢钾5g/L，丙氨酸0.01g/L，甲硫氨酸0.01g/L，苏氨酸0.1g/L，微量无机盐(组成为：CoCl 2·6H 2O 150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L)0.02g/L，pH 7.0±0.5。 The composition of the preparation medium is: cyanobacteria extract 50g/L, multivitamins (30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% vitamin E) 0.01g/L, magnesium sulfate 0.01g/L, phosphate diphosphate Potassium hydrogen 5g/L, alanine 0.01g/L, methionine 0.01g/L, threonine 0.1g/L, trace inorganic salts (composition: CoCl 2 6H 2 O 150mg/L, CuCl 2 5H 2 O 200mg/L, H 3 BO 3 100mg/L, MnCl 2 4H 2 O 150mg/L, Na 2 MoO 4 2H 2 O 150mg/L, ferric citrate 100mg/L) 0.02g/L, pH 7.0 ± 0.5.
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，当可利用上述揭示的方法及技术内容作出些许的更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。The above are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. The skilled person, within the scope of the technical solution of the present invention, can make some changes or modifications to equivalent examples of equivalent changes by using the methods and technical contents disclosed above, but not departing from the content of the technical solution of the present invention, Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solutions of the present invention.

Claims (10)

  1. 一种用于筛选高产赤霉素菌种的培养基，其特征在于，所述培养基组成为：蓝藻提取物40～60g/L，复合维生素0.01～0.05g/L，矿物质0.01～0.1g/L，磷酸二氢钾1～5g/L，丙氨酸0.01～0.1g/L，甲硫氨酸0.01～0.1g/L，苏氨酸0.01～0.1g/L，微量无机盐0.01～0.02g/L。A medium for screening high-yielding gibberellin strains, characterized in that the medium is composed of: 40-60g/L of cyanobacteria extract, 0.01-0.05g/L of multivitamins, and 0.01-0.1g of minerals /L, potassium dihydrogen phosphate 1～5g/L, alanine 0.01～0.1g/L, methionine 0.01～0.1g/L, threonine 0.01～0.1g/L, trace inorganic salt 0.01～0.02 g/L.
  2. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述培养基组成为：蓝藻提取物50g/L，复合维生素0.03g/L，矿物质0.05g/L，磷酸二氢钾3g/L，丙氨酸0.04g/L，甲硫氨酸0.06g/L，苏氨酸0.05g/L，微量无机盐0.01g/L。The medium for screening high-yielding gibberellin strains according to claim 1, wherein the medium is composed of: cyanobacterial extract 50g/L, multivitamin 0.03g/L, mineral 0.05g/L L, potassium dihydrogen phosphate 3g/L, alanine 0.04g/L, methionine 0.06g/L, threonine 0.05g/L, trace inorganic salt 0.01g/L.
  3. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述蓝藻提取物按照以下方法制备得到：将经过诱变处理的赤红球菌(Rhodococcus ruber)按照菌藻比1:1000～1:2000的体积比接种到经过初步脱水得到的蓝藻浆泥中，在光照培养箱菌藻共同培养7～10天，然后将培养后的混合物经离心、过滤，得到沉淀物，然后将沉淀物在200～300℃热处理10～15min后，冷却至常温，得到蓝藻提取物。The medium for screening high-yielding gibberellin strains according to claim 1, wherein the cyanobacteria extract is prepared according to the following method: the mutagenized Rhodococcus ruber is prepared according to the bacterium algae The volume ratio of 1:1000~1:2000 is inoculated into the cyanobacteria slurry obtained by preliminary dehydration, and the bacteria and algae are co-cultivated in a light incubator for 7~10 days, and then the cultured mixture is centrifuged and filtered to obtain a precipitate , and then the precipitate is heat-treated at 200-300° C. for 10-15 min, and then cooled to room temperature to obtain the cyanobacterial extract.
  4. 根据权利要求3所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述所述经过诱变处理的赤红球菌(Rhodococcus ruber)是按照以下方法得到：取20μL浓度为1×10 7～1×10 8CFU/mL的赤红球菌孢子悬浮液涂布于载片上，然后将载片放到ARTP诱变育种仪内进行诱变处理。 The culture medium for screening high-yielding gibberellin strains according to claim 3, wherein the mutagenized Rhodococcus ruber is obtained according to the following method: take 20 μL of a concentration of 1 Rhodococcus rhodochrous spore suspension of ×10 7 to 1 × 10 8 CFU/mL was spread on the slides, and then the slides were put into the ARTP mutagenesis breeder for mutagenesis treatment.
  5. 根据权利要求4所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述所述诱变条件为：诱变功率设定为260～320W，气流量设定为8～12SLM，诱变处理时间设定为50～60s。The medium for screening high gibberellin-producing strains according to claim 4, wherein the mutagenesis conditions are: mutagenesis power is set to 260-320W, and air flow is set to 8- 12SLM, the mutagenesis treatment time was set to 50-60s.
  6. 根据权利要求3所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述所述经过初步脱水得到的蓝藻浆泥是按照以下方法得到：将从水中打捞上来的含水率大于99％的蓝藻浆液离心脱水机中处理，得到浓缩后的含水率约97％的蓝藻浆泥。The medium for screening high-yielding gibberellin strains according to claim 3, wherein the described cyanobacterial slurry obtained through preliminary dehydration is obtained according to the following method: The cyanobacterial slurry greater than 99% is processed in a centrifugal dehydrator to obtain a cyanobacterial slurry with a moisture content of about 97% after concentration.
  7. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述复合维生素的百分比组成为：30％维生素B1、25％维生素B12，30％维生素C，25％维生素E。The medium for screening high-producing gibberellin strains according to claim 1, wherein the percentage composition of the complex vitamins is: 30% vitamin B1, 25% vitamin B12, 30% vitamin C, 25% Vitamin E.
  8. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述 矿物质选自硫酸镁、硫酸锌、硫酸钠、硫酸钾中的一种或者两种以上的混合物。The medium for screening high-yielding gibberellin strain according to claim 1, wherein the mineral is selected from one or more of magnesium sulfate, zinc sulfate, sodium sulfate, potassium sulfate mixture.
  9. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述微量无机盐组成为：CoCl 2·6H 2O 150mg/L，CuCl 2·5H 2O 200mg/L，H 3BO 3 100mg/L，MnCl 2·4H 2O 150mg/L，Na 2MoO 4·2H 2O 150mg/L，柠檬酸铁100mg/L。 The medium for screening high-producing gibberellin strains according to claim 1, wherein the trace inorganic salts are composed of: CoCl 2 ·6H 2 O 150 mg/L, CuCl 2 ·5H 2 O 200 mg/L L, H 3 BO 3 100 mg/L, MnCl 2 ·4H 2 O 150 mg/L, Na 2 MoO 4 ·2H 2 O 150 mg/L, ferric citrate 100 mg/L.
  10. 根据权利要求1所述的用于筛选高产赤霉素菌种的培养基，其特征在于，所述培养基的pH为7.0±0.5。The medium for screening high gibberellin-producing strains according to claim 1, wherein the pH of the medium is 7.0±0.5.
PCT/CN2020/132546 2020-11-28 2020-11-28 Culture medium for screening high-yield gibberellin strain WO2022110088A1 (en)

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