CN108004150A - A kind of epicoccum nigrum LS10H and its application - Google Patents
A kind of epicoccum nigrum LS10H and its application Download PDFInfo
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Abstract
The invention discloses a kind of epicoccum nigrum LS10H and its application, belongs to microbial technology field, and the epicoccum nigrum LS10H has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center；Deposit number：CGMCC No.13871；Preservation date：On 06 06th, 2017；Preservation place：Institute of Microorganism, Academia Sinica；The strain is fast with the speed of growth, and vitality is vigorous, and pollution microbes are not easy during fermented and cultured, it is easy to pure culture, set out with the bacterial strain, natural biochrome rose pigment and carotenoid can be produced by the control of fermentating culturing process, be conducive to the production extraction of natural pigment.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of epicoccum nigrum LS10H and its application.
Background technology
One kind that natural food colour refers to extract from animal and plant tissue and microbial fermentation product eats color
Element.Compared with animal and plant tissue extraction, preparing natural pigment using microbial fermentation has economy, efficiently and from ground
The advantages such as reason, season or climatic effect, are one of important sources of natural pigment.Part microorganisms pigments are except lifting food color
Also there are certain nutrition health-care functions outside damp quality, as Monascouruarin also has the drug effect of lipid-loweringing, decompression and hypoglycemic at the same time
Function, lycopene have the function of higher oxidize-resistant physiology etc..The diversity of microorganism and its complexity of cometabolism are determined
The diversity of microorganisms pigments and its function is determined, it is also most to have development and one kind life of application potential that chromogenic element, which occurs, for microorganism
Look for plain production method.Presently found microorganisms pigments mainly have carotenoid, melanin, flavin and several major classes of quinones,
Such as by Blakeslea trispora (Blakeslea trispora) produced beta carotene and lycopene, Monascus
(Monascus spp.) produced Monascouruarin, the produced melanin of mould (mold) and phaffiafhodozyma (Phaffia
Rhodozyma) produced astaxanthin etc..
Epicoccum (Epicoccum Link) microorganism be a kind of safety, not generation toxin, there is applications well
The natural food colouring agent production bacterial strain of prospect, the pigment found at present in Epicoccum mainly have carotenoid pigment
(Carotenoids), flavipin (Flavipin), chromanone (Chromanone), polyketone class pigment
(Orevactaene) and pyridinone pigment (Epipyridone) etc.；Epicoccum nigrum is a kind of endogenetic fungus, belongs to Fungi Imperfecti
Guiding principle, the mould mesh of shell, Bei Mei sections, Epicoccum are wider in distributed in nature.Among research application, epicoccum nigrum and its spore master
It is to be applied to disclose one in biological control and the fermenting and producing of natural pigment, such as Chinese Patent Application No. 201110253331.2
Kind epicoccum nigrum DB3 and its answering in flax, hemp, jute or bastose technique are used in peroxide degumming preparation weaving
Epicoccum nigrum was reported first in 1997 with, Yue-Zhong Shu etc. can ferment produce a kind of new polyenoid (hydrocarbon) polyketone
Class pigment, Chinese patent publication No. CN106591156A disclose a kind of filamentous fungi epicoccum nigrum FXZ2 and its in heavy metals
Lead, cadmium pollution it is biological prosthetic in application.Application of the epicoccum nigrum in the pigment that ferments, that announces at present is less.
The content of the invention
According to above the deficiencies in the prior art, the technical problems to be solved by the invention are to propose a kind of epicoccum nigrum
LS10H and its application, in order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：
A kind of epicoccum nigrum LS10H, is named as epicoccum nigrum (Epicoccum nigrum) LS10H and has been preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, deposit number：CGMCC No.13871, preservation date：2017
06 day 06 month year, preservation place：Institute of Microorganism, Academia Sinica, and the screening of bacterial strain, separation are carried out in the following way
And identification：
1) bacterial strain screening and separation：The soil of a variety of trees rhizospheres in Wuhu City Mt. Mountain park is taken, is hanged with sterile water
Float and shake mixing, screening and culturing medium is coated on after 100 times of dilution, quiescent culture 4-7 days, pair can with chromogenesis it is Filamentous very
Bacterium bacterium colony further carries out line separation in screening and culturing medium, until obtaining the pure culture of purpose bacterial strain；
2) identification of bacterial strain：Using conventional DNA clone technology, extract the chromosomal DNA of bacterial strain, using the chromosomal DNA as
Template, using 5.8S rDNA-ITS region universal primers as amplimer, bacterial strain 5.8S rDNA-ITS bases are obtained using PCR method
Because of fragment, then PCR product is connected with plasmid pMD18-T and converted big after the detection of 1% agarose gel electrophoresis confirms
Enterobacteria JM109 competent cells, extracting recombinant plasmid carry out the sequencing of target gene, the DNA sequences that then will be sequenced out
Row, are compared by database Blast/blastp, then comparison result is utilized Neighbor-Joining methods structure system
Development tree, which is accredited as epicoccum nigrum by combining form observation result, and names epicoccum nigrum LS10H.
Preferably, the screening and culturing medium component is potato 200g/L, glucose 20g/L, ampicillin 0.3g/L,
Kanamycins 0.1g/L, agar powder 15g/L, deionized water 1000mL, pH6.7-7.2.
Preferably, the universal primer is ITS1：5'-TCCGTAGGTGAACCTGCGG-3' and ITS4：5'-
TCCTCCGCT TATTGATATGC-3' primers.
Preferably, the reaction system of the PCR method is：On the basis of 50 μ L, 0.5 μ L, dNTPs mixing of chromosomal DNA
4 μ L, rTaq archaeal dna polymerase of thing 0.5 μ L, ITS1 0.3 μ L, ITS4 0.3 μ L, 5 μ L of DNA polymerase buffer liquid, distilled water 39.4
μ L, PCR amplification program：95 DEG C of denaturation 4min, 94 DEG C of denaturation 30s, 55 DEG C of renaturation 45s, 72 DEG C of extension 90s, 30 circulate, and 72
DEG C re-extend 10min.
Preferably, the epicoccum nigrum LS10H optimum growth temperatures are 25-35 DEG C, and the most suitable growth pH is 3.5-7.5.
A kind of application of epicoccum nigrum LS10H, the epicoccum nigrum LS10H are applied to fermenting and producing water colo(u)r.
Preferably, the water colo(u)r is rose pigment and carotenoid.
Compared with prior art, beneficial effects of the present invention：
1. the epicoccum nigrum LS10H of separation and Extraction of the present invention, the speed of growth is fast, and vitality is vigorous, during fermented and cultured
Pollution microbes are not easy, are easy to pure culture, and using different culture mediums, incubation time and cultivation temperature, produce different colours
Water colo(u)r, is conducive to the production extraction of natural pigment.
2. the rose pigment that the present invention is prepared using epicoccum nigrum LS10H fermentations, there is ripe rose to be had
Some red shades, and it is soluble easily in water.
3. the present invention is easy to operate for technique using epicoccum nigrum LS10H fermentation preparation rose pigments, that is, it is suitable for solid
State fermented and cultured, and it is suitable for liquid state fermentation culture, fermentation process avoids polluting.
Brief description of the drawings
Fig. 1 is growth temperature curve map under epicoccum nigrum LS10H different temperatures；
Fig. 2 is growth temperature curve map under epicoccum nigrum LS10H differences pH；
Epicoccum nigrum LS10H cultivates 3 days bacterium colony photos in PDA culture medium under the conditions of Fig. 3 is 20 DEG C；
Epicoccum nigrum LS10H cultivates 6 days bacterium colony photos in PDA culture medium under the conditions of Fig. 4 is 30 DEG C；
Epicoccum nigrum LS10H cultivates 10 days bacterium colony back side photos in PDA culture medium under the conditions of Fig. 5 is 30 DEG C；
Fig. 6 is 400 times of microscopic morphology figures of epicoccum nigrum LS10H mycelia；
Fig. 7 is bacterial strain LS10H 5.8S rDNA-ITS phylogenetic evolution trees.
Embodiment
Below by the description to embodiment, it is described in further detail, to help those skilled in the art to this hair
Bright inventive concept, technical solution have more complete, accurate and deep understanding.
Embodiment 1
Epicoccum nigrum LS10H bacterial strain screenings, separation, identification：
1) bacterial strain screening and separation：0.5g is taken to pick up from the soil of a variety of trees rhizospheres and use in Wuhu City Mt. Mountain park
5mL sterile waters are suspended and shake mixing, and screening and culturing medium is coated on after 100 times of dilution, right in 30 DEG C of quiescent cultures 5 days
Line separation can further be carried out in screening and culturing medium with the filamentous fungi bacterium colony of chromogenesis, until obtaining purpose bacterial strain
Pure culture, wherein, screening and culturing medium contains potato 200g/L, and glucose 20g/L, ampicillin 0.3g/L, it is mould to block that
Plain 0.1g/L, agar powder 15g/L, deionized water 1000mL, pH7, and ampicillin and kanamycins in medium sterilization simultaneously
Bacterial strain is added after being cooled to 50 DEG C to be cultivated, according to the bacterial strain optimum growth temperature curve of culture studies separation and Extraction as schemed
Shown in 1, for the most suitable growth pH as shown in Fig. 2, bacterial strain Initial stage of culture under the conditions of 30 DEG C is usually first 3 days, mycelia, bacterium colony quality are soft
It is soft for white as shown in figure 3, microexamination shows that mycelia is very thin, has the tabula as shown in Figure 4；Late stage of culture the 4th to the 7th day, bacterium
Falling surface has yellow liquid exudate such as Fig. 5, while has a large amount of water-soluble yellow pigments to secrete into culture medium；Continue culture
8 to 15 days thalline can produce dark brown spot punctate substance, and the phase adjusts cultivation temperature to 10-25 DEG C of scope to bacterial strain after incubation
Interior, thalline can largely produce red water colo(u)r and secrete into culture medium, continue culture the 8 to 15th day after thalline can produce it is black
Moth patch punctate substance is as shown in Figure 6：
2) identification of bacterial strain：Bacterium is identified using molecular biology method, specially using DNA grams conventional
The chromosomal DNA of grand technology extraction bacterial strain LS10H, draws using the chromosomal DNA as template, with 5.8S rDNA-ITS regions are general
Thing ITS1：5'-TCCGTAGGTGAACCTGCGG-3' and ITS4：5'-TCCTCCGCTTATTGATATGC-3' is amplimer,
Bacterial strain 5.8S rDNA-ITS genetic fragments are obtained using PCR method, the reaction system of PCR method is as follows：With L benchmark of 50 μ,
0.5 μ L, dNTPs mixture of chromosomal DNA 4 μ L, rTaq archaeal dna polymerase, 0.5 0.3 0.3 μ L of μ L, ITS4 of μ L, ITS1 are added,
5 μ L of DNA polymerase buffer liquid, 39.4 μ L of distilled water, PCR amplification program：95 DEG C of denaturation 4min, 94 DEG C are denatured 30s, 55 DEG C of renaturation
45s, 72 DEG C of extension 90s, condition will carry out 30 circulations above, and finally re-extend 10min at 72 DEG C；By PCR product through 1% fine jade
Sepharose electrophoresis detection is connected with plasmid pMD18-T after confirming and converts e. coli jm109 competent cell, extracts
Recombinant plasmid simultaneously entrusts Sangon Biotech (Shanghai) Co., Ltd.) limited company complete target gene sequencing, the results showed that,
It is 511bp that bacterial strain rDNA-ITS regional DNAs, which effectively measure sequence length, specifically as shown in SEQ ID No.1.
Bacterial strain rDNA-ITS DNA sequence dnas are compared measure sequence by online database Blast/blastp, select
Take similar sequences and carry out multiple alignment using BioEdit softwares, comparison result utilizes Neighbor- by software MEGA 5.1
Joining (NJ) methods phylogenetic tree construction as described in Figure 7, observe the bacterial strain that result isolates this and identify by combining form
For epicoccum nigrum, and it is named as epicoccum nigrum (Epicoccum nigrum) LS10H.Freezed by being inoculated with inclined-plane and preparing glycerine
Obtained strains LS10H is carried out preservation by tube method under the conditions of 4 DEG C and -80 DEG C of ultralow temperature respectively.
Embodiment 2
Epicoccum nigrum LS10H is in the aborning application of rose pigment fermentation：
1) seed culture：By epicoccum nigrum LS10H by potato 200g/L, glucose 20g/L, deionized water 1000mL
The pH of composition is that line culture is carried out in 7 PDA culture medium, and culture uses mycelium 8cm under the sterile washings of 10mL after 3 days2, system
Obtain mycelium suspension, as fermentation seed liquor；
2) ferment：The above-mentioned mycelium suspensions of 5mL are taken to be inoculated in 35mL by glucose 25g/L, peptone 1g/L, NaCl
0.5g/L, K2HPO40.5g/L, MgSO40.5g/L, FeSO4The pH that 0.01g/L is formed is 5.5, through 121 DEG C of sterilizing 20min
On the fluid nutrient medium of cooling, after being cultivated 3 days under 30 DEG C, shaking speed 200r/min, by fermentation condition be set as 18 DEG C,
200r/min, continues culture 3 days；
3) ethanol extraction pigment：After fermentation, 30min, separating thallus and zymotic fluid are centrifuged in 4000r/min, then
The ethanol solution that concentration is 60% is added into wet thallus, the additive amount of ethanol is that additive amount is depending on the amount of wet thallus
10 mL/g, by both after mixing, leach 30min at 25 DEG C, after 4000r/min centrifuges 30min, obtain true ethanol and carry
Liquid is taken, 2.5mL zymotic fluids or ethanol extract is directly drawn and is transferred in 1cm cuvettes, under room temperature in 450nm
Absorbance value OD is measured at wavelength450, using nonvaccinated fermentation medium as blank control, under above-mentioned fermentation condition, fermentation
Pigment content can reach OD in liquid4502.1, pigment content accounts for the 20% of fermentation pigment total content in thalline.
Embodiment 3
Epicoccum nigrum LS10H is in the aborning application of rose pigment fermentation：
1) seed culture：By epicoccum nigrum LS10H by potato 200g/L, glucose 20g/L, deionized water 1000mL
The pH of composition is that line culture is carried out in 7 PDA culture medium, and culture uses mycelium 8cm under the sterile washings of 10mL after 3 days2, system
Obtain mycelium suspension, as fermentation seed liquor；
2) ferment：The above-mentioned mycelium suspensions of 5mL are taken to be inoculated in 45mL by glucose 25g/L, peptone 0.5g/L, NaCl
1g/L, K2HPO40.3g/L, MgSO40.4g/L, FeSO4The pH that 0.015g/L is formed is 5.5, through 121 DEG C of sterilizing 20min
On the fluid nutrient medium of cooling, after being cultivated 3 days under 30 DEG C, shaking speed 200r/min, by fermentation condition be set as 15 DEG C,
200r/min, continues culture 4 days；
3) ethanol extraction pigment：After fermentation, 30min, separating thallus and zymotic fluid are centrifuged in 4000r/min, then
The ethanol solution that concentration is 60% is added into wet thallus, the additive amount of ethanol is that additive amount is depending on the amount of wet thallus
10 mL/g, by both after mixing, leach 30min at 25 DEG C, after 4000r/min centrifuges 30min, obtain true ethanol and carry
Liquid is taken, 2.5mL zymotic fluids or ethanol extract is directly drawn and is transferred in 1cm cuvettes, under room temperature in 450nm
Absorbance value OD is measured at wavelength450, using nonvaccinated fermentation medium as blank control, under above-mentioned fermentation condition, fermentation
Pigment content can reach OD in liquid4501.8, pigment content accounts for the 27% of fermentation pigment total content in thalline.
Embodiment 4
Epicoccum nigrum LS10H is in the aborning application of fermentative carotenoid：
1) seed culture：By epicoccum nigrum LS10H by potato 200g/L, glucose 20g/L, deionized water 1000mL
The pH of composition is that line culture is carried out in 7 PDA culture medium, and culture uses mycelium 8cm under the sterile washings of 10mL after 4 days2, system
Obtain mycelium suspension, as fermentation seed liquor；
2) ferment：The above-mentioned mycelium suspensions of 5mL are taken to be inoculated in 100g by long rice 50g, ZnSO40.02g, NaCl 1g/
L, K2HPO40.4g/L, MgSO40.6g/L, deionized water 50mL form pH be 6, through 121 DEG C sterilizing 20min, cooling
In solid-state fermentation culture medium, quiescent culture 4 days under the conditions of 30 DEG C are stirred evenly, during which used sterile glass rod every 1 day
A uniform stirring processing is carried out to fermentation substrate；
3) purify：After fermentation, 500mL deionized waters are added by the quality of solid state fermentation, after soaking and stirring evenly
30min is extracted in 40 DEG C of water-baths, leaching liquor centrifuges 30min under the conditions of 4000r/min, removes solid content and collects supernatant
Liquid, by supernatant by vacuum freeze-drying method it is dry water-soluble carotenoid crude product, by water-soluble carotenoid crude product
It is dissolved at ambient temperature in 50mL absolute ethyl alcohols, centrifugation removes the solid content that cannot be dissolved in absolute ethyl alcohol and collects supernatant
Liquid, removes ethanol by rotary evaporation in vacuo method at 60 DEG C by the ethanol lysate of the pigment of acquisition and can obtain water-soluble class
Carrotene sterling, ultraviolet-uisible spectrophotometer Detection wavelength is 438nm, using deionized water as blank control, in above-mentioned fermentation
Under the conditions of, pigment content can reach OD in leaching liquor438 1.4。
Embodiment 5
Epicoccum nigrum LS10H is in the aborning application of fermentative carotenoid：
1) seed culture：By epicoccum nigrum LS10H by potato 200g/L, glucose 20g/L, deionized water 1000mL
The pH of composition is that line culture is carried out in 7 PDA culture medium, and culture uses mycelium 8cm under the sterile washings of 10mL after 4 days2, system
Obtain mycelium suspension, as fermentation seed liquor；
2) ferment：The above-mentioned mycelium suspensions of 5mL are taken to be inoculated in 50g by long rice 25g, ZnSO40.05g, NaCl 1g/L,
K2HPO40.5g/L, MgSO4The pH that 0.6g/L, deionized water 25mL are formed is 5.5, and through 121 DEG C of sterilizing 20min, cooling is consolidated
In state fermentation medium, quiescent culture 5 days under the conditions of 29 DEG C are stirred evenly, during which used sterile glass rod pair every 1 day
Fermentation substrate carries out a uniform stirring processing；
3) purify：After fermentation, 500mL deionized waters are added by the quality of solid state fermentation, after soaking and stirring evenly
90min is extracted in 50 DEG C of water-baths, leaching liquor centrifuges 30min under the conditions of 4000r/min, removes solid content and collects supernatant
Liquid, by supernatant by vacuum freeze-drying method it is dry water-soluble carotenoid crude product, by water-soluble carotenoid crude product
It is dissolved at ambient temperature in 50mL absolute ethyl alcohols, centrifugation removes the solid content that cannot be dissolved in absolute ethyl alcohol and collects supernatant
Liquid, removes ethanol by rotary evaporation in vacuo method at 60 DEG C by the ethanol lysate of the pigment of acquisition and can obtain water-soluble class
Carrotene sterling, ultraviolet-uisible spectrophotometer Detection wavelength is 438nm, using deionized water as blank control, in above-mentioned fermentation
Under the conditions of, pigment content can reach OD in leaching liquor438 3.2。
Embodiment 6
The measure of epicoccum nigrum LS10H optimum growth temperatures：
By epicoccum nigrum LS10H by potato 200g/L, the pH that glucose 20g/L, deionized water 1000mL are formed is 7
PDA culture medium in into line cultivate, culture 4 days after use the sterile washings of 10mL under mycelium 8cm2, mycelium suspension is made,
5mL bacterial suspension inoculations are taken in 35mL PDA culture mediums, by shaking table temperature be set in respectively 15 DEG C, 20 DEG C, 25 DEG C, 28 DEG C, 30
DEG C, 35 DEG C and 40 DEG C, shaking speed 200r/min, under these conditions respectively cultivate 4 days after, culture is all transferred to pre-
First dry to the 50mL centrifuge tubes of constant weight and somatic cells are collected by centrifugation, further by centrifuge tube and somatic cells together 65
It is dried in DEG C baking oven, every 3 hours weigh once in assay balance, and the front and rear weight change of gained twice is less than 0.001g
It is considered as sample and is dried to constant weight, utilizes calculating dry cell weight of poor quality.Size according to dry cell weight judges the most suitable of strain
Growth temperature range, dry cell weight is bigger, shows the growth of more suitable strain.
It should be noted that：(1) optimum growth temperature, refers to a relative temperature range of suitable thalli growth, (2) quality
Difference, which refers to dry cell weight and is equal to the gross mass of the dry sample containing centrifuge tube and somatic cells to constant weight, to be subtracted and is pre-dried to
The quality of the centrifuge tube of constant weight.
The present invention is exemplarily described above in conjunction with specific embodiment, it is clear that the present invention implements and from upper
The limitation of mode is stated, if the improvement of the various unsubstantialities of inventive concept and technical scheme of the present invention progress is employed, or
It is not improved by the present invention design and technical solution directly apply to other occasions, protection scope of the present invention it
It is interior.Protection scope of the present invention should be determined by the scope of protection defined in the claims.
Sequence table
<110>Anhui Polytechnic University
<120>A kind of epicoccum nigrum LS10H and its application
<130> 2017.12.20
<141> 2017-12-23
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 511
<212> DNA
<213> Epicoccum nigrum
<400> 1
gggttacctg gcagttagta gacttcggtc tgctacctct tacccatgtc ttttgagtac 60
cttcgtttcc tcggcgggtc cgcccgccga ttggacaaca ttcaaaccct ttgcagttgc 120
aatcagcgtc tgaaaaaact taatagttac aactttcaac aacggatctc ttggttctgg 180
catcgatgaa gaacgcagcg aaatgcgata agtagtgtga attgcagaat tcagtgaatc 240
atcgaatctt tgaacgcaca ttgcgcccct tggtattcca tggggcatgc ctgttcgagc 300
gtcatttgta ccttcaagct ctgcttggtg ttgggtgttt tgtctcgcct ctgcgtgtag 360
actcgcctta aaacaattgg cagccggcgt attgatttcg gagcgcagta catctcgcgc 420
tttgcactca taacgacgac gtccaaaagt acatttttac actcttgacc tcggatcagg 480
tagggatacc cgctgaactt aagcatatca t 511
Claims (7)
1. a kind of epicoccum nigrum LS10H, it is characterised in that the epicoccum nigrum LS10H is named as epicoccum nigrum
(Epicoccum nigrum) LS10H, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number：CGMCC No.13871, preservation date：On 06 06th, 2017, preservation place：Chinese Academy of Sciences's microbe research
Institute, and screening, separation and the identification of bacterial strain are carried out in the following way：
1) bacterial strain screening and separation：The soil of a variety of trees rhizospheres in Wuhu City Mt. Mountain park is taken, is suspended simultaneously with sterile water
Concussion mixes, and screening and culturing medium is coated on after 100 times of dilution, quiescent culture 4-7 days, pair can be with the filamentous fungi bacterium of chromogenesis
Fall and line separation is further carried out in screening and culturing medium, until obtaining the pure culture of purpose bacterial strain；
2) identification of bacterial strain：Using conventional DNA clone technology, the chromosomal DNA of bacterial strain is extracted, using the chromosomal DNA as template,
Using 5.8S rDNA-ITS region universal primers as amplimer, bacterial strain 5.8S rDNA-ITS gene pieces are obtained using PCR method
PCR product, is then connected and converts large intestine bar by section after the detection of 1% agarose gel electrophoresis confirms with plasmid pMD18-T
Bacterium JM109 competent cells, extracting recombinant plasmid carry out the sequencing of target gene, and the DNA sequence dna that then will be sequenced out, leads to
Cross database Blast/blastp to be compared, then comparison result is utilized into the structure systematic growth of Neighbor-Joining methods
Tree, which is accredited as epicoccum nigrum by combining form observation result, and names epicoccum nigrum LS10H.
2. epicoccum nigrum LS10H according to claim 1, it is characterised in that the screening and culturing medium component is potato
200g/L, glucose 20g/L, ampicillin 0.3g/L, kanamycins 0.1g/L, agar powder 15g/L, deionized water
1000mL, pH6.7-7.2.
3. epicoccum nigrum LS10H according to claim 1, it is characterised in that the universal primer is ITS1：5'-
TCCGTAGGTGAACCTGCGG-3' and ITS4：5'-TCCTCCGCTTATTGATATGC-3' primers.
4. epicoccum nigrum LS10H according to claim 1, it is characterised in that the reaction system of the PCR method is：With
On the basis of 50 μ L, 0.5 μ L, dNTPs mixture of chromosomal DNA, 4 μ L, rTaq archaeal dna polymerase 0.5 μ L, ITS1 0.3 μ L, ITS4
0.3 μ L, 5 μ L of DNA polymerase buffer liquid, 39.4 μ L of distilled water, PCR amplification program：95 DEG C denaturation 4min, 94 DEG C denaturation 30s, 55
DEG C renaturation 45s, 72 DEG C of extension 90s, 30 circulations, 72 DEG C re-extend 10min.
5. epicoccum nigrum LS10H according to claim 1, it is characterised in that the epicoccum nigrum LS10H the most suitable growth temperature
Spend for 25-35 DEG C, the most suitable growth pH is 3.5-7.5.
A kind of 6. application of any epicoccum nigrum LS10H of claim 1-5, it is characterised in that the epicoccum nigrum
LS10H is applied to fermenting and producing water colo(u)r.
7. the application of epicoccum nigrum LS10H according to claim 5, it is characterised in that the water colo(u)r is rose
Red pigments and carotenoid.
Priority Applications (1)
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| CN112358974A (en) * | 2020-12-09 | 2021-02-12 | 昆明理工大学 | Endophytic fungus epicoccum nigrum FZT214 and application thereof |
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| CN1377956A (en) * | 2001-04-05 | 2002-11-06 | 国家海洋局第一海洋研究所 | Ocean bacteria capable of producing haematochrome and method for producing haematochrome using said bacteria |
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| CN112358974A (en) * | 2020-12-09 | 2021-02-12 | 昆明理工大学 | Endophytic fungus epicoccum nigrum FZT214 and application thereof |
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