CN115478026B - Radioresistant coccus and application thereof - Google Patents
Radioresistant coccus and application thereof Download PDFInfo
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Abstract
The invention discloses a radioresistant coccus and application thereof, belonging to the technical field of microbial fermentation production. The radioresistant coccus is classified and named (Deinococcus sp.) 43, and has been preserved in China Center for Type Culture Collection (CCTCC), with a preservation number of CCTCC No: m2022653, the preservation time is 2022, 5 and 17 days. The strain is obtained by screening and separating from ginkgo rhizosphere soil, and is cultured for 24 hours to reach the growth stabilization period. The strain is used for flavone fermentation, and can be used for rapidly and efficiently fermenting and producing quercetin in a short time, and the yield of the quercetin is as high as 1.37mg/L. The strain realizes the production of quercetin by bacterial fermentation, so that the strain has great application prospect in the aspect of industrial fermentation production of quercetin.
Description
Technical Field
The invention belongs to the technical field of flavonoid substances produced by microbial fermentation, and particularly relates to a radioresistant coccus and application thereof.
Background
Flavonoids are a group of polyphenolic compounds produced in plants as secondary metabolites, widely found in fruits, vegetables and other food crops. Because of their extremely potent antioxidant activity, they have positive effects on a variety of diseases (e.g., cardiovascular diseases and atherosclerosis, etc.) as well as other biological activities (e.g., anti-inflammatory and anti-aging, etc.). Numerous in vitro and in vivo bioactivity studies have demonstrated their powerful therapeutic and prophylactic benefits for many degenerative diseases and conditions, including neurodegeneration, diabetes, inflammation, autoimmune diseases and cancer; for example, quercetin can effectively inhibit proliferation and apoptosis of tumor cells, while apigenin has low intrinsic toxicity and significant inhibition on cancer cells. Compared with the traditional synthetic medicines, the adverse side effects of flavonoid treatment are almost negligible, so that the flavonoid is an indispensable component in a plurality of nutritional health care products at present.
Most terrestrial plants have evolved over long periods of environmental acclimation to produce flavonoids, which are commonly found in flowers, leaves and seeds. As secondary metabolites, flavonoids in plants play a vital role in mediating plant responses to biological and non-biological environmental factors. For example, anthocyanins are involved in the coloration of fruits and flowers to attract animals and to protect plants from biotic stress factors such as herbivores, bacteria, fungi and the like and abiotic environmental stress sources (such as ultraviolet light absorption). Thus, flavonoids are ubiquitous in all plants. At present, most of flavonoid substances circulated in the market are extracted from plants such as ginkgo, soybean and celery, but because plant growth is limited by environmental factors such as seasons, regions and the like and the market demand increases year by year, the flavonoid substances extracted from the plants can not meet the market demand, and meanwhile, the production of the flavonoid substances by conventional chemical synthesis is complex, a plurality of problems still face, and the large-scale industrial production of the flavonoid substances is still a certain distance.
In recent years, the production of flavones by means of synthetic biology has been rapidly developed due to the development of genetic sequencing technology and the development and application of a series of molecular biology tools, but there is no report on the discovery of bacteria that naturally produce quercetin. Most researches are still focused on the construction of engineering bacteria for producing flavonoid substances, the engineering bacteria are modified by utilizing a synthetic biotechnology to produce flavonoid framework substances, and the efficient heterologous synthesis of the flavonoid substances in the engineering bacteria is realized through a series of synthetic biological methods and strategies such as the construction of a pathway from the head design, the optimization of a modularized modification pathway, the modification of a thallus metabolic network of a gene silencing system and the like. However, there are a number of problems to be solved in the establishment of engineering strains, e.g., C 4 H is a P responsible for hydroxylation of trans-cinnamic acid to coumaric acid 450 Cytochrome monooxygenases, due to their instability and lack of specific cytochrome P 450 Reductase, ultimately leading to its non-function in E.coli; yeast is also a useful relatively large groupOne of the engineering chassis bacteria, but there is also a problem in designing secondary metabolite pathways with yeast hosts because they lack the ability to support multiple gene (multi-cis) transcriptional units; in addition, since genes in genetic engineering technology can only be derived from existing genes in nature and often involve multiple genes at the same time, it means that a lot of time is required to screen ideal genes from nature, and multiple factors such as suitability are considered when assembling these genes. The journal of nature has issued a "five major challenges faced by synthetic biology" to point out that further developments in synthetic biology face the bottleneck of technological innovation and technological integration, and five challenges need to be overcome: (1) Many biological components are not accurately described and therefore are difficult to apply; (2) Even if the function of each component is known, when multiple components are assembled together, it may not work as imagined in advance, and the gene network is difficult to predict; (3) As gene networks become larger, the process of construction and testing of gene networks becomes more laborious; (4) Once constructed and placed into a cell, the synthetic gene network may have an unexpected effect on its host cell, many components beginning to exhibit incompatibilities; (5) The molecular activity in the cell is easy to randomly fluctuate or form noise, the variation of the growth condition can also affect the behavior, and the gene mutation generated randomly for a long time can damage the function of the gene network. These all can lead to the eventual breakdown of the artificially synthesized living system, and thus these existing problems and challenges need to be addressed. The factors together lead to a plurality of problems in constructing engineering strains for producing target flavones to be solved; the invention discovers a radiation-resistant coccus which can be used for producing quercetin by industrial fermentation and has great potential in industrial fermentation of flavonoid substances.
Disclosure of Invention
The invention mainly aims at solving the problem that natural quercetin-producing bacteria are not found at present, and provides a radiation-resistant coccus for producing flavonoid quercetin, which can be used for efficiently producing quercetin and has great application prospect for producing quercetin by industrial fermentation.
In order to achieve the above purpose, the present invention mainly provides the following technical solutions:
the invention provides a radioresistant coccus, which is classified and named Deinococcus sp.43, and has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No: m2022653, the preservation time is 2022, 5 and 17 days.
Furthermore, the radioresistant coccus is separated from Yue Lushan gingko rhizosphere soil in the tsaoko city of Hunan province, the growth speed is high, pink is presented in a flat plate, the surface of the bacterium is smooth, the bacterium is light pink in a liquid culture medium, and the bacterium can grow rapidly within the range of pH 6-8 and 28-30 ℃.
The invention also provides an acquisition method of the radioresistant coccus, which comprises the following steps:
(1) Dissolving a gingko rhizosphere soil sample with sterile water, stirring, standing, and taking supernatant for gradient dilution;
(2) Coating the diluted solution on a glucose peptone yeast extract (TGY) solid plate culture medium for culturing to obtain pink single colony;
(3) Adding agar powder into TGY solid plate culture medium, sterilizing at high temperature, transferring Shan Junla, coating on TGY culture medium, culturing, selecting single colony, and streaking for multiple times to separate pure bacteria;
(4) And (3) performing 16SrDNA amplification sequencing on the red single colony obtained after purification, and performing homology analysis to obtain the radioresistant coccus.
In the method, in the step (2), 1mL of 10 obtained is taken respectively -5 、10 -6 、10 -7 Three gradients of dilutions were plated on TGY solid medium for cultivation.
In the method, in the step (2), the composition of the TGY solid medium is as follows: 5g/L of tryptone, 3g/L of yeast extract powder, 1g/L of glucose and 15g/L of agar; the TGY solid medium has a pH of 6.8-7.2 at 25 ℃.
In the method, in the step (2), the culture time is 24 hours, and the culture temperature is 30 ℃.
In the method, in the step (3), the addition amount of the agar powder is 2% (w/v), the high-temperature sterilization temperature is 115 ℃, and the sterilization time is 30min.
In the method, in the step (3), the culture time is 12-48 hours, and the culture temperature is 28-30 ℃.
In the method, in the step (4), the single colony is purified by using 30% sterilized glycerol.
Further, in the step (4), a phylogenetic tree was constructed by a contiguous method using MEGA 7.0 software, and the obtained strain Deinococcus sp.43 had 98.72% homology with the sequence of Deinococcus sp.N5 of P.radiodurans.
The invention also provides application of the radioresistant coccus for fermenting and producing flavonoid substances.
The steps for producing flavonoid substances by fermentation of the radioresistant coccus are as follows:
(1) Sucking the radioresistant coccus fungus liquid, and adding the radioresistant coccus fungus liquid into a TGY liquid culture medium for culture to obtain fermentation liquor;
(2) Placing the fermentation liquor on a cell disruption instrument for disruption to obtain fermentation liquor with completely broken thalli;
(3) Extracting the fermentation broth after crushing treatment by adopting an organic solvent, and concentrating.
In the step (1), inoculating the strain separated and purified on the solid culture medium into a liquid culture medium for culture, and obtaining an OD < 600 > = 1 bacterial liquid for fermentation culture;
in the step (1), bacterial liquid is sucked according to the inoculum size of 1 percent and added into a TGY liquid culture medium, the culture time of the fermentation liquid is 60-72h, the culture temperature is 28-30 ℃, and the rotating speed is 180-200rpm.
In the step (1), phenylalanine is added according to the concentration of 1-3g/L to be used as a precursor of flavonoid.
In the step (2), the crushing temperature is 28 ℃, the amplitude transformer is 06, the total crushing duration is 20min, wherein each ultrasonic wave is 2s, and the gap is 3s.
In the step (3), the fermentation liquor after the crushing treatment is mixed with ethyl acetate, and after standing and standing, an upper organic phase is separated to obtain a primary extraction fermentation liquor.
In the step (3), the primary extraction fermentation liquor is mixed with ethyl acetate again, an upper organic phase is taken after standing, and the organic phases collected by the two ethyl acetate extractions are mixed.
In the step (3), the organic phase collected by extraction is concentrated to obtain a concentrated solution.
Further, the method comprises the steps of,
cell disruption: 200mL of the fermentation broth is placed on a cell disrupter for disruption, and the fermentation broth with completely disrupted cells is obtained to release target substances possibly existing in cells of the cells.
Primary extraction of fermentation liquor: mixing the crushed fermentation liquor with ethyl acetate according to the equal proportion of 1:1, shaking to fully contact the fermentation liquor, standing for 12 hours, and separating to obtain an upper organic phase.
Secondary extraction of fermentation liquor: and fully mixing the fermentation liquor after primary extraction with ethyl acetate according to the proportion of 2:1, standing and taking an upper organic phase. The organic phases collected from the two extractions are mixed.
The organic phase is concentrated: the temperature of the rotary steaming water is set to 65 ℃, the temperature of a cooling tower is set to-10 ℃, the distillation bottle is fully cleaned by methanol and then is used, after the rotary steaming is finished, the inner wall of the distillation bottle is washed by 5mL of methanol for three times (1, 2 and 2 mL), and the three washing solutions are collected in a 5mL centrifuge tube to obtain concentrated solution, and the concentrated solution is marked; the flask was rinsed three times with 2mL of methanol before the next extract was spin distilled, and the rinse was discarded.
Liquid phase HPLC assay:
pretreatment of experiment: taking 1mL of the obtained concentrated solution, passing through a 0.22 mu m oil system filter membrane, and collecting in a liquid phase sample bottle; distilled water and acetonitrile are filtered by a 0.22 mu m organic phase filter membrane on a suction filter, collected in a brown bottle, and after the suction filtration is finished, the filtrate is placed in an ultrasonic water bath for 30min.
Compared with the prior art, the invention has the following advantages:
the invention discovers that the Deinococcus sp.43 of the radioresistant coccus has short culture period, simple culture medium components and low cost for the first time, and can quickly and efficiently ferment and produce quercetin in a short time, and the yield is as high as 1.37mg/L.
Drawings
FIG. 1 shows the morphology of the strain Deinococcus sp.43 in TGY solid plates (a) and liquid medium (b);
FIG. 2 is a phylogenetic tree of the strain Deinococcus sp.43;
FIG. 3 is a graph (c) showing the yields of quercetin (a), kaempferol and isorhamnetin (a), a graph (b) obtained by extraction of Deinococcus sp.43 fermentation broth, and a graph (b) obtained by conversion of the Deinococcus sp.43 fermentation broth and a control thereof.
Detailed Description
The invention is further illustrated, but not limited, by the following examples.
Example 1
The embodiment is the separation, purification and identification of a strain Deinococcus sp.43, wherein the Deinococcus sp.43 is separated from Yue Lushan gingko rhizosphere soil in Hunan province, and the specific steps are as follows:
dissolving appropriate amount of semen Ginkgo rhizosphere soil sample with sterile water, stirring to dissolve large granule soil particles, standing, gradient diluting 1mL supernatant, and respectively collecting 10 -5 、10 -6 、10 -7 Three gradient dilutions 1mL were spread evenly on TGY solid plate medium consisting of: 5g/L of tryptone, 3g/L of yeast extract powder, 1g/L of glucose and 15g/L of agar; the TGY solid medium had a pH of 7.0 at 25 ℃. Culturing at 30℃for 24 hours, to obtain a pink single colony. Adding 2% (w/v) agar powder into the culture medium, sterilizing at 115deg.C for 30min, pouring into a plate, transferring the single colony onto solid TGY culture medium, spreading, culturing at 30deg.C for 24 hr, and selecting single colony for streaking for multiple times to separate pure bacteria. Finally, the separated single bacteria are stored in a refrigerator at the temperature of minus 80 ℃ by 30 percent of sterilized glycerol.
FIG. 1 (a) shows the morphology of the strain Deinococcus sp.43 in TGY solid plate medium; as can be seen from the figure, deinococcus sp.43 of the radioresistant coccus grows on solid medium plates to form round pink colonies with smooth edges.
Bacterial strain Deinococcus sp.43 was picked from fresh solid medium into centrifuge tubes, DNA was extracted with bacterial genome extraction kit and PCR amplified using 16S universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTACCTTGTTACGACTT-3'). PCR products were sequenced by Shanghai Biolimited, spliced using Bioedit software, then uploaded to NCBI database (https:// www.ncbi.nlm.nih.gov /), BLAST aligned, homology analyzed, and phylogenetic tree constructed in a contiguous manner using MEGA 11.0 software, the results of which are shown in FIG. 2. The results showed that the strain Deinococcus sp.43 had 98.72% homology with the sequence of Deinococcus sp.N5 of the radiation-resistant bacterium.
The 16S rDNA sequencing splice results were as follows:
>GGGGAAGGGCGGGTGCTTAGAATGCAGTCGAACGGCAGTCTTCGGACTGTAGTGGCGCACGGGTGAGTAACGCGTAACTGACCTACCCCAAAGTCGCGGATAACGATTCGAAAGAATCGCTAATACGTGATGTGCTGTCAGATTGTGTTCTGCCAGTAAAGATTGATTGCTTTGGGATGGGGTTGCGTTCCATCAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGACGGATTAGCCCGGCCCTGAGGAGGGGGGCCGGCCACAAGGGGCAACTGAAAACACGGGTCCCAACTCCTACGGGAGGCAGCAGTTAGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCGACGCCGCGTGAGGGATGAAGGTCTTCGGATCGTAAACCTCTGAATCAGGGACGAAAGACACGTTATGTGGGATGACGGTACCTGAGTAATAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACCCGGAATCACTGGGCGTAAAGGGCGTGTAGGCGGACACTTAAGTCTGGTTTTAAAGACTGCGGCTCAACCGCAGGGATGGACTGGATACTGGGTGTCTTGACCTCTGGAGAGAGAACTGGAATTCCTGGTGTAGCGGTGGAATGCGTAGATACCAGGAGGAACACCAATGGCGAAGGCAGGTTCTTGGACAGAAGGTGACGCTGAGGCGCGAAAGTGTGGGGAGCGAACCGGATTAGATACCCGGGTAGTCCACACCCTAAACGATGTACGTTGGCTAACCGCAGGATGCTGTGGTTGGCGAAGCTAACGCGATAAACGTACCGCCTGGGAAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATGCTAGGAACTCCTGAGAGATCAGGAGGTGCCCTTCGGGGAACCTAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTACCTTTAGTTGCCAGCATTGAGTTGGGCACTCTAGAGGGACTGCCTATGAAAGTAGGAGGAAGGCGGGGATGACGTCTAGTCAGCATGGTCCTTACGACCTGGGCTAACACACGTGCTACAATGGATGGGACAACGCGCAGCCAACTTGCGAAAGTGAGCGAATCGCTGAAACCCATCCCCAGTTCAGATCGGAGTCTGCAACTCGACTCCGTGAAGTTGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTAAATTGCAGCTGAAACCGCCGGGAGCCTCACGGCAGGCGTCTAGA。
example 2
The embodiment is a case of producing quercetin by a strain Deinococcus sp.43, and the specific steps are as follows:
fermentation: the culture medium used in the experimental fermentation is TGY culture medium, a proper amount of bacterial liquid is selected from a bacterial preservation tube with the preservation number of Deinococcus sp.43 and coated on a TGY solid plate, and the culture is carried out at 30 ℃ overnight to obtain single bacterial colony. Picking single colony, placing in TGY liquid culture medium, culturing at 30deg.C and 200rpm for 24 hr to OD600 = 1; the morphology of the strain Deinococcus sp.43 in TGY broth is shown in FIG. 1 (b), which shows a yellow color when cultivated in broth to the log phase of growth; meanwhile, phenylalanine is added into the culture medium according to the concentration of 2g/L to be used as a precursor for producing flavonoid substances, and the fermentation system is 200mL; inoculating 1% of the seed solution into a fermentation system, culturing at 30deg.C and 200rpm for 72 hr, and setting blank culture medium without seed solution fermentation as blank control.
And (3) fermentation liquor extraction: placing 200mL of fermentation liquor on a cell disruption instrument, setting the total ultrasonic time to be 20min, wherein the ultrasonic treatment is carried out for 2s intermittently for 3s to obtain fermentation liquor with completely disrupted thalli so as to release target substances possibly existing in thalli cells; mixing the crushed fermentation liquor with ethyl acetate according to the equal proportion of 1:1, shaking to fully contact the fermentation liquor, standing for 12 hours, and separating to obtain an upper organic phase; and fully mixing the fermentation liquor after primary extraction with ethyl acetate again according to the proportion of 2:1, standing and taking an upper organic phase. Mixing the organic phases collected by the two extractions; carrying out rotary steaming treatment on 300mL of ethyl acetate extract, setting the rotary steaming water temperature to 65 ℃, setting the cooling tower temperature to-10 ℃, fully cleaning a distillation bottle with methanol before rotary steaming, then using the distillation bottle, flushing the inner wall of the distillation bottle with 5mL of methanol for three times (1, 2 and 2 mL) after rotary steaming, collecting the three times of washing liquid in a 5mL centrifuge tube to obtain concentrated liquid, and marking; the concentrated methanol solution is filtered by a 0.22 mu m filter membrane and then is sucked and placed into a brown high-efficiency liquid phase sample bottle in proper amount. The flask was rinsed three times with 2mL of methanol before the next extract was spin distilled, and the rinse was discarded.
Preparation of a standard sample: accurately weighing 0.020g, 0.020g and 0.010g of quercetin, kaempferol and isorhamnetin respectively, dissolving with methanol, and fixing the volume to 50mL to prepare mother solutions with the concentration of 0.4mg/mL, 0.4mg/mL and 0.2mg/mL respectively. The mother solutions were diluted separately to prepare standard solutions of three flavone standard substances in a series of concentrations of 0.100mg/mL, 0.050mg/mL, 0.025mg/mL, 0.0125mg/mL and 0.00625mg/mL. A series of standard solutions of three flavone standard substances were filtered through a 0.22 μm filter and placed in a brown high performance liquid sample bottle (-20 ℃ for preservation).
The detection conditions were set as follows: the autosampler was set to a sample volume of 10 μl, a total flow rate of 1.0mL/min (a: b=1:1); column temperature of the chromatographic column is 35 ℃; the detector is set to be 360nm in detection wavelength; the extraction and elution procedure was stopped at 0-10 min. Mobile phase a: acetonitrile, mobile phase B: double steaming; chromatographic column: YMC-TriartC18 (250 mm. Times.4.6 mm,5 μm).
Washing the chromatographic column: the column was washed with A, B phase for more than half an hour before the sample was assayed until no peak appeared.
Standards of 0.4mg/L, 0.4mg/L and 0.2mg/L of quercetin, kaempferol and isorhamnetin are prepared. HPLC detection is performed on the methanol concentrate and the standard, and the detection result is shown in FIG. 3.
FIG. 3 shows the liquid phase results of the fermentation broth of the strain Deinococcus sp.43 and the content of quercetin; FIG. 3 (a) shows a chromatogram of a standard sample of quercetin, kaempferol and isorhamnetin, in which three peaks appear from the front to the rear in this figure, and the peak-appearing times are about 4.4min, 6.0min and 6.2min in this order; FIG. 3 (b) is a chromatogram after extraction of Deinococcus sp.43 fermentation broth of a radioresistant bacterium, wherein obvious peak appearance can be seen in the graph at about 4.4min, which indicates that the bacterium ferments to produce quercetin; FIG. 3 (c) shows the amount of quercetin obtained after further conversion, wherein CK represents a blank medium (medium without added strain), D-43 represents a fermentation broth of the strain Deinococcus sp.43, and after 72 hours of fermentation, the yield of quercetin from the test group of the strain Deinococcus sp.43 is calculated to be 1.37mg/L, and the obtained quercetin has the ability to naturally produce quercetin, and is feasible for fermentation of quercetin.
Example 3
In this example, which is a metabolome of the fermentation product of the strain Deinococcus sp.43, deinococcus sp.43 was subjected to the same fermentation treatment as in example 2, and the fermentation broth was subjected to metabolite detection by UPLC-MS/MS, and the data acquisition instrument system mainly comprises ultra-high performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) (SHIMADZU Nexera X, https:// www.shimadzu.com.cn /) and tandem mass spectrometry (Tandem mass spectrometry, MS/MS) (Applied Biosystems 4500QTRAP, http:// www.appliedbiosystems.com.cn /).
The liquid phase conditions mainly comprise:
(1) Chromatographic column: agilent SB-C18.8 μm,2.1mm x 100mm;
(2) Mobile phase: phase a is ultrapure water (0.1% formic acid added) and phase B is acetonitrile (0.1% formic acid added);
(3) Elution gradient: the proportion of B phase is 5% in 0.00min, the proportion of B phase is linearly increased to 95% in 9.00min and maintained at 95%1min, the proportion of B phase is reduced to 5% in 10.00-11.10min, and the proportion is balanced to 14min at 5%;
(4) The flow rate is 0.35mL/min; column temperature 40 ℃; the sample injection amount was 4. Mu.L.
The mass spectrum conditions mainly comprise:
electrospray ion source (electrospray ionization, ESI) temperature 550 ℃; an ion spray voltage (IS) 5500V (positive ion mode)/-4500V (negative ion mode); ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set to 50, 60 and 25psi, respectively, with the impact induced ionization parameter set high. Instrument tuning and mass calibration were performed with 10 and 100. Mu. Mol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scanning uses MRM mode and sets the collision gas (nitrogen) to medium. DP and CE for each MRM ion pair were completed by further declustering voltage (declustering potential, DP) and Collision Energy (CE) optimization. A specific set of MRM ion pairs is monitored at each time period based on the metabolites eluted during each time period.
The mass spectrum data are processed by using software analysis 1.6.3, and a sample off-machine mass spectrum file is opened by using multi Quant software to perform integration and correction work of chromatographic peaks.
As shown in Table 1, 77 flavonoids including flavonols, isoflavones, anthocyanidins, flavanols, and flavonols were detected, and quercetin was contained therein, indicating that the strain has quercetin-producing ability; secondly, the metabonomic data also show that quercetin related derivatives such as quercetin marigold exist, and the solubility of the quercetin is poor, so that the bioavailability of the quercetin is poor, which suggests that the bacterium possibly has related enzymes capable of converting the quercetin into substances which are more favorable for absorption and utilization of organisms, and has wide application prospects.
TABLE 1 Metabolic group quercetin yield profile for fermentation broths
Sequence listing
<110> university of south-middle school
<120> a radioresistant coccus and use thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1408
<212> DNA
<213> radiation-resistant cocci (Deinococcus sp.)
<400> 1
ggggaagggc gggtgcttag aatgcagtcg aacggcagtc ttcggactgt agtggcgcac 60
gggtgagtaa cgcgtaactg acctacccca aagtcgcgga taacgattcg aaagaatcgc 120
taatacgtga tgtgctgtca gattgtgttc tgccagtaaa gattgattgc tttgggatgg 180
ggttgcgttc catcagctag ttggtggggt aaaggcccac caaggcgacg acggattagc 240
ccggccctga ggaggggggc cggccacaag gggcaactga aaacacgggt cccaactcct 300
acgggaggca gcagttagga atcttccaca atgggcgaaa gcctgatgga gcgacgccgc 360
gtgagggatg aaggtcttcg gatcgtaaac ctctgaatca gggacgaaag acacgttatg 420
tgggatgacg gtacctgagt aatagcaccg gctaactccg tgccagcagc cgcggtaata 480
cggagggtgc aagcgttacc cggaatcact gggcgtaaag ggcgtgtagg cggacactta 540
agtctggttt taaagactgc ggctcaaccg cagggatgga ctggatactg ggtgtcttga 600
cctctggaga gagaactgga attcctggtg tagcggtgga atgcgtagat accaggagga 660
acaccaatgg cgaaggcagg ttcttggaca gaaggtgacg ctgaggcgcg aaagtgtggg 720
gagcgaaccg gattagatac ccgggtagtc cacaccctaa acgatgtacg ttggctaacc 780
gcaggatgct gtggttggcg aagctaacgc gataaacgta ccgcctggga agtacggccg 840
caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900
attcgaagca acgcgaagaa ccttaccagg tcttgacatg ctaggaactc ctgagagatc 960
aggaggtgcc cttcggggaa cctagacaca ggtgctgcat ggctgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt acctttagtt gccagcattg 1080
agttgggcac tctagaggga ctgcctatga aagtaggagg aaggcgggga tgacgtctag 1140
tcagcatggt ccttacgacc tgggctaaca cacgtgctac aatggatggg acaacgcgca 1200
gccaacttgc gaaagtgagc gaatcgctga aacccatccc cagttcagat cggagtctgc 1260
aactcgactc cgtgaagttg gaatcgctag taatcgcagg tcagcatact gcggtgaata 1320
cgttcccggg ccttgtacac accgcccgtc acaccatggg agtaaattgc agctgaaacc 1380
gccgggagcc tcacggcagg cgtctaga 1408
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ggctaccttg ttacgactt 19
Claims (10)
1. The radioresistant coccus is classified and named (Deinococcus sp.) 43, and the preservation number is CCTCC No: m2022653.
2. Use of a radioresistant coccus as claimed in claim 1 for the fermentative production of flavonoids.
3. Use of a radioresistant coccus as claimed in claim 2 for the fermentative production of quercetin.
4. Use of a radioresistant coccus as claimed in claim 2 or 3, wherein the step of fermentation production of flavonoids by the radioresistant coccus comprises the steps of:
(1) Sucking the radioresistant coccus fungus liquid, and adding the radioresistant coccus fungus liquid into a TGY liquid culture medium for culture to obtain fermentation liquor;
(2) Placing the fermentation liquor on a cell disruption instrument for disruption to obtain fermentation liquor with completely broken thalli;
(3) Extracting the fermentation broth after crushing treatment by adopting an organic solvent, and concentrating.
5. The use of a radioresistant coccus as claimed in claim 4, wherein in step (1), the strain isolated and purified on the solid medium is inoculated into a liquid medium for cultivation, and an od600=1 bacterial liquid is obtained for fermentation cultivation.
6. The use of a radioresistant coccus as claimed in claim 4, wherein in step (1) the bacterial liquid is added to the TGY liquid medium in an inoculum size of 1%, the culture time of the fermentation liquid is 60-72 hours, the culture temperature is 28-30 ℃, and the rotation speed is 180-200rpm.
7. The use of a radioresistant coccus as claimed in claim 4, wherein in step (1) phenylalanine is added as a precursor of flavonoid production at a concentration of 1-3 g/L.
8. The use of a radioresistant coccus as claimed in claim 4, wherein in step (3), the fermentation broth after the crushing treatment is mixed with ethyl acetate, left to stand, and separated to obtain an upper organic phase, thereby obtaining a primary extraction fermentation broth.
9. The use of a radioresistant coccus as claimed in claim 8, wherein in step (3), the primary extracted fermentation broth is again mixed with ethyl acetate, the upper organic phase is taken after standing, and the organic phases collected by two ethyl acetate extractions are mixed.
10. The use of a radioresistant coccus as claimed in claim 4, wherein in step (3) the organic phase collected by the extraction is concentrated to give a concentrate.
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