CN110551638B - Penicillium citrinum and application thereof in production of citrinin - Google Patents
Penicillium citrinum and application thereof in production of citrinin Download PDFInfo
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Abstract
The invention discloses penicillium citrinum and application thereof in citrinin production, belonging to the technical field of microorganisms and fermentation. The invention screens out a Penicillium citrinum (Penicillium citrinum) HR-087 strain, and the Penicillium citrinum (Penicillium citrinum) HR-087 can produce citrinin with high yield; the citrinin content in the fermentation liquor can reach 4.1g/L by shaking the culture flask of the Penicillium citrinum HR-087 for 96 h; fermenting the Penicillium citrinum HR-087 fermentation tank for 96h to make the content of citrinin in the fermentation liquid reach 9.62 g/L.
Description
Technical Field
The invention relates to penicillium citrinum and application thereof in citrinin production, belonging to the technical field of microorganisms and fermentation.
Background
Citrinin (CIT), also known as Citrinin, has a molecular formula C13H14O5The pure product is yellow crystal, sensitive to fluorescence, can be pyrolyzed in acidic and alkaline solutions, is originally discovered to be produced by penicillium in 30 years of 20 th century, is highly regarded as antibiotic, is excluded from the possibility of being used as therapeutic antibiotic due to the discovery of nephrotoxicity, and is considered as mycotoxin.
The research shows that the citrinin has strong renal toxicity, and the administration of the citrinin can enlarge the kidney of various experimental animals, and finally cause renal failure. Research also found that citrinin has teratogenic, hepatic metabolism impairing, carcinogenic and mutagenic effects and may inhibit the central nervous system. Therefore, in the reference published by the international cancer research institution of the world health organization in 2017 for the preliminary arrangement of carcinogen lists, citrinin is defined as a class 3 carcinogen, and the limited standard of citrinin in food has also been strictly regulated in japan and the european union.
At present, the detection of the content of citrinin is mainly realized by taking citrinin as a standard sample and by using a high performance liquid chromatography, so that a large amount of citrinin is required to be used as the standard sample in the detection of the content of citrinin in food in the market.
However, the existing methods for producing citrinin have low yield, mainly because the existing citrinin producing strains have low yield, for example, Li Hui Fang et al uses Penicillium expansum as a producing strain to produce citrinin by shake flask fermentation for 14d, the yield is only 123.3 μ g/L (see the specific literature: Li Hui Fang, an expansin producing citrinin and the research on the production conditions thereof [ J ] food science, 2018,39(24): 162-; chen cautios et al use monascus strain L as a production strain to produce citrinin by shake flask fermentation for 7 days, wherein the yield is only 75.24mg/L (see the specific literature: Chen cautios. Penicillium expansum producing citrinin and the research on the production conditions thereof [ J ] food science, 2018,39(24): 162-; blanc et al, produced citrinin by Monascus strain by shake flask fermentation 14d with yield of only 370mg/L using Monascus ruber as the production strain (see in particular, blank. production of citrinin by vacuum species of Monascus [ J ]. Biotechnology Letters,1995,17: 291-.
Therefore, it is urgently needed to find a strain capable of producing citrinin with high yield so as to improve the yield of the citrinin standard sample.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide Penicillium citrinum (Penicillium citrinum) capable of producing citrinin at a high yield.
[ solution ]
In order to solve the problems, the invention provides Penicillium citrinum (Penicillium citrinum), which is preserved in China center for type culture collection in 23.05.2019 with the preservation number of CCTCC NO: M2019384 and the preservation address of Wuhan university in Wuhan, China.
The Penicillium citrinum is separated from a wild jujube sample from Fenghuangshan in Shanxi province, the strain is subjected to sequencing analysis, the 18S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in NCBI, and the result shows that the strain is Penicillium citrinum and is named as Penicillium citrinum HR-087.
The colony of the Penicillium citrinum HR-087 on the PDA culture medium has ciliated edges, the mycelial layer is soft, white and flat when in initial growth, the center of the colony is grayish green, the edges of the colony are white to light yellow, and the back of the colony is yellow.
The invention also provides a method for producing citrinin, which comprises the steps of inoculating the Penicillium citrinum into a fermentation medium for fermentation to obtain fermentation liquor containing the citrinin, and then extracting the fermentation liquor to obtain the citrinin.
In one embodiment of the present invention, the fermentation temperature is 20 to 37 ℃, the rotation speed is 100 to 1000rpm, and the ventilation amount is 1 to 5 vvm.
In one embodiment of the invention, the fermentation temperature is 30 ℃, the rotation speed is 800rpm, and the aeration is 4 vvm.
In one embodiment of the invention, the fermentation medium comprises 20-80 g/L of glycerol, 1-10 g/L of peptone and 50-400 g/L of potato.
In one embodiment of the invention, the fermentation medium comprises 30-50 g/L of glycerol, 2-8 g/L of peptone and 150-250 g/L of potato.
In one embodiment of the invention, the components of the fermentation medium comprise 40g/L glycerol, 5g/L peptone and 200g/L potato.
In one embodiment of the invention, glycerol is fed to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation.
In one embodiment of the invention, glycerol is fed to the fermentation medium at a feed rate of 0.4mL/h throughout the fermentation.
The invention also provides the application of the Penicillium citrinum or the method for producing citrinin in producing citrinin.
[ advantageous effects ]
The invention screens out a Penicillium citrinum (Penicillium citrinum) HR-087 strain, and the Penicillium citrinum (Penicillium citrinum) HR-087 can produce citrinin with high yield; the citrinin content in the fermentation liquor can reach 4.1g/L by shaking the culture flask of the Penicillium citrinum HR-087 for 96 h; fermenting the Penicillium citrinum HR-087 fermentation tank for 96h to make the content of citrinin in the fermentation liquid reach 9.62 g/L.
Biological material preservation
A Penicillium citrinum (Penicillium citrinum) HR-087, which is classified and named as Penicillium citrinum, has been preserved in China center for type culture Collection in 23.05.2019 with the preservation number of CCTCC NO: M2019384, and the preservation unit address is Wuhan, Wuhan university, China.
Drawings
FIG. 1: influence of the rotational speed on the biomass and the citrinin yield in the shake flask fermentation of Penicillium citrinum HR-087.
FIG. 2: the effect of glycerol feed rate on biomass and citrinin production during fermentation in a Penicillium citrinum (HR-087) fermentor.
Detailed Description
The invention is further illustrated by the following specific examples.
The media involved in the following examples are as follows:
PDA liquid culture medium: 200g/L of potato and 20g/L of glucose.
PDA solid medium: 200g/L of potato, 20g/L of glucose and 20g/L of agar.
The detection methods referred to in the following examples are as follows:
method for determining biomass: collecting fermentation liquid, centrifuging for 15min under 14000r/min, removing supernatant after centrifugation, drying precipitate to constant weight, and weighing.
The method for measuring the content of the citrinin comprises the following steps:collecting fermentation liquor, centrifuging for 15min under 14000r/min, collecting supernatant 100 μ L, adding 900 μ L methanol, mixing, and measuring by Agilent 1100 High Performance Liquid Chromatography (HPLC); HPLC detection conditions: a chromatographic column: ZORBAX Eclipse XDB C18column; mobile phase: 450mL/L methanol and 650mL/L water; flow rate: 1 mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; a detector: an ultraviolet detector; detection wavelength: 290 nm.
The method for measuring the concentration of glycerol comprises the following steps: collecting fermentation liquid, centrifuging for 15min under 14000r/min, collecting supernatant 100 μ L, adding 900 μ L distilled water, mixing, and measuring by Shimadzu 20A High Performance Liquid Chromatography (HPLC); HPLC detection conditions: a chromatographic column: ZORBAX Aminex HPX-87H column; mobile phase: 5mmol/L HCl; flow rate: 0.5 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; a detector: ultraviolet and parallax refraction detectors; detection wavelength: 190 nm.
Purifying and extracting citrinin: collecting fermentation liquor, adding ethyl acetate into the fermentation liquor according to the ratio of 1:3 (V/V) for extraction, collecting supernatant, performing rotary evaporation to obtain yellow oily substance, and freeze-drying the oily substance to obtain a primarily purified citrinin product in yellow powder.
Example 1: screening and identification of Penicillium citrinum
The method comprises the following specific steps:
1. preliminary screening
Taking wild jujube from Yanan Fenghuang mountain in Shaanxi as a sample; pretreating a sample, sequentially soaking the sample in 95% alcohol for 1-5 min, then washing with sterile water for 4-5 times, soaking the sample in 0.1% mercuric chloride for 3-8 min, then washing with sterile water for 7-8 times, soaking the sample in 75% alcohol for 3-5 min, then washing with sterile water for 4-5 times for surface disinfection, finally cutting the sample into blocks under sterile conditions, inoculating the blocks into a PDA solid culture medium, and standing and culturing the blocks at constant temperature of 30 ℃ for 48 hours; after 48 hours, selecting mycelium, inoculating the mycelium into a new PDA solid culture medium for culture, and separating and purifying to obtain two endophytes, namely a strain HR-087 and a strain CG-035.
2. Double sieve
Respectively inoculating the obtained strain HR-087 and strain CG-035 into PDA solid culture medium, culturing at 30 deg.C for 48h, respectively inoculating 1mg mycelium into 250mL shake flask containing 50mL PDA liquid culture medium, culturing at 28 deg.C and 220r/min for 72h to obtain seed solution; transferring the seed solution to a 250mL shake flask with 20mL fermentation medium by an inoculation amount of 1%, and culturing for 96h at 30 ℃ and 220r/min to obtain a fermentation solution; wherein the components of the fermentation medium comprise 40g/L of glycerol, 5g/L of peptone and 200g/L of potato.
The content of citrinin in the fermentation liquor obtained by fermenting the strain HR-087 and the strain CG-035 is respectively measured, and the fact that the fermentation liquor obtained by fermenting the strain HR-087 contains 4.1g/L of citrinin and the fermentation liquor obtained by fermenting the strain CG-035 does not contain the citrinin is found. Therefore, only the strain HR-087 can produce citrinin with high yield.
3. Identification
Extracting a genome of the strain HR-087, amplifying and sequencing 18S rDNA of the strain HR-087 (the nucleotide sequence of the 18S rDNA amplification of the strain HR-087 is shown as SEQ ID NO. 1), and comparing the nucleotide sequence in NCBI to show that the strain is Penicillium citrinum which is named as Penicillium citrinum HR-087.
Example 2: cultivation of Penicillium citrinum
The method comprises the following specific steps:
after Penicillium citrinum (Penicillium citrinum) HR-087 is inoculated into a PDA solid culture medium and cultured for 48 hours at 30 ℃, colonies are observed, and are found to have ciliated edges, a mycelium layer is soft, white and flat when growing, the center of the colonies is gray green, the edges are white to light yellow, and the back of the colonies is yellow.
Inoculating Penicillium citrinum (Penicillium citrinum) HR-087 into PDA liquid culture medium, culturing at 15-40 deg.C for 48 HR, and determining OD of the culture solution600The suitable growth temperature is found to be 20-37 ℃.
Respectively inoculating Penicillium citrinum (Penicillium citrinum) HR-087 into a PDA liquid culture medium only containing a carbon source and additionally adding 1-25 g/LNH4Measuring OD of the culture solution after culturing for 48h at 30 ℃ in a PDA liquid culture medium of Cl600It was found that it adds only a carbon sourceGrowth under conditions, but not under conditions rich in inorganic nitrogen sources.
Example 3: influence of fermentation Medium on the yield of Citrinin in the Shake-flask fermentation Process of Penicillium citrinum
The method comprises the following specific steps:
penicillium citrinum (Penicillium citrinum) HR-087 was used as the production strain, and the fermentation medium was replaced with the one shown in Table 1 based on example 3.
The content of citrinin in the fermentation broth obtained by fermentation using different fermentation media was measured, and the results are shown in table 1.
As can be seen from Table 1, when the ingredients comprising 40g/L glycerol, 5g/L peptone and 200g/L potato were used, the yield of citrinin from Penicillium citrinum HR-087 was the highest, up to 4.1 g/L.
TABLE 1 different fermentation media and the corresponding citrinin yields
Example 4: fermentation of Penicillium citrinum in a fermenter
The method comprises the following specific steps:
inoculating Penicillium citrinum (Penicillium citrinum) HR-087 obtained in example 1 into a PDA solid culture medium, culturing at 30 ℃ for 48h, inoculating 1mg of mycelia into 250mL shake flasks filled with 50mLPDA liquid culture medium, and culturing at 28 ℃ and 220r/min for 72h to obtain a seed solution; transferring the seed solution to a 5L fermentation tank filled with 2.5L fermentation medium at an inoculum size of 1%, and culturing at 28 deg.C and 800r/min for 96 hr to obtain fermentation liquid; wherein the fermentation medium comprises 40g/L glycerol, 5g/L peptone and 200g/L potato, and the glycerol is fed to the fermentation medium at a feed rate of 0.4mL/h during the whole fermentation period.
The content of citrinin in the fermentation liquor obtained by fermenting Penicillium citrinum HR-087 shows that the fermentation liquor obtained by fermenting Penicillium citrinum HR-087 contains 9.62g/L of citrinin, and the yield is high.
Example 5: influence of rotational speed on biomass and citrinin yield during fermentation in Penicillium citrinum fermenter
The method comprises the following specific steps:
penicillium citrinum HR-087 was used as a production strain, and the rotation speeds were replaced by 400r/min, 600r/min and 800r/min, respectively, based on example 5.
The content of citrinin in the fermentation broth obtained by fermentation at different rotation speeds was determined, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the yield of citrinin from Penicillium citrinum HR-087 was the highest, up to 9.62g/L, when a rotation speed of 800r/min was used.
Example 6: effect of Glycerol feeding Rate on Biomass and citrinin production during fermentation in Penicillium citrinum fermenter
The method comprises the following specific steps:
with Penicillium citrinum HR-087 as the production strain, glycerol feeding rates were changed to 0.4mL/h, 0.5mL/h, and 0.7mL/h, respectively, based on example 5.
The content of citrinin in the fermentation broth obtained by fermentation with different glycerol feeding rates was determined and the results are shown in FIG. 2.
As can be seen from FIG. 2, the yield of citrinin from Penicillium citrinum (HR-087) was the highest, up to 9.62g/L, when a glycerol feed rate of 0.4mL/h was used.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> penicillium citrinum strain and application thereof in citrinin production
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 537
<212> DNA
<213> Penicillium citrinum
<400> 1
tgaacctgcg gaaggatcat taccgagtgc gggcccctcg gggcccaacc tcccacccgt 60
gttgcccgaa cctatgttgc ctcggcgggc cccgcgcccg ccgacggccc ccctgaacgc 120
tgtctgaagt tgcagtctga gacctataac gaaattagtt aaaactttca acaacggatc 180
tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga taactaatgt gaattgcaga 240
attcagtgaa tcatcgagtc tttgaacgca cattgcgccc tctggtattc cggagggcat 300
gcctgtccga gcgtcattgc tgccctcaag cccggcttgt gtgttgggcc ccgtcccccc 360
cgccgggggg acgggcccga aaggcagcgg cggcaccgcg tccggtcctc gagcgtatgg 420
ggcttcgtca cccgctctag taggcccggc cggcgccagc cgacccccaa cctttaatta 480
tctcaggttg acctcggatc aggtagggat acccgctgaa cttaagcata tcaataa 537
Claims (21)
1. Penicillium citrinum (B)Penicillium citrinum) Characterized in that the Penicillium citrinum (A)Penicillium citrinum) Has been preserved in China Center for Type Culture Collection (CCTCC) No. M2019384 in 23.05.2019, and the preservation address is Wuhan, Wuhan university, China.
2. A process for the production of citrinin, said process comprising contacting the Penicillium citrinum (B) of claim 1Penicillium citrinum) Inoculating to fermentation medium, fermenting to obtain fermentation liquid containing citrinin, and mixingAnd extracting the fermentation liquor to obtain the citrinin.
3. The method for producing citrinin according to claim 2, wherein the fermentation temperature is 20-37 ℃, the rotation speed is 100-1000 rpm, and the ventilation volume is 1-5 vvm.
4. The method of claim 2 or 3, wherein the fermentation is at a temperature of 30 ℃, a speed of 800rpm, and a ventilation of 4 vvm.
5. The method for producing citrinin according to claim 2 or 3, wherein the fermentation medium comprises 20-80 g/L glycerol, 1-10 g/L peptone and 50-400 g/L potato.
6. The method for producing citrinin according to claim 2 or 3, wherein the fermentation temperature is 30 ℃, the rotation speed is 800rpm, and the aeration is 4 vvm; the fermentation medium comprises 20-80 g/L of glycerol, 1-10 g/L of peptone and 50-400 g/L of potatoes.
7. The method of claim 2, wherein the fermentation is at a temperature of 30 ℃, a speed of 800rpm, and an aeration rate of 4 vvm; the fermentation medium comprises 30-50 g/L of glycerol, 2-8 g/L of peptone and 150-250 g/L of potatoes.
8. The method of claim 4, wherein the fermentation medium comprises 30-50 g/L glycerol, 2-8 g/L peptone and 150-250 g/L potato.
9. The method of any one of claims 2-3 and 7, wherein the fermentation medium comprises 40g/L glycerol, 5g/L peptone and 200g/L potato.
10. The method of claim 4, wherein the fermentation medium comprises 40g/L glycerol, 5g/L peptone and 200g/L potato.
11. The method of any one of claims 2-3,8, and 10, wherein glycerol is fed to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation period.
12. The method of claim 5, wherein the glycerol is added to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation period.
13. The method of claim 6, wherein the glycerol is fed to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation period.
14. The method of claim 7, wherein the glycerol is added to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation period.
15. The method of claim 9, wherein the glycerol is added to the fermentation medium at a feed rate of 0.4-1.0 mL/h throughout the fermentation period.
16. The method of any one of claims 2-3,8,10, 12-15, wherein glycerol is fed to the fermentation medium at a feed rate of 0.4mL/h throughout the fermentation.
17. The method of claim 4, wherein glycerol is added to the fermentation medium at a feed rate of 0.4mL/h throughout the fermentation.
18. The method for producing citrinin according to claim 2 or 3, wherein the fermentation medium comprises 20-80 g/L glycerol, 1-10 g/L peptone and 50-400 g/L potato; throughout the fermentation, glycerol was fed to the fermentation medium at a feed rate of 0.4 mL/h.
19. The method for producing citrinin according to claim 2 or 3, wherein the fermentation temperature is 30 ℃, the rotation speed is 800rpm, and the aeration is 4 vvm; the fermentation medium comprises 30-50 g/L of glycerol, 2-8 g/L of peptone and 150-250 g/L of potatoes; throughout the fermentation, glycerol was fed to the fermentation medium at a feed rate of 0.4 mL/h.
20. The method of claim 2, wherein the fermentation is at a temperature of 30 ℃, a speed of 800rpm, and an aeration rate of 4 vvm; the components of the fermentation medium comprise 40g/L of glycerol, 5g/L of peptone and 200g/L of potato; throughout the fermentation, glycerol was fed to the fermentation medium at a feed rate of 0.4 mL/h.
21. The Penicillium citrinum (C) of claim 1Penicillium citrinum) The application in producing citrinin.
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