CN106479900A - 25 bacterial strain of high yield monascus purpureus penicillium oxalicum Po and application thereof - Google Patents
25 bacterial strain of high yield monascus purpureus penicillium oxalicum Po and application thereof Download PDFInfo
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 77
- 241000985513 Penicillium oxalicum Species 0.000 title claims abstract description 40
- 244000113306 Monascus purpureus Species 0.000 title description 2
- 235000002322 Monascus purpureus Nutrition 0.000 title description 2
- 229940057059 monascus purpureus Drugs 0.000 title description 2
- PCZOHLXUXFIOCF-BXMDZJJMSA-N lovastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 PCZOHLXUXFIOCF-BXMDZJJMSA-N 0.000 claims abstract description 56
- PCZOHLXUXFIOCF-UHFFFAOYSA-N Monacolin X Natural products C12C(OC(=O)C(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 PCZOHLXUXFIOCF-UHFFFAOYSA-N 0.000 claims abstract description 55
- QLJODMDSTUBWDW-UHFFFAOYSA-N lovastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(C)C=C21 QLJODMDSTUBWDW-UHFFFAOYSA-N 0.000 claims abstract description 53
- 229960004844 lovastatin Drugs 0.000 claims abstract description 52
- 238000000855 fermentation Methods 0.000 claims abstract description 39
- 230000004151 fermentation Effects 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 244000061456 Solanum tuberosum Species 0.000 claims description 14
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 235000015097 nutrients Nutrition 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000012546 transfer Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 235000015278 beef Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 235000015895 biscuits Nutrition 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 230000028070 sporulation Effects 0.000 abstract description 3
- 238000005457 optimization Methods 0.000 abstract description 2
- 241000228143 Penicillium Species 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 13
- 108020004463 18S ribosomal RNA Proteins 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 244000007853 Sarothamnus scoparius Species 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 230000000877 morphologic effect Effects 0.000 description 5
- 241000228150 Penicillium chrysogenum Species 0.000 description 4
- 235000012000 cholesterol Nutrition 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
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- 239000005416 organic matter Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 240000007817 Olea europaea Species 0.000 description 2
- 241000228153 Penicillium citrinum Species 0.000 description 2
- 241000228127 Penicillium griseofulvum Species 0.000 description 2
- 241000222350 Pleurotus Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- CABVTRNMFUVUDM-VRHQGPGLSA-N (3S)-3-hydroxy-3-methylglutaryl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C[C@@](O)(CC(O)=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 CABVTRNMFUVUDM-VRHQGPGLSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 208000029386 Apodia Diseases 0.000 description 1
- 241001465318 Aspergillus terreus Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- -1 Hydroxymethylglutaryl Chemical group 0.000 description 1
- 241000228347 Monascus <ascomycete fungus> Species 0.000 description 1
- 241000031003 Monascus ruber Species 0.000 description 1
- 241001247959 Omphalotus olearius Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000115273 Penicillium meleagrinum Species 0.000 description 1
- 241001503951 Phoma Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- SHTFZHTWSLHVEB-BDNRQGISSA-N lineatin Chemical compound O1C(C)(C)[C@H]2[C@@]3(C)C[C@@H]1O[C@@H]2C3 SHTFZHTWSLHVEB-BDNRQGISSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
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- 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
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to biotechnology microorganism field, is related to the penicillium oxalicum that 1 plant of liquid fermentation produces Lovastatin（Penicillium oxalicum）25 bacterial strain of Po, purposes and fermentation method for producing.Penicillium oxalicum（Penicillium oxalicum）25 bacterial strain of Po, the bacterial strain are preserved in China typical culture collection center, and deposit number is：CCTCC M 2016381, preservation date is：On July 7th, 2016.25 bacterial strain of mould Po of the present invention has the good characteristics such as high yield Lovastatin, fast growth, sporulation quantity are many, culture is simple.It is 5.0 ~ 5.5 in the initial pH of fermentation medium, under the conditions of cultivation temperature is 28 DEG C, after liquid fermentation medium and fermentation condition optimization, fermentation 81h ~ 105h, Lovastatin yield is up to more than 260mg/L for 25 bacterial strain of mould Po.
Description
Technical field
The invention belongs to biotechnology microorganism field, is related to the penicillium oxalicum that 1 plant of liquid fermentation produces Lovastatin
（Penicillium oxalicum）Po-25 bacterial strain, purposes and fermentation method for producing.
Background technology
Lovastatin（Lovastatin）It is the favourable agents of the reduction cholesterol that various countries generally acknowledge, with efficient, low toxicity, peace
Full feature, it can Selective depression Hydroxymethylglutaryl coacetylases（HMG-CoA）Reductase activity, and it is biological to block cholesterol
Synthesis, is applied quite varied in recent years in terms of clinical treatment.From 1979, the remote Teng Zhang of Japanese scholars（Akira Endo）Professor
From red monascus（Monascus ruber）Nutrient solution in isolate a kind of activity that can strongly suppress cholesterol biosynthesis
Material, is named as Monacolin K（Monacolin K）Start, after 1 year, U.S. Albert etc. was from Aspergillus terreus
（Aspergillus terrers）In be found that similar material, and be named as Mevinolin, now claim Lovastatin.Hereafter
It was found that Lovastatin is present in including Pleurotus（Pleurotus）, stem point Pseudomonas（Phoma）, Penicillium
（Penicillium）, light class navel mushroom（Omphalotus olearius）In interior multiple fungies.This series of discovery
The sensation in the world is caused, has started the research boom of fungi Lovastatin.
Mould（Penicillium）Generally on citrus and other fruit, the cheese of refrigeration and the spore contamination by them
Other foods on can all find, its conidium is also present in the air and rotten material in soil everywhere.Mould is sought
Saprogenesis, its source of nutrition are extremely wide, are class omnivorousness fungies, can be grown in any matrix containing organic matter.Blue or green
Mould（Penicillium）It is the important industrial microorganism resource of China, the use in human lives is extensive.Since nineteen twenty-nine not
Lay is bright（Fleming）, find point mould（Penicillium notatum）Produce bacteria growing inhibiting antibiotics penicillin with
Come, many kinds to the category bacterium have made intensive studies.Common mould species has：Point mould（Penicillium notatum）, penicillium chrysogenum（P. chrysogenum）, penicillium oxalicum（P. oxalicum）, Penicillium citrinum（P.citrinum）、
Spot mould（P. meleagrinum）, Penicillium griseofulvum（P. griseofulvum）Deng.Mould（Penicillium）In growth
During can produce multiple desirable metabolites, such as Lovastatin, cholesterol biosynthesis can be suppressed；Cellulase, pectase, sugar
Change enzyme, etc. multiple enzymes, can be used for prepare enzyme preparation；Organic acid such as citric acid, gluconic acid etc., can be used for industrial production etc., all
Cause the extensive concern of domestic and international researcher.
Content of the invention
In order to solve above-mentioned technical problem, first purpose of the present invention is to provide penicillium oxalicum（Penicillium oxalicum）Po-25 bacterial strain, with high yield Lovastatin, growth is fast, sporulation quantity is big, cultivate the good characteristic such as simple.This
Second bright purpose is to provide above-mentioned penicillium oxalicum（Penicillium oxalicum）Po-25 bacterial strain cuts down him in preparation Lip river
The application in spit of fland.
In order to above-mentioned first purpose is realized, following technical scheme is present invention employs：
Penicillium oxalicum（Penicillium oxalicum）Po-25 bacterial strain, the bacterial strain are preserved in China typical culture collection
The heart, deposit number is：CCTCC M 2016381, preservation date is：On July 7th, 2016.
The spontaneous fermentation sample that the present invention is collected from Zhejiang Province various places different niches（Food, soil, air, organic matter etc.）
In isolate and purify and obtain the pure bacterial strain of 163 plants of moulds；The numbering for obtaining 1 plant height product Lovastatin through screening is Po-25 quality strains, should
Strain isolation is from the orange for rotting；According to morphological feature and 18S rDNA gene order, identify Po-25 bacterial strain for penicillium oxalicum
（Penicillium oxalicum）；Po-25 bacterial strain is preserved in China typical culture collection center, and deposit number is：CCTCC
M 2016381, preservation date is：On July 7th, 2016.
The morphological feature of Po-25 bacterial strain：In PDA culture medium, 28 DEG C of 3 d of culture form more typical bacterium colony.At the beginning of bacterium colony
Phase is white fluffy, and 2 d rear center mycelia start greening, gradually become dirty-green after 3 d of culture；Bacterium colony is flat, quality suede
Shape, aerial hyphae are close and short and small, and conidium structure is produced in a large number, in dark olive green color, easy to fall off（Accompanying drawing 1）；Microscope is seen
The thread intertexture of hyphal cell is examined, has barrier film, 6 ~ 8 μm of diameter；Conidiophore betides matrix, has tabula, and wall is smoothed, base
Portion's amacrine, after branch, produces the asymmetric stigma of two wheels, referred to as shape such as broom, broom shape body；The usual two-wheel of broom shape body
Raw, metulae is often taken turns 2 ~ 3,13 ~ 20 μm × 3.2 ~ 3.7 μm, is generally relatively close to each other；Bottle stalk often take turns 5 ~ 8 or
More, 10 ~ 15 2.5 ~ 3.2 μm of μ m, assume ampuliform during young age or needle-like is draped over one's shoulders, closely cylindric when fully ripe（Accompanying drawing 2）；Bottle
Stalk top produces the conidium of chain, and conidium is oval, 3.0 ~ 4.0 μm of 4.5 ~ 5.5 μ m of size, and wall is smooth, is in
Blue-green（Accompanying drawing 3）.
18S rDNA gene sequencing result shows that its length is 1231 bp（Accompanying drawing 4）, with penicillium oxalicum
（Penicillium oxalicum）Evolutionary distance nearest（Accompanying drawing 5）.According to the morphological feature of Po-25 bacterial strain, in conjunction with mould
Category（Penicillium）Key to species, Po-25 identification of strains is penicillium oxalicum（Penicillium oxalicum）.
In order to above-mentioned second purpose is realized, following technical scheme is present invention employs：
A kind of preparation method of Lovastatin, the method comprise the following steps：
1）With the penicillium oxalicum Po-25 bacterial strain mycelia described in a small amount of claim 1 of transfer needle picking, transfer flat in PDA culture medium
In ware, 3 d of activation culture；
2）2 pure culture biscuits involvng inoculations are taken in first order seed nutrient solution, 2 d of shaking table culture, primary seed solution is obtained；
3）In secondary seed nutrient solution, primary seed solution is added by 10%, 200 r/min shaking table culture, 1 d becomes secondary seed
Liquid；
Secondary seed solution is accessed by 20% in 5 L ferment tank nutrient solutions, the initial pH of fermentation culture is 5.0 ~ 5.5, culture
Under the conditions of temperature is 28 DEG C, fermentation time is 81 h ~ 105 h.
Preferably, described PDA culture medium is made up of following component：Potato 20%, glucose 2%, agar powder
2.0%th, 1 L, pH 5.0 ~ 5.5 of water.
Preferably, described first order seed nutrient solution is made up of following component：Potato 20%, glucose 2%, water 1
L、pH 5.0~5.5.
Preferably, described secondary seed nutrient solution is made up of following component：Potato 20%, glucose 2%, beef
Cream 0.5%, 1 L, pH 5.0 ~ 5.5 of water.
Preferably, described fermentation culture is made up of following component：Potato 20%, glucose 2%, yeast extract
0.5%th, NaCl 0. 2%, 1 L, pH 5.0 ~ 5.5 of water.
Mould Po-25 bacterial strain has the excellent spies such as high yield Lovastatin, fast growth, sporulation quantity are many, culture is simple
Property.It is 5.0 ~ 5.5 in the initial pH of fermentation medium, under the conditions of cultivation temperature is 28 DEG C, mould Po-25 bacterial strain passes through liquid fermentation
After culture medium and fermentation condition optimization, 81 h ~ 105 h, Lovastatin yield of fermentation are up to 260 more than mg/L（Accompanying drawing 6）；
With the Lovastatin in LC-MS-MS separation detection zymotic fluid, as a result show structure, the molecule of separator
Amount is consistent with the quasi- product of Lovastatin（Accompanying drawing 7）.Mould Po-25 bacterial strain can provide excellent species for the production of Lovastatin.
Description of the drawings
3 d bacterium colony fronts are cultivated in Fig. 1 mould Po-25 bacterial strain PDA culture medium.
Fig. 2 mould Po-25 bacterial strain conidial head microscopic features（400 X）.
Fig. 3 mould Po-25 bacterial strain conidium microscopic features（400 X）.
Fig. 4 mould Po-25 bacterial strain 18S rDNA gene order（1231 bp）.
The mould Po-25 bacterial strain phylogenetic tree that Fig. 5 is set up based on 18S rDNA gene order.
Biomass of Fig. 6 mould Po-25 bacterial strain in 5 L fermentation tanks and Lovastatin yield change with fermentation time
Curve.
Fig. 7 mould Po-25 bacterial strain fermentation liquor Lovastatin liquid chromatograph mass spectrography detection figure.
The yield of Lovastatin in the representative penicillium bacterial strain zymotic fluid of 50 plants of Fig. 8.
Specific embodiment
Below the specific embodiment of the invention is made a detailed explanation.
1 penicillium oxalicum of embodiment（Penicillium oxalicum）The separation of Po-25 bacterial strain, screening and identification
1 culture medium
1. PDA culture medium：Potato 20%, glucose 2%, agar powder 2.0%, 1 L, pH 5.5 ~ 6.0 of water.For penicillium bacterial strain
Isolate and purify and identify.
2. PD culture medium：Potato 20%, glucose 2%, beef extract 0.5%, 1 L, pH 5.5 ~ 6.0 of water.For high yield
The screening of Lovastatin penicillium bacterial strain.
2 experimental techniques
The the isolating and purifying of 2.1 penicillium bacterial strains
From the spontaneous fermentation sample that different niches are collected（Food, soil, fruit, organic matter etc.）Surface picking is accessed by a small amount of mycelia
PDA culture medium planar surface, 28 DEG C of 24 h of culture, after white fluffy mycelia grows, take a little mycelia and transfer in another PDA
After continuing culture 3 d generation spore on culture medium flat plate, micro- sem observation has the characteristic feature of mould, a little edge bacterium of picking
Silk purifying 3 times, obtains the pure bacterial strain of the homogeneous mould of proterties.The penicillium bacterial strain for isolating and purifying acquisition is numbered and is stored in 25% glycerine,
It is placed in 4 DEG C of refrigerators standby.
The screening of 2.2 high yield Lovastatin penicillium bacterial strains
Use high performance liquid chromatography（HPLC）In the pure bacterial strain fermentation liquor of 163 plants of moulds of detection, Lovastatin yield is screened.
After mould respectively preserves 28 DEG C of 3 d of PDA plate culture of bacterial strain, 1 bacteria cake is taken with card punch（0.8 mm of diameter）Turn and be inoculated in PD culture
In liquid, 28 DEG C, 200 r/min shaking table culture, 5 d.Take 1 mL of zymotic fluid, plus 4 mL of methyl alcohol（By 1:4 ratio）, ultrasonically treated
20 min, 50 DEG C of 2 h of water-bath, 3000 r/min are centrifuged 3 min, take supernatant, through 0.45 μm of membrane filtration, using HPLC method
Detection Lovastatin yield, screens the penicillium bacterial strain of high yield Lovastatin.
The identification of 2.3 Po-25 bacterial strains
With a small amount of bacterial strain mycelia of tweezers picking, transfer in PDA culture medium flat board, 3 d of activation culture；Make even 1 bacteria cake in ware
（0.8 mm of diameter）It is inoculated on new PDA culture medium flat board, 28 DEG C, 3 d of culture, the mycelia of micro- sem observation Po-25 bacterial strain,
The morphological feature of conidial head, conidiophore, conidium etc., and take pictures.The genomic DNA of Po-25 bacterial strain is extracted, is expanded
Increase 18S rDNA gene order, serve the sequencing of marine growth engineering company.
3 experimental results
The screening isolated and purified with high yield Lovastatin penicillium bacterial strain of 3.1 penicillium bacterial strains
By isolating and purifying the spontaneous fermentation sample that collects from Zhejiang Province various places different niches（Food, soil, fruit, organic matter
Deng）In isolate and purify altogether and obtain the pure bacterial strain of 163 plants of moulds.
Using efficient liquid phase（HPLC）Method carries out Lovastatin yield detection to the pure bacterial strain fermentation liquor of 163 plants of moulds, knot
Fruit shows that different Aspergillus strain Lovastatin yield have significant difference（Accompanying drawing 8）.1 plant of Lovastatin yield is obtained through screening
Higher penicillium bacterial strain, numbering are Po-25 bacterial strain, and Lovastatin yield is that 186.5 mg/L, Po-25 strain isolation rot certainly
Orange.
The qualification result of 3.2 Po-25 penicillium bacterial strains
The morphological feature of Po-25 bacterial strain：In PDA culture medium, 28 DEG C of 3 d of culture form more typical bacterium colony.The bacterium colony initial stage is
White fluffy, 2 d rear center mycelia start greening, gradually become dirty-green after 3 d of culture；Bacterium colony is flat, and quality is velvet-like, gas
Raw mycelia is close and short and small, and conidium structure is produced in a large number, in dark olive green color, easy to fall off（Accompanying drawing 1）；Micro- sem observation mycelia
The thread intertexture of cell, has barrier film, 6 ~ 8 μm of diameter；Conidiophore betides matrix, has tabula, and wall is smoothed, and base portion apodia is thin
Born of the same parents, after branch, produce the asymmetric stigma of two wheels, referred to as shape such as broom, broom shape body；The usual two-wheel life of broom shape body, metulae
Often 2 ~ 3 are taken turns, 13 ~ 20 μm × 3.2 ~ 3.7 μm, be generally relatively close to each other；Bottle stalk often takes turns 5 ~ 8 or more, and 10
~ 15 2.5 ~ 3.2 μm of μ m, assume ampuliform or drape over one's shoulders needle-like during young age, closely cylindric when fully ripe（Accompanying drawing 2）；Bottle stalk top
The conidium of chain is produced, conidium is oval, 3.0 ~ 4.0 μm of 4.5 ~ 5.5 μ m of size, and wall is smooth, in blue-green
（Accompanying drawing 3）.
The 18S rDNA gene order of Po-25 bacterial strain：Analysis result shows, the 18S rDNA gene order of Po-25 bacterial strain
Length is 1231 bp（Accompanying drawing 4）, based on the mould that 18S rDNA gene order is set up（Penicillium）Systematic growth tree table
Bright, with penicillium oxalicum（Penicillium oxalicum）Evolutionary distance nearest（Accompanying drawing 5）.Form according to Po-25 bacterial strain
Feature and 18S rDNA gene order, Po-25 identification of strains is penicillium oxalicum（Penicillium oxalicum）.
2 penicillium oxalicum of embodiment（Penicillium oxalicum）Growth of the Po-25 bacterial strain in 5 L fermentation tanks is bent
Line and Lovastatin produce rule
1 bacterial strain：Penicillium oxalicum（Penicillium oxalicum）Po-25 bacterial strain.
2 culture mediums
1. PDA culture medium：Potato 20%, glucose 2%, agar powder 2.0%, 1 L, pH 5.0 ~ 5.5 of water.For penicillium bacterial strain
Activation culture.
2. first order seed nutrient solution：Potato 20%, glucose 2%, 1 L, pH 5.0 ~ 5.5 of water.For penicillium oxalicum Po-
The first order seed culture of 25 bacterial strains.
3. secondary seed nutrient solution：Potato 20%, glucose 2%, beef extract 0.5%, 1 L, pH 5.0 ~ 5.5 of water.For
The secondary seed culture of penicillium oxalicum Po-25 bacterial strain.
4. fermentation culture：Potato 20%, glucose 2%, yeast extract 0.5%, NaCl 0. 2%, water 1 L, pH 5.0 ~
5.5.Lovastatin is produced for penicillium oxalicum Po-25 strain fermentation.
3 experimental techniques
The culture of 3.1 mould Po-25 bacterial strains
A small amount of Po-25 bacterial strain mycelia is chosen with transfer needle, transfer in PDA culture medium plate, 3 d of activation culture；Make even 2 bacterium in ware
Cake（8 mm of diameter）It is inoculated in first order seed nutrient solution, 2 d of shaking table culture, primary seed solution is obtained；In secondary seed culture
In liquid, primary seed solution is accessed by 10%, 200 r/min shaking table culture, 1 d becomes secondary seed solution；In 5 L ferment tank cultures
Fermented and cultured is carried out by 20% access secondary seed solution in liquid.
The measure of 3.2 mould Po-25 bacterial strain dry mycelial weight
1.5 mL sky centrifuge tube is put in dries in 85 DEG C of baking oven, weighs sky centrifuge tube quality for m；Often 1 mL culture drawn by pipe
Liquid, at 4 DEG C, 10000 r/min are centrifuged 10 min, abandon supernatant；Plus sterile distilled water is washed 3 times, 10000 r/min centrifugation 10
Min, abandons supernatant, is put in 85 DEG C of baking ovens and dries to constant weight, and weighs quality for M；Dry mycelial weight quality is M-m.1 mL is taken every 3 h
Po-25 bacterial strain fermentation liquor determines dry mycelial weight.With fermentation time as abscissa, Po-25 bacterial strain dry mycelial weight is drawn for ordinate
Growth curve.
Lovastatin volume analysis during 3.3 mould Po-25 strain fermentations
1 mL of zymotic fluid, plus 4 mL of methyl alcohol are taken every 3 h（By 1:4 ratio）, ultrasonically treated 20 min, 50 DEG C of 2 h of water-bath,
3000 r/min are centrifuged 3 min, take supernatant, and through 0.45 μm of membrane filtration, HPLC method detects Lovastatin yield, to send out
The ferment time is abscissa, and the Lovastatin yield of Po-25 bacterial strain is ordinate, draws Po-25 bacterial strain Lovastatin yield
With fermentation time change curve.
The identification of Lovastatin in 3.4 mould Po-25 bacterial strain fermentation liquors
With the Lovastatin structure in LC-MS-MS separation detection zymotic fluid, molecular weight etc..
4 experimental results
The growth curve of 4.1 mould Po-25 bacterial strains
Dry mycelial weight is measured by sampling every 3 h, mould Po-25 strain growth curve is shown in Fig. 6.0 ~ 18 h of mould Po-25 bacterial strain gives birth to
Long speed is slow, is lag phase；18 h ~ 51 h fast-growth, biomass increases, and is exponential phase；Ferment to 54 h, growing way
Tend towards stability, biomass reaches highest, be to stablize growth period, dry mycelial weight reaches 240.5 g/L；Occur foam after 90 h, enter
Decline phase.
In 4.2 mould Po-25 bacterial strain fermentation liquors, Lovastatin yield is with fermentation time change curve
Every 3 h sampling with HPLC method determine mould Po-25 bacterial strain fermentation liquor Lovastatin yield, Lovastatin yield with
The change curve of fermentation time is shown in Fig. 6, ferments to 81 h, and in zymotic fluid, Lovastatin yield is basicly stable, and maximum is
263.9 mg/L.
The identification of Lovastatin in 4.3 mould Po-25 bacterial strain fermentation liquors
With the Lovastatin in LC-MS-MS separation detection zymotic fluid, as a result show separator structure, divide
Son amount is consistent with the quasi- product of Lovastatin, and molecular formula is C24H36O5, molecular weight is 404.45（Accompanying drawing 7）.
Claims (7)
1. penicillium oxalicum（Penicillium oxalicum）Po-25 bacterial strain, the bacterial strain are preserved in China typical culture collection
Center, deposit number is：CCTCC M 2016381, preservation date is：On July 7th, 2016.
2. the penicillium oxalicum Po-25 bacterial strain described in claim 1 is used for producing Lovastatin（Lovastatin）.
3. a kind of preparation method of Lovastatin, it is characterised in that the method comprises the following steps：
1）With the penicillium oxalicum Po-25 bacterial strain mycelia described in a small amount of claim 1 of transfer needle picking, transfer flat in PDA culture medium
In ware, 3 d of activation culture；
2）2 pure culture biscuits involvng inoculations are taken in first order seed nutrient solution, 2 d of shaking table culture, primary seed solution is obtained；
3）In secondary seed nutrient solution, primary seed solution is added by 10%, 200 r/min shaking table culture, 1 d becomes secondary seed
Liquid；
Secondary seed solution is accessed by 20% in 5 L ferment tank nutrient solutions, the initial pH of fermentation culture is 5.0 ~ 5.5, culture
Under the conditions of temperature is 28 DEG C, fermentation time is 81 h ~ 105 h.
4. the preparation method of a kind of Lovastatin according to claim 3, it is characterised in that PDA culture medium is by following group
Divide and constitute：Potato 20%, glucose 2%, agar powder 2.0%, 1 L, pH 5.0 ~ 5.5 of water.
5. the preparation method of a kind of Lovastatin according to claim 3, it is characterised in that first order seed nutrient solution by with
Under component constitute：Potato 20%, glucose 2%, 1 L, pH 5.0 ~ 5.5 of water.
6. the preparation method of a kind of Lovastatin according to claim 3, it is characterised in that secondary seed nutrient solution by with
Under component constitute：Potato 20%, glucose 2%, beef extract 0.5%, 1 L, pH 5.0 ~ 5.5 of water.
7. the preparation method of a kind of Lovastatin according to claim 3, it is characterised in that fermentation culture is by following
Component is constituted：Potato 20%, glucose 2%, yeast extract 0.5%, NaCl 0. 2%, 1 L, pH 5.0 ~ 5.5 of water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182142A (en) * | 2018-09-28 | 2019-01-11 | 浙江师范大学 | Skin falls mould and its application |
| CN109608517A (en) * | 2019-01-18 | 2019-04-12 | 三峡大学 | A kind of novel antimicrobial peptide and the application in citrus is fresh-keeping |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999050434A1 (en) * | 1998-03-30 | 1999-10-07 | Eduard Sardaryan | Strain of the microorganism penicillium oxalicum var. armeniaca and its application |
| CN102154124A (en) * | 2011-03-28 | 2011-08-17 | 浙江师范大学 | Penicillium oxallcum Po-5 strain and application thereof |
-
2016
- 2016-09-30 CN CN201610867245.3A patent/CN106479900B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999050434A1 (en) * | 1998-03-30 | 1999-10-07 | Eduard Sardaryan | Strain of the microorganism penicillium oxalicum var. armeniaca and its application |
| CN102154124A (en) * | 2011-03-28 | 2011-08-17 | 浙江师范大学 | Penicillium oxallcum Po-5 strain and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ABDEL-NASER, A.ZOHRI: "REVIEW ARTICLE Production of some therapeutic compounds by fungi cultivated on agro-industrial by-products", 《INTERNATIONAL CONFERENCE ON:》 * |
| SADIA JAVED,ET AL.: "Screening of Indigenously Isolated Fungi for Lovastin Production and its in vivo Evaluation", 《CURRENT PHARMACEUTICAL BIOTECHNOLOGY》 * |
| ZHEN-JUN ZHAO,ET AL.: "Exposure assessment of lovastatin in Pu-erh tea", 《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182142A (en) * | 2018-09-28 | 2019-01-11 | 浙江师范大学 | Skin falls mould and its application |
| CN109608517A (en) * | 2019-01-18 | 2019-04-12 | 三峡大学 | A kind of novel antimicrobial peptide and the application in citrus is fresh-keeping |
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