CN106479900B - High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof - Google Patents
High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof Download PDFInfo
- Publication number
- CN106479900B CN106479900B CN201610867245.3A CN201610867245A CN106479900B CN 106479900 B CN106479900 B CN 106479900B CN 201610867245 A CN201610867245 A CN 201610867245A CN 106479900 B CN106479900 B CN 106479900B
- Authority
- CN
- China
- Prior art keywords
- culture
- lovastatin
- bacterial strain
- penicillium oxalicum
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 78
- 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 45
- 230000004151 fermentation Effects 0.000 claims abstract description 45
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 16
- 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
- 238000011218 seed culture Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 239000000284 extract Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 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
- 239000007788 liquid Substances 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 11
- 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 17
- 241000894006 Bacteria Species 0.000 description 11
- 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
- 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
- 238000001514 detection method Methods 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
- 235000013305 food Nutrition 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
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 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
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000005416 organic matter Substances 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 239000000126 substance Substances 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
- 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
- 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
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 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
- 230000003698 anagen phase Effects 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
- 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
- 238000009776 industrial production Methods 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
- 238000005259 measurement Methods 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
- 230000008569 process Effects 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
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
Abstract
The invention belongs to biotechnology microorganism field, be related to 1 plant of liquid fermentation production Lovastatin penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain, purposes and fermentation method for producing.Penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain, which is preserved in China typical culture collection center, deposit number are as follows: CCTCC M 2016381, preservation date are as follows: on July 7th, 2016.The good characteristics such as mould Po-25 bacterial strain of the present invention has high yield Lovastatin, the speed of growth is fast, sporulation quantity is more, 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 mould Po-25 bacterial strain is by liquid fermentation medium and fermentation condition optimization, 81 h ~ 105 h, Lovastatin yield of fermenting is up to 260 mg/L or more.
Description
Technical field
The invention belongs to biotechnology microorganism fields, are related to the penicillium oxalicum of 1 plant of liquid fermentation production Lovastatin
(Penicillium oxalicum) Po-25 bacterial strain, purposes and fermentation method for producing.
Background technique
Lovastatin (Lovastatin) is the favourable agents for the reduction cholesterol that various countries generally acknowledge, has efficient, low toxicity, peace
Full feature, its energy selective depression Hydroxymethylglutaryl coacetylase (HMG-CoA) reductase activity, and block cholesterol biological
Synthesis, in recent years using very extensive in terms of clinical treatment.From 1979, the remote Teng Zhang of Japanese scholars (Akira Endo) was taught
From red monascus (Monascus ruber) culture solution in isolate a kind of activity that can strongly inhibit cholesterol biosynthesis
Substance is named as Monacolin K(Monacolin K) start, after 1 year, U.S. Albert etc. was from Aspergillus terreus
(Aspergillus terrers) in have found similar substance, and be named as Mevinolin, now claim Lovastatin.Hereafter
It was found that Lovastatin be present in including Pleurotus (Pleurotus), stem point Pseudomonas (Phoma), Penicillium
(Penicillium), shine class navel mushroom (Omphalotus olearius) including a variety of fungies in.This series of discovery
The sensation for causing the world has started the research boom of fungi Lovastatin.
Mould (Penicillium) usually on citrus and other fruit, the cheese of refrigeration and the spore contamination by them
Other foods on can find, conidium also exists in air and on rotten substance everywhere in soil.Mould battalion
Saprogenesis, source of nutrition is extremely wide, is a kind of omnivorousness fungi, can be grown in any matrix containing organic matter.It is green
Mould (Penicillium) it is the important industrial microorganism resource in China, the use in human lives is extensively.Since nineteen twenty-nine not
Lay is bright (Fleming), discovery point mould (Penicillium notatum) generate inhibit bacterial growth antibiotics penicillin with
Come, many kinds of the category bacterium are had made intensive studies.Common mould type 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) etc..Mould (Penicillium) growing
A variety of desirable metabolites, such as Lovastatin can be generated in the process, can inhibit cholesterol biosynthesis；Cellulase, pectase, sugar
Change enzyme, etc. a variety of enzymes, can be used for preparing enzyme preparation；Organic acid such as citric acid, gluconic acid etc. can be used for industrial production etc.,
Cause the extensive concern of domestic and international researcher.
Summary of the invention
In order to solve the above technical problems, the first purpose of the invention is to provide penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain, there are the good characteristics such as high yield Lovastatin, growth are fast, sporulation quantity is big, culture is simple.This hair
Second bright purpose be to provide above-mentioned penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain preparation Lip river cut down him
The application in spit of fland.
In order to realize first above-mentioned purpose, present invention employs technical solutions below:
Penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain, the bacterial strain be preserved in Chinese Typical Representative culture guarantor
Hiding center, deposit number are as follows: CCTCC M 2016381, preservation date are as follows: on July 7th, 2016.
The spontaneous fermentation sample (food, soil, air, organic matter etc.) that the present invention is collected from Zhejiang Province various regions different niches
In isolate and purify and obtain 163 plants of pure bacterial strains of mould；Obtaining 1 plant height to produce the number of Lovastatin through screening is Po-25 quality strains, should
The orange that strain isolation rots certainly；According to morphological feature and 18S rDNA gene order, identify Po-25 bacterial strain be penicillium oxalicum
(Penicillium oxalicum)；Po-25 bacterial strain is preserved in China typical culture collection center, deposit number are as follows: CCTCC
M 2016381, preservation date are as follows: on July 7th, 2016.
The morphological feature of Po-25 bacterial strain: 28 DEG C of 3 d of culture form more typical bacterium colony in PDA culture medium.At the beginning of bacterium colony
Phase is white fluffy, and 2 d rear center mycelia start greening, gradually becomes dirty-green after cultivating 3 d；Bacterium colony is flat, quality suede
Shape, aerial hyphae is close and short and small, and conidium structure largely generates, and is in dark olive green color, (attached drawing 1) easy to fall off；Microscope is seen
It examines hyphal cell filiform to interweave, tool diaphragm, 6 ~ 8 μm of diameter；Conidiophore betides matrix, there is tabula, and wall is smooth, base
Portion's amacrine generates the two asymmetric stigmas of wheel, shaped like broom, referred to as broom shape body after branch；The usual two-wheel of broom shape body
Raw, every 2 ~ 3, the wheel of metulae, is usually relatively close to each other by 13 ~ 20 μm × 3.2 ~ 3.7 μm；Bottle stalk it is every wheel 5 ~ 8 or
More, 2.5 ~ 3.2 μm of 10 ~ 15 μ m, present when young age ampuliform or drape over one's shoulders it is needle-shaped, when sufficiently mature nearly cylindric (attached drawing 2)；Bottle
Obstruct the conidium that top generates chain, conidium ellipse, 3.0 ~ 4.0 μm of 4.5 ~ 5.5 μ m of size, wall is smooth, is in
Blue-green (attached drawing 3).
18S rDNA gene sequencing the result shows that, the length is 1231 bp(attached drawings 4), with penicillium oxalicum
(Penicillium oxalicum) evolutionary distance recently (attached drawing 5).According to the morphological feature of Po-25 bacterial strain, in conjunction with mould
Belong to (Penicillium) Key to species, by Po-25 bacterial strain be accredited as penicillium oxalicum (Penicillium oxalicum).
In order to realize second above-mentioned purpose, present invention employs technical solutions below:
A kind of preparation method of Lovastatin, this method include the following steps:
1) with transfer needle picking, penicillium oxalicum Po-25 bacterial strain mycelia described in claim 1, switching are cultivated in PDA on a small quantity
In base plate, 3 d of activation culture；
2) take 2 pure culture biscuits involvng inoculations in first order seed culture solution, primary seed solution is made in 2 d of shaking table culture；
3) in secondary seed culture solution, primary seed solution is added by 10%, 200 r/min shaking table culture, 1 d is at second level kind
Sub- liquid；
By 20% access secondary seed solution in 5 L ferment tank culture solutions, the initial pH of fermentation culture is 5.0 ~ 5.5,
Under the conditions of cultivation temperature is 28 DEG C, fermentation time is the h of 81 h ~ 105.
Preferably, the PDA culture medium is made of component below: potato 20%, glucose 2%, agar powder
2.0%, 1 L, pH 5.0 ~ 5.5 of water.
Preferably, the first order seed culture solution is made of component below: potato 20%, glucose 2%, water 1
L、pH 5.0~5.5。
Preferably, the secondary seed culture solution is made of component below: potato 20%, glucose 2%, beef
Cream 0.5%, 1 L, pH 5.0 ~ 5.5 of water.
Preferably, the fermentation culture is made of component below: potato 20%, glucose 2%, yeast extract
0.5%, NaCl 0. 2%, 1 L, pH 5.0 ~ 5.5 of water.
The excellent spies such as mould Po-25 bacterial strain has high yield Lovastatin, the speed of growth is fast, sporulation quantity is more, 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 fermenting is up to 260 mg/L or more (attached drawing 6)；
With the Lovastatin in liquid chromatography-mass spectrometry separation detection fermentation liquid, the results showed that the structure of isolate, molecule
It measures (attached drawing 7) consistent with the quasi- product of Lovastatin.Mould Po-25 bacterial strain can provide excellent species for the production of Lovastatin.
Detailed description of the invention
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 established based on 18S rDNA gene order.
Biomass and Lovastatin yield of Fig. 6 mould Po-25 bacterial strain in 5 L fermentors 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 50 plants of Fig. 8 representative penicillium bacterial strain fermentation liquids.
Specific embodiment
The specific embodiment of the invention is made a detailed explanation below.
1 penicillium oxalicum of embodiment (Penicillium oxalicum) Po-25 bacterial strain separation, screening and identification
1 culture medium
1. PDA culture medium: potato 20%, glucose 2%, agar powder 2.0%, water 1 L, pH 5.5 ~ 6.0.For mould
Bacterial strain isolating and purifying and identifying.
2. PD culture medium: potato 20%, glucose 2%, beef extract 0.5%, water 1 L, pH 5.5 ~ 6.0.For high yield
The screening of Lovastatin penicillium bacterial strain.
2 experimental methods
2.1 penicillium bacterial strains isolate and purify
The a small amount of mycelia of spontaneous fermentation sample (food, soil, fruit, organic matter etc.) surface picking collected from different niches
PDA culture medium planar surface is accessed, 28 DEG C of 24 h of culture take a little mycelia switching in another after white fluffy mycelia grows
Continue after cultivating 3 d generation spore on one PDA culture medium plate, micro- sem observation has the characteristic feature of mould, and picking is a little
Edge mycelia purifies 3 times, obtains the pure bacterial strain of the uniform mould of character.The penicillium bacterial strain for isolating and purifying acquisition number is stored in 25%
In glycerol, it is spare to be placed in 4 DEG C of refrigerators.
The screening of 2.2 high yield Lovastatin penicillium bacterial strains
Lovastatin yield in 163 plants of pure bacterial strain fermentation liquors of mould is detected with high performance liquid chromatography (HPLC) to be sieved
Choosing.After mould respectively saves 28 DEG C of 3 d of PDA plate culture of bacterial strain, 1 bacteria cake (0.8 mm of diameter) is taken to turn to be inoculated in PD with punch
In culture solution, 28 DEG C, 200 r/min shaking table culture, 5 d.1 mL of fermentation liquid is taken, adds 4 mL(of methanol in the ratio of 1:4), ultrasound
20 min are handled, 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, utilization
HPLC method detects 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 plate, 3 d of activation culture；It makes even 1 in ware
Bacteria cake (0.8 mm of diameter) is inoculated on new PDA culture medium plate, 28 DEG C, cultivates 3 d, micro- sem observation Po-25 bacterial strain
The morphological feature of mycelia, conidial head, conidiophore, conidium etc., and take pictures.Extract the genome of Po-25 bacterial strain
DNA expands 18S rDNA gene order, Shanghai bio-engineering corporation is sent to be sequenced.
3 experimental results
The screening of 3.1 penicillium bacterial strains isolated and purified with high yield Lovastatin penicillium bacterial strain
By isolating and purifying the spontaneous fermentation sample collected from Zhejiang Province various regions different niches, (food, fruit, has soil
Machine matter etc.) in isolate and purify and obtain 163 plants of pure bacterial strains of mould altogether.
The detection of Lovastatin yield, knot are carried out to the pure bacterial strain fermentation liquor of 163 plants of moulds using efficient liquid phase (HPLC) method
Fruit shows different Aspergillus strain Lovastatin yield, and there are significant difference (attached drawings 8).1 plant of Lovastatin yield is obtained through screening
Higher penicillium bacterial strain, number are Po-25 bacterial strain, and Lovastatin yield is 186.5 mg/L, and Po-25 strain isolation rots certainly
Orange.
The qualification result of 3.2 Po-25 penicillium bacterial strains
The morphological feature of Po-25 bacterial strain: 28 DEG C of 3 d of culture form more typical bacterium colony in PDA culture medium.At the beginning of bacterium colony
Phase is white fluffy, and 2 d rear center mycelia start greening, gradually becomes dirty-green after cultivating 3 d；Bacterium colony is flat, quality suede
Shape, aerial hyphae is close and short and small, and conidium structure largely generates, and is in dark olive green color, (attached drawing 1) easy to fall off；Microscope is seen
It examines hyphal cell filiform to interweave, tool diaphragm, 6 ~ 8 μm of diameter；Conidiophore betides matrix, there is tabula, and wall is smooth, base portion
Amacrine generates the two asymmetric stigmas of wheel, shaped like broom, referred to as broom shape body after branch；The usual two-wheel of broom shape body
Raw, every 2 ~ 3, the wheel of metulae, is usually relatively close to each other by 13 ~ 20 μm × 3.2 ~ 3.7 μm；Bottle stalk it is every wheel 5 ~ 8 or
More, 2.5 ~ 3.2 μm of 10 ~ 15 μ m, present when young age ampuliform or drape over one's shoulders it is needle-shaped, when sufficiently mature nearly cylindric (attached drawing 2)；Bottle
Obstruct the conidium that top generates chain, conidium ellipse, 3.0 ~ 4.0 μm of 4.5 ~ 5.5 μ m of size, wall is smooth, is in
Blue-green (attached drawing 3).
The 18S rDNA gene order of Po-25 bacterial strain: analysis the result shows that, the 18S rDNA gene order of Po-25 bacterial strain
Length be 1231 bp(attached drawings 4), based on 18S rDNA gene order establish mould (Penicillium) systematic growth tree table
It is bright, with penicillium oxalicum (Penicillium oxalicum) evolutionary distance recently (attached drawing 5).According to the form of Po-25 bacterial strain
Feature and 18S rDNA gene order, by Po-25 bacterial strain be accredited as penicillium oxalicum (Penicillium oxalicum).
2 penicillium oxalicum of embodiment (Penicillium oxalicum) growth song of the Po-25 bacterial strain in 5 L fermentors
Line and Lovastatin generate 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 mould
The activation culture of bacterial strain.
2. first order seed culture 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 culture 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 methods
The culture of 3.1 mould Po-25 bacterial strains
A small amount of Po-25 bacterial strain mycelia is chosen with transfer needle, is transferred in PDA culture medium plate, 3 d of activation culture；It makes even ware
In 2 bacteria cakes (8 mm of diameter) be inoculated in first order seed culture solution, 2 d of shaking table culture, be made primary seed solution；In secondary seed
In culture solution, by 10% access primary seed solution, 200 r/min shaking table culture, 1 d is at secondary seed solution；In 5 L ferment tanks
Fermented and cultured is carried out by 20% access secondary seed solution in culture solution.
The measurement of 3.2 mould Po-25 bacterial strain dry mycelial weights
1.5 mL sky centrifuge tubes are put in dries in 85 DEG C of baking oven, and weighing sky centrifuge tube quality is m；Every pipe draws 1 mL
Culture solution, at 4 DEG C, 10000 r/min are centrifuged 10 min, abandon supernatant；Sterile distilled water is added to wash 3 times, 10000 r/min centrifugation
10 min abandon supernatant, are put into 85 DEG C of baking ovens and dry to constant weight, and weighing quality is M；Dry mycelial weight quality is M-m.1 is taken every 3 h
ML Po-25 bacterial strain fermentation liquor measures dry mycelial weight.Using fermentation time as abscissa, Po-25 bacterial strain dry mycelial weight is drawn for ordinate
Growth curve processed.
Lovastatin volume analysis during 3.3 mould Po-25 strain fermentations
1 mL of fermentation liquid is taken every 3 h, adds 4 mL(of methanol in the ratio of 1:4), it is ultrasonically treated 20 min, 50 DEG C of water-baths 2
H, 3000 r/min are centrifuged 3 min, take supernatant, and through 0.45 μm of membrane filtration, HPLC method detects Lovastatin yield, with
Fermentation time is abscissa, and the Lovastatin yield of Po-25 bacterial strain is ordinate, draws Po-25 bacterial strain Lovastatin and produces
Amount is with fermentation time change curve.
The identification of Lovastatin in 3.4 mould Po-25 bacterial strain fermentation liquors
With Lovastatin structure, the molecular weight etc. in liquid chromatography-mass spectrometry separation detection fermentation liquid.
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.Mould Po-25 bacterial strain 0 ~ 18
The h speed of growth is slow, is lag phase；The h fast-growth of 18 h ~ 51, biomass increase, and are logarithmic growth phase；After fermentation to 54 h,
Growing way tends towards stability, and biomass reaches highest, and to stablize growth period, dry mycelial weight is up to 240.5 g/L；There is foam after 90 h,
Into decline phase.
Lovastatin yield is with fermentation time change curve in 4.2 mould Po-25 bacterial strain fermentation liquors
Mould Po-25 bacterial strain fermentation liquor Lovastatin yield is measured every 3 h sampling HPLC method, Lovastatin is produced
Amount is shown in Fig. 6 with the change curve of fermentation time, and after fermentation to 81 h, Lovastatin yield is basicly stable in fermentation liquid, maximum
Value is 263.9 mg/L.
The identification of Lovastatin in 4.3 mould Po-25 bacterial strain fermentation liquors
With the Lovastatin in liquid chromatography-mass spectrometry separation detection fermentation liquid, the results showed that isolate knot
Structure, molecular weight are consistent with the quasi- product of Lovastatin, molecular formula C24H36O5, molecular weight is 404.45(attached drawing 7).
Claims (7)
1. penicillium oxalicum (Penicillium oxalicum) Po-25 bacterial strain, which is preserved in China typical culture collection
Center,
Deposit number are as follows: CCTCC M 2016381, preservation date are as follows: on July 7th, 2016.
2. application of the penicillium oxalicum Po-25 bacterial strain described in claim 1 in production Lovastatin (Lovastatin).
3. a kind of preparation method of Lovastatin, it is characterised in that this method includes the following steps:
1) the penicillium oxalicum Po-25 bacterial strain mycelia described in a small amount of claim 1 of transfer needle picking is transferred in PDA culture medium
In plate, 3 d of activation culture；
2) step 1) is taken to obtain pure culture biscuits involvng inoculation in first order seed culture solution, primary seed solution is made in 2 d of shaking table culture；
3) in secondary seed culture solution, according to weight percent content, primary seed solution is added by 10%, 200 r/min shake
1 d is at secondary seed solution for bed culture；
By 20% access secondary seed solution in 5 L ferment tank culture solutions, the initial pH of fermentation culture is 5.0 ~ 5.5, training
Under the conditions of feeding temperature is 28 DEG C, fermentation time is the h of 81 h ~ 105.
4. a kind of preparation method of Lovastatin according to claim 3, it is characterised in that PDA culture medium is according to weight
Degree meter is made of component below: potato 20%, glucose 2%, agar powder 2.0%, 1 L, pH 5.0 ~ 5.5 of water.
5. a kind of preparation method of Lovastatin according to claim 3, it is characterised in that first order seed culture solution according to
Weight percent content meter is made of component below: potato 20%, glucose 2%, 1 L, pH 5.0 ~ 5.5 of water.
6. a kind of preparation method of Lovastatin according to claim 3, it is characterised in that secondary seed culture solution by with
Under be made of according to weight percent content meter following components: potato 20%, glucose 2%, beef extract 0.5%, water 1 L, pH
5.0~5.5。
7. a kind of preparation method of Lovastatin according to claim 3, it is characterised in that fermentation culture is according to weight
Degree meter is made of component below: potato 20%, glucose 2%, yeast extract 0.5%, NaCl 0. 2%, 1 L of water,
pH 5.0~5.5。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610867245.3A CN106479900B (en) | 2016-09-30 | 2016-09-30 | High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610867245.3A CN106479900B (en) | 2016-09-30 | 2016-09-30 | High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106479900A CN106479900A (en) | 2017-03-08 |
| CN106479900B true CN106479900B (en) | 2019-04-16 |
Family
ID=58268053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610867245.3A Active CN106479900B (en) | 2016-09-30 | 2016-09-30 | High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106479900B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182142B (en) * | 2018-09-28 | 2020-11-03 | 浙江师范大学 | Penicillium dermatum and application thereof |
| 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 (1)
| Title |
|---|
| Exposure assessment of lovastatin in Pu-erh tea;Zhen-Jun Zhao,et al.;《International Journal of Food Microbiology》;20130326;第164卷;第26–31页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106479900A (en) | 2017-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109266562A (en) | One plant height produces ethyl acetate exception Brunswick Durham yeast and its cultural method and application | |
| CN107699499B (en) | One Aspergillus oryzae ZA127 and its application | |
| CN107190026B (en) | Method for improving secondary metabolite of monascus | |
| CN105062895B (en) | The monascus strain and its selection of a kind of high extracellular uranidin and application | |
| CN106978350A (en) | One plant of aspergillus niger and its application in the preparation of Pu'er tea chlorins compound | |
| CN109370929A (en) | A kind of application of S. cervisiae in wine brewing | |
| CN104928202A (en) | Fermentation culture method of bacillus | |
| CN108707556A (en) | A kind of horizontal stalk fungal strain and its application in producing cellulase | |
| CN101993847B (en) | Bacterial cellulose strain | |
| CN107267401A (en) | One plant of Mucor bacteria strain and the application on fermentation green brick tea | |
| CN102119631A (en) | Grifola frondosa strain for producing polysaccharide with composite raw material of rice bran and wheat bran | |
| KR101056952B1 (en) | Mass production method of black scale mushroom using lumber cultivation | |
| CN105219653B (en) | Produce Aspergillusclavatus (Aspergillus clavatus) Ac-32 bacterial strains and its application of Lovastatin | |
| CN108315265B (en) | Aspergillus versicolor Av-2 strain and application thereof | |
| CN108841889B (en) | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation | |
| CN108823110B (en) | Strain for producing griseofulvin and application thereof | |
| CN106479900B (en) | High yield monascus purpureus penicillium oxalicum Po-25 bacterial strain and application thereof | |
| CN104277985A (en) | Monascus purpureus M-24 bacterial strain and application thereof for preparation of Monacolin K | |
| CN106754430A (en) | Dendritic bacterial strains of cladosporium Cc 106 of high yield monascus purpureus and application thereof | |
| CN101275152A (en) | Application of rhizoctonia solani mycelium to improving trichodermin in trichoderma harzianum | |
| CN109136100A (en) | One plant of Tabin aspergillus bacterial strain and the application on fermentation green brick tea | |
| CN106085880B (en) | A kind of separation method and used medium of smut | |
| CN110438015A (en) | The method that the dried immature fruit of citron orange endogenetic fungus and its fermentation for producing hesperidinase produce hesperidinase | |
| CN106399121B (en) | A kind of purple red yeast rice bacteria strain | |
| CN101508960B (en) | Rhizopus and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240131 Address after: 250000 Hisense Tianchen, Tianchen Road, Jinan City, Shandong Province Patentee after: Zhuofan (Jinan) Technology Innovation Co.,Ltd. Country or region after: China Address before: 321004 No. 688 Yingbin Road, Zhejiang, Jinhua Patentee before: ZHEJIANG NORMAL University Country or region before: China |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240208 Address after: 710100 Building 6, No. 381 Kunchi 1st Road, Xiliu Street Office, High tech Zone, Xi'an City, Shaanxi Province Patentee after: Shaanxi Didu Pharmaceutical and Chemical Co.,Ltd. Country or region after: China Address before: 250000 Hisense Tianchen, Tianchen Road, Jinan City, Shandong Province Patentee before: Zhuofan (Jinan) Technology Innovation Co.,Ltd. Country or region before: China |
|
| TR01 | Transfer of patent right |