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
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Abstract
本发明属于生物技术微生物领域，涉及1株液体发酵生产Lovastatin的草酸青霉（Penicillium oxalicum）Po‑25菌株、用途和发酵生产方法。草酸青霉（Penicillium oxalicum）Po‑25菌株，该菌株保藏于中国典型培养物保藏中心，保藏编号为：CCTCC M 2016381，保藏日期为：2016年7月7日。本发明青霉Po‑25菌株具有高产Lovastatin、生长速度快、产孢量多、培养简单等优良特性。在发酵培养基初始pH为5.0~5.5，培养温度为28℃条件下，青霉Po‑25菌株通过液体发酵培养基和发酵条件优化后，发酵81 h~105 h，Lovastatin产量可达260 mg/L以上。
The invention belongs to the field of biotechnology microorganisms, and relates to a Penicillium oxalicum Po-25 strain for liquid fermentation to produce Lovastatin, uses and a fermentation production method. Penicillium oxalicum Po-25 strain, the strain is deposited in the China Center for Type Culture Collection, the preservation number is: CCTCC M 2016381, and the preservation date is: July 7, 2016. The Penicillium Po-25 strain of the invention has the excellent characteristics of high Lovastatin production, fast growth rate, large spore production, simple culture and the like. Under the condition that the initial pH of the fermentation medium is 5.0~5.5 and the culture temperature is 28℃, the Penicillium Po-25 strain can be fermented for 81 h~105 h after the liquid fermentation medium and fermentation conditions are optimized, and the yield of Lovastatin can reach 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)
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| CN117866784B (en) * | 2024-01-15 | 2024-11-05 | 延边大学 | Penicillium oxalicum D5 and application thereof in dry-process mature beef hip meat processing |
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| 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 |
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| 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 |
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| 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 * |
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