CN108004277A - One kind prepares linoleic method using bacillus megaterium - Google Patents
One kind prepares linoleic method using bacillus megaterium Download PDFInfo
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- CN108004277A CN108004277A CN201810067168.2A CN201810067168A CN108004277A CN 108004277 A CN108004277 A CN 108004277A CN 201810067168 A CN201810067168 A CN 201810067168A CN 108004277 A CN108004277 A CN 108004277A
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- bacillus megaterium
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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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
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a kind of linoleic method is prepared using bacillus megaterium, including by bacillus megaterium L2（Bacillus megateriumL2）Inoculation is in NA solid slope culture mediums, and 30 ± 1 DEG C are cultivated 2 days, are activated 3 times, then are accessed in NB liquid seed culture mediums, 30 ± 1 DEG C, 130 150 r/min, and 20 24h of shaken cultivation, obtains seed liquor, with 6%（v/v）Ratio seed liquor is transferred in 250mL NB fermentation mediums, 30 ± 1 °C, 130 150 r/min, cultivate 3 days terminate fermentation.Zymotic fluid centrifuges 15 20min under 5,000 6000 r/min, 4 °C, carries out bacterium solution separation, removes zymotic fluid, collects somatic cells, to obtain the final product.The present invention production linoleic conditional stability of polyunsaturated fatty acid, culture substrate are readily available, and fermentation condition is stable, technique is simple.
Description
Technical field
The present invention relates to biological technical field, relates in particular to a kind of bacillus megaterium using the sub- oil of beef extract production
The method of acid.
Background technology
Bacillus megaterium（Bacillus megaterium）Belong to bacillus, be a kind of very promising function
Type bacterium, has the advantages that nutritional requirement is low, condition of culture is easy, breeding is rapid, safe and non-toxic, and it is anti-to be widely used in biology
Control, purify water, the synthesis of degradation of pesticide, microbial-bacterial fertilizer, antibiotic, the field such as enzyme industry.Contain a variety of more insatiable hungers in bacterium
And aliphatic acid, mainly including linoleic acid (LA), conjugated linoleic acid (CLA), γ -2 leukotrienes (GLA), arachidonic acid (ARA),
Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) etc., are the important composition components of biomembrane, are bioactive substances
Precursor, be of great significance to the health of human body.
Linoleic acid（Linoleic acid）It is that a kind of human body oneself cannot produce, it is necessary to the required fat obtained by diet
Fat acid, as a kind of polyunsaturated fatty acid（PUFA）, it is the dietary constituents more healthy than meat or dairy products saturated fat,
Blood fat, softening blood vessel can be effectively reduced, reduces blood pressure, promote microcirculation, can prevent or reduce the incidence of cardiovascular disease；Conjugation
Linoleic acid has anticancer, anti arteriosclerosis, kills or suppress the effect of cancer cell produces as linoleic isomers, moreover it is possible to
Promote lipid metabolism, participate in energy and distribute and promote to grow, there is wide purposes in terms of animal feeding.At present, prepare sub-
Oleic acid after hydrolysis rich in linoleic grease mostly as raw material, to obtain fatty acid mixed, and fatty acid mixed is through separating, purifying
After obtain linoleic acid.The method for industrially preparing fatty acid mixed is more, has been summed up three classes：First, synthetic method, that is, paraffin
Oxidizing process or a hydrocarbon oxidizing process, second, recycle ready denier oil acid from papermaking wastewater, third, from natural oil non-catalysis hydrolyzation
Or produced through enzyme hydrolysis, its complex production process, of high cost, energy consumption is big, stability is poor.Polyunsaturated fatty acid in microorganism
Rich content, bacterium polyunsaturated fatty acid are mainly present on film with phosphatide or other liquid forms, and microorganism grows
From the influence of season, region and weather, fermentation period is short, and adaptable strong, growth and breeding rapidly, is easily cultivated, from original
The features such as expecting place of production limitation.Therefore, it is an important channel using micro-organisms polyunsaturated fatty acid linoleic acid, bacterium produces
PUFA can be used as biological feed, be introduced into marine aquaculture food chain, will have to raising aquatic products PUFA qualities important
Meaning.And the genetic mechanism of bacterium is relatively easy, the method for researching and producing polyunsaturated fatty acid is beneficial to illustrate its PUFA
Synthesis mechanism, help to improve the ability of bacterium production PUFA, also have carrying out transgenic research field to other biological
Good prospect.
The content of the invention
A kind of production linoleic condition of polyunsaturated fatty acid that it is an object of the invention to overcome disadvantages mentioned above and provide
Stablize, culture substrate is readily available, and fermentation condition is stable, technique simply prepares linoleic side using bacillus megaterium
Method.
One kind prepares linoleic method using bacillus megaterium, including：
（1）Actication of culture
By bacillus megaterium L2（Bacillus megateriumL2）Bacterial strain is connected in NA solid slope culture mediums, and 30 ± 1
DEG C quiescent culture 1-2 days, activates 3 times, it is spare to be stored in 4 DEG C of refrigerators；
（2）The preparation of seed liquor
The bacillus megaterium L2 bacterial strains of activation are scraped into two rings using oese and are inoculated in 250mL NB liquid seed culture mediums
In, 30 ± 1 DEG C, 130-150r/min, Shaking culture 20-24h, obtains seed liquor；
（3）Fermented and cultured
With 6%（v/v）Ratio seed liquor is transferred in 250mL NB fermentation mediums, 30 ± 1 °C, 130-150r/min, training
Support and terminate within 3 days fermentation, zymotic fluid centrifuges 15-20min under 5000-6000r/min, 4 °C, carries out bacterium solution separation, removes fermentation
Liquid, collects somatic cells, to obtain the final product.
Above-mentioned bacillus megaterium produces linoleic method, the wherein system of NA solid slope culture mediums using beef extract
Preparation Method is as follows：10 g of peptone, 5 g of glucose, 5 g of sodium chloride, 3 g of beef extract, 20 g of agar, distilled water 1000 ml, PH
7.0-7.2；Sterilising conditions：121 DEG C, 15-20min.
Above-mentioned bacillus megaterium produces linoleic method, the wherein system of NB liquid seed culture mediums using beef extract
Preparation Method is as follows：10 g of peptone, 5 g of glucose, 5 g of sodium chloride, 3 g of beef extract, distilled water 1000 ml, PH 7.0-7.2；
Sterilising conditions：121 DEG C, 15-20min.
Above-mentioned bacillus megaterium produces linoleic method, wherein the preparation side of NB fermentation mediums using beef extract
Method is as follows：15 g/L of glucose, 25 g/L of peptone, 4.58 g/L of beef extract, 3.23 g/L of sodium chloride, 1000 ml of distilled water,
PH value is 7.0-7.2；Sterilising conditions：121 DEG C, 15-20min.
The strain is deposited in China typical culture collection center on 26th in September in 2012（Address：Wuhan, China is military
Chinese university）, preserving number：CCTCC NO：M2012381, it is entitled：Bacillus megaterium L2（Bacillus megaterium
L2）.
Compared with prior art, the present invention there is obvious beneficial effect, as can be known from the above technical solutions：Utilize the sub- oil of production
Sour bacillus megaterium L2 carries out the culture medium based on beef extract biodegradable and conversion, and fermenting and producing linoleic acid, can be
Solve polyunsaturated fatty acid source and one new way is provided.And bacillus megaterium L2（Bacillus megateriumL2）
Bacterium source is reliable, and to the wide adaptation range of the natural environmental conditions such as temperature, pH, easily culture and preservation, produce how unsaturated fat
The linoleic conditional stability of fat acid, can be as the bacterial strain of extraction polyunsaturated fatty acid, expanding resource storehouse.
Embodiment
One kind prepares linoleic method using bacillus megaterium, comprises the following steps：
（1）Actication of culture
Bacillus megaterium L2 bacterial strains are connected in NA solid slope culture mediums, 30 ± 1 DEG C of quiescent cultures 1-2 days, activate 3 times,
It is spare to be stored in 4 DEG C of refrigerators；
（3）The preparation of seed liquor
The bacillus megaterium L2 bacterial strains of activation are scraped into two rings using oese and are inoculated in 250mLNB liquid seed culture mediums
In, 30 ± 1 DEG C, 130-150r/min, Shaking culture 20-24h, obtains seed liquor；
（3）Fermented and cultured
With 6%（v/v）Ratio seed liquor is transferred in 250mL NB fermentation mediums, 30 ± 1 °C, 130-150r/min, training
Support and terminate within 3 days fermentation, zymotic fluid centrifuges 15-20min at 5000-6000 r/min, 4 DEG C, carries out bacterium solution separation, removes fermentation
Liquid, collects somatic cells, to obtain the final product.
Bacillus megaterium（Bacillus megaterium）The identification of polyunsaturated fatty acid in L2 thalline：
（1）The preparation of medicinal extract
Above-mentioned gained somatic cells are taken to be placed in air dry oven, 50-55 DEG C dries to constant weight, milling, crosses 50-60 mesh sieves and obtains bacterium
Powder, is extracted with ethyl acetate, and solvent is recovered under reduced pressure, and obtains test sample ethyl acetate portion and water section, and by ethyl acetate
Part concentrated by rotary evaporation obtains medicinal extract.
（2）The separation of sample
Acetic acid ethyl ester extract is taken to carry out silica gel column chromatography, by petroleum ether：Ethyl acetate（100:1,50:1,20:1,10:1,5:
1,2:1,1:1）, ethyl acetate：Methanol（30:1,20:1,10:1,5:1,2:1,1:1）, v/v, pure methanol system rushes column, thin layer
Ultraviolet 254nm detections, are divided into 16 parts（LE1-16）, the moderate LE4 of polarity is chosen, silica gel column chromatography is carried out again, by stone
Oily ether：Ethyl acetate（60:1,40:1）, v/v, thin layers of ultraviolet 254nm detection, are divided into 7 parts（LE4-1-LE4-7）, take oil
Shape material LE4-5 components carry out GC-MS analyses, are fatty acid component.
（3）GC-MS analysis conditions
GC conditions：Chromatographic column is AB-INOWAX（30m×0.25μm×0.25mm）Capillary column, 50 DEG C of initial temperature
（Keep 2min）, it is warming up to 240 DEG C（5℃/min）, keep 15min, run time：55 min；250 DEG C of temperature of vaporization chamber；Carry
Gas is high-purity He (99.999%)；7.65psi, carrier gas flux 1.0mL/min are pressed before column；1 μ L of sample size；Do not shunt.
Mass Spectrometry Conditions：Ion gun is EI sources；230 DEG C of ion source temperature；150 DEG C of quadrupole rod temperature；Electron energy 70eV；Hair
34.6 μ A of radio stream；Multiplier voltage 1624V；280 DEG C of interface temperature；29 ~ 500amu of mass range；The solvent delay time：
5.0min。
（4）Conclusion：Fatty acid composition in bacillus megaterium L2 thalline is mainly linoleic acid, octadecenoic acid, 15
Alkanoic acid and palmitic acid, for its Linoleic acid as unrighted acid, content highest, is 23.607%.
The above described is only a preferred embodiment of the present invention, not make limitation in any form, Ren Hewei to the present invention
Disengaging technical solution of the present invention content, any simple modification made according to technical spirit of the invention to above example, etc.
With change and modification, in the range of still falling within technical solution of the present invention.
Claims (5)
1. one kind prepares linoleic method using bacillus megaterium, including：
（1）Actication of culture
Bacillus megaterium L2 bacterial strains are connected in NA solid slope culture mediums, 30 ± 1 DEG C of quiescent culture 48h, activated 3 times, protected
It is spare to be stored in 4 DEG C of refrigerators；
（2）The preparation of seed liquor
The bacillus megaterium L2 bacterial strains of activation are scraped into two rings using oese and are inoculated in 250mL NB liquid seed culture mediums
In, 30 ± 1 DEG C, 130-150 r/min, Shaking culture 20-24h, obtains seed liquor；
（3）Fermented and cultured
Seed liquor is taken 6% to be inoculated in by volume in 250mL NB fermentation mediums, 30 ± 1 °C, 130-150 r/min, culture 3
It terminates fermentation, and zymotic fluid centrifuges 15-20min under 5000-6000 r/min, 4 °C, carries out bacterium solution separation, removes zymotic fluid,
Collect somatic cells, to obtain the final product.
2. bacillus megaterium as claimed in claim 1 produces linoleic method, wherein NA solid slopes using beef extract
The preparation method of culture medium is as follows：10 g of peptone, 5 g of glucose, 5 g of sodium chloride, 3 g of beef extract, 20 g of agar, distilled water
1000 ml, PH 7.0-7.2；Sterilising conditions：121 DEG C, 15-20min.
3. bacillus megaterium as claimed in claim 1 or 2 produces linoleic method, wherein NB liquid strains using beef extract
The preparation method of sub- culture medium is as follows：10 g of peptone, 5 g of glucose, 5 g of sodium chloride, 3 g of beef extract, distilled water 1000
Ml, PH 7.0-7.2；Sterilising conditions：121 DEG C, 15-20min.
4. bacillus megaterium as claimed in claim 3 produces linoleic method, wherein NB fermented and cultureds using beef extract
The preparation method of base is as follows：15 g/L of glucose, 25 g/L of peptone, 4.58 g/L of beef extract, 3.23 g/L of sodium chloride, distillation
Water 1000 ml, pH value 7.0-7.2；Sterilising conditions：121 DEG C, 15-20min.
5. bacillus megaterium as claimed in claim 4 produces linoleic method using beef extract, wherein：Huge gemma
Bacillus L2 bacterial strain preserving numbers：CCTCC NO：M2012381, it is entitled：Bacillus megaterium L2（Bacillus megaterium
L2）.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110092809A (en) * | 2019-06-14 | 2019-08-06 | 贵州大学 | A method of utilizing bacillus megaterium separating and extracting beta-sitosterol |
| CN110194722A (en) * | 2019-04-28 | 2019-09-03 | 贵州大学 | A method of utilizing bacillus megaterium separation and Extraction erucyl amide |
| CN110330425A (en) * | 2019-08-06 | 2019-10-15 | 贵州大学 | A method of utilizing bacillus megaterium separation and Extraction behenic acid |
| CN110563575A (en) * | 2019-07-25 | 2019-12-13 | 贵州大学 | Method for separating and extracting phenylacetic acid by using bacillus megaterium |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110194722A (en) * | 2019-04-28 | 2019-09-03 | 贵州大学 | A method of utilizing bacillus megaterium separation and Extraction erucyl amide |
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| CN110563575A (en) * | 2019-07-25 | 2019-12-13 | 贵州大学 | Method for separating and extracting phenylacetic acid by using bacillus megaterium |
| CN110330425A (en) * | 2019-08-06 | 2019-10-15 | 贵州大学 | A method of utilizing bacillus megaterium separation and Extraction behenic acid |
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