CN101979624A - Preparation method of microbial oil rich in gamma-linolenic acid - Google Patents
Preparation method of microbial oil rich in gamma-linolenic acid Download PDFInfo
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- CN101979624A CN101979624A CN201010505418.XA CN201010505418A CN101979624A CN 101979624 A CN101979624 A CN 101979624A CN 201010505418 A CN201010505418 A CN 201010505418A CN 101979624 A CN101979624 A CN 101979624A
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- linolenic acid
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- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 title claims abstract description 60
- 235000020664 gamma-linolenic acid Nutrition 0.000 title claims abstract description 60
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 229960002733 gamolenic acid Drugs 0.000 title claims abstract description 47
- 230000000813 microbial effect Effects 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 20
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 238000011218 seed culture Methods 0.000 claims abstract description 15
- 241000306282 Umbelopsis isabellina Species 0.000 claims abstract description 11
- 239000004519 grease Substances 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 7
- 239000003895 organic fertilizer Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
- 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 16
- 229940041514 candida albicans extract Drugs 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 239000012138 yeast extract Substances 0.000 claims description 16
- 239000002054 inoculum Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000010899 nucleation Methods 0.000 claims description 4
- 230000011218 segmentation Effects 0.000 claims description 4
- 238000000638 solvent extraction Methods 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 241000235575 Mortierella Species 0.000 abstract description 2
- 239000002921 fermentation waste Substances 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 2
- 241000233866 Fungi Species 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- HOBAELRKJCKHQD-UHFFFAOYSA-N (8Z,11Z,14Z)-8,11,14-eicosatrienoic acid Natural products CCCCCC=CCC=CCC=CCCCCCCC(O)=O HOBAELRKJCKHQD-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- HOBAELRKJCKHQD-QNEBEIHSSA-N dihomo-γ-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCCCC(O)=O HOBAELRKJCKHQD-QNEBEIHSSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- GMVPRGQOIOIIMI-UHFFFAOYSA-N (8R,11R,12R,13E,15S)-11,15-Dihydroxy-9-oxo-13-prostenoic acid Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CCCCCCC(O)=O GMVPRGQOIOIIMI-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 235000021298 Dihomo-γ-linolenic acid Nutrition 0.000 description 2
- 241000907999 Mortierella alpina Species 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 244000196929 Oenothera glazioviana Species 0.000 description 2
- 241000235401 Phycomyces blakesleeanus Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000134363 Umbelopsis ramanniana Species 0.000 description 2
- 229960000711 alprostadil Drugs 0.000 description 2
- 235000021342 arachidonic acid Nutrition 0.000 description 2
- 229940114079 arachidonic acid Drugs 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 150000002617 leukotrienes Chemical class 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 description 2
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 241000235389 Absidia Species 0.000 description 1
- 241001149952 Amylomyces rouxii Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
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- 235000007689 Borago officinalis Nutrition 0.000 description 1
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- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101000862089 Clarkia lewisii Glucose-6-phosphate isomerase, cytosolic 1A Proteins 0.000 description 1
- 241000235555 Cunninghamella Species 0.000 description 1
- 241001290628 Cunninghamella echinulata Species 0.000 description 1
- LMHIPJMTZHDKEW-XQYLJSSYSA-M Epoprostenol sodium Chemical compound [Na+].O1\C(=C/CCCC([O-])=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 LMHIPJMTZHDKEW-XQYLJSSYSA-M 0.000 description 1
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- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241000219925 Oenothera Species 0.000 description 1
- 235000004496 Oenothera biennis Nutrition 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 235000001466 Ribes nigrum Nutrition 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of bioengineering and discloses a preparation method of microbial oil rich in gamma-linolenic acid, which comprises the steps of sequentially carrying out primary seed culture, secondary seed culture, tertiary seed culture and four-stage fermentation on Mortierella pusilla (Mortierella isabellina), then collecting thalli, and extracting the microbial oil rich in gamma-linolenic acid from the thalli, wherein the four-stage fermentation adopts a mode of regulating and controlling the fermentation temperature in sections. The invention provides a preparation method of microbial oil rich in gamma-linolenic acid. The content of the gamma-linolenic acid in the grease is improved through two-step fermentation temperature regulation and control, the quality of the grease is improved, meanwhile, the organic solvent is used for extraction, the environmental pollution is reduced, the fungus cake can be used as an organic fertilizer and a carbon-nitrogen source for secondary utilization by microorganisms, the comprehensive utilization of fermentation waste is realized, and the positive promotion effect on the industrial production of the gamma-linolenic acid is achieved.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of preparation method who is rich in the microbial oil of gamma-linolenic acid.
Background technology
Gamma-linolenic acid (γ-linolenic acid or Gamma-linolenic acid are called for short GLA) is all-cis formula 6,9,12-punicic acid, molecular formula C
18H
30O
2, be the serial polyunsaturated fatty acid of a kind of ω-6, be colourless oil liquid, very easily oxidation in air.It is a kind of indispensable fatty acid of human body, in human body, be transformed by linolic acid, then be converted into dihomogammalinolenic acid (Dihomo-γ-linolenic acid, DGLA) and arachidonic acid (Arachidonic acid, ARA), be transformed into prostaglandin E1 (PGE1), leukotriene (LT) and Epoprostenol Sodium (PGI2) again.Gamma-linolenic acid has widely physiologically active and significantly pharmacological action, and it can reducing blood-fat, antithrombotic cardiovascular and cerebrovascular diseases, prevention and treatment hypertension, atherosclerosis.Simultaneously again can be antibiotic, anti-inflammatory, antitumor, anti-diabetic, anti-HIV infection etc., aspect beauty treatment, the makeup application is being arranged also, the premenstrua syndromes is had certain curative effect, by spoken highly of into " 21 century functional foodstuff leading role " (single Changhai, Zhao Dan. Sichuan food and fermentation, 2006, (1): 17-20).
1919, Hei-duschka from Oenotheraceae (Evening Primrose Flamily) plant root of Redsepal Eveningprimrose, find first gamma-linolenic acid (Wu Guangli, Wang Xijun, Li Jun, etc. agricultural and technology, 1994, (3): 19-22).At present known have in more than the 80 kind of higher plant seed grease contain a certain amount of gamma-linolenic acid, wherein content is higher (Tian Xinzhen, Wang Xianlei such as root of Redsepal Eveningprimrose, borage, Ribes nigrum L., blueweed, micropore grass, Sun Guilin, Deng. biotechnology, 2008,18 (1): 89-92).But from plant, extract gamma-linolenic acid and be subjected to all multifactor restrictions; the growth cycle of plant is long; floor space is big, and is subjected to the restriction of natural condition, and the collection of plant seed is difficult; the oil-contg instability; be difficult to satisfy the growing demand of people (Hai Hua, Shang Dejing, Li Qingwei. industrial microorganism; 2002,3 (24): 46-50).Bernhard in 1948 and Albercht (Bernhard K, Albrecht H.Helv Chim Acta, 1948,31 (4): 977-988) at first from the thalline fat of phycomyces blakesleeanus (Phycomyces blakesleeanus), identify gamma-linolenic acid, opened the prelude of Production by Microorganism Fermentation gamma-linolenic acid.Cloth Laplace palpus Rhizopus mould and that find afterwards, Mucor, Aspergillus, absidia, Syncephalastrum etc. have stronger gamma-linolenic acid synthesis capability.The more bacterial strain of research is Mortierella isabellina (Mortierella isabellina), mortierella ramanniana (Mortierella ramanniana), short mortierella (Mortierella nana), Mortierella alpina (Mortierella alpina), cunninghamella echinulata (Cunninghamella echinlata), western Shandong Mucor (Li Zhongling such as (Mucor rouxii) at present, Qiu Shuning, Wang Weiwei, Deng. JOURNAL OF MICROBIOLOGY, 2008,28 (1): 94-97).Although can produce more than the bacterial classification of gamma-linolenic acid has so, because it is numerous to influence the factor of gamma-linolenic acid output, is difficult to obtain a kind of suitable method and improves the content of gamma-linolenic acid in grease, cause this type of greasy of low quality.The contriver finds when research one strain Mortierella isabellina ME-L16 (CCTCC No.M 2010195) (number of patent application 201010281243.9), temperature is huge to the content influence of GLA in its grease, so carried out the present invention, set up a kind of microbial oil preparation method who is rich in gamma-linolenic acid, the GLA that this method is suitable for other equally produces bacterial classification.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method who is rich in the microbial oil of gamma-linolenic acid.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of preparation method who is rich in the microbial oil of gamma-linolenic acid, with Mortierella isabellina (Mortierella isabellina) successively through first order seed cultivate, secondary seed is cultivated, three grades of seed culture and level Four fermentation, collect thalline then, extract grease and promptly get the microbial oil that is rich in gamma-linolenic acid from thalline, wherein the mode of segmentation regulation and control leavening temperature is adopted in the level Four fermentation.
Wherein, the microbial strains that described method adopted is Mortierella isabellina (Mortierella isabellina, CCTCCNo.M 2010195).
Wherein, described first order seed is cultivated and is slant culture, and substratum is the PDA solid medium, and culture condition is 25～28 ℃, cultivates 3～5 days, to the spore growth and maturity.
Wherein, described secondary seed is cultivated, and substratum is: glucose 30～40g/L, yeast extract paste 6～8g/L, KH
2PO
43～4g/L, MgSO
47H
2O 0.5～1g/L, all the other are water, pH 7.0; Culture condition is: first order seed is cultivated the spore that obtains wash with sterilized water, with 10
5～10
6Individual spore/mL inoculation, air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, 28～30 ℃ (preferred 28 ℃) were cultivated 2～4 days.
Wherein, described three grades of seed culture, substratum is: glucose 60～80g/L, yeast extract paste 7～9g/L, KH
2PO
44～5g/L, MgSO
47H
2O 1～2g/L, all the other are water, pH 7.0; Culture condition is: with secondary seed three grades of seeding tanks of inoculum size access with 8～10% (v/v), air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, and 28～30 ℃ (preferred 28 ℃) were cultivated 4～5 days.
Wherein, described level Four fermentation, substratum is: glucose 100～120g/L, yeast extract paste 8～9g/L, KH
2PO
45～7g/L, MgSO
47H
2O 2～3g/L, all the other are water, pH 7.0; Culture condition is: with the inoculum size access level Four fermentor tank of three grades of seeds with 8～10% (v/v), air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, cultivate and collect thalline in 8 days; Wherein, the mode of segmentation regulation and control leavening temperature is adopted in the level Four fermentation, and preceding 6 days culture temperature are 28～30 ℃ (preferred 28 ℃), and the 7th to 8 day culture temperature is 20～22 ℃ (preferred 20 ℃).
Wherein, the method for from thalline, extracting microbial oil be thalline through vacuum-drying, mechanical disintegration, organic solvent extraction, solvent recuperation promptly gets the microbial oil that is rich in gamma-linolenic acid, the organic solvent of recovery can further recycle, and has reduced environmental pollution.Described organic solvent is one or more the mixing in sherwood oil, ethanol, normal hexane, the chloroform.
Wherein, the thalline behind the extraction grease can also be used as organic fertilizer, or fodder additives, or utilizes for microorganism as nitrogenous source.
Analytical procedure in the inventive method is as follows:
Chromatographic condition: Thermo Finnigan TRACE DSQ type gas chromatograph-mass spectrometer, adopt DB-5MS capillary column (30m * 0.25mm * 0.25 μ m).50 ℃ of initial column temperatures are warmed up to 250 ℃ with 20 ℃/min, keep 2min.200 ℃ of sample introduction temperature, carrier gas are helium, flow rate of carrier gas 1ml/min, not split stream sampling.Total run time is 12min.Extracting head at injection port in 200 ℃ of following desorption 5min.
Mass spectrometric detection condition: EI source 70eV, 250 ℃ of ion source temperatures, 250 ℃ of transmission line temperature, sweep limit 50～400aum.
All do not adopt water as solvent when the solvent of used substratum clearly indicates in the inventive method.
The per-cent of the inoculum size in the inventive method is meant the ratio of seed liquor and the volume of fermention medium.
PH regulator mode in the inventive method: regulate pH with the sulfuric acid of 1mol/L and the sodium hydroxide of 2mol/L.
Beneficial effect:
The present invention has set up a kind of preparation method who is rich in the microbial oil of gamma-linolenic acid.By the two-step fermentation temperature adjusting, improved the content of gamma-linolenic acid in the grease, improved greasy quality, use organic solvent extraction simultaneously, reduced environmental pollution, the bacterium cake can be used as organic fertilizer and utilizes for microorganism once more as carbon nitrogen source, has realized the comprehensive utilization of fermentation wastes, will play positive pushing effect to the GLA suitability for industrialized production.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: temperature is produced the influence of gamma-linolenic acid to Mortierella isabellina Mortierella isabellina ME-L16.
The ME-L16 bacterial strain is transferred on the PDA solid medium, cultivates 2～4 days for 25～28 ℃, and spore is growth and maturity.With transfering loop picking 2 ring spores in the liquid seed culture medium of 50mL 25 ℃ cultivated 2 days.Cultivated 6～8 days with 130rpm shaking table in the inoculum size access fermention medium of 10% (v/v), wherein culture temperature is made as 15 ℃ respectively, and 18 ℃, 20 ℃, 23 ℃, 25 ℃, 28 ℃, 30 ℃, survey dry cell weight, detect the content of GLA simultaneously with GC-MS.Analyze culture temperature and strain fermentation is produced the influence of GLA.
Liquid seed culture medium is: glucose 30g/L, yeast extract paste 6g/L, KH
2PO
43g/L, MgSO
47H
2O 0.5g/L, all the other are water, pH 7.0.
Fermention medium is: glucose 80g/L, yeast extract paste 6g/L, KH
2PO
43g/L, MgSO
47H
2All the other are water for O 0.5g/L.
Table 1 differing temps is to producing the influence of gamma-linolenic acid
Embodiment 2: the foundation of high yield gamma-linolenic acid two-step fermentation temperature adjusting strategy.
According to embodiment 1, temperature is huge to the influence of gamma-linolenic acid content, so set up two Buwen's degree regulation and control strategy to improve the content of gamma-linolenic acid in the tunning.The ME-L16 bacterial strain is transferred on the PDA solid medium, cultivates 2～4 days for 25 ℃, and spore is growth and maturity.With transfering loop picking 2 ring spores in the liquid seed culture medium of 50mL 28 ℃ cultivated 2 days.Treat that ripe inoculum size with 10% (v/v) inserts 130rpm in the fermention medium, 28 ℃ of shaking tables were cultivated 6 days, began that at the 7th day temperature is transferred to 20 ℃ and cultivated 2 days, surveyed dry cell weight during fermentation termination, detected the content of GLA simultaneously with GC-MS.Other sets up control group, and except that leavening temperature remained at 28 ℃ of cultivations, other conditions were identical with two-step approach, relatively the content of regulation and control of two Buwen's degree and control group GLA.
Liquid seed culture medium is: glucose 30g/L, yeast extract paste 6g/L, KH
2PO
43g/L, MgSO
47H
2O 0.5g/L, all the other are water, pH 7.0.
Fermention medium is: glucose 80g/L, yeast extract paste 6g/L, KH
2PO
43g/L, MgSO
47H
2All the other are water for O 0.5g/L.
Table 2 liang Buwen is controlled the comparison of strategy and control group product gamma-linolenic acid
Under low temperature environment, saturated fatty acid is because zero pour is low, easily solidify, make the flowability of cell membranes variation to be unfavorable for growth, the breeding of cell, in order to resist this environmental stress, cell trends towards a part of saturated fatty acid desaturation is become polyunsaturated fatty acid, increase the flowability of film fat, keeping normal physiological function, thereby increased the output of GLA.
Embodiment 3: level Four fermentative production gamma-linolenic acid.
The ME-L16 bacterial strain through first order seed cultivate, secondary seed is cultivated, three grades of seed culture and level Four are fermented cultivates.
First order seed is cultivated it and is slant culture, and substratum is the PDA solid medium, and culture condition is 25 ℃, cultivates 3～5 days, to the spore growth and maturity.
Secondary seed is cultivated, and its substratum is: glucose 30g/L, yeast extract paste 6g/L, KH
2PO
43g/L, MgSO
47H
2O 0.5g/L, all the other are water, pH 7.0.Culture condition is: the spore on inclined-plane is washed with sterilized water, with 10
6Individual spore/mL inoculation, air flow 1V/ (Vmin), rotating speed are 120rpm, cultivate 4 days for 28 ℃.
Three grades of seed culture, its substratum is: glucose 60g/L, yeast extract paste 7g/L, KH
2PO
44g/L, MgSO
47H
2O1g/L, all the other are water, pH 7.0.Culture condition is: will grow good secondary seed three grades of seeding tanks of inoculum size access with 10% (v/v), air flow 1V/ (Vmin), rotating speed are 120rpm, cultivate 4 days for 28 ℃.
The level Four fermentation, its substratum is: glucose 100g/L, yeast extract paste 8g/L, KH
2PO
45g/L, MgSO
47H
2O2g/L, all the other are water, pH 7.0.Culture condition is: will grow the inoculum size access level Four fermentor tank of good secondary seed with 10% (v/v), air flow 1V/ (Vmin), rotating speed are 120rpm, cultivating 8 natural gift carries out for two sections, 7th day temperature reduce to 20 ℃ and continue cultivate 2 day in 28 ℃ of cultivations 6 days early stage, collects thalline.
The result: dry cell weight is 35g/L, and total lipid content is 49%, and GLA content is 22%, and GLA output is 3.773g/L.Embodiment 4: level Four fermentative production gamma-linolenic acid.
The ME-L16 bacterial strain through first order seed cultivate, secondary seed is cultivated, three grades of seed culture and level Four are fermented cultivates.
First order seed is cultivated it and is slant culture, and substratum is the PDA solid medium, and culture condition is 25 ℃, cultivates 3～5 days, to the spore growth and maturity.
Secondary seed is cultivated, and its substratum is: glucose 40g/L, yeast extract paste 8g/L, KH
2PO
43g/L, MgSO
47H
2O 0.5g/L, all the other are water, pH 7.0.Culture condition is: the spore on inclined-plane is washed with sterilized water, with 10
6Individual spore/mL inoculation, air flow 1V/ (Vmin), rotating speed are 130rpm, cultivate 4 days for 28 ℃.
Three grades of seed culture, its substratum is: glucose 80g/L, yeast extract paste 9g/L, KH
2PO
44g/L, MgSO
47H
2O1g/L, all the other are water, pH 7.0.Culture condition is: will grow good secondary seed three grades of seeding tanks of inoculum size access with 10% (v/v), air flow 1V/ (Vmin), rotating speed are 130rpm, cultivate 5 days for 28 ℃.
The level Four fermentation, its substratum is: glucose 120g/L, yeast extract paste 9g/L, KH
2PO
45g/L, MgSO
47H
2O2g/L, all the other are water, pH 7.0.Culture condition is: will grow the inoculum size access level Four fermentor tank of good secondary seed with 10% (v/v), air flow 1V/ (Vmin), rotating speed are 130rpm, cultivating 8 natural gift carries out for two sections, 7th day temperature reduce to 20 ℃ and continue cultivate 2 day in 28 ℃ of cultivations 6 days early stage, collects thalline.
The result: dry cell weight is 37g/L, and total lipid content is 47%, and GLA content is 22.5%, and GLA output is 3.913g/L.
Embodiment 5: the extraction of microbial oil.
With fermented liquid in the centrifugal collection thalline of whizzer 6000～8000rpm.The mycelium of results is sent into vacuum drier again, and 70～90 ℃ of following vacuum-drying 15～25min get dry mycelium.Dry mycelium passes through mechanical disintegration again, organic solvent extraction, and solvent recuperation promptly gets the high microbial oil that contains gamma-linolenic acid.Wherein extraction agent is sherwood oil and ethanol (volume ratio is 5: 2) extraction 12h, has extracted and by underpressure distillation extraction agent has been reclaimed, recycle.The bacterium cake can be used as fodder additives, organic fertilizer and utilizes for microorganism once more as carbon nitrogen source.
Claims (8)
1. preparation method who is rich in the microbial oil of gamma-linolenic acid, it is characterized in that with Mortierella isabellina (Mortierella isabellina) successively through first order seed cultivate, secondary seed is cultivated, three grades of seed culture and level Four fermentation, collect thalline then, extract the microbial oil that is rich in gamma-linolenic acid from thalline, wherein the mode of segmentation regulation and control leavening temperature is adopted in the level Four fermentation.
2. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1 is characterized in that it is slant culture that described first order seed is cultivated, and substratum is the PDA solid medium, and culture condition is 25～28 ℃, cultivates 3～5 days.
3. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1 is characterized in that described secondary seed cultivation, and substratum is: glucose 30～40g/L, yeast extract paste 6～8g/L, KH
2PO
43～4g/L, MgSO
47H
2O 0.5～1g/L, all the other are water, pH 7.0; Culture condition is: first order seed is cultivated the spore that obtains wash with sterilized water, with 10
5～10
6Individual spore/mL inoculation, air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, cultivate 2～4 days for 28～30 ℃.
4. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1 is characterized in that described three grades of seed culture, and substratum is: glucose 60～80g/L, yeast extract paste 7～9g/L, KH
2PO
44～5g/L, MgSO
47H
2O 1～2g/L, all the other are water, pH 7.0; Culture condition is: with secondary seed three grades of seeding tanks of inoculum size access with 8～10% (v/v), air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, cultivate 4～5 days for 28～30 ℃.
5. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1 is characterized in that described level Four fermentation, and substratum is: glucose 100～120g/L, yeast extract paste 8～9g/L, KH
2PO
45～7g/L, MgSO
47H
2O 2～3g/L, all the other are water, pH 7.0; Culture condition is: with the inoculum size access level Four fermentor tank of three grades of seeds with 8～10% (v/v), air flow 1～1.5V/ (Vmin), rotating speed are 120～150rpm, cultivate and collect thalline in 8 days; Wherein, the mode of segmentation regulation and control leavening temperature is adopted in the level Four fermentation, and culture temperature was 28～30 ℃ in preceding 6 days, and culture temperature was 20～22 ℃ in the 7th to 8 day.
6. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1, the method that it is characterized in that extraction microbial oil from thalline is that thalline is through vacuum-drying, mechanical disintegration, organic solvent extraction, solvent recuperation promptly gets the microbial oil that is rich in gamma-linolenic acid.
7. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 6 is characterized in that described organic solvent is one or more the mixing in sherwood oil, ethanol, normal hexane, the chloroform.
8. the preparation method who is rich in the microbial oil of gamma-linolenic acid according to claim 1 is characterized in that extracting thalline behind the grease as organic fertilizer, or fodder additives, or utilizes for microorganism as nitrogenous source.
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| CN104531342A (en) * | 2014-12-18 | 2015-04-22 | 北京化工大学 | Method for gently and efficiently extracting microbial oil |
| CN106047956A (en) * | 2016-08-05 | 2016-10-26 | 广东海洋大学 | Method for preparing gamma-linolenic acid-rich grease through sea squirt-associated Penicillium citrinum Asc2-4 fermentation |
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| CN1415754A (en) * | 2002-12-13 | 2003-05-07 | 南开大学 | Method for producing soybean containg content of high gamma-linolenic acid |
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| CN101445815A (en) * | 2007-11-26 | 2009-06-03 | 北京有容建业科技发展有限责任公司 | Microbial synthesis method of gamma-linolenic acid oil |
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| 《大连轻工业学院学报》 20060930 孟晓敏 等 深黄被孢霉H3,3410-5gamma-亚麻酸发酵的实验研究 168-171 1-8 第25卷, 第3期 * |
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| CN104531342A (en) * | 2014-12-18 | 2015-04-22 | 北京化工大学 | Method for gently and efficiently extracting microbial oil |
| CN106047956A (en) * | 2016-08-05 | 2016-10-26 | 广东海洋大学 | Method for preparing gamma-linolenic acid-rich grease through sea squirt-associated Penicillium citrinum Asc2-4 fermentation |
| CN106047956B (en) * | 2016-08-05 | 2019-11-26 | 广东海洋大学 | A method of the Penicillium citrinum Asc2-4 that grown nonparasitically upon another plant altogether using ascidian fermentation preparation is rich in the grease of gamma-Linolenic acid |
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