CN103031350B - Method for producing PUFA (polyunsaturated fatty acid) - Google Patents
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- 235000020777 polyunsaturated fatty acids Nutrition 0.000 title claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 20
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 13
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims abstract description 7
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- 238000002360 preparation method Methods 0.000 claims description 13
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- 239000000725 suspension Substances 0.000 claims description 9
- 241000228212 Aspergillus Species 0.000 claims description 6
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- 229920001817 Agar Polymers 0.000 claims description 3
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- 239000012467 final product Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 238000012807 shake-flask culturing Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 abstract description 5
- 150000004670 unsaturated fatty acids Chemical class 0.000 abstract description 5
- 239000001965 potato dextrose agar Substances 0.000 abstract 4
- 238000012258 culturing Methods 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 238000009423 ventilation Methods 0.000 abstract 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 4
- 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
- 239000000284 extract Substances 0.000 description 4
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- 235000019198 oils Nutrition 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 239000002253 acid Substances 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
- 230000006837 decompression Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- DVSZKTAMJJTWFG-UHFFFAOYSA-N docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCCC=CC=CC=CC=CC=CC=CC(O)=O DVSZKTAMJJTWFG-UHFFFAOYSA-N 0.000 description 2
- MBMBGCFOFBJSGT-KUBAVDMBSA-N docosahexaenoic acid Natural products CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 2
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- 238000011084 recovery Methods 0.000 description 2
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- QXJSBBXBKPUZAA-CMDGGOBGSA-N (e)-octadec-10-enoic acid Chemical compound CCCCCCC\C=C\CCCCCCCCC(O)=O QXJSBBXBKPUZAA-CMDGGOBGSA-N 0.000 description 1
- ZONJATNKKGGVSU-UHFFFAOYSA-N 14-methylpentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCC(O)=O ZONJATNKKGGVSU-UHFFFAOYSA-N 0.000 description 1
- 240000004904 Amorphophallus paeoniifolius Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- HXWJFEZDFPRLBG-UHFFFAOYSA-N Timnodonic acid Natural products CCCC=CC=CCC=CCC=CCC=CCCCC(O)=O HXWJFEZDFPRLBG-UHFFFAOYSA-N 0.000 description 1
- 235000021322 Vaccenic acid Nutrition 0.000 description 1
- UWHZIFQPPBDJPM-FPLPWBNLSA-M Vaccenic acid Natural products CCCCCC\C=C/CCCCCCCCCC([O-])=O UWHZIFQPPBDJPM-FPLPWBNLSA-M 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 239000012259 ether extract Substances 0.000 description 1
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- 235000019197 fats Nutrition 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
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- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
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- 239000004519 grease Substances 0.000 description 1
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- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- CASUWPDYGGAUQV-UHFFFAOYSA-M potassium;methanol;hydroxide Chemical compound [OH-].[K+].OC CASUWPDYGGAUQV-UHFFFAOYSA-M 0.000 description 1
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- UWHZIFQPPBDJPM-BQYQJAHWSA-N trans-vaccenic acid Chemical compound CCCCCC\C=C\CCCCCCCCCC(O)=O UWHZIFQPPBDJPM-BQYQJAHWSA-N 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing PUFA (polyunsaturated fatty acid) by utilizing potato in an Aspergillus niger xj strain. The method comprises the following steps of inoculating the Aspergillus niger xj strain in a PDA (potato dextrose agar) solid agarslantculture-medium to culture for three days at 27 DEG C and activate for three times, inoculating in a PDA liquid seed culture medium again to culture in a shake flask for three days at 27 DEG C and150rpm to obtain a seed liquid, transplanting the seed liquid in a 13L PDA fermentation medium according to a proportion of 5%(v/v) at 28+/-1 DEG C and a stirring speed of 200-300rpm, controlling the ventilation amount within 1-2L/min, culturing for five days and stopping fermenting; and filtering through four layers of sterile gauze to remove mycelium, and collecting fermentation liquid to obtain the PUFA. According to the method for producing the PUFA, disclosed by the invention, the content of the obtained unsaturated fatty acid is high, the culture medium is easy to obtain, the fermentation conditions are stable, and the process is simple.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of Aspergillus niger strain and utilize potato to produce the method for polyunsaturated fatty acid.
Background technology
Aspergillus niger (
aspergillus niger)belong to a Common Species in fungi, be distributed widely in grain, plant product and soil all over the world, because its institute's enzyme that produces enriches, so be one of modal bacterial classification in industrial application.In fungi, containing multiple polyunsaturated fatty acid, is the important sources of the lipid acid such as arachidonic acid (ARA), gamma-linolenic acid (GLA), linolic acid (LA), timnodonic acid (EPA), docosahexenoic acid (DHA).Polyunsaturated fatty acid (PUFAs) can regulate the lipid metabolism of human body, in antianaphylaxis, anti-inflammatory, reduces the sickness rate of cardiovascular disorder, auxiliary improvement of memory, assisting child intelligent growth, and the aspect such as delay senility has important physiological action.Current commercial polyunsaturated fatty acid especially omega-3 polyunsaturated fatty acids mainly extracts from fish oil, in higher plant and animal, seldom contain omega-3 polyunsaturated fatty acids, its complex manufacturing, cost are high, poor stability, are also subject to the restriction of weather condition and resource.And content is the abundantest in microorganism, mainly exist with the form of grease and film fat, and microorganism has strong adaptability, growth and breeding is rapid, growth cycle is short, easy cultivation, be not subject to the features such as places of origin of raw materials restriction.Therefore, utilizing microorganisms producing polyunsaturated fatty acid is an important channel, seeks superior strain and research zymotechnique, and the new selection of carbon source and the application of low-cost resource are just becoming research direction.
Long-term cultivated area 1,800 ten thousand t of world potato, the average fresh potato l5t/hmz of per unit area yield.Within 1999, world's potato yield has reached 28,978 ten thousand t.Potato processing is playing an important role aspect the benign development of the whole Potato Industry chain of drive.Entered climax as the potato processing industry development that realizes one of value-added main path of grain, potato, substantially for eating or be processed into bean vermicelli, vermicelli and starch raw, belongs to the elementary process segment at present, and its increment amplitude is limited.
Summary of the invention
The unsaturated fatty acid content of a kind of acquisition that the object of the invention is to overcome the problems referred to above and provide is high, and culture medium easily obtains, the method that fermentation condition is stable, technique is simply produced polyunsaturated fatty acid.
A method of producing polyunsaturated fatty acid, comprising:
(1) actication of culture
By aspergillus niger (
aspergillus nigerxj) bacterial strain is connected on PDA solid slant culture base, and 27 DEG C leave standstill cultivation 3 days, activate 3 times, are stored in 4 DEG C of refrigerators for subsequent use;
(2) preparation of spore suspension
By the bacterial strain having activated, with containing spore under the aseptic washing of 0.05% tween 80, proceed in the 100 mL sterilized waters with granulated glass sphere, after stirring, on vortex oscillator, shake again, fully break up spore, remove by filter mycelia with 4 layers of aseptic lens paper, make homogeneous spore suspension;
(3) preparation of seed liquor
By spore suspension, be inoculated in 100mLPDA liquid seed culture medium with volume ratio 5% inoculum size, 27 DEG C, 150rpm, shake-flask culture 3 days, obtains seed liquor;
(4) fermentation culture
Get seed liquor and 5% be inoculated in 13L PDA fermention medium by volume, 28 ± 1 ° of C, stirring velocity 200-300rpm, air flow is controlled at 1-2L/min, cultivates and stops fermentation in 5 days, and 4 layers of sterile gauze remove by filter mycelium, collect fermented liquid, to obtain final product.
The method of above-mentioned production polyunsaturated fatty acid, wherein the preparation method of PDA solid slant culture base is as follows: potato 200g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 20g, agar 20g, water is supplied 1000mL, pH nature.Sterilising conditions: 121 DEG C, 30min.
The method of above-mentioned production polyunsaturated fatty acid, wherein the preparation method of PDA liquid seed culture medium is as follows: potato 200g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 20g, water is supplied 1000mL, pH nature.Sterilising conditions: 121 DEG C, 30min.
The method of above-mentioned production polyunsaturated fatty acid, wherein the preparation method of PDA fermention medium is as follows: potato 2600g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 260g, water is supplied 13000mL, pH nature, 121 DEG C, sterilizing 30min.
This bacterial classification is deposited in Chinese Typical Representative culture collection center (address: Wuhan, China Wuhan University) on March 7th, 2005, preserving number: CCTCC NO:M 206021, and name is called: aspergillus niger (
aspergillus nigerxj).
The present invention compared with prior art, there is obvious beneficial effect, as can be known from the above technical solutions: utilize and produce polyunsaturated fatty acid aspergillus niger xj to carrying out biological degradation and conversion taking potato as main substratum, fermentative production polyunsaturated fatty acid, the restraining factors that can be solution polyunsaturated fatty acid source and the development of potato secondary industry provide a new way.And aspergillus niger (
aspergillus nigerxj) bacterium source is reliable, to the wide accommodation of the natural environmental condition such as temperature, pH, easily cultivates and preserves, and produces the conditional stability of unsaturated fatty acids, can be used as the bacterial strain that extracts unsaturated fatty acids, expanding resource storehouse.The potato of using in culture system, aboundresources, from the horse's mouth, safety.This technological line is reasonable, can large-scale industrial production.
Embodiment
A method of producing polyunsaturated fatty acid, comprises the following steps:
1) actication of culture
By aspergillus niger (
aspergillus nigerxj) bacterial strain is connected to PDA solid slant culture base (potato 200g stripping and slicing, uses water boil 30min, crosses leaching juice, adds glucose 20g, agar 20g, water is supplied 1000mL, pH nature, 121 DEG C, sterilizing 30min) upper, 27 DEG C leave standstill cultivation 3 days, activate 3 times, are stored in 4 DEG C of refrigerators for subsequent use;
2) preparation of spore suspension
By the bacterial strain having activated, with containing spore under the aseptic washing of 0.05% tween 80 (Toween-80), proceed in the 250 mL triangular flasks that 100 mL sterilized waters are housed with granulated glass sphere, after stirring with glass rod, on vortex oscillator, shake again, fully break up spore, remove by filter mycelia with 4 layers of aseptic lens paper, make homogeneous spore suspension;
3) preparation of seed liquor
By spore suspension, with 5%(v/v) inoculum size is inoculated in and 100mLPDA liquid seed culture medium is housed (potato 200g stripping and slicing, uses water boil 30min, crosses leaching juice, add glucose 20g, water is supplied 1000mL, pH nature, 121 DEG C, sterilizing 30min) 500mL triangular flask in, 27 DEG C, 150rpm, shake-flask culture 3 days
4) fermentation culture
Get seed liquor 5%(v/v) be inoculated in and 13L PDA fermention medium be housed (potato 2600g stripping and slicing, uses water boil 30min, crosses leaching juice, add glucose 260g, water is supplied 13000mL, pH nature, 121 DEG C, sterilizing 30min) 19L fermentor tank in, 28 ± 1 ° of C, stirring velocity 200-300rpm, air flow is controlled at 1-2L/min, cultivates and stops fermentation in 5 days, and 4 layers of sterile gauze remove by filter mycelium, collect fermented liquid, to obtain final product.
Aspergillus niger
(Aspergillus nigerxj) qualification of polyunsaturated fatty acid in fermented liquid:
(1) preparation of medicinal extract
Get above-mentioned gained fermented liquid, with petroleum ether extraction, decompression and solvent recovery, obtains test sample sherwood oil part and water section, and sherwood oil part concentrated by rotary evaporation is obtained to medicinal extract.
2) separation of sample
Getting petroleum ether extract and carry out silica gel column chromatography, with V (sherwood oil) ﹕ V (ethyl acetate)=1:0-8:1 carries out wash-out, obtains oily mater, through TLC(thin layer chromatography) analysis, is fatty acid composition.
3) esterification of sample
Get 54mg oily mater in Erlenmeyer flask, add 20ml benzene-sherwood oil (1:1, v/v) to dissolve, then add 0.2mol/l KOH-MeOH solution 10ml, vibration evenly, constant temperature 30min in 40 DEG C of water-baths, cooling, add 20ml distilled water, shake up, leave standstill, after layering is clear, get supernatant liquor decompression and solvent recovery, obtain esterification product.
(4) GC-MS analysis condition
GC conditions: HP-5MS (0.25mm × 30m, 0.25 μ is m); 50 DEG C of column temperatures (retaining 2min), are warming up to 280 DEG C (5 DEG C/min), keep 3min; Temperature of vaporization chamber is 250 DEG C; Taking high-pure helium as carrier gas; Before post, press 7.62psi, carrier gas flux 1.0mL/min; Sample size 1 μ L; Splitting ratio 50:1.
Mass spectrum condition: ion source is EI source; 280 DEG C of interface temperature; 230 DEG C of ion source temperatures; Multiplier voltage 1785V; 150 DEG C of quadrupole temperature; Electron energy 70eV; Transmitter current 34.6 μ A; Mass range 10～550amu; Solvent delay 3min.
(5) conclusion: the fatty acid component in fermented liquid is mainly linolic acid, 10-octadecenoic acid, 14-methyl pentadecylic acid and palmitinic acid, wherein vaccenic acid acid content is the highest, and content is 48.112%, is secondly linolic acid, content is 41.919%, and unsaturated fatty acids accounts for 90.031%.
Claims (4)
1. a method of producing polyunsaturated fatty acid, comprising:
(1) actication of culture
Be the aspergillus niger of CCTCC NO:M 206021 by preserving number,
aspergillus nigerxj bacterial strain is connected on PDA solid slant culture base, and 27 DEG C leave standstill cultivation 3 days, activate 3 times, are stored in 4 DEG C of refrigerators for subsequent use;
(2) preparation of spore suspension
By the bacterial strain having activated, with containing spore under the aseptic washing of 0.05% tween 80, proceed in the 100 mL sterilized waters with granulated glass sphere, after stirring, on vortex oscillator, shake again, fully break up spore, remove by filter mycelia with 4 layers of aseptic lens paper, make homogeneous spore suspension;
(3) preparation of seed liquor
By spore suspension, be inoculated in 100mLPDA liquid seed culture medium with volume ratio 5% inoculum size, 27 DEG C, 150rpm, shake-flask culture 3 days, obtains seed liquor;
(4) fermentation culture
Get seed liquor and 5% be inoculated in 13L PDA fermention medium by volume, 28 ± 1 ° of C, stirring velocity 200-300rpm, air flow is controlled at 1-2L/min, cultivates and stops fermentation in 5 days, and 4 layers of sterile gauze remove by filter mycelium, collect fermented liquid, to obtain final product.
2. the method for production polyunsaturated fatty acid as claimed in claim 1, wherein the preparation method of PDA solid slant culture base is as follows: potato 200g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 20g, agar 20g, water is supplied 1000mL, pH nature; Sterilising conditions: 121 DEG C, 30min.
3. the method for production polyunsaturated fatty acid as claimed in claim 1 or 2, wherein the preparation method of PDA liquid seed culture medium is as follows: potato 200g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 20g, water is supplied 1000mL, pH nature; Sterilising conditions: 121 DEG C, 30min.
4. the method for production polyunsaturated fatty acid as claimed in claim 3, wherein the preparation method of PDA fermention medium is as follows: potato 2600g stripping and slicing, use water boil 30min, cross leaching juice, add glucose 260g, water is supplied 13000mL, pH nature, 121 DEG C, sterilizing 30min.
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| CN103865805B (en) * | 2014-01-28 | 2016-05-04 | 李祝 | Fermented black aspergillus suppresses the purposes of tobacco ralstonia solanacearum |
| CN106906258A (en) * | 2017-03-01 | 2017-06-30 | 贵州大学 | A kind of method for producing 5 hydroxymethylfurfurals |
| CN107022585A (en) * | 2017-06-12 | 2017-08-08 | 贵州大学 | A kind of method that Aspergillus niger strain produces furfural |
| CN110004193B (en) * | 2018-12-04 | 2022-09-20 | 贵州大学 | Method for separating and extracting 5, 5' -oxo-di (methylene) difuran-2-formaldehyde by using aspergillus niger |
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| CN1847388A (en) * | 2006-04-21 | 2006-10-18 | 贵州大学 | Aspergillus niger strain with Agrobacterium tumefaciens inhibiting effect and its use |
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| CN1847388A (en) * | 2006-04-21 | 2006-10-18 | 贵州大学 | Aspergillus niger strain with Agrobacterium tumefaciens inhibiting effect and its use |
Non-Patent Citations (2)
| Title |
|---|
| 李元森等.真菌发酵生产保健油脂的研究进展.《食品研究与开发》.2009, |
| 真菌发酵生产保健油脂的研究进展;李元森等;《食品研究与开发》;20090131;全文 * |
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