CN103540623A - Method for producing gamma linolenic acid (GLA) through fermenting high-density cultured cunninghamellaceae - Google Patents
Method for producing gamma linolenic acid (GLA) through fermenting high-density cultured cunninghamellaceae Download PDFInfo
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- CN103540623A CN103540623A CN201310531470.6A CN201310531470A CN103540623A CN 103540623 A CN103540623 A CN 103540623A CN 201310531470 A CN201310531470 A CN 201310531470A CN 103540623 A CN103540623 A CN 103540623A
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Abstract
The invention discloses a method for producing gamma linolenic acid (GLA) through fermenting high-density cultured cunninghamellaceae. The method comprises the following steps: according to a volume ratio of preserved glycerin strain to shake flask activation culture medium being 1:25, inoculating a cunninghamellaceae strain to a first-level shake flask which contains an activation culture medium and carrying out activation cultivation; according to an inoculum size of 5-10vol%, transferring the cunninghamellaceae strain to a second-level shake flask for enlarging cultivation; according to the inoculum size of 5-10vol%, inoculating the cunninghamellaceae strain subjected to cultivation enlargement into a first-level seeding tank for fermentation; and according to the inoculum size of 5-10vol%e, inoculating the cunninghamellaceae strain subjected to cultivation enlargement in the seeding tank into a fermenting tank for fermentation cultivation, and after finishing the fermentation, measuring that the dry weight of hyphae in a fermentation liquor reaches 60-80g/L, the oil content reaches 20-25g/L and the GLA content reaches 4.8-6.1g/L. The method can be used for lowering the production cost of GLA and is suitable for the industrial production of GLA.
Description
Technical field
The present invention relates to the production method of a kind of GLA, the method for little gram of Mildy Way fermentative production GLA of especially a kind of high-density culture, is to adopt the method for microorganism fermentation to produce GLA, can be applicable to the large-scale industrial production of GLA.
Background technology
Gamma-linolenic acid (GLA) is polyunsaturated fatty acid a kind of who belongs to n-6 series, the relative molecular weight of GLA is 278.4, under room temperature, be liquid, water insoluble and be soluble in the non-polar solvents such as ethanol, ether, trichloromethane, sherwood oil, the purity of GLA reaches 35% and is very easily oxidized when above and is difficult for preserving, and can only exist with siccative oil state.
GLA has a lot of important physiological functions, GLA is one of essential fatty acid, it is one of important component forming each tissue biological's film of human body, it passes through complicated metabolic process in vivo, produce multiple secondary metabolite, main formation dihomo-gamma-linolenic acid (DHGLA) or arachidonic acid (AA), and then be converted into prostaglandin(PG) (PGS), leukotriene (LT) and thromboxane (TXB).GLA has anti-microbial effect because suppressing the growth of Gram-negative bacteria, positive bacteria; GLA can breed by inhibition tumor cell; GLA is to rheumatoid arthritis, enteritis, and the inflammation such as ephritis have obvious curative effect and improvement effect; Thereby GLA and meta-bolites thereof can reduce the effect that cholesterol in blood fat, triglyceride level and low-density lipoprotein have treatment hypertension, reducing blood-fat; GLA also has certain promoter action to fat-reducing.Exactly because GLA has so numerous physiological action, its potential pharmaceutical use receives publicity.At present GLA aspect medical, functional foodstuff aspect, makeup aspect, feed aspect be with a wide range of applications.
Along with the exploitation of GLA using value, the demand of GLA also increases day by day.GLA finds in the seed of root of Redsepal Eveningprimrose, after this at other plant, also finds its existence in as plants such as borage, black currant, Microula sikkimensis.At present, evening primrose seed is still the main source of GLA.The plant of being rich in gamma-linolenic acid mostly is wild, due to the impact of the conditions such as climate, the place of production, exists oil offtake unstable, and the problem such as the production cycle is long, and oneself can not meet the demand in market therefore from plant, to extract GLA.Because microorganism has, fecundity is strong, growth is fast, mycelium is easily collected and extracts, not affected by the conditions such as the place of production, raw material, weather, in composition and human milk, lubricant component approaches, can increase substantially the output of gamma-linolenic acid and the advantage such as reduce production costs by animal nutrition, therefore, people begin one's study and utilize Microbial resources to produce GLA.
Summary of the invention
The object of the present invention is to provide the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture, adopt microbe fermentation method, by little gram of Mildy Way production GLA of high-density culture, replace the GLA in other sources to meet the need of market.
In order to reach above-mentioned purpose, solution of the present invention is:
A method of little gram of Mildy Way fermentative production GLA of high-density culture, its step is as follows:
The first step, according to preservation glycerine bacterial classification: shaking flask activation medium is that 1: 25 volume ratio is inoculated in little gram of Mildy Way bacterial classification in the one-level shaking flask that activation medium is housed, in 20-25 ℃ of shaking table with 120-170rpm rotating speed activation culture 24-48h;
Second step, is transferred to little gram of Mildy Way activated spawn in secondary shaking flask according to the volume percent inoculum size of 5-10%, in 20-25 ℃ of shaking table with 120-170rpm rotating speed enlarged culturing 24-48h;
The 3rd step, is inoculated in little gram of Mildy Way bacterial classification of enlarged culturing in seeding tank according to the volume percent inoculum size of 5-10%, adopts the ammoniacal liquor of 28-30% to control between pH5.5-6.0, culture temperature 20-25 ℃, air flow 8-10m in whole fermenting process
3/ h, dissolved oxygen is controlled between 30-50%, cultivates 24-48h, until biomass reaches 20-25g/L;
The 4th step, according to the volume percent inoculum size of 5-10%, little gram of Mildy Way bacterial classification of enlarged culturing in seeding tank is inoculated in to fermentation cylinder for fermentation again and cultivates 144-168h, in whole fermenting process, adopt the ammoniacal liquor of 28-30% to be controlled between pH5.5-6.0, culture temperature 20-25 ℃, air flow 20-25m
3/ h, dissolved oxygen is controlled between 30-50%, adds glucose control glucose concn between 5-10g/L by stream, after finishing fermentation, records mycelia dry weight in fermented liquid and reaches 60-80g/L, and fat content reaches 20-25g/L, and GLA content reaches 4.8-6.1g/L.
Above-mentioned shaking flask activation culture based formulas is: glucose 50-100g/L, KH
2pO
42-5 g/L, MgSO
47H
2o 0.5-3g/L.
The culture medium prescription of above-mentioned shaking flask enlarged culturing is: glucose 50-100g/L, KH
2pO
42-5 g/L, MgSO
47H
2o 0.5-3g/L.
The culture medium prescription of above-mentioned seed fermentation tank is: glucose 30-100g/L, KH
2pO
43-8g/L, MgSO
47H
2o 1-4g/L, yeast extract paste 10-20g/L, NaNO
31-5g/L.
The culture medium prescription of above-mentioned large fermentor tank is: glucose 30-100g/L, KH
2pO
43-8g/L, MgSO
47H
2o 1-4g/L, yeast extract paste 10-20g/L; NaNO
31-5g/L.
Above-mentioned little gram of Mildy Way fermentation culture temperature is 20-25 ℃.
Above-mentioned little gram of Mildy Way fermentation culture pH is 5.5-6.0.
Adopt after such scheme, the present invention utilizes little gram of Mildy Way of high-density culture method fermentation to produce GLA resource as an alternative, and the advantage that GLA is extracted in replacement from plant, marine alga, marine fishes is as follows:
One, can whole year production, be not subject to the impact of the factors such as place, climatope, there is no the dependency of season or weather;
Two, adopt microorganism culturing fermentative production GLA, there is growth cycle short, stable yield, production process can manual control etc. has advantage;
Three, adopt microorganism culturing fermentative production GLA, its mycelium is easy to collect, and GLA is easy to extract, and production cost is low, is applicable to suitability for industrialized production;
Four, adopt microorganism culturing fermentative production GLA, can significantly improve by the optimization of animal nutrition and fermentation condition output and the quality of product, volume increase space is large, and quality product controllability is good;
Five, adopt culture of microorganism to produce GLA, can reduce because of the market requirement and plant in a large number occupying cultivated land that root of Redsepal Eveningprimrose brings, save soil, and alleviate marine fishes are catched and killed to the resource exhaustion bringing in a large number.
Embodiment
Embodiment
The first step, presses glucose: 50g/L, KH
2pO
4: 2 g/L, MgSO
47H
2o 0.5g/L configures activation medium, and 121 ℃, 25min sterilizing is standby;
Get the little gram of Mildy Way glycerine pipe that 2mL is preserved in Ultralow Temperature Freezer and be inoculated in the 250mL one-level shaking flask that 50mL activation medium is housed, in 22 ℃ of shaking tables with 170rpm rotating speed activation culture 48h;
Second step, is inoculated into little gram of Mildy Way bacterial classification of activation culture in the secondary shaking flask that the sterilized activation medium of 200mL is housed according to 10% volume percent inoculum size, in 22 ℃ of shaking tables with 170rpm rotating speed enlarged culturing 48h;
The 3rd step is pressed glucose: 100g/L, KH in 1000L seeding tank
2pO
4: 8g/L, MgSO
47H
2o:4g/L, yeast extract paste: 20g/L; NaNO
3: 5g/L configuration 500L seed tank culture base, configured latter 121 ℃, sterilizing 30min is standby;
Little gram of Mildy Way bacterial classification of secondary shaking flask enlarged culturing is inoculated in the 1000L seeding tank that contains 500L substratum according to 5% volume percent inoculum size, in whole fermenting process, adopt 28% ammoniacal liquor to control between pH5.5-6.0, control 22 ± 1 ℃ of culture temperature, air flow 10m
3/ h, dissolved oxygen is controlled between 30-50%, cultivates 48h artifact amount and reaches 24.2g/L;
The 4th step is pressed glucose: 100g/L, KH in 8000L fermentor tank
2pO
4: 8g/L, MgSO
47H
2o:4g/L, yeast extract paste: 20g/L; NaNO
3: 5g/L configuration 5000L fermention medium, configured latter 121 ℃, sterilizing 30min is standby;
Little gram of Mildy Way bacterial classification in 1000L seeding tank is inoculated in the 8000L fermentor tank that contains 5000L fermention medium according to 10% volume percent inoculum size, in whole fermenting process, adopt 28% ammoniacal liquor to control between pH5.5-6.0,22 ± 1 ℃ of culture temperature, air flow 20m
3/ h, dissolved oxygen is controlled between 30-50%, adds glucose control glucose concn between 5-10g/L by stream, cultivate 144h after finishing fermentation, record mycelium dry weight in fermented liquid and reach 70.5g/L, fat content reaches 22.23g/L, and GLA content reaches 5.81g/L.
Claims (7)
1. a method of little gram of Mildy Way fermentative production GLA of high-density culture, is characterized in that step is as follows:
The first step, according to preservation glycerine bacterial classification: shaking flask activation medium is that 1: 25 volume ratio is inoculated in little gram of Mildy Way bacterial classification in the one-level shaking flask that activation medium is housed, in 20-25 ℃ of shaking table with 120-170rpm rotating speed activation culture 24-48h;
Second step, is transferred to little gram of Mildy Way activated spawn in secondary shaking flask according to the volume percent inoculum size of 5-10%, in 20-25 ℃ of shaking table with 120-170rpm rotating speed enlarged culturing 24-48h;
The 3rd step, is inoculated in little gram of Mildy Way bacterial classification of enlarged culturing in seeding tank according to the volume percent inoculum size of 5-10%, adopts the ammoniacal liquor of 28-30% to control between pH5.5-6.0, culture temperature 20-25 ℃, air flow 8-10m in whole fermenting process
3/ h, dissolved oxygen is controlled between 30-50%, cultivates 24-48h, until biomass reaches 20-25g/L;
The 4th step, according to the volume percent inoculum size of 5-10%, little gram of Mildy Way bacterial classification of enlarged culturing in seeding tank is inoculated in to fermentation cylinder for fermentation again and cultivates 144-168h, in whole fermenting process, adopt the ammoniacal liquor of 28-30% to be controlled between pH5.5-6.0, culture temperature 20-25 ℃, air flow 20-25m
3/ h, dissolved oxygen is controlled between 30-50%, adds glucose control glucose concn between 5-10g/L by stream, after finishing fermentation, records mycelia dry weight in fermented liquid and reaches 60-80g/L, and fat content reaches 20-25g/L, and GLA content reaches 4.8-6.1g/L.
2. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: above-mentioned shaking flask activation culture based formulas is: glucose 50-100g/L, KH
2pO
42-5 g/L, MgSO
47H
2o 0.5-3g/L.
3. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: the culture medium prescription of above-mentioned shaking flask enlarged culturing is: glucose 50-100g/L, KH
2pO
42-5 g/L, MgSO
47H
2o 0.5-3g/L.
4. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: the culture medium prescription of above-mentioned seed fermentation tank is: glucose 30-100g/L, KH
2pO
43-8g/L, MgSO
47H
2o 1-4g/L, yeast extract paste 10-20g/L, NaNO
31-5g/L.
5. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: the culture medium prescription of above-mentioned large fermentor tank is: glucose 30-100g/L, KH
2pO
43-8g/L, MgSO
47H
2o 1-4g/L, yeast extract paste 10-20g/L; NaNO
31-5g/L.
6. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: above-mentioned little gram of Mildy Way fermentation culture temperature is 20-25 ℃.
7. the method for little gram of Mildy Way fermentative production GLA of a kind of high-density culture as claimed in claim 1, is characterized in that: above-mentioned little gram of Mildy Way fermentation culture pH is 5.5-6.0.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1236628A (en) * | 1998-05-22 | 1999-12-01 | 尤俊 | Medicinal preparation containing high content of alpha-linolenic acid and its preparing process |
| CN1827770A (en) * | 2005-03-01 | 2006-09-06 | 孙善林 | Process for producing gamma-linolenic acid by microbe microorganism fermentation |
-
2013
- 2013-11-01 CN CN201310531470.6A patent/CN103540623B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1236628A (en) * | 1998-05-22 | 1999-12-01 | 尤俊 | Medicinal preparation containing high content of alpha-linolenic acid and its preparing process |
| CN1827770A (en) * | 2005-03-01 | 2006-09-06 | 孙善林 | Process for producing gamma-linolenic acid by microbe microorganism fermentation |
Non-Patent Citations (1)
| Title |
|---|
| 周立新等: "α-亚麻酸与γ-亚麻酸", 《西部粮油科技 》, vol. 25, no. 6, 30 December 2000 (2000-12-30), pages 46 - 48 * |
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