CN1827770A - Process for producing gamma-linolenic acid by microbe microorganism fermentation - Google Patents
Process for producing gamma-linolenic acid by microbe microorganism fermentation Download PDFInfo
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- CN1827770A CN1827770A CN 200510045965 CN200510045965A CN1827770A CN 1827770 A CN1827770 A CN 1827770A CN 200510045965 CN200510045965 CN 200510045965 CN 200510045965 A CN200510045965 A CN 200510045965A CN 1827770 A CN1827770 A CN 1827770A
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Abstract
The method comprises the following steps: expansive culturing the bacterial; then fermenting, adding sugar liquid, getting the thalline containing r-linolenic acid, dewatering and drying. The sugar content in liquid is 30-40%, the time is 40-50 hours and the fermentation time is 120-180 hours. Use the cunninghamellaechinulata to mutagenesis strain and carry out continuous flow invoice method, improving the productivity, increasing the thalline productivity by 26.36%, fats by 7.63% and r-linolenic acid 22.24%, decreasing the bacterial pollution, shortening the fermentation cycle and reducing the production cost.
Description
Technical field:
The invention belongs to microbial technology field, be specifically related to the linolenic technology of a kind of microbial fermentation r-.
Background technology:
The r-linolenic acid is essential fatty acid (vitamin F), is again the medicine of treatment hyperlipidemia, arteriosclerosis, the heart, cerebro-vascular diseases and diabetes, is mostly to extract from plant (month bamboo water pipe grass seed).Because chemical substance is residual in the restriction of raw material sources and the extraction, microbial fermentation r-linolenic acid is the focus of countries in the world research in recent years.
Microbial fermentation r-linolenic acid product, r-linolenic acid yield height has no side effect, and is rich in abundant nutrition materials such as multiple amino acids, VITAMIN, also contain a large amount of SOD antibiotic senescence-factors such as (superoxide-dismutases), therefore be widely used in medicine, nourishing function foods and cosmetics.
Microbial fermentation be with sugar as the linolenic matrix of the main r-of conversion, and the height that sugar is measured has directly influenced the linolenic transformation efficiency of r-.
The sugar amount of disposable input high density in the microbial fermentation, the high osmotic pressure of generation has influenced microbial growth, has also influenced r-linolenic acid high conversion simultaneously.
It is long that microbiological industry production at present exists the microorganism adaptive phase, and microorganism growth is slow, and biomass is low, and r-linolenic acid yield is low, the long problem of fermentation period, defectives such as production cost height.
Technical problem to be solved by this invention is to solve the above problems and weak point, designs a kind of fermentation process of being convenient to industrialization.
The invention provides a kind of microorganism and add the linolenic method of fermentation r-in Continuous Flow, this method is, bacterial classification is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), the common micro-organisms center preservation bacterial classification AS3.154 of China Committee for Culture Collection of Microorganisms is an original strain, preserving number is CGMCCNO.0232, comprises the following steps:
(1) bacterial classification with screening carries out the seed enlarged culturing
(2) ferment at fermention medium with the bacterium of enlarged culturing, it is characterized in that: after the fermentation, Continuous Flow is fully fermented with liquid glucose, collect after the fermentation of adding of Continuous Flow obtain containing the linolenic thalline of r-and dewater, drying.The liquid glucose that Continuous Flow adds is preferably the liquid glucose that contains 30-40% sugar, and Continuous Flow adds 40-50 hour or divided minor tick to add liquid glucose in 40-50 hour, and after continuously fermenting 120-180 hour, collects thalline.Like this, microorganism is under suitable sugared concentration, and along with the utilization and the consumption of sugar, constantly Continuous Flow adds certain sugar amount material and makes microorganism obtain higher biomass and the linolenic output of r-at the suitable condition bottom fermentation of the best.The present invention further optimization embodiment is:
1, seed enlarged culturing
Dress 60-70% seed culture medium in the 50-100L seeding tank, bacterial classification (5-10%) is inserted in the sterilization back, at 25-30 ℃, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-250rpm, tank pressure 0.03-0.06MP fermented 40-60 hour, was first order seed.The 60-70% seed culture medium of packing in the 500L jar again after the sterilization, inserts first order seed (5-10%), at air flow 1: 0.2-1: 1 (V, V, M), and mixing speed 100-250rp; Tank pressure 0.03-0.06MP, fermented 40-60 hour, for secondary enlarges seed by temperature 25-30 ℃.
The 60-70% seed culture medium of packing in the 5000L jar after the sterilization, inserts secondary seed, and inoculum size is 5-10%, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-250rpm, tank pressure 0.03-0.06MP; Temperature 25-30 ℃, fermented 40-60 hour.
Seed culture medium: 2-4% sucrose, 0.1-0.5% yeast extract paste, 0.1-0.5% peptone, 0.2-0.5% urea, 0.1-0.5% potassium primary phosphate, 0.05-0.1% lime carbonate, 0.1-0.2% sal epsom, 0.00002-0.00004VB
1, 0.0002-0.0005% zinc sulfate, PH5.5-6.0.
2, the linolenic fermenting process of adding of Continuous Flow r-:
R-linolenic acid Continuous Flow adds fermentation, and the 50-60% basis fermention medium of packing in the 30000-50000L fermentor tank after the sterilization, is inoculated three grades of seed 5-10%, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-200rpm, tank pressure 0.03-0.06MP; Temperature 25-30 ℃.After above-mentioned fermentation 40-50 hour, add a certain amount of liquid glucose continuously, stream adds 40-50 hour, ferments 120-190 hour, ferments after the termination, collects thalline, and dehydration, oven dry, pulverizing get dry thalline, are formulation products.
Continuous Flow adds in the fermentation, fermention medium: 2-4% sucrose, 0.2-0.4% urea, 0.2-0.6% yeast extract paste, 0.2-0.6% peptone, 0.2-0.5% potassium primary phosphate, 0.1-0.15% lime carbonate, 0.02-0.05% sal epsom, 0.00001-0.00003%VB
1, 0.0001-0.0005% copper sulfate, 0.0002-0.0005% zinc sulfate, 0.0001-0.0005% manganous sulfate, 0.0001-0.0005% ferrous sulfate, PH5.5-6.0.
Thalline provided by the present invention is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCCNO.0232, be with Institute of Microorganism, Academia Sinica, it is original strain that bacterium protects storehouse bacterial classification AS3.154, preferably the new dissociant that obtains through mutagenesis screening is numbered M10.
The present invention compared with prior art has following characteristics:
1, the present technology linolenic bacterial classification that is mainly belonged to by the spore enzyme of r-that is used to ferment, what the present invention adopted is little Ke Yinhan mould and mutagenesis bacterial classification thereof.This bacterium is introduced by Beijing Institute of Micro-biology bacterial classification center, through the mutagenesis gained.This strain classification name Cunninghamella echinulata As 3.2006 under fermentation condition of the present invention, can obtain higher dry mycelium, reaches as high as 4.25%, and total fatty acids can reach 41.85%, and r-linolenic acid yield accounts for more than 21.98% of total fat.
2, fermentation technique of the present invention saved that bacterial classification adapts to and the adjustment period, therefore shortened fermentation period, reduced the energy and manpower consumption, lowered production cost.
3, fermentation technique of the present invention has been avoided forming at a large amount of organic acids of fermentation, prevents that acidity is low excessively, influence fermentation and the linolenic formation of r-.
4, technology of the present invention, the fermentation concentration utmost point adapt to the growth of zymophyte seed, stronger inhibition living contaminants, reduce the tank switching rate, reduced the cost loss.
The technology of the present invention and existing fermentation technique have improved biomass and the linolenic yield of r-, and fermentation period is short, reduce energy consumption, reduce cost, and have improved productivity.
Embodiment:
Embodiment one,
Bacterial classification is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), and the common micro-organisms center preservation bacterial classification AS3.154 of China Committee for Culture Collection of Microorganisms is an original strain, and preserving number is CGMCCNO.0232.In the 500L fermentor tank, drop into fermention medium 50%, after the sterilization, insert one-level and enlarge seed liquor 15L, air flow 1: 0.8 (V, V, M), stirring velocity 240rpm, 29 ℃ of leavening temperatures, ferment after 46 hours, with the liquid glucose of the 40% concentration flow acceleration with 1L/ hour, stream adds 45 hours, the termination in 190 hours of fermenting, is collected thalline, dehydration, oven dry, pulverizing, detection yield at discharging.Dry mycelium yield 3.93%, fatty yield 39.92%, r-linolenic acid 19.01%.
Seed culture medium: 2.5% sucrose, 0.1% yeast extract paste, 0.1% peptone, 0.5% urea, 0.4% potassium primary phosphate, 0.06% lime carbonate, 0.2% sal epsom, 0.00004%VB
1, 0.0005% zinc sulfate, PH5.5.
Fermention medium: 3% sucrose, 0.3% urea, 0.6% yeast extract paste, 0.3% peptone, 0.4% potassium primary phosphate, 0.11% lime carbonate, 0.04% sal epsom, 0.00001%VB
1, 0.0004% copper sulfate, 0.0002% zinc sulfate, 0.0005% manganous sulfate, 0.0005% ferrous sulfate, PH5.7.
Embodiment two,
Bacterial classification is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), the common micro-organisms center preservation bacterial classification AS3.154 of China Committee for Culture Collection of Microorganisms is an original strain, preserving number is CGMCCNO.0232, in the 1000L fermentor tank, drop into 50% fermention medium, secondary seed solution 50L is inserted in the sterilization back, ventilation is 1: 0.8 (V, V, M), 28 ℃ of leavening temperatures, stirring velocity is 210rpm, fermented 48 hours, stream adds 40 hours with liquid glucose (40%) with 2L/ hour flow acceleration stream, stopped fermentation, discharging in 168 hours, collect thalline, dehydration, oven dry, pulverize, detect yield.Obtain dry mycelium 4.23%, fat 40.52%, r-linolenic acid 20.90%.
Seed culture medium: 2% sucrose, 0.3% yeast extract paste, 0.3% peptone, 0.25% urea, 0.5% potassium primary phosphate, 0.07% lime carbonate, 0.1% sal epsom, 0.000025%VB
1, 0.0004% zinc sulfate, PH5.8.
Fermention medium: 2.5% sucrose, 0.4% urea, 0.3% yeast extract paste, 0.5% peptone, 0.2% potassium primary phosphate, 0.13% lime carbonate, 0.02% sal epsom, 0.00003%VB
1, 0.0005% copper sulfate, 0.0005% zinc sulfate, 0.0003% manganous sulfate, 0.0003% ferrous sulfate, PH5.8.
Embodiment three,
Bacterial classification is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), the common micro-organisms center preservation bacterial classification AS3.154 of China Committee for Culture Collection of Microorganisms is an original strain, preserving number is CGMCCNO.0232, in the 10000L fermentor tank, drop into fermention medium 50%, the sterilization back is inserted three grades and is enlarged strain liquid 250L, ventilation is 1: 0.7, stirring velocity is 180rpm, 25 ℃ of leavening temperatures, fermented 46 hours, 40% liquid glucose that stream adds sterilization adds 46 hours with 20L/ hour flow acceleration stream, fermentation in 150 hours stops discharging, collect thalline, dehydration, oven dry, pulverize, detect yield, dry mycelium yield 4.21%, fat yield 41.85%, r-linolenic acid 21.00%.
Seed culture medium: 4% sucrose, 0.2% yeast extract paste, 0.2% peptone, 0.2% urea, 0.2% potassium primary phosphate, 0.1% lime carbonate, 0.15% sal epsom, 0.00003%VB
1, 0.0002% zinc sulfate, PH6.0.
Fermention medium: 4% sucrose, 0.25% urea, 0.5% yeast extract paste, 0.6% peptone, 0.5% potassium primary phosphate, 0.14% lime carbonate, 0.05% sal epsom, 0.00002%VB
1, 0.0001% copper sulfate, 0.0004% zinc sulfate, 0.0002% manganous sulfate, 0.0002% ferrous sulfate, PH5.9.
Embodiment four,
Thalline is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCCNO.0232, be with Institute of Microorganism, Academia Sinica, it is original strain that bacterium protects storehouse bacterial classification AS3.154, preferably the new dissociant that obtains through mutagenesis screening is numbered M10.In the 30000L fermentor tank, drop into 50% fermented liquid, behind the sterilization, insert level Four seed liquor 800L, air flow is 1: 0.5 (V., V, M), stirring velocity is 120rpm, and leavening temperature is 26 ℃, ferments after 45 hours, stream adds 40% sterilization liquid glucose and adds 48 hours with 60L/ hour flow acceleration stream, fermentation in 144 hours stops, discharging, collection thalline, dehydration, oven dry, pulverizing, detection yield, and obtaining the dry mycelium yield is 4.25%, fat 41.33%, r-linolenic acid 21.98%.
Seed culture medium: 3.5% sucrose, 0.5% yeast extract paste, 0.5% peptone, 0.4% urea, 0.1% potassium primary phosphate, 0.08% lime carbonate, 0.2% sal epsom, 0.000035%VB
1, 0.00035% zinc sulfate, PH5.9.
Fermention medium: 2.0% sucrose, 0.2% urea, 0.4% yeast extract paste, 0.2% peptone, 0.5% potassium primary phosphate, 0.12% lime carbonate, 0.03% sal epsom, 0.00002%VB
1, 0.0003% copper sulfate, 0.0003% zinc sulfate, 0.0001% manganous sulfate, 0.0001% ferrous sulfate, PH6.0.
Embodiment five,
Thalline is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCCNO.0232, be with Institute of Microorganism, Academia Sinica, it is original strain that bacterium protects storehouse bacterial classification AS3.154, preferably the new dissociant that obtains through mutagenesis screening is numbered M10.In the 50000L fermentor tank, drop into 50% fermented liquid, behind the sterilization, insert level Four seed liquor 1500L, air flow is 1: 0.2 (V, V, M), stirring velocity is 100rpm, and leavening temperature is 30 ℃, ferments after 45 hours, stream adds 40% sterilization liquid glucose and adds 50 hours with 80L/ hour flow acceleration stream, fermentation in 120 hours stops, discharging, collection thalline, dehydration, oven dry, pulverizing, detection yield, and obtaining the dry mycelium yield is 4.19%, fat 40.9%, r-linolenic acid 21.01%.
Seed culture medium: 3% sucrose, 0.4% yeast extract paste, 0.4% peptone, 0.3% urea, 0.3% potassium primary phosphate, 0.05% lime carbonate, 0.15% sal epsom, 0.00002%VB
1, 0.0003% zinc sulfate, PH5.7.
Fermention medium: 3.5% sucrose, 0.35% urea, 0.2% yeast extract paste, 0.4% peptone, 0.3% potassium primary phosphate, 0.15% lime carbonate, 0.05% sal epsom, 0.00003%VB
1, 0.0002% copper sulfate, 0.0002% zinc sulfate, 0.0004% manganous sulfate, 0.0004% ferrous sulfate, PH5.5.
Claims (4)
1, a kind of method of producing gamma-linolenic acid by microbe microorganism fermentation, bacterial classification is the mould Cunninghamella echinulata of little Ke Yinhan (Thaxter), the common micro-organisms center preservation bacterial classification AS3.154 of China Committee for Culture Collection of Microorganisms is an original strain, preserving number is CGMCCNO.0232, comprises the following steps:
(1) bacterial classification with screening carries out the seed enlarged culturing
(2) ferment at fermention medium with the bacterium of enlarged culturing, it is characterized in that: after the fermentation, Continuous Flow is fully fermented with liquid glucose, collect after the fermentation of adding of Continuous Flow obtain containing the linolenic thalline of r-and dewater, drying.
2, as the method for the said producing gamma-linolenic acid by microbe microorganism fermentation of claim 1, it is characterized in that: the liquid glucose that Continuous Flow adds is the liquid glucose that contains 30-40% sugar, Continuous Flow adds 40-50 hour or divided minor tick to add liquid glucose in 40-50 hour, and after continuously fermenting 120-180 hour, collect thalline.
3, as the method for the said producing gamma-linolenic acid by microbe microorganism fermentation of claim 1, it is characterized in that: dress 60-70% seed culture medium in (1) 50-100L seeding tank, bacterial classification (5-10%) is inserted in the sterilization back, at 25-30 ℃, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-250rpm, tank pressure 0.03-0.06MP, fermented 40-60 hour, and be first order seed.The 60-70% seed culture medium of packing in the 500L jar again after the sterilization, inserts first order seed (5-10%), at air flow 1: 0.2-1: 1 (V, V, M), and mixing speed 100-250rp; Tank pressure 0.03-0.06MP, fermented 40-60 hour, for secondary enlarges seed by temperature 25-30 ℃; The 60-70% seed culture medium of packing in the 5000L jar after the sterilization, inserts secondary seed, and inoculum size is 5-10%, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-250rpm, tank pressure 0.03-0.06MP; Temperature 25-30 ℃, fermented 40-60 hour;
(2) the linolenic fermenting process of adding of Continuous Flow r-:
R-linolenic acid Continuous Flow adds fermentation, and the 50-60% basis fermention medium of packing in the 30000-50000L fermentor tank after the sterilization, is inoculated three grades of seed 5-10%, air flow 1: 0.2-1: 1 (V, V, M), mixing speed 100-200rpm, tank pressure 0.Q3-0.06MP; Temperature 25-30 ℃.After above-mentioned fermentation 40-50 hour, Continuous Flow adds a certain amount of liquid glucose (30-40%) stream and adds 40-50 hour, ferments 120-180 hour, ferments after the termination, collects thalline, and dehydration, oven dry, pulverizing get dry thalline, are formulation products.
4, according to claim 1 or the linolenic method of 2 or 3 described a kind of microorganism continuous stream addition fermentation r-, it is characterized in that in the described step:
(1) in the seed enlarged culturing, seed culture medium is formed: 2-4% sucrose, 0.1-0.5% yeast extract paste, the 0.1-0.5% peptone, 0.2-0.5% urea, 0.1-0.5% potassium primary phosphate, 0.05-0.1% lime carbonate, 0.1-0.2% sal epsom, 0.00002-0.00004%VB
1, 0.0002-0.0005% zinc sulfate, PH5.5-6.0.
(2) Continuous Flow adds in the fermentation, fermention medium: 2-4% sucrose, 0.2-0.4% urea, the 0.2-0.6% yeast extract paste, 0.2-0.6% peptone, 0.2-0.5% potassium primary phosphate, 0.1-0.15% lime carbonate, 0.02-0.05% sal epsom, 0.00001-0.00003%V B
1, 0.0001-0.0005% copper sulfate, 0.0002-0.0005% zinc sulfate, 0.0001-0.0005% manganous sulfate, 0.0001-0.0005% ferrous sulfate, PH5.5-6.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548674B (en) * | 2009-05-15 | 2013-08-21 | 山西省农业科学院旱地农业研究中心 | Microorganism drought-resisting inducer and preparing method thereof |
| CN103540623A (en) * | 2013-11-01 | 2014-01-29 | 厦门汇盛生物有限公司 | Method for producing gamma linolenic acid (GLA) through fermenting high-density cultured cunninghamellaceae |
-
2005
- 2005-03-01 CN CN 200510045965 patent/CN1827770A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548674B (en) * | 2009-05-15 | 2013-08-21 | 山西省农业科学院旱地农业研究中心 | Microorganism drought-resisting inducer and preparing method thereof |
| CN103540623A (en) * | 2013-11-01 | 2014-01-29 | 厦门汇盛生物有限公司 | Method for producing gamma linolenic acid (GLA) through fermenting high-density cultured cunninghamellaceae |
| CN103540623B (en) * | 2013-11-01 | 2016-03-09 | 厦门汇盛生物有限公司 | The method of a kind of high-density culture little gram of Mildy Way fermentative production GLA |
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