CN101353610A - Method for preparing microbial oil by irradiation of microorganism with 60Co gamma-ray - Google Patents
Method for preparing microbial oil by irradiation of microorganism with 60Co gamma-ray Download PDFInfo
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- CN101353610A CN101353610A CNA2008102230799A CN200810223079A CN101353610A CN 101353610 A CN101353610 A CN 101353610A CN A2008102230799 A CNA2008102230799 A CN A2008102230799A CN 200810223079 A CN200810223079 A CN 200810223079A CN 101353610 A CN101353610 A CN 101353610A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for preparing PUFAs by using <60>Co Gamma ray for irradiating microorganism, which belongs to the technical field of biochemical engineering. The method adopts the <60>Co Gamma ray to irradiate oil-yielding microorganism mycelium or protoplast thereof so as to obtain high-yielding strain for oil production to prepare the PUFAs. The method has the advantages that the technological operation is simple, the irradiated mycelium and protoplast have high forward-mutation rate, and the method can obviously enhance the oil productivity of the oil-yielding microorganism, thus reducing the production cost of the PUFAs.
Description
Technical field
The invention belongs to technical field of biochemical industry, particularly a kind of
60Co gamma-ray irradiation microorganism prepares the method for microbial oil.
Background technology
Biofuel is the renewable and clean energy resource of a kind of alternative fossil oil of growing up based on environmental degradation and energy dilemma in recent years.The domestic and international at present research for biodiesel manufacture mainly concentrates on vegetables oil or food and drink waste grease aspect, but utilize vegetables oil to be the raw materials cost height, its cost accounts for 70%～85% of total cost of production, with the food and drink abendoned oil is raw material, also exist raw material to be difficult to the difficulty of collecting and transporting, these have all seriously restricted the industrialization process of biofuel, and the raw material of therefore seeking a kind of cheapness becomes the key of biofuel industrialization.
Microbial oil (microbial oil) claims Unicell Oils and Fats (single cell oil) again, be under certain conditions by microorganisms such as yeast, mould, bacterium and algae, utilize carbohydrate, hydrocarbon polymer and common grease as carbon source, a large amount of greases that in thalline, produce.Biofuel by various animal and plant greases through esterification or transesterification metallization processes and get, and the lipid acid of most of microbial oil is formed and generally vegetables oil is close, so the alternative Vegetable oil lipoprotein production of microbial oil biofuel (Li Xiaosong, surplus setting sail. microbial oil. food science and technology, 1997, (5): 8-9.Das UN.Essential fatty acid metabolism in patients with essentialhypertension, diabetes mellitus and coronary heart disease.Prostaglandins, Leukotrienes ﹠amp; Essential Fatty Acids, 1995,52 (6): 387-391.)
In the produce oil bacterial strain, yeast and mould are considered to good product fat microorganism.Development along with modern biotechnology, may obtain more Microbial resources, as by wild bacterium being carried out the mutant strain that means such as mutagenesis, Protoplast Technique and orthogenesis all may obtain to have higher oil-producing capacity, thereby improve the application efficiency of oleaginous microorganism.Nineteen ninety-five, (Luo Yuping, Yang Rongying such as Luo Yuping, Li Siguang, etc. produce saccharomycetic separation of Zoomeric acid and evaluation. microorganism journal, 1995,35 (6): 400-403.) be separated to the yeast that a plant height produces Zoomeric acid, Zoomeric acid content is up to 50.14% in total fat; Fungus system report in 1998, with mortierella ramanniana SM541 is original strain, through ultraviolet compounded lithium chloride mutagenic treatment, obtain mutant strain SM541-9, its biomass is brought up to 28.8g/L by 12.6g/L, and fat content is brought up to 15.7g/L by 5.8g/L, arachidonic acid content is increased to 623mg/L (Xue Zhaohui by 321mg/L, Wu Moucheng. the microbial oil progress. Shanxi foodstuffs industry, 2002, (2): 10-11.).The Wu Qing of Tsing-Hua University is surplus, Miu Xiaoling has obtained the heterotrophism frustule (Miu Xiaoling of lipid content up to dry cell weight 55% by heterotrophism transformant engineering, the research of the utilization of algae renewable energy source and the anti-environment-stress of frustule [D], Beijing: Tsing-Hua University, 2004.).Qu Wei etc. are starting strain with this Da Shi saccharomyces oleaginosus, have gone out a plant height through ultraviolet mutation breeding and have produced greasy quality yeast bacterial strain, and biomass can reach 24.2g/L; Oil quantity 14.6g/L (Qu Wei, Liu Bo, Lv Jianzhou, etc. the seed selection of this Da Shi yeast strain of high yield grease and the preliminary study of conditions of flask fermentation. Liaoning Normal University's journal (natural science edition), 2006,29 (1): 88-92.).
60The Co gamma-rays is a kind of as ionizing ray in the physical mutagen, can cause biological gene sudden change and chromosome aberration, is a kind of effective means that obtains the high yield aimed strain.And with the direct selection by mutation of protoplastis, simple possible can improve the variation amplitude of positive mutant again.At present, also of no use
60The report of Co gamma-ray irradiation microorganisms producing microbial oil.
Summary of the invention
The object of the present invention is to provide a kind of
60Co gamma-ray irradiation microorganism prepares the method for microbial oil.Adopt
60Co gamma-ray irradiation oleaginous microorganism thalline or its protoplastis obtain the grease production superior strain, prepare the method for microbial oil.
Its concrete processing step is as follows:
1, with the rhodotorula glutinis of slant culture, the Si Dakaiyi yeast, mortierella, oleaginous microorganisms such as mortierella ramanniana wash thalline with sterilized water, and it is 5-7 * 106/mL that cell concn is regulated in microscopic counting, the preparation bacteria suspension.Or with the rhodotorula glutinis of slant culture, the Si Dakaiyi yeast, mortierella, oleaginous microorganisms such as mortierella ramanniana insert in the liquid nutrient medium, shaking culture 14-16h, 3000-5000r/min is centrifugal, abandoning supernatant, with the high osmotic buffer washing, with the EDTA pre-treatment thalline of the thalline after centrifugal with 0.1-0.3% beta-mercaptoethanol and 0.1-0.5%; And then handle bacteria suspension 10-20h with 1-2% helicase and 0.1-0.5% N,O-Diacetylmuramidase and 0.1-0.5% cellulolytic enzyme mixed enzyme solution and prepare protoplastis, protoplast formation rate was greater than 90% o'clock, the centrifugal enzyme liquid that goes is with high osmotic buffer washing, suspension protoplastis.
2, cell suspension or the protoplastis with preparation is suspended in the high osmotic buffer, uses
60The Co gamma-ray irradiation, irradiation dose 100-1000Gy, dose rate 3-5Gy/min dilutes 10 after the mutagenesis
-3-10
-6Coat on the perfect medium, lucifuge is cultivated 2-3d under 30-35 ℃ of dark condition.
3, cultured bacterium colony is carried out primary dcreening operation, from lethality rate greater than the mutant strain of selecting in the regenerated bacterium colony under 90% the irradiation dose greater than 3mm.
4, the bacterium colony that primary dcreening operation is obtained inserts 30-35 ℃ of seed culture medium, and 100-250r/min cultivates 18-24h, with (percent by volume) inoculum size access fermention medium of 5%-10%.Shaking culture 48-72h under 30-35 ℃, 100-250rmp, after the fermentation ends, the centrifugal collection thalline of fermented liquid 3000-5000r/min, and extract microbial oil, the screening superior strain.
Oleaginous microorganism of the present invention comprises rhodotorula glutinis, Si Dakaiyi yeast, mortierella or mortierella ramanniana.
Its composition of high osmotic buffer of the present invention is: 0.1mol/L pH 6.0 phosphoric acid buffers, 0.8mol/L N.F,USP MANNITOL.
Beneficial effect of the present invention
Adopt
60Co gamma-ray irradiation oleaginous microorganism thalline or its protoplastis, obtain the grease production superior strain, and preparation microbial oil, its technological operation is simple, irradiation thalline and protoplastis positive variation rate height, can significantly improve the grease production ability of oleaginous microorganism, thereby reduce the microbial oil production cost.
Embodiment
The invention provides a kind of
60Co gamma-ray irradiation microorganism prepares the method for microbial oil.Adopt
60Co gamma-ray irradiation oleaginous microorganism thalline or its protoplastis obtain the grease production superior strain, prepare the method for microbial oil.
Lifting specific embodiment is below again further specified the present invention.
Example 1:
(1) bacterial classification: glutinous rhodotorula (Rhodotorula gutini As2.107), available from Chinese science research institute institute of microbiology.
(2) substratum:
1. solid slant culture base (perfect medium)
Yeast powder: 10g/L; Peptone: 20g/L; Glucose: 20g/L; Agar: 20g/L; PH=5.
2. seed culture medium
Glucose: 15g/L; (NtH
4)
2SO
4: 2g/L; Yeast powder: 1g/L; KH
2PO
4: 7g/L; Na
2HPO
4: 2g/L; MgSO
4: 1.5g/L; PH=5.5.
3. fermention medium
Glucose: 50g/L; KNO
3: 0.38g/L; Yeast powder: 4g/L; KH
2PO
4: 6g/L; Na
2HPO
4: 2g/L; MgSO
4: 2g/L; CaCl
2: 0.1g/L; FeCl
3: 0.07g/L; PH=5.5; 115 ℃ of sterilization 20min.
(3) preparation of yeast suspension
The glutinous rhodotorula of preserving is transferred on the slant medium activates, wash thalline with the 10ml sterilized water as far as possible, microscopic counting adjusting cell concn is 7 * 10
6Individual/mL.
(4)
60The Co gamma-ray irradiation
The bacteria suspension for preparing is moved into sterile test tube.With
60The Co gamma-rays carries out irradiation, and irradiation dose is respectively 0GY, 200GY, 500GY, 800GY, 1000GY.Dilute 10 behind the irradiation
-3-10
-6Doubly coat on the perfect medium, lucifuge is cultivated 3d under 30 ℃ of dark conditions.
(5) screening of high yield strain
Select big and slick bacterium colony to insert seed culture medium, shaking culture 24h under 30 ℃, 180r/min uses the sudan black dyeing microscopic examination, and it is big to select fat granule, and the bacterial strain that density is high carries out multiple sieve.The bacterium colony of primary dcreening operation is inserted 30 ℃ of seed culture mediums, 180r/min cultivates 24h, and the inoculum size with 5% is inoculated in the fermention medium, shaking culture 72h under 30 ℃, 180r/min, the centrifugal collection thalline of 5000r/min after the fermentation ends adopts the acid heat method to extract grease.Through repeated screening, finally obtain a strain dissociant Rhodotorula gutiniW1.
(6) The selection result
The high yield strain that screening is obtained inserts 30 ℃ of seed culture mediums, 180r/min cultivates 24h, inoculum size with 5% is inoculated in the fermention medium, and shaking culture 72h compares with control group under 30 ℃, 180r/min, and its fat content and output are compared respectively according to improving 50% and 42.2%.
Example 2:
(1) bacterial classification: with example 1.
(2) substratum:
1. solid slant culture base (perfect medium): with example one.
2. seed culture medium: with example one.
3. fermention medium: with example one.
4. perfect medium (g/L): yeast soaks powder 3.0, extractum carnis 10.0, and Tryptones 10.0, Zulkovsky starch 1.0, glucose 5.0, L-halfcystine 0.50, NaCI 5.0, and NaAc 3.0, agar powder 15, pH8.5.
5. the perfect medium of regenerating
In perfect medium, add 0.5mol/L sucrose (or 0.8mol/L N.F,USP MANNITOL or 1mol/L sorbyl alcohol).
6. double-deck regeneration perfect medium
The upper strata needs to add 0.6% agar in perfect medium; Bottom is perfect medium.
(3) preparation of protoplastis
Starting strain activation with preserving inserts in the liquid nutrient medium, and shaking culture 16h is centrifugal, abandoning supernatant, and with the high osmotic buffer washing, the EDTA that adds 0.2% β, one mercaptoethanol and 0.5% carries out pre-treatment 30min to bacteria suspension; And carry out enzymolysis processing 10h with 1% helicase+0.5% N,O-Diacetylmuramidase+0.5% cellulolytic enzyme mixed enzyme solution, the rate of formation of protoplastis reaches 93.3%
(4)
60The Co gamma-ray irradiation
The protoplasma body fluid for preparing is added aseptic high sepage suspend, the microscopy counting is diluted to 106/mL with high sepage, moves into sterile test tube, uses
60The Co gamma-rays carries out irradiation, and irradiation dose is respectively 0GY, 50GY, 100GY, 200GY, 500GY, 800GY.Behind the irradiation, be diluted to 10
-4-10
-6Coat on perfect medium and the regeneration culture medium, lucifuge is cultivated 3d under 30 ℃ of dark conditions.
(5) screening of high yield strain
Select big and slick bacterium colony to insert seed culture medium, shaking culture 24h under 30 ℃, 180r/min uses the sudan black dyeing microscopic examination, and it is big to select fat granule, and the bacterial strain that density is high carries out multiple sieve.The bacterium colony of primary dcreening operation is inserted 30 ℃ of seed culture mediums, 180r/min cultivates 24h, and the inoculum size with 5% is inoculated in the fermention medium, shaking culture 72h under 30 ℃, 180r/min, the centrifugal collection thalline of 5000r/min after the fermentation ends adopts the acid heat method to extract grease.The final strain dissociant Rhodotorula gutiniW2 that obtains.
(6) preparation of microbial oil
With sieve the grease high yield strain bacterium colony of primary dcreening operation is inserted 30 ℃ of seed culture mediums, 180r/min cultivates 24h, the inoculum size with 5% is inoculated in the fermention medium, shaking culture 60h under 30 ℃, 180r/min.Compare with control group, its fat content and output are compared respectively according to improving 40% and 34%.
Claims (3)
1, a kind of
60Co gamma-ray irradiation microorganism prepares the method for microbial oil, it is characterized in that processing step comprises:
(1) oleaginous microorganism is washed thalline with sterilized water, it is 5-7 * 10 that cell concn is regulated in microscopic counting
6Individual/mL, the preparation bacteria suspension; Or in the oleaginous microorganism access liquid nutrient medium with slant culture, shaking culture 14-16h, 3000-5000r/min is centrifugal, abandoning supernatant, with the high osmotic buffer washing, with the EDTA pre-treatment thalline of the thalline after centrifugal with 0.1-0.3% beta-mercaptoethanol and 0.1-0.5%; And then handle bacteria suspension 10-20h with 1-2% helicase and 0.1-0.5% N,O-Diacetylmuramidase and 0.1-0.5% cellulolytic enzyme mixed enzyme solution and prepare protoplastis, protoplast formation rate was greater than 90% o'clock, the centrifugal enzyme liquid that goes is with high osmotic buffer washing, suspension protoplastis;
(2) cell suspension or the protoplastis with preparation is suspended in the high osmotic buffer, uses
60The Co gamma-ray irradiation, irradiation dose 100-1000Gy, dose rate 3-5Gy/min dilutes 10 after the mutagenesis
-3-10
-6Coat on the perfect medium, lucifuge is cultivated 2-3d under 30-35 ℃ of dark condition;
(3) cultured bacterium colony is carried out primary dcreening operation, carry out multiple sieve greater than the mutant strain of selecting in the regenerated bacterium colony under 90% the irradiation dose greater than 3mm from lethality rate.
(4) bacterium colony that primary dcreening operation is obtained inserts 30-35 ℃ of seed culture medium, and 100-250r/min cultivates 18-24h, inserts fermention medium with 5%-10% volume inoculum size; Shaking culture 48-72h under 30-35 ℃, 100-250rmp, after the fermentation ends, the centrifugal collection thalline of fermented liquid 3000-5000r/min, and extract microbial oil, the screening superior strain.
2. in accordance with the method for claim 1, it is characterized in that: described oleaginous microorganism comprises rhodotorula glutinis, Si Dakaiyi yeast, mortierella or mortierella ramanniana.
3. in accordance with the method for claim 1, it is characterized in that: its composition of described high osmotic buffer is: 0.1mol/L pH 6.0 phosphoric acid buffers, 0.8mol/L N.F,USP MANNITOL.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107208035A (en) * | 2014-12-31 | 2017-09-26 | 艾尼股份公司 | Saccharomyces olei variant, its preparation method and its purposes for lipid production |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107208035A (en) * | 2014-12-31 | 2017-09-26 | 艾尼股份公司 | Saccharomyces olei variant, its preparation method and its purposes for lipid production |
| CN107208035B (en) * | 2014-12-31 | 2021-06-04 | 艾尼股份公司 | Oleaginous yeast variants, method for obtaining same and use thereof for lipid production |
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