CN1255529C - Separating and screening method for arachindonic acid high yield strain - Google Patents
Separating and screening method for arachindonic acid high yield strain Download PDFInfo
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- CN1255529C CN1255529C CN 200410060623 CN200410060623A CN1255529C CN 1255529 C CN1255529 C CN 1255529C CN 200410060623 CN200410060623 CN 200410060623 CN 200410060623 A CN200410060623 A CN 200410060623A CN 1255529 C CN1255529 C CN 1255529C
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Abstract
The present invention discloses a method for separating and screening high yield strains of arachidonic acid, which comprises the following steps: (1) in the step of low temperature separation of producing strains, collected soil samples are separated on a flat plate after being diluted by aseptic water to be cultivated at the temperature of 0 to 10 DEG C, single colony is selected after the mildew colony grows, and then, the single colony is preserved after being separated and purified; (2) in the screening step of high yield strains, two processes are carried out; (2.1) in a preliminary screening process, the single colony which is treated by low temperature separation is cultivated for 5 to 12 days in liquid, wet strains are collected, the strains are put in a container after being washed, 0.2% to 0.8% of red tetrazoline solution whose pH value is from 7.0 to 9.0 is added to the container to be placed for 0.5 to 4 hours, the strains are milled into uniform pulp after being washed, triphenyl formazan is extracted from the uniform pulp, and then, the absorption value of the supernatant solution is treated by colorimetric determination at the wavelength of 485 nm to determine dyeing degree; (2.2) in a secondary screening process, the mildew of red tetrazoline with thick dyeing degree is selected, the strains are dried, and then, the grease of the strains is extracted to carry out gas chromatographic analysis. The method of the present invention can be used for quickly and efficiently screening high yield strains, and the content of the arachidonic acid in the grease of the mountain spore mildew of the high yield strain which is screened reaches 72.3%.
Description
Technical field
The invention belongs to the microbe to screen technology, be specifically related to the microorganism separating screening method of synthetic arachidonic acid oil.
Background technology
Arachidonic acid and linolic acid, linolenic acid have different physiological roles as 3 kinds of lipid acid of needed by human.Arachidonic acid mainly is present in organ muscle and the blood tissues, becomes struetural lipid with phospholipids incorporate, and human body is played an important role.Arachidonic acid is the direct precursor of many eicosylene acid derivatives, comprises prostaglandin E2 (PGE2), prostacyclin (PGI2), the plain A2 (TXA2) of thromboxane, leukotrienes B4 (LTB4) and C4 (LTC4) or the like.Metabolism, hemorheology, blood vessel elasticity, leukocyte function and the platelet activation etc. of these bio-active substance confrontation lipoprotein have important regulatory role.Arachidonic acid also has esterified cholesterol, a series of physiologically actives such as anticoagulant, increase blood vessel elasticity, blood viscosity lowering, adjusting leukocyte function, raising immunizing power.
Arachidonic acid is the natural component in the breast milk, is baby's brain and amphiblestroid important composition composition, and is extremely important to infant development.Arachidonic acid is in the last March of pregnancy, this lipid acid can be deposited on the brain and retina of fetus, and its relative content in brain and eye also can continue to increase in postnatal some months, so eyesight is grown and neurodevelopmental baby is especially important to being in.Existing a large amount of research reports show that arachidonic acid helps baby's growth, the growth of central nervous system, and amphiblestroid growth, the growth of intelligence and cognitive ability, vascular system is grown and immunity system is grown.Compare with the baby who feeds with ordinary powdered milk with the baby that arachidonic milk powder is fed with having replenished DHA, all show better eyesight accuracy, show in the test of dealing with problems better, the score in the neurodevelopment test is higher.
In vegetables oil, the overwhelming majority does not contain arachidonic acid, though arachidonic acid extensively is present in the animal body, the arachidonic acid that derives from animal tissues is on the output or all can't satisfy the demand in market on cost.People catch at the product that great amount of cost is low, arachidonic acid content is high for a long time.
Adopting fermentation method is a kind of substituting source by microorganism yield peanut in next life tetraenoic acid, has found that many microorganisms can both synthesize arachidonic acid, wherein with the tool application prospect of Mortierella alpina (Mortierella alpina).Mortierella alpina is a kind of filamentous fungus, when it is grown in the substratum that with the carbohydrate is carbon source, can accumulate more grease in the thalline, its greasy lipid acid contains abundant polyunsaturated fatty acid in forming, especially arachidonic acid content is higher, the bacterial classification that is considered to best production arachidonic acid oil, and Holland, Britain and U.S. FDA have successively passed through the authentication of wild mortierella and product edible safety thereof.
TCC is a kind of oxygenant, is the coenzyme of desaturase.TCC is absorbed by viable cell, is reduced with the hydrogen atom effect of desaturase release.We find that the TCC dye levels and the arachidonic acid content of thalline has positive correlation, and the bacterial strain TCC dye levels that arachidonic acid content is high is dark.
For arachidonic production, the generation bacterium that a strain is good is vital.It not only can utilize cheap substratum, obtains high output, and arachidonic acid content wants high in the product, because this will simplify the separation and purification process in the aftertreatment.The screening method that traditional arachidonic acid produces bacterium comprises and utilizes low temperature, acetylsalicylic acid selective medium to screen, but no matter adopt which kind of screening method, after separating a large amount of bacterial strains of acquisition, all need to carry out the mensuration of arachidonic acid content to seek superior strain wherein, this is a job very consuming time, comprises thalline drying, grease extraction and gas chromatographic analysis etc.
Summary of the invention
The object of the present invention is to provide a kind of arachidonic acid high yielding strain kind separating screening method, this method can filter out the arachidonic acid high yielding strain kind quickly and efficiently.
A kind of arachidonic acid high yielding strain kind separating screening method provided by the invention the steps include:
(1) low ternperature separation process of soil bacterial classification and purifying:
Adopting soil sample is carried out plate isolation by the sterilized water dilution, and 0 ℃ of-10 ℃ of cultivation, mold colony to be grown is chosen single bacterium colony, preserves after the separation and purification;
(2) arachidonic acid high yielding strain screening:
(2.1) primary dcreening operation: single bacterium colony of low ternperature separation process was carried out liquid culture 5-12 days, the results wet thallus, after the washing thalline is placed container, every 0.1g bacterial classification adds the TCC solution of the 0.2%-0.8% of 2ml or 4ml pH7.0-9.0, placed 0.5-4 hour 25 ℃ or room temperature dark place, be milled into homogenate after the thalline washing, extract San Ben Ji Jia Za from homogenate, supernatant liquor colorimetric estimation absorption value under the 485nm wavelength is determined dye levels;
(2.2) multiple sieve: choose the dark mould of TCC dye levels, thalline is carried out drying, extract the thalline grease and carry out gas chromatographic analysis, obtain the high bacterial classification of arachidonic acid content.
Owing to can be separated to genus mortierella mortierella subgenus fungi under the low temperature, and mortierella subgenus fungi can be synthesized arachidonic acid, and the dyeing of the arachidonic acid content in the thalline and TCC has positive correlation, so the present invention adopts low temperature screening and the TCC method that combines that dyes can screen arachidonic acid high yielding strain quickly and efficiently.The applicant utilizes screening method proposed by the invention, has obtained a strain arachidonic acid high yielding strain plant height mountain mortierella Mortierella alpina M
0223, wherein arachidonic acid residue content has been up to 72.8%.(the applicant on March 10th, 2003 at Chinese Academy of Sciences typical case culture collection council preservation arachidonic acid high yielding strain plant height mountain mortierella, deposit number is: CGMCC No.0903).
Embodiment
It is following that the present invention is further detailed explanation in conjunction with example.
Embodiment 1:
(1) utilize low temperature screening arachidonic acid to produce bacterium
Get in the Central China University of Science and Technology campus soil everywhere, mix.The soil sample of adopting is diluted by sterilized water, gets 10
-2, 10
-3Two extent of dilution respectively are coated with 6 flat boards, and plate culture medium is potato substratum (PDA, 20% murphy juice, 2% glucose, 2% agar, a pH nature).Flat board is positioned over cultivation under 4 ℃ of low temperature, obtains 72 strain fungal strains.These moulds all have very similar colonial morphology, and the bacterium colony surface color is a white, back side color yellow, and the spore color is white or yellow.Very thin when bacterium colony just grows, circle, expansion from level to level then forms petal-shaped (what have is the Rose flap, have as the chrysanthemum shape), and grows long hair shape mycelia.Microscopically is observed, and more fat particles is arranged in the mycelium.All these moulds can grow under 4 ℃ of low temperature and 25 ℃ of temperature, and growth is very fast under 25 ℃ of temperature.
Chosen wherein several strains bacterial classification faster of growing, carried out liquid culture 10 days, the results thalline extracts grease, carries out the greasy fatty acid analysis of thalline.The results are shown in Table 1.
A few strain bacterial classifications of table 1 low temperature screening and the arachidonic acid content in the grease thereof
| Strain number | Arachidonic acid content (%) |
| M4 M10 M23 M25 L31 | 42.8 15.6 42.5 5.7 30.8 |
| L24 H22 H25 H27 | 18.5 18.2 32.5 31.7 |
(2) utilize in the TCC dye levels of thalline and the thalline arachidonic acid content to have positive correlation and carry out primary dcreening operation
Single bacterium colony of low ternperature separation process was carried out liquid culture 7 days, the results wet thallus with distilled water wash twice, is got the 0.1g thalline and has been placed and cover test tube, add 0.2% TCC (Triphenyltetrazolium chloride) solution of 2mlpH8.5,25 ℃ place the dark place to place 1 hour.Thalline is milled into homogenate with behind twice of the distilled water wash, with the San Ben Ji Jia Za (triphenylformazan) of 2ml ethyl acetate room temperature extracting redness three times, merges extract, determines dye levels with spectrophotometer colorimetric estimation absorption value under the 485nm wavelength.The results are shown in Table 2, show that the TCC dye levels of thalline and the arachidonic acid content in the thalline grease have positive correlation.
Each bacterial strain of low temperature screening was carried out liquid culture 10 days, mycelia is carried out TCC dyeing and quantitative dye levels, choosing wherein, the darker 24 strain bacterial strains (the results are shown in Table 3) of dyeing carry out next step multiple sieve.
The TCC dye levels of table 2 different strains thalline and the relation of the arachidonic acid content in the grease
| Bacterial strain number | Dye levels (A485nm) | AA content in the grease |
| M23 M10 M4 L31 | 0.861 0.325 0.955 0.472 | 39.6 16.1 45.8 28.4 |
(3) utilize gas chromatography analysis method to carry out multiple sieve
The darker 24 strain bacterial strains (the results are shown in Table 3) of dyeing are carried out the thalline drying, extract the thalline grease, the promoting the circulation of qi analysis of hplc of going forward side by side is examined or check growth, produce oil and the arachidonic acid production ability of each bacterial strain.Have positive correlation from this arachidonic acid and thalline dye levels of showing the grease as can be seen, the method for utilizing TCC and low temperature to combine can effectively be screened the arachidonic acid high yielding strain kind.Bacterial strain M
0223Grease in arachidonic acid content reach 72.3%, the arachidonic acid yield reaches 4.82g/l, show good arachidonic acid production ability, this bacterial strain is Mortierella alpina (Mortierella alpina) through morphological specificity, physiological characteristic and rrna 18S rDNA Sequence Identification.
The screening of table 3 arachidonic acid high yielding strain
| Bacterial strain number | Dye levels A485 | Biomass (gL -1) | Fat content (%) | Grease yield (gL -1) | Arachidonic acid content (%) | Arachidonic acid yield (gL -1) |
| M2 M3 M4 M12 M20 M21 M28 M0223 Y1 Y8 Y12 H21 H24 H25 H27 L5 L8 L9 L23 L25 L31 G1 W51 | 0.637 0.673 0.966 0.442 0.519 0.451 0.450 1.065 0.451 0.624 0.700 0.440 0.403 0.416 0.462 0.615 0.663 0.754 0.408 0.405 0.454 0.520 0.415 | 14.4 18.9 20.1 15.4 19.7 12.7 15.1 19.5 21.2 20.5 22.1 13.5 14.2 17.1 20.1 21.2 18.8 19.2 14.8 18.2 17.5 12.9 16.1 | 33.1 44.8 39.8 33.3 35.7 40.1 51.0 34.2 39.6 41.2 36.3 37.6 33.5 42.1 43.2 39.9 43.7 42.6 33.3 47.2 41.9 32.8 38.1 | 4.77 8.47 8.01 4.92 7.00 5.11 7.70 6.67 8.47 8.45 8.02 5.08 4.76 7.20 8.63 8.43 8.21 8.18 4.92 8.61 7.34 4.22 6.12 | 35.1 44.5 51.2 30.4 42.5 34.1 34.2 72.3 36.0 41.2 45.5 34.6 29.5 32.6 34.3 41.5 44.8 49.6 30.8 29.5 33.2 38.8 31.7 | 1.67 3.76 4.10 1.50 2.98 1.74 2.63 4.82 3.05 3.48 3.65 1.76 1.40 2.35 2.96 3.50 3.68 4.06 1.51 2.95 2.43 1.63 1.94 |
Embodiment 2:
(1) utilize low temperature screening arachidonic acid to produce bacterium
Get Wuhan urban district soil everywhere, mix.The soil sample of adopting is diluted by sterilized water, gets 10
-2, 10
-3Two extent of dilution respectively are coated with 3 flat boards, and plate culture medium is czapek's solution (SODIUMNITRATE 2g/l, dipotassium hydrogen phosphate 1g/l, Repone K 0.5g/l, sal epsom 0.5g/l, ferrous sulfate 0.01g/l, glucose 30g/l, agar 20g/l, a pH nature).Flat board is positioned over cultivation under 10 ℃ of low temperature, obtains 42 strain fungal strains.These mold colony forms are similar to embodiment 1.
(2) utilize the TCC dye levels and the gas chromatography analysis method of thalline to screen
The single bacterium colony that is separated to was carried out liquid culture 5 days, the results wet thallus, washing twice is got the 0.1g thalline and has been placed and cover test tube, adds 0.4% the TCC solution of 4mlpH9.0, places the dark place to place under the room temperature 4 hours.Thalline is milled into homogenate with behind twice of the distilled water wash, with 2ml ethanol room temperature extracting three times, merges extract, determines dye levels with spectrophotometer colorimetric estimation absorption value under the 485nm wavelength.Choosing wherein, 5 darker strain bacterial strains of dyeing carry out the thalline drying, extract the thalline grease, the promoting the circulation of qi analysis of hplc of going forward side by side, examine or check growth, produce oil and the arachidonic acid production ability of each bacterial strain, obtain a plant height mountain mortierella Y6, arachidonic acid content reaches 42.5% in its thalline grease, and the arachidonic acid yield is 3.12g/L.
Embodiment 3:
(1) utilize low temperature screening arachidonic acid to produce bacterium
Get Wuhan urban district soil everywhere, mix.The soil sample of adopting is diluted by sterilized water, gets 10
-2, 10
-3Two extent of dilution respectively are coated with 3 flat boards, and plate culture medium is potato substratum (PDA, 20% murphy juice, 2% glucose, 2% agar, a pH nature).Flat board is positioned over cultivation under 0 ℃ of low temperature, obtains 10 strain fungal strains.These mold colony forms are similar to embodiment 1.
(2) utilize the TCC dye levels and the gas chromatography analysis method of thalline to screen
The single bacterium colony that is separated to was carried out liquid culture 12 days, the results wet thallus, washing twice is got the 0.1g thalline and has been placed and cover test tube, adds 0.8% the TCC solution of 2mlpH7.0, and 25 ℃ place the dark place to place 0.5 hour.Thalline is milled into homogenate with behind twice of the distilled water wash, with 2ml acetone room temperature extracting three times, merges extract, determines dye levels with spectrophotometer colorimetric estimation absorption value under the 485nm wavelength.Choosing wherein, 4 darker strain bacterial strains of dyeing carry out the thalline drying, extract the thalline grease, the promoting the circulation of qi analysis of hplc of going forward side by side, examine or check growth, produce oil and the arachidonic acid production ability of each bacterial strain, obtain a plant height mountain mortierella M6, arachidonic acid content reaches 62.5% in its thalline grease, and the arachidonic acid yield is 4.12g/L.
Claims (1)
1. arachidonic acid high yielding strain kind separating screening method, its step comprises:
(1) low ternperature separation process of soil bacterial classification and purifying:
Adopting soil sample is carried out plate isolation by the sterilized water dilution, and 0 ℃ of-10 ℃ of cultivation, mold colony to be grown is chosen single bacterium colony, preserves after the separation and purification;
(2) arachidonic acid high yielding strain screening:
(2.1) primary dcreening operation: single bacterium colony of low ternperature separation process was carried out liquid culture 5-12 days, the results wet thallus, after the washing thalline is placed container, every 0.1g bacterial classification adds the TCC solution of the 0.2%-0.8% of 2ml or 4ml pH7.0-9.0, placed 0.5-4 hour 25 ℃ or room temperature dark place, be milled into homogenate after the thalline washing, extract San Ben Ji Jia Za from homogenate, supernatant liquor colorimetric estimation absorption value under the 485nm wavelength is determined dye levels;
(2.2) multiple sieve: choose the dark mould of TCC dye levels, thalline is carried out drying, extract the thalline grease and carry out gas chromatographic analysis, obtain the high bacterial classification of arachidonic acid content.
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| CN101113410B (en) * | 2007-07-09 | 2010-05-19 | 南京工业大学 | Mortierella alpina and application thereof |
| CN101109015B (en) * | 2007-07-09 | 2011-05-04 | 南京工业大学 | Preparation method of arachidonic acid grease |
| CN101709311B (en) * | 2009-11-25 | 2013-01-02 | 南京工业大学 | Rapid high-yield method of arachidonic acid |
| CN105861339B (en) * | 2016-06-16 | 2019-10-18 | 江南大学 | Recombination Mortierella alpina, its construction method and the application of one plant of overexpression GTP cyclohydrolase gene |
| CN110331099A (en) * | 2019-07-31 | 2019-10-15 | 江南大学 | A kind of rapid screening method of oil-producing filamentous fungi genetic modification bacterial strain |
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