CN1255529C - Separating and screening method for arachindonic acid high yield strain - Google Patents

Abstract

The invention discloses a method for isolating and screening high-yield strains of arachidonic acid, the steps of which are: (1) low-temperature separation of producing bacteria: the collected soil samples are diluted with sterile water, separated on plates, and cultured at 0-10°C , to grow mold colonies, pick out a single colony, separate and purify and save; (2) Screening of high-yield bacteria: (2.1) Primary screening: carry out liquid culture of single colonies separated at low temperature for 5-12 days, harvest wet cells, wash Finally, put the bacteria in the container, add 0.2%-0.8% red tetrazolium solution with pH 7.0-9.0, let it stand for 0.5-4 hours, wash the bacteria and grind them into a homogenate, and extract three Phenylformazan, the supernatant was colorimetrically measured at a wavelength of 485nm to determine the degree of staining; (2.2) re-screening: select the mold with a deep staining degree of red tetrazolium, dry the thallus, extract the fat of the thallus for gas phase spectrum analysis. This method can be used to quickly and efficiently screen high-yield strains, and the content of arachidonic acid in the oil of Mortierella alpina strains of the screened high-yield strain reached 72.3%.

Description

Isolation and screening method of arachidonic acid high-yielding strains

technical field

The invention belongs to microorganism screening technology, in particular to a microorganism separation and screening method for synthesizing arachidonic acid oil.

Background technique

Arachidonic acid, linoleic acid, and linolenic acid are three kinds of fatty acids essential to the human body and have various physiological functions. Arachidonic acid mainly exists in organ muscles and blood tissues, and combines with phospholipids to form structural lipids, which plays an important role in the human body. Arachidonic acid is the immediate precursor of many eicosenoic acid derivatives, including prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane A2 (TXA2), leukotriene B4 (LTB4), and C4 (LTC4) and so on. These biologically active substances have important regulatory effects on lipoprotein metabolism, blood rheology, blood vessel elasticity, leukocyte function and platelet activation. Arachidonic acid also has a series of physiological activities such as esterifying cholesterol, inhibiting platelet aggregation, increasing blood vessel elasticity, reducing blood viscosity, regulating white blood cell function, and improving immunity.

Arachidonic acid is a natural component in breast milk, an important component of the baby's brain and retina, and is very important for baby development. Arachidonic acid in the last three months of pregnancy, this fatty acid will be deposited on the brain and retina of the fetus, and its relative content in the brain and eyes will continue to increase in the months after birth, so it is important for vision Especially important for developmental and neurodevelopmental infants. A large number of research reports have shown that arachidonic acid is beneficial to the growth of infants, the development of the central nervous system, the development of the retina, the development of intelligence and cognitive ability, the development of the vascular system and the development of the immune system. Infants fed formula supplemented with DHA and arachidonic acid all showed better visual accuracy, performed better on a problem-solving test, and performed better on a neurodevelopmental test than infants fed regular formula. Score higher.

Most vegetable oils do not contain arachidonic acid. Although arachidonic acid is widely found in animals, arachidonic acid derived from animal tissues cannot meet the needs of the market either in terms of output or cost. need. There has been a long-felt desire to obtain large quantities of low-cost products high in arachidonic acid.

The production of arachidonic acid by microorganisms by fermentation is an alternative source. It has been found that many microorganisms can synthesize arachidonic acid, among which Mortierella alpina has the most application prospect. Mortierella alpina is a filamentous fungus. When it grows in a medium with carbohydrates as a carbon source, it will accumulate more oil in the fungus. The fatty acid composition of its oil contains rich polyunsaturated fatty acids, especially It has a high content of arachidonic acid, and is considered to be the best strain for producing arachidonic acid oil, and the FDA of the Netherlands, the United Kingdom and the United States have successively passed the certification of wild Mortierella and its products for food safety.

Red tetrazolium is an oxidizing agent and a coenzyme of dehydrogenase. Red tetrazole is absorbed by living cells and reduced by the interaction with hydrogen atoms released by dehydrogenase. We found that the red tetrazolium staining degree of the bacteria was positively correlated with the arachidonic acid content, and the red tetrazolium staining degree of strains with high arachidonic acid content was deeper.

For the production of arachidonic acid, a good producing strain is crucial. It can not only use cheap medium to obtain high yield, but also have high arachidonic acid content in the product, because this will simplify the separation and purification process in post-processing. The traditional screening methods for arachidonic acid-producing bacteria include the use of low temperature and aspirin-selective media to screen, but no matter which screening method is used, it is necessary to measure the content of arachidonic acid after a large number of strains are isolated to find out which This is a very time-consuming work, including bacterial cell drying, oil extraction and gas chromatography analysis.

Contents of the invention

The purpose of the present invention is to provide a method for isolating and screening high-arachidonic acid-producing strains, which can quickly and efficiently screen high-arachidonic acid-producing strains.

A kind of arachidonic acid high-yielding strain separation and screening method provided by the invention, its steps are:

(1) Low-temperature separation and purification of soil bacteria:

Dilute the collected soil samples with sterile water for plate separation, culture at 0°C-10°C, wait for mold colonies to grow, pick out a single colony, separate and purify and store;

(2) Screening of arachidonic acid high-yielding bacteria:

(2.1) Preliminary screening: liquid culture the single colony isolated at low temperature for 5-12 days, harvest the wet bacteria, put the bacteria in a container after washing, add 2ml or 4ml of pH7.0-9.0 per 0.1g of bacteria 0.2%-0.8% red tetrazolium solution, placed in a dark place at 25°C or room temperature for 0.5-4 hours, the bacteria were washed and ground into a homogenate, and triphenylformazan was extracted from the homogenate, and the supernatant was at 485nm Colorimetrically measure the absorption value at the wavelength to determine the degree of dyeing;

(2.2) Re-screening: select the fungus with deep red tetrazolium staining, dry the fungus, extract fat from the fungus for gas chromatography analysis, and obtain the fungus with high arachidonic acid content.

Mortierella subgenus fungi can be isolated at low temperature, and Mortierella subgenus fungi can synthesize arachidonic acid, and the content of arachidonic acid in the fungus has a positive correlation with red tetrazolium staining Therefore, the present invention can quickly and efficiently screen arachidonic acid high-yielding bacteria by adopting the method of combining low-temperature screening and red tetrazolium staining. Using the screening method proposed by the present invention, the applicant obtained a high-yielding arachidonic acid strain, Mortierella alpina M ₀₂₂₃ , in which the content of arachidonic acid residues reached up to 72.8%. (The applicant has deposited the arachidonic acid high-yielding strain Mortierella alpina in the Type Culture Collection Committee of the Chinese Academy of Sciences on March 10, 2003, and the preservation number is: CGMCC No.0903).

Detailed ways

The following in conjunction with examples the present invention is described in further detail.

Example 1:

(1) Using low temperature to screen arachidonic acid-producing bacteria

Take soil from various places on the campus of Huazhong University of Science and Technology and mix it. The collected soil samples were diluted with sterile water, and two dilutions of 10 ^-2 and 10 ^-3 were used to smear 6 plates respectively. The plate medium was potato medium (PDA, 20% potato juice, 2% glucose, 2% agar , pH natural). Place the plate at 4°C for culture and obtain 72 mold strains. These molds all have very similar colony morphology, the surface color of the colony is white, the back color is yellow, and the spore color is white or yellow. When the colony first grows, it is very thin and round, and then expands layer by layer, forming petals (some are rose petals, some are like chrysanthemums), and grow long hairy hyphae. Observed under a microscope, there are more grease particles in the mycelia. All these molds can grow at a low temperature of 4°C and at a temperature of 25°C, and grow faster at a temperature of 25°C.

A few strains with faster growth were selected and cultured in liquid for 10 days. The bacteria were harvested, the oil was extracted, and the fatty acid analysis of the bacteria oil was carried out. The results are shown in Table 1.

Table 1 Several strains screened at low temperature and the content of arachidonic acid in their oils Strain number Arachidonic acid content (%) M4M10M23M25L31 42.815.642.55.730.8 L24H22H25H27 18.518.232.531.7

(2) The degree of red tetrazolium staining of the bacteria is positively correlated with the content of arachidonic acid in the bacteria for preliminary screening

The single colony isolated at low temperature was subjected to liquid culture for 7 days, the wet thallus was harvested, washed twice with distilled water, 0.1 g of the thallus was placed in a test tube with a cover, and 2 ml of 0.2% red tetrazolium (Triphenyltetrazolium chloride) of pH 8.5 was added The solution was placed in the dark at 25°C for 1 hour. The cells were washed twice with distilled water and ground into a homogenate, and the red triphenylformazan (triphenylformazan) was extracted three times with 2ml ethyl acetate at room temperature, and the extracts were combined, and the color was measured with a spectrophotometer at a wavelength of 485nm The absorbance is measured to determine the degree of staining. The results are shown in Table 2, which shows that the red tetrazolium staining degree of the bacteria is positively correlated with the content of arachidonic acid in the fat of the bacteria.

The strains screened at low temperature were cultured in liquid for 10 days, the mycelium was stained with red tetrazolium and the degree of staining was quantified, and 24 strains with darker staining were selected (results shown in Table 3) for the next step of re-screening.

Table 2 Relationship between red tetrazolium staining degree of different bacterial strains and arachidonic acid content in oil Strain number Staining degree (A485nm) AA content in oil M23M10M4L31 0.8610.3250.9550.472 39.616.145.828.4

(3) Re-screening by gas chromatography analysis method

The 24 strains with darker staining (results in Table 3) were dried to extract fat from the cells and analyzed by gas chromatography to examine the growth, oil production and arachidonic acid production capacity of each strain. It can be seen from the table that the arachidonic acid in the oil has a positive correlation with the staining degree of the bacteria, and the method of combining red tetrazole and low temperature can effectively screen the high-yield strains of arachidonic acid. The content of arachidonic acid in the oil of strain M ₀₂₂₃ reached 72.3%, and the yield of arachidonic acid reached 4.82g/l, showing good arachidonic acid production capacity. The sequence was identified as Mortierella alpina.

Table 3 Screening of arachidonic acid high-yielding bacteria Strain number Staining degree A485 Biomass (gL ^-1 ) Oil content (%) Oil production (gL ^-1 ) Arachidonic acid content (%) Arachidonic acid production (gL ^-1 ) M2M3M4M12M20M21M28M0223Y1Y8Y12H21H24H25H27L5L8L9L23L25L31G1W51 0.6370.6730.9660.4420.5190.4510.4501.0650.4510.6240.7000.4400.4030.4160.4620.6150.6630.7540.4080.4050.4540.5200.415 14.418.920.115.419.712.715.119.521.220.522.113.514.217.120.121.218.819.214.818.217.512.916.1 33.144.839.833.335.740.151.034.239.641.236.337.633.542.143.239.943.742.633.347.241.932.838.1 4.778.478.014.927.005.117.706.678.478.458.025.084.767.208.638.438.218.184.928.617.344.226.12 35.144.551.230.442.534.134.272.336.041.245.534.629.532.634.341.544.849.630.829.533.238.831.7 1.673.764.101.502.981.742.634.823.053.483.651.761.402.352.963.503.684.061.512.952.431.631.94

Example 2:

(1) Using low temperature to screen arachidonic acid-producing bacteria

Take the soil from various places in the urban area of Wuhan and mix it. The collected soil samples were diluted with sterile water, and two dilutions of ^10-2 and ^10-3 were applied to three plates respectively. , potassium chloride 0.5g/l, magnesium sulfate 0.5g/l, ferrous sulfate 0.01g/l, glucose 30g/l, agar 20g/l, pH natural). Place the plate at a low temperature of 10°C for cultivation, and obtain 42 mold strains. These mold colony forms are similar to embodiment 1.

(2) Use the red tetrazolium staining degree of the bacteria and the gas chromatography analysis method to screen

The isolated single colony was carried out liquid culture for 5 days, the wet thalline was harvested, washed twice, 0.1 g thalline was placed in a test tube with a cover, 4 ml of 0.4% red tetrazolium solution of pH 9.0 was added, and placed in Leave in the dark for 4 hours. The cells were washed twice with distilled water and ground into a homogenate, extracted three times with 2ml ethanol at room temperature, combined the extracts, and measured the absorbance with a spectrophotometer at a wavelength of 485nm to determine the degree of staining. Among them, 5 strains with darker staining were selected to dry the cells, extract the fat of the cells, and conduct gas chromatography analysis to examine the growth, oil production and arachidonic acid production capacity of each strain, and obtain a strain of Mortierella alpina Y6, The content of arachidonic acid in the bacterial fat reaches 42.5%, and the output of arachidonic acid is 3.12g/L.

Example 3:

(1) Using low temperature to screen arachidonic acid-producing bacteria

Take the soil from various places in the urban area of Wuhan and mix it. The collected soil samples were diluted with sterile water, and two dilutions of 10 ^-2 and 10 ^-3 were used to smear three plates respectively. The plate medium was potato medium (PDA, 20% potato juice, 2% glucose, 2% agar , pH natural). Place the plate at 0°C for culture and obtain 10 mold strains. These mold colony forms are similar to embodiment 1.

(2) Use the red tetrazolium staining degree of the bacteria and the gas chromatography analysis method to screen

The isolated single colony was subjected to liquid culture for 12 days, the wet thalline was harvested, washed twice, 0.1 g thalline was placed in a test tube with a cover, 2 ml of 0.8% red tetrazolium solution of pH 7.0 was added, and placed at 25°C Place in the dark for 0.5 hours. The cells were washed twice with distilled water and ground into a homogenate, extracted three times with 2ml acetone at room temperature, combined the extracts, and measured the absorbance with a spectrophotometer at a wavelength of 485nm to determine the degree of staining. Four strains with darker staining were selected for cell drying, cell fat was extracted, and gas chromatographic analysis was performed to examine the growth, oil production and arachidonic acid production capacity of each strain, and a strain of Mortierella alpina M6 was obtained. The content of arachidonic acid in the bacterial fat reaches 62.5%, and the output of arachidonic acid is 4.12g/L.

Claims (1)

1. A method for isolation and screening of arachidonic acid high-yield strains, the steps comprising: (1) Low-temperature separation and purification of soil bacteria: Dilute the collected soil samples with sterile water for plate separation, culture at 0°C-10°C, wait for mold colonies to grow, pick out a single colony, separate and purify and store; (2) Screening of arachidonic acid high-yielding bacteria: (2.1) Preliminary screening: liquid culture the single colony isolated at low temperature for 5-12 days, harvest the wet bacteria, put the bacteria in a container after washing, add 2ml or 4ml of pH7.0-9.0 per 0.1g of bacteria 0.2%-0.8% red tetrazolium solution, placed in a dark place at 25°C or room temperature for 0.5-4 hours, the bacteria were washed and ground into a homogenate, and triphenylformazan was extracted from the homogenate, and the supernatant was at 485nm Colorimetrically measure the absorption value at the wavelength to determine the degree of dyeing; (2.2) Re-screening: select the fungus with deep red tetrazolium staining, dry the fungus, extract fat from the fungus for gas chromatography analysis, and obtain the fungus with high arachidonic acid content.