CN102925503B - Method for preparing ARA (Arachidonic Acid) by culturing mortierella alpina by utilizing solid material culture medium - Google Patents
Method for preparing ARA (Arachidonic Acid) by culturing mortierella alpina by utilizing solid material culture medium Download PDFInfo
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- CN102925503B CN102925503B CN201210385420.7A CN201210385420A CN102925503B CN 102925503 B CN102925503 B CN 102925503B CN 201210385420 A CN201210385420 A CN 201210385420A CN 102925503 B CN102925503 B CN 102925503B
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Abstract
The invention discloses a method for preparing an ARA (Arachidonic Acid) by culturing a mortierella alpina by utilizing a solid material culture medium. The method comprises the following steps of using rice bran and niblet as material, adding inorganic salt in defined amount, using the inorganic salt as the solid material culture medium, collecting and smashing thallus after culturing strain in liquid, inoculating smashed thallus to the solid material culture medium, controlling culture temperature, mosturizing and controlling relative humidity during a culture process and appropriately stirring, and generating a large amount of spores. pH (Potential Of Hydrogen) segmentation regulation and control can be carried out during a fermentation process by using a citric acid and citrate. According to the method disclosed by the invention, the amount of seed spores of is large, the sunchronism is good, the activity is strong, the fermentation period of the mortierella alpina can be shortened, the growth of the mortierella alpine and the accumulation of ARA grease can be facilitated through the pH segmentation regulation and control, and the production and the quality of the ARA can be increased.
Description
Technical field
The present invention relates to arachidonic preparation method in microbial technology field, refer in particular to the Mortierella alpina thalline after liquid culture, after shattering, be seeded to solid material substratum, cultivate a large amount of spores of breeding, then prepare arachidonic method through liquid fermenting.
Technical background
Arachidonic acid (ARA) is the synthetic important precursor of human body prostaglandin(PG), has esterified cholesterol, anticoagulant, increase blood vessel elasticity, reduces blood viscosity, regulates leukocyte function, improves a series of physiologically actives such as immunizing power.Past, business ARA was mainly derived from pork liver and fish oil, but content is extremely low and unstable, and development cost costliness, is difficult to meet social demand, particularly milk preparation and the required ARA of pharmaceutical industry measures very large.At the eighties initial stage, people find that some filamentous fungus and microalgae contain arachidonic acid in succession.Because microorganism has, fast growth, grease and ARA content are high, fermentation preparation technology is fairly simple and be not subject to the advantages such as raw material restriction, utilize microbial method to produce the focus that arachidonic acid becomes various countries' research.
Mortierella alpina (Mortierella alpina) is considered to best ARA and produces bacterial strain, and it has the high and PUFAs of ARA content and forms the features such as reasonable.At present, utilize the research of alpina fermentative production ARA mainly to concentrate on the aspects such as strain improvement, medium optimization.Study a kind of from yeast culture, the directed regulation and control of combining with fermentation process, the arachidonic acid preparation method who effectively improves fermentation level has market outlook.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of utilization and admittedly expects that culture medium culturing Mortierella alpina prepares arachidonic method, this method is the seed culture method of Mortierella alpina, and in conjunction with suitable zymotechnique, to shorten its fermentation period, improve its output and quality.
The common Mortierella alpina that the bacterial classification that present method is used is yield peanut tetraenoic acid.
The present invention prepare method that spore adopts be thalline after liquid culture, collect thalline and pulverize, inoculate to solid material substratum.Ripe thalline after liquid culture, can produce spore after being seeded to solid material substratum in a large number.
The present invention's product spore substratum used is for admittedly expecting substratum.This substratum is characterised in that, take rice bran, corn grain is raw material, adds appropriate inorganic salt formulated.
The present invention is in fermentation culture regulation process, and institute's substance is trisodium citrate and Citric acid monohydrate Food grade.Trisodium citrate is alkalescence, and it is acid that citric acid is, and at thalline amount reproduction growth phase, makes pH value stabilization between 6.0～6.5, is conducive to the growth and breeding of ARA somatic cells.And add in the ARA fermentation produce oil stage, can pH value be regulated, make pH value stabilization between 7.5～8.0, be conducive to improve activity of acetyl coenzyme A carboxylase.The reaction of acetyl-CoA carboxylase catalysis is the rate-limiting step in lipid acid building-up process, is the key point of the synthetic regulation and control of lipid acid.Simultaneously citric acid is absorbed by thalline, in cytosol, is subject to citrate lyase effect and ruptures, and generates acetyl-CoA.And acetyl-CoA is the synthetic crucial initial substance of lipid acid.
The technical problem to be solved in the present invention is realized by following scheme: under the bacterial classification of having grown on PDA substratum is washed by 20mL stroke-physiological saline solution, make bacteria suspension, in the liquid shaking bottle substratum that to be seeded to loading amount that sterilizing is good be 50mL, in 28 ℃, 180rpm constant-temperature table is cultivated 2 days, in liquid shaking bottle, cultured thalline is collected thalline by filtration, through sterilized water, wash, with electrical grinding machine, be ground into length at 1000～2000 μ m, inoculate to the good solid material substratum of sterilizing, fully stir bacterial classification is uniformly distributed in substratum, and spread out even material, in 28 ℃, cultivate 3～5 days, between incubation period, during every 24h, moisturizing 2mL is to maintain suitable relative humidity, and stir stirring, 400mL stroke-physiological saline solution is poured in cultured Kolle flask of admittedly expecting seed, fully vibration, to wash lower spore, spore liquid is by carrying out amplification culture in 1.2% inoculum size access 30L seeding tank, secondary seed solution is carried out fermentative production arachidonic acid with 10% inoculum size access 100L fermentor tank again, described liquid shaking bottle substratum: glucose 20～60g/L, yeast powder 5～20g/L, magnesium sulfate 0.1～1g/L, potassium primary phosphate 0.1～2g/L, describedly admittedly expect substratum: take rice bran, corn particle is raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration mixes by 10: 1～5 mass ratio, in Stainless Steel Kettle after boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilizing 30min.
Concrete steps are as follows:
1, utilize liquid nutrient medium to cultivate Mortierella alpina thalline:
1. liquid shaking bottle substratum: glucose 20～60g/L, yeast powder 5～20g/L, magnesium sulfate 0.1～1g/L, potassium primary phosphate 0.1～2g/L.Substratum is through 121 ℃ of sterilizing 30min.
2. under the bacterial classification of having grown on PDA substratum being washed by 20mL stroke-physiological saline solution, make bacteria suspension, in the liquid shaking bottle substratum that to be seeded to loading amount that above-mentioned sterilizing is good be 50mL, in 28 ℃, 180rpm constant-temperature table is cultivated 2 days.
2, collect above-mentioned cultured thalline, after shattering, be seeded in solid material substratum:
1. take rice bran, corn particle is raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration mixes by 10: 1～5 mass ratio, in Stainless Steel Kettle after boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilizing 30min, makes solid material substratum.
2. will by filtration, collect thalline at the cultured thalline of liquid shaking bottle, through sterilized water, wash, with electrical grinding machine, be ground into length at 1000～2000 μ m, inoculate to the good solid material substratum of above-mentioned sterilizing, fully stir bacterial classification is uniformly distributed in substratum, and spread out even material, in 28 ℃, cultivate 3～5 days.Between incubation period, during every 24h, moisturizing 2mL, to maintain suitable relative humidity, and stirs stirring.
3, above-mentioned utilization expects that culture medium culturing Mortierella alpina prepares arachidonic method admittedly, it is characterized in that: described take carbon source be main solid materials as: rice bran, corn particle, the mass ratio of rice bran, corn particle is 10: 1～5.
4, arachidonic acid is prepared in fermentation: 400mL stroke-physiological saline solution is poured in cultured Kolle flask of admittedly expecting seed, and fully vibration, to wash lower spore.Spore liquid is by carrying out amplification culture in 1.2% inoculum size access 30L seeding tank, and secondary seed solution is carried out fermentative production arachidonic acid with 10% inoculum size access 100L fermentor tank again.
5, fermentation pH carries out segmentation control, it is characterized in that, front 48h makes pH value stabilization between 6.0～6.5, then makes pH value stabilization between 7.0～9.0.
6, in fermenting process, be used in that to regulate the material of pH value be citric acid and Citrate trianion beneficial effect of the present invention:
1, spore cultural method of the present invention is conducive to a large amount of generations of spore;
2, of the present inventionly admittedly expect that substratum sedimentation is good, the spore of production is easy to separated with mycelium with substratum, and the seed liquor miospore amount providing for fermentative production is large, and synchronism is good, can shorten whole fermentation period;
3, seed provided by the invention has higher vigor in fermentation, can improve the quantity and quality of product;
4, the present invention uses trisodium citrate and Citric acid monohydrate Food grade to carry out fermentation control to be conducive to the synthetic of lipid acid, can to improve lipid acid resultant velocity and resultant quantity, thereby improve ARA fermentation level.
Accompanying drawing explanation
Fig. 1 is zymotechnique schema of the present invention.
Embodiment
1, liquid seeds and admittedly expect the lab scale fermentation results contrast of seed
1. liquid seeds lab scale fermentation flow process: PDA inclined-plane seed → liquid shaking bottle cultivation → 30L seeding tank (secondary seed) → 100L fermentor tank.
PDA inclined-plane makes, and is that industry professional is familiar with.
Liquid shaking bottle substratum: glucose 30g/L, yeast powder 10g/L, magnesium sulfate 0.5g/L, potassium primary phosphate 1.0g/L.
Secondary seed medium: glucose 30g/L, yeast powder 10g/L, magnesium sulfate 0.5g/L, potassium primary phosphate 1.0g/L.
Fermention medium: glucose 30g/L, yeast powder 15g/L, ammonium sulfate 0.5g/L, magnesium sulfate 0.5g/L, zinc sulfate 0.01g/L, potassium primary phosphate 1.0g/L, Sodium Glutamate 0.8g/L.
Idiographic flow: wash lower PDA slant pore by 20mL stroke-physiological saline solution, access in the shaking flask that 400mL seed culture medium is housed that sterilizing is good, in 28 ℃ of constant-temperature tables, 180rpm cultivates 3 days; Cultured shake-flask seed, by 1.2% inoculum size access 30L seeding tank, is inoculated rear nutrient solution volume 15L, and 28 ℃, pH value 6.0～6.5, rotating speed 100rpm, tank pressure 0.03Mpa, air flow 1.0m
3/ h cultivates 2 days; Secondary seed solution, by 10% inoculum size access 100L fermentor tank, is inoculated to rear nutrient solution volume 60L, 28 ℃, pH value 6.0～6.5, rotating speed 60rpm, tank pressure 0.03MPa, air flow 2.0～3.0m
3/ h, fermentation culture 8 days.After fermentation 72h, every 24h sampling detects total grease weight in unit nutrient solution, and ARA purity.
2. admittedly expect seed lab scale fermentation flow process: admittedly PDA inclined-plane seed → liquid shaking bottle cultivate → is expected seed culture → 30L seeding tank (secondary seed) → 100L fermentor tank.
Admittedly expect substratum: take rice bran, corn particle is raw material, the mass ratio of rice bran, corn particle is 3: 1, being 2g/L dipotassium hydrogen phosphate solution with concentration mixes by the mass ratio of 5: 1, in Stainless Steel Kettle after boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilizing 30min.
PDA substratum, liquid shaking bottle substratum, secondary seed medium and fermention medium are as previously mentioned.
Idiographic flow: under the bacterial classification of having grown on PDA substratum is washed by 20mL stroke-physiological saline solution, make bacteria suspension, in the liquid shaking bottle substratum that to be seeded to loading amount that above-mentioned sterilizing is good be 50mL, in 28 ℃, 180rpm constant-temperature table is cultivated 2 days.At the cultured thalline of liquid shaking bottle, by sterile gauze, filter and collect thalline, through sterilized water washing 2 times, with electrical grinding machine, be ground into length at 1400～1600 μ m, inoculate to the good solid material substratum of above-mentioned sterilizing, fully stir bacterial classification is uniformly distributed in substratum, and spread out even material, in 28 ℃, cultivate 3 days.Between incubation period, during every 24h, moisturizing 2mL, to maintain suitable relative humidity, and stirs stirring.400mL stroke-physiological saline solution is poured in cultured Kolle flask of admittedly expecting seed, and fully vibration, to wash lower spore.Spore liquid, by 1.2% inoculum size access 30L seeding tank, is inoculated to rear nutrient solution volume 15L, 28 ℃, pH value 6.0～6.5, rotating speed 100rpm, tank pressure 0.03MPa, air flow 1.0m
3/ h cultivates 2 days; Secondary seed solution, by 10% inoculum size access 100L fermentor tank, is inoculated to rear nutrient solution volume 60L, 28 ℃, pH value 6.0～6.5, rotating speed 60rpm, tank pressure 0.03MPa, air flow 2.0～3.0m
3/ h.Fermentation culture 8 days.After fermentation 72h, every 24h sampling detects total grease weight in unit nutrient solution, and ARA purity.
Two groups of experimental programs all adopt stream liquid feeding sugar, potassium primary phosphate during the fermentation, with the concentration of controlling sugar and phosphorus respectively at 35～45g/L, 0.08～0.12g/L.Before air flow quantity 72h, be 2.0m
3/ h, is adjusted to 3.0m after 72h
3/ h.Experimental result is as table 1.
Table 1 liquid seeds with admittedly expect seed lab scale fermentation results
Compare with traditional liquid seed culture method, the seed culture mode that present method adopts has following advantage: 1. the growth vigor of bacterium cell is strong, synchronism is better; 2. bacterial classification physiological status is stable, can keep stable throughput; Although 3. increased the seed culture time, can shorten fermentation period, energy efficient, and can obtain higher ARA output and purity.
2, admittedly expect under seed culture method, in fermenting process, effect is controlled in the segmentation of pH value:
Seed and fermention medium are as previously mentioned.
Idiographic flow: under the bacterial classification of having grown on PDA substratum is washed by 20mL stroke-physiological saline solution, make bacteria suspension, in the liquid shaking bottle substratum that to be seeded to loading amount that above-mentioned sterilizing is good be 50mL, in 28 ℃, 180rpm constant-temperature table is cultivated 2 days.At the cultured thalline of liquid shaking bottle, by sterile gauze, filter and collect thalline, through sterilized water washing 2 times, with electrical grinding machine, be ground into length at 1400～1600 μ m, inoculate to the good solid material substratum of above-mentioned sterilizing, fully stir bacterial classification is uniformly distributed in substratum, and spread out even material, in 28 ℃, cultivate 3 days.Between incubation period, during every 24h, moisturizing 2mL, to maintain suitable relative humidity, and stirs stirring.400mL stroke-physiological saline solution is poured in cultured Kolle flask of admittedly expecting seed, and fully vibration, to wash lower spore.Spore liquid, by 1.2% inoculum size access 30L seeding tank, is inoculated to rear nutrient solution volume 15L, 28 ℃, pH value 6.0～6.5, rotating speed 100rpm, tank pressure 0.03MPa, air flow 1.0m
3/ h cultivates 2 days; Secondary seed solution, by 10% inoculum size access 100L fermentor tank, is inoculated to rear nutrient solution volume 60L, 28 ℃, pH value 6.0～6.5, rotating speed 60rpm, tank pressure 0.03MPa, air flow 2.0～3.0m
3/ h.In fermenting process, adopt stream liquid feeding sugar, potassium primary phosphate, with the concentration of controlling sugar and phosphorus respectively at 35～45g/L, 0.08～0.12g/L.Before air flow quantity 72h, be 2.0m
3/ h, is adjusted to 3.0m after 72h
3/ h.
Organize 1.: 48h before fermentation, use sodium hydroxide and dilute hydrochloric acid to regulate pH value, make it to be stabilized between 6.0～6.5.After fermentation 48h, be controlled between 7.5～8.0.Fermentation culture 8 days.After fermentation 72h, every 24h sampling detects total grease weight in unit nutrient solution, and ARA purity.
Organize 2.: 48h before fermentation, use trisodium citrate and Citric acid monohydrate Food grade to regulate pH value, make it to be stabilized between 6.0～6.5.After fermentation 48h, be controlled between 7.5～8.0.Fermentation culture 8 days.After fermentation 72h, every 24h sampling detects total grease weight in unit nutrient solution, and ARA purity.Experimental result is as table 2.
Table 2 fermenting process regulates pH value lab scale fermentation results
By table 2, obtained, fermenting process carries out the segmentation of pH value to be controlled, and is conducive to the accumulation of ARA grease; And it is better to use trisodium citrate and Citric acid monohydrate Food grade to carry out pH value regulating effect.
Claims (4)
1. utilize and admittedly expect that culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: under the bacterial classification of having grown on PDA substratum is washed by 20mL stroke-physiological saline solution, make bacteria suspension, in the liquid shaking bottle substratum that to be seeded to loading amount that sterilizing is good be 50mL, in 28 ℃, 180rpm constant-temperature table is cultivated 2 days, in liquid shaking bottle, cultured thalline is collected thalline by filtration, through sterilized water, wash, with electrical grinding machine, be ground into length at 1000～2000 μ m, inoculate to the good solid material substratum of sterilizing, fully stir bacterial classification is uniformly distributed in substratum, and spread out even material, in 28 ℃, cultivate 3～5 days, between incubation period, during every 24h, moisturizing 2mL is to maintain suitable relative humidity, and stir stirring, 400mL stroke-physiological saline solution is poured in cultured Kolle flask of admittedly expecting seed, fully vibration, to wash lower spore, spore liquid is by carrying out amplification culture in 1.2% inoculum size access 30L seeding tank, secondary seed solution is carried out fermentative production arachidonic acid with 10% inoculum size access 100L fermentor tank again, described liquid shaking bottle substratum: glucose 20～60g/L, yeast powder 5～20g/L, magnesium sulfate 0.1～1g/L, potassium primary phosphate 0.1～2g/L, describedly admittedly expect substratum: take rice bran, corn particle is raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration mixes by 10: 1～5 mass ratio, in Stainless Steel Kettle after boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilizing 30min.
2. by utilization claimed in claim 1, admittedly expect that culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: in described solid material substratum, the mass ratio of material rice bran used, corn particle is 10: 1～5.
3. by utilization claimed in claim 1, admittedly expect that culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: described fermentation pH carries out segmentation control, front 48h makes pH value stabilization between 6.0～6.5, then makes pH value stabilization between 7.0～9.0.
4. by utilization claimed in claim 3, admittedly expect that culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: described fermenting process carries out the segmentation of pH value and controls and be used in that to regulate the material of pH value be citric acid and Citrate trianion.
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| CN103571896B (en) * | 2013-11-18 | 2016-05-25 | 嘉必优生物技术（武汉）股份有限公司 | A kind of Mortierella alpina mutant strain that utilizes is produced the method for arachidonic acid oil and the arachidonic acid oil of production thereof |
| CN104326780B (en) * | 2014-10-24 | 2016-08-31 | 汤阴森奇生物技术有限公司 | A kind of technique utilizing superseded maize seed to make edible fungus species culture medium |
| CN105112466A (en) * | 2015-09-23 | 2015-12-02 | 润科生物工程（福建）有限公司 | Method for preparing arachidonic acid through fermentation with addition of product accelerating agent |
| CN110129384B (en) * | 2018-02-08 | 2022-07-01 | 浙江海正药业股份有限公司 | Preparation method of arachidonic acid |
| CN109673815B (en) * | 2018-12-29 | 2022-08-09 | 嘉必优生物技术（武汉）股份有限公司 | Functional feed rich in arachidonic acid and beta carotene prepared by fermentation and preparation method thereof |
| WO2022104673A1 (en) * | 2020-11-20 | 2022-05-27 | 内蒙古金达威药业有限公司 | Method for producing arachidonic acid |
| CN112322507A (en) * | 2020-12-09 | 2021-02-05 | 盛世荣恩生物科技有限公司 | Strain culture medium of Mortierella pseudomorpha and culture method thereof |
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| US5658767A (en) * | 1991-01-24 | 1997-08-19 | Martek Corporation | Arachidonic acid and methods for the production and use thereof |
| CN101109015B (en) * | 2007-07-09 | 2011-05-04 | 南京工业大学 | Method of preparing arachidonic acid oil and fat |
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