CN102925503A - 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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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 the microbial technology field, refer in particular to the Mortierella alpina thalline after liquid culture, be seeded to solid material substratum after shattering, cultivate a large amount of spores of breeding, prepare arachidonic method through liquid fermenting again.
Technical background
Arachidonic acid (ARA) is the synthetic important precursor of human body prostaglandin(PG), has esterified cholesterol, anticoagulant, increase blood vessel elasticity, reduction blood viscosity, regulates a series of physiologically actives such as leukocyte function, raising immunizing power.Past, commercial ARA was mainly derived from pork liver and fish oil, but content is extremely low and unstable, and development cost are expensive, and the demand, particularly milk preparation and the required ARA of pharmaceutical industry that are difficult to satisfied society measure very large.At the eighties initial stage, people find that in succession some filamentous fungus and microalgae contain arachidonic acid.Fast growth, grease and ARA content are high, fermentation preparation technology is fairly simple and be not subjected to the advantage such as raw material restriction because microorganism has, and 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 characteristics 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 of Effective Raise fermentation level has market outlook.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of utilization and expects that admittedly the 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 employed bacterial classification of present method is the common Mortierella alpina of 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 used product spore substratum of the present invention is for admittedly expecting substratum.This substratum is characterised in that, take rice bran, corn grain as raw material, adds an amount of inorganic salt formulated.
The present invention is in the 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 the 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 the pH value be regulated, make the 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 the 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 subjected to the 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: will wash with the 20mL stroke-physiological saline solution at the long good bacterial classification of PDA substratum, make bacteria suspension, good loading amount is in the liquid shaking bottle substratum of 50mL to be seeded to sterilization, in 28 ℃, the 180rpm constant-temperature table was cultivated 2 days, cultured thalline is collected thalline by filtering in liquid shaking bottle, wash through sterilized water, be ground into length at 1000～2000 μ m with electrical grinding machine, inoculate to the good solid material substratum of sterilization, fully stirring is uniformly distributed in the substratum bacterial classification, and the even material in stand, cultivated 3～5 days in 28 ℃, between incubation period, moisturizing 2mL is to keep suitable relative humidity during every 24h, and stirring stirring, the 400mL stroke-physiological saline solution is poured in the cultured Kolle flask of admittedly expecting seed, fully vibration, washing spore, spore liquid is by carrying out amplification culture in 1.2% the inoculum size access 30L seeding tank, and secondary seed solution is carried out the 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, sal epsom 0.1～1g/L, potassium primary phosphate 0.1～2g/L; Describedly admittedly expect substratum: take rice bran, corn particle as raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration compares mixing by 10: 1～5 quality, in Stainless Steel Kettle behind the boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in the 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilization 30min.
Concrete steps are as follows:
1, utilize liquid nutrient medium to cultivate the Mortierella alpina thalline:
1. liquid shaking bottle substratum: glucose 20～60g/L, yeast powder 5～20g/L, sal epsom 0.1～1g/L, potassium primary phosphate 0.1～2g/L.Substratum is through 121 ℃ of sterilization 30min.
2. will wash with the 20mL stroke-physiological saline solution at the long good bacterial classification of PDA substratum, and make bacteria suspension, and be seeded in the liquid shaking bottle substratum that the good loading amount of above-mentioned sterilization is 50mL, in 28 ℃, the 180rpm constant-temperature table was cultivated 2 days.
2, collect above-mentioned cultured thalline, be seeded to after shattering in the solid material substratum:
1. take rice bran, corn particle as raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration compares mixing by 10: 1～5 quality, in Stainless Steel Kettle behind the boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in the 1000mL Kolle flask, every bottled amount is 200g, makes solid material substratum in 121 ℃ of sterilization 30min.
2. will collect thalline by filtering at the cultured thalline of liquid shaking bottle, wash through sterilized water, be ground into length at 1000～2000 μ m with electrical grinding machine, inoculate to the good solid material substratum of above-mentioned sterilization, fully stirring is uniformly distributed in the substratum bacterial classification, and the even material in stand, cultivated 3～5 days in 28 ℃.Between incubation period, moisturizing 2mL and stirs stirring keeping suitable relative humidity during every 24h.
3, above-mentioned utilization expects that admittedly the culture medium culturing Mortierella alpina prepares arachidonic method, is characterized in that: described take carbon source as main solid materials as: rice bran, corn particle, the mass ratio of rice bran, corn particle is 10: 1～5.
4, fermentation preparation arachidonic acid: the 400mL stroke-physiological saline solution is poured in the cultured Kolle flask of admittedly expecting seed, and fully vibration is to wash spore.Spore liquid is by carrying out amplification culture in 1.2% the inoculum size access 30L seeding tank, and secondary seed solution is carried out the 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 the pH value stabilization between 6.0～6.5, then makes the pH value stabilization between 7.0～9.0.
6, being used in the material of regulating the pH value in the fermenting process is 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 expect that admittedly the substratum sedimentation is good, the spore of production is easy to separate with mycelium with substratum, and the seed liquor miospore amount that provides 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 the ARA fermentation level.
Description of drawings
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.
The PDA inclined-plane makes, and is that industry personage is familiar with.
Liquid shaking bottle substratum: glucose 30g/L, yeast powder 10g/L, sal epsom 0.5g/L, potassium primary phosphate 1.0g/L.
Secondary seed medium: glucose 30g/L, yeast powder 10g/L, sal epsom 0.5g/L, potassium primary phosphate 1.0g/L.
Fermention medium: glucose 30g/L, yeast powder 15g/L, ammonium sulfate 0.5g/L, sal epsom 0.5g/L, zinc sulfate 0.01g/L, potassium primary phosphate 1.0g/L, Sodium Glutamate 0.8g/L.
Idiographic flow: wash the PDA slant pore with the 20mL stroke-physiological saline solution, in the access sterilization shaking flask that the 400mL seed culture medium is housed well, in 28 ℃ of constant-temperature tables, 180rpm cultivated 3 days; Cultured shake-flask seed is inoculated rear nutrient solution volume 15L by in 1.2% the inoculum size access 30L seeding tank, and 28 ℃, pH value 6.0～6.5, rotating speed 100rpm, tank pressure 0.03Mpa, air flow 1.0m
3/ h cultivated 2 days; Secondary seed solution by in the 10% inoculum size access 100L fermentor tank, is inoculated 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.Behind the fermentation 72h, every 24h sampling detects total grease weight in the 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 as raw material, the mass ratio of rice bran, corn particle is 3: 1, being the 2g/L dipotassium hydrogen phosphate solution with concentration compares mixing by 5: 1 quality, in Stainless Steel Kettle behind the boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in the 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilization 30min.
PDA substratum, liquid shaking bottle substratum, secondary seed medium and fermention medium are as previously mentioned.
Idiographic flow: will wash with the 20mL stroke-physiological saline solution at the long good bacterial classification of PDA substratum, and make bacteria suspension, and be seeded in the liquid shaking bottle substratum that the good loading amount of above-mentioned sterilization is 50mL, in 28 ℃, the 180rpm constant-temperature table was cultivated 2 days.Filter the collection thalline at the cultured thalline of liquid shaking bottle by sterile gauze, through sterilized water washing 2 times, be ground into length at 1400～1600 μ m with electrical grinding machine, inoculate to the good solid material substratum of above-mentioned sterilization, fully stirring is uniformly distributed in the substratum bacterial classification, and the even material in stand, cultivated 3 days in 28 ℃.Between incubation period, moisturizing 2mL and stirs stirring keeping suitable relative humidity during every 24h.The 400mL stroke-physiological saline solution is poured in the cultured Kolle flask of admittedly expecting seed, and fully vibration is to wash spore.Spore liquid by in 1.2% the inoculum size access 30L seeding tank, is inoculated 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 cultivated 2 days; Secondary seed solution by in the 10% inoculum size access 100L fermentor tank, is inoculated 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.Behind the fermentation 72h, every 24h sampling detects total grease weight in the 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.Be 2.0m before the air flow quantity 72h
3/ h is adjusted to 3.0m behind the 72h
3/ h.Experimental result such as table 1.
Table 1 liquid seeds with admittedly expect seed lab scale fermentation results
Compare with the 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. the 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 the seed culture method, in the fermenting process, pH value segmentation control effect:
Seed and fermention medium are as previously mentioned.
Idiographic flow: will wash with the 20mL stroke-physiological saline solution at the long good bacterial classification of PDA substratum, and make bacteria suspension, and be seeded in the liquid shaking bottle substratum that the good loading amount of above-mentioned sterilization is 50mL, in 28 ℃, the 180rpm constant-temperature table was cultivated 2 days.Filter the collection thalline at the cultured thalline of liquid shaking bottle by sterile gauze, through sterilized water washing 2 times, be ground into length at 1400～1600 μ m with electrical grinding machine, inoculate to the good solid material substratum of above-mentioned sterilization, fully stirring is uniformly distributed in the substratum bacterial classification, and the even material in stand, cultivated 3 days in 28 ℃.Between incubation period, moisturizing 2mL and stirs stirring keeping suitable relative humidity during every 24h.The 400mL stroke-physiological saline solution is poured in the cultured Kolle flask of admittedly expecting seed, and fully vibration is to wash spore.Spore liquid by in 1.2% the inoculum size access 30L seeding tank, is inoculated 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 cultivated 2 days; Secondary seed solution by in the 10% inoculum size access 100L fermentor tank, is inoculated 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.Adopt stream liquid feeding sugar, potassium primary phosphate in the fermenting process, with the concentration of controlling sugar and phosphorus respectively at 35～45g/L, 0.08～0.12g/L.Be 2.0m before the air flow quantity 72h
3/ h is adjusted to 3.0m behind the 72h
3/ h.
Organize 1.: 48h before the fermentation, use sodium hydroxide and dilute hydrochloric acid to regulate the pH value, make it to be stabilized between 6.0～6.5.Behind the fermentation 48h, be controlled between 7.5～8.0.Fermentation culture 8 days.Behind the fermentation 72h, every 24h sampling detects total grease weight in the unit nutrient solution, and ARA purity.
Organize 2.: 48h before the fermentation, use trisodium citrate and Citric acid monohydrate Food grade to regulate the pH value, make it to be stabilized between 6.0～6.5.Behind the fermentation 48h, be controlled between 7.5～8.0.Fermentation culture 8 days.Behind the fermentation 72h, every 24h sampling detects total grease weight in the unit nutrient solution, and ARA purity.Experimental result such as table 2.
Table 2 fermenting process is regulated pH value lab scale fermentation results
Got by table 2, fermenting process carries out pH value segmentation control, 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 expect that admittedly the culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: will wash with the 20mL stroke-physiological saline solution at the long good bacterial classification of PDA substratum, make bacteria suspension, good loading amount is in the liquid shaking bottle substratum of 50mL to be seeded to sterilization, in 28 ℃, the 180rpm constant-temperature table was cultivated 2 days, cultured thalline is collected thalline by filtering in liquid shaking bottle, wash through sterilized water, be ground into length at 1000～2000 μ m with electrical grinding machine, inoculate to the good solid material substratum of sterilization, fully stirring is uniformly distributed in the substratum bacterial classification, and the even material in stand, cultivated 3～5 days in 28 ℃, between incubation period, moisturizing 2mL is to keep suitable relative humidity during every 24h, and stirring stirring, the 400mL stroke-physiological saline solution is poured in the cultured Kolle flask of admittedly expecting seed, and fully vibration is to wash spore, spore liquid is by carrying out amplification culture in 1.2% the inoculum size access 30L seeding tank, secondary seed solution is carried out the fermentative production arachidonic acid, described liquid shaking bottle substratum with 10% inoculum size access 100L fermentor tank again: glucose 20～60g/L, yeast powder 5～20g/L, sal epsom 0.1～1g/L, potassium primary phosphate 0.1～2g/L; Describedly admittedly expect substratum: take rice bran, corn particle as raw material, being 1～3g/L dipotassium hydrogen phosphate with concentration compares mixing by the quality of 10:1～5, in Stainless Steel Kettle behind the boiling 30min, spread the cleaved surface moisture that dries in the air out, be sub-packed in the 1000mL Kolle flask, every bottled amount is 200g, in 121 ℃ of sterilization 30min.
2. expect admittedly that by utilization claimed in claim 1 the culture medium culturing Mortierella alpina prepares arachidonic method, is characterized in that: the used material of described solid medium is: rice bran, corn particle, the mass ratio of rice bran, corn particle is 10: 1～5.
3. expect admittedly that by utilization claimed in claim 1 the culture medium culturing Mortierella alpina prepares arachidonic method, it is characterized in that: described fermentation pH carries out segmentation control, front 48h makes the pH value stabilization between 6.0～6.5, then makes the pH value stabilization between 7.0～9.0.
4. expect admittedly that by utilization claimed in claim 3 the culture medium culturing Mortierella alpina prepares arachidonic method, is characterized in that: described fermenting process carries out pH value segmentation control, and to be used in the material of regulating the pH value be citric acid and Citrate trianion.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103571896A (en) * | 2013-11-18 | 2014-02-12 | 嘉必优生物工程（武汉）有限公司 | Method for producing arachidonic acid grease by utilizing mortierella alpina mutant strain, and arachidonic acid grease produced by method |
| CN104326780A (en) * | 2014-10-24 | 2015-02-04 | 汤阴森奇生物技术有限公司 | Technique for making edible fungus strain culture medium from obsolete eliminated corn seeds |
| 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 |
| CN109673815A (en) * | 2018-12-29 | 2019-04-26 | 嘉必优生物技术（武汉）股份有限公司 | Fermentation preparation is rich in the functional feed and preparation method of arachidonic acid and bata-carotene |
| CN110129384A (en) * | 2018-02-08 | 2019-08-16 | 浙江海正药业股份有限公司 | A kind of arachidonic preparation method |
| 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 |
| 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 |
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| CN103571896A (en) * | 2013-11-18 | 2014-02-12 | 嘉必优生物工程（武汉）有限公司 | Method for producing arachidonic acid grease by utilizing mortierella alpina mutant strain, and arachidonic acid grease produced by method |
| CN103571896B (en) * | 2013-11-18 | 2016-05-25 | 嘉必优生物技术（武汉）股份有限公司 | Method for producing arachidonic acid oil by utilizing mortierella alpina mutant strain and arachidonic acid oil produced by method |
| CN104326780A (en) * | 2014-10-24 | 2015-02-04 | 汤阴森奇生物技术有限公司 | Technique for making edible fungus strain culture medium from obsolete eliminated corn seeds |
| 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 |
| CN110129384A (en) * | 2018-02-08 | 2019-08-16 | 浙江海正药业股份有限公司 | A kind of arachidonic preparation method |
| 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 |
| CN109673815A (en) * | 2018-12-29 | 2019-04-26 | 嘉必优生物技术（武汉）股份有限公司 | Fermentation preparation is rich in the functional feed and preparation method of arachidonic acid and bata-carotene |
| 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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