CN101538592B - Method for producing DHA by Crypthecodinium cohnii industrial fermentation - Google Patents
Method for producing DHA by Crypthecodinium cohnii industrial fermentation Download PDFInfo
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- CN101538592B CN101538592B CN2009100618338A CN200910061833A CN101538592B CN 101538592 B CN101538592 B CN 101538592B CN 2009100618338 A CN2009100618338 A CN 2009100618338A CN 200910061833 A CN200910061833 A CN 200910061833A CN 101538592 B CN101538592 B CN 101538592B
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Abstract
The invention discloses a method for producing grease containing triglyceride type DHA by Crypthecodinium cohnii industrial fermentation. The method comprises the following steps: taking the Crypthecodinium cohnii as a strain, employing a culture medium comprising a carbon source, a nitrogen source and inorganic salts, and obtaining the DHA grease by fermentation; the nitrogen source contains hydrolytic liquor of slow-release bean pulp powder as nitrogen source. The grease produced by the fermentation contains more than 50% of DHA, among which EPA content is below 1%, and the DHA productivity reaches 4.6g/L per day. The DHA has high output, high purity, safe and reliable quality, low production cost, stable production, and little or no EPA; The method can produce environment-friendly and safe high DHA content triglyceride type DHA grease in quantity with low cost.
Description
Technical field
The present invention relates to microbial fermentation production method, be specifically related to utilize the production of Kou Shi Crypthecodinium cohnii strain fermentation to contain the docosahexenoic acid compound lard.
Background technology
Docosahexenoic acid (cis-4,7,10; 13; 16,19-docosahexaenoic acid is called for short DHA) be Europe rice loud, high-pitched sound 3 serial long chain polyunsaturated fatty acidss; Be brain, amphiblestroid important structure material, can also regulate central nervous system function, prevention and treatment cardiovascular disorder, diminish inflammation, suppress cancer.
The commercial source of DHA mainly is fish oil and little algae at present.The output of fish oil and DHA content are all unstable, and extract is mingled with fishy smell, and containing of from fish oil, extracting also contain timnodonic acid (EPA) in the DHA compound lard, and according to research, EPA has adverse influence to infant's growth.Present marine fishery resources extracts DHA and causes great waste near exhausted from fish oil.The cultivation of little bath needs illumination and carbonic acid gas, and production technique is numerous and diverse, and cost is higher.Along with the increase of population, and people are to the active demand of health, and the whole world need be sought a large amount of DHA and newly originate the corresponding increase of demand of DHA, and preferably the DHA in this source does not contain EPA.
Application (patent) number: 200710025079.3 patent discloses a kind of from Crypthecodinium cohnii, the extraction and the method for refining DHA; Lay particular emphasis on from Crypthecodinium cohnii and extract and refining DHA; The fermentative prepn DHA of Crypthecodinium cohnii is related to less, and DHA content is 40-50% in its disclosed extract.
Application (patent) number: 200610125476.3 patent disclose a kind of from the Kou Shi Crypthecodinium cohnii method of fermented extracted DHA.Its disclosed output is that DHA content is at 30%-50% in the alginic cell, and the alginic cell of acquisition is 20-40g/L, and the alginic cell oleaginousness is 20-50%, and its productivity is no more than 3.5 grams per liters. day.
Summary of the invention
It is bacterial classification with Kou Shi Crypthecodinium cohnii (Crypthecodinium cohnii) original strain that problem to be solved by this invention provides a kind of; Industrial fermentation is produced the method for docosahexaenoic acid grease, and this method can low-cost, the high-load triglyceride type DHA of mass production grease.
Technical scheme provided by the invention is:
Produce the greasy method of docosahexenoic acid (DHA) with Kou Shi Crypthecodinium cohnii (Crypthecodinium cohnii) industrial fermentation; Comprise that with the Kou Shi Crypthecodinium cohnii be bacterial classification; Employing comprises the substratum of carbon source, nitrogenous source and inorganic salt, makes docosahexaenoic acid grease through fermentation; Contain slow nitrogenous source bean cake powder hydrolysis juice in the said nitrogenous source.Make DHA content through fermentation and be higher than 50% grease, wherein timnodonic acid (EPA) content is below 1%.
Above-mentioned slow nitrogenous source is obtained by laxative remedy: bean cake powder is dissolved in the water, regulates pH to 9.0-10.0, and be warming up to 70-80 ℃, insulation 8-10h crosses the leaching clear liquid and obtains the slow nitrogenous source; Wherein the consumption weight ratio of bean cake powder and water is 1: 3-1: 10; The consumption of slow nitrogenous source is the 4.0-8.0% of substratum gross weight.
Also contain growth factor bean sprouts juice in the above-mentioned substratum, the consumption of growth factor is the 2.0-4.0% of substratum gross weight; Growth factor is obtained by laxative remedy: soybean sprout is cleaned, drained, get bean sprouts juice with the squeezing machine squeezing and obtain growth factor.
The Kou Shi Crypthecodinium cohnii of bacterial classification for cultivating, transfer and go into to shake a bottle enlarged culturing, obtain through the one-level enlarged culturing more then through slant activation used in said fermentation; Or Kou Shi Crypthecodinium cohnii for cultivating, transfer and go into to shake a bottle enlarged culturing, after one-level enlarged culturing and secondary enlarged culturing, obtain again then through slant activation.
Above-mentioned shaking in the used substratum of bottle enlarged culturing contained bush sugar alcohol and l-arginine, and bush sugar alcohol and arginic consumption are respectively the 0.05-0.15% and the 0.05-0.15% of substratum gross weight.
The present invention can replenish bacterial classification when fermentation culture 48-60h, the benefit of bacterial classification is gone into the 8-10% that amount is a fermentating liquid volume.
The present invention also can be during the fermentation, when the reducing sugar content of fermented liquid supplementary carbon source during at 2-3wt%.
The present invention can increase dissolved oxygen gradually in the fermentation culture process: initial air flow is 0.5VVM, improves air flow 0.02VVM behind later on each supplementary carbon source.The mode that increases progressively through dissolved oxygen had both increased dissolved oxygen, stablized metabolic balance again.
In the fermentation whole process, keep the pH nature.
Preferably, the present invention gets into stationary phase in fermentation, and (it is 66-72h that the used bacterial classification of the present invention gets into the time of stationary phase) preceding controlled temperature is 29-31 ℃, and controlled temperature is 22-24 ℃ after getting into stationary phase.
The present invention utilizes the method for Kou Shi Crypthecodinium cohnii fermentative prodn DHA, and DHA content is 51-55% in its extract, and EPA content wherein is no more than 1%, and DHA productivity can reach 4.6 grams per liters. day, greatly reduce the fermentation costs of DHA.
Technical indicator of the present invention is all apparently higher than the technical indicator of prior art, and prepared DHA quality is more excellent, and cost is lower, helps the large-scale industrial production manufacturing, and reduces the consumption threshold of DHA, is beneficial to applying of DHA.
Embodiment
The present invention adopts conventional fermentation method to produce docosahexaenoic acid grease through optimizing substratum and culture condition.
The shake-flask culture based formulas of optimizing is following:
The shake-flask culture based formulas (in gram/100ml substratum) that table is optimized
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 2.0-4.0 | Bean sprouts juice | 2.0-4.0 |
| Yeast extract paste | 0.3-0.6 | Sodium Glutamate | 0.8-1.2 |
| Glucose | 2.0-5.0 | Molasses | 0.8-1.2 |
| Potassium primary phosphate | 0.2-0.6 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.2-0.8 |
| Sea salt | 0.3-1.2 | L-arginine | 0.05-0.15 |
| The bush sugar alcohol | 0.05-0.15 |
One-level, secondary and the fermentative medium formula optimized are following:
One-level, secondary and fermentative medium formula (in gram/100ml substratum) that table is optimized
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 4.0-8.0 | Bean sprouts juice | 2.0-4.0 |
| Yeast extract paste | 0.4-0.8 | Sodium Glutamate | 1.0-3.0 |
| Glucose | 4.0-8.0 | Steeping water | 0.2-0.7 |
| Potassium primary phosphate | 0.2-0.6 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.2-0.8 |
| Sea salt | 0.3-1.2 |
Get the good slant strains of activation after shaking bottle, firsts and seconds seeding tank enlarged culturing; Inoculum size according to 8-10% inserts in the fermentor tank, pH nature, 29-31 ℃ of initial controlling tank temperature; Regulate leavening temperature to 22-24 ℃ behind the fermentation 66-72h; The initial air flow is 0.5VVM, and concentration of reduced sugar is more than 2.0%, when the reducing sugar content of fermented liquid is mended sugar (supplementary carbon source) during at 2-3wt% in the omnidistance control of the fermentation fermented liquid; Each sugar back of mending increases air flow quantity 0.02VVM, when fermenting to 48-60h, in fermented liquid, replenishes the bacterial classification of 8-10%.
Zymotechnique is produced DHA thus, and oleaginousness is 60-80% in the dry mycelium, and DHA content is 51-55% in the grease, and wherein EPA content is no more than 1.0%, DHA productivity reach 4.6 grams/(rise. day)
In the methods of the invention, be suitable for producing cultivation Kou Shi Crypthecodinium cohnii under the condition that contains DHA oil.Generally, the fungus culture technology is well-known to those skilled in the art, and these technology can be used for the inventive method.Usually adopt the slant strains inoculation to shake bottle, shaking flask inoculation kind first class seed pot, first class seed pot inoculation secondary seed jar, the compound lard that the production of fermentation culture Kou Shi Crypthecodinium cohnii contains triglyceride type DHA is carried out in the technical process of secondary seed jar inoculation fermentation jar.
The composition of fermention medium can be inequality, but all contain carbon source and nitrogenous source.Carbon source is a glucose preferably, and its content is about 40-80 gram in every liter of fermention medium.Consumption can be with final cultures density difference.Operable carbon source comprises glucose, the low-cost conventional carbon source that molasses, maize treacle or other are used to ferment.Those skilled in the art are easy to confirm the suitable amounts of these carbon sources.Usually, in culturing process, need in batches supplementary carbon source in addition, to satisfy the consumption of mikrobe to carbon source, the whole carbon sources of disposable adding then can suppress microbial growth.
Nitrogenous source is selected organic and inorganic nitrogen-sourced usually.Organic nitrogen source comprises yeast extract paste, yeast extract, peptone etc., inorganic nitrogen-sourced SODIUMNITRATE, nitric acid ammonia, the ammonium sulfate etc. of comprising.Not only can satisfy the demand of thalli growth breeding but produce the slow organic nitrogen source bean cake powder hydrolysis juice of finding the use hydrolysis treatment in the DHA grease, and can effectively improve oil offtake, and improve the DHA content in the grease at this fermentation research.Employed bean cake powder hydrolysis juice can disposable adding when preparing culture medium.Wherein bean cake powder hydrolysis juice preparation method is: with bean cake powder and water according to 1: 3-1: after 10 the ratio preparation dissolving, regulate pH to 9.0-10.0, and be warming up to 70-80 ℃, and insulation 8-10h, it is subsequent use to cross the leaching clear liquid.
The present invention adds somatomedin in substratum, with the growth of promotion thalline, and improve DHA content and productive rate.Originally discover, in fermention medium, add the growth factor of 2-4% bean sprouts juice, not only can effectively promote the growth of thalline as the fermentation of Kou Shi Crypthecodinium cohnii, and content and the productive rate of raising DHA that can be higher.
Whether add trace element in the substratum of the present invention result of the present invention is not had substantial effect.Said trace element is a vitamins B
1, vitamins B
6And vitamins B
12In one or more, when when wherein a kind of, addition is the 0.002-0.01% of substratum weight; When for wherein several kinds the time, the addition of any one component is the 0.002-0.01% of substratum weight.
Usually, in shaking bottle, prepare the shake-flask culture base according to the culture medium prescription of optimizing.For example, can be according to adding 40 gram dregs of beans hydrolysis juice, 40 gram bean sprouts juice, 5 gram yeast extract pastes in every liter of substratum; 10 gram Sodium Glutamates, 50 gram glucose, 10 gram molasses, 4 gram potassium primary phosphates; 3 gram MAGNESIUM SULPHATE HEPTAHYDRATE 99.5s, 10 gram sea salt, 1 gram bush sugar alcohol; 1 gram l-arginine, and add water to volume requiredly, keep pH nature.
(121 ℃, 30min 0.1-0.12Mpa), and inserts cultured Kou Shi Crypthecodinium cohnii bacterial classification and cultivates after being cooled to about 30 ℃ through high-temperature sterilization with prepared culture medium.
Usually, in fermentor tank, prepare seed and fermention medium according to one-level, secondary and the fermentative medium formula optimized.For example, can be according to adding 60 gram dregs of beans hydrolysis juice, 40 gram bean sprouts juice, 5 gram yeast extract pastes in every liter of substratum; 20 gram Sodium Glutamates, 60 gram glucose, 5 gram steeping waters, 5 gram potassium primary phosphates; 6 gram MAGNESIUM SULPHATE HEPTAHYDRATE 99.5s, 10 gram sea salt, and add water to volume requiredly, keep pH nature.
(121 ℃, 30min 0.1-0.12Mpa), and inserts cultured Kou Shi Crypthecodinium cohnii bacterial classification and cultivates after being cooled to about 30 ℃ through high-temperature sterilization with prepared culture medium.
Bubbling air carries out gaseous interchange in fermented liquid, supplies with the needed oxygen of thalli growth, in the fermentation culture process, increases dissolved oxygen gradually, and the mode that increases progressively through dissolved oxygen had both increased dissolved oxygen, stablized metabolic balance again.Usually be 0.5VVM at fermentation initial air flow, each sugar back of mending, back increases air flow quantity 0.02VVM.
Inoculum size for well, is preferably used the inoculum size of about 10% volume with 8-10%.
Need monitoring nutrition consumption level in the fermenting process.Reducing sugar content preferably is not less than 2.0% in the fermented liquid; Suggestion is when the reducing sugar content of fermented liquid additional glucose during at 2-3wt%.In a preferred embodiment, a fermentation period consumes every liter of substratum 150 gram glucose.
Leavening temperature generally is controlled at 22 to 31 ℃.Different steps control different temperature in fermentation; Generally be controlled at high slightly temperature in the starting stage of fermentation; Be preferably and be controlled at 29-31 ℃; Be beneficial to the growth and the breeding of thalline, induce DHA preferentially to produce the value of accumulation, preferably be controlled at 22-24 ℃ and temperature is controlled in the stage of oil and fat accumulation.
The bacterial classification of afterfermentation liquid long-pending 10% when fermenting to 48-60h.
In the whole process of fermentation, keep the pH nature, need not to control.
Gather in the crops the frond cell through centrifugal or filtering method after the fermentation ends; Extract through adding organic solvent-normal hexane behind the method broken wall of extruding or enzymolysis; After removing solvent, obtain crude oil again, crude oil obtains refining DHA grease after alkali refining, decolouring, deodorization.
Carry out one or multinomial improvement, can ferment obtains containing the triglyceride level quasi-grease that is higher than the 50%DHA residue, can obtain containing the triglyceride level quasi-grease of 55% DHA residue under the top condition.
In the embodiment of the best, bean cake powder hydrolysis juice concentration is 60g/l in the fermention medium, the about 6g/l of yeast extract paste concentration; The bean sprouts juice concentration is 40g/l, and concentration of sodium glutamate is 20g/l, the about 80g/l of glucose concn; Steeping water concentration is 5g/l, and the biphosphate potassium concn is 5g/l, and MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 concentration is 4g/l; The sea salt addition is 10g/l, and adds water to volume requiredly, keeps pH nature.The initial air flow is 0.5VVM; When reducing sugar content in the fermented liquid is mended into glucose during at 2-3wt%; Mend the sugar back increases air flow quantity 0.02VVM at every turn, when fermenting to 48 hours, mends the bacterial classification into fermentating liquid volume 10%, keeps the pH nature in the whole fermentation process.When amino nitrogen content is lower than 0.035% in the fermented liquid, finish fermentation, so can obtain DHA content and be 55% compound lard, and wherein EPA content is no more than 0.5%.
Through above optimizing fermentation, can obtain higher living weight, wherein contain 60% to 80% grease, wherein the DHA residue of triglyceride level form accounts for 51% to 55%, and EPA content wherein is less than 1%.
More than the present invention has been carried out comprehensive description, following indefiniteness embodiment is used to further specify the present invention.
The screening of embodiment 1. shake-flask culture bases
Prescription a strengthens source of slow release nitrogen-bean cake powder hydrolysis juice (g/100ml substratum)
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 4.0 | Sodium Glutamate | 0.8 |
| Yeast extract paste | 0.6 | Molasses | 1.0 |
| Glucose | 5.0 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Potassium primary phosphate | 0.6 | Sea salt | 1.0 |
Prescription b strengthens growth factor-bean sprouts juice (g/100ml substratum)
| Project | Content | Project | Content |
| Bean sprouts juice | 3.0 | Sodium Glutamate | 0.8 |
| Yeast extract paste | 0.6 | Molasses | 1.0 |
| Glucose | 5.0 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Potassium primary phosphate | 0.6 | Sea salt | 1.0 |
Prescription c strengthens bush sugar alcohol and l-arginine (g/100ml substratum)
| Project | Content | Project | Content |
| Yeast extract paste | 0.6 | Sodium Glutamate | 0.8 |
| Glucose | 5.0 | Molasses | 1.0 |
| Potassium primary phosphate | 0.6 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 | L-arginine | 0.1 |
| The bush sugar alcohol | 0.1 | Vitamins B 1 | 0.005 |
Prescription d Comprehensive Experiment (g/100ml substratum)
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 4.0 | Bean sprouts juice | 3.0 |
| Yeast extract paste | 0.6 | Sodium Glutamate | 0.8 |
| Glucose | 5.0 | Molasses | 1.0 |
| Potassium primary phosphate | 0.6 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 | L-arginine | 0.1 |
| The bush sugar alcohol | 0.1 |
Prescription e blank experiment (g/100ml substratum)
| Project | Content | Project | Content |
| Yeast extract paste | 0.6 | Sodium Glutamate | 0.8 |
| Glucose | 5.0 | Molasses | 1.0 |
| Potassium primary phosphate | 0.6 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 | Vitamins B 1 | 0.005 |
| Vitamins B 12 | 0.005 |
Prescription f common fermentation culture medium prescription (g/100ml substratum)
| Project | Content | Project | Content |
| Yeast extract paste | 0.5 | Sodium Glutamate | 1.0 |
| Glucose | 6.0 | Steeping water | 0.5 |
| Potassium primary phosphate | 0.4 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 | Vitamins B 1 | 0.005 |
| Vitamins B 12 | 0.005 |
Above-mentioned bean cake powder hydrolysis juice is made by laxative remedy: bean cake powder is dissolved in the water, regulates pH to 9.0-10.0, and be warming up to 75 ℃, insulation 9h crosses the leaching clear liquid and obtains bean cake powder hydrolysis juice; Wherein the consumption weight ratio of bean cake powder and water is 1: 6.
Bean sprouts juice is obtained by laxative remedy: fresh soybean sprout is cleaned, drained, get bean sprouts juice with the squeezing machine squeezing and obtain bean sprouts juice.
According to above-mentioned substratum a/b/c/d/e prescription, shake preparation 100ml substratum in the bottle at 500ml, the pH nature through high-temperature sterilization, cools off subsequent use.
The Kou Shi Crypthecodinium cohnii slant strains that activation is good inserts in the above-mentioned a/b/c/d/e shake-flask culture base, 30 ℃ of holding temperatures, and rotating speed 180rpm cultivates 48h, makes Kou Shi Crypthecodinium cohnii seed liquid.
According to the prescription of substratum f, preparation one-level and fermention medium insert the seed liquor that makes in the first class seed pot, feed the air of 0.25VVM, and holding temperature is 30 ℃, keeps the pH nature, fermentation culture 48h.
The above-mentioned seed liquor that spreads cultivation is added 50 liters of fermentor tanks according to 10% inoculum size, feed the air of 0.5VVM, holding temperature is 30 ℃; Keep pH nature, keep in the fermenting process that concentration of reduced sugar is more than 3% in the fermented liquid, when the reducing sugar amount is lower than 3%, replenish glucose; Increase air flow quantity 0.02VVM immediately, replenish 10% fresh bacterial classification when fermenting to 48h, ferment to 70h adjustment leavening temperature be 23 ℃; Fermentation to 100 hour, amino nitrogen concentration is 0.03% in the fermented liquid, stops fermentation.
Through centrifugation method results frond cell, extract through adding organic solvent-normal hexane behind the method broken wall of enzymolysis after the fermentation ends, after removing solvent, obtain crude oil again, crude oil obtains refining DHA grease after alkali refining, decolouring, deodorization.
Fermentation is the result see the following form:
| Living weight (g/l) | Total lipid content (%) | DHA content (%) | EPA content (%) | |
| A (source of slow release nitrogen) | 35.7 | 52.9 | 52.8 | 0.59 |
| B (growth factor) | 33.4 | 50.3 | 51.1 | 0.79 |
| C (l-arginine and bush sugar alcohol) | 30.2 | 50.4 | 51.5 | 0.87 |
| D (comprehensively) | 36.6 | 54.8 | 54.3 | 0.42 |
| E (blank) | 29.1 | 46.5 | 43.6 | 1.27 |
Can find out that by last table the experiment of strengthening each factor all is superior to blank on index, and prescription each item technical indicator of composite factor is the highest, all is superior to other, preferably as the shake-flask culture based formulas.
2 50 liters of jar fermentation culture of embodiment Kou Shi Crypthecodinium cohnii is produced DHA
Culture medium prescription (g/100ml substratum)
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 6.0 | Sodium Glutamate | 1.0 |
| Yeast extract paste | 0.5 | Steeping water | 0.5 |
| Glucose | 6.0 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Potassium primary phosphate | 0.4 | Sea salt | 1.0 |
Above-mentioned bean cake powder hydrolysis juice is obtained by laxative remedy: bean cake powder is dissolved in the water, regulates pH to 9.0, and be warming up to 80 ℃, insulation 8 is crossed the leaching clear liquid and is obtained bean cake powder hydrolysis juice; Wherein the consumption weight ratio of bean cake powder and water is 1: 3.The preparation of bean sprouts juice is with embodiment 1.
Add water to volume required, pH nature, high-temperature sterilization, be cooled to 30 ℃ subsequent use.
Bottle bacterial classification that shakes that embodiment 1 prescription d is prepared inserts according to 10% inoculum size and contains in 50 liters of fermentor tanks of 30 liters of substratum, feeds the air of 0.5VVM, and holding temperature is 30 ℃; Keep pH nature, keep in the fermenting process that concentration of reduced sugar is more than 3% in the fermented liquid, when the reducing sugar amount is lower than 3%, replenish glucose; Increase air flow quantity 0.02VVM immediately, amount to and mend 4% glucose, replenish 10% fresh bacterial classification when fermenting to 48h; Ferment to 68h adjustment leavening temperature be 23 ℃; Fermentation to 96 hour, amino nitrogen concentration is 0.03% in the fermented liquid, stops fermentation.
Through centrifugation method results frond cell, extract through adding organic solvent-normal hexane behind the method broken wall of enzymolysis after the fermentation ends, after removing solvent, obtain crude oil again, crude oil obtains refining DHA grease after alkali refining, decolouring, deodorization.
Carrying out the index of correlation analysis obtains: living weight is 41g/l, and fat content is 60%, and gas chromatographic detection DHA content is 53.4%, and EPA content is 0.6%, and DHA productivity is the 4.2g/ liter. day.Below be the moity of fermentation back gained compound lard:
C12:0 0.1023
C14:0 4.4136
C16:0 18.9548
C16:1 1.1578
C18:0 0.6745
C18:1 0.3457
C18:2 0.3173
C18:3 0.1084
C20:4 1.6938
C20:5 0.4124
C22:5 18.3747
C22:6 53.2673
3 500 liters of jar fermentation culture of embodiment Kou Shi Crypthecodinium cohnii is produced DHA
Culture medium prescription (g/100ml substratum)
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 8.0 | Bean sprouts juice | 3.0 |
| Yeast extract paste | 0.5 | Sodium Glutamate | 1.0 |
| Glucose | 6.0 | Steeping water | 0.5 |
| Potassium primary phosphate | 0.4 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 |
Above-mentioned bean cake powder hydrolysis juice is obtained by laxative remedy: bean cake powder is dissolved in the water, regulates pH to 10.0, and be warming up to 70 ℃, insulation 10h crosses the leaching clear liquid and obtains the slow nitrogenous source; Wherein the consumption weight ratio of bean cake powder and water is 1: 10.The preparation of bean sprouts juice is with embodiment 1.
Add water to volume required, pH nature, high-temperature sterilization, be cooled to 30 ℃ subsequent use.
Bottle bacterial classification that shakes that embodiment 1 prescription d is prepared inserts according to 10% inoculum size and contains in 50 liters of fermentor tanks of 30 liters of substratum, feeds the air of 0.25VVM, and holding temperature is 29 ℃, keeps the pH nature, and fermentation culture 48h obtains the seed liquor that spreads cultivation.
The above-mentioned seed liquor that spreads cultivation is inserted 500 liters of fermentor tanks that contain 300 liters of substratum according to 10% inoculum size, feed the air of 0.5VVM, holding temperature is 31 ℃; Keep pH nature, keep in the fermenting process that concentration of reduced sugar is more than 3% in the fermented liquid, when the reducing sugar amount is lower than 3%, replenish glucose; Increase air flow quantity 0.02VVM immediately, amount to and mend 4.5% glucose, replenish 10% fresh bacterial classification when fermenting to 48h; Ferment to 70h adjustment leavening temperature be 23 ℃; Fermentation to 100 hour, amino nitrogen concentration is 0.03% in the fermented liquid, stops fermentation.
Through centrifugation method results frond cell, extract through adding organic solvent-normal hexane behind the method broken wall of enzymolysis after the fermentation ends, after removing solvent, obtain crude oil again, crude oil obtains refining DHA grease after alkali refining, decolouring, deodorization.
Carrying out the index of correlation analysis obtains: living weight is 51g/l, and fat content is 72%, and gas chromatographic detection DHA content is 55.3%, and EPA content is 0.2%, and DHA productivity is the 4.6g/ liter. day.Below be the moity of fermentation back gained compound lard:
C12:0 0.1015
C14:0 4.4264
C16:0 17.2475
C16:1 1.1723
C18:0 0.6612
C18:1 0.3245
C18:2 0.3194
C18:3 0.1218
C20:4 1.6836
C20:5 0.2144
C22:5 18.3592
C22:6 55.3345
45 tons of jar fermentation culture of embodiment Kou Shi Crypthecodinium cohnii is produced DHA
Culture medium prescription (g/100ml substratum)
| Project | Content | Project | Content |
| Bean cake powder hydrolysis juice | 8.0 | Bean sprouts juice | 4.0 |
| Yeast extract paste | 0.7 | Sodium Glutamate | 1.0 |
| Glucose | 8.0 | Steeping water | 0.7 |
| Potassium primary phosphate | 0.4 | MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 | 0.3 |
| Sea salt | 1.0 | Vitamins B 1 | 0.005 |
| Vitamins B 12 | 0.005 |
The preparation of bean cake powder hydrolysis juice and bean sprouts juice is with embodiment 1.
Add water to volume required, pH nature, high-temperature sterilization, be cooled to 30 ℃ subsequent use.
Bottle bacterial classification that shakes that embodiment 1 prescription d is prepared inserts according to 10% inoculum size and contains in 50 liters of fermentor tanks of 30 liters of substratum, feeds the air of 0.25VVM, and holding temperature is 30 ℃, keeps the pH nature, and fermentation culture 48h obtains the one-level seed liquor that spreads cultivation.
The above-mentioned one-level seed liquor that spreads cultivation is inserted 500 liters of fermentor tanks that contain 300 liters of substratum according to 10% inoculum size, feed the air of 0.25VVM, holding temperature is 30 ℃, keeps the pH nature, and fermentation culture 48h obtains the secondary seed liquor that spreads cultivation.
The above-mentioned secondary seed liquor that spreads cultivation is inserted 5 tons of fermentor tanks that contain 3 tons of substratum according to 10% inoculum size, feed the air of 0.5VVM, holding temperature is 30 ℃; Keep pH nature, keep in the fermenting process that concentration of reduced sugar is more than 3% in the fermented liquid, when the reducing sugar amount is lower than 3%, replenish glucose; Increase air flow quantity 0.02VVM immediately, amount to and mend 4.0% glucose, replenish 10% fresh bacterial classification when fermenting to 55h; Ferment to 70h adjustment leavening temperature be 22 ℃; Fermentation to 115 hour, amino nitrogen concentration is 0.03% in the fermented liquid, stops fermentation.
Through centrifugation method results frond cell, extract through adding organic solvent-normal hexane behind the method broken wall of enzymolysis after the fermentation ends, after removing solvent, obtain crude oil again, crude oil obtains refining DHA grease after alkali refining, decolouring, deodorization.
Carrying out the index of correlation analysis obtains: living weight is 53g/l, and fat content is 68%, and gas chromatographic detection DHA content is 54.7%, and EPA content is 0.3%, and DHA productivity is the 4.5g/ liter. day.Below be the moity of fermentation back gained compound lard:
C12:0 0.1426
C14:0 4.4482
C16:0 17.5257
C16:1 1.1246
C18:0 0.6857
C18:1 0.3572
C18:2 0.3146
C18:3 0.1923
C20:4 1.6367
C20:5 0.3163
C22:5 18.6045
C22:6 54.7253
Claims (6)
1. produce the method for docosahexaenoic acid grease with Kou Shi Crypthecodinium cohnii (Crypthecodinium cohnii) industrial fermentation; Comprise that with the Kou Shi Crypthecodinium cohnii be bacterial classification; Employing comprises the substratum of carbon source, nitrogenous source and inorganic salt, makes docosahexaenoic acid grease through fermentation; It is characterized in that: said every 100ml substratum contains: 4.0g bean cake powder hydrolysis juice, 0.6g yeast extract paste, 5.0g glucose, 0.6g potassium primary phosphate, 1.0g sea salt, 0.1g bush sugar alcohol, 3.0g bean sprouts juice, 0.8g Sodium Glutamate, 1.0g molasses, 0.3g MAGNESIUM SULPHATE HEPTAHYDRATE 99.5,0.1g l-arginine; Perhaps said every 100ml substratum contains: 6.0g bean cake powder hydrolysis juice, 0.5g yeast extract paste, 6.0g glucose, 0.4g potassium primary phosphate, 1.0g Sodium Glutamate, 0.5g steeping water, 0.3g MAGNESIUM SULPHATE HEPTAHYDRATE 99.5,1.0g sea salt; Perhaps said every 100ml substratum contains: 8.0g bean cake powder hydrolysis juice, 0.5g yeast extract paste, 6.0g glucose, 0.4g potassium primary phosphate, 1.0g sea salt, 3.0g bean sprouts juice, 1.0g Sodium Glutamate, 0.5g steeping water, 0.3g MAGNESIUM SULPHATE HEPTAHYDRATE 99.5; Perhaps said every 100ml substratum contains: 8.0g bean cake powder hydrolysis juice, 0.7g yeast extract paste, 8.0g glucose, 0.4g potassium primary phosphate, 1.0g sea salt, 0.005g vitamins B
12, 4.0g bean sprouts juice, 1.0g Sodium Glutamate, 0.7g steeping water, 0.3g MAGNESIUM SULPHATE HEPTAHYDRATE 99.5,0.005g vitamins B
1Bean cake powder hydrolysis juice in the above-mentioned substratum is obtained by laxative remedy: bean cake powder is dissolved in the water; The consumption weight ratio of bean cake powder and water is 1: 3-1: 10, and regulate pH to 9.0-10.0, and be warming up to 70-80 ℃; Insulation 8-10h crosses the leaching clear liquid and obtains bean cake powder hydrolysis juice; Bean sprouts juice in the above-mentioned substratum is obtained by laxative remedy: soybean sprout is cleaned, drained, get soybean sprout juice with the squeezing machine squeezing and obtain bean sprouts juice.
2. method according to claim 1 is characterized in that: the Kou Shi Crypthecodinium cohnii of bacterial classification for cultivating, transfer and go into to shake a bottle enlarged culturing, obtain through the one-level enlarged culturing more then through slant activation used in said fermentation; Or Kou Shi Crypthecodinium cohnii for cultivating, transfer and go into to shake a bottle enlarged culturing, after one-level enlarged culturing and secondary enlarged culturing, obtain again then through slant activation.
3. method according to claim 1 and 2 is characterized in that: when fermentation culture 48-60h, replenish bacterial classification, the benefit of bacterial classification is gone into the 8-10% that amount is a fermentating liquid volume.
4. method according to claim 1 and 2 is characterized in that: during the fermentation, and when the reducing sugar content of fermented liquid supplementary carbon source during at 2-3wt%.
5. method according to claim 4 is characterized in that in the fermentation culture process, increasing gradually dissolved oxygen: initial air flow is 0.5VVM, improves air flow 0.02VVM behind later on each supplementary carbon source.
6. method according to claim 1 and 2 is characterized in that: get into early stage stationary phase in fermentation, controlled temperature is 29-31 ℃, and controlled temperature is 22-24 ℃ after getting into stationary phase.
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| CN103468575B (en) * | 2013-09-18 | 2015-05-06 | 武汉轻工大学 | Crypthecodinium cohnii mutant strain with high yield of DHA (docosahexaenoic acid), as well as fermentation method and application thereof |
| CN105646189A (en) * | 2016-01-14 | 2016-06-08 | 青岛琅琊台集团股份有限公司 | Method for extracting EPA from marine microalgae |
| CN106010970B (en) * | 2016-07-13 | 2019-07-26 | 中国科学院青岛生物能源与过程研究所 | A kind of hidden dinoflagellate of Kou Shi and its application |
| CN109055448B (en) * | 2018-09-25 | 2020-08-04 | 广西小藻农业科技有限公司 | Production, extraction and purification process of DHA-rich algae oil |
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