CN103614427A - Method for producing docosahexaenoic acid (DHA) through fermenting straw hydrolyzate - Google Patents
Method for producing docosahexaenoic acid (DHA) through fermenting straw hydrolyzate Download PDFInfo
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Abstract
The invention discloses a method for producing docosahexaenoic acid (DHA) through fermenting a straw hydrolyzate. The method comprises the following steps of (1) pretreating straw powder by using an ammonia gas blasting method, so as to obtain pretreated straw powder; (2) carrying out enzymolysis on the pretreated straw powder by using cellulase, beta-galactosidase and pectinase, so as to obtain the straw hydrolyzate; (3) adding a culture medium into the straw hydrolyzate, then, inoculating Aurantiochytrium to carry out fermentation culture, so as to obtain Aurantiochytrium fermentation liquor; (4) extracting the Aurantiochytrium fermentation liquor by using mixed liquid of ethanol and n-hexane, so as to obtain an extract, and drying the extract, thereby obtaining the DHA. According to the DHA prepared by the method, the yield is stable; in the case of not adding exogenous glucose, by adopting the method, the dry weight of obtained thalli can reach 58-66g/L, oil and fat account for 40.8-53.3% of the dry weight of cells, the DHA accounts for 42.1-45.3% of the dry weight of the oil and fat, and the DHA yield is 10.3-15.4g/L.
Description
Technical field
The present invention relates to a kind of method of utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid.
Background technology
Docosahexenoic acid (Docosahexaenoic acid, be called for short DHA) be a kind of long-chain highly unsaturated fatty acid of needed by human, have significant anti-cardiovascular disease, reducing blood-fat, hypotensive, prevent various cancers, promote infant intelligent development, improve grownup's cerebral function and prevent the important physiological function such as senile dementia, be developed to various medicines, healthcare products, superior cosmetics, food and feeds etc., market is huge.The traditional resource of DHA is fish oil, but owing to excessively developing and fishing for abyssal pelagic fishes, fish oil resource is difficult to meet people's demand growing to DHA.In recent years, utilize the grease that Production by Microorganism Fermentation is rich in DHA to become focus.The not only binary fission breeding as bacterium of thalassiomycetes-schizochytrium limacinum, fast growth, and also its fat content is high, extracts simply, has industry development future.Domestic and international many mechanisms or are applying for the Patents of schizochytrium limacinum fermentative production DHA: the patent that application number is 201010104774.0 has been announced the technique of utilizing dregs of beans hydrolyzate to produce schizochytrium limacinum thalline, and its carbon source of using is glucose 70～80g/L; Application number is that 200910130628.2 patent has been announced the technique of utilizing producing DHA by Schizochytrium in high-density culture through fermentation, and the culture medium carbon source using is glucose 120～125g/L; Application number is that 201010150460.4 patent has been announced full-synthetic culture medium for cultivating the method for schizochytrium limacinum, and the culture medium carbon source using is glucose 60～150g/L; Application number is that 201010237946.1 patent has announced that to take schizochytrium limacinum be to produce the method that strain fermentation is produced polyene unsaturated fatty acid, it is characterized in that adding glycinebetaine or trehalose to improve fat content in fermention medium.Above-mentioned application disclosed patent, mainly utilize glucose or starchy material fermentative production DHA with schizochytrium limacinum, and utilize cheap carbon source, and the method for producing DHA as lignocellulose hydrolyzate there is not yet report.
Stalk is the common lignocellulosic material in rural area, at present China rural area to the common processing mode of stalk have burning, compost, also field, make the modes such as simple and easy material, utilization ratio is low and be unfavorable for industrialization promotion.In addition, the method for comprehensive utilization of stalk resource also has direct fermentation to produce biogas, producing fuel ethyl alcohol by ferment, lactic acid etc., but added value is lower, cannot really realize the high-efficiency comprehensive utilization of stalk resource.
Summary of the invention
The object of this invention is to provide a kind of method of utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid; the present invention utilizes cheap straw lignocellulose for raw material; through hydrolysis, obtain water-soluble carbohydrate; through schizochytrium limacinum, transform and obtain DHA; the production cost of DHA will significantly be reduced; form farmland saving, can produce continuously the technique of DHA, and be conducive to environment protection and Sustainable Socioeconomic Development.
A kind of method of utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid provided by the present invention, comprises the steps:
(1) utilize ammonia blasting procedure pretreated straw powder, obtain pretreated straw powder;
(2) by pretreated straw powder described in cellulase, beta-galactosidase enzymes and pectinase enzymatic hydrolysis, obtain stalk hydrolyzed solution;
(3) in described stalk hydrolyzed solution, add substratum, then access schizochytrium limacinum (Aurantiochytrium) and carry out fermentation culture, obtain schizochytrium limacinum fermented liquid;
(4) with the mixed solution of ethanol and normal hexane, extract described schizochytrium limacinum fermented liquid, obtain extracting solution; Described extracting solution drying obtains docosahexenoic acid.
In above-mentioned method, in step (1), in maize straw, rice straw, sugarcane stalk, wheat stalk, cotton stalk and broomcorn straw, at least one makes described straw powder;
The particle diameter of described straw powder is less than 5mm;
Before the described straw powder of preparation, preferably stalk is dried, as being dried approximately 2 days at 80 ℃ in convection oven.
In above-mentioned method, in step (1), described ammonia blasting procedure comprises the steps:
By described straw powder regulate and control to mass water content be 40%～60%, specifically can be 40%, 45%, 50%, 55% or 60%, then add in high-pressure reactor; Then utilize ammonia under 3.0～4.0MPa, to process described straw powder, can adopt the mode of jacket steam heating to carry out, specifically can under 3.0MPa, 3.5MPa or 4.0MPa, carry out;
In above-mentioned method, in step (1), the temperature of described processing can be 80 ℃～100 ℃, specifically can be 80 ℃, 90 ℃, 95 ℃ or 100 ℃, and the time can be 30～60 minutes, specifically can be 30 minutes, 45 minutes or 60 minutes;
The mass water content of described pretreated straw powder can be 60%～70%, specifically can be 60%, 65% or 70%;
After described processing finishes, can open purging valve, allow the pressure pcl in high-pressure reactor fall; Described pretreated straw powder is placed and spent the night, allow remaining ammoniacal liquor evaporate, be stored in 4 ℃ of preservations, for next step reaction.
In above-mentioned method, in step (2), described cellulase, the consumption of beta-galactosidase enzymes and pectinase enzymatic hydrolysis can be: described in 1Kg, pretreated straw powder need to add cellulase described in 125000～225000CMC U, polygalacturonase described in beta-galactosidase enzymes and 1550～3100U described in 32500～58500pNPG U, as described in 1Kg, pretreated straw powder adds cellulase as described in 125000CMC U, polygalacturonase described in beta-galactosidase enzymes and 1550U described in 32500pNPG U, cellulase described in 175000CMC U, polygalacturonase described in beta-galactosidase enzymes and 2325U described in 45500pNPG U, cellulase described in 225000CMC U, polygalacturonase described in beta-galactosidase enzymes and 2325U described in 58500pNPG U, cellulase described in 150000CMC U, cellulase described in polygalacturonase or 150000CMC U described in beta-galactosidase enzymes and 1550U described in 39000pNPG U, polygalacturonase described in beta-galactosidase enzymes and 3100U described in 39000pNPG U, specifically can adopt and add the mode of Accellerase1500 and Multifect pectinase to carry out enzymolysis,
The definition of Mei Huo unit: 1CMC U cellulase activity is under the condition that is 4.8 at 50 ℃, pH, and 1 minute hydrolyzed carboxymethylcellulo, e generates the required enzyme amount of 1 μ mol glucose equivalent; 1pNPG U beta-galactosidase enzymes is lived as under the condition that is 4.8 at 50 ℃, pH, and within 1 minute, hydrolysis p-nitrophenol-beta-glucoside generates the required enzyme amount of 1 μ mol p-nitrophenol; 1U polygalacturonase is lived as under the condition that is 4.3 at 50 ℃, pH, and within 1 minute, decompose pectin produces the required enzyme amount of 1 μ mol glucose equivalent.
In above-mentioned method, the condition of described enzymolysis is as follows:
PH be 4.0～5.5 and temperature be to process 6～7 days under the condition of 50 ℃～55 ℃, specifically can pH be 4.0 and temperature be under the condition of 50 ℃, process 7 days, pH be 4.5 and temperature be under the condition of 55 ℃, process 6 days, pH be 5.0 and temperature be under the condition of 50 ℃, process 7 days, pH be 5.0 and temperature be under the condition of 55 ℃, process 6 days, pH be 5.5 and temperature be under the condition of 55 ℃, process 7 days or pH be 5.0 and temperature be to process 6 days under the condition of 50 ℃.
In above-mentioned method, in step (3), substratum is composed as follows described in every 100mL:
1.0～2.0g peptone; 1.0～2.0g yeast extract powder; 2.0～3.0g sea crystal; 0.04～0.06g KH
2pO
4; 0.02～0.03g (NH
4)
2sO
4; 0.2～0.4g Na
2sO
4; Water with surplus;
Described in every 100mL the composition of substratum specifically can be following any:
1) 1.0～1.8g peptone; 1.0～1.8g yeast extract powder; 2.0～2.5g sea crystal; 0.04～0.06gKH
2pO
4; 0.02～0.03g (NH
4)
2sO
4; 0.2～0.4g Na
2sO
4; Water with surplus;
2) 1.0g peptone; 1.0g yeast extract powder; 2.0g sea crystal; 0.04g KH
2pO
4; 0.02g (NH
4)
2sO
4; 0.2g Na
2sO
4; Water with surplus;
3) 1.5g peptone; 1.5g yeast extract powder; 2.0g sea crystal; 0.06g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus;
4) 2.0g peptone; 2.0g yeast extract powder; 3.0g sea crystal; 0.04g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.3g Na
2sO
4; Water with surplus;
5) 1.0g peptone; 1.0g yeast extract powder; 2.0g sea crystal; 0.06g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus;
6) 1.8g peptone; 1.8g yeast extract powder; 2.5g sea crystal; 0.04g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus.
In above-mentioned method, in step (3), the starting point concentration of described schizochytrium limacinum (Aurantiochytrium) access is schizochytrium limacinum bacterial classification described in substratum access 10～12mL described in every 100mL.
In above-mentioned method, in step (3), the condition of described fermentation culture is as follows:
In described fermentation culture starts to 75～85 hours, the temperature of controlling described fermentation culture is 27.5～28.5 ℃, continues in fermentation culture to 85～95 hour, and the temperature of controlling described fermentation culture is 24.5～25.5 ℃; Continue to be cultured to fermentation ends, the temperature of controlling described fermentation culture is 19.5～20.5 ℃;
And in described fermentation culture starts to 55～65 hours, by adding ammoniacal liquor, take maintenance system pH as 5.8～6.2, continue in fermentation culture to 95～105 hour, by adding described stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain schizochytrium limacinum fermented liquid.
In above-mentioned method, in step (4), the volume ratio of described ethanol and described normal hexane can be 1:2～5, as 1:2.6.
In above-mentioned method, in step (4), before the mixed solution with ethanol and normal hexane extracts described schizochytrium limacinum fermented liquid, described method also comprises the steps:
Described schizochytrium limacinum fermented liquid is carried out to centrifugation, obtain supernatant liquor; After washing described supernatant liquor with physiological saline, be suspended from hydrochloric acid soln again.
The present invention compared with prior art, has the following advantages:
(1) raw material is easily purchased, cheap.
Stalk is agriculture production by product, and China's annual production reaches hundred million tons according to statistics, is not yet fully used at present, and environmental pollution has also been caused in some areas.Stalk is easy to collect, and processing pulverizes and be hydrolyzed relatively easy, and hydrolyzed solution is mainly the sugar of Mierocrystalline cellulose and hemicellulose degraded, can be used as the carbon source of schizochytrium limacinum production high added value DHA.
(2) the DHA stable yield that prepared by the present invention, do not adding in Exogenous Glucose situation, the dry cell weight that adopts the present invention to obtain can reach 58～66g/L, and grease accounts for 40.8～53.3% of dry cell weight, DHA accounts for 42.1～45.3% of grease dry weight, and DHA output is 10.3～15.4g/L.
(3) hydrolysis is simple, easy and simple to handle with fermenting process, easily controls, and is applicable to suitability for industrialized production.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Schizochytrium limacinum used in following embodiment (Aurantiochytrium limacinum SR21) is collected center (ATCC) purchased from US mode bacterial classification, and bacterial strain number is MYA-1381.
Embodiment 1, corn stalk hydrolysis are produced DHA
(1) ammonia blasting procedure pre-treatment maize straw
By maize straw in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain corn stalk powder.Get 100kg dry straw powder, with distilled water, regulate water content to 40%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 3.0MPa its pressure, passes into the 1m that corn stalk powder is housed
3high-pressure reactor.With jacket steam, be heated to 80 ℃, maintain 60min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis maize straw
The corn stalk powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 60%), with the phosphate buffered saline buffer of 1M pH4.0, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 50 ℃, with 10mol/L HCl, regulate pH to 4.0, then add 15g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPG U/g, 1kg corn stalk powder need to add 125000CMC U cellulase and 32500pNPG U beta-galactosidase enzymes) and 3g/L Multifect pectinase(Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg corn stalk powder need to add 1550U polygalacturonase), process 7 days for 50 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the corn stalk hydrolysis obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
1.0g peptone; 1.0g yeast extract powder; 2.0g sea crystal; 0.04g KH
2pO
4; 0.02g (NH
4)
2sO
4; 0.2g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 80 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 80 hours to 90 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 90 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.Simultaneously, in fermentation culture starts to 60 hours, by adding ammoniacal liquor, take maintenance system pH as 6.0 ± 0.2, and in fermentation culture starts to 100 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when in system being detected, the concentration of reducing sugar is lower than 1g/L, stop fermentation, obtain the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 62g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 80 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 41.5% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 ℃ of constant temperature backflow esterification 1 hour, get 0.5mL supernatant GC-MS and analyze.GC-MS condition is: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio 20:1; Sample size 1 μ L; Injector temperature is 280 ℃; Detector temperature is 280 ℃; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 6.9%, and hexadecanoic acid (palmitinic acid) content is 28.2%, Content of Eicosapentaenoic Acid is 15.2%, DHA content is 44.7%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 5.0%, DHA output is 11.5g/L.
Embodiment 2, rice straw hydrolyzed solution are produced DHA
(1) ammonia blasting procedure pretreated water rice straw
By rice straw in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain rice straw powder.Get 100kg dry straw powder, with distilled water, regulate water content to 45%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 3.5MPa its pressure, passes into the 1m that rice straw powder is housed
3high-pressure reactor.With jacket steam, be heated to 90 ℃, maintain 45min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis rice straw
The corn stalk powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 60%), with the phosphate buffered saline buffer of 1M pH4.5, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 55 ℃, with 10mol/L HCl, regulate pH to 4.5, add 21g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, 1kg corn stalk powder need to add 175000CMC U cellulase and 45500pNPG U beta-galactosidase enzymes) and 4.5g/L Multifect pectinase(Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg corn stalk powder need to add 2325U polygalacturonase), process 6 days for 55 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the rice straw hydrolyzed solution obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
1.5g peptone; 1.5g yeast extract powder; 2.5g sea crystal; 0.06g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 12mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 75 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 75 hours to 85 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 85 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.In fermentation culture starts to 55 hours, by adding ammoniacal liquor, take maintenance system pH as 6.0 ± 0.2, and in fermentation culture starts to 95 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 66g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 70 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 53.5% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 ℃ of constant temperature backflow esterification 1 hour, get 0.5mL supernatant GC-MS and analyze.GC-MS condition is: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio 20:1; Sample size 1 μ L; 280 ℃ of injector temperatures; 280 ℃ of detector temperatures; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 6.7%, and hexadecanoic acid (palmitinic acid) content is 28.5%, Content of Eicosapentaenoic Acid is 14.9%, DHA content is 43.7%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 6.2%, DHA output is 15.4g/L.
Embodiment 3, sugarcane stalk hydrolyzed solution are produced DHA
(1) ammonia blasting procedure pre-treatment sugarcane stalk
By sugarcane stalk in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain sugarcane straw powder.Get 100kg dry straw powder, with distilled water, regulate water content to 50%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 4.0MPa its pressure, passes into the 1m that sugarcane straw powder is housed
3high-pressure reactor.With jacket steam, be heated to 95 ℃, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis sugarcane stalk
The corn stalk powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 65%), with the phosphate buffered saline buffer of 1M pH5.0, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 50 ℃, with 10mol/L HCl, regulate pH to 5.0, add 27g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, 1kg sugarcane straw powder need to add 225000CMC U cellulase and 58500pNPG U beta-galactosidase enzymes) and 4.5g/L Multifect pectinase(Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg sugarcane straw powder need to add 2325U polygalacturonase), process 7 days for 50 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the sugarcane stalk hydrolyzed solution obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
2.0g peptone; 2.0g yeast extract powder; 3.0g sea crystal; 0.04g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.3g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 11mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 85 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 85 hours to 95 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 95 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.And in fermentation culture starts to 65 hours, by adding ammoniacal liquor, take maintenance system pH as 6.0 ± 0.2, and in fermentation culture starts to 105 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 61g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 75 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 51.6% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 ℃ of constant temperature backflow esterification 1 hour, get 0.5mL supernatant GC-MS and analyze.GC-MS condition is: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio 20:1; Sample size 1 μ L; 280 ℃ of injector temperatures; 280 ℃ of detector temperatures; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 7.2%, and hexadecanoic acid (palmitinic acid) content is 28.6%, Content of Eicosapentaenoic Acid is 15.1%, DHA content is 45.3%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 3.8%, DHA output is 14.3g/L.
Embodiment 4, wheat stalk hydrolyzed solution are produced DHA
(1) ammonia blasting procedure pre-treatment wheat stalk
By wheat stalk in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain wheat stalk powder.Get 100kg dry straw powder, with distilled water, regulate water content to 60%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 4.0MPa its pressure, passes into the 1m that wheat stalk powder is housed
3high-pressure reactor.With jacket steam, be heated to 100 ℃, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis wheat stalk
The wheat stalk powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 70%), with the phosphate buffered saline buffer of 1M pH5.0, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 55 ℃, with 10mol/L HCl, regulate pH to 5.0, add 15g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, 1kg sugarcane straw powder need to add 125000CMC U cellulase and 32500pNPG U beta-galactosidase enzymes) and 3.0g/L Multifect pectinase(Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg sugarcane straw powder need to add 1550U polygalacturonase), process 6 days for 55 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the wheat stalk hydrolyzed solution obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
1.0g peptone; 1.0g yeast extract powder; 2.0g sea crystal; 0.06g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 12mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 80 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 80 hours to 90 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 90 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.In fermentation culture starts to 60 hours, by adding ammoniacal liquor, take maintenance system pH as 6.0 ± 0.2, and in fermentation culture starts to 100 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, in system being detected, the concentration of reducing sugar, lower than 1g/L, stops fermentation, obtains the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 65g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 70～80 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 50.7% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH
3oH62 ℃ of constant temperature backflow esterification 1 hour, gets 0.5mL supernatant GC-MS and analyzes.GC-MS condition: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio 20:1; Sample size is 1 μ L; Injector temperature is 280 ℃; Detector temperature is 280 ℃; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 6.3%, and hexadecanoic acid (palmitinic acid) content is 29.5%, Content of Eicosapentaenoic Acid is 14.8%, DHA content is 42.1%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 7.3%, DHA output is 13.9g/L.
Embodiment 5, cotton stalk hydrolyzed solution are produced DHA
(1) ammonia blasting procedure pre-treatment cotton stalk
By cotton stalk in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain cotton stalk powder.Get 100kg dry straw powder, with distilled water, regulate water content to 55%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 3.5MPa its pressure, passes into the 1m that cotton stalk powder is housed
3high-pressure reactor.With jacket steam, be heated to 100 ℃, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis cotton stalk
The cotton stalk powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 65%), with the phosphate buffered saline buffer of 1M pH5.5, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 55 ℃, with 10mol/L HCl, regulate pH to 5.5, add 18g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, 1kg sugarcane straw powder need to add 150000CMC U cellulase and 39000pNPG U beta-galactosidase enzymes) and 3.0g/L Multifect pectinase (Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg sugarcane straw powder need to add 1550U polygalacturonase), process 7 days for 55 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the cotton stalk hydrolyzed solution obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
1.8g peptone; 1.8g yeast extract powder; 2.5g sea crystal; 0.04g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 75 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 75 hours to 85 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 85 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.And in fermentation culture starts to 55 hours, by adding ammoniacal liquor, take maintenance system pH as 6.0 ± 0.2, and in fermentation culture starts to 95 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 58g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 80 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 48.5% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 ℃ of constant temperature backflow esterification 1 hour, get 0.5mL supernatant GC-MS and analyze.GC-MS condition is: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio is 20:1; Sample size is 1 μ L; Injector temperature is 280 ℃; Detector temperature is 280 ℃; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 6.2%, and hexadecanoic acid (palmitinic acid) content is 28.5%, Content of Eicosapentaenoic Acid is 13.8%, DHA content is 43.6%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 7.9%, DHA output is 12.3g/L.
Embodiment 6, broomcorn straw hydrolyzed solution are produced DHA
(1) ammonia blasting procedure pre-treatment broomcorn straw
By broomcorn straw in 80 ℃ of convection oven dry approximately 2 days, with stalk crasher (Henan good luck engineering works), grind, with the mesh screen in 5mm aperture, filter and obtain broomcorn straw powder.Get 100kg dry straw powder, with distilled water, regulate water content to 50%, be placed in 1m
3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, heating reaches after 4.0MPa its pressure, passes into the 1m that broomcorn straw powder is housed
3high-pressure reactor.With jacket steam, be heated to 95 ℃, maintain 45min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.Straw powder that pretreatment with agueous Ammonia is crossed is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 ℃ standby.
(2) enzymatic hydrolysis broomcorn straw
The broomcorn straw powder 30kg(water content that step (1) pretreatment with agueous Ammonia is crossed is about 60%), with the phosphate buffered saline buffer of 1M pH5.0, regulate phosphoric acid salt final concentration to 50mM, adding distil water is to 100L, in 121 ℃ of sterilizing 30min, be cooled to after 50 ℃, with 10mol/L HCl, regulate pH to 5.0, add 18g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, 1kg sugarcane straw powder need to add 150000CMC U cellulase and 39000pNPG U beta-galactosidase enzymes) and 6.0g/L Multifect pectinase (Jie Nengke biotechnology company limited, polygalacturonase content is about 155U/g, 1kg sugarcane straw powder need to add 3100U polygalacturonase), process 6 days for 50 ℃.Tubular-bowl centrifuge for hydrolyzed solution (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, be stored in 4 ℃.In 2 months, can use.Before use, with NaOH, adjust pH to 6.0.
(3) DHA fermentation
In the broomcorn straw hydrolyzed solution obtaining in step (2), add substratum, substratum is composed as follows described in every 100mL:
2.0g peptone; 2.0g yeast extract powder; 2.5g sea crystal; 0.06g KH
2pO
4; 0.03g (NH
4)
2sO
4; 0.4g Na
2sO
4; Water with surplus.
In 121 ℃ of sterilizing 30min, cooling after according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn, cultivate approximately 120 hours.Wherein, in fermentation starts by 85 hours, controlling culture temperature is 28.0 ± 0.5 ℃; In 85 hours to 95 hours, controlling culture temperature is 25.0 ± 0.5 ℃; Within 95 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 ℃.And in fermentation culture starts to 65 hours, by add ammoniacal liquor with maintenance system pH 6.0 ± 0.2, and in fermentation culture starts to 105 hours, by adding stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain the schizochytrium limacinum fermented liquid that is rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermented liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, resuspended centrifugal with deionized water, repeat to move to after 2 times the flat board of known constant weight, in 85 ℃ of oven for drying to constant weight, measuring dry cell weight in fermented liq is 60g/L.
Get 5mL schizochytrium limacinum fermented liquid in 10mL centrifuge tube, 8000r/min, 4 ℃ of frozen centrifugation 10min, abandon supernatant, use physiological saline centrifuge washing, in triplicate.Obtain wet thallus and be resuspended in 4mL10mol/L hydrochloric acid, 70～80 ℃ of water-baths are about 1 hour, after cooling, add 0.75mL dehydrated alcohol and 2mL normal hexane, fully shake up, 1200r/min, 2min centrifuging and taking supernatant, in constant weight screw socket pipe, repeats 3 times, schizochytrium limacinum grease is drained and obtained to vacuum, records grease and account for 40.8% of dry cell weight.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 ℃ of constant temperature backflow esterification 1 hour, get 0.5mL supernatant GC-MS and analyze.GC-MS condition: 6890C-5975N gas chromatograph-mass spectrometer (Agilent), HP-INNOWAX polarity capillary column, 0.25 μ m * 250 μ m * 30m; Carrier gas: helium; Carrier gas flux is: 1.0mL/min, constant rate; Loading pattern: shunting, splitting ratio is 20:1; Sample size is 1 μ L; 280 ℃ of injector temperatures; Detector temperature is 280 ℃; Heating schedule: keep 1min at 150 ℃, 10 ℃/min is warming up to 200 ℃, and 2 ℃/min is warming up to 220 ℃, keeps 5min.After 240 ℃, move 3min.
Record in grease, tetradecanoic acid content is 7.2%, and hexadecanoic acid (palmitinic acid) content is 29.5%, Content of Eicosapentaenoic Acid is 13.2%, DHA content is 42.1%, and other heteroacid (pentadecylic acid, margaric acid and octadecanoic acid) content is that 8.0%, DHA output is 10.3g/L.
Claims (10)
1. a method of utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid, comprises the steps:
(1) utilize ammonia blasting procedure pretreated straw powder, obtain pretreated straw powder;
(2) by pretreated straw powder described in cellulase, beta-galactosidase enzymes and pectinase enzymatic hydrolysis, obtain stalk hydrolyzed solution;
(3) in described stalk hydrolyzed solution, add substratum, then access schizochytrium limacinum (Aurantiochytrium) and carry out fermentation culture, obtain schizochytrium limacinum fermented liquid;
(4) with the mixed solution of ethanol and normal hexane, extract described schizochytrium limacinum fermented liquid, obtain extracting solution; Described extracting solution drying obtains docosahexenoic acid.
2. method according to claim 1, is characterized in that: in step (1), in maize straw, rice straw, sugarcane stalk, wheat stalk, cotton stalk and broomcorn straw, at least one makes described straw powder;
The particle diameter of described straw powder is less than 5mm.
3. method according to claim 1 and 2, is characterized in that: in step (1), described ammonia blasting procedure comprises the steps:
By described straw powder regulate and control to mass water content be 40%～60%, then add in high-pressure reactor; Then utilize ammonia under 3.0～4.0MPa, to process described straw powder.
4. method according to claim 3, is characterized in that: in step (1), the temperature of described processing is 80 ℃～100 ℃, and the time is 30～60 minutes;
The mass water content of described pretreated straw powder is 60%～70%.
5. according to the method described in any one in claim 1-4, it is characterized in that: in step (2), the consumption of described cellulase, described beta-galactosidase enzymes and described polygalacturonase is: described in 1Kg, pretreated straw powder need to add described in cellulase described in 125000～225000CMC U, 32500～58500pNPG U polygalacturonase described in beta-galactosidase enzymes and 1550～3100U.
6. method according to claim 5, is characterized in that: the condition of described enzymolysis is as follows:
PH be 4.0～5.5 and temperature be to process 6～7 days under the condition of 50 ℃～55 ℃.
7. according to the method described in any one in claim 1-6, it is characterized in that: in step (3), substratum is composed as follows described in every 100mL:
1.0～2.0g peptone; 1.0～2.0g yeast extract powder; 2.0～3.0g sea crystal; 0.04～0.06g KH
2pO
4; 0.02～0.03g (NH
4)
2sO
4; 0.2～0.4g Na
2sO
4; Water with surplus.
8. according to the method described in any one in claim 1-7, it is characterized in that: in step (3), the starting point concentration of described schizochytrium limacinum (Aurantiochytrium) access is schizochytrium limacinum described in every 100mL substratum access 10～12mL.
9. according to the method described in any one in claim 1-8, it is characterized in that: in step (3), the condition of described fermentation culture is as follows:
In described fermentation culture starts to 75～85 hours, the temperature of controlling described fermentation culture is 27.5～28.5 ℃, continues in fermentation culture to 85～95 hour, and the temperature of controlling described fermentation culture is 24.5～25.5 ℃; Continue to be cultured to fermentation ends, the temperature of controlling described fermentation culture is 19.5～20.5 ℃;
And in described fermentation culture starts to 55～65 hours, by adding ammoniacal liquor, take maintenance system pH as 5.8～6.2, continue in fermentation culture to 95～105 hour, by adding described stalk hydrolyzed solution, with the concentration of reducing sugar in maintenance system, be greater than 10g/L, afterwards, when the concentration of reducing sugar is lower than 1g/L in system being detected, stop fermentation, obtain schizochytrium limacinum fermented liquid.
10. according to the method described in any one in claim 1-9, it is characterized in that: in step (4), before the mixed solution with ethanol and normal hexane extracts described schizochytrium limacinum fermented liquid, described method also comprises the steps:
Described schizochytrium limacinum fermented liquid is carried out to centrifugation, obtain supernatant liquor; After washing described supernatant liquor with physiological saline, be suspended from hydrochloric acid soln again.
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| ES2617081A1 (en) * | 2015-12-15 | 2017-06-15 | Interquim, S.A. | Procedure for the production of dha (Machine-translation by Google Translate, not legally binding) |
| CN108048333A (en) * | 2017-12-15 | 2018-05-18 | 福建省金燕海洋生物科技股份有限公司 | Utilize the technique of culture medium fermenting and producing schizochytrium limacinum prepared by seaweed slag |
| CN111944700A (en) * | 2020-08-12 | 2020-11-17 | 苏州聚维元创生物科技有限公司 | Thraustochytrium and application thereof in production of DHA (docosahexaenoic acid) by taking straws as raw material |
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