CN103614427B - Method for producing docosahexaenoic acid (DHA) through fermenting straw hydrolyzate - Google Patents

Method for producing docosahexaenoic acid (DHA) through fermenting straw hydrolyzate Download PDF

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CN103614427B
CN103614427B CN201310571413.0A CN201310571413A CN103614427B CN 103614427 B CN103614427 B CN 103614427B CN 201310571413 A CN201310571413 A CN 201310571413A CN 103614427 B CN103614427 B CN 103614427B
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fermentation
dha
straw powder
schizochytrium limacinum
stalk
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CN103614427A (en
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黄建忠
江贤章
张明亮
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Fujian Normal University
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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

Utilize the method for stalk hydrolyzed solution fermentative production docosahexenoic acid
Technical field
The present invention relates to a kind of method 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 cancer, promote infant intelligent development, improve the cerebral function of grownup and prevent the important physiological function such as senile dementia, be developed to various medicine, healthcare products, superior cosmetics, food and feeds etc., market is huge.The traditional resource of DHA is fish oil, but fishes for abyssal pelagic fishes owing to excessively developing, and fish oil resource is difficult to meet people's demand growing to DHA.In recent years, the grease that Production by Microorganism Fermentation is rich in DHA is utilized 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 simple, has industry development future.Domestic and international many mechanisms or are applying for that schizochytrium limacinum fermentation produces the Patents of DHA: to be the patent disclosure of 201010104774.0 utilize dregs of beans hydrolyzate to produce the technique of schizochytrium limacinum thalline to application number, and its carbon source used is glucose 70 ~ 80g/L; The technique of application number to be the patent disclosure of 200910130628.2 utilize producing DHA by Schizochytrium in high-density culture through fermentation, the culture medium carbon source used is glucose 120 ~ 125g/L; The application number full-synthetic culture medium that has been the patent disclosure of 201010150460.4 is for cultivating the method for schizochytrium limacinum, and the culture medium carbon source used is glucose 60 ~ 150g/L; Application number has been the patent disclosure of 201010237946.1 with schizochytrium limacinum is produce the method that strain fermentation produces polyene unsaturated fatty acid, it is characterized in that adding in the fermentation medium glycinebetaine or trehalose to improve fat content.Above-mentioned application disclosed patent, mainly utilize glucose or starchy material fermentative production DHA with schizochytrium limacinum, and utilize cheap carbon source, and as lignocellulose hydrolysate produces the method for DHA, there is not been reported.
Stalk is the common lignocellulosic material in rural area, and the common processing mode of current China rural area to stalk has burning, compost, also field, makes the modes such as simple and easy material, and 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, really cannot 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 utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid; the present invention utilizes cheap straw lignocellulose to be raw material; water-soluble carbohydrate is obtained through hydrolysis; transform through schizochytrium limacinum and obtain DHA; to significantly reduce the production cost of DHA; formed farmland saving, can the technique of continuous seepage DHA, and be conducive to environment protection and Sustainable Socioeconomic Development.
A kind of method 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, stalk hydrolyzed solution is obtained;
(3) in described stalk hydrolyzed solution, add substratum, then access schizochytrium limacinum (Aurantiochytrium) and carry out fermentation culture, obtain schizochytrium limacinum fermentation liquid;
(4) extract described schizochytrium limacinum fermentation liquid with the mixed solution of ethanol and normal hexane, obtain extracting solution; Namely described extracting solution drying obtains docosahexenoic acid.
In above-mentioned method, in step (1), described straw powder is made up of at least one in maize straw, rice straw, sugarcane stalk, wheat stalk, cotton stalk and broomcorn straw;
The particle diameter of described straw powder is less than 5mm;
Before the described straw powder of preparation, preferably stalk is carried out drying, as can drying about 2 days at 80 DEG C in convection oven.
In above-mentioned method, in step (1), described ammonia blasting procedure comprises the steps:
Regulating and controlling described straw powder to mass water content is 40% ~ 60%, specifically can be 40%, 45%, 50%, 55% or 60%, then adds in high-pressure reactor; Then utilize ammonia to process described straw powder under 3.0 ~ 4.0MPa, the mode that jacket steam can be adopted to heat is carried out, and specifically can carry out under 3.0MPa, 3.5MPa or 4.0MPa;
In above-mentioned method, in step (1), the temperature of described process can be 80 DEG C ~ 100 DEG C, and specifically can be 80 DEG C, 90 DEG C, 95 DEG C or 100 DEG C, 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 process terminates, can purging valve be opened, allow the pressure pcl in high-pressure reactor fall; Described pretreated straw powder is placed and spends the night, allow remaining ammoniacal liquor evaporate, be stored in 4 DEG C 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: pretreated straw powder described in 1Kg needs to add cellulase described in 125000 ~ 225000CMC U, polygalacturonase described in beta-galactosidase enzymes described in 32500 ~ 58500pNPG U and 1550 ~ 3100U, as as described in 1Kg, pretreated straw powder adds cellulase as described in 125000CMC U, polygalacturonase described in beta-galactosidase enzymes described in 32500pNPG U and 1550U, cellulase described in 175000CMC U, polygalacturonase described in beta-galactosidase enzymes described in 45500pNPG U and 2325U, cellulase described in 225000CMC U, polygalacturonase described in beta-galactosidase enzymes described in 58500pNPG U and 2325U, cellulase described in 150000CMC U, cellulase described in polygalacturonase described in beta-galactosidase enzymes described in 39000pNPG U and 1550U or 150000CMC U, polygalacturonase described in beta-galactosidase enzymes described in 39000pNPG U and 3100U, the mode adding Accellerase1500 and Multifect pectinase specifically can be adopted to carry out enzymolysis,
The definition of Mei Huo unit: 1CMC U cellulase activity be 50 DEG C, under pH is the condition of 4.8, within 1 minute, hydrolyzed carboxymethylcellulo, e generates the enzyme amount needed for 1 μm of ol glucose equivalent; 1pNPG U beta-galactosidase enzymes live for 50 DEG C, under pH is the condition of 4.8, within 1 minute, hydrolysis p-nitrophenol-beta-glucoside generates the enzyme amount needed for 1 μm of ol p-nitrophenol; 1U polygalacturonase live for 50 DEG C, under pH is the condition of 4.3, within 1 minute, decompose pectin produces the enzyme amount needed for 1 μm of ol glucose equivalent.
In above-mentioned method, the condition of described enzymolysis is as follows:
Under pH is 4.0 ~ 5.5 and temperature is the condition of 50 DEG C ~ 55 DEG C process 6 ~ 7 days, specifically can process 7 days under pH is 4.0 and temperature is the condition of 50 DEG C, pH be 4.5 and temperature be the condition of 55 DEG C under process 6 days, under pH is 5.0 and temperature is the condition of 50 DEG C process 7 days, under pH is 5.0 and temperature is the condition of 55 DEG C process 6 days, under pH is 5.5 and temperature is the condition of 55 DEG C process 7 days or under pH is 5.0 and temperature is the condition of 50 DEG C process 6 days.
In above-mentioned method, in step (3), described in every 100mL, substratum is composed as follows:
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; With the water of surplus;
The composition of substratum described in every 100mL specifically can be following any one:
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; With the water of 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; With the water of 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; With the water of 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; With the water of 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; With the water of 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; With the water of surplus.
In above-mentioned method, in step (3), the starting point concentration that described schizochytrium limacinum (Aurantiochytrium) accesses 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:
Start in 75 ~ 85 hours in described fermentation culture, the temperature controlling described fermentation culture is 27.5 ~ 28.5 DEG C, and continue in fermentation culture to 85 ~ 95 hour, the temperature controlling described fermentation culture is 24.5 ~ 25.5 DEG C; Continue to be cultured to fermentation ends, the temperature controlling described fermentation culture is 19.5 ~ 20.5 DEG C;
And start in 55 ~ 65 hours in described fermentation culture, by adding ammoniacal liquor with maintenance system pH for 5.8 ~ 6.2, continue in fermentation culture to 95 ~ 105 hour, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding described stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L, stopping fermentation, namely obtaining schizochytrium limacinum fermentation 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 extracting described schizochytrium limacinum fermentation liquid with the mixed solution of ethanol and normal hexane, described method also comprises the steps:
Described schizochytrium limacinum fermentation liquid is carried out centrifugation, obtains supernatant liquor; Be suspended from again in hydrochloric acid soln with after supernatant liquor described in brine.
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, and be not yet fully used at present, some areas also create environmental pollution.Stalk is easy to collect, processing pulverize and hydrolysis relatively easy, the sugar of hydrolyzed solution mainly Mierocrystalline cellulose and hemicellulose degradation, can be used as the carbon source of schizochytrium limacinum production high added value DHA.
(2) the DHA stable yield prepared of the present invention, do not adding in Exogenous Glucose situation, the dry cell weight adopting the present invention to obtain can reach 58 ~ 66g/L, and grease accounts for 40.8 ~ 53.3% of dry cell weight, 42.1 ~ 45.3%, the DHA output that DHA accounts for grease dry weight are 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 used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Schizochytrium limacinum (Aurantiochytrium limacinum SR21) purchased from American Type Culture Collection (ATCC) used in following embodiment, bacterial strain number is MYA-1381.
Embodiment 1, corn stalk hydrolysis produce DHA
(1) ammonia blasting procedure pre-treatment maize straw
By maize straw drying about 2 days in 80 DEG C of convection oven, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain corn stalk powder.Get 100kg dry straw powder, regulate water content to 40% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 3.0MPa, pass into the 1m that corn stalk powder is housed 3high-pressure reactor.Be heated to 80 DEG C with jacket steam, maintain 60min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis maize straw
The corn stalk powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 60%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH4.0, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 50 DEG C, regulate pH to 4.0 with 10mol/L HCl, then add 15g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPG U/g, then 1kg corn stalk powder needs 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, then 1kg corn stalk powder needs to add 1550U polygalacturonase), 50 DEG C process 7 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the corn stalk hydrolysis that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 80 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 80 hours, in 90 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 90 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.Simultaneously, start in 60 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH for 6.0 ± 0.2, and start in 100 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L by adding stalk hydrolyzed solution, stop fermentation, obtain the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 62g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 41.5%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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 DEG C; Detector temperature is 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 5.0%, DHA output is 11.5g/L.
Embodiment 2, rice straw hydrolyzed solution produce DHA
(1) ammonia blasting procedure pretreated water rice straw
By rice straw drying about 2 days in 80 DEG C of convection oven, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain rice straw powder.Get 100kg dry straw powder, regulate water content to 45% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 3.5MPa, pass into the 1m that rice straw powder is housed 3high-pressure reactor.Be heated to 90 DEG C with jacket steam, maintain 45min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis rice straw
The corn stalk powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 60%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH4.5, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 55 DEG C, regulate pH to 4.5 with 10mol/L HCl, add 21g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, then 1kg corn stalk powder needs 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, then 1kg corn stalk powder needs to add 2325U polygalacturonase), 55 DEG C process 6 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the rice straw hydrolyzed solution that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 12mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 75 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 75 hours, in 85 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 85 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.Start in 55 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH for 6.0 ± 0.2, and start in 95 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L, stopping fermentation, obtaining the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 66g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 53.5%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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 280 DEG C; Detector temperature 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 6.2%, DHA output is 15.4g/L.
Embodiment 3, sugarcane stalk hydrolyzed solution produce DHA
(1) ammonia blasting procedure pre-treatment sugarcane stalk
By the drying about 2 days in 80 DEG C of convection oven of sugarcane stalk, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain sugarcane straw powder.Get 100kg dry straw powder, regulate water content to 50% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 4.0MPa, pass into the 1m that sugarcane straw powder is housed 3high-pressure reactor.Be heated to 95 DEG C with jacket steam, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis sugarcane stalk
The corn stalk powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 65%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH5.0, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 50 DEG C, regulate pH to 5.0 with 10mol/L HCl, add 27g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, then 1kg sugarcane straw powder needs 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, then 1kg sugarcane straw powder needs to add 2325U polygalacturonase), 50 DEG C process 7 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the sugarcane stalk hydrolyzed solution that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 11mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 85 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 85 hours, in 95 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 95 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.And start in 65 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH for 6.0 ± 0.2, and start in 105 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L, stopping fermentation, obtaining the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 61g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 51.6%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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 280 DEG C; Detector temperature 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 3.8%, DHA output is 14.3g/L.
Embodiment 4, wheat stalk hydrolyzed solution produce DHA
(1) ammonia blasting procedure pre-treatment wheat stalk
By wheat stalk drying about 2 days in 80 DEG C of convection oven, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain wheat stalk powder.Get 100kg dry straw powder, regulate water content to 60% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 4.0MPa, pass into the 1m that wheat stalk powder is housed 3high-pressure reactor.Be heated to 100 DEG C with jacket steam, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis wheat stalk
The wheat stalk powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 70%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH5.0, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 55 DEG C, regulate pH to 5.0 with 10mol/L HCl, add 15g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, then 1kg sugarcane straw powder needs 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, then 1kg sugarcane straw powder needs to add 1550U polygalacturonase), 55 DEG C process 6 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the wheat stalk hydrolyzed solution that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 12mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 80 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 80 hours, in 90 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 90 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.Start in 60 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH for 6.0 ± 0.2, and start in 100 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding stalk hydrolyzed solution, afterwards, when detecting that the concentration of reducing sugar in system is lower than 1g/L, stopping fermentation, obtaining the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 65g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 50.7%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH 3oH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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 DEG C; Detector temperature is 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 7.3%, DHA output is 13.9g/L.
Embodiment 5, cotton stalk hydrolyzed solution produce DHA
(1) ammonia blasting procedure pre-treatment cotton stalk
By cotton stalk drying about 2 days in 80 DEG C of convection oven, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain cotton stalk powder.Get 100kg dry straw powder, regulate water content to 55% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 3.5MPa, pass into the 1m that cotton stalk powder is housed 3high-pressure reactor.Be heated to 100 DEG C with jacket steam, maintain 30min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis cotton stalk
The cotton stalk powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 65%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH5.5, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 55 DEG C, regulate pH to 5.5 with 10mol/L HCl, add 18g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, then 1kg sugarcane straw powder needs 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, then 1kg sugarcane straw powder needs to add 1550U polygalacturonase), 55 DEG C process 7 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the cotton stalk hydrolyzed solution that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 75 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 75 hours, in 85 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 85 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.And start in 55 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH for 6.0 ± 0.2, and start in 95 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L, stopping fermentation, obtaining the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 58g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 48.5%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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 DEG C; Detector temperature is 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 7.9%, DHA output is 12.3g/L.
Embodiment 6, broomcorn straw hydrolyzed solution produce DHA
(1) ammonia blasting procedure pre-treatment broomcorn straw
By broomcorn straw drying about 2 days in 80 DEG C of convection oven, grind with stalk crasher (Henan good luck engineering works), carry out filtration with the mesh screen in 5mm aperture and obtain broomcorn straw powder.Get 100kg dry straw powder, regulate water content to 50% with distilled water, be placed in 1m 3in high-pressure reactor.Get 100kg ammoniacal liquor in 200L stainless steel pressure tank, after heating makes its pressure reach 4.0MPa, pass into the 1m that broomcorn straw powder is housed 3high-pressure reactor.Be heated to 95 DEG C with jacket steam, maintain 45min.Then open purging valve, allow the pressure pcl in high-pressure reactor fall.The straw powder crossed by pretreatment with agueous Ammonia is placed and is spent the night, and allows remaining ammoniacal liquor evaporation, be stored in 4 DEG C for subsequent use.
(2) enzymatic hydrolysis broomcorn straw
The broomcorn straw powder 30kg(water content crossed by step (1) pretreatment with agueous Ammonia is about 60%), regulate phosphoric acid salt final concentration to 50mM with the phosphate buffered saline buffer of 1M pH5.0, adding distil water is to 100L, in 121 DEG C of sterilizing 30min, after being cooled to 50 DEG C, regulate pH to 5.0 with 10mol/L HCl, add 18g/L Accellerase1500(Jie Nengke biotechnology company limited, cellulase content 2500CMC U/g; Beta-galactosidase enzymes content is about 650pNPGU/g, then 1kg sugarcane straw powder needs 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, then 1kg sugarcane straw powder needs to add 3100U polygalacturonase), 50 DEG C process 6 days.Hydrolyzed solution tubular-bowl centrifuge (sky, Shanghai this tubular-bowl centrifuge GQ145) is carried out centrifugal, is stored in 4 DEG C.Can use in 2 months.Before use, adjust pH to 6.0 with NaOH.
(3) DHA fermentation
In the broomcorn straw hydrolyzed solution that step (2) obtains, add substratum, described in every 100mL, substratum is composed as follows:
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; With the water of surplus.
In 121 DEG C of sterilizing 30min, according to every 100mL substratum access 10mL schizochytrium limacinum liquid spawn after cooling, cultivate about 120 hours.Wherein start in 85 hours in fermentation, controlling culture temperature is 28.0 ± 0.5 DEG C; Within 85 hours, in 95 hours, controlling culture temperature is 25.0 ± 0.5 DEG C; Within 95 hours, to fermentation ends, controlling culture temperature is 20.0 ± 0.5 DEG C.And start in 65 hours in fermentation culture, by adding ammoniacal liquor with maintenance system pH 6.0 ± 0.2, and start in 105 hours in fermentation culture, 10g/L is greater than with the concentration of reducing sugar in maintenance system by adding stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1g/L, stopping fermentation, obtaining the schizochytrium limacinum fermentation liquid being rich in DHA.
(4) extraction of DHA
Get the schizochytrium limacinum fermentation liquid that obtains in 20mL step (3) in 50mL centrifuge tube, 8000r/min, 4 DEG C of frozen centrifugation 10min, resuspended centrifugal with deionized water, move to the flat board of known constant weight after repeating 2 times, in 85 DEG C of oven for drying to constant weight, measuring dry cell weight in fermented liq is 60g/L.
Get 5mL schizochytrium limacinum fermentation liquid in 10mL centrifuge tube, 8000r/min, 4 DEG C 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 DEG C of water-bath 1 hours, 0.75mL dehydrated alcohol and 2mL normal hexane is added after cooling, 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, and record that grease accounts for dry cell weight 40.8%.
Take 100mg gained schizochytrium limacinum grease, add 2mL n-hexane dissolution, then add 2mL10%HCl/CH3OH62 DEG C of constant temperature refuxing 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 μm × 250 μm × 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; Injector temperature 280 DEG C; Detector temperature is 280 DEG C; Heating schedule: keep 1min at 150 DEG C, 10 DEG C/min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min is run after 240 DEG C.
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 8.0%, DHA output is 10.3g/L.

Claims (1)

1. utilize a method for stalk hydrolyzed solution fermentative production docosahexenoic acid, comprise the steps:
(1) utilize ammonia blasting procedure pretreated straw powder, obtain pretreated straw powder; The particle diameter of described straw powder is less than 5 mm;
Described ammonia blasting procedure comprises the steps:
Regulating and controlling described straw powder to mass water content is 40% ~ 60%, then adds in high-pressure reactor; Then ammonia is utilized to process described straw powder under 3.0 ~ 4.0 MPa;
The temperature of described process is 80 DEG C ~ 100 DEG C, and the time is 30 ~ 60 minutes;
The mass water content of described pretreated straw powder is 60% ~ 70%;
(2) by pretreated straw powder described in cellulase, beta-galactosidase enzymes and pectinase enzymatic hydrolysis, stalk hydrolyzed solution is obtained;
The consumption of described cellulase, described beta-galactosidase enzymes and described polygalacturonase for: pretreated straw powder described in 1 Kg needs to add polygalacturonase described in beta-galactosidase enzymes described in cellulase described in 125 000 ~ 225 000 CMC U, 32 500 ~ 58 500 pNPG U and 1550 ~ 3100 U;
The condition of described enzymolysis is as follows:
6 ~ 7 days are processed under pH is 4.0 ~ 5.5 and temperature is the condition of 50 DEG C ~ 55 DEG C;
(3) in described stalk hydrolyzed solution, add substratum, then access schizochytrium limacinum ( aurantiochytrium) carry out fermentation culture, obtain schizochytrium limacinum fermentation liquid;
(4) extract described schizochytrium limacinum fermentation liquid with the mixed solution of ethanol and normal hexane, obtain extracting solution; Namely described extracting solution drying obtains docosahexenoic acid.
2, method according to claim 1, is characterized in that: in step (1), and described straw powder is made up of at least one in maize straw, rice straw, sugarcane stalk, wheat stalk, cotton stalk and broomcorn straw.
3, the method according to any one of claim 1-2, is characterized in that: in step (3), and described in every 100 mL, substratum is composed as follows:
1.0 ~ 2.0 g peptones; 1.0 ~ 2.0 g yeast extract powders; 2.0 ~ 3.0 g sea crystals; 0.04 ~ 0.06 g KH 2pO 4; 0.02 ~ 0.03 g (NH 4) 2sO 4; 0.2 ~ 0.4 g Na 2sO 4; With the water of surplus.
4, method according to claim 1, is characterized in that: in step (3), described schizochytrium limacinum ( aurantiochytrium) starting point concentration that accesses is schizochytrium limacinum described in every 100 mL substratum access 10 ~ 12 mL.
5, method according to claim 1, is characterized in that: in step (3), the condition of described fermentation culture is as follows:
Start in 75 ~ 85 hours in described fermentation culture, the temperature controlling described fermentation culture is 27.5 ~ 28.5 DEG C, and continue in fermentation culture to 85 ~ 95 hour, the temperature controlling described fermentation culture is 24.5 ~ 25.5 DEG C; Continue to be cultured to fermentation ends, the temperature controlling described fermentation culture is 19.5 ~ 20.5 DEG C;
And start in 55 ~ 65 hours in described fermentation culture, by adding ammoniacal liquor with maintenance system pH for 5.8 ~ 6.2, continue in fermentation culture to 95 ~ 105 hour, 10 g/L are greater than with the concentration of reducing sugar in maintenance system by adding described stalk hydrolyzed solution, afterwards, when the concentration of reducing sugar in system being detected lower than 1 g/L, stopping fermentation, namely obtaining schizochytrium limacinum fermentation liquid.
6, method according to claim 1, is characterized in that: in step (4), and before extracting described schizochytrium limacinum fermentation liquid with the mixed solution of ethanol and normal hexane, described method also comprises the steps:
Described schizochytrium limacinum fermentation liquid is carried out centrifugation, obtains supernatant liquor; Be suspended from again in hydrochloric acid soln with after supernatant liquor described in brine.
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