CN103882072A - Method for producing docosahexaenoic acid by using schizochytrium limacinum - Google Patents
Method for producing docosahexaenoic acid by using schizochytrium limacinum Download PDFInfo
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Abstract
The invention discloses a method for producing a docosahexaenoic acid (DHA) by using schizochytrium limacinum. The method comprises the following steps of (1) activating and culturing schizochytrium limacinum into a schizochytrium limacinum seed solution; (2) introducing the schizochytrium limacinum seed solution into a fermentation tank for culturing under the culture condition of 25-28 DEG C, aerating and stirring at a constant rotation speed, meanwhile fluidly feeding materials including carbon sources, nitrogen sources and glycerin, and keeping the concentration of glycerin at 0-10.0 g/liter; (3) controlling the temperature of a fermentation stable period at 18-23 DEG C, aerating and stirring at a constant rotation speed; and (4) when the concentration of the carbon source in a fermentation liquor is less than 5.0 g/liter, completing fermentation and collecting wet cells to obtain docosahexaenoic acid-rich greases. After the method is applied to large-scale fermentation and production of docosahexaenoic acid, the biomass of schizochytrium limacinum and the DHA content can increase, the quality of a DHA grease product is greatly improved, the grease quality is further improved and the energy consumption and the production cost are reduced.
Description
Technical field
The invention belongs to fermentation field, more specifically, relate to a kind of method of utilizing schizochytrium limacinum to produce docosahexenoic acid.
Background technology
Docosahexenoic acid (DHA) belongs to polyunsaturated fatty acid, has very important physiological function, is that human body self can not synthesize but indispensable important nutrient in a large number.DHA is present in human body retina and corticocerebral nervous tissue in a large number, has the brain of promotion and visual acuity, improves intelligence, improves the effect of memory and eyesight; In addition, it can also stop cholesterol to deposit on vessel wall, prevents or alleviate the generation of atherosclerosis and coronary heart disease.
The production ways of tradition DHA is mainly derived from the extraction purifying of fish oil; and fish oil resource is very limited; production cost is high; quality is unstable; output far can not be met the need of market; and ocean environment is seriously damaged in recent years, make its security be subject to serious query, in the urgent need to finding the new way of a kind of DHA of being produced on a large scale.At present, some low grade marine bacteria, thalassiomycetes and microalgaes are found, as Shewanella (Shewanella), thraustochytriale (Thraustochytrium), schizochytrium limacinum (Schizochytrium), Kou Shi Crypthecodinium cohnii (Crythecodinium cohnii) etc. can synthesize DHA.Due to microorganism, have that growth is fast, nutritional requirement is low and be easy to carry out the features such as large scale culturing, become the first-selected approach of large-scale production DHA.Especially schizochytrium limacinum, growth cycle is short, and biomass and fat content are high, and target product DHA productivity is higher, on DHA produces, is used widely
But utilize at present schizochytrium limacinum to produce the method DHA of DHA, DHA content is no more than 50%, further improves DHA content, will significantly improve product quality, thus in the situation that not increasing cost additional income significantly.Meanwhile, prior art, for maintaining thalline normal growth, therefore needs to maintain fermentor tank and ventilates in a large number, and energy consumption is larger.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of method of utilizing schizochytrium limacinum to produce docosahexenoic acid, its object is to induce schizochytrium limacinum to increase the resultant quantity of DHA by glycerine, further improve thus DHA output, DHA oil and fat product quality is improved greatly, reduce DHA production cost.
For achieving the above object, according to one aspect of the present invention, provide a kind of method of utilizing schizochytrium limacinum to produce docosahexenoic acid, comprised the following steps:
(1) schizochytrium limacinum kind activation culture is become to schizochytrium limacinum seed liquor;
(2) schizochytrium limacinum seed liquor is cultivated according to volume percent 2% to 4% access fermentor tank, 25 degrees Celsius to 28 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, flow feeding simultaneously, described feed supplement comprises carbon source, nitrogenous source and glycerine, maintains glycerol concentration in the scope of 0 to 10.0 grams per liter;
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, between 18 degrees Celsius to 23 degrees Celsius, is ventilated and keeps constant rotational speed to stir;
(4) when carbon source concentration in fermented liquid finishes fermentation during lower than 5.0 grams per liter, thereby collect wet thallus and obtain being rich in the grease of docosahexenoic acid.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, in step described in it (2) flow feeding be according to carbon source concentration level in fermented liquid in batches stream add, described carbon source is preferably glucose and/or maltose, described nitrogenous source is preferably yeast powder and/or Sodium Glutamate.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, step described in it (2) point three flow feedings, each flow feeding, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, starting stream adds, when carbon source concentration in fermented liquid is between 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops stream and adds in the time of 5.0 grams per liter to 10.0 grams per liter.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, described in it, schizochytrium limacinum is schizochytrium limacinum (Schizochytrium sp) S056, be preserved in Chinese Typical Representative culture collection center C CTCC on September 29th, 2013, depositary institution is Wuhan, China Wuhan University, and deposit number is CCTCC M2013459.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, and its step (2) is controlled air flow between 10 cubic meters/hour to 50 cubic meters/hour, and step (3) is controlled air flow between 10 cubic meters/hour to 30 cubic meters/hour.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, and schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by step described in it (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 1% to 5%, under the condition of 150 revs/min to 220 revs/min of shaking speed, 22 degrees Celsius to 28 degrees Celsius of culture temperature, carry out shake-flask culture, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed liquor;
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 1% to 5%, 150 revs/min to 300 revs/min of mixing speed, 0.2 cubic meter/hour to 15 cubic meters/hour of air flow, under the condition that culture temperature is 22 degrees Celsius to 28 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, seed culture medium component in step described in it (a): glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, peptone 1.0 grams per liter to 5.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, seed enlarged culturing base component in its step (b) in seed culture tank: glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, soybean-cake flour 0 to 3.0 grams per liter, SODIUMNITRATE 1.0 grams per liter to 2.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
Preferably, the described schizochytrium limacinum that utilizes is produced the method for docosahexenoic acid, and in its step (a), the culture cycle of seed liquor is 20-24 hour, and in step (b), the culture cycle of seed enlarged culturing is 20-24 hour.
According to another aspect of the present invention, a kind of application is provided, glycerine is applied to induction schizochytrium limacinum increases docosahexenoic acid resultant quantity.
In general, the above technical scheme of conceiving by the present invention compared with prior art, can obtain following beneficial effect:
(1) method of utilizing schizochytrium limacinum to produce docosahexenoic acid provided by the present invention, by complex optimum schizochytrium limacinum culture condition, can improve the biomass of schizochytrium limacinum, thereby improves DHA output.
(2) the present invention can be applicable to the fermentation of more than 10 tons large scale fermentation tank, the in the situation that of scale of mass production, still can maintain the high biological yield of schizochytrium limacinum.
(3) the present invention induces schizochytrium limacinum to increase the resultant quantity of docosahexenoic acid by glycerine, during large scale fermentation, add glycerine and maintain suitable concn, thereby induction schizochytrium limacinum increases synthetic docosahexenoic acid, in the situation that biomass is certain, further improve the content of docosahexenoic acid, thereby improved the output of docosahexenoic acid, DHA oil and fat product quality is improved greatly.
(4) method provided by the invention, by Optimizing Process Parameters and selection bacterial classification, air flow while compared with prior art significantly having reduced fermentation, be only 1/10th left and right of prior art air flow, thereby reduced cost of energy and Financial cost for ventilating, environmental friendliness and production cost significantly reduce.
(5) the present invention by comprehensive later stage low temperature fermentation, reduce air flow and flow feeding etc. fermentation condition, further improved the output of docosahexenoic acid, DHA oil and fat product quality is improved greatly
Accompanying drawing explanation
Fig. 1 is the method flow diagram that utilizes schizochytrium limacinum to produce docosahexenoic acid provided by the invention.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.In addition,, in each embodiment of described the present invention, involved technical characterictic just can combine mutually as long as do not form each other conflict.
The method of utilizing schizochytrium limacinum to produce docosahexenoic acid provided by the invention, comprises the following steps:
(1) schizochytrium limacinum activation culture is become to schizochytrium limacinum seed liquor; Described schizochytrium limacinum is preferably schizochytrium limacinum (Schizochytrium sp) S056, is preserved in Chinese Typical Representative culture collection center C CTCC on September 29th, 2013, and deposit number is CCTCC M2013459.
(2) schizochytrium limacinum seed liquor is cultivated according to volume percent 2% to 4% access fermentor tank, 25 degrees Celsius to 28 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, with time-division three flow feedings, described feed supplement comprises carbon source, nitrogenous source and glycerine, carbon source is preferably glucose and/or maltose, more preferably glucose; Nitrogenous source is preferably yeast powder and/or Sodium Glutamate, and more preferably yeast powder maintains in the scope of glycerol concentration 0 to 10 grams per liter.Each flow feeding, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, starting stream adds, when carbon source concentration in fermented liquid is between 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops stream and adds in the time of 5.0 grams per liter to 10.0 grams per liter.
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, between 18 degrees Celsius to 23 degrees Celsius, is controlled air flow between 10 cubic meters/hour to 100 cubic meters/hour, keeps constant rotational speed to stir.
(4) when carbon source concentration in fermented liquid finishes fermentation during lower than 5.0 grams per liter, thereby collect wet thallus and obtain being rich in the grease of docosahexenoic acid.
Schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by described step (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 1% to 5%, under the condition of 150 revs/min to 220 revs/min of shaking speed, 22.0 degrees Celsius to 28.0 degrees Celsius of culture temperature, carry out shake-flask culture, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed liquor.Described seed culture medium preferred ingredient: glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, peptone 1.0 grams per liter to 5.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 1% to 5%, 150 revs/min to 300 revs/min of mixing speed, 0.2 cubic meter/hour to 15.0 cubic meters/hour of air flow, under the condition that culture temperature is 22.0 degrees Celsius to 28.0 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.Seed enlarged culturing base preferred ingredient in described seed culture tank: glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, soybean-cake flour 0 to 3.0 grams per liter, SODIUMNITRATE 1.0 grams per liter to 2.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
Be below embodiment:
Embodiment 1
Utilize schizochytrium limacinum to produce a method for docosahexenoic acid, comprise the following steps:
(1) by schizochytrium limacinum (Schizochytrium sp) S056, activation culture becomes schizochytrium limacinum seed liquor.
(2) schizochytrium limacinum seed liquor being accessed to fermentor tank according to volume ratio 2% cultivates, 28 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, 10 cubic meters/hour to 50 cubic meters/hour of air flows, flow feeding simultaneously, described feed supplement comprises carbon source, nitrogenous source and glycerine, maintains glycerol concentration at 0 to 10 grams per liter.Described carbon source is glucose, and described nitrogenous source is yeast powder.Flow feeding, divide three streams to add, each stream adds, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, starting stream adds, when carbon source concentration in fermented liquid is at 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops stream and adds in the time of 5.0 grams per liter to 10.0 grams per liter.
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, at 23 degrees Celsius, is controlled air flow between 10 cubic meters/hour to 30 cubic meters/hour, keeps constant rotational speed to stir.
(4) in the time that glucose concn in fermented liquid is 0.0 grams per liter, finish fermentation, thereby collection wet thallus obtains being rich in the grease of docosahexenoic acid.
Schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by described step (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 1%, under the condition of 220 revs/min of shaking speed, 22 degrees Celsius of culture temperature, carries out shake-flask culture, makes schizochytrium limacinum growth arrive logarithmic phase, obtains seed liquor.Described seed culture medium preferred ingredient: glucose 30 grams per liters, yeast powder 8 grams per liters, peptone 3 grams per liters, magnesium sulfate 0.5 grams per liter, potassium primary phosphate 2 grams per liters.
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 1%, 300 revs/min of mixing speed, 0.2 cubic meter/hour to 0.5 cubic meter/hour of air flow, under the condition that culture temperature is 28 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.Seed enlarged culturing base preferred ingredient in described seed culture tank: glucose 30 grams per liters, yeast powder 8 grams per liters, soybean-cake flour 0 grams per liter, SODIUMNITRATE 1.6 grams per liters, magnesium sulfate 0.5 grams per liter, potassium primary phosphate 2 grams per liters.
Fermentation results, fermentation biomass, DHA content and DHA output are in table 1.
Embodiment 2
Utilize schizochytrium limacinum to produce a method for docosahexenoic acid, comprise the following steps:
(1) schizochytrium limacinum activation culture is become to schizochytrium limacinum seed liquor.Described schizochytrium limacinum, obtain in accordance with the following methods: get a certain amount of water sample, coated YPD solid medium (the glucose 20.0g/L of high salt, yeast powder 10.0g/L, peptone 20.0g/L, agar 20.0g/L, sea crystal 10.0 g/L) on, cultivate 48 hours for 25.0 degrees Celsius, obtain a large amount of single bacterium colonies, picking list bacterium colony is in liquid nutrient medium, 180 revs/min of shaking speed, under the condition that culture temperature is 25.0 degrees Celsius, carry out shake-flask culture after 72 hours, respectively thalline is dyeed, microscopic examination, screening obtains the bacterial strain that oil offtake is high, utilize gas chromatograph to analyze the fatty acid component of above bacterial strain grease, choose and in fatty acid component, contain DHA and the higher strain of content, further by colonial morphology and displaing microstructure observing, and the Molecular Evolutionary Analysis of 18s rDNA, identify that this bacterial strain is schizochytrium limacinum.
(2) schizochytrium limacinum seed liquor being accessed to fermentor tank according to volume ratio 4% cultivates, 25 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, 120 cubic meters/hour to 150 cubic meters/hour of air flows, flow feeding simultaneously, described feed supplement comprises carbon source, nitrogenous source and glycerine, maintains glycerol concentration in the scope of 0 to 10 grams per liter.Described carbon source is maltose, and described nitrogenous source is Sodium Glutamate.Flow feeding, divide three streams to add, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, start flow feeding, when carbon source concentration in fermented liquid is between 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops flow feeding in the time of 5.0 grams per liter to 10.0 grams per liter.
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, at 19 degrees Celsius, is controlled air flow between 80 cubic meters/hour to 100 cubic meters/hour, keeps constant rotational speed to stir.
(4) when carbon source concentration in fermented liquid finishes fermentation during lower than 5.0 grams per liter, thereby collect wet thallus and obtain being rich in the grease of docosahexenoic acid.
Schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by described step (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 3%, under the condition of 180 revs/min of shaking speed, 26 degrees Celsius of culture temperature, carries out shake-flask culture, makes schizochytrium limacinum growth arrive logarithmic phase, obtains seed liquor.Described seed culture medium preferred ingredient: glucose 50 grams per liters, yeast powder 10 grams per liters, peptone 1 grams per liter, magnesium sulfate 1.2 grams per liters, potassium primary phosphate 1 grams per liter.
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 5%, 220 revs/min of mixing speed, 12 cubic meters/hour to 15 cubic meters/hour of air flows, under the condition that culture temperature is 22 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.Seed enlarged culturing base preferred ingredient in described seed culture tank: glucose 60.0 grams per liters, yeast powder 10 grams per liters, soybean-cake flour 2 grams per liters, SODIUMNITRATE 1 grams per liter, magnesium sulfate 1 grams per liter, potassium primary phosphate 1 grams per liter.
Fermentation results, fermentation biomass, DHA content and DHA output are in table 1.
Embodiment 3
Utilize schizochytrium limacinum to produce a method for docosahexenoic acid, comprise the following steps:
(1) schizochytrium limacinum activation culture is become to schizochytrium limacinum seed liquor.Described schizochytrium limacinum, obtains in accordance with the following methods:
By the schizochytrium limacinum gathering in embodiment 2 through ultraviolet ray-nitrosoguanidine complex mutation repeatedly, mutafacient system is to utilize 0.5mL PBS damping fluid (100mM, pH7.0) to make schizochytrium limacinum bacteria suspension, add again 0.5mL2.5mg/mL nitroso guanidine solution, process 1 hour for 30 degrees Celsius, centrifugal above-mentioned treatment solution under aseptic condition, makes its bacterium liquid OD with distilled water wash thalline
650after reaching 0.4 to 0.6, pour culture dish into, make this bacterium liquid become the thin layer liquid of thickness 1mm to 5mm, again under 25W ultraviolet lamp, 40mm distance is irradiated 30 seconds, the centrifugal thalline through above-mentioned ultraviolet processing under aseptic condition, and by its Eddy diffusion and 1mL liquid YPD substratum, coat solid YPD flat board, under 25 ℃ of conditions, dark culturing 48 hours is to growing single bacterium colony, the single bacterium colony growing after mutagenesis differs in size, screen the schizochytrium limacinum bacterial strain that wherein cell dia is larger, carry out rapid screening using this as superior strain morphological specificity, access is containing in 96 hole high flux screening equipment of 1.5mL liquid YPD substratum respectively, under the condition of 200 revs/min of shaking speed, concussion is cultivated 72 hours, the higher bacterial strain of centrifugal acquisition biomass.
(2) schizochytrium limacinum seed liquor being accessed to fermentor tank according to volume ratio 3% cultivates, 26 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, 60 cubic meters/hour to 80 cubic meters/hour of air flows, flow feeding simultaneously, described feed supplement comprises carbon source, nitrogenous source and glycerine, maintains glycerol concentration in the scope of 0 to 10 grams per liter.Described carbon source is the mixture of glucose and maltose mass ratio 1:1, and described nitrogenous source is the mixture of yeast powder and Sodium Glutamate mass ratio 1:1.Flow feeding, divide three streams to add, each stream adds, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, starting stream adds, when carbon source concentration in fermented liquid is between 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops stream and adds in the time of 5.0 grams per liter to 10.0 grams per liter.
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, at 18 degrees Celsius, is controlled air flow between 50 cubic meters/hour to 70 cubic meters/hour, keeps constant rotational speed to stir.
(4) when carbon source concentration in fermented liquid finishes fermentation during lower than 3.0 grams per liter, thereby collect wet thallus and obtain being rich in the grease of docosahexenoic acid.
Schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by described step (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 5%, under the condition of 150 revs/min of shaking speed, 28 degrees Celsius of culture temperature, carries out shake-flask culture, makes schizochytrium limacinum growth arrive logarithmic phase, obtains seed liquor.Described seed culture medium preferred ingredient: glucose 60 grams per liters, yeast powder 5 grams per liters, peptone 5 grams per liters, magnesium sulfate 2 grams per liters, potassium primary phosphate 3 grams per liters.
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 3%, 150 revs/min of mixing speed, 8 cubic meters/hour to 10 cubic meters/hour of air flows, under the condition that culture temperature is 26 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.Seed enlarged culturing base preferred ingredient in described seed culture tank: glucose 50.0 grams per liters, yeast powder 5 grams per liters, soybean-cake flour 3 grams per liters, SODIUMNITRATE 2 grams per liters, magnesium sulfate 2 grams per liters, potassium primary phosphate 3 grams per liters.
Fermentation results, fermentation biomass, DHA content and DHA output are in table 1.
Embodiment 4
For embodiment 1, embodiment 2, embodiment 3, carry out respectively blank test; Use identical schizochytrium limacinum, according to identical fermentation process, only feed supplement is corresponding carbon source, nitrogenous source, and does not comprise that glycerine is contrast, and the blank fermentation results of embodiment 1, embodiment 2, embodiment 3 is in table 1.Results of comparison shows, to three kinds of schizochytrium limacinums, adding after glycerine, biomass, DHA content, DHA output etc. are in table 1, show that glycerine can induce schizochytrium limacinum to increase the synthetic of docosahexenoic acid.
Table 1 fermentation results
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. utilize schizochytrium limacinum to produce a method for docosahexenoic acid, it is characterized in that, comprise the following steps:
(1) schizochytrium limacinum kind activation culture is become to schizochytrium limacinum seed liquor;
(2) schizochytrium limacinum seed liquor is cultivated according to volume percent 2% to 4% access fermentor tank, 25 degrees Celsius to 28 degrees Celsius of culture temperature, ventilate and keep constant rotational speed to stir, flow feeding simultaneously, described feed supplement comprises carbon source, nitrogenous source and glycerine, maintains glycerol concentration in the scope of 0 to 10.0 grams per liter;
(3) enter fermentation stability after date, after oxygen dissolved no longer declines, controlled fermentation temperature, between 18 degrees Celsius to 23 degrees Celsius, is ventilated and keeps constant rotational speed to stir;
(4) when carbon source concentration in fermented liquid finishes fermentation during lower than 5.0 grams per liter, thereby collect wet thallus and obtain being rich in the grease of docosahexenoic acid.
2. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 1, it is characterized in that, in described step (2) flow feeding be according to carbon source concentration level in fermented liquid in batches stream add, described carbon source is glucose and/or maltose, and described nitrogenous source is yeast powder and/or Sodium Glutamate.
3. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 2, it is characterized in that, described step (2) point three flow feedings, each flow feeding, within the scope of carbon source concentration in fermented liquid drops to 20.0 grams per liter to 30.0 grams per liters time, starting stream adds, when carbon source concentration in fermented liquid is between 30.0 grams per liter to 40.0 grams per liters, nitrogen concentration is between 2.0 grams per liter to 4.0 grams per liters, and glycerol concentration stops stream and adds in the time of 5.0 grams per liter to 10.0 grams per liter.
4. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 1, it is characterized in that, described schizochytrium limacinum is schizochytrium limacinum (Schizochytrium sp) S056, be preserved in Chinese Typical Representative culture collection center C CTCC on September 29th, 2013, deposit number is CCTCC M2013459.
5. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 4, it is characterized in that, step (2) is controlled air flow between 10 cubic meters/hour to 50 cubic meters/hour, and step (3) is controlled air flow between 10 cubic meters/hour to 30 cubic meters/hour.
6. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 1, is characterized in that, schizochytrium limacinum kind activation culture is become schizochytrium limacinum seed liquor by described step (1), comprises following sub-step:
(a) seed culture:
In seed culture medium, access schizochytrium limacinum, inoculum size volume percent is 1% to 5%, under the condition of 150 revs/min to 220 revs/min of shaking speed, 22 degrees Celsius to 28 degrees Celsius of culture temperature, carry out shake-flask culture, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed liquor;
(b) seed enlarged culturing:
Seed enlarged culturing base in the shake-flask seed liquid access seed culture tank that step (a) is obtained carries out enlarged culturing, inoculum size volume percent is 1% to 5%, 150 revs/min to 300 revs/min of mixing speed, 0.2 cubic meter/hour to 15 cubic meters/hour of air flow, under the condition that culture temperature is 22 degrees Celsius to 28 degrees Celsius, carry out seed enlarged culturing, make schizochytrium limacinum growth arrive logarithmic phase, obtain seed and expand seed liquor.
7. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 6, it is characterized in that, seed culture medium component in described step (a): glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, peptone 1.0 grams per liter to 5.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
8. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 6, it is characterized in that, seed enlarged culturing base component in step (b) in seed culture tank: glucose 30.0 grams per liter to 60.0 grams per liters, yeast powder 5.0 grams per liter to 10.0 grams per liters, soybean-cake flour 0 to 3.0 grams per liter, SODIUMNITRATE 1.0 grams per liter to 2.0 grams per liters, magnesium sulfate 0.5 grams per liter to 2.0 grams per liter, potassium primary phosphate 1.0 grams per liter to 3.0 grams per liters.
9. the method for utilizing schizochytrium limacinum to produce docosahexenoic acid as claimed in claim 6, is characterized in that, in step (a), the culture cycle of seed liquor is 20-24 hour, and in step (b), the culture cycle of seed enlarged culturing is 20-24 hour.
10. glycerine is applied to induction schizochytrium limacinum increases docosahexenoic acid resultant quantity.
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| CN106834370A (en) * | 2017-01-20 | 2017-06-13 | 武汉净宇微藻科技有限公司 | A kind of method of raising schizochytrium limacinum DHA and carotenoids yield |
| CN106676127A (en) * | 2017-02-14 | 2017-05-17 | 厦门大学 | Construction method and application of schizochytrium limacinum engineering bacteria for KS (Beta-ketoacyl Synthase) gene knockout |
| CN106676127B (en) * | 2017-02-14 | 2020-01-17 | 厦门大学 | Construction method and application of ketone group synthetase gene knocked-out schizochytrium limacinum engineering bacteria |
| CN112481189A (en) * | 2020-12-29 | 2021-03-12 | 嘉必优生物技术（武汉）股份有限公司 | Method for domesticating schizochytrium limacinum and application thereof |
| CN112481189B (en) * | 2020-12-29 | 2022-08-09 | 嘉必优生物技术（武汉）股份有限公司 | Method for domesticating schizochytrium limacinum and application thereof |
| WO2022198990A1 (en) * | 2021-03-22 | 2022-09-29 | 华南理工大学 | Method for increasing yield of eicosapentaenoic acid in schizochytrium sp. |
| CN114940947A (en) * | 2022-04-29 | 2022-08-26 | 深圳古他生物科技有限公司 | DHA-producing schizochytrium GT-D1 and application thereof, DHA-rich grease and preparation method thereof |
| CN114940947B (en) * | 2022-04-29 | 2024-03-29 | 深圳古他生物科技有限公司 | Schizochytrium limacinum GT-D1 capable of producing DHA, application thereof, DHA-rich grease and preparation method thereof |
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