CN101096640A - Method for producing arachidonic acid with fresh water green algae cultivation by photoautotroph mode - Google Patents
Method for producing arachidonic acid with fresh water green algae cultivation by photoautotroph mode Download PDFInfo
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Abstract
The invention relates to a technology for culturing and producing microalgae in particular to a method for producing arachidonic acid (tetracosenoic acid)by culturing fresh water algae with photoautotroph method. The technical project comprises the following steps: (1)domesticating and screening green algae seed; (2)culturing the domesticated green algae seed in the culture medium with photo biological reactor; (3)accumulating the quick growth of the algae cell and polyunsaturated fatty acid; proceeding with a large scale culture of the green algae; (4)collecting when the biomass in the culture liquid and the arachidonic acid in the algae reach the requirements; (5)getting arachidonic acid by collecting the algae cell; (6)circulating the collected breeding waste water. The invention is used to produce arachidonic acid.
Description
Technical field
The present invention relates to little algae and cultivate and production technology, relate in particular to the method for cultivating the long arachidonic acid of green algate of fresh water production (eicosatetraenoic acid) in the photoautotrophy mode.
Background technology
Green alga Neochloris belongs to or the Parietochloris genus is the unicellular algae that belongs to Chlorophyta, cell circle or oval, diameter is 10-15um, under the certain culture condition, can accumulate arachidonic acid in a large number in the frond, reach as high as 20% of dry weight, and the arachidonic acid major part in this algae all is distributed in the oil droplet in the frond, arachidonic acid distribution mode unanimity with in the milk is easy to be absorbed by the body, utilize.Arachidonic acid is a kind of important unsaturated fatty acids, is the precursor of many class eicosenoic acids, and as prostaglandin(PG), blood coagulation flavine and blood triolefin element etc. have the important physical function.Docosatetratenoic acid still is the important component part of cell and cytolemma, plays an important role in prevention and treatment multiple disease of human body and infant's growth.Simultaneously, arachidonic acid still is one of important component of phosphatide in human brain cytolemma and other neurocyte, and brain and retinal development to the infant particularly play an important role in the growth of infant's cognitive ability and eyesight.But the mankind itself can not synthesize these lipid acid, must obtain from food, and arachidonic content are very low in the general food.International food and agricultural organization (FAO) and The World Health Organization (WHO) declare, grow for guaranteeing baby's normal growth, add the arachidonic acid of per kilogram infant weight 60mg in the suggested formula milk powder.U.S. Wyeth adds arachidonic formula milk to it claims, adds arachidonic acid and can make baby's IQ increase by 7 fens.
In addition, arachidonic acid is with a wide range of applications at aspects such as food, healthcare products, biochemical industry and aquacultures.
Relevant arachidonic physiological function and Research on Practical Application thereof have more report, and this also is the theoretical foundation that people pay attention to edible fish oil.Mostly traditional living tetraenoic acid is to extract from fish oil.But, because the bathypelagic fish oil composition is very complicated, particularly contain a lot of harmful saturated fatty acids, in addition, with fish oil is that the arachidonic acid that raw material obtains has a lot of shortcomings, as have fishy smell, mouthfeel is very poor, also is subjected to the pollution of heavy metal simultaneously easily, and its application is very restricted.Simultaneously, Fu Za separation and purification procedures also cause the cost of lipid acid very high.At last, also be faced with the problem that there is lack of raw materials, cause its output and price to fluctuate along with the output of fish and price because fishing resources exhausted day by day is these lipid acid of raw material with fish oil.At present except coming from fish oil, utilize fermentation method to cultivate the arachidonic acid product of mortierella (Mortierelal) production on the market in addition, adopt the fermentative Production arachidonic acid to have complex process, invest big, as need to prevent bacterium pollution, need adding external source organic carbon source and cost to cross high unavoidable problem.And little algae both can have been cultivated under sealing condition as photo-autotroph, also can cultivate under open condition, only needed to add in substratum some inorganic nutritive salt in culturing process.Simultaneously,, therefore not only can extract arachidonic acid because little algae work is the category that belongs to plant, but also can be directly edible with the form of algae powder.
Before the present patent application; arachidonic report of a lot of productions and patent have been arranged; as patent CN1175956, CN1255545C, CN1323904, CN1362522, CN15399825, CN1537378 etc.; produce arachidonic acid but all be confined to utilize the heterotrophic fermentation method to cultivate; because the arachidonic acid content in general little algae is very low, therefore utilizes the photoautotrophy method to change the cultivation green alga on a large scale, commercially produce arachidonic acid and still belong to blank.
Summary of the invention
For overcoming the deficiencies in the prior art, the objective of the invention is to: provide a kind of and cultivate green algate of fresh water in the photoautotrophy mode and produce arachidonic method, the technical solution used in the present invention is:
Cultivate green algate of fresh water with photoautotrophy and produce arachidonic method, comprise the following steps:
1., green alga algae kind is tamed, is screened;
2., domestication back green algae adopts the optical-biological reaction reactor to carry out the pre-cultivation of algae kind in substratum;
3., after pre-the cultivation, carry out the quick growth of frustule and the accumulation of polyunsaturated fatty acid, i.e. the large scale culturing of green alga;
4., biomass in the above-mentioned nutrient solution and the arachidonic acid in the frond reach when necessarily requiring and can gather;
Thereby 5., collect frustule and obtain needed arachidonic acid;
6., the breeding wastewater after recycle is gathered.
Described substratum comprises carbon source, nitrogenous source, phosphorus source, iron source, inorganic salt, and carbon source is yellow soda ash, sodium bicarbonate, carbonic acid, carbonic acid gas or its mixture; Nitrogenous source is selected from organic nitrogen compound, inorganic nitrogen compound or its mixture; The phosphorus source is phosphoric acid salt, hydrophosphate or its mixture; The iron source is organic iron compound, inorganic iron compound or its mixture; Inorganic salt are selected from following material for one or more: sodium salt, sylvite, magnesium salts, calcium salt, manganese salt, brill salt, zinc salt, mantoquita, molybdate, boride and composition thereof.
Described carbon source is yellow soda ash, sodium bicarbonate, carbonic acid, carbonic acid gas or its mixture, and described inorganic nitrogen compound comprises nitrate, its mixture of ammonium salt, and organic nitrogen compound is a urea.
Described carbon source concentration is 0-50mM/L, and used nitrogen concentration is 0-50mM/L, and described phosphorus source concentration is 0.01-1.0mM, and described iron source concentration is 0.01-50uM, and described inorganic salt concentration is a standard with the sodium salt, is 0.01-100ppm.
The pH value of substratum is 5.0-10.0.
Described culture temperature is 5-45 ℃, is preferably 10-30 ℃, more preferably 15-25 ℃.
Described cultivation is carried out under ventilation or stuffiness and agitation condition.
Described cultivation is cultivated under source of artificial light or lamp.
Be refined as and comprise the following steps:
1., green alga is carried out acclimation and screening under cold condition;
2., the green alga algae after domestication is cultivated in the substratum of one of claim 1-5 in advance;
3., after pre-the cultivation, inoculate in the substratum of one of above-mentioned claim 1-5 and cultivate, wherein the concentration of nitrogenous source be the 1/100-1/2 of nitrogen concentration in the pre-cultivation in this step substratum.
Wherein pre-incubated temperature is 10-45 ℃ and is preferably 10-30 ℃, more preferably 15-25 ℃.，。
The above-mentioned culture temperature after pre-the cultivation is 5-45 ℃, is preferably 10-30 ℃, more preferably 15-25 ℃.
The cultivation facility of indication is enclosed or the cultivation facility that do not seal.
Cell harvesting is meant bubble separation, sedimentation, filtration or the method such as centrifugal of adopting.
Substratum behind the collection frustule is handled the combination that refers to adopt sedimentation, filtration, foam separation or three kinds of methods.
The present invention can bring following technique effect:
By adjusting culture condition, impel green alga to accumulate arachidonic acid in a large number, wait arachidonic acid content to reach 10% and gather when above.
Adopt filtration method or whizzer to separate green alga, any chemical substance is not added in sepn process, and the stillness of night after the separation can be returned and culture the pond and use, and has solved the edible green alga problem of gathering.
Adopt foamet or MF method to remove algae liquid gather organism and solid particulate in the breeding wastewater of back, water can recycling, can the conserve water resource.
The present invention has prolonged the green alga breeding production phase, has improved the output and the quality of culturing green alga greatly; Produce food grade algae powder, develop the second product of high added value.
Product of the present invention itself is an environmental protective hygienic food, can be processed into capsule or adds to such as in milk preparation and other food with the form of foodstuff additive.
Algae powder among the present invention can adopt the arachidonic acid in organic solvent or the vegetable oil extraction algae powder, uses as foodstuff additive.
Description of drawings
Embodiment
The invention provides the substratum that a kind of green algate of fresh water is used, comprise carbon source, nitrogenous source, phosphorus source, iron source, inorganic salt, wherein said carbon source comprises yellow soda ash, sodium bicarbonate, carbonic acid, carbonic acid gas or its mixture; Described nitrogenous source is selected from organic nitrogen compound, inorganic nitrogen compound or its mixture; Described inorganic nitrogen compound comprises nitrate, ammonium salt or its mixture, and organic nitrogen compound is a urea.Described iron source is organic iron compound, inorganic iron compound or its mixture; This is selected from following material for one or more described inorganic salt: sodium salt, sylvite, magnesium salts, calcium salt, manganese salt, brill salt, molybdate, zinc salt, boride, mantoquita and composition thereof.
In substratum of the present invention, wherein said carbon source concentration is 0-50mM/L, and used nitrogen concentration is 0-50mM; Described phosphorus source concentration is 0-0.5mM; Described iron source concentration is 0.1-50uM; Described inorganic salt concentration is a standard with the sodium salt, is 0.01-100ppm.Its pH value is 5.0-11.0.
The present invention also provides a kind of method of cultivating little algae in above-mentioned substratum.This method is to carry out in ventilation and/or mechanical stirring condition.Described little algae is that Neochloris or Parietochloris belong to algae.The algae kind is kept in solid inclined-plane or the liquid nutrient medium.
The used freshwater microalgae of the present invention is to produce arachidonic little algae, comprises the kind that can wait acquisition from UTEX (Texas ,Usa university protect kind of a center) and CCAP (Britain algae and protozoon preservation center), as UTEX1707, and CCAP254/6.
The purpose of this invention is to provide a kind of cultivation green alga and produce arachidonic method, it is to adopt closed or open culture systems, cultivate green alga and produce arachidonic acid, this method adopts the photoautotrophy method to cultivate little algae, compares with other method, only needs to add some inorganic salt in substratum, mustn't add organism as nutrition, as the energy, have advantages such as less investment, cost is low, cultivation is simple with luminous energy, and can be in saline and alkaline beach and desert etc. the area produce.
The present invention adopts method and the facility of big area closed culture green alga, and closed cultivation is meant the transparent vessel in sealing.Open cultivation refer in the open or the cultivation pool in the greenhouse in cultivate.The pond that the pond body has Steel Concrete or soil to build up covers or the film of plastic covering or rubber mass not, and there is the isolation strip centre, and whipping appts is arranged in the pond, and nutrient solution is circulated; The pond is provided with by steel or other material etc. and makes skeleton, makes the awning on wall, top with glass reinforced plastic, glass, synthetic glass or plastics film, around the canopy by the ventilation window.This breed pond need not be keeped in repair all the year round, can reduce nutrient solution and pollute, and improves algae opaque amount and output, prolongs the breeding production phase.
Further specify the present invention below in conjunction with embodiment.
Embodiment 1:
Disinfectant fresh water is put into the duct type bioreactor of one-level breeding, and adjusting PH is 6～8, and nutrient concentration is that 1.5g/ rises NaNO
3, 36mg/L CaCl2,20mg/L Na2CO3,75mg/L MgSO4,30mg/L K2HPO4,1mg/L Fe-EDTA and trace element solution, inoculate according to 0.1g biomass/L, and pour the filtrated air that contains the 1-5% carbonic acid gas, algae liquid is circulated in the pipeline of sealing.Cultured 5～10 days; Enter the secondary breeding phase, thin waterway in glasshouse is cultured and is placed above-mentioned breed nutrient solution in the pond, culture adopt lamp, inoculation culture liquid from above-mentioned one-level bioreactor, inoculum density be the 0.1g biomass/liter, cultured 5-10 days; Enter three grades of breeding phases, according to 0.1g biomass/L, density change above-mentioned secondary expanding species liquid in plastic greenhouse thin waterway and culture in the above-mentioned breed nutrient solution in the pond, cultured 7～10 days; Under field conditions (factors), disinfectant fresh water is put into the outdoor breeding pond that has whipping appts, adjust water body, requiring wherein is pH6～8, adds three grades of expanding species liquid in above-mentioned water body, and inoculum density is 0.1g biomass/L, add above-mentioned element, NaNO is kept to the 1/3-1/10 of original content, and stirred 0.5 hour every 2～3 hours daytime, cultured 7～10 days; Treat that above-mentioned nutrient solution biomass reaches 1-2g biomass/L, arachidonic acid content reaches at 10% o'clock can be adopted centrifugal or micro-filtration is gathered; Algae after the separation is stuck with paste spraying drying in spray-dryer, and inlet temperature is 165 ℃, and temperature out is 80 ℃, get final product green algae powder.
Embodiment 2:
According to embodiment 1 described step, wherein use the condition of culturing nutrient solution to be: temperature is 6-8,0.25g/L NaNO at 25 ℃, pH value
3, 2.5mg/L CaCl2,25mg/L NaCl, 75mg/L MgSO4,25mg/L K2HPO4,12.5mg/LFeSO4-EDTA and trace element solution.
Embodiment 3:
According to embodiment 1 described step, wherein use the condition of culturing nutrient solution to be: temperature is 1.0/ liter of NaNO at 15 ℃, pH value as 6-8, nutrient concentration
3, 2.5mg/L CaCl2,25mg/L NaCl, 75mg/L MgSO4,25mg/L K2HPO4,12.5mg/L FeSO4-EDTA and trace element solution.
Embodiment 4:
With disinfectant fresh water or put into the pillar bioreactor and cultivate in advance, adjusting PH is 6～8, and nutrient concentration is that 1.5g/ rises NaNO
3, 36mg/L CaCl2,20mg/L Na2CO3,75mg/L MgSO4,30mg/L K2HPO4,1mg/L Fe-EDTA and trace element solution, inoculate according to 0.1g biomass/L, and pour the filtrated air that contains the 0-5% carbonic acid gas and stir, continuous illumination, intensity of illumination is 150W, and culture temperature is 25 ℃.Cultured 5～10 days; Collect frustule when reaching 5g/L Deng biomass, continue in the NaNO3 nutrient solution to cultivate above-mentioned not the containing that is transferred to new configuration, cultured 5～10 days, waits arachidonic acid content to reach at 10% o'clock can to adopt centrifugal or micro-filtration is gathered; Algae after the separation is stuck with paste spraying drying in spray-dryer, and inlet temperature is 165 ℃, and temperature out is 80 ℃, get final product green algae powder.
Embodiment 5:
According to embodiment 1 described substratum, cultivation facility wherein is board-like bioreactor, wherein use the condition of culturing nutrient solution to be: temperature is at 20-30 ℃, pH value is 6-8, illumination is 120W, pour the filtrated air of carbonated 1-5% at reactor bottom, cultivated 5-10 days, stop inflation when reaching 5g/L Deng biomass, treat that the frustule natural subsidence is to reactor bottom, discard supernatant liquid liquid, and add and to continue inflation in the nutrient solution that does not contain SODIUMNITRATE of new configuration and cultivate, cultured 5～10 days, wait arachidonic acid content to reach at 10% o'clock can to adopt centrifugal or micro-filtration is gathered; Algae after the separation is stuck with paste spraying drying in spray-dryer, and inlet temperature is 165 ℃, and temperature out is 80 ℃, get final product the fresh green algae powder.
Embodiment 6: cultivate according to embodiment 1 described cultivation, illumination changes 250W into, cultures altogether and cultivates after 14 days; Reaching 5g/L and arachidonic acid content Deng biomass reaches 10% and can adopt centrifugal or micro-filtration is gathered; Algae after the separation is stuck with paste spraying drying in spray-dryer, and inlet temperature is 165 ℃, and temperature out is 80 ℃, get final product green algae powder.
Outstanding substantial characteristics of the present invention and positively effect can be embodied from following embodiment, but they are not that the present invention is imposed any restrictions.
Therefore by the above, according to method of the present invention, cultivate green alga suitability for industrialized production arachidonic acid in the autotrophy mode, can low-cost, stably provide the arachidonic acid that does not have fishy smell, substitute the existing extracting method expensive from fish oil or the method for heterotrophic fermentation.
In addition, the present invention also comprises yellow soda ash, sodium bicarbonate in carbon source, carbonic acid, carbonic acid gas; Organic nitrogen compound, inorganic nitrogen compound; Inorganic nitrogen compound comprises nitrate, ammonium salt, and organic nitrogen compound comprises urea.Described iron source is organic iron compound, inorganic iron compound; This is selected from following material for one or more described inorganic salt: test under the condition of sodium salt, clock salt, magnesium salts, calcium salt, manganese salt, brill salt, molybdate, zinc salt, boride, mantoquita, still can obtain certain effect.
In substratum of the present invention, wherein said carbon source concentration is 0,50mM/L, and used nitrogen concentration is 0,50mM; Described phosphorus source concentration is 0.01,1.0mM; Described iron source concentration is 0.01,50uM; Described inorganic salt concentration is a standard with the sodium salt, is 0.01,100ppm.Its pH value is 5,10 to test respectively, still can obtain certain effect.
The pre-incubated temperature of the present invention is 5,45 ℃; Test, still can obtain certain effect.
The concentration of nitrogenous source is tested for 1/100,1/5 of nitrogen concentration in pre-the cultivation, still can obtain certain effect.
More than invention has been described with embodiment.Those of ordinary skill in the art can make multiple change on above-mentioned basis, these changes still belong within this specification sheets institute covering scope.
Claims (9)
1, a kind ofly cultivates green algate of fresh water in the photoautotrophy mode and produce arachidonic method, it is characterized in that, comprise the following steps:
1., green alga algae kind is tamed, is screened;
2., domestication back green algae adopts the optical-biological reaction reactor to carry out the pre-cultivation of algae kind in substratum;
3., after pre-the cultivation, carry out the quick growth of frustule and the accumulation of polyunsaturated fatty acid, i.e. the large scale culturing of green alga;
4., biomass in the above-mentioned nutrient solution and the arachidonic acid in the frond reach when necessarily requiring and can gather;
Thereby 5., collect frustule and obtain needed arachidonic acid;
6., the breeding wastewater after recycle is gathered.
2, according to claim 1ly a kind ofly cultivate green algate of fresh water in the photoautotrophy mode and produce arachidonic method, it is characterized in that, described substratum, comprise carbon source, nitrogenous source, phosphorus source, iron source, inorganic salt, carbon source is yellow soda ash, sodium bicarbonate, carbonic acid, carbonic acid gas or its mixture; Nitrogenous source is selected from organic nitrogen compound, inorganic nitrogen compound or its mixture; The phosphorus source is phosphoric acid salt, hydrophosphate or its mixture; The iron source is organic iron compound, inorganic iron compound or its mixture; Inorganic salt are selected from following material for one or more: sodium salt, sylvite, magnesium salts, calcium salt, manganese salt, brill salt, zinc salt, mantoquita, molybdate, boride and composition thereof.
3, according to claim 2ly a kind ofly cultivate green algate of fresh water in the photoautotrophy mode and produce arachidonic method, it is characterized in that, described carbon source is yellow soda ash, sodium bicarbonate, carbonic acid, carbonic acid gas or its mixture, described inorganic nitrogen compound comprises nitrate, its mixture of ammonium salt, and organic nitrogen compound is a urea.
4, according to claim 2ly a kind ofly cultivate green algate of fresh water in the photoautotrophy mode and produce arachidonic method, it is characterized in that, described carbon source concentration is 0-50mM/L, used nitrogen concentration is 0-50mM/L, and described phosphorus source concentration is 0.01-1.0mM, and described iron source concentration is 0.01-50uM, described inorganic salt concentration, with the sodium salt is standard, is 0.01-100ppm, and the pH value of substratum is 5.0-10.0.
5, according to claim 1ly a kind ofly cultivate green algate of fresh water with photoautotrophy and produce arachidonic method, it is characterized in that described culture temperature is 5-45 ℃, be preferably 10-30 ℃, more preferably 15-25 ℃.
6, according to claim 1ly a kind ofly cultivate green algate of fresh water in the photoautotrophy mode and produce arachidonic method, it is characterized in that, described cultivation is carried out under ventilation or stuffiness and agitation condition, and described cultivation is cultivated under source of artificial light or lamp.
7, a kind ofly cultivate green algate of fresh water with photoautotrophy and produce arachidonic method according to claim 1-4 is described, it is characterized in that, be refined as and comprise the following steps: 1., green alga is carried out acclimation and screening under cold condition; 2., the green alga algae after domestication is cultivated in the substratum of one of claim 1-4 in advance; 3., after pre-the cultivation, inoculate in the substratum of one of above-mentioned claim 1-4 and cultivate, wherein the concentration of nitrogenous source be the 1/100-1/2 of nitrogen concentration in the pre-cultivation in this step substratum.
8, according to claim 7ly a kind ofly cultivate green algate of fresh water with photoautotrophy and produce arachidonic method, it is characterized in that, wherein pre-incubated temperature is 10-45 ℃ and is preferably 10-30 ℃, more preferably 15-25 ℃, the above-mentioned culture temperature after pre-the cultivation is 5-45 ℃, be preferably 10-30 ℃, more preferably 15-25 ℃.
9, according to claim 1ly a kind ofly cultivate green algate of fresh water with photoautotrophy and produce arachidonic method, it is characterized in that, the cultivation of indication is to carry out in enclosed or the cultivation facility that do not seal, cell harvesting is meant and adopts bubble separation, sedimentation, filtration or method such as centrifugal, and collecting that substratum behind the frustule handles is the combination of adopting sedimentation, filtration, foam separation or three kinds of methods.
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| CN104446691A (en) * | 2013-09-24 | 2015-03-25 | 中国科学院上海高等研究院 | Micro algae-biochar composite bio-fertilizer and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104446691A (en) * | 2013-09-24 | 2015-03-25 | 中国科学院上海高等研究院 | Micro algae-biochar composite bio-fertilizer and preparation method thereof |
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