CN104480178A - Method for rapidly accumulating astaxanthin by forcing haematococcus pluvialis - Google Patents
Method for rapidly accumulating astaxanthin by forcing haematococcus pluvialis Download PDFInfo
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Abstract
The invention discloses a method for rapidly accumulating astaxanthin by forcing haematococcus pluvialis, belonging to the field of aquaculture. Formerly, haematococcus pluvialis is cultured by virtue of an annular track type cement pit or a photobioreactor, so that astaxanthin is low in yield and easily polluted, the process is complex, the operation is troublesome, and the investment of capital construction and the cost are high. According to the method, a two-stage culture method is utilized; in the first stage, the culture is carried out in a conical flask, relatively low temperature and illumination intensity are set, so that the growth of algal cells is benefited, and the damage caused by highlight to the cells is avoided; in the second stage, the aeration culture is carried out by virtue of a white plastic bucket, so that the temperature and the illumination intensity are increased, the illumination time is prolonged, and the accumulation of astaxanthin is benefited. A formula of traditional culture liquid is further optimized, so that nutrients are relatively comprehensive and balanced, the cost is low, the operation is flexible and convenient, and the yield of astaxanthin is increased by 120%.
Description
Technical field
The invention belongs to aquaculture field, particularly relate to a kind of method of coercing Haematocoocus Pluvialls Rapid Accumulation astaxanthin.
Background technology
Haematocoocus Pluvialls is named again the raw haematococcus pulvialis in lake or lake green blood ball algae, is extensively present in organic abundant poisons in freshwater.Be subordinate to Chlorophyta, Chlorophyceae, volvocales, haematococcus pulvialis section, haematococcus.Determination of Astaxanthin in Haematococcus Pluvialis content is 1.5-3.0%, is counted as the concentrate of natural astaxanthin.Astaxanthin is a kind of natural antioxidant efficiently, and major function is scavenging free radicals, improves flight against senium of human body ability.Large quantity research show the Haematocoocus Pluvialls accumulation speed of astaxanthin and output biological higher than other, and the proportioning of Haematocoocus Pluvialls institute's astaxanthin-containing and ester class thereof (about 70% monoesters, 25% dibasic acid esters and 5% monomer) and aquatic animal self proportioning fairly similar, the advantage that the astaxanthin having chemosynthesis and utilize phaffiafhodozyma etc. to extract does not have.The structure of Determination of Astaxanthin in Haematococcus Pluvialis is based on 3S-3 ' S type, basically identical with astaxanthin structure in the aquatic living things bodies such as salmon; Astaxanthin in phaffiarhodozyma structure is then 3R-3R type.Haematocoocus Pluvialls is acknowledged as the best biology that occurring in nature produces natural astaxanthin, cultivates Haematocoocus Pluvialls extraction astaxanthin and has vast potential for future development.In addition, Haematocoocus Pluvialls extract is gorgeous redness, there is extremely strong pigment deposition ability, functional pigmented as one, can be used as the fodder additives of abalone, sturgeon, salmon, rainbow trout, porgy, Crustacean and fancy fishes and various bird, live pig, effect be anti-oxidant, eliminate free radical, the generation of enhancing antibody, strengthen animal immunizing power, improve its ornamental value.Haematocoocus Pluvialls is many at organic matter, grows vigorous, optimum growth temperature 25-30 DEG C, optimal light intensity 4000-18000lux, advantageous pH range 7.5-8.5 in the fresh water that particularly nitrogen, microcosmic salt enrich.
My research shows, haematococcus pluvialis growing stage and astaxanthin accumulation stage require significantly different to environmental factors such as illumination, temperature, salinity, nutritive salt, the aging synthesis being conducive to astaxanthin of high light intensity, high-temperature and nutrient solution.Accordingly, the present invention proposes the two-stage cultivation method of Haematococcus pluvialis production, namely first with lower temperature and light according to and higher nitrogen salt condition algae is bred to comparatively ideal density, then improve temperature and light according to the accumulation of coercing astaxanthin.
About the method for coercing Haematocoocus Pluvialls and accumulate in a large number astaxanthin, past, Yan little Jun discloses a kind of method (application number 201310038104) of haematococcus pluvialis to produce astaxanthin, first Haematocoocus Pluvialls is naturally cultivated 6-8 days in the water of cultivation pool pond, allow it breed, then in pool water within, add potassium primary phosphate and SODIUMNITRATE, and be covered with red pigment on pond, wavelength is under 630-760nm light-wave irradiation, allows Haematocoocus Pluvialls frustule grow to conversion state fast; Transform the Chi Shuizhong that state frustule is the potassium primary phosphate of 0.02-0.05 grams per liter in concentration, and be covered with blue membrane on pond, wavelength is under 430-490nm light-wave irradiation, allows Haematocoocus Pluvialls frustule support to maturation, obtains the Haematocoocus Pluvialls being rich in astaxanthin; Potassium primary phosphate, SODIUMNITRATE and ruddiness can make frustule grow fast, and the short period reaches conversion state; Potassium primary phosphate, blue light can make the quick growth and maturity of frustule and accumulation astaxanthin; The method less investment, cultivate simple, better results, but troublesome poeration, easily pollute, be difficult to apply aborning.In addition, Xu Qingshan have also been invented a kind of method for transformation (application number 201210561399) cultivating haematococcus pluvialis to produce astaxanthin, comprise the preparation of immobilization transformant, immobilization transformant combine to cell of nourishing and growing, relevant envrionment conditions control etc., step is as follows: 1) prepare highdensity Haematocoocus Pluvialls transformant; 2) immobilization gel carrier is prepared; 3) immobilization Haematocoocus Pluvialls transformant gel carrier is prepared; 4) algae kind concentration as required, adds Haematocoocus Pluvialls transformant gel carrier prepared by step 3, immobilization gel carrier and the culturing cell needing to transform; 5) temperature controls at 24-26 DEG C, and controlled light is at 8000Lux, and by regulating the method for water level, controlling water level, at 0.5m, adopts the method for flowing water, realizes the Induction Transformation of immobilization transformant; Method inducing effect provided by the invention is obvious, stable and reliable operation, but complex process, troublesome poeration, and initial cost is large, and cost is high, of poor benefits.
In order to overcome the deficiency of current technology, the present invention adopts two-stage cultivation method, first stage carries out in Erlenmeyer flask, lower temperature and light is provided to shine intensity, be conducive to frustule growth and avoid high light to cause light to injure to cell, subordinate phase adopts white plastic tank air-charging incubation in microdisk electrode room, improves temperature and light and shines intensity and extend light application time, be conducive to the accumulation of astaxanthin.The present invention is also optimized traditional nutrient solution formula, supplements Ca (NO
3)
2, NaH
2pO
4, MgSO
47H
2o, CuSO
45H
2o, MnCl
24H
2o, FeSO
47H
2o, urea, people urinates, calcium pantothenate, medical stone powder, α-naphthaleneacetic acid, inositol, folic acid, Nicotinicum Acidum, vitamin H, EDTA, the compositions such as soil extract, nutrition more comprehensively, balanced, not easily pollute, cost is low, easy handling, and astaxanthin yield improves 120%.
Summary of the invention
In order to overcome the deficiency of current technology, the invention provides a kind of method of coercing Haematocoocus Pluvialls Rapid Accumulation astaxanthin, concrete grammar is as follows:
The preparation nutrient solution composition of step 1 nutrient solution: Ca (NO
3)
20.4 part, NaH
2pO
40.1 part, MgSO
47H
2o 0.05 part, CuSO
45H
2o 0.02 part, MnCl
24H
2o 0.1 part, FeSO
47H
2o 0.04 part, 0.5 part, urea, people urinates 1 part, calcium pantothenate 0.03 part, medical stone powder 0.04 part, α-naphthaleneacetic acid 0.002 part, inositol 0.001 part, 0.004 part, folic acid, Nicotinicum Acidum 0.001 part, vitamin H 0.008 part, EDTA 0.04 part, soil extract 1.2 parts, 1000 parts, cold boiling water; Load in brown grinding port plug reagent bottle, shake up, sealing, it is for subsequent use to insert 4 DEG C of Refrigerator stores;
Step 2 Erlenmeyer flask is cultivated and is got one 3000 milliliters of Erlenmeyer flasks, add nutrient solution described in 1500 milliliters of steps 1, choosing growth is vigorous, pollution-free, be in the Haematocoocus Pluvialls algae kind inoculation of exponential phase of growth, inoculation will be carried out at 9-10 point in the morning, inoculum density 40,000 cells/every milliliter, shake up gently, newspaper and bungee sealing, be placed in microdisk electrode room and cultivate, air-conditioning temperature control, fluorescent tube provides illumination, do not inflate, adjust the temperature to 26 DEG C, intensity of illumination is to 5000lux, light dark period=12h/L+12h/D, namely intermittent light is provided, 12 h light, 12 h dark, every day manual shaking flask 3 times, each 45 seconds, cultured continuously is after 7 days, proceed to next step,
One 65 liters of large spoken parts in an opera plastic tanks (commercially available) with cover are got in the cultivation of step 3 white plastic tank, and add nutrient solution described in step 1 40 liters, then add algae liquid described in step 2 1800 milliliters, shake 3 minutes gently, cover lid, cultivates.Lid is stamped 2 holes, each bore dia 10 millimeters, insert 2 pipes, one is plastics tubing, long 3 meters, diameter 8 millimeters, and one end connects air compressor, and the other end is inserted into bottom algae liquid, in order to inflation, is filled with mixed gas, gaseous constituent=pure CO of air 98%+
22%, uninterrupted inflation continuously, tolerance should not be too large, can not float to allow frustule; Another root is shorter, and long 30 centimetres, diameter 10 millimeters is harder material pipe, in order to exhaust; This plastic tank is put into microdisk electrode room cultivate, room conditioning temperature control, fluorescent tube throws light on, controlled light intensity is carried out with the quantity opening fluorescent tube, there is provided continuous unbroken light photograph, cultured continuously 8 days, within first 4 days, adjust the temperature to 28 DEG C, intensity of illumination 18000lux is (because of stop and the sorption of plastic tank wall, the actual intensity of illumination arriving algae liquid is about 12000lux), latter 4 days, adjust the temperature to 30 DEG C, intensity of illumination 20000lux, after proceed to next step;
Step 4 frustule is collected and is got algae liquid described in step 3 1000 parts, adds 0.5 part of chitin, fully stirs 15 minutes, then adds 0.8 part of alum, stir 20 minutes, leaves standstill, precipitates 36 hours, abandon supernatant liquor, and get the red algae mud in bottom, frustule is collected complete.
Beneficial outcomes
In the past, Haematocoocus Pluvialls adopted endless track formula cement pit or bioreactor to cultivate, and shortcoming is that astaxanthin productive rate is low, easily pollutes, complex process, troublesome poeration, and initial cost is large, and cost is high, deficiency in economic performance.In order to overcome the deficiency of current technology, the present invention adopts two-stage cultivation method, first stage carries out in Erlenmeyer flask, lower temperature and light is provided to shine intensity, be conducive to frustule growth and avoid high light to cause light to injure to cell, subordinate phase adopts white plastic tank air-charging incubation in microdisk electrode room, improves temperature and light and shines intensity and extend light application time, be conducive to the accumulation of astaxanthin.The present invention is also optimized traditional nutrient solution formula, supplements Ca (NO
3)
2, NaH
2pO
4, MgSO
47H
2o, CuSO
45H
2o, MnCl
24H
2o, FeSO
47H
2o, urea, people urinates, calcium pantothenate, medical stone powder, α-naphthaleneacetic acid, inositol, folic acid, Nicotinicum Acidum, vitamin H, EDTA, the compositions such as soil extract, nutrition more comprehensively, balanced, not easily pollute, cost is low, easy handling, and astaxanthin yield improves 120%.
Accompanying drawing explanation
Fig. 1 is the Haematocoocus Pluvialls form of a large amount of accumulation astaxanthin under field of microscope;
Fig. 2 cultivates Haematocoocus Pluvialls white plastic tank figure used in step 3;
Fig. 3 is the growing state of Haematocoocus Pluvialls, wherein X-coordinate be incubation time (my god), ordinate zou is cell density, and unit is ten thousand cells/every milliliter of algae liquid.
Embodiment
The preparation method of soil extract described in step 1: get loose in the woods, to be rich in organic matter upper layer of soil 1 kilogram, add 5 liters, tap water, boil 20 minutes, mud is poured in beaker, leave standstill 24 hours, rear Aspirate supernatant, pour in Erlenmeyer flask, add cotton plug, boil 15 minutes, cooling, staticly settles 12 hours, gets supernatant liquor and namely obtain soil extract, load grinding port plug reagent bottle, sealing, labelled, be placed in 4 DEG C of refrigerators for subsequent use;
Described in step 1, people urinates preparation method: get one 3000 milliliters of Erlenmeyer flasks, loads people and urinates 1500 milliliters, leave standstill, spontaneous fermentation 48 hours, be placed on electric furnace and heat, boil 3 minutes, cooling, load the brown reagent bottle of grinding port plug, sealing, is placed in 4 DEG C of refrigerator cold-storages for subsequent use;
The Haematocoocus Pluvialls of inoculation carries out purifying in advance (this area ordinary method, as long as can realize purifying, such as Gao Shi
Extraction method), during to guarantee inoculation, algae kind is purebred and is in eugonic exponential phase of growth;
In step 1, various nutritive salt will add by the order provided in formula, to avoid chemical reaction;
In step 1, various inorganic nutrient salt and VITAMIN can be made into mother liquor in advance and be placed in 4 DEG C of refrigerator cold-storages and save backup, and do not exceed 30 days storage period;
In step 1 and 2 glassware such as Erlenmeyer flask used all need to wash be placed on dry in 85 DEG C of baking ovens after use;
Seeded process described in step 2 need at 9-10 point and carrying out on aseptic operating platform in the morning;
In step 3, plastic tank used will select light transmission good and ductile strength is high, to improve the efficiency of light energy utilization and plastic tank work-ing life;
In step 1, pharmaceutical chemicals purity used all needs chemical pure rank.
Coerce a method for Haematocoocus Pluvialls Rapid Accumulation astaxanthin, concrete grammar is as follows:
The preparation nutrient solution composition of step 1 nutrient solution: Ca (NO
3)
20.4 part, NaH
2pO
40.1 part, MgSO
47H
2o 0.05 part, CuSO
45H
2o 0.02 part, MnCl
24H
2o 0.1 part, FeSO
47H
2o 0.04 part, 0.5 part, urea, people urinates 1 part, calcium pantothenate 0.03 part, medical stone powder 0.04 part, α-naphthaleneacetic acid 0.002 part, inositol 0.001 part, 0.004 part, folic acid, Nicotinicum Acidum 0.001 part, vitamin H 0.008 part, EDTA 0.04 part, soil extract 1.2 parts, 1000 parts, cold boiling water; Load in brown grinding port plug reagent bottle, shake up, sealing, it is for subsequent use to insert 4 DEG C of Refrigerator stores;
Step 2 Erlenmeyer flask is cultivated and is got one 3000 milliliters of Erlenmeyer flasks, add nutrient solution described in 1500 milliliters of steps 1, choosing growth is vigorous, pollution-free, be in the Haematocoocus Pluvialls algae kind inoculation of exponential phase of growth, inoculation will be carried out at 9-10 point in the morning, inoculum density 40,000 cells/every milliliter, shake up gently, newspaper and bungee sealing, be placed in microdisk electrode room and cultivate, air-conditioning temperature control, fluorescent tube provides illumination, do not inflate, adjust the temperature to 26 DEG C, intensity of illumination is to 5000lux, light dark period=12h/L+12h/D, namely intermittent light is provided, 12 h light, 12 h dark, every day manual shaking flask 3 times, each 45 seconds, cultured continuously is after 7 days, proceed to next step,
One 65 liters of large spoken parts in an opera plastic tanks (commercially available) with cover are got in the cultivation of step 3 white plastic tank, and add nutrient solution described in step 1 40 liters, then add algae liquid described in step 2 1800 milliliters, shake 3 minutes gently, cover lid, cultivates.Lid is stamped 2 holes, each bore dia 10 millimeters, insert 2 pipes, one is plastics tubing, long 3 meters, diameter 8 millimeters, and one end connects air compressor, and the other end is inserted into bottom algae liquid, in order to inflation, is filled with mixed gas, gaseous constituent=pure CO of air 98%+
22%, uninterrupted inflation continuously, tolerance should not be too large, can not float to allow frustule; Another root is shorter, and long 30 centimetres, diameter 10 millimeters is harder material pipe, in order to exhaust; This plastic tank is put into microdisk electrode room cultivate, room conditioning temperature control, fluorescent tube throws light on, controlled light intensity is carried out with the quantity opening fluorescent tube, there is provided continuous unbroken light photograph, cultured continuously 8 days, within first 4 days, adjust the temperature to 28 DEG C, intensity of illumination 18000lux is (because of stop and the sorption of plastic tank wall, the actual intensity of illumination arriving algae liquid is about 12000lux), latter 4 days, adjust the temperature to 30 DEG C, intensity of illumination 20000lux, after proceed to next step;
Step 4 frustule is collected and is got algae liquid described in step 3 1000 parts, adds 0.5 part of chitin, fully stirs 15 minutes, then adds 0.8 part of alum, stir 20 minutes, leaves standstill, precipitates 36 hours, abandon supernatant liquor, and get the red algae mud in bottom, frustule is collected complete.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in previous embodiment, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of embodiment of the present invention technical scheme.
Claims (1)
1. coerce a method for Haematocoocus Pluvialls Rapid Accumulation astaxanthin, it is characterized in that concrete steps are as follows:
The preparation of step (1) nutrient solution
Nutrient solution composition: Ca (NO
3)
20.4 part, NaH
2pO
40.1 part, MgSO
47H
2o 0.05 part, CuSO
45H
2o 0.02 part, MnCl
24H
2o 0.1 part, FeSO
47H
2o 0.04 part, 0.5 part, urea, people urinates 1 part, calcium pantothenate 0.03 part, medical stone powder 0.04 part, α-naphthaleneacetic acid 0.002 part, inositol 0.001 part, 0.004 part, folic acid, Nicotinicum Acidum 0.001 part, vitamin H 0.008 part, EDTA 0.04 part, soil extract 1.2 parts, 1000 parts, cold boiling water; Load in brown grinding port plug reagent bottle, shake up, sealing, it is for subsequent use to insert 4 DEG C of Refrigerator stores;
Step (2) Erlenmeyer flask is cultivated
Get one 3000 milliliters of Erlenmeyer flasks, add nutrient solution described in 1500 milliliters of steps 1, choosing growth is vigorous, pollution-free, be in the Haematocoocus Pluvialls algae kind inoculation of exponential phase of growth, inoculation will be carried out at 9-10 point in the morning, inoculum density 40,000 cells/every milliliter, shake up gently, newspaper and bungee sealing, be placed in microdisk electrode room and cultivate, air-conditioning temperature control, fluorescent tube provides illumination, do not inflate, adjust the temperature to 26 DEG C, intensity of illumination is to 5000lux, light dark period=12h/L+12h/D, namely intermittent light is provided, 12 h light, 12 h dark, every day manual shaking flask 3 times, each 45 seconds, cultured continuously 7 days, proceed to next step,
The white plastic tank of step (3) is cultivated
Get one 65 liters of large spoken parts in an opera plastic tanks (commercially available) with cover, add nutrient solution described in step 1 40 liters, then add algae liquid described in step 2 1800 milliliters, shake 3 minutes gently, cover lid, cultivates.Lid is stamped 2 holes, each bore dia 10 millimeters, insert 2 pipes, one is plastics tubing, long 3 meters, diameter 8 millimeters, and one end connects air compressor, and the other end is inserted into bottom algae liquid, in order to inflation, is filled with mixed gas, gaseous constituent=pure CO of air 98%+
22%, uninterrupted inflation continuously, tolerance should not be too large, can not float to allow frustule; Another root is shorter, and long 30 centimetres, diameter 10 millimeters is harder material pipe, in order to exhaust; This plastic tank is put into microdisk electrode room cultivate, room conditioning temperature control, fluorescent tube throws light on, controlled light intensity is carried out with the quantity opening fluorescent tube, there is provided continuous unbroken light photograph, cultured continuously 8 days, within first 4 days, adjust the temperature to 28 DEG C, intensity of illumination 18000lux is (because of stop and the sorption of plastic tank wall, the actual intensity of illumination arriving algae liquid is about 12000lux), latter 4 days, adjust the temperature to 30 DEG C, intensity of illumination 20000lux, after proceed to next step;
Step (4) frustule is collected
Get algae liquid described in step 3 1000 parts, add 0.5 part of chitin, fully stir 15 minutes, then add 0.8 part of alum, stir 20 minutes, leave standstill, precipitate 36 hours, abandon supernatant liquor, get the red algae mud in bottom, frustule is collected complete.
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| CN105524838A (en) * | 2016-03-07 | 2016-04-27 | 临沂大学 | Method for two-stage culture of porphyridium by using urine as main nitrogen source |
| CN110117541A (en) * | 2019-05-06 | 2019-08-13 | 哈尔滨师范大学 | A kind of high-efficiency antimicrobial culture medium and preparation method thereof of monoshell seam class diatom |
| CN110144296A (en) * | 2019-06-19 | 2019-08-20 | 北京林业大学 | A kind of cultural method and its application for cultivating the culture medium for producing astaxanthin microalgae, a kind of economical production astaxanthin microalgae |
| CN115449485A (en) * | 2022-11-14 | 2022-12-09 | 天津长芦汉沽盐场有限责任公司 | Method for culturing marine chlorella |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110144296A (en) * | 2019-06-19 | 2019-08-20 | 北京林业大学 | A kind of cultural method and its application for cultivating the culture medium for producing astaxanthin microalgae, a kind of economical production astaxanthin microalgae |
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