CN108949888B - Method for promoting dunaliella algae to accumulate carotenoids and beta-carotene by utilizing beta-ionone - Google Patents
Method for promoting dunaliella algae to accumulate carotenoids and beta-carotene by utilizing beta-ionone Download PDFInfo
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Abstract
The invention belongs to the technical field of food science and discloses a method for promoting dunaliella algae to accumulate carotenoids and beta-carotene by utilizing beta-ionone. The method comprises the following steps: performing induction culture on Dunaliella cells to be induced in a culture medium containing beta-ionone, accumulating beta-carotene in carotenoid and carotenoid in the algae cells, and then extracting pigment; the Dunaliella cells to be induced are Dunaliella cells in logarithmic growth phase and/or stationary phase. The method is simple and easy to implement, has low cost, can obviously improve the yield of the total carotenoid and the beta-carotene, has simple culture conditions because the Dunaliella is extremely salt-tolerant unicellular green algae, is not easily polluted by other microorganisms, is favorable for large-scale culture, and meets the standard of industrialized large-scale production.
Description
Technical Field
The invention belongs to the technical field of food science, and particularly relates to a method for promoting dunaliella algae to accumulate carotenoids and beta-carotene by utilizing beta-ionone.
Background
Carotenoids are a general term for an important group of natural pigments, which are widely distributed in higher plants, algae and certain photosynthetic and non-photosynthetic bacteria, fungi. Carotenoids have important physiological functions for animals and humans, and have effects of improving eyesight, resisting oxidation, enhancing immunity, preventing cardiovascular diseases, etc. Carotenoids are useful in industry as nutritional supplements, cosmetics, food colors and colorants. The beta-carotene is an orange natural pigment, rich in carrot, pawpaw, mango, green vegetables and the like, is a precursor of vitamin A, is a main source of the vitamin A of a human body, and has the effects of resisting oxidation (removing free radicals), enhancing the immunity of the human body, protecting the eyesight, delaying senescence, preventing cancers and the like.
At present, the production method of beta-carotene mainly comprises chemical synthesis, natural extraction, microbial fermentation and the like. In the past, the chemically synthesized beta-carotene products are basically exclusive to the market, and the currently used beta-carotene is mostly a chemically synthesized product, because the chemically produced beta-carotene has low cost, almost all trans-isomers, low biological activity and chromosome aberration effect, does not have many physiological functions of natural beta-carotene and reduces the efficacy of the product. In recent years, Blakeslea trispora (Blakesleatrispora) is utilized to ferment and produce natural beta-carotene, the all-trans structure of the beta-carotene produced by the method accounts for more than 85 percent, the cis structure accounts for 5 to 10 percent, and the beta-carotene has certain biological activity. The natural beta-carotene extracted from Dunaliella containing 40% cis-structure has good biological activity but higher cost. Dunaliella is an extremely salt-tolerant eukaryotic unicellular green alga, which is mainly found in salt pan, salt lake and sea, has strong stress resistance and simple culture conditions, can be cultured in a high-salt environment, can avoid the pollution of other microorganisms, and is beneficial to large-scale outdoor culture; under the conditions of high light, high salt, nutrient stress and the like, Dunaliella bardawil (Dunaliella bardawil) can accumulate a large amount of beta-carotene, the maximum weight can reach 14 percent of the dry weight, and the Dunaliella bardawil is an excellent biological source of natural beta-carotene. However, the long period for cultivating the dunaliella is relatively long, the yield of the beta-carotene is relatively limited, and the finding of a substance for promoting the dunaliella to largely accumulate the beta-carotene becomes a key technical problem in the field.
Disclosure of Invention
In order to quickly accumulate beta-carotene in carotenoid and improve the content of beta-carotene in carotenoid and carotenoid in Dunaliella, the invention provides a method for promoting Dunaliella to accumulate carotenoid and beta-carotene in carotenoid by utilizing beta-ionone.
The purpose of the invention is realized by the following technical scheme:
a method for promoting Dunaliella algae to accumulate carotenoids and beta-carotene by using beta-ionone comprises the following steps: the Dunaliella cell to be induced is induced and cultured in a culture medium containing beta-ionone, the carotenoid and beta-carotene in the carotenoid are accumulated in the algal cell, and then pigment is extracted.
The Dunaliella cells to be induced are Dunaliella cells in logarithmic growth phase (including prophase, metaphase and anaphase) and/or stationary phase. OD of algal fluid when Dunaliella algae cells are in logarithmic growth phase (including prophase, metaphase and anaphase) and/or stationary phase630≥0.7。
The Dunaliella is preferably Dunaliella bardawil.
The addition amount of the beta-ionone is 1-20 mg/L (namely 1-20 mg of beta-ionone is added into 1L of culture medium), preferably 1-18 mg/L, and more preferably 1-15 mg/L; the culture medium is a liquid culture medium, preferably a Dunaliella liquid culture medium.
The time of the induction culture is 2 h-7 days.
The induction culture conditions are that the culture is performed with shaking at the rotation speed of 10-200r/min under the conditions that the illumination intensity is 1000-8000Lx, the light-dark period is 6-24h:6-24h, the temperature is 20-35 ℃, and preferably at the rotation speed of 50r/min under the conditions that the illumination intensity is 8000Lx, the light-dark period is 14h:10h, the temperature is 26 ℃.
The dunaliella liquid culture medium comprises the following components: NaNO3 0.420g/L，NaCl 87.690g/L，NaH2PO4·2H2O 0.015g/L，NaHCO3 0.840g/L，KCl 0.074g/L，MgSO4·7H2O 1.230g/L，CaCl2·2H20.044g/L of O, 0.5mL/L of Fe-EDTA solution and 1mL/L of A5 trace element solution.
Fe-EDTA solution: na (Na)2EDTA 1.804g/L，FeCl36H2O 0.483 g/L; a5 trace element solution: h3BO32.860g/L，MnCl2·4H2O 1.810g/L，ZnSO4·7H2O 0.220g/L，CuSO4·5H2O 0.079g/L，(NH4)6Mo7O24·4H2O 0.039g/L。
The method comprises the following specific steps:
adding Dunaliella cell into liquid culture medium, culturing until OD of algae liquid630When the content is more than or equal to 0.7, adding beta-ionone to continue culturing, and extracting pigment to obtain carotenoid and beta-carotene. What is needed isThe culture conditions are that the culture is performed under the conditions of illumination intensity of 1000-.
The method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
The organic solvent is acetone, ethanol, methanol or acetonitrile and the like; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
The beneficial effect of the invention is that the content of carotenoid in Dunaliella algae and the content of beta-carotene in the carotenoid can be effectively improved after the beta-ionone is added, probably because the beta-ionone can promote the synthesis of isoprenoid which is a precursor substance for synthesizing the carotenoid. The dosage of the beta-ionone is very small, and the production cost is not obviously increased while the Dunaliella is stimulated to accumulate a large amount of beta-carotene. Because the beta-ionone is a volatile aroma component, the effect is better (namely the algae cells are in a logarithmic growth phase or a stable phase) by adding the beta-ionone 2-6 hours before the beta-carotene is collected and extracted from the dunaliella.
Compared with the prior art, the invention has the advantages that:
(1) the Dunaliella utilized by the invention belongs to eukaryotic autotrophic microorganisms, has simple culture conditions and strong stress resistance, can be cultured in a high-salt environment, can avoid the pollution of other microorganisms, and is beneficial to large-scale outdoor culture. And the Dunaliella is nontoxic and harmless, and can be directly used as a natural health product.
(2) The beta-ionone used in the invention has little dosage, and the production cost is not obviously increased while the Dunaliella is stimulated to accumulate a large amount of beta-carotene. The beta-ionone is a volatile aromatic substance, can be rapidly diffused, and can promote the dunaliella to accumulate the beta-carotene in a short time.
(3) Under the stress of extreme environments such as high salt, strong light, nitrogen deficiency and the like, the beta-carotene has higher yield and higher benefit.
The method is simple and easy to implement, has low cost, can obviously improve the yield of total carotenoid and beta-carotene, is extremely salt-tolerant unicellular green algae, has simple culture conditions, is not easily polluted by other microorganisms, is more beneficial to large-scale culture compared with bacteria and heterotrophic microorganisms such as Blakeslea trispora, and meets the standard of industrial large-scale production.
Drawings
FIG. 1 shows the treatment of Dunaliella bardawil (OD) with 2-20 mg/L beta-ionone6300.714) total carotenoid and β -carotene content for 3 hours;
FIG. 2 shows the treatment of Dunaliella bardawil (OD) with 2-20 mg/L beta-ionone6300.714) total carotenoid and β -carotene content for 6 hours;
FIG. 3 shows the treatment of Dunaliella bardawil (OD) with 1-50 mg/L of beta-ionone6301.421) total carotenoid and β -carotene content for 4 days.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto.
The method for measuring the pigment content comprises the following steps: measuring the absorbance values at the wavelengths of 665.2nm, 652.4nm and 470nm by using a spectrophotometer or a microplate reader, and calculating by using the following formula:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
The method for measuring the content of the beta-carotene comprises the following steps: firstly, a beta-carotene standard curve is made, 100 mu g/mL beta-carotene standard solutions are respectively diluted by acetone, and the following beta-carotene solutions with different concentrations are prepared: 0.1, 0.2, 0.3, 0.4, 0.5 and 1. mu.g/mL, and then measured at 453nm with a microplate readerTo obtain a standard curve y of 0.795x +0.0098, R20.999, where y is the absorbance at 453 nm; x is the beta-carotene content (. mu.g/mL).
Example 1
(1) Activated culture
Firstly, preparing a Dunaliella liquid culture medium, which comprises the following components: NaNO3 0.420g/L，NaCl 87.690g/L，NaH2PO4·2H2O 0.015g/L，NaHCO3 0.840g/L，KCl 0.074g/L，MgSO4·7H2O 1.230g/L，CaCl2·2H2O0.044 g/L, Fe-EDTA solution (Na)2EDTA 1.804g/L，FeCl3·6H2O0.483 g/L)0.5mL/L, A5 microelement solution (H)3BO3 2.860g/L，MnCl2·4H2O 1.810g/L，ZnSO4·7H2O 0.220g/L，CuSO4·5H2O 0.079g/L，(NH4)6Mo7O24·4H2O 0.039g/L)1mL/L；
Then, the Dunaliella bardawil cells are inoculated in the liquid culture medium and cultured under the conditions of illumination intensity of 8000Lx, light-dark period of 14h:10h (illumination 14h, no light 10h) (light-dark alternate culture) and temperature of 26 ℃ and shaking at the rotating speed of 50r/min to obtain the algae liquid with synchronous growth.
(2) Induced culture
When the algae cells in the algae liquid are in logarithmic growth phase (OD)6300.714), 0, 2, 5, 10 and 20mg/L β -ionone were added to the algal solution, and the culture was continued (8000 Lx of illuminance, 14h of light dark cycle: 10h (14 h of light, 10h of no light), 26 ℃ temperature, shaking culture at 50 r/min) for 3 hours.
(3) Pigment extraction
Centrifuging 2mL of algae solution at 8000r/min for 5min, discarding supernatant, adding 2mL of acetone into algae mud, vortex dispersing, leaching for 20min, centrifuging at 10000r/min for 15min, collecting upper acetone phase, transferring to new centrifuge tube, and filtering with 0.45 μm filter membrane.
(4) Determination of content
Measuring the absorbance values of the acetone phase solution at 665.2nm, 652.4nm and 470nm respectively by using a microplate reader, and calculating by using the following formula:
chlorophyll a (μ g/mL) ═ 16.72 · a665.2-9.16·A652.4
Chlorophyll b (μ g/mL) ═ 34.09. A652.4-15.28·A665.2
Total carotenoids (μ g/mL) ═ 1000. a470-1.63·Chl a-104.96·Chl b)/221
The method for measuring the content of the beta-carotene comprises the following steps: firstly, a beta-carotene standard curve is made, 100 mu g/mL beta-carotene standard solutions are respectively diluted by acetone, and the following beta-carotene solutions with different concentrations are prepared: 0.2, 0.5, 1, 2, 3, 4, 5 and 10 μ g/mL, and then measuring the absorbance at 453nm with a microplate reader to obtain a standard curve y of 0.795x +0.0098 and R2 of 0.999, where y is the absorbance at 453 nm; x is the beta-carotene content (. mu.g/mL).
The total carotenoid and beta-carotene contents are calculated according to the formula, and the total carotenoid and beta-carotene contents are respectively improved by 14.3-33.3% and 29.1-43.8% compared with a control group which is not treated by the beta-ionone after the Dunaliella bardawil is treated by the beta-ionone with the content of 2-20 mg/L for 3 hours (see figure 1).
Example 2
(1) Activated culture (same as example 1)
(2) Induced culture
The same as example 1, except that the culture was continued for 6 hours after the addition of beta-ionone.
(3) Pigment extraction (same as example 1)
(4) Determination of content
The method for measuring the contents of total carotenoid and beta-carotene is the same as that in example 1, and the results show that after the Dunaliella bardawil is treated by adding 2-20 mg/L beta-ionone for 6 hours, the contents of total carotenoid and beta-carotene are respectively improved by 9.3-33.0% and 16.2-38.1% compared with the control group which is not treated by adding beta-ionone (see figure 2).
Example 3
(1) Activated culture (same as example 1)
(2) Induced culture
When the Dunaliella bardawil cells are in stationary phase (OD)6301.421), 0, 1, 2, 5, 20 and 50mg/L β -ionone was added to the algal solution, and the culture was continued for 4 days.
(3) Pigment extraction (same as example 1)
(4) Determination of content
According to calculation, after the Dunaliella bardawil is treated by adding 1, 2 and 5mg/L beta-ionone for 4 days, the total carotenoid and beta-carotene contents are respectively improved by 13.9-28.6% and 31.0-39.3% compared with a control group which is not treated by adding the beta-ionone, and after the Dunaliella bardawil is treated by high-concentration beta-ionone, namely 50mg/L for 4 days, the total carotenoid and beta-carotene contents are obviously reduced compared with the control group due to the reduction of the cell biomass of the Dunaliella bardawil (see figure 3). The low concentration of beta-ionone can promote the Dunaliella algae to accumulate total carotenoids and beta-carotene.
Claims (5)
1. A method for promoting Dunaliella to accumulate carotenoids and beta-carotene by utilizing beta-ionone is characterized by comprising the following steps: the method comprises the following steps: adding Dunaliella cell into liquid culture medium, culturing until OD of algae liquid630When the content is more than or equal to 0.7, adding beta-ionone to continue culturing, and extracting pigment to obtain carotenoid and beta-carotene;
the Dunaliella cells are Dunaliella cells in logarithmic growth phase and/or stationary phase; the Dunaliella cell is a Dunaliella bardawil cell;
the addition amount of the beta-ionone is 1-20 mg/L;
the conditions of continuous culture, namely induction culture, are that the culture is performed with shaking at the rotating speed of 50r/min under the conditions that the illumination intensity is 8000Lx, the light-dark period is 14h:10h and the temperature is 26 ℃;
the culture conditions are that the culture is performed under the conditions that the illumination intensity is 1000-.
2. The method for promoting the accumulation of carotenoids and β -carotene by dunaliella using β -ionone according to claim 1, wherein: the induction culture time is 2 h-7 days.
3. The method for promoting the accumulation of carotenoids and β -carotene by dunaliella using β -ionone according to claim 1, wherein: the culture medium is a liquid culture medium; the liquid culture medium comprises the following components: NaNO3 0.420 g/L，NaCl 87.690 g/L，NaH2PO4·2H2O 0.015 g/L，NaHCO3 0.840 g/L，KCl 0.074 g/L，MgSO4·7H2O 1.230 g/L，CaCl2·2H20.044g/L of O, 0.5mL/L of Fe-EDTA solution and 1mL/L of A5 trace element solution;
the Fe-EDTA solution is Na2EDTA 1.804 g/L，FeCl3·6H2O0.483 g/L; the A5 trace element solution is H3BO32.860 g/L，MnCl2·4H2O 1.810 g/L，ZnSO4·7H2O 0.220 g/L，CuSO4·5H2O 0.079g/L，(NH4)6Mo7O24·4H2O 0.039 g/L。
4. The method for promoting the accumulation of carotenoids and β -carotene by dunaliella using β -ionone according to claim 1, wherein: the method for extracting the pigment comprises the following steps: centrifuging the cultured algae solution, discarding the supernatant, mixing the algae mud with an organic solvent, leaching, centrifuging, and filtering the supernatant with a filter membrane.
5. The method for promoting the accumulation of carotenoids and β -carotene by dunaliella using β -ionone according to claim 4, wherein: the organic solvent is acetone, ethanol, methanol or acetonitrile; the leaching time is 15-30 min, and the filter membrane is a 0.45-micrometer filter membrane.
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| CN101573318A (en) * | 2006-11-10 | 2009-11-04 | 帝斯曼知识产权资产管理有限公司 | Process for the preparation of belta-ionones and vitamin A, vitamin A derivatives, carotenes and carotenoids |
| CN104450849A (en) * | 2014-11-25 | 2015-03-25 | 临沂大学 | Method for forcing dunaliella tertiolecta to accumulate beta-carotene |
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| CN101573318A (en) * | 2006-11-10 | 2009-11-04 | 帝斯曼知识产权资产管理有限公司 | Process for the preparation of belta-ionones and vitamin A, vitamin A derivatives, carotenes and carotenoids |
| CN104450849A (en) * | 2014-11-25 | 2015-03-25 | 临沂大学 | Method for forcing dunaliella tertiolecta to accumulate beta-carotene |
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