CN108676768B - Chlorella growth promoter and preparation method thereof - Google Patents
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Abstract
The invention discloses a chlorella growth promoter and a preparation method thereof, wherein the chlorella growth promoter comprises the following raw materials in parts by weight: 0.1-10 parts of regulator, 0.001-2 parts of quercetin, 0.005-1 part of nano selenium and 987-999.894 parts of polyethylene glycol; wherein the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of (2-3) to (5-7); the invention opens up a new industrial culture way for microalgae growth, and has the advantages of simple material acquisition, convenient operation and low cost; the product is green, environment-friendly and safe, and the plant-derived flavonoid can be used as a natural plant stimulator to promote the photosynthetic metabolic reaction of chlorella cells and improve the growth potential of chlorella and the accumulation of carbon source substrates in the chlorella cells, so that the growth rate of the chlorella is improved, and the problem of industrial culture of rapid growth of microalgae is solved.
Description
Technical Field
The invention belongs to the field of biomass recycling and biological energy, and particularly relates to a chlorella growth promoter and a preparation method thereof.
Background
The chlorella has the characteristics of stable and rapid propagation speed and low cultivated land requirement of a culture system, and can be industrially cultured in an efficient bioreactor for large-scale production and application. Chlorella biomass has also been reported to be produced at rates exceeding those of many terrestrial plants. The method has high economic value, can be used for producing biodiesel, protein, secondary metabolites, such as carotenoid, astaxanthin, particularly unsaturated fatty acid and the like, and is widely applied to the development of green sustainable biological energy. When the chlorella culture medium is added with exogenous biological stimulin, stress pressure can be caused to the chlorella culture medium, the growth potential of the chlorella culture medium is improved, and meanwhile, the photosynthesis efficiency and biomass are promoted. The high-efficiency productivity offsets the cost of organic carbon sources necessary for the photosynthetic reaction of the cultured chlorella to a certain extent, provides a research basis for the subsequent high-density culture of the chlorella and the development of a series of metabolites with high added values, and provides theoretical support for optimizing the culture conditions and the culture technology of the chlorella.
The flavonoid quercetin is mainly present in fruits, leaves and bark of plants, and is also found in some vegetables and fruits. Quercetin contains a plurality of phenolic hydroxyl groups, belongs to natural flavonoid compounds, and has the functions of resisting oxidation and eliminating the activity of free radicals in cells and the pharmacological activities of resisting bacteria, viruses, cancers and tumors through a certain in-vitro oxidation model verification. Most of flavonoids of plant sources can be used as a natural phytohormone to influence the growth and metabolism of microalgae cells.
The industrial early-stage exploration of the chlorella in the market at the present stage mainly adopts the following three methods: (1) mutation breeding by irradiation; (2) the key gene expression in the growth and metabolism process of the chlorella is regulated and controlled by utilizing a genetic engineering technical means, so that the growth rate of the chlorella is improved; (3) optimizing the culture condition of chlorella. Some irradiation treatment and stress treatment can affect the growth metabolic state of algae cells, even lead to the reduction of the total yield of microalgae, and the algae cells need to be transferred in multi-stage culture, so the operation process is strict, and more culture cost is consumed. (2) In the absence of a stable and effective chlorella genetic transformation isolation system, the expression of the recombinant vector in the nucleus or chloroplast is transient and random. Therefore, the development of an efficient and environment-friendly chlorella growth promoter is urgently needed in the current industrial production of chlorella.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to develop an efficient and environment-friendly chlorella growth promoter to overcome the defects of the prior art.
The invention adopts the following technical scheme to solve the technical problems:
a chlorella growth promoter comprises the following raw materials in parts by weight: 0.1-10 parts of regulator, 0.001-2 parts of quercetin, 0.005-1 part of nano selenium and 987-999.894 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of (2-3) to (5-7).
Further, the chlorella growth promoter comprises the following raw materials in parts by weight: 7 parts of regulator, 0.005 part of quercetin, 0.06 part of nano-selenium and 992.935 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 2.5: 5.
Further, the chlorella growth promoter comprises the following raw materials in parts by weight: 2 parts of regulator, 0.015 part of quercetin, 0.7 part of nano-selenium and 997.285 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 3: 5.5.
Further, the chlorella growth promoter comprises the following raw materials in parts by weight: 4 parts of regulator, 0.035 part of quercetin, 0.1 part of nano-selenium and 995.865 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 2.5: 6.
Further, the chlorella growth promoter comprises the following raw materials in parts by weight: 10 parts of regulator, 0.02 part of quercetin, 0.5 part of nano-selenium and 989.48 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 3:5.
Further, the preparation method of the chlorella growth promoter comprises the following steps:
(1) stirring and uniformly mixing hexadecyl trimethyl ammonium bromide and ammonia water at 70-80 ℃ to obtain a clear solution, adding tetraethyl silicate, aging for 20-24h, drying the mixed solution, calcining at 550-600 ℃ for 5-6h to obtain silicon dioxide powder, performing intermittent irradiation treatment on the obtained silicon dioxide powder under a vacuum condition by using plasma with the temperature of 400-800 ℃, the power of 2-4KW and the source of oxygen and ammonia gas, and keeping the total effective irradiation time for 40-90min to obtain micro-nano biological silicon powder;
(2) placing the collected pistacia chinensis bunge leaves in an oven for 110-90 ℃ sterilization, collecting, drying the leaves, crushing, sieving, heating, performing ultrasonic extraction at 60-90 ℃ for 2 times, performing constant volume on the crushed mixture, performing reduced pressure filtration, centrifuging at 3000-6000rpm for 15-20min, performing filtration sterilization by using a filter membrane, keeping the supernatant, and performing freeze drying to obtain pistacia chinensis bunge leaf extract dry powder;
(3) mixing the micro-nano biological silicon powder obtained in the step (1) and the pistacia chinensis bunge leaf extract dry powder obtained in the step (2) according to the mass ratio to obtain a regulator;
(4) culturing providencia sp enriched with nano-selenium, adding 100mg/L sodium selenite into the fermentation liquid, culturing for 24-48h to obtain zymocyte liquid,wherein the effective viable count is 1.8 × 1010-2.6×1010cfu/mL; the providencia stuartii is providencia stuartii, and is purchased from China general microbiological culture collection center; the preservation number is CGMCC NO. 1.1528;
(5) performing ultrasonic treatment with the power of 100-;
(6) and (4) fully shaking and uniformly mixing the regulator obtained in the step (3), the quercetin, the nano-selenium obtained in the step (5) and the polyethylene glycol to prepare the chlorella growth promoter.
Further, the volume ratio of the oxygen to the ammonia in the step (1) is (2-3) to (5-7).
Further, the silicon dioxide powder prepared in the step (1) is 150-300 micron silicon dioxide powder.
Further, the intermittent irradiation treatment mode in the step (1) is as follows: irradiating for 5-10min, and stopping for 5-10 min.
Further, the fully shaking and uniformly mixing mode in the step (6) is as follows: processing at 3000-.
The invention has the following beneficial effects: compared with the prior art, the invention has the advantages that:
(1) the preparation method is simple, the materials are simple, the operation is simple and convenient, the cost is low, the large-scale production can be carried out, and a new way for industrial culture of microalgae growth is developed;
(2) promoting the growth of chlorella by using a regulator, nano-selenium, a plant source biological stimulator and polyethylene glycol to prepare a plant source nano-composite material; the plant-derived flavonoid substance can be used as a natural phytostimulin to regulate the growth metabolic process of algae cells, improve the antioxidant performance and growth potential of the algae cells and the accumulation of carbon source substrates in chlorella cells, thereby influencing the biomass accumulation of the algae cells, improving the growth rate of the algae cells and solving the problem of industrial culture of rapid growth of microalgae.
(3) The product is natural, green, environment-friendly, safe and free of side effects, and can improve the growth of chlorella by 1.5 times, effectively improve photosynthesis and biomass of chlorella, and improve the potential of industrial application.
Drawings
FIG. 1 is a graph showing the effect of a chlorella growth promoter according to an embodiment of the present invention on the growth of chlorella.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The chlorella growth promoter provided by the embodiment comprises the following raw materials in parts by weight: 7 parts of regulator, 0.005 part of quercetin, 0.06 part of nano-selenium and 992.935 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 2.5: 5. Compared with the blank control group, the growth promoting rate of chlorella on day 4 of the treatment group added with the extract can reach 3.43 times that of the common treatment group.
Example 2
The chlorella growth promoter provided by the embodiment comprises the following raw materials in parts by weight: 2 parts of regulator, 0.015 part of quercetin, 0.7 part of nano-selenium and 997.285 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 3: 5.5. Compared with the blank control group, the growth promoting rate of chlorella on day 4 of the treatment group added with the extract can reach 1.93 times that of the common treatment group.
Example 3
The chlorella growth promoter provided by the embodiment comprises the following raw materials in parts by weight: 4 parts of regulator, 0.035 part of quercetin, 0.1 part of nano-selenium and 995.865 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 2.5: 6. Compared with the blank control group, the growth promoting rate of chlorella on day 4 of the treatment group added with the extract can reach 2.47 times of that of the common treatment group.
Example 4
The chlorella growth promoter provided by the embodiment comprises the following raw materials in parts by weight: 10 parts of regulator, 0.02 part of quercetin, 0.5 part of nano-selenium and 989.48 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 3:5. Compared with the blank control group, the growth promoting rate of the chlorella on the 4 th day of the treatment group added with the extract can reach 1.97 times of that of the common treatment group.
Example 5
A method for preparing the chlorella growth promoter of the above embodiment comprises the following steps:
(1) stirring and uniformly mixing hexadecyl trimethyl ammonium bromide and ammonia water at 80 ℃ to obtain a clear solution, adding tetraethyl silicate, aging for 24 hours, drying the mixed solution, calcining at 600 ℃ for 6 hours to obtain 150-micron silicon dioxide powder, and performing intermittent irradiation treatment on the obtained silicon dioxide powder under a vacuum condition by using plasma with the temperature of 400 ℃, the power of 2KW and the volume ratio of oxygen to ammonia gas (2: 5) as a gas source, wherein the irradiation treatment is performed for 5 minutes, the stopping is performed for 5 minutes, and the total effective irradiation time is kept for 50 minutes to obtain micro-nano biological silicon powder;
(2) sterilizing collected pistacia chinensis bunge leaves in an oven at 120 ℃, collecting the pistacia chinensis bunge leaves, drying the leaves at 60 ℃, crushing, sieving, heating, performing ultrasonic extraction at 70 ℃ for 2 times, performing constant volume on the crushed mixture, performing reduced pressure filtration, centrifuging the mixture at the rotating speed of 4000rpm for 20min, performing filtration sterilization by using a filter membrane, keeping the supernatant, and performing freeze-drying to obtain dry powder of the pistacia chinensis bunge leaf extract;
(3) mixing the micro-nano biological silicon powder obtained in the step (1) and the pistacia chinensis bunge leaf extract dry powder obtained in the step (2) according to the mass ratio to obtain a regulator;
(4) culturing providencia sp enriched with nano-selenium, adding sodium selenite 100mg/L into the fermentation liquid, culturing for 24h to obtain zymocyte liquid with effective viable count of 1.8 × 1010cfu/mL; the providencia stuartii is providencia stuartii, and is purchased from China general microbiological culture collection center; the preservation number is CGMCC NO. 1.1528;
(5) carrying out ultrasonic treatment with the power of 100W on the zymocyte liquid obtained in the step (4) for 20min, and centrifuging at the rotating speed of 9000rpm for 30min to obtain nano selenium;
(6) and (4) treating the regulator obtained in the step (3), quercetin, nano-selenium obtained in the step (5) and polyethylene glycol at the rotating speed of 3000rpm for 20min, fully shaking and uniformly mixing to obtain the chlorella growth promoter.
Example 6
A method for preparing the chlorella growth promoter of the above embodiment comprises the following steps:
(1) stirring and uniformly mixing cetyl trimethyl ammonium bromide and ammonia water at 70 ℃ to obtain a clear solution, adding tetraethyl silicate, aging for 20 hours, drying the mixed solution, calcining at 580 ℃ for 5 hours to obtain 300 micron-sized silicon dioxide powder, and performing intermittent irradiation treatment on the obtained silicon dioxide powder under a vacuum condition by using plasma with the temperature of 700 ℃, the power of 3KW and the volume ratio of oxygen to ammonia gas (2.5: 6) as a gas source, wherein the irradiation treatment is performed for 8 minutes and is stopped for 10 minutes, and the total effective irradiation time is kept for 90 minutes to obtain the micro-nano biological silicon powder;
(2) placing the collected pistacia chinensis bunge leaves in an oven for sterilizing at 115 ℃, collecting, drying the leaves at 60 ℃, crushing, sieving, heating, performing ultrasonic extraction at 80 ℃ for 2 times, performing constant volume and reduced pressure filtration on the crushed mixture, centrifuging the crushed mixture at 5000rpm for 18min, performing filtration sterilization by using a filter membrane, keeping the supernatant, and performing freeze-drying to obtain dry powder of the pistacia chinensis bunge leaf extract;
(3) mixing the micro-nano biological silicon powder obtained in the step (1) and the pistacia chinensis bunge leaf extract dry powder obtained in the step (2) according to the mass ratio to obtain a regulator;
(4) culturing providencia sp enriched with nano-selenium, adding sodium selenite 300mg/L into the fermentation liquid, culturing for 48h to obtain zymocyte liquid with effective viable count of 2.6 × 1010cfu/mL; the providencia stuartii is providencia stuartii, and is purchased from China general microbiological culture collection center; the preservation number is CGMCC NO. 1.1528;
(5) carrying out ultrasonic treatment on the zymocyte liquid obtained in the step (4) with the power of 300W for 60min, and centrifuging for 50min at the rotating speed of 10000rpm to obtain nano selenium;
(6) and (4) treating the regulator obtained in the step (3), quercetin, nano-selenium obtained in the step (5) and polyethylene glycol at the rotating speed of 4000rpm for 40min, fully shaking and uniformly mixing to obtain the chlorella growth promoter.
In the embodiment, the plant source nano-stimulin is prepared by using materials such as a regulator, nano-selenium, quercetin, polyethylene glycol and the like, and the growth of chlorella is promoted. The chlorella growth promoter is added into a culture medium required by the growth of the chlorella, and the growth potential of the chlorella and the accumulation of carbon source substrates in chlorella cells are improved by promoting the photosynthetic metabolic reaction of chlorella cells, so that the growth rate of the chlorella is improved, and the problem of industrial culture of rapid growth of the microalgae is solved.
FIG. 1 is a graph showing the effect of the chlorella growth promoting agent of this example on the growth of chlorella, wherein the Control group corresponds to the algal cells of the treated group without using the growth promoting agent, and the CW1-CW4 treated groups correspond to the experimental groups using the chlorella growth promoting agent of examples 1-4; the results show that: the chlorella growth promoter of the embodiment can effectively improve the biomass of chlorella.
Claims (5)
1. The chlorella growth promoter is characterized by comprising the following raw materials in parts by weight: 7 parts of regulator, 0.005 part of quercetin, 0.06 part of nano-selenium and 992.935 parts of polyethylene glycol; the regulator is formed by mixing micro-nano biological silicon powder and pistacia chinensis bunge extract dry powder according to a mass ratio of 2.5: 5;
the chlorella growth promoter is prepared by the following steps:
(1) stirring and uniformly mixing hexadecyl trimethyl ammonium bromide and ammonia water at 70-80 ℃ to obtain a clear solution, adding tetraethyl silicate, aging for 20-24h, drying the mixed solution, calcining at 550-600 ℃ for 5-6h to obtain silicon dioxide powder, performing intermittent irradiation treatment on the obtained silicon dioxide powder under a vacuum condition by using plasma with the temperature of 400-800 ℃, the power of 2-4KW and the source of oxygen and ammonia gas, and keeping the total effective irradiation time for 40-90min to obtain micro-nano biological silicon powder;
(2) placing the collected pistacia chinensis bunge leaves in an oven for 110-90 ℃ sterilization, collecting, drying the leaves, crushing, sieving, heating, performing ultrasonic extraction at 60-90 ℃ for 2 times, performing constant volume on the crushed mixture, performing reduced pressure filtration, centrifuging at 3000-6000rpm for 15-20min, filtering and sterilizing by using a filter membrane, keeping the supernatant, and performing freeze drying to obtain pistacia chinensis bunge extract dry powder;
(3) mixing the micro-nano biological silicon powder obtained in the step (1) and the pistacia chinensis bunge extract dry powder obtained in the step (2) according to a mass ratio of 2.5:5 to obtain a regulator;
(4) culturing providencia rich in nano-seleniumProvidencia.spAdding 100mg/L sodium selenite and 300mg/L sodium selenite into the fermentation liquor, culturing for 24-48h to obtain the zymocyte liquid, wherein the effective viable count is 1.8 multiplied by 1010-2.6×1010cfu/mL；
(5) Performing ultrasonic treatment with the power of 100-;
(6) and (4) fully shaking and uniformly mixing the regulator obtained in the step (3), the quercetin, the nano-selenium obtained in the step (5) and the polyethylene glycol to prepare the chlorella growth promoter.
2. The method for preparing a chlorella growth promoter according to claim 1, wherein the volume ratio of oxygen to ammonia in step (1) is (2-3) to (5-7).
3. The method for preparing chlorella growth promoter as claimed in claim 1, wherein the silica powder prepared in step (1) is 150-300 micron silica powder.
4. The method for preparing a chlorella growth promoter according to claim 1, wherein the intermittent irradiation treatment in step (1) comprises: irradiating for 5-10min, and stopping for 5-10 min.
5. The method for preparing a chlorella growth promoter according to claim 1, wherein the fully shaking and blending manner in the step (6) is as follows: processing at 3000-.
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