CN108004195B - Method for promoting rapid growth of chlorella by using rhus chinensis leaf extract - Google Patents
Method for promoting rapid growth of chlorella by using rhus chinensis leaf extract Download PDFInfo
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- CN108004195B CN108004195B CN201711478930.8A CN201711478930A CN108004195B CN 108004195 B CN108004195 B CN 108004195B CN 201711478930 A CN201711478930 A CN 201711478930A CN 108004195 B CN108004195 B CN 108004195B
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention discloses a method for promoting chlorella to grow rapidly by using a rhus chinensis leaf extract, which comprises the following specific steps: adding the extract of the leaves of the Pimenta chinensis Bak with the extraction concentration of 0.015-0.05 g/L into the cultured chlorella. The method has the advantages that the rhus chinensis leaves are used for promoting the rapid growth of the chlorella, the process is simple, the source is wide, the cost is low, the method is green and environment-friendly, the preparation effect is good, and a new way for utilizing the forest residue is developed by utilizing the energy forest residue as the raw material; provides a new technical idea for industrialized and rapid mass culture of chlorella.
Description
Technical Field
The invention relates to a method for promoting chlorella to grow rapidly, in particular to a method for promoting chlorella to grow rapidly by using a rhus chinensis leaf extract.
Background
The chlorella has the characteristics of photoautotrophic growth, rapid and stable propagation, no cultivated land occupation of a culture system, wide distribution, easy culture, capability of mass culture in a high-efficiency bioreactor, and particular suitability for industrial large-scale production. Chlorella has been reported to be used for producing biodiesel, unsaturated fatty acid, vitamin, and secondary metabolite carotenoid, astaxanthin and amino acid, etc. Especially, after the bioactive substance extract from the chlorella is compounded with common nutrient fertilizers, the biological medicine fertilizer with high attached value is developed, is a huge resource bank to be developed, and has potential practical production application value. In addition, the bioactive extract of the chlorella contains various active amino acids and a large amount of secondary metabolites, can be used as an organic nutritional feed for animals and a fish bait additive, has the function of improving the immunity of animals and plants, is used for developing agricultural, animal and fishery products with high green organic nutritional value, and has huge development prospect. However, chlorella has traditionally been cultured by accumulating metabolites using organic carbon sources as the main photosynthetic raw material. When the exogenous stimulant is used as the photosynthetic raw material of the chlorella, the growth rate of the chlorella can be improved, and a large amount of biological metabolites can be effectively accumulated, so that the chlorella can reach ultrahigh chlorella density and biomass, the cost of organic carbon sources required by the chlorella photosynthetic culture can be offset to a certain extent by high-efficiency productivity, and a research basis is provided for the subsequent high-density culture of the chlorella and the development of high-added-value metabolites. However, the existing chlorella culture period is long, and large-scale industrial production cannot be realized.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for promoting the rapid growth of chlorella by using a rhus chinensis leaf extract, and the method utilizes a crude extract of forest source plant leaves to promote the rapid growth of chlorella.
The invention is realized by the following technical scheme: the specific process is as follows: adding the extract of the leaves of the Pimenta chinensis Bak with the extraction concentration of 0.015-0.05 g/L into the cultured chlorella.
The preparation method of the rhus chinensis leaf extract comprises the following steps:
(1) crushing Rhus chinensis Mill leaves into uniform granules, and sieving;
(2) mixing the powder of the leaves of the rhus chinensis with the extracting solution according to the proportion of 1: and (2) carrying out hot leaching and ultrasonic-assisted extraction for 1-4 times at a material-liquid ratio of 5-25, treating the mixture at the extraction temperature of 40-80 ℃ for 1-4 h, carrying out suction filtration and centrifugation, collecting supernatant, combining filtrate, carrying out reduced pressure filtration on the filtrate, removing mixed particulate matters in the extract, and carrying out centrifugal filtration and sterilization on the extract to obtain the crude extract of rhus chinensis leaves.
And (3) mixing and extracting the rhus chinensis leaves by adopting different auxiliary extracting solutions in the step (2), wherein the auxiliary extracting solution is water, the extracting temperature is 40-60 ℃, ultrasonic extraction is carried out for 1-2 hours for 1-3 times, the residue obtained after the previous extraction and centrifugation is subjected to the next extraction under the same condition as the previous extraction, the supernatant obtained after the centrifugation is combined to complete the extraction, the mixed particle substances in the extracting solution are removed by reduced pressure filtration after the volume is fixed, and the extracting solution is subjected to centrifugal filtration and sterilization to obtain the crude rhus chinensis leaf extracting solution.
And (3) performing mixed extraction on the rhus chinensis leaves by adopting different auxiliary extraction methods in the step (2), wherein the auxiliary extraction solution is water, the extraction temperature is 60-80 ℃, the hot extraction is performed for 2-4 times for 3-4 hours, the mixed particulate matters in the extraction solution are removed by reduced pressure filtration after the volume is constant, and the extraction solution is subjected to centrifugal filtration sterilization to obtain the crude rhus chinensis leaf extraction solution.
And (3) mixing and extracting the rhus chinensis leaves by adopting different auxiliary extracting solutions in the step (2), wherein the auxiliary extracting solution is ethanol, the extracting temperature is 50-70 ℃, hot leaching is carried out for 1-3 h for 1-2 times, mixed particle substances in the extracting solution are removed by reduced pressure filtration after constant volume is realized, and the extracting solution is subjected to centrifugal filtration sterilization to obtain the crude rhus chinensis leaf extracting solution.
And (3) mixing and extracting the rhus chinensis leaves by adopting different auxiliary extracting solutions in the step (2), wherein the auxiliary extracting solution is ethanol, the extracting temperature is 40-70 ℃, ultrasonic extraction is performed for 2-4 times every 2-4 hours, mixed particle substances in the extracting solution are removed by reduced pressure filtration after constant volume is performed, and the extracting solution is subjected to centrifugal filtration sterilization to obtain the crude rhus chinensis leaf extracting solution.
The ultrasonic extraction method can control extraction time. The extraction rate of polyphenol is related to the extraction time, and the excessive extraction time can cause chemical reactions such as oxidation, degradation and the like of polyphenol in the extracting solution, and the extraction rate is reduced.
When the auxiliary extracting solution is ethanol, the volume concentration of the ethanol is 60 percent. When the volume fraction of the ethanol is lower, more water-soluble protein and more saccharides are dissolved in the mixed solution; when the volume fraction of ethanol is higher, the fat-soluble substances are more dissolved, which affects the extraction rate of polyphenol in the mixture.
The culture conditions of the chlorella are as follows: inoculating and culturing a BG11 culture medium subjected to filtration sterilization in a triangular flask subjected to high-temperature and high-pressure sterilization, and placing the inoculated chlorella into an artificial climate incubator for culturing at the temperature of 23 +/-1 ℃, the illumination intensity of 2000-6000 lx and the light-dark ratio of 12 h: and (4) inoculating once every 12-16 days for 12h, and uniformly distributing the chlorella to obtain enough illumination.
The crude extract of the rhus chinensis leaves contains natural flavonoid compounds with a plurality of phenolic hydroxyl groups, and the in-vitro oxidation model verifies that the crude extract has the effects of resisting oxidation and removing the activity of free radicals in cells, and most of the flavonoid substances from plant sources can be used as natural phytostimulin to influence the biochemical metabolic reaction process of algae cells. The flavonoids can be used as biological stimulin to promote accumulation of acetylcholine in algae cells, influence oxygen absorption of in vitro chloroplast, and inhibit chloroplast expansion caused by light stimulation; stimulates the outflow of sodium ions and potassium ions from chloroplasts and regulates and controls the membrane-to-ion of the chloroplastsPermeability and coupling between electron transfer and ATP synthesis. In addition, the compound can also participate in regulating and controlling the metabolic pathways of phosphatidylethanolamine and phosphatidylcholine in algae cells, regulating and controlling the permeability of cell membranes to ions, generating a second messenger to open calcium channels and improve intracellular Ca2+The concentration of the algae-derived protein affects various physiological processes of algae cells. Meanwhile, the expression of various kinases regulated and controlled by calcium ions can be activated and improved, protein kinase C combined with plasma membranes is activated, phosphorylation reaction is activated, cell proliferation and differentiation of chlorella cells are promoted, and the biomass of chlorella is improved.
Compared with the prior art, the invention has the following advantages: the method has the advantages that the rhus chinensis leaves are used for promoting the rapid growth of the chlorella, the process is simple, the source is wide, the cost is low, the method is green and environment-friendly, the preparation effect is good, and a new way for utilizing the forest residue is developed by utilizing the energy forest residue as the raw material; provides a new technical idea for industrialized and rapid mass culture of chlorella.
Drawings
FIG. 1 is a histogram of growth comparison of Chlorella species;
FIG. 2 is a graph showing the comparison of the growth of chlorella before and after the treatment in example 3.
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
After the rhus chinensis leaves are collected, the leaves are immediately placed in an oven, sterilization treatment is carried out for 5-10 min at 120 ℃, then the leaves are dried within 60 ℃, smashed into uniform leaf particles by a pulverizer, and sieved to 30-100 meshes.
The obtained rhus chinensis leaf particles are stored in a brown closed container for drying and collection.
Weighing a certain amount of rhus chinensis leaf powder, placing the powder in a 1L glass container, uniformly mixing a solution and the rhus chinensis leaf powder according to a material ratio of 1:15g/ml, wherein the solution is water, heating the mixture after uniform mixing for ultrasonic extraction at the extraction temperature of 80 ℃, and placing the mixed solution in an ultrasonic crusher for ultrasonic extraction for 2 times for 1 hour. Performing secondary extraction on the residues after the centrifugation of the first extraction under the same conditions as the first extraction, combining the two supernatants after the centrifugation to finish the secondary extraction, performing volume-constant volume separation on the crushed mixture, performing reduced pressure filtration to remove mixed particulate matters in the extract, centrifuging the extract at 5000rpm for 20min, performing filtration sterilization by using a filter membrane, and retaining the supernatant. And (3) taking a part of supernatant, and freeze-drying to obtain the rhus chinensis leaf extract dry powder.
In this example, the treatment group to which the extract was added was designated as CVW 1.
Example 2
After the rhus chinensis leaves are collected, the leaves are immediately placed in an oven, sterilization treatment is carried out for 5-10 min at 120 ℃, then the leaves are dried within 60 ℃, smashed into uniform leaf particles by a pulverizer, and sieved to 30-100 meshes.
The obtained rhus chinensis leaf particles are stored in a brown closed container for drying and collection.
Weighing a certain amount of Rhus chinensis Mill leaf powder, placing in a 1L glass container, and hot-extracting for 1 time at 80 deg.C for 2h with water as auxiliary extractive solution and Rhus chinensis Mill leaf powder at a ratio of 1: 15. And (3) after volume fixing, filtering under reduced pressure to remove mixed particulate matters in the extracting solution, finally centrifuging the extracting solution at 5000rpm for 20min, and filtering and sterilizing by using a filter membrane to obtain a crude extract of the rhus chinensis leaves. And (3) taking a part of supernatant, and freeze-drying to obtain the rhus chinensis leaf extract dry powder.
The treatment group with the extract added in this example was designated CVW 2.
Example 3
Hot-dipping the extract solution and the Rhus chinensis Mill leaf powder at a feed-liquid ratio of 1:15g/ml and an auxiliary extract of 60% ethanol at an extraction temperature of 60 ℃ for 2h for 2 times. The solubility of polyphenols in ethanol can increase with increasing temperature, but when the temperature is too high, chemical reactions such as oxidative decomposition of polyphenols can also occur. In view of the problems of thermal stability, solvent volatilization and impurity dissolution of the phenolic substances, the extraction conditions are optimized by adopting a method of extracting for multiple times at 60 ℃. Performing secondary extraction on the residues after the centrifugation of the first extraction under the same conditions as the first extraction, combining the two supernatants after the centrifugation to finish the secondary extraction, performing constant volume, performing reduced pressure filtration to remove mixed particulate matters in the extracting solution, performing sexual centrifugation on the extracting solution at 5000rpm for 20min, collecting, performing filter sterilization by using a filter membrane, and keeping the supernatant. Freeze drying part of the supernatant to obtain dried powder of ethanol extract of Rhus chinensis Mill leaves. And (3) taking a part of supernatant, and freeze-drying to obtain the rhus chinensis leaf extract dry powder.
The treatment group with the added extract in this example was designated CVE 1.
Example 4
The material-liquid ratio of the extraction solution to the rhus chinensis leaf powder is 1:15g/ml, the auxiliary extraction solution is 60% ethanol, and ultrasonic extraction is carried out for 4 times every 1h at the extraction temperature of 60 ℃. And (4) performing secondary extraction on the residues obtained after the centrifugation of the first extraction by adopting the same conditions as the first extraction, combining the supernatants obtained after the centrifugation for the second extraction, and so on. And (3) carrying out reduced pressure filtration after volume fixing to remove mixed particulate matters in the extracting solution, carrying out sexual centrifugation on the extracting solution for 20min at 5000rpm, collecting, filtering and sterilizing through a filter membrane, and keeping a supernatant. Freeze drying part of the supernatant to obtain dried powder of ethanol extract of Rhus chinensis Mill leaves. And (3) taking a part of supernatant, and freeze-drying to obtain the rhus chinensis leaf extract dry powder.
The treatment group with the added extract in this example was designated CVE 2. Compared with the blank control group, when the addition concentration of the rhus chinensis is 0.025g/L, the growth promotion rate of the chlorella on the 3 rd day can reach 2.34 times of that of the common treatment group.
The ratio of the chlorella prepared in examples 1 to 4 to the blank control group was as shown in FIG. 1:
compared with a blank Control, the CVW1 has the advantage that when the added concentration of the rhus chinensis is 0.015g/L, the growth promotion rate of chlorella on the 3 rd day can reach 1.87 times of that of a common treatment group.
Compared with blank Control, CVW2 has the growth promoting rate of chlorella on 3 days of 1.98 times that of common treatment group when the added concentration of Rhus chinensis is 0.025 g/L.
Compared with the blank Control, the CVE1 has the growth promoting rate of the chlorella on the 3 rd day which is 2.34 times that of the common treatment group when the added concentration of the rhus chinensis is 0.035 g/L.
Compared with a blank control, the growth promotion rate of the chlorella on the 3 rd day can reach 2.18 times of that of a common treatment group when the added concentration of the rhus chinensis is 0.05g/L by CVE 2.
As shown in fig. 2, the chlorella treated in example 3 is a photograph on the right side of the figure, and a photograph before treatment is shown on the left side of the figure, so that the chlorella grows quickly and the color of the whole container is obviously deepened.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A method for promoting the growth of chlorella by using a rhus chinensis leaf extract is characterized by comprising the following specific steps: adding the extract of the leaves of the halodendron delavayi with the extraction concentration of 0.015-0.05 g/L into the cultured chlorella;
the preparation method of the rhus chinensis leaf extract comprises the following steps: (1) crushing Rhus chinensis Mill leaves into uniform granules, and sieving; (2) mixing the powder of the leaves of the rhus chinensis with the extracting solution according to the proportion of 1: carrying out hot extraction or ultrasonic-assisted extraction for 1-4 times at a material-liquid ratio of 5-25, treating the mixture at an extraction temperature of 40-80 ℃ for 1-4 h, carrying out suction filtration and centrifugation, collecting supernatant, combining filtrate, carrying out reduced pressure filtration on the filtrate, removing mixed particulate matters in the extract, and carrying out centrifugal filtration sterilization on the extract to obtain a crude extract of rhus chinensis leaves; the extract is water or ethanol.
2. The method for promoting the growth of chlorella by using the rhus chinensis leaf extract as claimed in claim 1, wherein different auxiliary extracting solutions are adopted in the step (2) to perform mixed extraction on the rhus chinensis leaves, the auxiliary extracting solution is water, the extraction temperature is 40-60 ℃, ultrasonic extraction is performed for 1-2 hours for 1-3 times, the residue after centrifugation of the previous extraction is subjected to the next extraction under the same conditions as the previous extraction, the supernatant liquid obtained after the centrifugation of the previous extraction is combined after the centrifugation to complete the extraction, the mixed particulate matter in the extracting solution is removed by reduced pressure filtration after the volume is fixed, and the extracting solution is subjected to centrifugal filtration sterilization to obtain the crude rhus chinensis leaf extract.
3. The method for promoting the growth of chlorella by using the rhus chinensis leaf extract as claimed in claim 1, wherein different auxiliary extraction methods are adopted in the step (2) to carry out mixed extraction on rhus chinensis leaves, the auxiliary extraction solution is water, the extraction temperature is 60-80 ℃, the hot extraction is carried out for 2-4 times for 3-4 hours, the mixed particulate matter in the extraction solution is removed by reduced pressure filtration after the volume is constant, and the extraction solution is subjected to centrifugal filtration sterilization to obtain the crude rhus chinensis leaf extract.
4. The method for promoting the growth of chlorella by using the rhus chinensis leaf extract as claimed in claim 1, wherein different auxiliary extracting solutions are adopted in the step (2) to carry out mixed extraction on the rhus chinensis leaves, the auxiliary extracting solution is ethanol, the extraction temperature is 50-70 ℃, the hot extraction is carried out for 1-3 hours for 1-2 times, the mixed particulate matter in the extracting solution is removed by reduced pressure filtration after the volume is constant, and the extracting solution is subjected to centrifugal filtration sterilization to obtain the crude extract of the rhus chinensis leaves.
5. The method for promoting the growth of chlorella by using the rhus chinensis leaf extract as claimed in claim 1, wherein different auxiliary extracting solutions are adopted in the step (2) to perform mixed extraction on the rhus chinensis leaves, the auxiliary extracting solution is ethanol, the extraction temperature is 40-70 ℃, ultrasonic extraction is performed for 2-4 times every 2-4 hours, the mixed particulate matter in the extracting solution is removed by reduced pressure filtration after volume fixing, and the extracting solution is subjected to centrifugal filtration sterilization to obtain the crude extract of the rhus chinensis leaves.
6. The method of using Rhus chinensis Mill leaf extract for promoting growth of Chlorella vulgaris as claimed in any one of claims 4 or 5 wherein when the auxiliary extractive solution is ethanol, the volume concentration of ethanol is 60%.
7. The method of using a rhus chinensis leaf extract to promote the growth of chlorella as claimed in claim 1, wherein the chlorella is cultured under conditions selected from the group consisting of: inoculating and culturing a BG11 culture medium subjected to filtration sterilization in a triangular flask subjected to high-temperature and high-pressure sterilization, and placing the inoculated chlorella into an artificial climate incubator for culturing at the temperature of 23 +/-1 ℃, the illumination intensity of 2000-6000 lx and the light-dark ratio of 12 h: and (4) inoculating once every 12-16 days for 12h, and uniformly distributing the chlorella to obtain enough illumination.
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| CN108892563A (en) * | 2018-07-26 | 2018-11-27 | 中国科学院合肥物质科学研究院 | A kind of production selenium promotor and preparation method thereof for improving chlorella and producing organic selenium |
| CN108676768B (en) * | 2018-07-27 | 2022-03-04 | 中国科学院合肥物质科学研究院 | Chlorella growth promoter and preparation method thereof |
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| CN102864117A (en) * | 2012-09-25 | 2013-01-09 | 北京林业大学 | Method for promoting rapid growth of chlorella by using Chinese parasol fallen leaves |
| FR2985183A1 (en) * | 2011-12-30 | 2013-07-05 | Dermance | Topical composition, useful for treating skin aging, comprises a combination of enzymatic exfoliant action composition and active agent present in a flushable mask applied on skin |
| CN103768109A (en) * | 2012-10-25 | 2014-05-07 | 奇复康药物研发(苏州)有限公司 | Extraction method for Chinese parasol tree bark flavonoid components |
| CN106381269A (en) * | 2016-08-30 | 2017-02-08 | 浙江工商大学 | Method for increasing yield of chlorella by ultrasonic extraction of rice leaf |
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| FR2985183A1 (en) * | 2011-12-30 | 2013-07-05 | Dermance | Topical composition, useful for treating skin aging, comprises a combination of enzymatic exfoliant action composition and active agent present in a flushable mask applied on skin |
| CN102864117A (en) * | 2012-09-25 | 2013-01-09 | 北京林业大学 | Method for promoting rapid growth of chlorella by using Chinese parasol fallen leaves |
| CN103768109A (en) * | 2012-10-25 | 2014-05-07 | 奇复康药物研发(苏州)有限公司 | Extraction method for Chinese parasol tree bark flavonoid components |
| CN106381269A (en) * | 2016-08-30 | 2017-02-08 | 浙江工商大学 | Method for increasing yield of chlorella by ultrasonic extraction of rice leaf |
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