Application of Polygonatum extract in promoting the growth of Chlorella

Abstract

This invention discloses the application of Polygonatum sibiricum extract in promoting the growth of Chlorella, belonging to the field of Chlorella cultivation technology. This invention effectively promotes the growth of Chlorella by mixing Polygonatum sibiricum extract with Chlorella culture medium. This application uses natural plant-derived extracts to replace traditional chemical additives or organic carbon sources, avoiding the problems of poor environmental compatibility and high production costs associated with chemical substances. It provides a green and low-cost growth promotion method for the large-scale cultivation of Chlorella. Furthermore, the algal biomass obtained from the cultivation of Chlorella containing Polygonatum sibiricum extract can serve as a natural delivery carrier for the active ingredients of Polygonatum sibiricum, and can be used in the production of functional foods or cosmetics, realizing the synergistic utilization of microalgae cultivation and the development of traditional Chinese medicine resources.

Description

Technical Field

[0001] This invention relates to the field of Chlorella cultivation technology, and in particular to the application of Polygonatum extract in promoting Chlorella growth. Background Technology

[0002] Chlorella possesses highly efficient carbon fixation and the ability to synthesize high-value bioactive substances, demonstrating significant application prospects in ecological protection, bioenergy, and medicine. Achieving large-scale, low-cost cultivation of Chlorella is crucial for advancing these applications. Currently, methods to promote Chlorella growth largely rely on chemical additives or organic carbon sources. These methods suffer from poor environmental compatibility and high production costs, hindering the development of green cultivation technologies.

[0003] Plant-derived extracts have attracted attention as natural biostimulants, but existing studies show that the effects of different plant extracts on Chlorella growth are highly dependent on plant species, extraction methods, concentrations, and culture conditions. Their effects exhibit diverse results, including promotion, inhibition, or no significant impact, lacking a unified understanding. How to screen for active substances that can stably promote Chlorella growth from numerous plant-derived extracts is an urgent technical problem to be solved. Summary of the Invention

[0004] In view of this, the present invention provides the application of Polygonatum extract in promoting the growth of Chlorella, so as to provide a plant-derived biostimulant that can stably promote the growth of Chlorella.

[0005] In a first aspect, the present invention provides the application of Polygonatum sibiricum extract in promoting the growth of Chlorella vulgaris, wherein the method of application is as follows: Chlorella culture medium and Polygonatum extract were mixed to obtain a mixed culture medium, which was then cultured.

[0006] Preferably, the concentration of Polygonatum sibiricum crude drug in the mixed culture medium is 0.1% to 0.4%.

[0007] Preferably, the preparation method of the Polygonatum extract includes: pulverizing Polygonatum medicinal material, adding water, and then extracting by reflux, filtering, and adjusting the volume to obtain the extract.

[0008] Furthermore, the ratio of the Polygonatum odoratum medicinal material to water is 1g : (3~8) mL.

[0009] Furthermore, the reflux extraction time is 20-60 min.

[0010] Preferably, the concentration of raw Polygonatum rhizome in the Polygonatum extract is 100~300 g / L.

[0011] Preferably, the culture is carried out under static conditions at a temperature of 22-28°C.

[0012] Preferably, the culture is carried out under alternating light and dark conditions, with a light-dark cycle of 12-15 hours of light and 9-12 hours of darkness.

[0013] Furthermore, the light intensity of the illumination is 2000~3000 lux.

[0014] Secondly, the present invention provides a method for promoting the growth of Chlorella, comprising the following steps: mixing Chlorella culture medium and Polygonatum extract to obtain a mixed culture medium, and then culturing it.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention effectively promotes the growth of Chlorella by mixing Polygonatum extract with Chlorella culture medium. This application uses natural plant extracts instead of traditional chemical additives or organic carbon sources, avoiding the problems of poor environmental compatibility and high production costs associated with chemical substances. It provides a green and low-cost growth-promoting method for the large-scale cultivation of Chlorella. Furthermore, the algal biomass obtained from the cultivation of Chlorella containing Polygonatum extract can serve as a natural delivery carrier for the active ingredients of Polygonatum, and can be used in the production of functional foods or cosmetics, achieving the synergistic utilization of microalgae cultivation and the development of traditional Chinese medicine resources. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 These are digital photographs of the Chlorella cultivation process in the experimental examples of this invention; Figure 2 These are the growth curves of Examples 1-3 and Comparative Example 1 of the present invention; Figure 3 These are the growth curves of comparative examples 1-4 of this invention; Figure 4 These are comparison charts of the dry weight of Chlorella in Examples 1-3 and Comparative Example 1 of this invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] Chlorella ( ChlorellaChlorella (S.) possesses characteristics such as efficient carbon fixation and synthesis of high-value bioactive substances, showing significant application prospects in ecological protection, bioenergy, and medicine and health. Achieving large-scale, low-cost cultivation of Chlorella is a key step in promoting these applications. Existing technologies for promoting Chlorella growth largely rely on chemical additives or organic carbon sources, which suffer from poor environmental compatibility and high production costs. Plant-derived extracts have attracted attention as natural biostimulants, but the effects of different plant extracts on Chlorella growth are highly dependent on plant species, extraction methods, concentrations, and cultivation conditions, exhibiting diverse results of promotion, inhibition, or no significant effect, lacking a unified understanding of the underlying principles. This invention applies Polygonatum extract to Chlorella cultivation. By mixing Polygonatum extract with Chlorella culture medium, it effectively promotes Chlorella growth, providing a green and low-cost growth-promoting method. Furthermore, the algal biomass obtained from Chlorella cultivation containing Polygonatum extract can serve as a natural delivery carrier for the active ingredients of Polygonatum, used in the production of functional foods or cosmetics, achieving synergistic utilization of microalgae cultivation and traditional Chinese medicine resource development.

[0020] This invention provides the application of Polygonatum extract in promoting the growth of Chlorella. The application method is as follows: Chlorella culture medium and Polygonatum extract are mixed to obtain a mixed culture medium, and then cultured.

[0021] In this invention, "Polygonatum" refers to the dried rhizome of the plant Polygonatum sibiricum, belonging to the Liliaceae family. The extract of Polygonatum sibiricum contains abundant active ingredients such as polysaccharides, saponins, flavonoids, and amino acids. Among these, polysaccharides may serve as a supplementary organic carbon source for Chlorella, directly absorbed and utilized by algal cells, promoting cell metabolism. Simultaneously, polysaccharides possess a certain metal ion chelating ability, which can improve the bioavailability of trace elements such as iron and magnesium in the culture medium, promoting chlorophyll synthesis and photosynthetic efficiency. Furthermore, the antioxidant active ingredients (such as flavonoids) in the extract may alleviate oxidative stress generated during the cultivation of Chlorella, maintaining cell membrane integrity and thus promoting cell division and growth. This invention, by directly adding the extract to the culture medium, utilizes the above multiple mechanisms to synergistically promote the growth of Chlorella, avoiding the environmental risks of chemical additives and achieving green cultivation.

[0022] The concentration of Polygonatum sibiricum extract in the mixed culture medium of this invention is 0.1% to 0.4% (w / v). For example, this concentration can be 0.1%, 0.2%, 0.3%, or 0.4%, preferably 0.1% to 0.4%, and more preferably 0.4%. Within this concentration range, the Polygonatum sibiricum extract can significantly promote cell division and biomass accumulation of Chlorella. If the concentration is too low, the growth-promoting effect is not significant; if the concentration is too high, it may lead to an excessively high concentration of organic matter in the culture medium, which may inhibit the growth of Chlorella or increase the risk of contamination. Those skilled in the art can calculate the required amount of Polygonatum sibiricum extract to be added according to the target concentration based on the actual culture scale.

[0023] In this invention, "crude drug" refers to the dried medicinal material of Polygonatum sibiricum. "Concentration of crude Polygonatum sibiricum" refers to the weight-volume percentage concentration (w / v) of the dried Polygonatum sibiricum, that is, the number of grams of dried Polygonatum sibiricum contained in every 100 mL of culture medium. For example, 0.1% means that every 100 mL of culture medium contains 0.1 g of crude Polygonatum sibiricum.

[0024] The extract of Polygonatum used in this invention is prepared by the following method: after pulverizing Polygonatum medicinal material, water is added, followed by reflux extraction, filtration, and volume adjustment to obtain the extract.

[0025] Specifically, the dried Polygonatum sibiricum herb is pulverized into coarse powder. Purified water is added at a ratio of 1 g: (3-8) mL. For example, the ratio can be 1 g: 3 mL, 1 g: 5 mL, or 1 g: 8 mL, preferably 1 g: 5 mL. The mixture of Polygonatum sibiricum and water is placed in a round-bottom flask, fitted with a reflux condenser, and heated to a gentle boil for reflux extraction. The reflux extraction time is 20-60 min, for example 20 min, 30 min, 40 min, 50 min, or 60 min, preferably 30 min. After extraction, the mixture is filtered, the filtrate is collected, and the residue is washed with a small amount of pure water. The washings and filtrate are combined and transferred to a volumetric flask, then diluted to a predetermined volume with pure water to obtain the Polygonatum sibiricum extract.

[0026] The concentration of Polygonatum sibiricum extract prepared in this invention is 100-300 g / L, meaning that each liter of extract contains 100-300 g of Polygonatum sibiricum extract. For example, this concentration can be 100 g / L, 150 g / L, 200 g / L, 250 g / L, or 300 g / L, preferably 200 g / L. Within this concentration range, the extract has suitable viscosity and effective component content, facilitating precise subsequent addition to Chlorella culture medium.

[0027] In this invention, the Chlorella culture medium can be a conventional culture medium suitable for Chlorella growth, preferably BG-11 medium. The preparation method of BG-11 medium is well known to those skilled in the art. Exemplarily, BG-11 medium powder is weighed and added to pure water at a ratio of 1.5~2.0 g of BG-11 medium powder per 1.0 L of pure water, stirred until completely dissolved, and the pH is adjusted to 6.8~7.2, preferably 7.0; then it is sterilized by moist heat at 118~122℃ for 15~25 min, for example, sterilization at 121℃ for 20 min, and then cooled for later use. The components of BG-11 medium include sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid (EDTA), sodium carbonate, and trace element solution, which can provide sufficient nitrogen, phosphorus, trace elements, and carbon source for Chlorella.

[0028] Specifically, Chlorella fuciformis extract in its logarithmic growth phase is mixed with the Polygonatum sibiricum extract prepared above in a specific ratio.

[0029] The well-mixed culture medium is placed under suitable conditions for cultivation. The cultivation is carried out under static conditions. Static cultivation avoids damage to algal cells from mechanical shear forces and promotes algal cell settling and light energy utilization, making it suitable for small-scale laboratory and large-scale industrial cultivation. The cultivation temperature is 22–28°C. For example, the temperature can be 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, or 28°C, preferably 25±1°C. This temperature range is suitable for the mesophilic growth characteristics of Chlorella and can maintain the activity of enzymes and metabolic rates within the algal cells.

[0030] In this invention, the cultivation is carried out under alternating light and dark conditions. *Chlorella vulgaris* is a photosynthetic autotrophic microalga, and the alternating light and dark conditions simulate the natural light cycle, which is beneficial for the effective conduct of photosynthesis and the synthesis of substances in the dark reaction stage. The light-dark cycle consists of 12-15 hours of light and 9-12 hours of darkness. For example, the light duration can be 12 h, 13 h, 14 h, or 15 h, and the corresponding dark duration can be 12 h, 11 h, 10 h, or 9 h, preferably 14 h of light and 10 h of darkness. Further, the light intensity is 2000-3000 lux. For example, the light intensity can be 2000 lux, 2200 lux, 2500 lux, 2800 lux, or 3000 lux, preferably 2500 lux. This light intensity provides sufficient light energy to drive photophosphorylation while avoiding photoinhibition.

[0031] This invention also provides a method for promoting the growth of Chlorella, comprising the following steps: mixing Chlorella culture medium and Polygonatum extract to obtain a mixed culture medium, and then culturing it. The specific operation of this method, the selection of various parameters (such as Polygonatum concentration, culture temperature, light-dark cycle, light intensity, etc.), and the preparation method of Polygonatum extract are all the same as described above, and will not be repeated here. This method can efficiently and environmentally promote the proliferation of Chlorella, increase the biomass yield per unit volume of culture medium, shorten the culture cycle, and reduce production costs.

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0033] Example 1 This embodiment provides the application of Polygonatum extract in promoting the growth of Chlorella.

[0034] (1) Preparation of Polygonatum extract Take 10 g of dried Polygonatum sibiricum, grind it into powder, place it in a 250 mL round-bottom flask, add 50 mL of purified water, assemble a reflux condenser, and heat it to a gentle boiling state on a constant-temperature heating mantle for reflux extraction for 30 min. After extraction, filter the mixture, collect the filtrate, rinse the inner wall of the flask with a small amount of pure water, combine the rinsing liquid and filtrate, transfer it to a 50 mL volumetric flask, and dilute to the mark with pure water to obtain the Polygonatum sibiricum extract. The concentration of Polygonatum sibiricum in this extract is 200 g / L (i.e., each liter of extract is equivalent to 200 g of Polygonatum sibiricum).

[0035] (2) Preparation of Chlorella culture medium Preparation of BG-11 medium: Weigh out BG-11 medium powder and add it to pure water at a ratio of 1.7 g of BG-11 medium powder per 1.0 L of pure water. Stir until completely dissolved, adjust the pH to 7.0, and then sterilize by moist heat at 121℃ for 20 min. After cooling, it is ready for use.

[0036] (3) Mixed culture Take Chlorella in its logarithmic growth phase ( Chlorella200 mL of *Sp. FACHB-8* algal solution was diluted to 1.5 L with sterile BG-11 medium and dispensed into sterile Erlenmeyer flasks, 20 mL per flask. 100 μL of the *Polygonatum sibiricum* extract stock solution prepared in step (1) and 300 μL of sterile BG-11 medium were added to the Erlenmeyer flasks to achieve a concentration of 0.1% (w / v) of *Polygonatum sibiricum* crude drug in the mixed culture medium. The Erlenmeyer flasks were then placed in a constant temperature and light incubator for static incubation. The incubation conditions were: temperature 25℃, light intensity 2500 lux, light-dark cycle of 14 h light / 10 h darkness, and a culture period of 5 days. Three replicates were set up for each group.

[0037] Example 2 The difference between this embodiment and Example 1 is that 200 μL of the Polygonatum extract stock solution and 200 μL of sterile BG-11 culture medium were added to the conical flask, so that the concentration of Polygonatum crude drug in the mixed culture medium was 0.2% (w / v). The remaining steps are the same as in Example 1.

[0038] Example 3 The difference between this embodiment and Example 1 is that 400 μL of the Polygonatum extract stock solution was added to the conical flask, so that the concentration of Polygonatum crude drug in the mixed culture medium was 0.4% (w / v). The remaining steps are the same as in Example 1.

[0039] Comparative Example 1 The difference between this comparative example and Example 1 is that no Polygonatum extract was added; instead, 400 μL of sterile BG-11 culture medium was added to the conical flask to maintain the same final volume as in Example 1. The remaining steps are the same as in Example 1.

[0040] Comparative Example 2 The difference between this comparative example and Example 1 is that American ginseng is used instead of Polygonatum in this comparative example.

[0041] Comparative Example 3 The difference between this comparative example and Example 2 is that American ginseng is used instead of Polygonatum in this comparative example.

[0042] Comparative Example 4 The difference between this comparative example and Example 3 is that American ginseng is used instead of Polygonatum in this comparative example.

[0043] Test case 1. Effects on the growth performance of Chlorella Digital photos of the Chlorella cultivation process, such as Figure 1 As shown. Starting from day 0 of the culture, 200 μL of sample was aseptically taken from the conical flasks of Examples 1-3 and Comparative Examples 1-4 at a fixed time (15:00) every day and transferred to the corresponding wells of a 96-well plate. The optical density (OD) value at a wavelength of 630 nm was measured using a microplate reader. 630After the measurement, 200 μL of sterile BG-11 medium was added to the original conical flask to maintain a constant culture volume. Measurements were performed continuously for 5 days, and OD values ​​were recorded. 630 Values ​​were used to plot growth curves, and the results are as follows: Figure 2 and Figure 3 As shown. OD on day 5 of Examples 1-3 and Comparative Example 1. 630 The values ​​are shown in Table 1. Based on OD... 630 The relative growth constant (K) and doubling time (G) of Chlorella can be calculated using the following formulas: K = (lnN t lnN0) / t;G = 0.693 / μ; Where N t OD of Chlorella algae solution on day t 630 Value, N0 is the OD value of the Chlorella culture solution in the early stage of cultivation. 630 The values ​​are: t is the culture time, μ is the specific growth rate, which is approximated by the relative growth constant K in this invention. The results are summarized in Table 1.

[0044] Table 1 OD 630 Value, relative growth constant K and doubling time G

[0045] from Figure 2 and Figure 3 It can be seen that the OD of Examples 1-3 630 The values ​​were all higher than those of the blank control group (Comparative Example 1), and increased with increasing concentration of Polygonatum odoratum, showing a clear concentration-dependent positive correlation. Among them, the OD values ​​of Example 3 (0.4%) were... 630 The highest OD value indicates the most significant growth-promoting effect. In contrast, the low concentrations of American ginseng (0.1% and 0.2%) showed almost no promoting effect on the growth of Chlorella, while the high concentration (0.4%) showed a significant inhibitory effect, with the final OD value lower than that of control group 1. Furthermore, Table 1 shows that the 0.4% Polygonatum treatment group had the highest K value and the shortest G value, indicating the highest algal cell proliferation efficiency, further confirming its strongest promoting effect. The 0.4% American ginseng treatment group had the lowest K value and the longest G value, with the K value being the lowest in the later stages of culture. Compared to the control group, the algal cells grew slowly, even ceasing proliferation, which is completely consistent with the growth stagnation reflected by the OD value, fully demonstrating its inhibitory effect.

[0046] 2. Determination of dry weight of algae Algal cell dry weight is a direct quantitative indicator reflecting the biomass accumulation of Chlorella, and its measurement results can further verify the reliability of the OD value analysis conclusions. After cultivation, the algal solutions from Examples 1-3 and Comparative Example 1 were transferred to 50 mL centrifuge tubes, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The precipitate was washed 2-3 times with pure water and transferred to pre-weighed 5 mL centrifuge tubes. After pre-freezing at -40℃ for 12 h, it was freeze-dried for 24 h until constant weight, weighed, and the dry weight of Chlorella was calculated. The results are as follows: Figure 4 As shown.

[0047] It can be seen that the dry weight measurement results are consistent with the OD values. 630 The values ​​showed a consistent trend. The dry weight of the 0.4% Polygonatum treatment group (Example 3) was 9.6 mg, which was significantly higher than the 4.2 mg of the blank group (Comparative Example 1), representing an increase of approximately 128.6%, confirming that this concentration had the strongest promoting effect on the growth of Chlorella.

[0048] The above experimental results show that Polygonatum extract can effectively promote the growth of Chlorella in the concentration range of 0.1% to 0.4%, and the promoting effect increases with increasing concentration. The optimal concentration is 0.4% (w / v), providing an effective biostimulant for the green and large-scale cultivation of Chlorella.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of Polygonatum sibiricum extract in promoting the growth of Chlorella, characterized in that, The method of application is as follows: Chlorella culture medium and Polygonatum extract were mixed to obtain a mixed culture medium, which was then cultured.

2. The application as described in claim 1, characterized in that, The concentration of Polygonatum sibiricum crude drug in the mixed culture medium is 0.1%~0.4%.

3. The application as described in claim 1, characterized in that, The preparation method of the Polygonatum extract includes: pulverizing Polygonatum medicinal material, adding water, extracting by reflux, filtering, and adjusting the volume to obtain the extract.

4. The application as described in claim 3, characterized in that, The ratio of the medicinal material Polygonatum to water is 1g : (3~8)mL.

5. The application as described in claim 3, characterized in that, The reflux extraction time is 20-60 min.

6. The application as described in claim 1, characterized in that, The concentration of raw Polygonatum rhizome in the extract is 100~300 g / L.

7. The application as described in claim 1, characterized in that, The culture was carried out under static conditions at a temperature of 22-28°C.

8. The application as described in claim 1, characterized in that, The culture was carried out under alternating light and dark conditions, with a light-dark cycle of 12-15 hours of light and 9-12 hours of darkness.

9. The application as described in claim 8, characterized in that, The light intensity of the illumination is 2000~3000 lux.

10. A method for promoting the growth of Chlorella, characterized in that, The process includes the following steps: mixing Chlorella culture medium and Polygonatum extract to obtain a mixed culture medium, and then culturing it.

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