Method for high-density proliferation of undaria pinnatifida gametophytes

Abstract

The invention discloses a method for high-density proliferation of undaria pinnatifida gametophytes, which comprises the following steps: S1, crushing a part of fresh undaria pinnatifida gametophytes into algae segments, and transferring the algae segments into a culture bottle filled with a part of culture medium, S2, placing the culture bottle into an illumination incubator, and carrying out high-density proliferation on the algae segments in the illumination incubator, so as to obtain the high-density proliferation of the undaria pinnatifida gametophytes. And S3, filtering the culture body by using a filter screen, picking out the gametophyte by using tweezers, absorbing excessive moisture by using sterile absorbent paper, weighing the mass of the gametophyte, and putting the gametophyte into an illumination incubator for illumination, so that the gametophyte is obtained. The nitrogen concentration and the phosphorus concentration in the illumination incubator are 11-150 mg / L and 1-10 mg / L respectively. Compared with the prior art, the method for high-density proliferation of the undaria pinnatifida gametophyte has the advantages that the biomass of the undaria pinnatifida gametophyte is increased to be 1.3 times or more of that of the undaria pinnatifida gametophyte when the culture condition is not optimized, large-scale rapid seedling raising of the undaria pinnatifida is facilitated, and the production efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine biochemical engineering, and specifically relates to a method for high-density proliferation of Undaria pinnatifida gametophytes. Background Art

[0002] Undaria pinnatifida is an important economic seaweed in China, containing various active substances such as fucoidan sulfate, phlorotannins, fucoxanthin, and mycosporine-like amino acids. These active substances have effects such as reducing blood lipids, antiviral, anti-tumor, and antioxidant, and have broad application prospects in the fields of health products, cosmetics, and food. The life history of Undaria pinnatifida consists of a sporophyte generation and a gametophyte generation. Among them, gametophytes can maintain a vigorous cell division state through vegetative proliferation under laboratory conditions without entering the developmental stage. It is not only an important form of germplasm resource preservation but also a way for the directional cultivation and efficient production of high-value active substances through metabolic regulation. Currently, there is no research on the biological preparation of high-value active substances based on the vegetative proliferation system of Undaria pinnatifida gametophytes at home and abroad.

[0003] China has made remarkable progress in the fields of artificial seedling raising and genetic breeding of Undaria pinnatifida. The survival rate and robustness of Undaria pinnatifida seedlings have been significantly improved, and multiple varieties with excellent traits such as strong stress resistance, fast growth rate, and good algal body quality have been cultivated. However, the biomass of Undaria pinnatifida gametophytes is not high enough, restricting its use as a raw material source for high-value active substances.

[0004] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for high-density proliferation of Undaria pinnatifida gametophytes, which can solve the technical problems raised in the above background art.

[0006] To achieve the above purpose, the technical solution provided by a specific embodiment of the present invention is as follows:

[0007] A method for high-density proliferation of Undaria pinnatifida gametophytes, comprising the following steps:

[0008] S1. Crush a portion of fresh Undaria pinnatifida gametophytes into algal segments, and transfer the algal segments to a culture flask containing a portion of culture medium.

[0009] S2. Place the culture flask in a light incubator, and set the temperature, light intensity, and photoperiod in the light incubator to 14°C to 22°C, 20 μmol·m -2 ·s -1~50 μmol·m -2 ·s -1 、 12L:12D to 16L:08D, and carry out aerated culture to obtain a culture,

[0010] S3. Filter the culture with a filter screen, pick out the gametophytes with tweezers and blot off the excess moisture with sterile blotting paper, then weigh the mass of the gametophytes,

[0011] The nitrogen and phosphorus concentrations in the light incubator are 11 - 150 mg / L and 1 - 10 mg / L respectively.

[0012] In one or more embodiments of the present invention, the mass of one portion of the fresh gametophytes of Undaria pinnatifida is 1 - 50 g, 0.5 - 5 g of the fresh gametophytes of Undaria pinnatifida are broken into algal segments of 150 - μm, the volume of the culture bottle is 500 mL, and the volume of the culture medium is 300 mL.

[0013] In one or more embodiments of the present invention, the components of the culture medium include NO 3- concentration 150 mg / L, initial PO4 3- concentration 10 mg / L, trace elements and vitamins.

[0014] In one or more embodiments of the present invention, in the step S3, the mesh number of the filter screen is 80 mesh, and the filter screen is a nylon filter screen.

[0015] In one or more embodiments of the present invention, the light quality used in the light incubator is one of white light, blue light, yellow light, red light, and green light.

[0016] In one or more embodiments of the present invention, in the step S2, the number of days of aerated culture is 16 days, and the culture medium is replaced every 4 days.

[0017] In one or more embodiments of the present invention, in the step S2, the number of days of aerated culture is 16 days, and the culture medium is replaced every 4 days.

[0018] In one or more embodiments of the present invention, the light quality used in the light incubator is one of white light, blue light, yellow light, red light, and green light.

[0019] In one or more embodiments of the present invention, in the step S3, the active substance mycosporine-like amino acids in the culture are extracted, and whether the biomass of the gametophytes of Undaria pinnatifida increases is judged by the mycosporine-like amino acids.

[0020] In one or more embodiments of the present invention, the ratio of the volume of the culture bottle to the mass of the culture medium is (1:1 - 1000:500).

[0021] Compared with the prior art, the method for high-density proliferation of Undaria pinnatifida gametophytes of the present invention determines the suitable light quality, temperature, nitrogen and phosphorus nutrient salt concentration, light intensity and photoperiod for significantly improving the gametophyte biomass, enabling the biomass of Undaria pinnatifida gametophytes to be increased by more than 1.3 times that of the biomass under unoptimized culture conditions, providing technical support for the biological preparation of high-value active substances by using Undaria pinnatifida gametophytes, and also being conducive to realizing large-scale and rapid seedling raising of Undaria pinnatifida, improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 For the initial NO 3- concentration of 50 mg / L in an embodiment of the present invention,

[0024] Figure 2 For the initial NO 3- concentration of 150 mg / L in an embodiment of the present invention,

[0025] Figure 3 For the initial PO4 3- concentration of 1 mg / L in an embodiment of the present invention,

[0026] Figure 4 For the initial PO4 3- concentration of 10 mg / L in an embodiment of the present invention,

[0027] Figure 5 Schematic diagram showing the effect of light quality on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention,

[0028] Figure 6 Schematic diagram showing the effect of temperature on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention,

[0029] Figure 7 For NO3 - concentration on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention,

[0030] Figure 8 For PO4 3- on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention,

[0031] Figure 9 Schematic diagram showing the effect of light intensity on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention,

[0032] Figure 10 Schematic diagram of the effect of light cycle on the biomass of Undaria pinnatifida gametophytes in an embodiment of the present invention

[0033] Figure 11 Uniform experimental design table in an embodiment of the present invention

[0034] Figure 12 Usage table of the uniform experimental design table in an embodiment of the present invention

[0035] Figure 13 Uniform experimental design table in an embodiment of the present invention

[0036] Figure 14 Uniform experimental result table in an embodiment of the present invention Detailed implementation manners

[0037] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0038] Embodiment 1

[0039] The method for high-density proliferation of Undaria pinnatifida gametophytes in an embodiment of the present invention includes the following steps:

[0040] S1. Crush a portion of fresh Undaria pinnatifida gametophytes into algal segments, and transfer the algal segments to a culture flask containing a portion of culture medium.

[0041] S2. Place the culture flask in a light incubator, and set the temperature, light intensity, and light cycle in the light incubator to 14°C - 22°C, 20 μmol·m -2 ·s -1 -50 μmol·m -2 ·s -1 , 12L:12D - 16L:08D, and perform aeration culture to obtain a culture body. The number of days of aeration culture is 16 days, and the culture medium is changed every 4 days.

[0042] S3. Filter the culture body with a filter screen, pick out the gametophytes with forceps, and blot off the excess water with sterile absorbent paper, and then weigh the mass of the gametophytes. In other words, extract the active substance mycosporine-like amino acids from the culture body, and judge whether the biomass of Undaria pinnatifida gametophytes has increased through mycosporine-like amino acids.

[0043] The nitrogen and phosphorus concentrations in the light incubator are 11 - 150 mg / L and 1 - 10 mg / L respectively.

[0044] Among them, the mass of a portion of fresh gametophytes of Undaria pinnatifida is 1 - 50 g. 0.5 - 5 g of fresh gametophytes of Undaria pinnatifida are broken into algal segments of 150 - 200 μm. The volume of the culture flask is 500 mL, and the volume of the culture medium is 300 mL. The components of the culture medium include NO 3- concentration of 150 mg / L, initial PO4 3- concentration of 10 mg / L, trace elements and vitamins. The mesh number of the filter screen is 80 meshes, and the filter screen is a nylon filter screen.

[0045] Among them, the light quality used in the light incubator is one of white light, blue light, yellow light, red light, and green light.

[0046] Among them, the ratio of the volume of the culture flask to the mass of the culture medium is (1:1 - 1000:500).

[0047] Correlation analysis shows that within the set temperature range (14°C - 22°C), light quality (white light, blue light, yellow light, red light, green light), and nitrogen and phosphorus concentration range (11 - 150 mg / L, 1 - 10 mg / L), the relative growth rate of gametophytes is positively correlated with temperature, light quality, and nitrogen and phosphorus concentration.

[0048] Using the method of the present invention, that is, the initial NO 3- concentration is set to 150 mg / L, the initial PO4 3- concentration is set to 10 mg / L, the light cycle is 12L:12D, the temperature is 22°C, green light, and the light intensity is 36 μmol·m -2 ·s -1 , to culture gametophytes of Undaria pinnatifida. Using 300 mL, 500 mL, and 4 L culture systems in sequence, inoculating 1 g, 2 g, and 8 g of gametophytes respectively, after culturing for 16 days, the biomass of gametophytes can reach 2.60 g, 5.51 g, and 21.2 g (wet weight), which is more than 1.3 times that of the unoptimized experimental group.

[0049] Inoculating 4 g of gametophytes of Undaria pinnatifida and culturing for 16 days under optimized conditions for extracting the active substance mycosporine-like amino acids (MAAs) from gametophytes. At the end of the experiment, the biomass of gametophytes obtained is 11.16 ± 0.11 g (wet weight), and the content of MAAs extract is 322.09 ± 3.67 mg / g (based on dry gametophyte powder). This value is higher than the MAAs content in many large brown algae and red algae thalli, such as Laminaria japonica, Sargassum fusiforme, and Gracilaria lemaneiformis, etc.

[0050] Example 2:

[0051] Break 1 g of fresh gametophytes of Undaria pinnatifida into algal segments of 150 - 200 μm, and transfer them to a 500 mL conical flask containing 300 mL of culture medium. The culture medium is prepared with artificial seawater, and the initial NO 3- concentration is set at 150 mg / L, the initial PO4 3- concentration is set at 10 mg / L, and trace elements and vitamins are added according to the f / 2 culture medium formula. Place the culture flask in a light incubator, and set the temperature, light quality, light intensity, and photoperiod to 22 °C, 36 μmol / (m 2 ·s), 12 h:12 h (L:D), and green light respectively, and aerate and culture for 16 d. Replace the culture medium every 4 d to avoid the growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, filter the culture system through an 80-mesh nylon filter screen, pick out the gametophytes with forceps, and blot off the excess water with sterile absorbent paper, then weigh the mass of the gametophytes, and the value is 2.60 g (wet weight).

[0052] Example 3:

[0053] Break 2 g of fresh gametophytes of Undaria pinnatifida into algal segments of 150 - 200 μm, and transfer them to a 1000 mL conical flask containing 500 mL of culture medium. The culture medium is prepared with artificial seawater, and the initial NO 3- concentration is set at 150 mg / L, the initial PO4 3- concentration is set at 10 mg / L, and trace elements and vitamins are added according to the f / 2 culture medium formula. Place the culture flask in a light incubator, and set the temperature, light quality, light intensity, and photoperiod to 22 °C, 36 μmol / (m 2 ·s), 12 h:12 h (L:D), and green light respectively, and aerate and culture for 16 d. Replace the culture medium every 4 d to avoid the growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, filter the culture system through an 80-mesh nylon filter screen, pick out the gametophytes with forceps, and blot off the excess water with sterile absorbent paper, then weigh the mass of the gametophytes, and the value is 5.51 g (wet weight).

[0054] Example 4:

[0055] Break 8 g of fresh gametophytes of Undaria pinnatifida into algal segments of 150 - 200 μm, and transfer them to a 5 L culture flask containing 4 L of culture medium. The culture medium is prepared with artificial seawater, and the initial NO 3- concentration is set at 150 mg / L, the initial PO4 3- concentration is set at 10 mg / L, and trace elements and vitamins are added according to the f / 2 culture medium formula. Place the culture flask in a light incubator, and set the temperature, light quality, light intensity, and photoperiod to 22 °C, 36 μmol / (m 2·s), 12 h:12 h (L:D), and green light, and cultured aerobically for 16 d. The culture medium was changed every 4 d to avoid growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, the culture was filtered through an 80-mesh nylon sieve, and the gametophytes were picked out with forceps and excess water was blotted off with sterile absorbent paper. Then the mass of the gametophytes was weighed, and the value was 21.20 g (wet weight).

[0056] The following are the data of multiple comparative examples in which the gametophytes of Undaria pinnatifida were cultured under unoptimized culture conditions.

[0057] Comparative Example 1:

[0058] 2 g of fresh Undaria pinnatifida gametophytes were broken into algal segments of 150 - 200 μm and transferred to a 1000 mL conical flask containing 500 mL of culture medium. The culture medium was prepared with artificial seawater, and the initial NO 3- concentration was set at 11 mg / L, the initial PO4 3- concentration was set at 5 mg / L, and trace elements and vitamins were added according to the f / 2 culture medium formula. The culture flask was placed in a light incubator, and the temperature, light quality, light intensity, and photoperiod were set at 18 °C, 30 μmol / (m 2 ·s), 12 h:12 h (L:D), and a white cold light source, and cultured aerobically for 16 d. The culture medium was changed every 4 d to avoid growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, the culture system was filtered through an 80-mesh nylon sieve, and the gametophytes were picked out with forceps and excess water was blotted off with sterile absorbent paper. Then the mass of the gametophytes was weighed, and the value was 3.54 g (wet weight).

[0059] Comparative Example 2:

[0060] 2 g of fresh Undaria pinnatifida gametophytes were broken into algal segments of 150 - 200 μm and transferred to a 1000 mL conical flask containing 500 mL of culture medium. The culture medium was prepared with artificial seawater, and the initial NO 3- concentration was set at 75 mg / L, the initial PO4 3- concentration was set at 1 mg / L, and trace elements and vitamins were added according to the f / 2 culture medium formula. The culture flask was placed in a light incubator, and the temperature, light quality, light intensity, and photoperiod were set at 18 °C, 30 μmol / (m 2 ·s), 12 h:12 h (L:D), and a white cold light source, and cultured aerobically for 16 d. The culture medium was changed every 4 d to avoid growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, the culture system was filtered through an 80-mesh nylon sieve, and the gametophytes were picked out with forceps and excess water was blotted off with sterile absorbent paper. Then the mass of the gametophytes was weighed, and the value was 3.48 g (wet weight).

[0061] Comparative Example 3:

[0062] The 2 g of fresh gametophytes of Undaria pinnatifida were broken into algal segments of 150 - 200 μm and transferred to a 1000 mL conical flask containing 500 mL of culture medium. The culture medium was prepared with artificial seawater, and the initial NO 3- concentration was set at 75 mg / L, the initial PO4 3- concentration was set at 5 mg / L, and trace elements and vitamins were added according to the f / 2 culture medium formula. The culture flask was placed in a light incubator, and the temperature, light quality, light intensity, and photoperiod were set at 16 °C, 30 μmol / (m 2 ·s), 12 h:12 h (L:D), and a white cold light source, respectively, and aerated for 16 days. The culture medium was changed every 4 days to avoid the growth retardation or death of gametophytes caused by nutrient deficiency. After the experiment, the culture system was filtered through an 80-mesh nylon filter, and the gametophytes were picked out with forceps and excess water was blotted off with sterile absorbent paper, and then the mass of the gametophytes was weighed, and the value was 3.55 g (wet weight).

[0063] From the above examples and comparative examples, it can be seen that the present invention significantly improves the biomass of gametophytes by changing the temperature, light intensity, and initial nitrogen and phosphorus concentrations, which shows obvious advantages when used as a raw material for extracting high-value active substances. And this method is easy to set up and convenient to operate.

[0064] As Figures 1 to 10 shown, by optimizing the culture conditions of Undaria pinnatifida gametophytes, the present invention determines the suitable light quality, temperature, nitrogen and phosphorus nutrient salt concentrations, light intensity, and photoperiod for significantly improving the biomass of gametophytes, so that the biomass of Undaria pinnatifida gametophytes is increased to more than 1.3 times that under the unoptimized culture conditions, providing technical support for the biological preparation of high-value active substances using Undaria pinnatifida gametophytes, and also facilitating the large-scale and rapid seedling raising of Undaria pinnatifida, improving the production efficiency.

[0065] As Figures 11 to 14 shown, three factors of light quality (A), temperature (B), and light (C) were selected, and a uniform experiment and an experimental design table were selected, and the 1st, 2nd, and 3rd columns were selected as the test conditions according to its use design table to determine a three-factor five-level uniform design test table, and the results are shown in Figure 14 .

[0066] As Figures 11 to 14As shown, the DPS statistical software was used to perform multiple linear regression and stepwise regression analysis of quadratic polynomials on the response value growth rate and each factor value. The model was evaluated through the correlation coefficient R, F value, and P value. Fitting with the multiple linear equation regression and performing the t-test on the regression coefficient, the P values of the regression coefficient tests for independent variables B and C were both > 0.05, indicating that the change between the factor and the response value is not a simple linear relationship, and the multiple linear regression model cannot be used for regression analysis. Using the stepwise regression method of quadratic polynomials and comprehensively considering the R, F values, and P values, after incorporating and excluding the parameters of the regression equation, a suitable quadratic polynomial regression equation and the combined values of each factor when Y takes the maximum value were obtained, resulting in: Y = -165.893 + 9.344A + 14.863AC + 5.685BC, R = 0.9992, F = 220.3239, P = 0.0495 < 0.05. When Y takes the maximum value, the light quality A is green, the temperature B is 22 °C, and the light intensity C is 36 μmol·m -2 ·s -1 , and the predicted relative growth rate is 6.203%. The verification test shows that when the light quality is green light, the temperature is 22 °C, and the light intensity is 36 μmol·m -2 ·s -1 (the initial NO3- concentration is set to 150 mg / L, and the initial PO4 3- concentration is set to 10 mg / L), the relative growth rate of the gametophyte is 6.414%.

[0067] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights involved.

[0068] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for high-density proliferation of Undaria pinnatifida gametophytes, characterized in that, It includes the following steps: S1. Break a portion of fresh gametophytes of Undaria pinnatifida into algal segments, and transfer the algal segments into a culture flask containing a portion of culture medium. S2. Place the culture flask in a light incubator, and set the temperature, light intensity, and photoperiod in the light incubator to 14°C to 22°C, 20 μmol·m -2 ·s -1 ~50 μmol·m -2 ·s -1 , 12L:12D to 16L:08D, and perform aerated culture to obtain a culture body. S3. Filter the culture with a filter screen, pick out the gametophytes with forceps, and absorb the excess moisture with sterile blotting paper, then weigh the mass of the gametophytes. The nitrogen and phosphorus concentrations in the light incubator are 11 - 150 mg / L and 1 - 10 mg / L respectively.

2. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 1, characterized in that, The mass of a portion of the fresh gametophytes of Undaria pinnatifida is 1 - 50 g. Break 0.5 - 5 g of the fresh gametophytes of Undaria pinnatifida into algal segments of 150 - 200 μm. The volume of the culture flask is 500 mL, and the volume of the culture medium is 300 mL.

3. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 2, characterized in that, The components of the medium include NO 3- at a concentration of 150 mg / L, initial PO4 3- at a concentration of 10 mg / L, trace elements and vitamins.

4. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 2 or 3, characterized in that, In step S3, the mesh number of the filter screen is 80 mesh, and the filter screen is a nylon filter screen.

5. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 4, characterized in that, The light quality used in the light incubator is one of white light, blue light, yellow light, red light, and green light.

6. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 4, characterized in that, In step S2, the number of days for aerated culture is 16 days, and the culture medium is replaced every 4 days.

7. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 1, characterized in that, In step S2, the number of days for aerated culture is 16 days, and the culture medium is replaced every 4 days.

8. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 1, characterized in that, The light quality used in the light incubator is one of white light, blue light, yellow light, red light, and green light.

9. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 1, characterized in that, In step S3, extract the active substance mycosporine-like amino acids from the culture, and determine whether the quality of the gametophytes of Undaria pinnatifida has improved based on the mycosporine-like amino acids.

10. The method for high-density proliferation of Undaria pinnatifida gametophytes according to claim 1, characterized in that, The ratio of the volume of the culture flask to the mass of the culture medium is (1:1 - 1000:500).

Images