CN113817649B - Method for producing pseudo-cladophyllin by inducing hair weeds to suspend and culture cells by using solar radiation - Google Patents

Abstract

The invention relates to a method for producing pseudo-cladophyllin by utilizing sun radiation to induce hair weeds suspension culture cells, which comprises the steps of inoculating the hair weeds suspension culture cells into a liquid culture medium to obtain a culture system A; then the culture system A is radiated for 0.5 to 1 hour under the sunlight to obtain a culture system B; then shake culturing the culture system B at constant temperature by using an LED fluorescent lamp for 1 day, 2 days or 3 days to obtain a culture system C; then treating the culture system C by a mode of treating the culture system A in the second step, and treating the obtained culture system by a mode of treating the culture system B in the third step to obtain a culture system D, so as to form a second cycle treatment on the hair weeds suspension culture cells; the method is used for continuously carrying out circulation treatment on the hair weeds suspension culture cells for 8-14 days, then the algae are collected by the centrifugal culture solution, and the pseudo-branch algae are extracted from the algae, so that the preparation of the pseudo-branch algae is completed.

Description

A method that uses sunlight radiation to induce Nostoc suspension culture cells to produce pseudophysin method

Technical field

The present invention relates to the technical field of induced synthesis of secondary metabolites of cyanobacteria, and is specifically a method for inducing the production of pseudophysin by using sunlight radiation to induce Nostoc suspension culture cells.

Background technique

Nostoc is a nitrogen-fixing cyanobacteria that grows in arid and semi-arid areas. It can synthesize a variety of biologically active substances and has high edible and medicinal value. Due to being subjected to various abiotic stresses all year round, especially high-intensity ultraviolet radiation, Nostoc has developed a variety of protective mechanisms during its evolution. Among them, the synthesis of pseudomycin is one of the most important ways for Nostoc cells to resist UV radiation damage.

Pseudophycein (C ₃₆ H ₂₀ N ₂ O ₄ ), whose English name is scytonemin, is a small brown-yellow fat-soluble pigment molecule that is distributed in the extracellular polysaccharide sheaths of some cyanobacteria. Pseudophycein molecules have strong stability and can withstand the effects of various adverse environments such as temperature, oxidative stress and ultraviolet radiation. In addition to its strong UV absorption ability, pseudophysin also has a variety of biological activities, such as antioxidant, anti-inflammatory and anti-proliferative activities, and has great application potential in the fields of medicine and cosmetics.

The synthesis of pseudophyton is mainly induced by ultraviolet radiation and is also affected by high light, salt stress and nutritional status. However, the current method of producing pseudophyton has the problems of low yield and high energy consumption.

Contents of the invention

Aiming at the problems of high energy consumption and low yield in the production of pseudophysin in the prior art, the present invention provides a method for inducing pseudophysin production by using sunlight radiation to induce Nostoc suspension culture cells. The method uses sunlight to perform periodic short-term Irradiation treatment is simple, easy, low-cost, and can achieve efficient induced synthesis of pseudomycodin, and can be applied to industrial production.

The present invention is realized through the following technical solutions:

A method for using sunlight radiation to induce Nostoc suspension culture cells to produce pseudophysin, including the following steps:

Step 1: First inoculate Nostoc suspension culture cells into the liquid medium to obtain culture system A;

Step 2: Radiate culture system A under sunlight for 0.5 to 1 hour to obtain culture system B;

Step 3: Culture system B on a shaking table at a constant temperature using an LED fluorescent lamp with a light intensity of 40 to 45 μmol photons m ^-2 s ^-1 for 1, 2 or 3 days to obtain culture system C;

Step 4: Treat culture system C in the same way as culture system A in step 2, and treat the resulting culture system in the same way as culture system B in step 3 to obtain culture system D, forming a second cycle for Nostoc suspension culture cells. deal with;

Step 5: Continue to cycle the Nostoc suspension culture cells according to the method of step 4. The total processing time is 8 to 14 days. Afterwards, the culture solution is centrifuged to collect the algae, and pseudophysin is extracted from it to complete the preparation of pseudophysin. .

Preferably, during the radiation treatment in step 2, the intensities of UV-A, UV-B and visible light are 1.4~23W m ^-2 , 0.1~1.6W m ^-2 and 76~1298 μmol photons m ^-2 s ^-1 respectively.

Preferably, the temperature during the radiation treatment in step 2 is 20-32°C.

Preferably, in step 1, Nostoc suspension culture cells are first inoculated into the liquid medium, and then L-tryptophan is added so that the concentration of L-tryptophan in the resulting system is 0.5 to 5 mmol/L, and then step 2 is performed. The radiation treatment described.

Preferably, in step 3, culture system B is cultured on a shaking table at a constant temperature of 24 to 26°C.

Further, step 1 is to inoculate Nostoc suspension culture cells into BG-11 liquid medium to obtain culture system A.

Preferably, when performing shaker culture in step 2, the rotation speed is 120-130 rpm.

Preferably, step 5 separates pseudomycodin in the culture solution according to the following process:

The culture solution is first centrifuged to obtain precipitated algal bodies, which are then freeze-dried and then extracted with acetone to obtain an extract. Finally, the extract is freeze-dried to obtain pseudomycodin.

Compared with the existing technology, the present invention has the following beneficial technical effects:

The invention is a method for inducing the production of pseudophysin by using natural sunlight radiation to induce suspension culture cells of Nostoc. As a renewable resource, sunlight can induce some cyanobacteria cells to synthesize pseudophysin, and the sunlight radiation can be reasonably used to induce pseudophysin. Vegetable suspension culture cells can synthesize pseudophysin in large quantities, which can solve the problems of high production cost and low yield of existing methods, and is also in line with the principles of economy, greenness and environmental protection. Long-term exposure to sunlight will inhibit the growth and photosynthesis of Nostoc suspension culture cells. Therefore, the use of periodic short-term sunlight irradiation of Nostoc suspension culture cells can reduce the adverse effects of sunlight radiation on the growth of cell biomass. , and can also efficiently induce the synthesis of pseudophysin. The method saves energy consumption, is simple and easy to implement, is suitable for large-scale production, can significantly improve economic benefits, and the harvested pseudophyton can be used in the fields of medicine and cosmetics.

Further, the present invention adds 0.5 to 5mM L-tryptophan to the culture medium, and then proceeds according to the subsequent process. Compared with the culture medium without adding L-tryptophan, after the same periodic irradiation treatment, The pseudophysin content can be further increased significantly.

Description of the drawings

Figure 1 is a diagram showing the influence of L-tryptophan (Try) on Nostoc biomass in Example 5 of the present invention.

Figure 2 is a graph showing the influence of L-tryptophan (Try) on the pseudophysin content of Nostoc in Example 5 of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to specific examples, which are explanations rather than limitations of the present invention.

The present invention will be described in detail below with reference to the drawings and specific embodiments.

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step 1: Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium, and use natural light for subsequent periodic short-term treatment to induce efficient synthesis of pseudophysin; in addition, Adding L-tryptophan to 100mL BG-11 liquid culture medium at a concentration of 0.5-5mmol/L, and also subjecting it to sunlight radiation treatment can further increase pseudophysin production.

Step 2, perform periodic short-term radiation treatment under natural light, each radiation treatment lasts for 0.5 to 1 hour. In the two adjacent radiations, from the beginning of the previous radiation to the beginning of the second radiation, it lasts for 1 day, 2 days, or 3 days; when adding L-tryptophan, each irradiation treatment is 0.5 hours. In the two adjacent irradiations, it takes 1 day from the beginning of the previous irradiation to the beginning of the second irradiation.

Step 3: After the treatment is completed, it is cultured under LED fluorescent lamp and constant temperature shaker for 1 day, 2 days or 3 days, and then short-term radiation treatment and shaker culture are carried out according to the above steps. The total treatment time is 8 to 14 days, and the light intensity is 40μmol photonsm ^-2 s ^-1 , temperature is 25℃, shaker speed is 120~130rpm.

Step 4: During the culture period, take 10 mL of culture solution, collect the materials by centrifugation, freeze-dry, and measure the cell biomass and pseudophysin content of Nostoc.

Step 5. Determination of pseudophycein content. Use 100% acetone to extract the dried algae overnight at 4°C in the dark. The extraction time is 12 to 18 hours to obtain an extract. Measure the OD ₃₃₄ in the extract. According to The molar absorption coefficient is 112.6Lg ^-1 ·cm ^-1 to calculate the pseudophysin content.

Step 6: freeze-dry the extract to obtain pseudophysin.

Example 1

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step (1): Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium. The initial OD ₇₃₀ is 0.30.

In step (2), the Nostoc suspension culture cells in step (1) are radiated in the sun. During the treatment period, the intensity and temperature of ultraviolet UV-A, ultraviolet UV-B, and visible light are: 1.4~23W m ^-2 and 0.1~ 1.6W m ^-2 , 76～1298μmolphotons m ^-2 s ^-1 and 20～32℃.

Step (3): irradiate the Nostoc suspension culture cells in step (1) under sunlight for 0.5 hours.

In step (4), the Nostoc suspension culture cells after irradiation in step (3) are cultured on a shaker under an LED fluorescent lamp and a constant temperature light room for 1 day, and then short-term radiation treatment and shaker culture are performed according to the above steps. The total processing time It is 12 days, the rotation speed is 130rpm, the LED fluorescent lamp intensity is 40μmol photons m ^-2 s ^-1 , and the temperature is 25℃.

In step (5), take samples from step (4), take 10 mL of the culture solution and centrifuge for 20 minutes to collect the samples, freeze-dry them, and measure the Nostoc cell biomass and pseudophysin content.

Step (6), determine the content of pseudophyton. Extract the dried algae with 100% acetone overnight at 4°C in the dark, and measure the absorbance of the acetone solution containing pseudophyton at a wavelength of 334 nm. According to the molar absorption coefficient The calculated content was 112.6L g ^-1 ·cm ^-1 . On the 12th day, the pseudophysin content in dried Nostoc was measured to be 11.1mg g ^-1 .

In step (7), the remaining acetone solution in step (6) is freeze-dried to obtain pseudomycodin.

Example 2

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step (1): Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium. The initial OD ₇₃₀ is 0.30.

In step (2), the Nostoc suspension culture cells in step (1) are radiated in the sun. During the treatment period, the intensity and temperature of ultraviolet UV-A, ultraviolet UV-B, and visible light are: 1.4~23W m ^-2 and 0.1~ 1.6W m ^-2 , 76～1298μmolphotons m ^-2 s ^-1 and 20～32℃.

Step (3): irradiate the Nostoc suspension culture cells in step (1) under sunlight for 0.5 hours.

In step (4), the Nostoc suspension culture cells after irradiation in step (3) are cultured in an LED fluorescent lamp and a shaker at a constant temperature for 2 days, and then short-term radiation treatment and shaker culture are performed according to the above steps. The total treatment time is 12 day, the rotation speed is 130rpm, the LED fluorescent lamp intensity is 40μmol photons m ^-2 s ^-1 , and the temperature is 25°C.

In step (5), take samples from step (4), take 10 mL of the culture solution and centrifuge for 20 minutes to collect the samples, freeze-dry them, and measure the Nostoc cell biomass and pseudophysin content.

Step (6), to measure the content of pseudophysin, extract the dried algae with 100% acetone overnight at 4°C in the dark, and measure the absorbance of the acetone solution containing pseudophysin at a wavelength of 334 nm. According to the molar coefficient of 112.6 The content was calculated by L g ^-1 cm ^-1 . On the 12th day, the pseudophysin content in dried Nostoc was measured to be 9.05mg g ^-1 .

In step (7), the remaining acetone solution in step (6) is freeze-dried to obtain pseudomycodin.

Example 3

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step (1): Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium. The initial OD ₇₃₀ is 0.27.

In step (2), the Nostoc suspension culture cells in step (1) are radiated in the sun. During the treatment period, the intensity and temperature of ultraviolet UV-A, ultraviolet UV-B, and visible light are: 1.4~23W m ^-2 and 0.1~ 1.6W m ^-2 , 76～1298μmolphotons m ^-2 s ^-1 and 20～32℃.

Step (3): irradiate the Nostoc suspension culture cells in step (1) under sunlight for 0.5 hours.

In step (4), the Nostoc suspension culture cells after irradiation in step (3) are cultured in an LED fluorescent lamp and a shaker at a constant temperature for 3 days, and then short-term radiation treatment and shaker culture are performed according to the above steps. The total treatment time is 12 day, the rotation speed is 130rpm, the LED fluorescent lamp intensity is 40μmol photons m ^-2 s ^-1 , and the temperature is 25°C.

In step (5), take samples from step (4), take 10 mL of the culture solution and centrifuge for 20 minutes to collect the samples, freeze-dry them, and measure the Nostoc cell biomass and pseudophysin content.

Step (6), determine the content of pseudophyton. Extract the dried algae with 100% acetone overnight at 4°C in the dark, and measure the absorbance of the acetone solution containing pseudophyton at a wavelength of 334 nm. According to the molar absorption coefficient The content was calculated as 112.6L g ^-1 ·cm ^-1 . On the 12th day, the pseudophysin content in dried Nostoc was measured to be 7.61 mg g ^-1 .

In step (7), the remaining acetone solution in step (6) is freeze-dried to obtain pseudomycodin.

Example 4

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step (1): Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium. The initial OD ₇₃₀ is 0.28.

In step (2), the Nostoc suspension culture cells in step (1) are radiated in the sun. During the treatment period, the intensity and temperature of ultraviolet UV-A, ultraviolet UV-B, and visible light are: 1.4~23W m ^-2 and 0.1~ 1.6W m ^-2 , 76～1298μmolphotons m ^-2 s ^-1 and 20～32℃.

Step (3): irradiate the Nostoc suspension culture cells in step (1) under sunlight for 1 hour.

Step (4), culture the Nostoc suspension culture cells in a shaker for 1 day after the radiation in step (3), and then perform short-term radiation treatment and shaker culture according to the above steps. The total treatment time is 12 days, and the rotation speed is 130 rpm. The LED fluorescent lamp intensity is 40 μmol photons m ^-2 s ^-1 and the temperature is 25°C.

In step (5), take samples from step (4), take 10 mL of the culture solution and centrifuge for 20 minutes to collect the samples, freeze-dry them, and measure the Nostoc cell biomass and pseudophysin content.

Step (6), to measure the content of pseudophyton, extract the dried algae with 100% acetone at 4°C overnight in the dark, and measure the absorbance of the acetone solution containing pseudophyton at a wavelength of 334 nm. According to the molar coefficient of 112.6 The content was calculated as L g ^-1 ·cm ^-1 . On the 12th day, the pseudophysin content in dried Nostoc was measured to be 3.58 mg g ^-1 .

In step (7), the remaining acetone solution in step (6) is freeze-dried to obtain pseudomycodin.

Example 5

The invention is a method for inducing pseudophysin production in Nostoc suspension culture cells using sunlight radiation, which includes the following steps:

Step (1): Inoculate Nostoc suspension culture cells in good growth status into a quartz flask containing 100 mL of BG-11 liquid culture medium. The initial OD ₇₃₀ is 0.28, and 5mM L-tryptophan is added.

Step (2): irradiate the Nostoc suspension culture cells in step (1) under sunlight. The intensity and temperature of ultraviolet UV-A, ultraviolet UV-B, and visible light are: 1.2 to 35W m ^-2 and 0.1 to 2.6W respectively. m ^-2 , 51～1720μmol photons m ^-2 s ^-1 and 27～39℃.

Step (3): irradiate the Nostoc suspension culture cells in step (1) under sunlight for 0.5 hours.

Step (4): Cultivate the Nostoc suspension culture cells in a shaker for 1 day after the radiation in step (3), and then perform short-term radiation treatment and shaker culture according to the above steps. The total treatment time is 12 days, and the rotation speed is 130 rpm. The light intensity is 40 μmol photons m ^-2 s ^-1 and the temperature is 25°C.

In step (5), take samples from step (4), take 10 mL of the culture solution and centrifuge for 20 minutes to collect the samples, freeze-dry them, and measure the Nostoc cell biomass and pseudophysin content.

Step (6), to measure the content of pseudophysin, extract the dried algae with 100% acetone overnight at 4°C in the dark, and measure the absorbance of the acetone solution containing pseudophysin at a wavelength of 334 nm. According to the molar absorbance The coefficient is 112.6L g ^-1 ·cm ^-1 to calculate the content.

As shown in Figure 1, the biomass after adding 5mM L-tryptophan was 1.23g L ^-1 , which was slightly lower than the treatment group without adding L-tryptophan (1.43g L ^-1 ), and the impact on biomass was small. As shown in Figure 2, after adding 5mM L-tryptophan, the pseudophysin content in dried Nostoc was measured to be 8.78mg g ^-1 , which was significantly higher than the treatment group without L-tryptophan (3.44mg g ^-1 ).

It can be seen from Table 1 that the biomass and pseudophysin content of Nostoc are different under different treatment frequencies. When the irradiation time is 0.5 hours and the treatment frequency is once a day, the pseudophysin content in dried Nostoc can reach 11.11 mg g ^-1 , and the effect is the best. If L-tryptophan substrate is added to the culture medium, the production of pseudomycin will be further increased.

Table 1 Biomass and pseudophysin content of Nostoc suspension culture cells under different treatment frequencies

Claims (3)

1. A method for producing pseudo-cladophyllin by using sun radiation to induce hair weeds to suspend cultured cells, which is characterized by comprising the following steps:

step 1, inoculating a hair weeds suspension culture cell into a BG-11 liquid culture medium to obtain a culture system A;

step 2, the culture system A is subjected to radiation treatment for 0.5 hour in the sun, the temperature is 20-32 ℃ during the radiation treatment, and the intensity of UV-A, UV-B and visible light is 1.4-23W m respectively ^-2 、0.1~1.6 W m ^-2 And 76-1298 mu mol of photons m ^-2 s ^-1 Obtaining a culture system B;

step 3, the culture system B is subjected to light intensity of 40-45 mu mol of photons m at the constant temperature of 24-26 DEG C ^-2 s ^-1 Carrying out shaking culture for 1 day or 2 days on the LED fluorescent lamp to obtain a culture system C;

step 4, treating the culture system C in a mode of treating the culture system A in the step 2, and treating the obtained culture system in a mode of treating the culture system B in the step 3 to obtain a culture system D, so as to form second circulation treatment on the hair weeds suspension culture cells;

and 5, continuously performing circulation treatment on the hair weeds suspension culture cells in the mode of the step 4, wherein the total treatment time is 12 days, and then, collecting algae by using a centrifugal culture solution, and extracting pseudo-cladophyllin from the algae to finish the preparation of the pseudo-cladophyllin.

2. The method for preparing pseudo-cladosporium cucumerinum by using solar radiation according to claim 1, wherein the rotation speed is 120-130 rpm when the shaking culture is performed in the step 3.

3. The method for preparing pseudo-branch phycin using solar radiation according to claim 1, wherein step 5 separates pseudo-branch phycin in the culture solution according to the following procedure:

centrifuging the culture solution to obtain precipitated algae, freeze-drying the algae, extracting with acetone to obtain extractive solution, and freeze-drying the extractive solution to obtain pseudo-branch phycocyanin.