CN112725232B - Culture medium and method for culturing spirulina with high phycocyanin content - Google Patents
Culture medium and method for culturing spirulina with high phycocyanin content Download PDFInfo
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- CN112725232B CN112725232B CN202110002344.6A CN202110002344A CN112725232B CN 112725232 B CN112725232 B CN 112725232B CN 202110002344 A CN202110002344 A CN 202110002344A CN 112725232 B CN112725232 B CN 112725232B
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Abstract
The invention belongs to the technical field of algae culture, and particularly relates to a culture medium and a method for culturing spirulina with high phycocyanin content. The invention provides a culture medium for culturing spirulina with high phycocyanin content, which comprises a spirulina basic culture medium and a reduced graphene oxide-titanium dioxide composite material. In the invention, the reduced graphene oxide-titanium dioxide composite material in the culture medium enters into spirulina cells to be agglomerated or combined with the spirulina cells, and the content of phycocyanin in the spirulina cells is improved through a shading effect and an oxidative stress effect.
Description
Technical Field
The invention belongs to the technical field of algae culture, and particularly relates to a culture medium and a method for culturing spirulina with high phycocyanin content.
Background
Phycocyanin (PC) is a dark blue powder isolated from spirulina. Phycocyanin has antioxidant and anti-inflammatory activities, can inhibit inflammation induced by glucose oxidase, and has obvious inhibition on acute and chronic tissue and organ inflammation; phycocyanin also has anticancer and antitumor activities, and the phycocyanin inhibits human cancer cell lines HL-60, K-562 and mu-937 to different degrees; the phycocyanin also has immunocompetence, and the phycocyanin can promote the regeneration of animal blood cells, improve the activity of lymphocytes, further improve the immune function of an organism and comprehensively enhance the disease prevention and disease resistance of the organism; the phycocyanin also has photosensitive activity and fluorescence activity, can be used as a photosensitizer in tumor laser treatment, and can be used as a fluorescent probe for medical diagnosis, clinical medicine and other fields.
The spirulina grown under natural conditions has low phycocyanin content (generally 10-20%) and is unfavorable for extraction, so that the phycocyanin yield is low, the price is high, and the application field is limited.
Disclosure of Invention
In view of the above, the present invention provides a culture medium and a method for culturing spirulina with high phycocyanin content, and spirulina obtained by culturing spirulina with the culture medium provided by the present invention has high phycocyanin content.
In order to solve the technical problems, the invention provides a culture medium for culturing spirulina with high phycocyanin content, which comprises a spirulina basic culture medium and a reduced graphene oxide-titanium dioxide composite material.
Preferably, the mass ratio of the reduced graphene oxide-titanium dioxide composite material to the spirulina basic culture medium is (50-70) mg (650-710) mL.
Preferably, the particle size of the reduced graphene oxide-titanium dioxide composite material is nano-scale.
Preferably, the preparation method of the reduced graphene oxide-titanium dioxide composite material comprises the following steps:
mixing graphene oxide, tetrabutyl titanate, isopropyl alcohol and water to obtain a dispersion liquid to be reacted;
and carrying out solvothermal reaction on the dispersion liquid to be reacted to obtain the reduced graphene oxide-titanium dioxide composite material.
Preferably, the volume ratio of the mass of the graphene oxide to the tetrabutyl titanate is (27-32) mg (45-55) mu L.
Preferably, the spirulina basal medium is a Zarrouk medium.
The invention also provides a method for culturing spirulina with high phycocyanin content, which comprises the following steps:
inoculating spirulina in a culture medium, wherein the culture medium is the culture medium according to the technical scheme;
culturing the inoculated spirulina to obtain the spirulina with high phycocyanin content.
Preferably, the inoculation amount of the inoculation is 0.2-0.4 g/L.
Preferably, the culturing conditions include: the temperature is 25-35 ℃, and the illumination intensity is 163-167 mu mol/m 2 /s。
Preferably, the time of the cultivation is 9 to 10 days.
The invention provides a culture medium for culturing spirulina with high phycocyanin content, which comprises a spirulina basic culture medium and a reduced graphene oxide-titanium dioxide composite material. In the invention, the reduced graphene oxide-titanium dioxide composite material enters the spirulina cells to be agglomerated or combined with the spirulina cells, so that a shading effect occurs, the absorption of the spirulina cells to light is blocked, the capturing of the antenna pigment to light is not facilitated, and the utilization rate of the antenna pigment to light is reduced; therefore, in order to optimize the absorption of light, the spirulina cells generate more antenna pigments by the compensation effect in the cells, and the phycocyanin is the main antenna pigment, so that the content of the phycocyanin in the spirulina cells is improved. Meanwhile, when the reduced graphene oxide-titanium dioxide composite material enters the spirulina cells, oxidation stress reaction can be generated on the spirulina cells (the conclusion can be obtained by obviously reducing the content of glutathione in the cells according to the detection result and obviously improving the content of oxidized glutathione), more oxidation-resistant substances are generated by metabolism in the spirulina cells in order to remove free radicals and lipid peroxides generated by the oxidation stress reaction in the cells, and phycocyanin is a main oxidation-resistant substance, so that the content of phycocyanin is further improved.
The invention also provides a method for culturing spirulina with high phycocyanin content, which comprises the following steps: inoculating spirulina in a culture medium, wherein the culture medium is the culture medium according to the technical scheme; culturing the inoculated spirulina to obtain the spirulina with high phycocyanin content. According to the invention, spirulina is cultivated in a culture medium containing the reduced graphene oxide-titanium dioxide composite material, and after the original graphene oxide-titanium dioxide composite material enters spirulina cells, the content of phycocyanin in the spirulina cells is improved through a shading effect and an oxidative stress effect.
Drawings
FIG. 1 is a schematic reaction diagram of a reduced graphene oxide-titanium dioxide composite prepared from graphene oxide, tetrabutyl titanate and water;
FIG. 2 is an XRD spectrum of the reduced graphene oxide-titanium dioxide composite material prepared in example 1;
FIG. 3 is a scanning electron microscope image of the reduced graphene oxide-titanium dioxide composite material prepared in example 1;
FIG. 4 is a photograph of spirulina obtained by observing spirulina cultured in example 2, comparative example 1 and comparative example 2 under a microscope of 10X 40 times; wherein a is the picture of spirulina cultivated in example 2, b is the picture of spirulina cultivated in comparative example 1, c is the picture of spirulina cultivated in comparative example 2.
Detailed Description
The invention provides a culture medium for culturing spirulina with high phycocyanin content, which comprises a spirulina basic culture medium and a reduced graphene oxide-titanium dioxide composite material.
In the invention, the particle size of the reduced graphene oxide-titanium dioxide composite material is preferably nano-scale, more preferably 10-20 nm; the reduced graphene oxide-titanium dioxide composite material comprises reduced graphene oxide and titanium dioxide particles loaded on the reduced graphene oxide sheets. In the invention, the mass percentage of the titanium dioxide in the reduced graphene oxide-titanium dioxide composite material is preferably 15-30%, more preferably 18-25%; the particle diameter of the titanium dioxide particles is preferably 1 to 5nm.
In the present invention, the preparation method of the reduced graphene oxide-titanium dioxide composite material preferably includes the steps of:
mixing graphene oxide, tetrabutyl titanate, isopropyl alcohol and water to obtain a dispersion liquid to be reacted;
and carrying out solvothermal reaction on the dispersion liquid to be reacted to obtain the reduced graphene oxide-titanium dioxide composite material.
According to the invention, graphene oxide, tetrabutyl titanate, isopropyl alcohol and water are mixed to obtain dispersion liquid to be reacted. In the present invention, the mixing preferably includes the steps of:
firstly mixing graphene oxide and isopropanol to obtain graphene oxide dispersion liquid;
carrying out second mixing on the graphene oxide dispersion liquid and tetrabutyl titanate to obtain a primary dispersion liquid to be reacted;
and thirdly mixing the primary to-be-reacted dispersion liquid and water.
According to the invention, graphene oxide and isopropanol are subjected to first mixing to obtain graphene oxide dispersion liquid. In the present invention, the ratio of the mass of the graphene oxide to the volume of the isopropyl alcohol is preferably 30 mg/23 to 27mL, more preferably 30 mg/25 mL. In the present invention, the first mixing is preferably performed under ultrasonic conditions, and the power of the ultrasonic waves is preferably 175 to 185W, more preferably 178 to 180W; the time is preferably 25 to 35 minutes, more preferably 30 to 33 minutes. In the invention, the graphene oxide preferably obtains thin-layer graphene oxide under ultrasonic conditions, and the number of layers of the thin-layer graphene oxide is preferably a single layer.
After the graphene oxide dispersion liquid is obtained, the graphene oxide dispersion liquid and tetrabutyl titanate are subjected to secondary mixing to obtain a primary dispersion liquid to be reacted. In the present invention, the ratio of the mass of the graphene oxide to the volume of the tetrabutyl titanate is preferably (27 to 32) mg (45 to 55) μl, more preferably 30mg (50 to 53) μl. In the present invention, the second mixing is preferably performed under stirring, and the stirring speed is preferably 500 to 700r/min, more preferably 580 to 600r/min; the time is preferably 30 to 60 minutes, more preferably 35 to 40 minutes.
After the primary to-be-reacted dispersion liquid is obtained, the primary to-be-reacted dispersion liquid and water are subjected to third mixing. In the present invention, the water is preferably ultrapure water. In the present invention, the volume ratio of tetrabutyl titanate to water is preferably 45 to 55:1000, more preferably 50 to 52:1000. In the present invention, the third mixing is preferably performed under stirring, and the stirring speed is preferably 500 to 700r/min, more preferably 580 to 600r/min; the time is preferably 30 to 60 minutes, more preferably 35 to 40 minutes. In the invention, the water is a solvent for the tetrabutyl titanate to undergo hydrolysis reaction.
According to the invention, graphene oxide can be dispersed into thin-layer graphene oxide through stepwise mixing, so that the surface area of the graphene oxide is increased, and the load capacity of titanium dioxide on the surface of the graphene oxide is improved.
After the dispersion liquid to be reacted is obtained, the dispersion liquid to be reacted is subjected to solvothermal reaction to obtain the reduced graphene oxide-titanium dioxide composite material. In the present invention, the temperature of the solvothermal reaction is preferably 175 to 185 ℃, more preferably 178 to 180 ℃; the time is preferably 7.5 to 8.5 hours, more preferably 7.8 to 8 hours.
In the invention, tetrabutyl titanate is subjected to hydrolysis reaction to generate titanium dioxide in the solvothermal reaction process and is loaded on the surface of graphene oxide, and meanwhile, the graphene oxide is reduced to reduced graphene oxide. In the present invention, the means of loading is preferably physical adsorption.
After the solvothermal reaction, the invention preferably reduces the temperature and centrifugates the product of the solvothermal reaction; and drying the precipitate obtained by centrifugation to obtain the reduced graphene oxide-titanium dioxide composite material. In the present invention, the temperature is preferably lowered to room temperature. The cooling mode is not particularly limited, as long as the required temperature can be reached. The present invention is not particularly limited as long as the centrifugation can achieve solid-liquid separation. In the present invention, the drying is preferably vacuum drying, and the vacuum degree of the vacuum drying is preferably 0.08 to 0.12MPa, more preferably 0.1MPa; the temperature is preferably 68 to 72 ℃, more preferably 70 ℃; the time is preferably 11 to 13 hours, more preferably 12 hours.
In the invention, a reaction schematic diagram of preparing the reduced graphene oxide-titanium dioxide composite material by taking graphene oxide, tetrabutyl titanate and water as raw materials is shown in fig. 1, wherein a red round substance represents tetrabutyl titanate and a green round substance represents titanium dioxide in fig. 1.
In the present invention, the spirulina basic medium is preferably a Zarrouk medium. The composition of the basic medium is not particularly limited as long as spirulina can be cultivated. In an embodiment of the invention, the Zarouk medium comprises the following components: 16.8g/L NaHCO 3 ，2.5g/L NaNO 3 ，1.00g/L K 2 SO 4 ，1.00g/L NaCl，0.20g/L MgSO 4 ·7H 2 O，0.01g/L FeSO 4· 7H 2 O，0.50g/LK 2 HPO 4 ，0.04g/L CaCL 2 ·2H 2 O，0.08g/L EDTA，2.86g/L H 3 BO 3 ，1.81g/L MnCl 2 ·4H 2 O，0.22g/LZnSO 4 ·4H 2 O，0.018g/LNaMoO 4 ，0.08g/L CuSO 4 ·5H 2 O。
In the invention, the mass ratio of the reduced graphene oxide-titanium dioxide composite material to the volume ratio of the spirulina basic culture medium is preferably (50-70) mg (650-710) mL, more preferably (55-60) mg (660-700) mL.
In the present invention, the preparation method of the culture medium for culturing spirulina according to the above technical scheme preferably comprises the following steps:
and adding the reduced graphene oxide-titanium dioxide composite material into a basic culture medium to obtain the culture medium for culturing the spirulina.
The invention preferably sterilizes the basal medium before adding the reduced graphene oxide-titanium dioxide composite material into the basal medium. The invention is not particularly limited to the sterilization, and the sterilization method conventional in the art can be adopted. The reduced graphene oxide-titanium dioxide composite material is preferably added to the basal medium under aseptic conditions.
The invention also provides a method for culturing spirulina with high phycocyanin content, which comprises the following steps:
inoculating spirulina in a culture medium, wherein the culture medium is the culture medium according to the technical scheme;
culturing the inoculated spirulina to obtain the spirulina with high phycocyanin content.
The invention inoculates spirulina in the culture medium, wherein the culture medium is the culture medium according to the technical scheme. In the present invention, the inoculation amount of the inoculation is preferably 0.2 to 0.4g/L, more preferably 0.3g/L. In the present invention, the inoculation is preferably performed under aseptic conditions.
After inoculating the spirulina, the invention cultures the inoculated spirulina to obtain the spirulina with high phycocyanin content. The invention is not particularly limited in the type of spirulina, and spirulina selected in the embodiment of the invention is spirulina platensis (FACHB-314), which is purchased from the institute of aquatic organisms of national academy of sciences.
The culture apparatus is not particularly limited in the present invention, and in the embodiment of the present inventionThe cultivation is carried out in an illumination incubator in a photobioreactor. In the present invention, the conditions of the culture include: the temperature is preferably 25 to 35 ℃, more preferably 28 to 30 ℃; the illumination intensity is preferably 163-167. Mu. Mol/m 2 Preferably 164 to 165. Mu. Mol/m 2 And/s. In the present invention, the time of the culture is preferably 9 to 10 days.
In the present invention, air is preferably introduced into the culture medium during the culture, and the flow rate of the air is preferably 0.8 to 1.2L/min, more preferably 1L/min.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Ultrasonic treatment is carried out on 30mg of graphene oxide and 25mL of isopropanol for 30min under the power of 180W to obtain a first solution;
stirring the first solution and 50 mu L of tetrabutyl titanate for 30min under the condition of 600r/min to obtain a second solution;
stirring the second solution and 1mL of ultrapure water for 30min under the condition of 600r/min to obtain a dispersion liquid to be reacted;
carrying out solvothermal reaction on the dispersion liquid to be reacted at 180 ℃ for 8 hours, naturally cooling to room temperature, and filtering; and drying the filter residue obtained by filtration for 12 hours under the condition that the vacuum degree is 0.1Mpa and the temperature is 70 ℃ to obtain the reduced graphene oxide-titanium dioxide composite material with the particle size of 10-20 nm.
Example 2
After the Zarouk culture medium is sterilized, 50mg of the reduced graphene oxide-titanium dioxide composite material prepared in the example 1 is added into 710mL of the Zarouk culture medium under the aseptic condition, and 0.213g of spirulina platensis is inoculated; the Zarouk medium included the following components: 16.8g/LNaHCO 3 ，2.5g/LNaNO 3 ，1.00g/LK 2 SO 4 ，1.00g/LNaCl，0.20g/LMgSO 4 ·7H 2 O，0.01g/LFeSO 4· 7H 2 O，0.50g/LK 2 HPO 4 ，0.04g/LCaCL 2 ·2H 2 O，0.08g/LEDTA，2.86g/LH 3 BO 3 ，1.81g/LMnCl 2 ·4H 2 O，0.22g/LZnSO 4 ·4H 2 O，0.018g/LNaMoO 4 ，0.08g/LCuSO 4 ·5H 2 O；
Placing the culture medium inoculated with spirulina in a light incubator of a photobioreactor at 25deg.C and 165 μmol/m 2 And/s, culturing for 9 days under the condition that the air flow rate is 1L/min, so as to obtain the spirulina with high phycocyanin content.
Comparative example 1
A spirulina with a high phycocyanin content was cultivated as in example 2, except that 50mg of reduced graphene oxide-titanium dioxide composite was replaced with 50mg of titanium dioxide.
Comparative example 2
Spirulina was cultivated as in example 2, except that the reduced graphene oxide-titanium dioxide composite was not added to the medium.
Test case
XRD detection is carried out on the reduced graphene oxide-titanium dioxide composite material prepared in the embodiment 1, and an XRD spectrum is shown in figure 2. From FIG. 2, tiO appears at 25.3 °, 37.8 °, 48 °, 53.9 °, 55.1 °, 62.7 ° 2 The characteristic peak of (2) shows that the titanium dioxide is successfully loaded on the surface of the reduced graphene oxide.
And (3) carrying out scanning electron microscope observation on the reduced graphene oxide-titanium dioxide composite material prepared in the embodiment 1 to obtain a scanning electron microscope image of the reduced graphene oxide-titanium dioxide composite material, wherein the scanning electron microscope image is shown in fig. 3. As can be seen from fig. 3, the reduced graphene oxide surface is loaded with titanium dioxide of nanoparticles; the particle size of the titanium dioxide is 1-5 nm.
The spirulina cultures of example 2, comparative example 1 and comparative example 2 were observed under a microscope of 10×40 times, respectively, to obtain a picture of spirulina, as shown in fig. 4, wherein a is a picture of spirulina culture of example 2, b is a picture of spirulina culture of comparative example 1, and c is a picture of spirulina culture of comparative example 2.
As can be seen from fig. 4, spirulina cultured without adding titanium dioxide or reduced graphene oxide-titanium dioxide composite material in the culture medium is divided along a single transverse plane, and the generated subcellular is still adhered, so that many cells are connected into a spiral filament shape, which is called an algae filament. After titanium dioxide is added into the culture medium, certain toxicity can be generated on spirulina cells, the heliciform of the algae filaments begins to disappear, and adhered cells are broken to destroy the morphology and structure of the algae filaments. After the reduced graphene oxide-titanium dioxide composite material is added into the culture medium, the morphology and the structure of the algae filament are hardly changed, which proves that the reduced graphene oxide-titanium dioxide composite material has higher compatibility with a spirulina system.
Taking 1mL of spirulina liquid cultured in example 2 and comparative examples 1 and 2 respectively, adding 2mL of deionized water, shaking uniformly, performing UV-Vis spectroscopic detection under 680nm wavelength, and recording the obtained data as A 680 The A is carried out 680 Multiplied by 3 to obtain OD 680 。
Taking 2mL of spirulina liquid cultured in example 2 and comparative examples 1 and 2 respectively, centrifuging at 5000r/min for 10min, mixing the precipitate obtained by centrifugation with 10mL of phosphate buffer solution, performing freeze thawing for 3 times, centrifuging, collecting supernatant, performing UV-Vis spectrophotometry under 620nm wavelength condition, and recording the obtained data as OD 620 The method comprises the steps of carrying out a first treatment on the surface of the Then UV-Vis spectroscopic detection was performed at 652nm, and the data obtained was recorded as OD 652 。
The spirulina cell dry weight (DCW) and phycocyanin yield (PC) were calculated according to the following formulas, the results of which are listed in table 1.
DCW＝0.5192×OD 680 ；
PC＝(OD 620 -0.474×OD 652 )/5.34；
Wherein, DCW is spirulina cell dry weight;
D 680 is the optical density value under the light wave with the wavelength of 680 nm;
PC is phycocyanin yield;
OD 620 is the optical density value under the light wave with the wavelength of 620 nm;
OD 652 optical density value at 652nm。
TABLE 1 phycocyanin content in spirulina cultured in example 2 and comparative examples 1 and 2
The intracellular PC content is the maximum value of the intracellular PC content in the culture period, and the dry weight is the dry weight of the day corresponding to the maximum value of the intracellular PC content.
As can be seen from the data in Table 1, the addition of the reduced graphene oxide-titanium dioxide composite material to the culture medium can increase the PC content in the spirulina cells, thereby increasing the PC yield, and the PC yield in example 2 is increased by 15.32% compared with that in comparative example 2.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (6)
1. A culture medium for culturing spirulina with high phycocyanin content comprises spirulina basic culture medium and reduced graphene oxide-titanium dioxide composite material; the volume ratio of the mass of the reduced graphene oxide-titanium dioxide composite material to the volume of the spirulina basic culture medium is (50-70) mg (650-710) mL;
the reduced graphene oxide-titanium dioxide composite material is prepared by the following steps:
ultrasonic treatment is carried out on 30mg of graphene oxide and 25mL of isopropanol for 30min under the power of 180W to obtain a first solution;
stirring the first solution and 50 mu L of tetrabutyl titanate for 30min under the condition of 600r/min to obtain a second solution;
stirring the second solution and 1mL of ultrapure water for 30min under the condition of 600r/min to obtain a dispersion liquid to be reacted;
carrying out solvothermal reaction on the dispersion liquid to be reacted at 180 ℃ for 8 hours, naturally cooling to room temperature, and filtering; and drying the filter residue obtained by filtration for 12 hours under the condition that the vacuum degree is 0.1Mpa and the temperature is 70 ℃ to obtain the reduced graphene oxide-titanium dioxide composite material with the particle size of 10-20 nm.
2. The culture medium of claim 1, wherein the spirulina basal medium is a Zarrouk medium.
3. A method of culturing spirulina with high phycocyanin content, comprising the steps of:
inoculating spirulina in a culture medium, wherein the culture medium is the culture medium of claim 1 or 2;
culturing the inoculated spirulina to obtain the spirulina with high phycocyanin content.
4. A method according to claim 3, wherein the inoculum size of the inoculum is in the range of 0.2 to 0.4g/L.
5. A method according to claim 3, wherein the conditions of the culture comprise: the temperature is 25-35 ℃, and the illumination intensity is 163-167 mu mol/m 2 /s。
6. The method according to claim 5, wherein the time of the culturing is 9 to 10 days.
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