Haematococcus pluvialis exopolysaccharide as well as preparation method and application thereof

Abstract

The invention discloses a haematococcus pluvialis exopolysaccharide and a preparation method and application thereof.The preparation method of the haematococcus pluvialis exopolysaccharide comprises the step that the exopolysaccharide in haematococcus pluvialis liquid which is cultured for 30 days or above in low light is extracted through an alcohol precipitation method, specifically, the haematococcus pluvialis liquid which is cultured for 30 days or above in low light is collected, and the haematococcus pluvialis exopolysaccharide is obtained. Carrying out centrifugal treatment on the algae liquid, and collecting supernate; adding absolute ethyl alcohol into the collected supernate to enable the final concentration of the ethyl alcohol to reach 70%-80%, standing in a refrigerator at the temperature of 4 DEG C overnight, centrifuging the supernate containing the ethyl alcohol, and collecting precipitate; adding 200mL of purified water into the precipitate, and then carrying out auxiliary dissolution under an ultrasonic condition; and finally, centrifuging again, collecting a faint yellow extracellular polysaccharide solution, and freeze-drying to obtain the haematococcus pluvialis extracellular polysaccharide dry powder. According to the haematococcus pluvialis exopolysaccharide prepared by the method disclosed by the invention, the accumulation of astaxanthin in haematococcus pluvialis cells can be remarkably accelerated, the yield of astaxanthin produced by large-scale commercial culture of haematococcus pluvialis is favorably improved, and the economic benefit is obviously improved.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biotechnology, and particularly relates to an extracellular polysaccharide of Haematococcus pluvialis, a preparation method thereof and an application thereof. Background Art

[0002] Astaxanthin is a carotenoid found widely in nature. As an effective colorant and strong oxidant, astaxanthin is widely used in health supplements and aquaculture. Currently, astaxanthin available on the market is primarily derived from synthetic and natural sources. With rising living standards and a growing awareness of food safety, naturally derived astaxanthin is attracting increasing attention due to its safety, stability, and high activity.

[0003] Haematococcus pluvialis, a single-celled green algae, can synthesize and accumulate large amounts of astaxanthin under adverse conditions and is considered the primary natural source of astaxanthin. However, the Haematococcus pluvialis astaxanthin industry currently faces challenges such as high production costs, low yields, and low efficiency. To meet growing market demand, rapidly and effectively increasing astaxanthin production from Haematococcus pluvialis has become a key goal for the industry.

[0004] Haematococcus pluvialis is susceptible to a variety of environmental factors during cultivation. Astaxanthin accumulation generally occurs under stress conditions that are unfavorable for its growth, while conditions that are favorable for its growth are generally unfavorable. In recent years, the combination of exogenous inducers and high light levels has been considered an effective strategy to promote astaxanthin accumulation in Haematococcus pluvialis. Therefore, finding an efficient exogenous inducer to increase astaxanthin production in Haematococcus pluvialis is of great significance for large-scale commercial cultivation of Haematococcus pluvialis. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an exopolysaccharide from Haematococcus pluvialis and a preparation method and application thereof, so as to achieve the purpose of rapidly increasing astaxanthin production and optimizing production efficiency.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for preparing extracellular polysaccharides of Haematococcus pluvialis comprises extracting the extracellular polysaccharides from an algae solution cultured in low light conditions for more than 30 days by using an alcohol precipitation method.

[0008] In the above scheme, algae liquid of Haematococcus pluvialis cultured under low light conditions for more than 30 days is collected, the algae liquid is centrifuged, and the supernatant is collected; then, anhydrous ethanol is added to the collected supernatant to make the final ethanol concentration reach 70%-80%, and then, after standing in a refrigerator at 4°C overnight, the ethanol-containing supernatant is centrifuged to collect the precipitate; 200 mL of pure water is added to the precipitate, and then the precipitate is assisted to dissolve under ultrasonic conditions; finally, centrifuged again, the light yellow exopolysaccharide solution is collected, and the Haematococcus pluvialis exopolysaccharide dry powder is obtained after freeze-drying.

[0009] The invention relates to an exopolysaccharide of Haematococcus pluvialis prepared by the preparation method described above.

[0010] In a further technical solution, guluronic acid is the monosaccharide component with the highest content in the extracellular polysaccharides of Haematococcus pluvialis.

[0011] In a further technical solution, the molecular weight of the Haematococcus pluvialis exopolysaccharide is 4.3 kDa.

[0012] A use of the above-mentioned Haematococcus pluvialis exopolysaccharide in promoting the accumulation of astaxanthin in Haematococcus pluvialis.

[0013] In a further technical solution, after the Haematococcus pluvialis cells are cultured to a plateau phase, the Haematococcus pluvialis cells are centrifuged and transferred to a new culture medium to form a Haematococcus pluvialis algae liquid, to which Haematococcus pluvialis extracellular polysaccharides are added to promote the accumulation of astaxanthin under light induction.

[0014] In a further technical solution, 110-330 mg of Haematococcus pluvialis exopolysaccharide is added to each liter of Haematococcus pluvialis algae liquid.

[0015] In a further technical solution, the new culture medium is a nitrogen-deficient culture medium.

[0016] In a further technical solution, the light intensity of the light-induced radiation is 100 to 500 μmol / m 2 / s, and the light-induced temperature was 25±2℃.

[0017] Through the above technical solution, the present invention provides an exopolysaccharide of Haematococcus pluvialis and its preparation method and application, which have the following beneficial effects:

[0018] The extracellular polysaccharide of Haematococcus pluvialis prepared by the present invention is a novel polysaccharide with a guluronic acid structure. By exogenously adding the extracellular polysaccharide of Haematococcus pluvialis during the astaxanthin accumulation process of the Haematococcus pluvialis, the astaxanthin production and the intracellular astaxanthin content of the Haematococcus pluvialis can be significantly increased under nitrogen-deficient culture conditions. The method uses a small amount of additives, has a short induction time, and has a good astaxanthin-promoting effect, which is conducive to the large-scale commercial utilization of Haematococcus pluvialis and improves the economic benefits of astaxanthin production by the Haematococcus pluvialis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0020] Figure 1 This is a photo of an exopolysaccharide sample of Haematococcus pluvialis prepared in an embodiment of the present invention;

[0021] Figure 2 This is a chromatogram of the molecular weight distribution of the exopolysaccharide of Haematococcus pluvialis prepared in an embodiment of the present invention;

[0022] Figure 3 The chromatograms of 15 monosaccharide standards are shown;

[0023] Figure 4 is a chromatogram of the exopolysaccharide of Haematococcus pluvialis prepared in an embodiment of the present invention;

[0024] Figure 5 The effects of exogenously adding exopolysaccharides of Haematococcus pluvialis at different final concentrations on the astaxanthin production of Haematococcus pluvialis in Examples 1-3 of the present invention and Comparative Examples 1-3 are shown.

[0025] Figure 6 The effects of exogenously adding different final concentrations of extracellular polysaccharides of Haematococcus pluvialis in Examples 1-3 of the present invention and Comparative Examples 1-3 on the astaxanthin content in Haematococcus pluvialis cells are shown. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.

[0027] The extraction process and structural characteristics of the exopolysaccharide of Haematococcus pluvialis according to the embodiment of the present invention are as follows:

[0028] 1. Extraction process

[0029] Collect the algae liquid of Haematococcus pluvialis that has been cultured under low light for more than 30 days, centrifuge the algae liquid, and collect the supernatant. Then, use the alcohol precipitation method to extract the extracellular polysaccharides in the supernatant. The specific operation method is: add anhydrous ethanol to the collected supernatant to make the final concentration of ethanol reach 70%-80%, then let it stand in a 4°C refrigerator overnight, centrifuge the supernatant containing ethanol, and collect the precipitate. Add 200mL of pure water to the precipitate, and then assist in dissolving it under ultrasonic conditions. Finally, centrifuge again, collect the light yellow extracellular polysaccharide solution, and obtain the extracellular polysaccharide dry powder sample of Haematococcus pluvialis after freeze-drying ( Figure 1 ).

[0030] 2. Structural features:

[0031] The method for detecting the molecular weight of Haematococcus pluvialis exopolysaccharide is as follows:

[0032] Take an appropriate amount of Haematococcus pluvialis exopolysaccharide and dissolve it in 0.05M NaCl solution to prepare a 5 mg / L Haematococcus pluvialis exopolysaccharide solution, vortex to dissolve and then centrifuge (12000rpm, 10min), aspirate the supernatant and then filter it with a 0.22μm water filter membrane into a sampling vial.

[0033] The exopolysaccharide solution of Haematococcus pluvialis was detected using a high-performance gel permeation chromatography system assembled with a differential detector RI-10A (Shimadzu, Japan). The chromatographic columns were a series of BRT 105-104-102 (8×300 mm), the mobile phase was a 0.05 M NaCl solution, the flow rate was 0.7 mL / min, the column temperature was 40°C, and the injection volume was 25 μL. A commercially available pullulan standard (Mw: 6491-883100 Da) was used for calibration. The standard curve of 1gMw-RT (weight-average molecular weight) was: y = -0.1932x + 11.5994, R 2 =0.9981, and the molecular weight of the sample can be obtained by substituting the retention time of the exopolysaccharide of Haematococcus pluvialis into the formula. The results show that the exopolysaccharide sample of Haematococcus pluvialis exhibits two characteristic peaks ( Figure 2 ), with molecular weights of 199.1 kDa and 4.3 kDa, and relative contents of 6.5% and 93.5%, respectively. The 4.3 kDa component is the main component.

[0034] The detection method of the monosaccharide composition of Haematococcus pluvialis exopolysaccharide is as follows:

[0035] Take 5mg of Haematococcus pluvialis extracellular polysaccharide sample and hydrolyze it with 3M trifluoroacetic acid at 120℃ for 3h. Prepare 15 kinds of monosaccharide standards, namely: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine, guluronic acid, mannuronic acid, and prepare standard mother liquor solutions. Then, take each monosaccharide standard solution to accurately prepare the concentration standard as a mixed standard. Use ion chromatograph (ICS5000, ThermoFisher) equipped with Dionex CarbopacTM PA20 (3*150mm) column for monosaccharide detection. The monosaccharide composition in HPEPS polysaccharide was identified according to the retention time of the standard, and the mass of each monosaccharide was determined according to the absolute quantitative method, and the molar ratio was calculated according to the molar mass of the monosaccharide. The results showed that by comparing with the monosaccharide standard ( Figure 3 ) compared, 10 monosaccharides were identified from the extracellular polysaccharides of Haematococcus pluvialis, including eight neutral sugars: fucose, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, and two uronic acids: guluronic acid, glucuronic acid ( Figure 4 Among them, guluronic acid has the highest content and is the main component, with a molar ratio of 0.374 (Table 1), indicating that the exopolysaccharide of Haematococcus pluvialis is a heteropolysaccharide rich in guluronic acid.

[0036] Table 1 Monosaccharide composition information of Haematococcus pluvialis exopolysaccharides

[0037] name Retention time (min) molar ratio Fucose 5.642 0.068 Rhamnose 10.875 0.025 arabinose 11.142 0.120 Glucosamine 12.775 0.002 Galactose 13.784 0.189 glucose 15.75 0.010 Xylose 18.284 0.108 Mannose 18.975 0.025 Guluonic acid 45.45 0.374 Glucuronic acid 48.184 0.079

[0038] The application of the exopolysaccharide extracted from Haematococcus pluvialis in the embodiment of the present invention in promoting the accumulation of astaxanthin in Haematococcus pluvialis is as follows:

[0039] The formulas of normal and nitrogen-deficient MCM culture media used in the embodiments of the present invention are as follows:

[0040] Table 2 Normal MCM culture medium formula

[0041] Element Concentration (mg / L) Element Concentration (μg / L) <![CDATA[KNO3]]> 200 <![CDATA[ZnCl2]]> 4.1 <![CDATA[KH2PO4]]> 20 <![CDATA[H3BO3]]> 61 <![CDATA[MgSO4·7H2O]]> 100 <![CDATA[CoCl2·6H2O]]> 5.1 <![CDATA[CaCl2]]> 40.5 <![CDATA[CuSO4·5H2O]]> 6.0 <![CDATA[Na2EDTA·2H2O]]> 3.36 <![CDATA[MnCl2·6H2O]]> 4.1 <![CDATA[FeCl3·6H2O]]> 2.44 <![CDATA[(NH4)6Mo7O4·4H2O]]> 38

[0042] Table 3 Nitrogen-deficient MCM culture medium formula

[0043]

[0044]

[0045] Example 1

[0046] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m 2 / s, and a light-dark ratio of 12h / 12h, and cultured to the plateau phase (after about 20 days of culture, the growth rate of the Haematococcus pluvialis cells no longer increased). The Haematococcus pluvialis cells in the plateau phase were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, exogenously added extracted Haematococcus pluvialis exopolysaccharides, so that the content of Haematococcus pluvialis exopolysaccharides in each liter of algae solution was 110 mg, at a temperature of 25°C and a light intensity of 200 μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0047] Example 2

[0048] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m 2 / s, and a light-dark ratio of 12h / 12h, and cultured to the stationary phase. The stationary phase Haematococcus pluvialis cells were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, exogenous Haematococcus pluvialis exopolysaccharide was added, so that the content of Haematococcus pluvialis exopolysaccharide per liter of algae solution was 220 mg. The culture medium was kept at a temperature of 25°C and a light intensity of 200 μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0049] On the 10th day of culture, the astaxanthin production was 22.2 mg / L ( Figure 5 ), the astaxanthin content per algal cell is 110.8 pg / cell ( Figure 6 ), which were increased by 82.2% and 156.3% respectively compared with the control group (Comparative Example 1). This shows that the exogenous addition of 220 mg / L Haematococcus pluvialis extracellular polysaccharide can promote the accumulation of astaxanthin in algal cells.

[0050] Example 3

[0051] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m 2 / s, and a light-dark ratio of 12h / 12h, and cultured to the stationary phase. The stationary phase Haematococcus pluvialis cells were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, exogenous Haematococcus pluvialis exopolysaccharide was added, so that the content of Haematococcus pluvialis exopolysaccharide per liter of algae solution was 330 mg. The culture medium was kept at a temperature of 25°C and a light intensity of 200 μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0052] Comparative Example 1

[0053] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m 2 / s, and a light-dark ratio of 12h / 12h to the stationary phase. The stationary phase Haematococcus pluvialis cells were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, the cells were cultured at a temperature of 25°C and a light intensity of 200μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0054] Comparative Example 2

[0055] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m2 / s, and a light-dark ratio of 12h / 12h, and cultured to the stationary phase. The stationary phase Haematococcus pluvialis cells were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, exogenous Haematococcus pluvialis exopolysaccharide was added, so that the content of Haematococcus pluvialis exopolysaccharide per liter of algae solution was 55 mg. The culture medium was kept at a temperature of 25°C and a light intensity of 200 μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0056] Comparative Example 3

[0057] First, the normal MCM medium (see Table 2) was used to culture the Haematococcus pluvialis cells at a temperature of 25°C and a light intensity of 20 μmol / m 2 / s, and a light-dark ratio of 12h / 12h, and cultured to the stationary phase. The stationary phase Haematococcus pluvialis cells were centrifuged and inoculated into a new nitrogen-deficient MCM medium (see Table 3) to obtain a Haematococcus pluvialis algae solution with a cell density of approximately 200,000 cells / mL. Subsequently, exogenous Haematococcus pluvialis exopolysaccharide was added, so that the content of Haematococcus pluvialis exopolysaccharide per liter of algae solution was 440 mg. The culture medium was kept at a temperature of 25°C and a light intensity of 200 μmol / m 2 / s continuous light conditions induced astaxanthin accumulation.

[0058] The effects of the exogenous addition of different final concentrations of extracellular polysaccharides from Haematococcus pluvialis in Examples 1-3 and Comparative Examples 1-3 on the astaxanthin production of Haematococcus pluvialis and the astaxanthin content in Haematococcus pluvialis cells are shown in Figure 2. Figure 5 and Figure 6 As shown. Figure 5 and Figure 6 It can be seen that when Example 1 was cultured to the 10th day, the astaxanthin production was 17.4 mg / L and the astaxanthin content per algal cell was 79.9 pg / cell, which were 42.9% and 84.9% higher than those in the control group (Comparative Example 1), respectively. This indicates that the exogenous addition of 110 mg / L of Haematococcus pluvialis exopolysaccharide can promote the accumulation of astaxanthin in algal cells. When Example 2 was cultured to the 10th day, the astaxanthin production was 22.2 mg / L and the astaxanthin content per algal cell was 110.8 pg / cell, which were 82.2% and 156.3% higher than those in the control group (Comparative Example 1), respectively. This indicates that the exogenous addition of 220 mg / L of Haematococcus pluvialis exopolysaccharide can promote the accumulation of astaxanthin in algal cells. In Example 3, on day 10 of culture, the astaxanthin production was 24.0 mg / L, and the astaxanthin content per algal cell was 121.5 pg / cell, representing increases of 98.3% and 181.2%, respectively, compared to the control group (Comparative Example 1). This indicates that exogenous addition of 330 mg / L of Haematococcus pluvialis exopolysaccharide can promote astaxanthin accumulation in algal cells.

[0059] When comparative example 1 was cultured to the 10th day, the astaxanthin production of the control group was 12.1 mg / L, and the astaxanthin content per algal cell was 43.2 pg / cell, both of which were significantly lower than those of the group treated with extracellular polysaccharides from Haematococcus pluvialis. When comparative example 2 was cultured to the 10th day, the astaxanthin production was 12.3 mg / L, and the astaxanthin content per algal cell was 45.9 pg / cell, which were not significantly different from those of the control group (comparative example 1). When comparative example 3 was cultured to the 10th day, in the Haematococcus pluvialis algae to which 440 mg / L extracellular polysaccharides from Haematococcus pluvialis were exogenously added, the astaxanthin production was 24.3 mg / L, and the astaxanthin content per algal cell was 127.1 pg / cell. Although these were 100.6% and 194.1% higher than those of the control group (comparative example 1), respectively, they were not significantly different from those of the 330 mg / L treatment group (Example 3), indicating that the promoting effect of higher concentrations on astaxanthin accumulation was limited. It can be seen that within the concentration range of 110-330 mg / L defined in the present invention, the exopolysaccharide of Haematococcus pluvialis can effectively promote the accumulation of astaxanthin. After the concentration exceeds 330 mg / L, the effect of promoting the accumulation of astaxanthin is no longer improved. When the concentration is lower than 110 mg / L, it cannot effectively promote the accumulation of astaxanthin.

[0060] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing exopolysaccharide from Haematococcus pluvialis, characterized in that: The extracellular polysaccharides from Haematococcus pluvialis cultured under low light conditions for more than 30 days were extracted using alcohol precipitation method.

2. The method for preparing exopolysaccharide from Haematococcus pluvialis according to claim 1, wherein: The algal liquid of Haematococcus pluvialis cultured under low light conditions for more than 30 days was collected, the algal liquid was centrifuged, and the supernatant was collected; anhydrous ethanol was then added to the collected supernatant to make the final ethanol concentration reach 70%-80%, and then after standing in a refrigerator at 4°C overnight, the ethanol-containing supernatant was centrifuged to collect the precipitate; 200 mL of pure water was added to the precipitate, and then the precipitate was assisted to dissolve under ultrasonic conditions; finally, the solution was centrifuged again to collect the light yellow extracellular polysaccharide solution, and the extracellular polysaccharide powder of Haematococcus pluvialis was obtained after freeze-drying.

3. An exopolysaccharide from Haematococcus pluvialis obtained by the method according to claim 1 or 2.

4. The exopolysaccharide of Haematococcus pluvialis according to claim 3, characterized in that Guluronic acid is the most abundant monosaccharide component in the extracellular polysaccharides of Haematococcus pluvialis.

5. The exopolysaccharide of Haematococcus pluvialis according to claim 3, characterized in that The molecular weight of the Haematococcus pluvialis exopolysaccharide is 4.3 kDa.

6. Use of the exopolysaccharide of Haematococcus pluvialis as claimed in claim 3 in promoting astaxanthin accumulation in Haematococcus pluvialis.

7. The use according to claim 6, characterized in that After the Haematococcus pluvialis cells reach a plateau phase, the Haematococcus pluvialis cells are centrifuged and transferred to a new culture medium to form a Haematococcus pluvialis algae liquid. Extracellular polysaccharides of Haematococcus pluvialis are added to the Haematococcus pluvialis algae liquid to promote the accumulation of astaxanthin under light induction.

8. The use according to claim 7, characterized in that 110-330 mg of Haematococcus pluvialis extracellular polysaccharide is added to each liter of Haematococcus pluvialis algae liquid.

9. The use according to claim 7, characterized in that The new culture medium is a nitrogen-deficient culture medium.

10. The use according to claim 7, characterized in that The light intensity of the light-induced 2 / s, and the light-induced temperature was 25±2℃.

Images