阅读说明：本技术 一种快速促进雨生红球藻内虾青素积累的方法 (Method for rapidly promoting accumulation of astaxanthin in haematococcus pluvialis ) 是由 刘建国 于文杰 张立涛 于 2021-11-11 设计创作，主要内容包括：本发明公开了一种快速促进雨生红球藻内虾青素积累的方法,将雨生红球藻细胞培养达到平台期后,将雨生红球藻细胞离心并接至新的培养基中,形成雨生红球藻藻液,向雨生红球藻藻液中添加草酰乙酸钠,在光诱导下促进虾青素的积累。本发明所公开的方法可极显著加快雨生红球藻细胞内积累虾青素,大幅缩短诱导时间和培养周期,有利于提升大规模商业化培养雨生红球藻生产虾青素的产量,明显提升经济效益。(The invention discloses a method for rapidly promoting astaxanthin accumulation in haematococcus pluvialis, which comprises the steps of culturing haematococcus pluvialis cells to a plateau period, centrifuging the haematococcus pluvialis cells and connecting the haematococcus pluvialis cells to a new culture medium to form haematococcus pluvialis algae liquid, adding sodium oxaloacetate into the haematococcus pluvialis algae liquid, and promoting the accumulation of astaxanthin under light induction. The method disclosed by the invention can remarkably accelerate the astaxanthin accumulation in haematococcus pluvialis cells, greatly shorten the induction time and the culture period, is favorable for improving the yield of astaxanthin produced by large-scale commercial culture of haematococcus pluvialis, and obviously improves the economic benefit.)

1. A method for rapidly promoting astaxanthin accumulation in haematococcus pluvialis is characterized in that haematococcus pluvialis cells are centrifuged and connected to a new culture medium after the haematococcus pluvialis cells are cultured to reach a plateau period to form haematococcus pluvialis algae liquid, sodium oxaloacetate is added into the haematococcus pluvialis algae liquid, and astaxanthin accumulation is promoted under light induction.

2. The method of claim 1, wherein 1-20mM sodium oxaloacetate per liter of Haematococcus pluvialis solution is added.

3. The method of claim 1, wherein the new medium is normal or nitrogen deficient.

4. The method of claim 3, wherein the medium is an MCM medium.

5. The method of claim 1, wherein the light-induced intensity of light is 50 μmol/m²More than s.

6. The method of claim 5, wherein the light-induced intensity of light is 100 μmol/m²/s。

7. The method of claim 1, wherein the temperature of light induction is 25 ± 3 ℃.

Technical Field

The invention belongs to the technical field of microalgae biology, and particularly relates to a method for rapidly promoting the accumulation of astaxanthin in haematococcus pluvialis.

Background

Astaxanthin is a carotenoid widely found in nature. As an effective colorant and a strong oxidant, the astaxanthin has application potential in various industries such as food nutrition, medicine health care, aquaculture, cosmetics and the like. Astaxanthin applied to the market at present is mainly divided into two sources of artificial synthesis and natural astaxanthin. With the improvement of living standard, people pay more attention to the food safety problem, and astaxanthin from natural sources has more attention due to the characteristics of safety, stability, strong activity and the like. Haematococcus pluvialis is a unicellular green alga that is capable of synthesizing and accumulating large amounts of astaxanthin under stress conditions, and is considered to be the most dominant source of natural astaxanthin. Due to the strong market demand for natural astaxanthin, the rapid and efficient increase of astaxanthin production by Haematococcus pluvialis has become a hot spot of current research.

At present, the most common method for promoting the accumulation of astaxanthin in haematococcus pluvialis is to apply an inducer from an external source and combine the inducer with stress conditions such as high light, nitrogen deficiency and the like. However, although many inducers can promote the synthesis of astaxanthin in Haematococcus pluvialis, large-scale commercial application cannot be realized due to the problems of high cost, difficult availability, potential safety hazard and the like. In addition, the induction time is also a major factor affecting the efficiency and cost of astaxanthin production by Haematococcus pluvialis. Therefore, the selection of a safe and economic inducer to quickly and efficiently improve the yield of astaxanthin in haematococcus pluvialis and the application of the inducer to industrial production have very important significance for realizing the large-scale development and utilization of astaxanthin.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for rapidly promoting the accumulation of astaxanthin in haematococcus pluvialis so as to achieve the aims of rapidly improving the yield of astaxanthin and optimizing the production efficiency.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for rapidly promoting astaxanthin accumulation in Haematococcus pluvialis comprises the steps of culturing Haematococcus pluvialis cells to a plateau period, centrifuging the Haematococcus pluvialis cells and connecting the Haematococcus pluvialis cells to a new culture medium to form Haematococcus pluvialis solution, adding sodium oxaloacetate into the Haematococcus pluvialis solution, and promoting astaxanthin accumulation under light induction.

In the scheme, 1-20mM sodium oxaloacetate is added into each liter of haematococcus pluvialis solution.

In the above scheme, the new culture medium is a normal or nitrogen-deficient culture medium.

Preferably, the medium is an MCM medium.

In the above scheme, the light-induced illumination intensity is50μmol/m²More than s.

Preferably, the light-induced illumination intensity is 100 μmol/m²/s。

In the scheme, the temperature of the light induction is 25 +/-3 ℃.

Through the technical scheme, the method for rapidly promoting the accumulation of astaxanthin in haematococcus pluvialis has the following beneficial effects:

according to the invention, sodium oxaloacetate is added exogenously in the process of culturing haematococcus pluvialis, and the synthesis of an astaxanthin precursor can be promoted by improving the level of substrates (pyruvic acid and 3-glyceraldehyde phosphate) under normal culture and nitrogen deficiency culture conditions, so that the content of astaxanthin in the haematococcus pluvialis is obviously improved; the method has the advantages of small additive amount, short induction time and low cost, is beneficial to large-scale commercial utilization of haematococcus pluvialis, and improves the economic benefit of producing astaxanthin by haematococcus pluvialis.

Drawings

FIG. 1 is a graph showing the effect of exogenous addition of sodium oxaloacetate at various concentrations on astaxanthin accumulation in Haematococcus pluvialis after inoculation into fresh normal MCM medium as described in examples 1-3 and comparative examples 1-2 of the present invention.

FIG. 2 is a graph showing the effect of exogenous addition of sodium oxaloacetate at various concentrations on astaxanthin accumulation in Haematococcus pluvialis after inoculation into fresh nitrogen deficient MCM medium as described in examples 4-6 and comparative examples 3-4 of the present invention.

FIG. 3 is a graph showing the effect of exogenously added sodium oxaloacetate at a final concentration of 10mM on the relative level of pyruvate in H.pluvialis on the day three after inoculation into new normal or nitrogen deficient MCM medium as described in examples 2 and 5 and comparative examples 1 and 3 of the invention.

FIG. 4 is a graph showing the effect of exogenously added sodium oxaloacetate at a final concentration of 10mM on the relative level of glyceraldehyde-3-phosphate in H.pluvialis on the third day after inoculation into new normal or nitrogen deficient MCM medium as described in examples 2 and 5 and comparative examples 1 and 3 of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below.

The normal and nitrogen deficient MCM medium formulations used in the examples of the invention were as follows:

TABLE 1 Normal MCM Medium formulation

TABLE 2 MCM medium formulation for nitrogen deficiency

Composition (I)	Concentration (mg/L)	Composition (I)	Concentration (μ g/L)
				KCl	147.48	ZnCl2	4.1
KH2PO4	20	H3BO3	61
				MgSO4·7H2O	100	CoCl2·6H2O	5.1
CaCl2	40.5	CuSO4·5H2O	6.0
				Na2EDTA·2H2O	3.36	MnCl2·6H2O	4.1
FeCl3·6H2O	2.44	(NH4)6Mo7O4·4H2O	38

Example 1

First, Haematococcus pluvialis cells were cultured in normal MCM medium (see Table 1) at 25 ℃ under a light intensity of 20. mu. mol/m²Culturing under the condition of 12 h/s light-dark ratio until the plateau stage (the growth rate of the haematococcus pluvialis cells is not increased any more). The haematococcus pluvialis cells in the plateau phase were centrifuged and inoculated into a new normal MCM medium (see table 1) to obtain a haematococcus pluvialis solution with a cell density of 5.9 ten thousand cells/mL. Subsequently, sodium oxaloacetate was exogenously added so that the content of sodium oxaloacetate per liter of the algal solution was 1mM, and the light intensity was 100. mu. mol/m at a temperature of 25 ℃²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

Example 2

The procedure is as in example 1, except that the sodium oxaloacetate content per liter of algal liquor is 10 mM.

Example 3

The procedure is as in example 1, except that the sodium oxaloacetate content per liter of algal liquor is 20 mM.

Example 4

Firstly, haematococcus pluvialis cells are cultured in normal MCM medium at 25 ℃ and 20 mu mol/m of light intensity²Culturing under the condition of 12 h/s light-dark ratio until the plateau stage. The haematococcus pluvialis cells in the plateau phase were centrifuged and inoculated into a new nitrogen deficient MCM medium (see table 2) to obtain a haematococcus pluvialis solution with a cell density of 6.3 ten thousand cells/mL. Subsequently, sodium oxaloacetate was added so that the content of sodium oxaloacetate per liter of the algal solution was 1mM, and the light intensity was 100. mu. mol/m at a temperature of 25 ℃ and a light intensity²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

Example 5

The procedure is as in example 4, except that the sodium oxaloacetate content per liter of algal liquor is 10 mM.

Example 6

The procedure is as in example 4, except that the sodium oxaloacetate content per liter of algal liquor is 20 mM.

Comparative example 1

The procedure is as in example 1, except that sodium oxaloacetate is not added as a control.

Comparative example 2

The procedure is as in example 1, except that the sodium oxaloacetate content per liter of algal liquor is 0.1 mM.

Comparative example 3

The procedure is as in example 4, except that sodium oxaloacetate is not added as a control.

Comparative example 4

The procedure is as in example 4, except that the sodium oxaloacetate content per liter of algal liquor is 0.1 mM.

And (3) detecting the astaxanthin content:

the astaxanthin content of Haematococcus pluvialis photo-induced in normal MCM medium in examples 1-3 and comparative examples 1 and 2 was measured by UV spectrophotometry on day 3 and day 7 of the cultivation, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the amount of astaxanthin per algal cell was increased by about 1.56-fold in Haematococcus pluvialis with exogenous addition of 1mM sodium oxaloacetate as compared with that in comparative example 1 (without addition of sodium oxaloacetate) in example 1 by the time of culturing to day 7, indicating that the addition of 1mM sodium oxaloacetate in the normal MCM medium significantly promotes the accumulation of astaxanthin in Haematococcus pluvialis cells under normal culture conditions, shortening the culture time.

In the case of Haematococcus pluvialis with exogenous addition of 10mM sodium oxaloacetate, the unit cell astaxanthin content was increased by about 0.96-fold by day 3 in example 2 compared to that of comparative example 1 (no addition of sodium oxaloacetate), and by day 7, the unit cell astaxanthin content was increased by about 2.5-fold in Haematococcus pluvialis with exogenous addition of 10mM sodium oxaloacetate, indicating that the addition of 10mM sodium oxaloacetate in the normal MCM medium significantly promotes the accumulation of Haematococcus pluvialis astaxanthin under normal culture conditions, shortening the culture time.

In comparison with comparative example 1 (no addition of sodium oxaloacetate), the astaxanthin content per algal cell in Haematococcus pluvialis with exogenous addition of 20mM sodium oxaloacetate increased by about 1.8 times by day 3 in example 3, and by day 7 by about 2.3 times by day 7 in Haematococcus pluvialis with exogenous addition of 20mM sodium oxaloacetate, indicating that the addition of 20mM sodium oxaloacetate in normal MCM medium can significantly promote the accumulation of Haematococcus pluvialis astaxanthin under normal culture conditions, shortening the culture time.

There was no significant difference between the astaxanthin content in the algal cells to which 0.1mM sodium oxaloacetate was added and the astaxanthin content in the algal cells to which no sodium oxaloacetate was added in comparative example 2 by the time of cultivation to day 7, indicating that in the normal MCM medium, the accumulation of astaxanthin was not promoted when the amount of sodium oxaloacetate added was too small.

The astaxanthin content of Haematococcus pluvialis photo-induced in the MCM medium lacking nitrogen in examples 4 to 6 and comparative examples 3 and 4 was measured by an ultraviolet spectrophotometer on the 3 rd and 7 th days of the culture, respectively, and the results are shown in FIG. 2.

As can be seen from FIG. 2, in the Haematococcus pluvialis with exogenous addition of 1mM sodium oxaloacetate, the unit cell astaxanthin content was increased by about 0.93 times in example 4 by day 3 compared to comparative example 3 (without addition of sodium oxaloacetate), and in the Haematococcus pluvialis with exogenous addition of 1mM sodium oxaloacetate, the unit cell astaxanthin content was increased by about 3.7 times by day 7, indicating that the addition of 1mM sodium oxaloacetate in the MCM medium lacking nitrogen can significantly promote the accumulation of Haematococcus pluvialis astaxanthin under normal culture conditions, shortening the culture time.

In comparison with comparative example 3 (no addition of sodium oxaloacetate), the astaxanthin content per algal cell in Haematococcus pluvialis with exogenous addition of 10mM sodium oxaloacetate increased by about 4.3 times by day 3 in example 5, and by day 7 by about 7.2 times by day 7 in Haematococcus pluvialis with exogenous addition of 10mM sodium oxaloacetate, indicating that the addition of 10mM sodium oxaloacetate to the nitrogen-deficient MCM medium can significantly promote the accumulation of Haematococcus pluvialis astaxanthin under normal culture conditions, shortening the culture time.

In comparison with comparative example 3 (no addition of sodium oxaloacetate), the astaxanthin content per algal cell in Haematococcus pluvialis with exogenous addition of 20mM sodium oxaloacetate increased by about 4.4 times by day 3 in example 6, and by day 7 by about 4.0 times by day 7 in Haematococcus pluvialis with exogenous addition of 20mM sodium oxaloacetate, indicating that the addition of 20mM sodium oxaloacetate in the nitrogen-deficient MCM medium can significantly promote the accumulation of Haematococcus pluvialis astaxanthin under normal culture conditions, shortening the culture time.

At the time of culturing to day 7, there was no significant difference between the astaxanthin content in the algal cells to which comparative example 4 added 0.1mM sodium oxaloacetate and the astaxanthin content in the algal cells to which no sodium oxaloacetate was added, indicating that in the nitrogen-deficient MCM medium, too small an amount of sodium oxaloacetate added did not contribute to the accumulation of astaxanthin.

In Haematococcus pluvialis, isopentenyl pyrophosphate (IPP), a synthetic precursor of astaxanthin, is synthesized through the methylerythritol phosphate (MEP) pathway using pyruvic acid and glyceraldehyde-3-phosphate as substrates. Based on an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) platform, the relative contents of pyruvic acid and 3-phosphoglyceraldehyde in haematococcus pluvialis cells from the culture days of the examples 2 and 5 and the comparative examples 1 and 3 to the 3 rd day are detected. As can be seen from FIGS. 3 and 4, the contents of pyruvic acid and glyceraldehyde 3-phosphate in Haematococcus pluvialis to which sodium oxaloacetate was exogenously added were significantly increased compared to the control group, regardless of the normal or nitrogen-deficient culture conditions, indicating that sodium oxaloacetate significantly increased the level of a substrate required for astaxanthin synthesis in Haematococcus pluvialis, and thus, within a suitable concentration range, sodium oxaloacetate rapidly promoted astaxanthin synthesis.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.