阅读说明：本技术 一种促进雨生红球藻内虾青素积累的方法 (Method for promoting accumulation of astaxanthin in haematococcus pluvialis ) 是由 刘建国 于文杰 张立涛 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种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后接至新的培养基中,形成雨生红球藻藻液,在进行光诱导时,向雨生红球藻藻液中添加延胡索酸钠,促进虾青素的积累。本发明所公开的方法可显著加快雨生红球藻细胞内积累虾青素、提高藻内虾青素含量,有利于增加规模化培养雨生红球藻生产虾青素的产量,提升综合经济效益。(The invention discloses a method for promoting astaxanthin accumulation in haematococcus pluvialis, which comprises the steps of culturing haematococcus pluvialis cells to a plateau period, centrifuging the haematococcus pluvialis cells, connecting the haematococcus pluvialis cells to a new culture medium to form haematococcus pluvialis algae liquid, and adding sodium fumarate into the haematococcus pluvialis algae liquid to promote the accumulation of astaxanthin during photoinduction. The method disclosed by the invention can obviously accelerate the astaxanthin accumulation in haematococcus pluvialis cells, improve the astaxanthin content in the haematococcus pluvialis cells, is beneficial to increasing the yield of astaxanthin produced by large-scale haematococcus pluvialis cultivation, and promotes the comprehensive economic benefit.)

1. A method for promoting astaxanthin accumulation in Haematococcus pluvialis is characterized in that after the Haematococcus pluvialis cells are cultured to reach a plateau period, the Haematococcus pluvialis cells are centrifuged and then are connected to a new culture medium to form Haematococcus pluvialis solution, and sodium fumarate is added into the Haematococcus pluvialis solution during photoinduction to promote the accumulation of astaxanthin.

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

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

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

5. The method of claim 4, wherein the light-induced intensity of light is 100 μmol/m²/s。

6. The method of claim 1, wherein the temperature of photoinduction is 25 ℃.

Technical Field

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

Background

Astaxanthin is a red fat-soluble carotenoid, and has wide market application prospect in the fields of aquaculture, cosmetics, foods, medical health products and the like due to extremely strong tinting strength and antioxidant activity. Astaxanthin has 3 optical isomers: the L-astaxanthin has stronger antioxidant activity and biological function than the other two configurations of astaxanthin. Haematococcus pluvialis is an organism with the highest content of astaxanthin known in nature, and accounts for about 1-5% of the dry weight of cells, and all the produced astaxanthin is of the levorotatory type, so that the Haematococcus pluvialis is regarded as a good source of natural astaxanthin. With the increasing market demand of natural astaxanthin, the large-scale cultivation of haematococcus pluvialis and the effective improvement of the astaxanthin yield are concerned by researchers at home and abroad, and become a research hotspot for astaxanthin production at present.

The haematococcus pluvialis is susceptible to various environmental factors in the culture process, the accumulation of astaxanthin generally occurs under the stress condition which is not favorable for the growth of the haematococcus pluvialis, and the culture condition which is favorable for the growth of the haematococcus pluvialis is not favorable for the accumulation of astaxanthin generally, so that the balance between the biomass and the accumulation of astaxanthin is the key for the efficient production of astaxanthin by using the haematococcus pluvialis. However, the problems of high production cost, low yield and efficiency, etc. still exist at present. Therefore, the search for economic and efficient measures to improve the yield of astaxanthin in haematococcus pluvialis is of great significance to the large-scale commercial culture of haematococcus pluvialis.

Disclosure of Invention

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

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

a method for promoting astaxanthin accumulation in Haematococcus pluvialis comprises culturing Haematococcus pluvialis cells to a plateau period, centrifuging the Haematococcus pluvialis cells, inoculating to a new culture medium to form Haematococcus pluvialis solution, and adding sodium fumarate into the Haematococcus pluvialis solution during photoinduction to promote astaxanthin accumulation.

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

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

In the scheme, the light-induced illumination intensity is 50 mu mol/m²More than s.

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

Preferably, the temperature at which light is induced is 25 ℃.

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

according to the method, sodium fumarate is added exogenously in the process of obtaining the astaxanthin in the haematococcus pluvialis, so that the content of the astaxanthin in the haematococcus pluvialis can be obviously improved under the conditions of normal culture and nitrogen deficiency culture; the additive is less in dosage, short in induction time and low in cost, and is beneficial to promoting the economic benefit of large-scale commercial astaxanthin production by utilizing haematococcus pluvialis.

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

Composition (I)	Concentration (mg/L)	Composition (I)	Concentration (μ g/L)
				KNO3	200	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

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

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a new normal MCM medium (see table 1) to form a haematococcus pluvialis solution at a cell density of 7.3 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 10mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells are cultured to the 12 th day, the content of the astaxanthin in the haematococcus pluvialis cells added with the sodium fumarate is 31.77 pg/cell, which is increased by 29.1% compared with the control group without the sodium fumarate, and the sodium fumarate can obviously promote the accumulation of the haematococcus pluvialis astaxanthin under normal culture conditions.

Example 2

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a new normal MCM medium (see table 1) to form a haematococcus pluvialis solution at a cell density of 7.3 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 20mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells are cultured to the 12 th day, the astaxanthin content of the algae cells added with the sodium fumarate is 62.34 pg/cell, and is increased by 153.3% compared with a control group without the sodium fumarate, which shows that the sodium fumarate can obviously promote the accumulation of haematococcus pluvialis astaxanthin under normal culture conditions.

Example 3

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a fresh nitrogen deficient MCM medium (see Table 2) to form a haematococcus pluvialis solution at a cell density of 7.5 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 1mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells are cultured to the 12 th day, the content of astaxanthin in the algae cells added with the sodium fumarate is 34.43 pg/cell, and is increased by 56.8% compared with a control group without the sodium fumarate, which shows that the sodium fumarate can obviously promote the accumulation of haematococcus pluvialis astaxanthin under the condition of nitrogen deficiency.

Example 4

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a fresh nitrogen deficient MCM medium (see Table 2) to form a haematococcus pluvialis solution at a cell density of 7.5 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 10mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells are cultured to the 12 th day, the content of astaxanthin in the algae cells added with the sodium fumarate is 54.28 pg/cell, and is increased by 147.2% compared with the control group without the sodium fumarate, which shows that the sodium fumarate can obviously promote the accumulation of haematococcus pluvialis astaxanthin under the condition of nitrogen deficiency.

Example 5

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. Then centrifuging the haematococcus pluvialis cellsAnd then inoculated into a new MCM medium (shown in a table 2) lacking nitrogen to form haematococcus pluvialis solution, and the cell density is 7.5 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 20mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells are cultured to the 12 th day, the content of astaxanthin in the algae cells added with the sodium fumarate is 49.07 pg/cell, and is increased by 123.5% compared with the control group without the sodium fumarate, which shows that the sodium fumarate can obviously promote the accumulation of haematococcus pluvialis astaxanthin under the condition of nitrogen deficiency.

Comparative example 1

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a new normal MCM medium (see table 1) to form a haematococcus pluvialis solution at a cell density of 7.3 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 0.1mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When cultured to day 12, there was no significant difference between the 25.35 pg/cell astaxanthin content in algal cells supplemented with 0.1mM sodium fumarate and the astaxanthin content in algal cells not supplemented with sodium fumarate.

Comparative example 2

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a new normal MCM medium (see table 1) to form a haematococcus pluvialis solution at a cell density of 7.3 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 25mM, at 25 deg.C and light intensity of 100 μmol/m²Inducing the freshwater shrimps under the conditions of/s and light-dark ratio of 12h/12hAnd (4) element accumulation.

When the cells were cultured until day 12, the astaxanthin content in the algae cells to which 25mM sodium fumarate had been added was about 64.82 pg/cell, which was not significantly different from that in the algae cells to which 20mM sodium fumarate had been added in example 2, but showed a significant inhibitory effect on cell growth.

Comparative example 3

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a fresh nitrogen deficient MCM medium (see Table 2) to form a haematococcus pluvialis solution at a cell density of 7.5 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 0.1mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When cultured to day 12, there was no significant difference between the astaxanthin content 22.75 pg/cell in algal cells supplemented with 0.1mM sodium fumarate and algal cells without sodium fumarate.

Comparative example 4

At a temperature of 25 ℃ and a light intensity of 20 [ mu ] mol/m²Culturing Haematococcus pluvialis cells with MCM medium (shown in Table 1) under the condition of 12 h/s and light-dark ratio to reach the plateau stage. The haematococcus pluvialis cells were then centrifuged and inoculated into a fresh nitrogen deficient MCM medium (see Table 2) to form a haematococcus pluvialis solution at a cell density of 7.5 ten thousand cells/mL. Then adding sodium fumarate to per liter Haematococcus pluvialis solution until the content of sodium fumarate is 25mM, at 25 deg.C and light intensity of 100 μmol/m²The astaxanthin accumulation is induced under the conditions of the light-dark ratio of 12 h/s.

When the cells were cultured until day 12, the content of astaxanthin in the algae cells to which 25mM sodium fumarate had been added was about 47.08 pg/cell, which was not significantly different from that in the algae cells to which 20mM sodium fumarate had been added in example 5, but showed a significant inhibitory effect on cell growth.

It can be seen that sodium fumarate can promote astaxanthin accumulation well within the concentration range of 1-20mM defined in the present invention, and when the concentration exceeds 20mM, the effect of promoting astaxanthin accumulation is not improved, but cell growth is inhibited, and when the concentration is less than 1mM, astaxanthin accumulation is not promoted well.

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.