阅读说明：本技术 一株产虾青素菌株及其应用 (Astaxanthin-producing strain and application thereof ) 是由 信丰学 周大伟 姜岷 章文明 蒋羽佳 于 2021-08-18 设计创作，主要内容包括：本发明涉及一株产虾青素菌株及其应用,所述产虾青素菌株分类命名为Xanthophyllomyces dendrorhous DW6,保藏号为CCTCC M2021732。本发明还提供了该产虾青素菌株在生产虾青素中的应用,所述酵母菌能够在以葡萄糖、蔗糖、果糖、阿拉伯糖等为碳源的培养基中发酵产生虾青素,具有极大的利用低劣生物质进行生产高价值产物的潜力。本发明为生物生产虾青素提供了新的高产菌株。(The invention relates to an astaxanthin-producing strain and application thereof, wherein the astaxanthin-producing strain is classified and named Xanthophyllomyces dendrorhous DW6 with preservation number of CCTCC M2021732. The invention also provides application of the astaxanthin-producing strain in astaxanthin production, the yeast can ferment and produce astaxanthin in a culture medium taking glucose, sucrose, fructose, arabinose and the like as carbon sources, and the astaxanthin-producing strain has great potential of producing high-value products by utilizing inferior biomass. The invention provides a new high-yield strain for biologically producing astaxanthin.)

1. An astaxanthin-producing strain, which is characterized in that the strain is classified and named as phaffia rhodozyma (Xanthophyllomyces dendrorhous) DW6 with the preservation number of CCTCC NO: m2021732.

2. Use of the strain of claim 1 for the production of astaxanthin.

3. The use of claim 2, wherein the strain is inoculated to YM solid culture medium for culture, after activation of the strain, single colony is inoculated to seed culture medium for second generation seed culture, and the seed solution is inoculated to fermentation culture medium for fermentation culture to obtain astaxanthin product.

4. The use according to claim 2 or 3, wherein the carbon source fermented by the strain is selected from one or more of glucose, sucrose, fructose, arabinose, glycerol, xylose, mannose, maltose, cellobiose.

5. The use according to claim 4, wherein the nitrogen source in the fermentation medium is ammonium sulfate, yeast powder and corn steep liquor powder.

6. The use according to claim 3, wherein the two generations of seeds are cultured in a manner that:

1) first-generation seed culture: taking a single colony from the YM solid culture medium, and inoculating the single colony to a seed culture medium for constant-temperature culture;

2) culturing second-generation seeds: inoculating the culture solution of the first generation seed culture to a new seed culture medium according to the inoculation amount of 10%, and culturing at constant temperature under the same conditions as the step 1) to obtain the seed solution for fermentation culture.

7. The use according to claim 6, wherein the incubation conditions are 20 ℃ to 22 ℃ at 200 rpm.

8. Use according to claim 3, wherein the temperature of the fermentation culture is 20 ℃ to 22 ℃.

9. Use according to claim 3, characterized in that the fermentation culture is carried out at 500 rpm.

10. The use according to claim 4, wherein the carbon source is supplemented to the initial concentration every day from the next day of the fermentative culture.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a strain for producing astaxanthin and application thereof.

Background

Astaxanthin, a natural carotenoid, has strong free radical scavenging activity and can protect lipid peroxidation and oxidative damage of LDL-cholesterol, cell membranes, cells and tissues. In addition, astaxanthin also has many basic biological functions including prevention of oxidation of essential polyunsaturated fatty acids and prevention of the effect of ultraviolet rays, support of good eyesight and eye health, and enhancement of immune response, and has been widely used in food additives, pigments or antioxidants and nutritional foods due to its high antioxidant activity.

Compared with other microorganisms, the yeast has the advantages of simple culture, large biomass, wide substrate utilization spectrum, strong adaptability in industrialization and the like. Since chemically synthesized astaxanthin cannot be used in humans, microorganisms capable of biosynthesizing astaxanthin have been a focus of research on astaxanthin production. Haematococcus pluvialis, phaffia rhodozyma and the like are astaxanthin-producing strains which are researched more at present, and microorganisms used for astaxanthin production are mainly Haematococcus pluvialis at present, but the Haematococcus pluvialis has a long production period and needs adverse conditions such as sufficient illumination and the like, but the phaffia rhodozyma has a short growth period and simple fermentation conditions, so the Haematococcus pluvialis is a potential strain for producing astaxanthin by a biological method. At present, higher astaxanthin yield is generally achieved by optimizing fermentation conditions or optimizing screening/mutagenesis in combination with fermentation conditions, but the breeding of astaxanthin-rich strains is still the most important research direction.

Disclosure of Invention

The invention aims to provide an astaxanthin-producing strain and application thereof.

The purpose of the invention is realized by the following technical scheme:

an astaxanthin-producing strain, which is classified and named as phaffia rhodozyma (Xanthophyllomyces dendrorhous) DW6 with the preservation number of CCTCC NO: m2021732.

Another object of the present invention is to provide the use of the above strain for the production of astaxanthin.

As a preferred embodiment, the present invention produces astaxanthin by means of microbial fermentation, comprising: inoculating the oleaginous yeast to a YM solid culture medium for culture, activating the strain, inoculating a single colony to a seed culture medium for second-generation seed culture, and inoculating a seed solution obtained by the seed culture to a fermentation culture medium for culture to obtain an astaxanthin product.

Further, the carbon source fermented by the strain is selected from one or more of glucose, sucrose, fructose, arabinose, glycerol, xylose, mannose, maltose and cellobiose.

Furthermore, the nitrogen source in the fermentation medium is ammonium sulfate, yeast powder and corn steep liquor powder.

Further, the two-generation seed culture mode is as follows:

1) first-generation seed culture: taking a single colony from the YM solid culture medium, and inoculating the single colony to a seed culture medium for constant-temperature culture;

2) culturing second-generation seeds: inoculating the culture solution of the first generation seed culture to a new seed culture medium according to the inoculation amount of 10%, and culturing at constant temperature under the same conditions as the step 1) to obtain the seed solution for fermentation culture.

Further, the constant temperature culture condition is 20-22 ℃ and the culture is carried out for 24h at 200 rpm.

Further, the temperature of fermentation culture is 20-22 ℃.

Further, the fermentation culture was carried out at 500 rpm.

Further, during the fermentation culture, the carbon source was supplemented to the initial concentration every day from the next day.

Further, astaxanthin-containing cells are obtained after the fermentation broth is centrifuged, and an astaxanthin product is obtained after separation.

There are many ways of extracting astaxanthin, and the extraction way of the invention is as follows: and (3) taking the fermentation liquor, centrifugally washing, collecting thalli, crushing the thalli, extracting, centrifugally collecting supernate, and obtaining the astaxanthin.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a phaffia rhodozyma for high yield of astaxanthin, provides a new high-yield strain for biological production of astaxanthin, the strain is fermented in a 5L fermentation tank, the astaxanthin content in the obtained product is 300mg/L, the dry cell weight reaches 36 g/L, the astaxanthin extracted by fermentation can be used for feeds, health products, cosmetics and the like, and the extracted thalli can be used as protein to be added into animal feeds, so that the quality of the feeds is improved, and the huge demand pressure of the feed production on grains is relieved.

Drawings

FIG. 1 is a schematic diagram of astaxanthin production by shake flask fermentation of the strain of example 2.

FIG. 2 is a graph showing the production of astaxanthin by the fermenter fermentation of the strain of example 3.

FIG. 3 is a schematic diagram of the fermentation production of astaxanthin by the strain of example 4 using different carbon sources.

The biological material of the present invention is classified and named as Phaffia rhodozyma (Xanthophyllomyces dendrorhous) DW6, preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 18 days in 2021, with the preservation number of CCTCC NO: m2021732, address: wuhan, Wuhan university.

Detailed Description

The present invention is described in further detail below with reference to examples, but the embodiments of the present invention are not limited thereto. Furthermore, the examples are described only to illustrate the present invention and should not be construed as limiting the invention as detailed in the claims.

The media used in the examples are as follows:

YM medium: 10g/L of glucose, 5g/L of peptone, 3g/L of yeast powder, 3g/L of malt extract and the balance of water; 20 g/L of agar powder is additionally added into the YM solid culture medium. The preparation method comprises the following steps: weighing glucose 1g, peptone 0.5 g, yeast powder 0.3 g, and malt extract 0.3 g, dissolving in distilled water 100 mL, 5M NaOH and 5M H₃PO₄The pH was adjusted to 5.5. + -. 0.2. Adding 2 g agar powder into YM solid culture medium, shaking, and sterilizing at 115 deg.C for 20 min.

Fermentation medium:

30 g/L of glucose, 1.5 g/L of ammonium sulfate, 1.5 g/L of potassium dihydrogen phosphate, 1.5 g/L of magnesium sulfate heptahydrate, 1.5 g/L of yeast powder and 10g/L of corn starch. The glucose is prepared into mother liquor and sterilized separately.

The seed culture medium and the fermentation medium are the same.

In the examples, the method for measuring the dry weight of the microbial cells and the astaxanthin content comprises the following steps:

the method for measuring the dry weight of the microbial cells comprises the following steps: taking a proper amount of fermentation liquor by using a centrifuge tube weighed in advance, centrifuging and washing twice, and then placing in an oven to dry at 65 ℃ to constant weight.

The extraction and determination method of astaxanthin comprises the following steps: measuring astaxanthin content by ultraviolet spectrophotometry.

Centrifuging 1mL of fermentation liquor at 12000rpm for 2min, washing twice with deionized water, collecting thalli, sucking water drops on the tube wall with filter paper, adding 1mL of dimethyl sulfoxide (DMSO) preheated at 55 ℃, oscillating for suspension, adding 2mL of acetone, oscillating for 20-30s with vortex, standing in the dark for 15 min, centrifuging at 12000r/m for 2min at 4 ℃, and measuring OD474 of a supernatant with a spectrophotometer. If the bacterial sludge still has color, the bacterial sludge can be repeatedly extracted until the bacterial sludge is white, and the calculation formula of the astaxanthin content is as follows:

astaxanthin (mg/L) =

In the formula: A-OD_474mmValue of

V₁-total volume of organic solvent (mL);

2150 specific extinction coefficient

V₂Volume of fermentation broth (mL);

10000: calculated amount per unit (mg/L)

Example 1

This example specifically describes the screening method of Phaffia rhodozyma strain DW 6.

The method comprises the steps of utilizing soil taken from the monarch lake of Nanjing industry university to carry out microbial resuspension, scribing on an YPD plate containing ampicillin resistance, selecting a colony with red color, scribing again, carrying out culture and separation at 20 ℃ to obtain a single colony, diluting and coating a bacterial liquid on a YM plate containing a proper amount of diphenylamine, selecting the colony with deep red color, carrying out fermentation determination to obtain a bacterial strain with high astaxanthin yield, and identifying the bacterial strain as phaffia rhodozyma (L.) (R.)Xanthophyllomyces dendrorhous) The strain is named DW 6.

Example 2

The phaffia rhodozyma strain DW6 is used for producing astaxanthin by shake-flask fermentation.

(1) Phaffia rhodozyma selected in example 1 (Xanthophyllomyces dendrorhous) DW6 is inoculated to YM solid culture medium, cultured at 20-22 deg.C for 2-3 days, and activated;

(2) seed culture, comprising:

a. first-generation seed culture: taking a single colony from the YM solid culture medium, inoculating the single colony to a seed culture medium, and culturing at a constant temperature of 20-22 ℃ and 200rpm for 48 hours;

b. culturing second-generation seeds: inoculating the culture solution of the first generation seed culture to a new seed culture medium according to the inoculation amount of 10%, and culturing at constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

(3) Inoculating a seed solution obtained by seed culture into a triangular flask filled with a fermentation medium for fermentation at 21 +/-1 ℃ at 200rpm, supplementing glucose to the concentration of 30 g/L every day from the next day of fermentation, culturing for five days, measuring the dry weight of cells, extracting astaxanthin in fermentation liquor, measuring the astaxanthin content, repeating the experiment, wherein the astaxanthin content can reach 87mg/L (the amount of astaxanthin produced in each L of fermentation liquor) after the fermentation for five days as shown in figure 1, and the yield is still increased.

Example 3

The fermentation tank of the Phaffia rhodozyma strain DW6 is used for producing astaxanthin by fermentation.

(1) Phaffia rhodozyma selected in example 1 (Xanthophyllomyces dendrorhous) DW6 is inoculated to YM solid culture medium, cultured at 20-22 deg.C for 2-3 days, and activated;

(2) seed culture, comprising:

a. first-generation seed culture: taking a single colony from the YM solid culture medium, inoculating the single colony to a seed culture medium, and culturing at a constant temperature of 20-22 ℃ and 200rpm for 48 hours;

b. culturing second-generation seeds: inoculating the culture solution of the first generation seed culture to a new seed culture medium according to the inoculation amount of 10%, and culturing at constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

(3) Inoculating the seed solution obtained by seed culture into a 5L fermentation tank filled with fermentation medium, at 21 ℃ +/-1 ℃, at 500rpm, supplementing glucose to a concentration of 30 g/L every day from the next day of fermentation, culturing for eight days, measuring the dry weight of cells, extracting astaxanthin in the fermentation broth, measuring the astaxanthin content, and repeating the experiment, wherein the astaxanthin content reaches 300mg/L (the amount of astaxanthin produced per L of fermentation broth) as shown in figure 2.

Example 4

The Phaffia rhodozyma strain DW6 utilizes different carbon sources to produce astaxanthin through fermentation.

(1) Phaffia rhodozyma selected in example 1 (Xanthophyllomyces dendrorhous) DW6 is inoculated to YM solid culture medium, cultured at 20-22 deg.C for 2-3 days, and activated;

(2) seed culture, comprising:

a. first-generation seed culture: taking a single colony from the YM solid culture medium, inoculating the single colony to a seed culture medium, and culturing at a constant temperature of 20-22 ℃ and 200rpm for 48 hours;

b. culturing second-generation seeds: inoculating the culture solution of the first generation seed culture to a new seed culture medium according to the inoculation amount of 10%, and culturing at constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

(3) Inoculating the seed solution obtained by seed culture into a 500mL shake flask fermentation tank filled with a fermentation medium, culturing for five days at 21 +/-1 ℃ and 200rpm, measuring the dry weight of cells, extracting astaxanthin from fermentation liquor, measuring the astaxanthin content, and repeating the experiment, wherein as shown in figure 3, glucose is used as a substrate, the astaxanthin content reaches 70 mg/L (the amount of astaxanthin produced in each L of fermentation liquor), and the dry weight of cells is similar to that of cells using glucose as the substrate.