阅读说明：本技术 一种提高虾青素产量的生产方法 (Production method for improving astaxanthin yield ) 是由 姜宗然 沈美雄 于 2019-10-31 设计创作，主要内容包括：本发明涉及一种提高虾青素产量的生产方法,该方法包括：取红法夫酵母菌发酵液于75℃恒温水浴中5h后,加入酶活浓度0.15U/mL的(1→3)-β-D-葡聚糖酶酶解0.5h,沸水浴灭活酶,离心,取上清液；向所述上清液中加入3mL 3mol/L HCl,沸水浴处理3min,冷却,离心分离洗涤两次,再添加5mL甲醇振荡提取1min,离心,取上清液,即获得虾青素。该方法可使红法夫酵母的破壁效率达90％,可提高虾青素含量。(The invention relates to a production method for improving the yield of astaxanthin, which comprises the steps of taking Phaffia rhodozyma fermentation liquor in a constant-temperature water bath at 75 ℃ for 5 hours, adding (1 → 3) - β -D-glucanase with the enzyme activity concentration of 0.15U/mL for enzymolysis for 0.5 hour, inactivating enzyme in a boiling water bath, centrifuging, taking supernate, adding 3mL of 3mol/L HCl into the supernate, treating in the boiling water bath for 3 minutes, cooling, centrifugally separating and washing twice, adding 5mL of methanol, oscillating and extracting for 1 minute, centrifuging, and taking the supernate to obtain the astaxanthin.)

1. The production method for improving the yield of astaxanthin is characterized by comprising the following steps:

(1) taking the fermentation liquor of the phaffia rhodozyma, adding (1 → 3) - β -D-glucanase with the enzyme activity concentration of 0.15U/mL for enzymolysis for 0.5h after 5h in a constant-temperature water bath at 75 ℃, inactivating the enzyme in a boiling water bath, centrifuging, and taking the supernatant;

(2) and adding 3mL of 3mol/L HCl into the supernatant, treating in a boiling water bath for 3min, cooling, performing centrifugal separation and washing twice, adding 5mL of methanol, oscillating and extracting for 1min, centrifuging, and taking the supernatant to obtain the astaxanthin.

2. The method for producing astaxanthin according to claim 1, wherein the step (1) further comprises:

preparing a culture medium, wherein the components of the culture medium comprise 30g/L of mixed carbon source and 7g/L, MgSO of mixed nitrogen source₄·7H₂O 1.5g/L、(NH₄)₃C₆H₅O₇2.0g/L、Na₂HPO₄2.0 g/L; wherein, the mixed carbon source package40% of molasses and 60% of starch sugar; the mixed nitrogen source comprises 40 percent of corn steep liquor and (NH)₄)₂SO₄60％；

Preparing Phaffia rhodozyma fermentation liquor, inoculating 1-ring Phaffia rhodozyma lawn into a 250mL triangular flask containing 30mL seed culture medium, and culturing at 20 ℃ and 160r/min for 48 h; then inoculating the shake flask with 10% inoculum size into a 250mL triangular flask containing 30mL fermentation medium, and culturing at 20 ℃ and 160r/min for 72h to obtain the fermentation liquid of the phaffia rhodozyma.

3. The method for producing astaxanthin according to claim 1, wherein the volume ratio of the fermentation broth of Phaffia rhodozyma to the (1 → 3) - β -D-glucanase in step (1) is 5: 1.

4. The method for producing astaxanthin according to claim 1, wherein the centrifugation in the step (1) is: centrifuging at 10000r/min for 10 min.

5. The method for producing astaxanthin according to claim 1, further comprising a step of HPLC analysis after the step (2), wherein the HPLC analysis is performed under the following chromatographic conditions: column temperature: 30 ℃; mobile phase: acetonitrile methanol-1: 9 (V/V); detection wavelength: 474 nm; sample introduction amount: 10 uL; flow rate: 1.0 mL/min; the chromatographic column is as follows: AgilentZORBAX-C18150X 4.6 mm.

Technical Field

The invention relates to the technical field of astaxanthin, in particular to a production method for improving the yield of astaxanthin.

Background

Astaxanthin (astaxanthin), also known as astaxanthin, is a nutritional pigment and antioxidant with great development potential, has a plurality of important biological functions of strong antioxidation, aging resistance, virus resistance, body coloration resistance, cancer resistance and the like, and has wide application prospect in the industries of food, medicine, cosmetics, aquaculture, livestock and poultry breeding, chemical industry and the like.

The production method of astaxanthin can be divided into chemical synthesis and biological synthesis. At present, the process for chemically synthesizing astaxanthin is mature and low in cost, but compared with natural astaxanthin, the chemically synthesized astaxanthin has lower biological activity. Phaffia rhodozyma is of great interest because of its remarkable astaxanthin synthesis capacity and is an important source of natural astaxanthin. In the related technology, the yield of astaxanthin is improved by breeding a high-yield Phaffia rhodozyma strain, optimizing astaxanthin fermentation conditions and optimizing an astaxanthin extraction process. However, the yield of astaxanthin obtained by culturing Phaffia yeast is not high at present, and the method is not suitable for industrialization.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the present invention aims to provide a production method for increasing the yield of astaxanthin. The method can make wall-breaking efficiency of Phaffia rhodozyma reach 90%, and can increase astaxanthin content.

To this end, in one aspect of the present invention, the present invention provides a production method for increasing the yield of astaxanthin, comprising the steps of:

(1) taking the fermentation liquor of the phaffia rhodozyma, adding (1 → 3) - β -D-glucanase with the enzyme activity concentration of 0.15U/mL for enzymolysis for 0.5h after 5h in a constant-temperature water bath at 75 ℃, inactivating the enzyme in a boiling water bath, centrifuging, and taking the supernatant;

(2) and adding 3mL of 3mol/L HCl into the supernatant, treating in a boiling water bath for 3min, cooling, performing centrifugal separation and washing twice, adding 5mL of methanol, oscillating and extracting for 1min, centrifuging, and taking the supernatant to obtain the astaxanthin.

According to the production method for improving the yield of the astaxanthin, the phaffia rhodozyma is subjected to wall breaking treatment by the (1 → 3) - β -D-glucanase, the wall breaking efficiency can reach 90%, the large-scale production of the wall-broken phaffia rhodozyma product and the astaxanthin product can be effectively realized, and the yield of the astaxanthin is improved.

In addition, the production method for improving the yield of astaxanthin according to the above embodiment of the invention may have the following additional technical features:

according to an embodiment of the present invention, the step (1) further comprises:

preparation of the culture MediumThe components comprise 30g/L of mixed carbon source and 7g/L, MgSO of mixed nitrogen source₄·7H₂O1.5g/L、(NH₄)₃C₆H₅O₇2.0g/L、Na₂HPO₄2.0 g/L; wherein the mixed carbon source comprises 40% of molasses and 60% of starch sugar; the mixed nitrogen source comprises 40 percent of corn steep liquor and (NH)₄)₂SO₄60％；

Preparing Phaffia rhodozyma fermentation liquor, inoculating 1-ring Phaffia rhodozyma lawn into a 250mL triangular flask containing 30mL seed culture medium, and culturing at 20 ℃ and 160r/min for 48 h; then inoculating the shake flask with 10% inoculum size into a 250mL triangular flask containing 30mL fermentation medium, and culturing at 20 ℃ and 160r/min for 72h to obtain the fermentation liquid of the phaffia rhodozyma.

According to the embodiment of the invention, in the step (1), the volume ratio of the phaffia rhodozyma fermentation broth to the (1 → 3) - β -D-glucanase is 5: 1.

According to an embodiment of the invention, in step (1), the centrifugation is: centrifuging at 10000r/min for 10 min.

According to the embodiment of the invention, after the step (2), the method further comprises a step of high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: column temperature: 30 ℃; mobile phase: acetonitrile methanol-1: 9 (V/V); detection wavelength: 474 nm; sample introduction amount: 10 uL; flow rate: 1.0 mL/min; the chromatographic column is as follows: agilent ZORBAX-C18150X 4.6 mm.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention provides a production method for improving the yield of astaxanthin, which comprises the following steps:

(1) taking Phaffia rhodozyma fermentation liquor in a 75 ℃ constant temperature water bath for 5h, adding (1 → 3) - β -D-glucanase with the enzyme activity concentration of 0.15U/mL for enzymolysis for 0.5h, inactivating enzyme in a boiling water bath, centrifuging, and taking supernatant, wherein the Phaffia rhodozyma fermentation liquor is prepared by the following steps of inoculating 1-ring Phaffia rhodozyma lawn into a 250mL triangular flask containing 30mL of seed culture medium, culturing for 48h at 20 ℃ and 160r/min, inoculating 10% of the inoculum size of the shake flask seed into the 250mL triangular flask containing 30mL of fermentation culture medium, and culturing for 72h at 20 ℃ and 160r/min to obtain the Phaffia rhodozyma fermentation liquor, wherein the culture medium comprises the components of 30g/L of mixed carbon source and 7g/L, MgSO g of mixed nitrogen source₄·7H₂O 1.5g/L、(NH₄)₃C₆H₅O₇2.0g/L、Na₂HPO₄2.0 g/L; wherein the mixed carbon source comprises 40% of molasses and 60% of starch sugar; the mixed nitrogen source comprises 40 percent of corn steep liquor and (NH)₄)₂SO₄60％。

(2) And adding 3mL of 3mol/L HCl into the supernatant, treating in a boiling water bath for 3min, cooling, performing centrifugal separation and washing twice, adding 5mL of methanol, oscillating and extracting for 1min, centrifuging, and taking the supernatant to obtain the astaxanthin.

According to the production method for improving the yield of the astaxanthin, the phaffia rhodozyma is subjected to wall breaking treatment by the (1 → 3) - β -D-glucanase, the wall breaking efficiency can reach 90%, the large-scale production of the wall-broken phaffia rhodozyma product and the astaxanthin product can be effectively realized, and the yield of the astaxanthin is improved.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.