CN109053520B

CN109053520B - Method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa

Info

Publication number: CN109053520B
Application number: CN201811096521.6A
Authority: CN
Inventors: 徐春厚; 刘颖; 谢为天; 康恺; 葛叶
Original assignee: Guangdong Ocean University
Current assignee: Shandong Bafu Bafu Food Technology Co ltd
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2020-09-01
Anticipated expiration: 2038-09-19
Also published as: CN109053520A

Abstract

The invention discloses a method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa. The method comprises the following steps: s1, breaking the walls of dry thalli cells of the rhodotorula mucilaginosa by adopting an acid-thermal method, centrifuging, removing supernate, washing, adding a leaching solution, and extracting for 2-4 times at 45-55 ℃ to obtain a crude astaxanthin extracting solution; s2, adding a KOH alcohol solution into the astaxanthin crude extract, and saponifying for 20 min-7 h at 4-50 ℃ in the dark to obtain a saponified astaxanthin extract; and S3, loading the astaxanthin extracting solution into a silica gel column chromatography, performing gradient elution by using a mobile phase, and separating to obtain an astaxanthin finished product. The invention improves the wall breaking method, the extraction solvent, the saponification method and the extraction conditions, so that the obtained astaxanthin has high yield and high purity, is favorable for reducing the damage of the astaxanthin in the wall breaking and saponification processes while improving the separation speed, has good physiological activity and can realize industrial production.

Description

Method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa

Technical Field

The invention belongs to the technical field of extraction and separation of microbial active ingredients. More particularly relates to a method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa.

Background

Astaxanthin is a natural carotenoid ubiquitous in the natural world, has multiple effects of resisting oxidation, aging, tumors and inflammation, enhancing immunity, coloring and promoting growth, and can be used for preventing diabetes, cardiovascular diseases and neurodegenerative diseases. The substance is widely applied to industries such as food, health care products, medicines, cosmetics, feed, breeding industry and the like, and countries such as the United states and the like allow the substance to be used as food pigment in animal and fish feed, so that the substance has wide market prospect.

At present, the prawns on the worldThe demand for melanin is large. The astaxanthin is mainly obtained from two sources, one is chemical synthesis, the other is biological extraction, and raw materials of the biological extraction mainly comprise aquatic product waste, haematococcus pluvialis, phaffia rhodozyma fermentation products and the like. However, the astaxanthin content in the aquatic product waste is low, the requirement on extraction conditions is strict, the production cost is high, and the yield is low; the haematococcus pluvialis autotrophic culture period is long, the technical level requirement is high, and the production process is complex; the biosynthesis of astaxanthin in phaffia rhodozyma cells is greatly influenced by fermentation conditions, the fermentation cost is high, and the extraction yield is low. In addition, the current biological extraction method mainly comprises supercritical CO₂Extraction method, chemical solvent extraction method, etc. the crude extract obtained by biological extraction is further saponified, separated and purified. The existing separation and purification method mainly comprises a column chromatography method, wherein the column chromatography method is divided into a normal-phase column chromatography method and a reverse-phase column chromatography method according to the difference of a fixed phase, wherein the normal-phase column chromatography mainly uses silica gel as a filler of the fixed phase, and dichloromethane-n-hexane is used as a mobile phase for carrying out column chromatography; the reverse phase column chromatography mainly uses bonded silica gel C18 as a filler and methanol-water as a mobile phase to carry out column chromatography. The astaxanthin with the purity of about 80-90% can be obtained by the method.

However, due to the limitations of wall breaking, extraction and separation processes of the existing extraction and separation methods, the astaxanthin prepared by the existing extraction and separation methods has low yield and low purity (less than or equal to 90%), and some of the technologies greatly increase the cost of separation and purification due to high-pressure, reduced-pressure and low-temperature technologies, and some of the technologies reduce the activity of the astaxanthin due to the adoption of high temperature, the use of organic solvents which are easy to remain and the like.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa. The method has the advantages of simple operation, high efficiency, high yield and purity of astaxanthin, reduced damage to astaxanthin structure, good physiological activity, large-scale extraction, and easy realization of industrial production.

The invention aims to provide a method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa.

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

a method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa comprises the following steps:

s1, breaking the walls of dry thalli cells of the rhodotorula mucilaginosa by adopting an acid-thermal method, centrifuging, removing supernate, washing, adding a leaching solution, and extracting for 2-4 times at 45-55 ℃ to obtain a crude astaxanthin extracting solution; wherein the leaching liquor is an acetone solution, or a mixed solution of ethyl acetate and ethanol, or a mixed solution of acetonitrile and methanol;

s2, adding a KOH alcohol solution into the astaxanthin crude extract, and saponifying for 20 min-7 h at 4-50 ℃ in the dark to obtain a saponified astaxanthin extract;

s3, loading the astaxanthin extracting solution into a silica gel column chromatography, performing gradient elution by using a mobile phase, and separating to obtain an astaxanthin finished product; wherein the mobile phase is selected from one or more of n-hexane, dichloromethane, acetone, petroleum ether or ethyl acetate.

Preferably, in the step S3, the mobile phase is a mixed solution of n-hexane, petroleum ether and ethyl acetate, and the volume ratio of the n-hexane, the petroleum ether and the ethyl acetate is 5-7: 1-2; or the mobile phase is a mixed solution of n-hexane and acetone, and the volume ratio of the n-hexane to the acetone is 2-7: 1-3.

More preferably, the volume ratio of the n-hexane, the petroleum ether and the ethyl acetate is 7:2: 1.

More preferably, the volume ratio of n-hexane to acetone is 5:2.

Preferably, in step S1, each extraction time is 25-35 min.

More preferably, in step S1, extraction is performed 3 times at 50 ℃; the extraction time is 30 min.

Preferably, in step S1, the conditions of the acid thermal method are as follows: adding 2-4 mol/L hydrochloric acid into dry rhodotorula mucilaginosa thalli cells, uniformly mixing, standing at room temperature for 30-50 min, and breaking the walls in boiling water bath for 1-5 min.

Preferably, the feed-liquid ratio of the dry rhodotorula mucilaginosa cells to the hydrochloric acid is 0.1: 3-7 g/mL.

More preferably, the feed-liquid ratio of the dry Rhodotorula mucilaginosa cells to hydrochloric acid is 0.1:5 g/mL.

Preferably, the concentration of the hydrochloric acid is 3 mol/L.

Preferably, standing at room temperature for 40min, and breaking cell wall in boiling water bath for 3 min.

Preferably, in step S1, the feed-liquid ratio of the dry Rhodotorula mucilaginosa cells to the leaching solution is 0.1: 5-10 g/mL.

More preferably, in step S1, the feed-liquid ratio of the dry Rhodotorula mucilaginosa cells to the leaching solution is 0.1:7 g/mL.

Preferably, in step S1, the leaching solution is a mixed solution of ethyl acetate and ethanol, and the volume ratio of ethyl acetate to ethanol is 0.5-3.5: 1.

More preferably, the volume ratio of ethyl acetate to ethanol is 2: 1.

Preferably, in step S2, the KOH alcohol solution is a KOH alcohol solution; the concentration of the KOH alcoholic solution is 10-20 g/L.

More preferably, in step S2, the concentration of the KOH alcoholic solution is 20 g/L.

Preferably, in step S2, the volume ratio of the astaxanthin crude extract to the KOH alcohol solution is 1: 0.6-2.

More preferably, in step S2, the volume ratio of the astaxanthin crude extract to the KOH alcohol solution is 1: 2.

Preferably, in step S2, the saponification is performed under the dark condition at 20-40 ℃ for 30-60 min.

More preferably, in step S2, the saponification condition is to allow the saponification to stand for 30min at 40 ℃ in the dark.

Preferably, the method for extracting, separating and purifying astaxanthin in the rhodotorula mucilaginosa further comprises the following steps of: centrifuging the rhodotorula mucilaginosa fermentation liquor, removing supernatant, washing with sterilized distilled water for 2-4 times, and drying the precipitated cells to constant weight at 40-60 ℃ (preferably 50 ℃), thus obtaining the dry somatic cells of the rhodotorula mucilaginosa.

More preferably, the centrifugation speed is 2000-6000 r/min, and the centrifugation time is 10-30 min.

Most preferably, the rate of centrifugation is 4000r/min and the time of centrifugation is 20 min.

Preferably, the preparation method of the rhodotorula mucilaginosa fermentation liquor comprises the following steps: inoculating 4-6% (preferably 5%) of Rhodotorula mucilaginosa seed liquid into a Rhodotorula mucilaginosa fermentation medium, and performing shaking culture at 25-35 ℃ and 130-150 r/min to obtain the Rhodotorula mucilaginosa fermentation liquid.

Preferably, the preparation method of the rhodotorula mucilaginosa seed liquid comprises the following steps: inoculating the Rhodotorula mucilaginosa slant preservation strain into a Rhodotorula mucilaginosa seed culture medium, and carrying out shake culture at 25-35 ℃ at 130-150 r/min to obtain the Rhodotorula mucilaginosa seed liquid.

More preferably, the shaking culture condition is shaking culture at 30 ℃ and 140r/min for 48-72 h.

Preferably, the rhodotorula mucilaginosa is a mutagenized strain of rhodotorula mucilaginosa ZTHY2(rhodotorula mucolytica ZTHY 2). The ZTHY2 strain is obtained by separating from the offshore area of the Leizhou peninsula, has the advantages of short growth period, large quantity of somatic cells, simple nutritional requirement and high astaxanthin content, and is an ideal astaxanthin producing strain. The strain is preserved in China center for type culture Collection in 2015 at 5-month and 13-month, and the preservation number is CCTCC NO: m2015296, the deposit address is: wuhan, Wuhan university, China.

Preferably, the silica gel particle size of the silica gel column chromatography is 200-300 meshes.

Preferably, the method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa further comprises the step of measuring the content of astaxanthin by using a high performance liquid chromatography.

More preferably, the conditions of the high performance liquid chromatography are:

novapac18 reverse phase chromatography column (3.9 mm. times.150 mm, 4 μm);

the sample injection amount is 10 mu L;

the mobile phase is as follows: a mixed solution of a solution A and a solution B, wherein the solution A is methanol-tetrahydrofuran-acetonitrile (53:36:11), and the solution B is water;

gradient elution conditions: 0-40 min, wherein the proportion of the liquid A is 65-100 percent; 40-45 min, wherein the proportion of the liquid A is 100-65%; 45-48 min, wherein the proportion of the liquid A is 65%;

the flow rate is 1.0 mL/min;

the column temperature was 35 ℃;

the detection wavelength was 475 nm.

The concentration of free astaxanthin in the astaxanthin finished product is 96.8 percent, and the extraction yield is 2.47mg/g (dry thalli). In addition, experiments show that the method improves the separation speed, is favorable for reducing the damage of the astaxanthin in the processes of wall breaking and saponification, and the extracted astaxanthin finished product has good activity, can improve the production performance of livestock and poultry, improves the quality of eggs, and reduces the content of cholesterol in the eggs.

The invention has the following beneficial effects:

the method takes dry rhodotorula mucilaginosa thalli as a raw material, adopts an acid-heat method to break the wall, takes a mixed solution of ethyl acetate and ethanol as a leaching liquor, extracts at 45-55 ℃, saponifies for 20 min-7 h under the dark condition of 4-50 ℃, selects a proper mobile phase to carry out silica gel column chromatography, and has high purity and yield of the prepared astaxanthin, the concentration of free astaxanthin is 96.8%, and the extraction yield is 2.47mg/g (dry thalli). In addition, the method has the advantages of good wall breaking effect and saponification effect, high extraction efficiency and high separation speed, is favorable for reducing the damage of the astaxanthin in the wall breaking and saponification processes while improving the separation speed, and has high extraction rate, high purity and high activity of the astaxanthin, thereby obtaining satisfactory purification effect. Moreover, the extraction and separation method is simple, has less requirements on reaction conditions and low cost, can be popularized and used on a large scale, and lays a foundation for development and application of pure astaxanthin products.

Drawings

FIG. 1 shows the standard curve of astaxanthin.

FIG. 2 is an HPLC chromatogram of an astaxanthin standard.

FIG. 3 is an HPLC chromatogram of an astaxanthin extraction sample.

FIG. 4 is a schematic diagram of thin layer chromatography of a saponified astaxanthin concentrate, wherein A is the KOH + methanol A method; b is KOH + methanol B method; and C is KOH + ethanol method.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

In the following examples, the astaxanthin-producing strain was a mutagenized strain of Rhodotorula mucilaginosa, ZTHY2, which was deposited in the China center for type culture Collection on 5/13/2015 with a collection number of CCTCC NO: m2015296, the deposit address is: wuhan, Wuhan university, China.

The rhodotorula seed culture medium comprises: peptone 2%, yeast extract 1%, glucose 2%, NaCl 2%, and natural pH value.

The rhodotorula fermentation medium comprises: 2% of peptone, 2% of yeast extract, 1% of beef extract, 2% of glucose, 2% of NaCl, 40.025% of MnSO40 and natural pH value.

Example 1A method for preparing Rhodotorula mucilaginosa dry somatic cells

A preparation method of dry somatic cells of rhodotorula mucilaginosa comprises the following steps:

(1) inoculating the slant-preserved strain of Rhodotorula mucilaginosa ZTHY2 into Rhodotorula mucilaginosa seed culture medium, and performing shake culture at 30 deg.C and 140r/min for 48h to obtain Rhodotorula mucilaginosa seed solution;

(2) inoculating the Rhodotorula mucilaginosa seed solution to a Rhodotorula mucilaginosa fermentation culture medium according to a ratio of 5%, and performing shaking culture at 30 ℃ and 140r/min for 72h to obtain Rhodotorula mucilaginosa fermentation liquor;

(3) centrifuging the Rhodotorula mucilaginosa fermentation liquor at 4000r/min for 20min, discarding the supernatant, washing with sterilized distilled water for 2 times, and drying the precipitated cells in a 50 ℃ incubator to constant weight to obtain dry somatic cells of the Rhodotorula mucilaginosa.

Example 2 method for extracting, separating and purifying astaxanthin from Rhodotorula mucilaginosa

1. A method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa comprises the following steps:

(1) weighing 0.1g of dry rhodotorula mucilaginosa cells of example 1, adding 5mL of 3mol/L hydrochloric acid, uniformly mixing, standing at room temperature for 40min, carrying out 3min in a boiling water bath, rapidly cooling, centrifuging at 4000r/min for 10min, removing supernate, and washing with distilled water for 2 times; adding 7mL of mixed solution of ethyl acetate and ethanol (the volume ratio of ethyl acetate to ethanol is 2:1), extracting in a water bath at 50 ℃ for 30min, centrifuging at 4000r/min for 15min, and repeatedly extracting for 2 times to obtain astaxanthin crude extract;

(2) placing 5mL of astaxanthin concentrated solution into a separating funnel, adding 5mL of ethyl acetate, adding 10mL of 20g/L potassium hydroxide ethanol solution, standing for 30min at 40 ℃ in the dark, adding a proper amount of saturated saline solution until phase separation occurs in the mixed solution, standing for 3h in a refrigerator at 4 ℃ and collecting an upper colored ethyl acetate phase to obtain a saponified astaxanthin extracting solution;

(3) filling the column by a wet method, filling silica gel (200-300 meshes) into a chromatographic column with a glass piston by taking n-hexane as a column filling solvent, loading an astaxanthin extracting solution into the silica gel column for chromatography, performing gradient elution by taking a mixed solution of n-hexane and acetone (the volume ratio of the n-hexane to the acetone is 5:2) as a mobile phase, and separating to obtain an astaxanthin finished product;

(4) measuring astaxanthin content by high performance liquid chromatography; wherein, the conditions of the high performance liquid chromatography are as follows:

novapac18 reverse phase chromatography column (3.9 mm. times.150 mm, 4 μm); the sample injection amount is 10 mu L; the mobile phase is as follows: a mixed solution of a solution A and a solution B, wherein the solution A is methanol-tetrahydrofuran-acetonitrile (53:36:11), and the solution B is water; gradient elution conditions: 0-40 min, wherein the proportion of the liquid A is 65-100 percent; 40-45 min, the proportion is 100% -65%; 45-48 min, wherein the proportion of the liquid A is 65%; the flow rate is 1.0 mL/min; the column temperature was 35 ℃; the detection wavelength was 475 nm.

2. Results

(1) The standard curve for astaxanthin is shown in FIG. 1. As can be seen from FIG. 1, astaxanthin has a good linear relationship in the concentration range of 1 to 6. mu.g/mL, the first regression equation is that Y is 3.182x +0.748, and the correlation coefficient R²＝0.992。

(2) HPLC chromatogram of elution components of astaxanthin standard and astaxanthin extractive solution are shown in FIG. 2 and FIG. 3. As can be seen from fig. 2 and 3, the retention times of the astaxanthin standard and the astaxanthin extraction sample under the present test conditions were 12.013min, but the extracted sample showed two very small peaks after the peak value of astaxanthin, which are presumed to be astaxanthin monoester and astaxanthin diester; the peak area determined by HPLC combined with the standard curve one-time regression equation for astaxanthin gave a free astaxanthin concentration of 96.8% in the astaxanthin extract sample. The extraction yield of astaxanthin according to the invention was 2.47mg/g (dry cell). In addition, experiments show that the astaxanthin finished product extracted by the method has good activity, and can improve the production performance of livestock and poultry, improve the quality of eggs and reduce the content of cholesterol in the eggs.

Example 3 wall breaking method and selection and optimization of extraction recipe

1. Wall breaking method and extraction method

Respectively adopting an acid-thermal method, an ultrasonic cracking method and a dimethyl sulfoxide (DMSO) + absolute ethyl alcohol extraction method to break the wall of the rhodotorula mucilaginosa, wherein the leaching liquor of the acid-thermal method is respectively 3, and respectively comprises the following steps: acetone solution, mixed solution of ethyl acetate and ethanol (2:1), and mixed solution of acetonitrile and methanol (7.5: 2.5).

The specific steps of the 3 wall-breaking extraction methods are respectively as follows:

(1) acid-heat method: weighing 0.1g of dry thallus cells in a centrifuge tube, adding 5mL of 3mol/L hydrochloric acid, uniformly mixing, standing at room temperature for 40min, boiling in a water bath for 3min, rapidly cooling, centrifuging for 10min at 4000r/min, removing supernatant, washing with distilled water for 2 times, adding 7mL of leaching liquor, extracting in a water bath at 50 ℃ for 30min, centrifuging for 15min at 4000r/min, and repeatedly extracting for 2 times; putting the supernatant into a 20mL graduated tube, and diluting to a constant volume of 15mL by using a leaching solution to obtain an astaxanthin crude extract; the leaching liquor is respectively 3 types, which are respectively: acetone solution, mixed solution of ethyl acetate and ethanol (2:1), and mixed solution of acetonitrile and methanol (7.5: 2.5).

(2) An ultrasonic cracking method: weighing 0.1g of dry thallus cells in a centrifuge tube, adding 2mL of distilled water, uniformly mixing, repeatedly freezing and thawing (-30 ℃ for cryopreservation, and dissolving at 37 ℃) for 3 times, then cracking by using ultrasonic waves (the frequency of the ultrasonic waves is 40kHz, the processing time is 10min, and the temperature is room temperature), adding 5mL of ethyl acetate in a cracking solution, extracting for 3h in a water bath at 50 ℃, centrifuging for 15min at 4000r/min, and repeatedly extracting for 2 times; and (3) putting the supernatant into a 20mL graduated tube, and metering the volume to 15mL by using ethyl acetate to obtain the astaxanthin crude extract.

(3) Dimethyl sulfoxide (DMSO) + absolute ethanol method: weighing 0.1g of dry thallus cells in a centrifuge tube, adding 5mL of DMSO preheated to 50 ℃, uniformly mixing, placing in a 50 ℃ water bath kettle for 5min, taking out the centrifuge tube, adding 2mL of absolute ethyl alcohol, standing and extracting for 15min in a light-closed manner, centrifuging for 15min at 4000r/min, and repeatedly extracting for 2 times; putting the supernatant into a 20mL graduated tube, and using absolute ethyl alcohol to fix the volume to 15mL to obtain the astaxanthin crude extract.

2. Measurement of astaxanthin content in the crude extract:

and detecting the light absorption value of the astaxanthin crude extract at 476nm by using a visible spectrophotometer, and qualitatively reflecting the content of the astaxanthin in the rhodotorula mucilaginosa crude extract by directly using the magnitude of the light absorption value.

3. Results

Experiments find that different wall-breaking methods have great difference on the extraction effect of the astaxanthin in the rhodotorula mucilaginosa. The results of the comparison of the extraction methods for the astaxanthin from the Rhodotorula mucilaginosa are shown in Table 1. As can be seen from Table 1, the absorbance value of astaxanthin extracted by the acid-thermal method using the mixed solution of ethyl acetate and ethanol (2:1) as the leaching solution is the highest, and is significantly better than that of the other four extraction methods. It is shown that the acid thermal method using the mixed solution of ethyl acetate and ethanol (2:1) as the leaching solution is better.

TABLE 1 comparison of the extraction methods for Rhodotorula mucilaginosa astaxanthin

EXAMPLE 4 selection of saponification method

Since natural astaxanthin is mostly present in the form of esters, and the esterified form of astaxanthin is relatively low in bioavailability and difficult to purify and detect, extraction of astaxanthin from rhodotorula mucilaginosa requires saponification and hydrolysis of the astaxanthin ester in the crude extract to free astaxanthin.

1. Method for saponifying astaxanthin concentrated solution

Under the condition of keeping out of the sun, 15mL of the astaxanthin crude extract is concentrated to 5mL by using a vacuum rotary evaporator (the temperature is 35 ℃, and the vacuum degree is 0.08MPa), and the astaxanthin concentrated solution is obtained. The astaxanthin concentrated solution was saponified by the following 3 methods to obtain free astaxanthin, and the running point of thin layer chromatography was used to reflect the degree of saponification.

(1) KOH + methanol a process: putting 5mL of astaxanthin concentrated solution into a centrifuge tube, adding 3mL of 10g/L potassium hydroxide methanol solution, standing for 7h at 4 ℃ in the dark, centrifuging at 10000r/min at 4 ℃ for 15min, collecting supernatant, namely saponified astaxanthin extracting solution, and performing thin-layer chromatography identification.

(2) KOH + methanol B Process: placing 2.5mL astaxanthin concentrated solution in a graduated test tube, adding 2.5mL methanol solution of 10g/L potassium hydroxide, and oscillating at 20 deg.C in the dark for 60 min; and (3) diluting the saponified solution to 10mL with methanol, filtering with a 0.45-micrometer microporous membrane to obtain a filtrate, namely the saponified astaxanthin extracting solution, and performing thin-layer chromatography identification.

(3) KOH + ethanol method: taking 5mL of astaxanthin concentrated solution, placing the astaxanthin concentrated solution in a separating funnel, adding 5mL of ethyl acetate, adding 10mL of 20g/L potassium hydroxide ethanol solution, standing for 30min at 40 ℃ under the dark condition, adding a proper amount of saturated saline solution until phase separation occurs in the mixed solution, standing for 3h in a refrigerator at 4 ℃, collecting an upper-layer colored ethyl acetate phase, namely the saponified astaxanthin extracting solution, and carrying out thin-layer chromatography identification.

2. Results

(1) The pattern diagram of the thin layer chromatography result is shown in FIG. 4 when the developing solvent is n-hexane + petroleum ether + ethyl acetate (7:1:2) solution. The ideal specific shift value should be between 0.2 and 0.8, and as can be seen from fig. 4, the specific shift value is at the position of 0.321, the point of the hydrolysate is obviously deepened and enlarged and is the running point of the free astaxanthin; and at the position of 0.768, the point of the hydrolysate becomes obviously shallow and small, and the point is close to the maximum value of the specific shift value, and the point is judged to be the running point of the astaxanthin ester. Compared with 3 saponification methods, the KOH + ethanol method has the most free astaxanthin, the most obvious running point and the highest separation degree.

(2) The type of saponification liquid, the concentration of alkali, the saponification temperature and the saponification time all have significant influence on the saponification effect. According to the method, the ethanol solution of potassium hydroxide is used as the astaxanthin ester saponification solution, and after saponification is performed for 20 min-7 h at 4-50 ℃ in the dark, the obtained sample is good, the reaction speed is improved, the damage of astaxanthin in the saponification process is reduced, and the ratio of free astaxanthin obtained through saponification detected by high performance liquid chromatography is 35%.

Example 5 thin layer chromatography identification and selection of developing Agents

1. Method of producing a composite material

(2) The developing solvent used for thin layer chromatography is: six combined developing agents such as n-hexane + dichloromethane + acetone (5:2:2) solution, n-hexane + petroleum ether + ethyl acetate (7:2:1) solution, n-hexane + petroleum ether + ethyl acetate (7:1:2) solution, n-hexane + acetone (7:3) solution, n-hexane + acetone (5:2) solution, petroleum ether + acetone (2:1) solution and methanol + acetonitrile (3:1) solution.

(2) And comparing the specific shift value, the separation degree and the separation number to select the optimal developing agent. Calculating the formula: the shift value is b/a; degree of separation 2d/(W1+ W2); in the formula, a: distance (cm) from origin to front of developer; b: distance (cm) from origin to center of each type of spot; d: the distance difference between the centers of two adjacent spots; w1, W2: the width of two adjacent spots.

2. Results

The results of the combination of developing agents, development time, specific shift values and degrees of separation used in thin layer chromatography are shown in Table 2. As shown in Table 2, the n-hexane + petroleum ether + ethyl acetate (7:2:1) solution and the n-hexane + acetone (5:2) solution have a high degree of separation, and the n-hexane + acetone solution has a moderate specific displacement value and a desirable development time. Therefore, the present experiment used a mixed solution of n-hexane + acetone (5:2) as the mobile phase (eluent) for column chromatography. Dropping the saponified astaxanthin extract liquid onto the surface of chromatographic column silica gel, selecting the optimal developing agent (n-hexane + acetone (5:2) solution) by thin layer chromatography as eluent, and collecting different elution components.

TABLE 2 comparison of thin-layer chromatography developing reagents

The above examples 3-5 show that the best wall breaking method for dry Rhodotorula mucilaginosa cells is acid-thermal method, the extraction solvent is mixed solution of ethyl acetate and ethanol (2:1), the saponification solution is ethanol solution of KOH (20g/L KOH), and the saponification condition is 40 deg.C, 30 min; the optimum developing agent and eluent are mixed solution of n-hexane and acetone (5: 2).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for extracting, separating and purifying astaxanthin in rhodotorula mucilaginosa is characterized by comprising the following steps of:

s1, breaking the walls of dry thalli cells of the rhodotorula mucilaginosa by adopting an acid-thermal method, centrifuging, removing supernate, washing, adding a leaching solution, and extracting for 2-4 times at 45-55 ℃ to obtain a crude astaxanthin extracting solution; the leaching solution is a mixed solution of ethyl acetate and ethanol, and the volume ratio of the ethyl acetate to the ethanol is 0.5-3.5: 1;

the acid-thermal method conditions are as follows: adding 2-4 mol/L hydrochloric acid into dry rhodotorula mucilaginosa thalli cells, uniformly mixing, standing at room temperature for 30-50 min, and breaking the walls in boiling water bath for 1-5 min;

s2, adding a KOH ethanol solution into the astaxanthin crude extract, and saponifying for 20min to 7h at the temperature of 4 to 50 ℃ in the dark to obtain a saponified astaxanthin extract; the concentration of the KOH ethanol solution is 10-20 g/L; the volume ratio of the astaxanthin crude extract to the KOH ethanol solution is 1:0.6 to 2;

s3, loading the astaxanthin extracting solution into a silica gel column chromatography, performing gradient elution by using a mobile phase, and separating to obtain an astaxanthin finished product; the mobile phase is a mixed solution of n-hexane, petroleum ether and ethyl acetate, and the volume ratio of the n-hexane to the petroleum ether to the ethyl acetate is 5-7: 1-2; or the mobile phase is a mixed solution of n-hexane and acetone, and the volume ratio of the n-hexane to the acetone is 2-7: 1-3.

2. The method according to claim 1, wherein the feed-to-liquid ratio of the dry Rhodotorula mucilaginosa cells to the hydrochloric acid is 0.1: 3-7 g/mL.

3. The method according to claim 1, wherein in step S1, the feed-liquid ratio of the dry somatic cells of Rhodotorula mucilaginosa to the leaching solution is 0.1: 5-10 g/mL.

4. The method according to claim 1, wherein in step S2, the saponification is performed under dark conditions at 20-40 ℃ for 30-60 min.

5. The method according to any one of claims 1 to 4, further comprising a step of collecting the dry somatic cells of Rhodotorula mucilaginosa: centrifuging the rhodotorula mucilaginosa fermentation liquor, removing supernatant, washing with sterilized distilled water for 2-4 times, and drying the precipitated cells at 40-60 ℃ to constant weight to obtain the dry somatic cells of the rhodotorula mucilaginosa.