CN110396478B - Sporobolomyces and method for optimizing and extracting carotenoid by using response surface method - Google Patents
Sporobolomyces and method for optimizing and extracting carotenoid by using response surface method Download PDFInfo
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Abstract
The invention relates to a spore-casting yeast and a method for optimally extracting carotenoid in the spore-casting yeast by utilizing a response surface method. The invention discloses a sporobolomyces strainSporobolomyces reseusCGMCC No. 17429. The invention utilizes Design expert8.0 software to carry out Box-Behnken Design to extract the technological conditions of the hydrochloric acid concentration, the hydrochloric acid dosage, the hydrochloric acid soaking time and the boiling water bath time of the carotenoid in the spore-casting yeast, and determines the optimal extraction technological parameters as follows: the boiling water bath time is 4.1min, the hydrochloric acid concentration is 4.5mol/L, the hydrochloric acid dosage is 3.6ml/g cell wet weight, the hydrochloric acid soaking time is 30min, and 1.0-3.0 g wet cells can be subjected to one-time acid heat treatment. Acid heat treatment can be carried out for 2 times on 3.0-4.0 g of wet cells, and higher extraction efficiency is still achieved by increasing the acid heat times after the acid heat treatment exceeds 4.0g of wet cells. The method for optimizing and extracting the carotenoid by utilizing the response surface method provided by the invention shortens the extraction time, reduces the energy consumption, and has the advantages of safety, high efficiency, stability and the like.
Description
Technical Field
The invention relates to a Sporobolomyces and a method for optimally extracting carotenoid by utilizing a response surface method, belonging to the fields of microbial fermentation and food processing.
Background
Carotenoids, represented by β -carotene, are yellow, orange-red or red polyene compounds, have biological activity of converting into vitamin a, and have various physiological functions of preventing cancer, cardiovascular diseases and the like. At present, carotenoids are recognized as a class a nutritional pigment by international organizations such as FAO and WHO, and are approved as a food additive with dual functions of nutrition and coloring in more than 50 countries and regions, and are widely used in health food and pharmaceutical and cosmetic industries. The carotenoid extracted from natural fruits and vegetables has the defects of high cost, high price, complex process, poor coloring power and the like.
The method for producing natural carotenoid by fermenting the sporogenous yeast is a problem which is researched by scholars in China more and has a better application prospect recently, and the sporogenous yeast is cultured to generate an intracellular secondary metabolite carotenoid, so that the key points for improving the yield, the quality and the production cost of the carotenoid produced by fermenting the sporogenous yeast are realized by fully crushing cells without damaging the loss of the carotenoid and completely extracting the carotenoid from the crushed cells. The currently reported methods for breaking the cell wall of the sporobolomyces are various, and the methods are commonly used in an acid-heat method, a grinding method, an enzyme digestion method, a freeze-thaw method, a dimethyl sulfoxide method and a cell autolysis method. The enzymatic method and the autolysis method can cause the reduction of carotenoid due to long wall breaking time (4-24 h); the grinding method has the limitations of low wall breaking efficiency (not higher than 80%), difficult temperature control, easy carotenoid oxidation and the like; although dimethyl sulfoxide can achieve a good wall-breaking effect, the organic solvent is very easy to generate residual toxicity, and the extracted carotenoid cannot be used as a raw material of food and medicines. The acid-heat method is the best method for breaking the cell wall of the spore-casting yeast. However, the research on the optimization of the carotenoid extraction process in the spore-casting yeast is very few, so that further research on the carotenoid extraction process is necessary.
Disclosure of Invention
The invention aims to provide a sporogenous yeast (Sporobolomyces reeus), and optimizes the extraction condition of carotenoid of the sporogenous yeast (Sporobolomyces reeus) by using a response surface analysis method on the basis of a single-factor test, thereby providing a reference basis for the comprehensive development and utilization of the sporogenous yeast.
In order to comprehensively examine the influence factors of acid-heat extraction of carotenoid in the spore-casting yeast, the technical scheme adopted by the invention for solving the technical problems is as follows:
sporobolomyces reesei (Sporobolomyces reeeus) is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 3 and 22 months, and the preservation number is CGMCC No. 17429.
Another objective of the present invention is to provide a method for optimizing and extracting carotenoids in sporophore (spoorobolomyces reeus) by using response surface method, wherein design expert8.0 software is used to perform Box-Behnken to design and extract process conditions of hydrochloric acid concentration, hydrochloric acid dosage, hydrochloric acid soaking time and boiling water bath time of carotenoids in sporophore (spoorobolomyces reeus), comprising the following steps:
1) fermentation culture of the sporobolomyces: adding fresh Sporobolomyces throw seed liquid into 30 triangular flasks of 1000ml containing 100ml of fermentation medium in an inoculation amount of about 5%, and performing shaking culture;
2) wet cell harvest: putting 3000ml of fermentation liquor into a centrifuge tube, freezing and centrifuging, pouring out supernatant, adding a little of sterile water for washing, freezing and centrifuging again, pouring out supernatant, combining a proper amount of centrifuged wet cells into another clean centrifuge tube with constant weight, inverting and drying water on the tube wall and the tube cover of the centrifuge tube until constant weight is achieved, and obtaining wet cells with constant weight;
3) acid heat crushing: transferring the constant-weight wet cells into a clean glass test tube by using a glass rod, performing acid-heat crushing on the wet cells under the conditions of 2.0-6.0mol/L hydrochloric acid concentration, 2.0-6.0ml/g hydrochloric acid dosage, 10-50min hydrochloric acid soaking time, 2-10min boiling water bath time and 1.0-5.0g wet weight of the cells, rapidly cooling, taking out the test tube, pouring cell mud into a centrifuge tube, centrifuging, discarding supernatant fluid to obtain cell fragment sediment, washing with water, centrifuging again, discarding supernatant fluid to obtain crushed cells;
4) extraction of carotenoids: adding 10ml of acetone into the crushed cells, extracting, oscillating by a vortex oscillator, centrifuging, and pouring the supernatant into another clean test tube to obtain acetone carotenoid extract;
5) quantitative determination of carotenoids: the carotenoid content was determined spectrophotometrically at 450 nm.
Further, the sporogenous yeast (Sporobolomyces reseus) in the step 1) is a wild strain, or various mutant strains, gene recombination strains or gene engineering strains.
Further, the fermentation medium in the step 1) is 20g/L of sucrose, 10g/L of peptone, 10g/L of yeast extract and pH6.0-6.5.
Further, the bacteria content of the fresh Sporobolomyces seed liquid in the step 1) is 5.0-10.0 multiplied by 108One per ml.
Further, the shaking culture temperature of the fresh Sporobolomyces in the step 1) is 12-32 ℃, the rotating speed is 180r/min, and the time is 48-72 h.
Further, the conditions of the two times of freezing and centrifugation in the step 2) are both 3000r/min and centrifugation is carried out for 15-20min at 4 ℃.
Further, the temperature of the two times of centrifugation in the step 3) is 4 ℃, the centrifugation speed is 4000r/min, and the centrifugation time is 15-20 min.
Further, the vortex time in the step 4) is 30min, the centrifugal temperature is 4 ℃, the centrifugal speed is 4000r/min, and the centrifugal time is 15-20 min.
Further, the extraction process of step 4) is performed until the wet cells have no pigment residue.
Through the technical scheme, compared with the prior art, the invention has the beneficial effects that: the method adopts a response surface method to determine variable parameters of the acid-heat extraction of the sporulation yeast carotenoid, selects four factors which have obvious influence on the extraction amount of the sporulation yeast carotenoid, namely the concentration of hydrochloric acid, the using amount of the hydrochloric acid, the soaking time of the hydrochloric acid and the boiling water bath time, and optimizes the optimal extraction process condition of the sporulation yeast carotenoid by a Plackett-Burman (PB) test-steepest climbing test-Response Surface (RSM) analysis method on the basis of a single-factor test. The method solves the defect that the orthogonal test which is frequently used in the extraction method of the carotenoid in the Sporobolomyces reeus so far can not continuously carry out comprehensive analysis on the experimental conditions of each level in the aspect of the optimized conditions, thereby causing isolation and incompleteness.
Detailed Description
The present invention will now be described in further detail with reference to specific examples.
As an effective method for optimizing process conditions, a Response Surface Method (RSM) is used for establishing a continuous variable curved Surface model, fitting the relationship between a plurality of Response variables and a series of variables through a regression equation, and investigating the influence of interaction of different test factor levels on a target Response value. The method can economically, effectively and quickly determine the optimal combination condition of multiple test factors. The research investigates the influence rule of different hydrochloric acid concentrations, hydrochloric acid dosage, hydrochloric acid soaking time and boiling water bath time on the extraction amount of the sporulation yeast carotenoid, and utilizes a response surface analysis method to obtain the optimization process of the extraction amount of the sporulation yeast carotenoid.
The invention provides a method for extracting carotenoid from Sporobolomyces reesei (Sporobolomyces reseus) by utilizing response surface optimization acid heat, which comprises the following steps:
1. fermentation culture of Sporobolomyces
Taking the bacteria content of 4.0-6.0 multiplied by 108Adding fresh Sporobolomyces sporophytes seed liquid of about 5% of inoculation amount into 30 1000ml triangular flasks containing 100ml fermentation medium (20g/L sucrose, 10g/L peptone, 10g/L yeast extract and pH6.0-6.5), and performing shaking culture at 12-32 deg.C and 180r/min for 48-72 h.
2. Wet cell harvesting
And (3) putting the 400ml fermentation liquor into a centrifuge tube, carrying out refrigerated centrifugation at 4 ℃ for 15-20min at 3000r/min, pouring out the supernatant, adding a little sterile water for washing, carrying out refrigerated centrifugation at 4 ℃ for 15-20min at 3000r/min again, pouring out the supernatant, combining a proper amount of centrifuged wet cells into another clean centrifuge tube with constant weight, inverting and drying the water on the tube wall and the tube cover of the centrifuge tube until the weight is constant, and obtaining the wet cells with constant weight.
3. Wet cell acid-Heat disruption Condition test
1) Single factor test
Taking 1.0g of wet cells with constant weight, transferring the wet cells into a clean glass test tube by using a glass rod, and carrying out single-factor tests on different hydrochloric acid concentrations (2.0, 3.0, 4.0, 5.0 and 6.0mol/L), different hydrochloric acid dosages (2.0, 3.0, 4.0, 5.0 and 6.0ml/g of wet weight of cells), different hydrochloric acid soaking times (10, 20, 30, 40 and 50min) and different boiling water bath times (2, 4, 6, 8 and 10min) respectively according to the principle that other factors are fixed on an average level and only one factor is changed. The average level of each factor specifically refers to: the concentration of hydrochloric acid is 4.0mol/L, the dosage of hydrochloric acid is 4.0ml/g of cell wet weight, the immersion time of hydrochloric acid is 30min, and the boiling water bath time is 6 min.
And (3) after acid-heat crushing, rapidly cooling, taking out the test tube, pouring the cell mud into a centrifuge tube, centrifuging for 15-20min at 4000r/min, discarding the supernatant to obtain cell fragment sediment, washing with appropriate amount of water, centrifuging for 15-20min at 4000r/min, and discarding the supernatant to obtain the crushed cells.
2) PB testing to determine major influencing factors
The method comprises the steps of taking the appropriate conditions of single-factor tests of cell wet weight, hydrochloric acid concentration, hydrochloric acid dosage, hydrochloric acid soaking time and boiling water bath time as central points, taking upper, lower, positive and negative 1-2 units as two horizontal points of a screening test design PB test, generating 12 PB test groups through response surface design software design expert8.0, completing the test according to the generated test groups and calculating the content of carotenoid. After the result is input in the software, the significance is obtained, and the single factor with the significance less than 0% is taken as the main influence factor.
3) Determination of central value in climbing test
Designing a steepest climbing test according to positive and negative effects of four main influence factors and factor estimation coefficients thereof determined by a PB test, wherein the numerical values of the positive effect influence factors are sequentially increased from a low level to a high level, the numerical values of the negative effect influence factors are sequentially decreased from the high level to the low level, measuring the extraction amount of the carotenoid content, and designing a subsequent response surface test by taking the condition corresponding to the maximum carotenoid content obtained in the climbing test as a central value.
4) Response surface test to obtain optimal condition
The method comprises the steps of taking numerical values of a plurality of significant factors determined by a finished steepest climbing test as central values of a response surface, using software design expert8.0 to carry out Box-Behnken design, taking positive and negative units before and after the central values as positive and negative levels of the response surface, generating a test group, finishing the test according to the test group generated by the software, calculating the content of carotenoid, inputting the numerical value of the content of the carotenoid into a corresponding position, and using the software to generate a corresponding response surface curved surface. And obtaining the optimal acid-heat crushing condition and a fitting equation of the wet cells through the response surface curved surface.
4. Extraction of carotenoids
Adding 10ml acetone into the crushed cells, extracting, shaking with vortex oscillator for 30min, centrifuging at 4000r/min for 15min, and pouring the supernatant into another clean test tube to obtain acetone carotenoid extract. The extraction process can be repeated as necessary until the wet cells are free of pigment residue.
5. Quantitative determination of carotenoids
Measuring the absorbance value of the properly diluted acetone extract at 450nm by using a spectrophotometer, and calculating the carotenoid content according to the following formula: X-A450 nm XRxV1/(0.16×V2)。
Wherein X is carotenoid content (mg/L), R is dilution factor, and V1Volume (ml) of acetone extract, V20.16 is the extinction coefficient, expressed in volume (ml) of the fermentation broth taken.
Example I Effect of hydrochloric acid concentration on Carotenoid extraction
Taking 5 clean sterile glass test tubes, adding 1.0g of wet cells which are fermented and cultured for 60 hours into the test tubes, respectively adding 2.0mol/L, 3.0mol/L, 4.0mol/L, 5.0mol/L and 6.0mol/L of hydrochloric acid, correspondingly adding the hydrochloric acid according to the proportion of 4ml/g of wet weight of the cells, controlling the soaking time of the hydrochloric acid to be 30min, then using a boiling water bath for 6min, finishing the test, only performing one-time extraction on the samples, and taking the average value of three parallel tests as the average content of the carotenoid. The results show that the content of carotenoids extracted showed a tendency of increasing first and then decreasing with increasing hydrochloric acid concentration, reaching a maximum of 3.61mg/L at 4.0 mol/L.
EXAMPLE II Effect of hydrochloric acid dosage on the amount of carotenoids extracted from Sporobolomyces
Taking 5 clean sterile glass test tubes, adding 1.0g of wet cells which are fermented and cultured for 60 hours into the test tubes, respectively, adding 4.0mol/L of hydrochloric acid, wherein the dosage of the hydrochloric acid is respectively added according to the proportion of 2.0ml/g of wet cell weight, 3.0ml/g of wet cell weight, 4.0ml/g of wet cell weight, 5.0ml/g of wet cell weight and 6.0ml/g of wet cell weight, controlling the soaking time of the hydrochloric acid to be 30min, then using a boiling water bath for 6min, extracting the samples once, and taking the average value of three parallel tests as the average content of the carotenoid. The results show that the content of the extracted carotenoid is in a trend of increasing firstly and then slowly decreasing with the increase of the dosage of the hydrochloric acid, and the maximum value is 3.68mg/L when the dosage of the hydrochloric acid is 3.0ml/g of the wet weight of the cells.
EXAMPLE III Effect of hydrochloric acid soaking time on Carotenoid extraction
Taking 5 clean sterile glass test tubes, adding 1.0g of wet cells which are subjected to fermentation culture for 60 hours, respectively, then adding 4.0mol/L of hydrochloric acid, wherein the amount of the hydrochloric acid is correspondingly added according to the wet weight ratio of 4.0ml/g of cells, controlling the soaking time of the hydrochloric acid to be 10min, 20min, 30min, 40min and 50min, respectively, then carrying out boiling water bath for 6min, extracting the samples once, and taking the average value of three parallel tests as the average content of the carotenoid. The result shows that the extraction amount of the extracted carotenoid content tends to increase and decrease along with the increase of the hydrochloric acid soaking time, and the maximum value is 3.40mg/L when the hydrochloric acid soaking time is 30 min.
EXAMPLE four Effect of boiling Water bath time on Carotenoid extraction
Taking 5 clean sterile glass test tubes, adding 1.0g of wet cells which are fermented and cultured for 60 hours into the test tubes, respectively adding 4.0mol/L hydrochloric acid, correspondingly adding the hydrochloric acid according to the proportion of 4.0ml/g of wet weight of the cells, controlling the soaking time of the hydrochloric acid to be 30min, respectively using boiling water bath for 2min, 4min, 6min, 8min and 10min, extracting the samples once, and taking the average value of three parallel tests as the average content of the carotenoid. The results show that the extraction amount of the extracted carotenoid content rapidly increases with the increase of the boiling water bath time, and reaches a maximum value of 3.61mg/L after 4min, and then tends to be stable.
Example five method for extracting carotenoids from Sporobolomyces reseus in response to the surface method
On the basis of the above single-factor examples, design expert8.0 software was used to perform a series of test design on the response surface, and the concentration of hydrochloric acid, the amount of hydrochloric acid used, the immersion time of hydrochloric acid and the boiling water bath time were subjected to Plackett-Burman test design and analysis with the carotenoid content as the response value Y, as shown in table 1. Wherein, the hydrochloric acid concentration, the hydrochloric acid dosage, the hydrochloric acid soaking time and the levels of "-1" and "1" during the boiling water bath time are respectively as follows: 3.0mol/L and 5.0mol/L, 2.0ml/g cell wet weight and 4.0ml/g cell wet weight, 20min and 40min, 3min and 5 min.
TABLE 1 Plackett-Burman test analysis results
As can be seen from Table 1, the single-factor time of boiling water bath, the concentration of hydrochloric acid, the amount of hydrochloric acid used and the time of hydrochloric acid soaking had a significant effect on the amount of carotenoid extracted in turn, and all of them were positive effects. The climbing test is designed according to the importance order and the effect relation, and the result shows that when the boiling water bath time is 3.5-4.5min, the hydrochloric acid concentration is 3.5-4.5mol/L, the hydrochloric acid dosage is 2.0-4.0ml/g cell wet weight and the hydrochloric acid soaking time is 20-40min, the extracted carotenoid content reaches the maximum balance and fluctuates up and down at 3.7 mg/L. These four factors were chosen as independent variables and the maximum yield area in each variable parameter was chosen to be suitable levels, where 1, 0, -1 represent high, medium and low levels of independent variable respectively. A four-factor three-level test protocol for carotenoid extraction in Sporobolomyces reesei (Sporobolomyces reeus) was designed by Box-Behnken, with the levels and codes of the test factors shown in Table 2.
TABLE 2 Box-Behnken design factors and levels
Box-Behnken test design and carotenoid extraction, protocol is shown in Table 3.
TABLE 3 Box-Behnken test design
According to the experimental results in table 3, the second polynomial regression model equation of the carotenoid content Y in the sporogenous yeast (sporogenous fungi research), the boiling water bath time a, the hydrochloric acid concentration B, the hydrochloric acid dosage C and the hydrochloric acid soaking time D is obtained by performing regression analysis on the experimental data with the carotenoid content (Y) as a response value:
Y＝4.05+0.19A+0.11B+0.29C+0.014D+0.083AB-0.058AC-0.035AD+0.12BC+0.034BD+0.012CD-0.41A2-0.27B2-0.27C2-0.10D2。
variance analysis is carried out on the regression model by using design expert8.0 software to determine the influence degree of each factor on the extraction content of carotenoid in the Sporobolomyces resis, and meanwhile, the effectiveness of the regression equation is also tested, and the result is shown in table 4.
TABLE 4 regression model and analysis of variance
As can be seen from table 4, the model selected in the experiment is significant (P is less than 0.0001), the mismatching term F is 1.59, and P is 0.3482 is greater than 0.05, which indicates that the proportion of the abnormal error in the equation obtained by the model and the actual fitting is small, indicating that the equation has good fitting condition and small error in the experiment; correction decision coefficient R of the model20.9734, the model can account for changes in 97.34% response valuesThe method has the advantages that the fitting degree of the equation is good, the predicted value and the measured value have good correlation, the test error is small, and the carotenoid content in the spore-casting yeast (Sporobolomyces reeus) can be analyzed and predicted by using the model; the mean square value of the error term of the model is small and is 1.030E-003, which indicates that the model is effective and the application of the response surface method to optimize the extraction process is feasible.
The optimization graph of the response surface analysis method is a three-dimensional graph formed by specific response values Y and corresponding factors and an equal-height graph on a two-dimensional plane, and can comprehensively and intuitively reflect the influence of each factor and the interaction among the factors on the response values. Each response surface in the optimization map was analyzed for two of the factors, the other two factors being fixed at zero level. The shape of the contour line can reflect the strength of the interaction, and the ellipse represents the obvious interaction.
From the response surface and the contour map or the coefficient of the regression equation and the equation analysis table, the factors influencing the carotenoid content in the spore-casting yeast (Sporobolomyces reeus) are mainly boiling water bath time A > hydrochloric acid concentration B > hydrochloric acid dosage C > hydrochloric acid soaking time D according to the value of the F value, and the effects are all significant levels.
EXAMPLE VI validation experiment of response surface optimization of Carotenoid extraction conditions in Sporobolomyces reeeus
In order to test the suitability and the effectiveness of the model equation, a verification test is carried out according to response surface data and according to the actually obtained optimal process conditions, and Design expert8.0 software automatically gives the optimal acid heat extraction conditions as follows: boiling water bath time 4.123min, hydrochloric acid concentration 4.484mol/L, hydrochloric acid dosage 3.645ml/g cell wet weight and hydrochloric acid soaking time 29.988min, and the carotenoid content is predicted to be the maximum under the optimal condition and is 4.166 mg/L. Taking the actually measured boiling water bath time as 4.1min, the hydrochloric acid concentration as 4.5mol/L, the hydrochloric acid dosage as 3.6ml/g cell wet weight and the hydrochloric acid soaking time as 30min, carrying out carotenoid extraction quantity measurement on 1.0-5.0g wet cells under the conditions, and taking the average content of three measurement results. The results show that when the wet weight of the cells is increased in the first extraction, the extraction amount of the carotenoid is in a trend of obvious reduction, and besides the wet weight of the cells is 1.0g and 2.0g, a small amount of pigment residues still remain in broken cells of other tubes with more wet weight after the acetone extraction, which indicates that the extraction process needs to be repeated. Therefore, after the 3.0g tube and the 4.0g tube with the pigment residue are extracted again and the 5.0g tube is extracted for three times, the broken cells are completely white, which shows that the carotenoid is completely extracted, the total carotenoid content of 1-3 times of extraction of 1.0 g-4.0 g wet cells fluctuates from 4.16mg/L to be constant, but the total carotenoid content of 3 times of extraction of 5.0g wet cells is in a descending trend. Therefore, the actually measured carotenoid content obtained under the optimized actually measured condition is 4.16mg/L and is very close to the predicted value, the fitting degree of the model is better, the analysis and prediction of the carotenoid extraction process in the sporogenous yeast (Sporobolomyces reeus) are reliable through the regression equation, and the carotenoid in the sporogenous yeast (Sporobolomyces reeus) can be extracted by utilizing the process condition.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (5)
1. A method for extracting carotenoid in Sporobolomyces is characterized in that: comprises the following steps of (a) preparing a solution,
1) fermentation culture of Sporobolomyces: adding fresh Sporobolomyces sporophore seed liquid cultured for 40-48 h into 30 1000ml triangular flasks containing 100ml fermentation medium in an inoculation amount of 5% respectively, and performing shaking culture; wherein the fermentation medium comprises 20g/L sucrose, 10g/L peptone and 10g/L yeast extract, the pH is 6.0-6.5, the temperature of shaking culture is 12-32 ℃, and the rotating speed is 180 r/min;
2) wet cell harvest: putting 3000ml of fermentation liquor into a centrifuge tube, freezing and centrifuging, pouring out supernatant, adding a little of sterile water for washing, freezing and centrifuging again, pouring out supernatant, combining a proper amount of centrifuged wet cells into another clean centrifuge tube with constant weight, inverting and drying water on the tube wall and the tube cover of the centrifuge tube until constant weight is achieved, and obtaining wet cells with constant weight;
3) acid heat crushing: taking wet cells with constant weight, transferring the wet cells into a clean glass test tube, carrying out acid-heat crushing under the conditions of 4.5mol/L hydrochloric acid concentration, 3.6ml/g cell wet weight hydrochloric acid dosage, 30min hydrochloric acid soaking time, 4.1min boiling water bath time and 1.0-4.0g cell wet weight, then rapidly cooling, taking out the test tube, pouring cell mud into a centrifuge tube, centrifuging, removing supernatant to obtain cell fragment precipitate, washing with water, centrifuging again, and removing supernatant to obtain crushed cells;
4) extraction of carotenoids: adding 10ml acetone aqueous solution into the crushed cells for extraction, oscillating with a vortex oscillator, centrifuging, pouring the supernatant into another clean test tube to obtain acetone carotenoid extract, and repeatedly extracting for 3 times;
the spore-casting yeast is preserved in the common microorganism center of China general microbiological culture Collection center in 2019, 3 and 22 months, and the preservation number is CGMCC No. 17429.
2. The method of extracting carotenoids from spore-casting yeast according to claim 1, wherein: the bacteria content of the fresh Sporobolomyces seed liquid in the step 1) is 5.0-10.0 multiplied by 108One per ml.
3. The method of extracting carotenoids from spore-casting yeast according to claim 2, wherein: the conditions of the two times of freezing and centrifuging in the step 2) are both 3000r/min and centrifuging for 15-20min at 4 ℃.
4. The method for extracting carotenoids from Sporobolomyces according to claim 3, wherein: in the step 3), the two-time centrifugation temperature is 4 ℃, the centrifugation rotating speed is 4000r/min, and the centrifugation time is 15-20 min.
5. The method of extracting carotenoids from spore-casting yeast according to claim 4, wherein: in the step 4), the vortex time is 30min, the centrifugal temperature is 4 ℃, the centrifugal speed is 4000r/min, and the centrifugal time is 15 min.
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