Method for preparing astaxanthin

Abstract

The invention discloses a method for preparing astaxanthin. The method comprises the following steps: (1) centrifuging thalli of phaffia rhodozyma, and discarding supernatant to obtain thalli; (2) adding a potassium phosphate buffer solution into the thalli, adding a compound enzyme (cellulase: pectinase = 1: 1, U / U), carrying out assisted ultrasonic treatment for 30 minutes, and carrying out enzymolysis wall breaking treatment; and (3) adding ethanol, extracting, centrifuging, and taking supernate to obtain the astaxanthin. By optimizing the culture medium, the astaxanthin yield of phaffia rhodozyma is increased. By adopting the extraction method disclosed by the invention, ultrasonic is assisted by the compound enzyme, the enzymolysis time is greatly shortened, the extraction rate of astaxanthin in phaffia rhodozyma can reach 90%, and the pollution is greatly reduced by replacing the conventionally used organic solvent acetone with the bio-based solvent ethanol.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting astaxanthin from Phaffia rhodozyma by utilizing cellulase and pectinase. Background Art

[0002] Astaxanthin is a carotenoid with strong antioxidant activity, anti-cancer ability, and significant coloring power. 95% of the global astaxanthin market is produced through chemical synthesis, but the growing preference for natural products is changing the market for natural astaxanthin, highlighting the production of microbial sources such as Phaffia rhodozyma.

[0003] Astaxanthin is an intracellular product and needs to be extracted from red Phaffia yeast through cell wall breaking and extraction steps. The methods for breaking the cell wall of red Phaffia yeast disclosed in the prior art include: mechanical method, chemical method and enzymatic method. The mechanical method is simple to operate, but the equipment is expensive. The chemical method is simple to operate but has high pollution. The enzymatic method has relatively mild treatment conditions and is more stable than other methods. It does not require special requirements for equipment and has less pollution, but generally requires a longer enzymatic hydrolysis time. The extraction method combining complex enzymes and ultrasound has a better effect. Considering the economic and social benefits, the enzymatic method is the best extraction method to achieve industrial production.

[0004] Due to the hydrophobic nature of astaxanthin, the extraction procedure is usually performed using conventional solid-liquid extraction (SLE) with acetone, which is considered harmful to the environment and unsafe for human health. In order to achieve the principles of green economy, biocompatible and low-cost solvents should be used to extract astaxanthin from Phaffia rhodozyma, such as the bio-based solvent ethanol. Summary of the invention

[0005] The present invention aims to provide a method for extracting astaxanthin from Phaffia rhodozyma by using cellulase and pectinase. The method uses composite enzyme to assist ultrasound, reduces the enzymolysis time, and the astaxanthin yield can reach 90%, and has no pollution to the environment.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for preparing astaxanthin comprises the following steps:

[0008] (1) fermenting Phaffia rhodozyma, centrifuging the fermentation liquid and discarding the supernatant to obtain bacterial cells;

[0009] (2) adding potassium phosphate buffer to the bacterial cells, and adding a complex enzyme composed of cellulase and pectinase, assisted by ultrasound, to perform enzymatic cell wall breaking treatment;

[0010] (3) Add low-grade alcohol for extraction, centrifuge, and take the supernatant to obtain astaxanthin.

[0011] As a preferred embodiment of the present invention, the fermentation medium for fermenting Phaffia rhodozyma is 20-30 g / L sucrose, 3-5 g / L yeast extract, 1-2 g / L magnesium sulfate, 0.5-1 g / L sodium chloride and 1-3 g / L potassium dihydrogen phosphate.

[0012] As a preferred embodiment of the present invention, the fermentation medium for fermenting Phaffia rhodozyma is 25 g / L sucrose, 3.5 g / L yeast extract, 1.5 g / L magnesium sulfate, 1 g / L sodium chloride and 1.5 g / L potassium dihydrogen phosphate.

[0013] As a preferred embodiment of the present invention, the fermentation conditions are 20-23°C, 170-220rpm, and the liquid volume is 15%-25%; the preferred fermentation conditions are 21°C, 180rpm, and the liquid volume is 20%.

[0014] As a preferred embodiment of the present invention, based on the initial fermentation liquid volume, the added amount of the complex enzyme is 1500-3500 U / mL.

[0015] As a preferred embodiment of the present invention, the ratio of cellulase to pectinase is 1:0.5-2 (U:U).

[0016] As a preferred embodiment of the present invention, the enzymatic hydrolysis time is 4 to 6 hours, the enzymatic hydrolysis temperature is 45 to 55° C., the enzymatic hydrolysis pH is 4.0 to 6.0, and the assisted ultrasound is 20 to 40 minutes.

[0017] As a preferred embodiment of the present invention, the lower alcohol is ethanol.

[0018] Beneficial effects:

[0019] (1) The present invention firstly improves the astaxanthin production of Phaffia rhodozyma by optimizing the culture medium;

[0020] (2) The present invention innovatively uses cellulase and pectinase to assist ultrasound, which reduces the enzymatic hydrolysis time, and the cell wall breaking conditions are mild and complete, which greatly improves the astaxanthin extraction rate, and the astaxanthin yield can reach 90%;

[0021] (3) The present invention uses the bio-based solvent ethanol instead of the conventionally used organic solvent acetone, which greatly reduces pollution;

[0022] (4) The present invention does not require large-scale equipment, is simple and convenient to operate, and does not pollute the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Embodiment 8 Technical route

[0024] Figure 2 Enzymatic hydrolysis process optimization results

[0025] Figure 3 Electron micrograph of yeast

[0026] A is an electron micrograph of intact yeast, B is an electron micrograph of yeast broken by enzymatic hydrolysis, and C is an electron micrograph of yeast after ethanol extraction. DETAILED DESCRIPTION

[0027] The present invention provides a method for extracting astaxanthin from Phaffia rhodozyma by using cellulase and pectinase, comprising the following steps:

[0028] (1) taking a fermentation broth of Phaffia rhodozyma, centrifuging and discarding the supernatant to obtain bacterial cells;

[0029] (2) Potassium phosphate buffer was added to the bacterial cells, and compound enzyme (cellulase: pectinase = 1:1, U / U) was added, and ultrasound was used for 30 min to perform enzymatic cell wall breaking treatment;

[0030] (3) Add ethanol to extract, centrifuge, and take the supernatant to obtain astaxanthin.

[0031] In the present invention, the fermentation culture conditions are as follows: the culture medium components include 25 g / L sucrose, 3.5 g / L yeast extract, 4 g / L sodium citrate, 1.5 g / L magnesium sulfate, 1 g / L sodium chloride and 1.5 g / L potassium dihydrogen phosphate; 21°C, 180 rpm, and a liquid volume of 20%.

[0032] In the present invention, the centrifugal speed is 4000 r / min, and the centrifugal time is 2 to 5 min, preferably 3 min.

[0033] In the present invention, the process of preparing potassium phosphate buffers of different pH values ​​is as follows: preparing 0.1M potassium phosphate buffer and adjusting the pH to 4.0-6.0.

[0034] In the present invention, during the enzymatic hydrolysis, potassium phosphate buffers of different pH values ​​are added to adjust the enzymatic hydrolysis pH, and the pH value is selected to be 4.0 to 6.0, preferably 4.0, 4.5, 5.0, 5.5, and 6.0. Considering the influence of acid and alkali on the stability of astaxanthin and the enzymatic hydrolysis effect, the pH value is further preferably 5.0.

[0035] In the present invention, the enzymatic hydrolysis temperature is 45 to 55°C, preferably 45°C, 50°C, 55°C, and considering the cost and enzymatic hydrolysis effect, 50°C is more preferred;

[0036] In the present invention, a composite enzyme (cellulase: pectinase = 1:1, U / U) is added, and the enzyme addition amount is 1500-3500U / mL, preferably 1500U / mL, 2000U / mL, 2500U / mL, 3000U / mL, 3500U / mL, and considering the cost and enzymolysis effect, 2500U / mL is further preferred;

[0037] In the present invention, the enzymatic hydrolysis time is 3 to 6 hours, preferably 3 hours, 4 hours, 5 hours, and 6 hours. Considering the time cost and the enzymatic hydrolysis effect, 4 hours is further preferred, with 30 minutes of assisted ultrasound.

[0038] In the present invention, the volume ratio of ethanol to the initial bacterial solution is 3 to 6:1, preferably 3:1, 4:1, 5:1, 6:1, and considering the cost and extraction effect, 5:1 is further preferred;

[0039] In the present invention, the ethanol extraction time is 10-50 min, preferably 10 min, 20 min, 30 min, 40 min, 50 min. Considering the cost and extraction effect, 30 min is more preferred.

[0040] In the following examples, the astaxanthin content was determined by high performance liquid chromatography, and the chromatographic conditions were as follows: mobile phase: phase A: methanol: water (1:9); phase B: acetonitrile: methanol: dichloromethane (21:21:8); flow rate: 0.8 mL / min; column temperature: 25°C; injection volume: 10 μL; detection wavelength: 475 nm; chromatographic column: Agilent C18 chromatographic column (4.6*2505 um).

[0041] The strain used in the examples and comparative examples of the present invention is Phaffia rhodozyma, numbered ATCC 74220, purchased from Guangdong Microbial Culture Collection Center.

[0042] Example 1

[0043] The fermentation medium was prepared as follows: 25 g / L sucrose, 3.5 g / L yeast extract, 4 g / L sodium citrate, 1.5 g / L magnesium sulfate, 1 g / L sodium chloride and 1.5 g / L potassium dihydrogen phosphate. Phaffia erythrorhizome ATCC 74220 was inoculated into the fermentation medium at a 5% inoculation rate. The fermentation conditions were 21°C, 180 rpm, 20% liquid volume, and the fermentation culture was carried out for 120 hours to obtain the fermentation liquid. Compared with the traditional YM medium, the astaxanthin yield was increased by 167%.

[0044] Example 2

[0045] 10 mL of the fermentation broth of Phaffia erythrorhiza ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, the enzymatic hydrolysis temperature was 45°C, the enzymatic hydrolysis time was 5 h, the enzyme addition amount was 1500 U / mL (based on the volume of the fermentation broth), and the ultrasound was assisted for 30 min. 10 mL of potassium phosphate buffer of different pH values ​​was added to adjust the effect of enzymatic hydrolysis pH 4.0, 4.5, 5.0, 5.5, and 6.0 on the extraction rate of astaxanthin from Phaffia erythrorhiza. The volume ratio of ethanol added to the initial bacterial solution was 4: 1, and the ethanol extraction time was 20 min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 81.2%, 75.7%, 80.7%, 78.2%, and 74.9%, respectively. Considering the influence of acid and alkali on the stability of astaxanthin and the enzymatic hydrolysis effect, pH 5.0 was selected for enzymatic hydrolysis.

[0046] Example 3

[0047] 10 mL of the fermentation broth of Phaffia erythrorhiza ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH of 5.0, the enzymatic hydrolysis time was 5 h, the enzyme addition amount was 1500 U / mL, and ultrasound was assisted for 30 min. The effects of enzymatic hydrolysis temperatures of 45°C, 50°C, and 55°C on the astaxanthin extraction rate of Phaffia erythrorhiza were determined respectively. The volume ratio of ethanol added to the initial bacterial solution was 4: 1, and the ethanol extraction time was 20 min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 79.8%, 82.9%, and 73.9%, respectively. Considering the cost and enzymatic hydrolysis effect, 50°C was selected for enzymatic hydrolysis.

[0048] Example 4

[0049] 10 mL of the fermentation broth of Phaffia erythrorhiza ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, and 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH of 5.0, the enzymatic temperature was 50°C, the enzymatic time was 5 h, and the assisted ultrasound was 30 min. The effects of enzyme addition amounts of 1500 U / mL, 2000 U / mL, 2500 U / mL, 3000 U / mL, and 3500 U / mL on the astaxanthin extraction rate of Phaffia erythrorhiza were determined respectively. The volume ratio of added ethanol to the initial bacterial solution was 4:1, and the ethanol extraction time was 20 min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 83.3%, 87.9%, 89.6%, 88.6%, and 85.9%, respectively. Considering the cost and enzymatic effect, 2500 U / mL was selected as the composite enzyme addition amount for enzymatic hydrolysis.

[0050] Example 5

[0051] 10 mL of red Phaffia fermentation broth was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1:1 (U / U) was selected, with an enzyme dosage of 2500 U / mL, and 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH at 5.0, the enzymatic temperature was 50°C, and ultrasound was used for 30 min. The effects of enzymatic hydrolysis time of 3 h, 4 h, 5 h, and 6 h on the astaxanthin extraction rate of red Phaffia were determined. The volume ratio of ethanol added to the initial bacterial solution was 4:1, and the ethanol extraction time was 20 min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 83.7%, 89.8%, 90.6%, and 91.7%, respectively. Considering the time cost and enzymatic hydrolysis effect, 4 h was selected as the enzymatic hydrolysis time.

[0052] Example 6

[0053] 10 mL of Phaffia rhodozyma fermentation broth was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1:1 (U / U) was selected, with an enzyme dosage of 2500 U / mL, and 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH at 5.0, the enzymatic temperature was 50 °C, the enzymatic time was 4 h, and the assisted ultrasound was 30 min. The volume ratio of ethanol added to the initial bacterial solution was 3:1, 4:1, 5:1, and 6:1, and the ethanol extraction time was 20 min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 85.7%, 89.5%, 90.6%, and 90.3%, respectively. Considering the cost and extraction effect, 5:1 was selected as the optimal volume ratio of ethanol to the initial bacterial solution.

[0054] Example 7

[0055] 10 mL of the fermentation broth of Phaffia erythrorhizome ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, the enzyme amount was 2500U / mL, 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH at 5.0, the enzymatic temperature was 50°C, the enzymatic time was 4h, and the assisted ultrasound was 30min. The volume ratio of ethanol to the initial bacterial solution was 5:1, 10min, 20min, 30min, 40min, and 50min. Three replicates were set for each treatment group. The astaxanthin extraction rates were calculated to be 83.6%, 90.1%, 91.3%, 90.5%, and 88.3%, respectively. Considering the cost and extraction effect, 30min was selected as the optimal extraction time.

[0056] Example 8

[0057] 10 mL of the fermentation broth of Phaffia erythrorhizome ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, and the enzyme amount was 2500 U / mL. 10 mL of pH 5.0 potassium phosphate buffer was added to fix the enzymatic pH at 5.0, the enzymatic temperature was 50°C, the enzymatic time was 4 h, and the assisted ultrasound was 30 min. The volume ratio of ethanol added to the initial bacterial solution was 5: 1, and the extraction time was 30 min. Three replicates were set for each treatment group. The astaxanthin extraction rate was calculated to be 91.2%.

[0058] Example 9

[0059] 10 mL of the fermentation broth of Phaffia erythrorhizome ATCC 74220 prepared in Example 1 was centrifuged to obtain wet cells, and a composite enzyme (cellulase: pectinase = 1: 1 (U / U) was selected, and the enzyme amount was 1500 U / mL. 10 mL of pH 6.0 potassium phosphate buffer was added to fix the enzymatic pH at 6.0, the enzymatic temperature was 45°C, the enzymatic time was 4h, and the assisted ultrasound was 30min. The volume ratio of ethanol added to the initial bacterial liquid was 3: 1, and the extraction time was 10min. Three replicates were set for each treatment group. The astaxanthin extraction rate was calculated to be 75.3%.

[0060] Comparative Example 1

[0061] According to the method of Example 1, the experiment of comparative example 1 was set up, and the comparative example used a single enzyme cellulase. Take 10mL of red Phaffia fermentation broth and centrifuge to obtain wet bacteria, add cellulase, add enzyme amount 5000U / mL, add 10mL pH5.5 potassium phosphate buffer to fix the enzymatic pH 5.5, enzymatic temperature 50°C, enzymatic time 4h, auxiliary ultrasound 30min. The volume ratio of ethanol added to the initial bacterial liquid is 5:1, and the extraction time is 30min. Each treatment group is set up with 3 replicates. The astaxanthin extraction rate is calculated to be 60.9%.

[0062] Comparative Example 2

[0063] According to the method of Example 1, the experiment of Comparative Example 2 was set up, and the comparative example used a single enzyme pectinase. Take 10mL of red Phaffia fermentation broth and centrifuge to obtain wet bacteria, add cellulase, add enzyme amount 5000U / mL, add 10mL pH5.0 potassium phosphate buffer to fix the enzymatic pH 5.0, enzymatic temperature 50°C, enzymatic time 4h, auxiliary ultrasound 30min. The volume ratio of ethanol added to the initial bacterial liquid is 5:1, and the extraction time is 30min. Each treatment group is set up with 3 replicates. The astaxanthin extraction rate is calculated to be 67.7%.

[0064] Comparative Example 3

[0065] The experiment of comparative example 3 was set up according to the method of Example 8, and the comparative example did not use ultrasound assistance. Take 10mL of red Phaffia fermentation broth and centrifuge to obtain wet bacteria, select composite enzyme (cellulase: pectinase = 1: 1 (U / U), add 2500U / mL of enzyme, add 10mL pH5.0 potassium phosphate buffer to fix the enzymatic pH 5.0, enzymatic temperature 50°C, enzymatic time 4h, no ultrasound. The volume ratio of ethanol added to the initial bacterial liquid is 5: 1, and the extraction time is 30min. Each treatment group is set up with 3 replicates. The astaxanthin extraction rate is calculated to be 83.7%.

[0066] As can be seen from the above embodiments, the present invention provides a method for extracting astaxanthin from red Phaffia yeast using cellulase and pectinase. The method of the present invention is used to extract astaxanthin from red Phaffia yeast. The enzymatic treatment conditions are relatively mild, more stable than other methods, no special requirements for equipment are required, and the pollution is small. However, generally a single enzyme requires a long enzymatic hydrolysis time, and the astaxanthin yield is also less than 70%. The composite enzyme combined with ultrasonic extraction method is used to reduce the enzymatic hydrolysis time. A good extraction effect can be obtained in 4 hours, and the astaxanthin yield can reach more than 90%. Instead of traditional organic solvents such as acetone to extract astaxanthin, the bio-based solvent ethanol is used for extraction, which greatly reduces pollution.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing astaxanthin, characterized in that: The steps include: (1) fermenting Phaffia rhodozyma, centrifuging the fermentation liquid and discarding the supernatant to obtain bacterial cells; (2) adding potassium phosphate buffer to the bacterial cells, and adding a complex enzyme composed of cellulase and pectinase, assisted by ultrasound, to perform enzymatic cell wall breaking treatment; (3) Add low-grade alcohol for extraction, centrifuge, and take the supernatant to obtain astaxanthin.

2. The method according to claim 1, characterized in that: The fermentation medium for fermenting red Phaffia yeast is 20-30 g / L sucrose, 3-5 g / L yeast extract, 1-2 g / L magnesium sulfate, 0.5-1 g / L sodium chloride and 1-3 g / L potassium dihydrogen phosphate.

3. The method according to claim 2, characterized in that The fermentation medium for fermenting Phaffia rhodozyma was 25 g / L sucrose, 3.5 g / L yeast extract, 1.5 g / L magnesium sulfate, 1 g / L sodium chloride and 1.5 g / L potassium dihydrogen phosphate.

4. The method according to claim 1, characterized in that: The fermentation conditions are 20-23°C, 170-220rpm, and a liquid volume of 15%-25%; the preferred fermentation conditions are 21°C, 180rpm, and a liquid volume of 20%.

5. The method according to claim 1, characterized in that Based on the initial fermentation broth volume, the amount of compound enzyme added is 1500-3500 U / mL, preferably 2500 U / mL.

6. The method according to claim 1, characterized in that The ratio of cellulase to pectinase is 1:0.5-2 (U:U), preferably 1:1 (U:U).

7. The method according to claim 1, characterized in that The enzymatic hydrolysis time is 4 to 6 hours, the enzymatic hydrolysis temperature is 45 to 55°C, the enzymatic hydrolysis pH is 4.0 to 6.0, and the assisted ultrasound is 20 to 40 minutes.

8. The method according to claim 1, characterized in that: The enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis temperature was 50 °C, the enzymatic hydrolysis pH was 5.0, and the assisted ultrasound was 30 min.

9. The method according to claim 1, characterized in that: The lower alcohol is ethanol.

10. The method according to claim 9, characterized in that The volume ratio of ethanol to the initial fermentation liquid is 4-6:1, and the extraction time is 10-60 minutes; preferably, the volume ratio of ethanol to the initial fermentation liquid is 5:1, and the extraction time is 30 minutes.

Images