Method for extracting goose carnosine and carnosine from tuna

Abstract

The invention provides a method for extracting goose carnosine and / or carnosine. The method comprises the steps of low-salt water extraction and high-salt water extraction under alkaline and heating conditions. The extraction method of the goose carnosine and / or carnosine provided by the invention is simple to operate, low in cost and easy for large-scale industrial use.

Description

Technical Field

[0001] The invention relates to a method for extracting anserine and carnosine from tuna, in particular to a method for extracting anserine and carnosine by using a two-stage salt water solution. Background Art

[0002] Tuna, also known as tuna, belongs to the class of bony fish, order of Perciformes, family of Scombridae, and is distributed in major warm waters around the world. It is an oceanic highly migratory fish. Tuna meat is tender, and its high-intensity athletic ability provides its muscles with a variety of nutrients, such as histidine dipeptide (angerine, carnosine), selenium, taurine, iron, zinc, magnesium, etc. Carnosine is a needle-shaped crystal that is easily soluble in water. Its melting point is 246-250℃, and its maximum absorption wavelength is 218nm. The H atom on the imidazole of anserine is replaced by CH 3 -substituted, is a derivative of carnosine, a highly water-soluble small molecule peptide with a molecular weight of about 250. This type of dipeptide naturally exists in the skeletal muscle of a variety of terrestrial vertebrates at millimolar concentrations, with the highest concentrations in fish, heart and central nervous system. Studies have shown that anserine has more prominent effects, such as lowering uric acid, anti-oxidation, anti-fatigue, regulating blood sugar, treating tumors, etc., and carnosine is relatively excellent in anti-oxidation and has been used in a variety of foods and cosmetics.

[0003] At present, there are few descriptions of carnosine extraction methods in the literature, especially the research on collecting anserine and carnosine from tuna at the same time. There are many studies on the extraction and preparation process of single anserine or other components, and the process route design is basically water extraction, enzymatic hydrolysis, and filtration. However, since anserine and carnosine are small molecular peptides, enzymatic hydrolysis will also hydrolyze the impurity proteins in the water extract into small molecular substances, which are difficult to separate from anserine and carnosine, bringing difficulties to subsequent separation and purification. Summary of the invention

[0004] The present invention provides a method for extracting anserine and / or carnosine, comprising the following steps: 1) Low-salt water extraction: add the first salt solution to the tuna meat homogenate to a final concentration of the first salt of 0.2-1.0 M, maintain the temperature above 45° C., and stir for more than 6 hours; 2) high salt water extraction: adding the second salt solution to the tuna meat homogenate to a final concentration of the second salt of 1.5-2.5 M, maintaining the temperature above 45° C., and stirring for more than 1 hour; and 3) Centrifugation: After the homogenate is cooled, centrifuge and collect the supernatant. The first salt is the same as or different from the second salt; in the low-salt water extraction and high-salt water extraction steps, the pH value of the tuna meat homogenate is monitored, and the pH value is maintained at not less than 9.0 by adding an alkaline solution.

[0005] In some embodiments, the first salt is selected from sodium chloride, sodium acetate, and combinations thereof, and the alkaline solution is sodium hydroxide solution.

[0006] In some embodiments, the tuna meat homogenate is prepared as follows: minced tuna meat and water are mixed in a volume ratio of 1:5 and then homogenized.

[0007] In some embodiments, the low-salinity extraction step is performed for more than 8 hours; and the high-salinity extraction step is performed for more than 2 hours.

[0008] In some embodiments, the low-salinity extraction step is performed at 45-60°C, and the high-salinity extraction step is performed at 75-85°C.

[0009] In some embodiments, the low-salt water extraction step comprises: adding a first salt solution to the tuna meat homogenate to a final concentration of the first salt of 0.5 M, maintaining the temperature at 55° C., and stirring for 8 hours.

[0010] In some embodiments, the high-salt water extraction step comprises: adding a second salt solution to the tuna meat homogenate to a final concentration of the second salt of 2.0 M, heating to 80° C., and stirring for 2 hours.

[0011] In some embodiments, the first salt is sodium chloride and the second salt is sodium acetate, or the first salt is sodium acetate and the second salt is sodium chloride.

[0012] In some embodiments, the method comprises the steps of: 1) Low-salt water extraction: put the tuna meat homogenate into a stirred tank, add sodium acetate solution to a final concentration of 0.5 M, and adjust the pH value to 9.5 with NaOH, turn on stirring, set the speed to 40 rpm, control the temperature in the tank to 55°C, and stir for 8 hours; 2) High-salt water extraction: add sodium acetate solution to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours; 3) Centrifugation: After the homogenate has cooled naturally, centrifuge at 1000g for 10 minutes and collect the supernatant. During the low-salinity extraction and high-salinity extraction processes, the pH value change was monitored and NaOH was added to maintain the pH not lower than 9.0.

[0013] In some embodiments, the method comprises the steps of: 1) Low-salt water extraction: put the tuna meat homogenate into a stirred tank, add sodium chloride solution to a final concentration of 0.5 M, and adjust the pH value to 9.5 with NaOH, turn on stirring, set the speed to 40 rpm, control the temperature in the tank to 55°C, and stir for 8 hours; 2) High-salt water extraction: add sodium acetate to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours; 3) Centrifugation: After the homogenate has cooled naturally, centrifuge at 1000g for 10 minutes and collect the supernatant. During the low-salinity extraction and high-salinity extraction processes, the pH value change was monitored and NaOH was added to maintain the pH not lower than 9.0.

[0014] The method for extracting anserine and / or carnosine provided by the invention has the advantages of simple operation, low cost and easy large-scale industrial use. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the detection of anserine content in the retentate are shown.

[0016] Figure 2 The results of the carnosine content test in the retentate are shown.

[0017] Figure 3 The HPLC spectrum of carnosine standard product is shown in Figure 1. In the figure, the vertical axis is the UV absorption intensity expressed as voltage signal (mV), and the horizontal axis is time (min), and the same applies to the following.

[0018] Figure 4 This is the HPLC spectrum of anserine standard.

[0019] Figure 5 HPLC spectrum of the retentate prepared from the supernatant of Example 2.

[0020] Figure 6 HPLC spectrum of the retentate prepared from the supernatant of Example 3.

[0021] Figure 7 HPLC spectrum of the retentate prepared from the supernatant of Example 4.

[0022] Figure 8 This is the HPLC spectrum of the retentate prepared from the supernatant of Example 5. DETAILED DESCRIPTION

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0024] The term "or" refers to a single element of the listed optional elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed optional elements.

[0025] The terms "comprising", "including", and "having" mean "including but not limited to", but also consist of only the listed elements.

[0026] The term "above" includes the number, for example, "above 2 hours" includes 2 hours and a duration greater than 2 hours.

[0027] The structural formula of Anserine is as follows:

[0028] The structural formula of L-Carnosine is as follows:

[0029] Unless otherwise specified, "low salt water extraction" herein refers to the extraction of anserine and carnosine from tuna meat under low salt conditions. The salt concentration of low salt conditions is, for example, 0.2-1.0 M, 0.3-0.9 M, 0.4-0.8 M, 0.5-0.7 M, 0.4-0.6 M, etc., such as 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M or 1.0 M. Unless otherwise specified, "high salt water extraction" herein refers to the extraction of anserine and carnosine from tuna meat under high salt conditions. The salt concentration of the high salt condition is, for example, 1.2-4.0 M, 1.5-3.0 M, 1.5-2.5 M, etc., such as 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2.0 M, 2.5 M, 2.8 M, 3.0 M or 4.0 M. Here, "salt" refers to small molecule inorganic salts or organic salts, such as sodium chloride, potassium chloride, sodium acetate, potassium acetate, etc., and the final concentration of salt is not considered when the tuna meat itself may contain salt. "Water extraction" in this article refers to separating the target substance (such as anserine and / or carnosine) from other components in an aqueous solution environment without using an organic solvent.

[0030] The inventors found that two-stage heated water extraction with different salt concentrations is beneficial to the separation of anserine and / or carnosine from other components in tuna meat homogenate, and subsequently studied other conditions such as temperature, water extraction time, salt type, pH value, etc.

[0031] The first stage of water extraction is preferably carried out at a relatively low salt concentration (low salt water extraction), such as 0.2-1.0 M, preferably 0.4-0.6 M. The water extraction temperature can be 45-60°C. The water extraction time is generally 6-8 h. Prolonging the water extraction time will improve the water extraction effect, but after 8 hours, the water extraction effect will not be significantly improved if the time is further extended.

[0032] The second stage of water extraction is preferably carried out at a relatively high salt concentration (high salt water extraction), such as 1.2-4.0 M, preferably 1.5-2.5 M. The water extraction temperature is above 45°C, preferably 75-85°C. The water extraction time is generally 1-2 h. After 2 h, if the time is further extended, the water extraction effect is not significantly improved.

[0033] During the low-salt water extraction and high-salt water extraction process, the pH value of the homogenate gradually decreased. The decrease in pH value will significantly affect the water extraction effect. Therefore, it is necessary to monitor the change in the pH value of the homogenate during the water extraction process, and add NaOH when necessary to maintain the pH value not lower than 9.0.

[0034] The present invention is further illustrated by means of specific examples below.

[0035] Example 1

[0036] 100 kg of frozen tuna meat was thawed and chopped, and water was added at a solid-liquid ratio of 1:5 for homogenization to obtain tuna meat homogenate for the following experiments.

[0037] Example 2

[0038] Anserine and carnosine extract were prepared by the following steps.

[0039] 1) Low-salt water extraction: 10 L of the tuna meat homogenate prepared in Example 1 was put into a stirred tank, sodium chloride was added to a final concentration of 0.5 M, and the pH value was adjusted to 9.5 with NaOH. Stirring was started, the speed was set to 40 rpm, the temperature in the tank was controlled at 55° C., and stirring was performed for 8 hours.

[0040] 2) High-salt water extraction: Add sodium chloride to a stirred tank to a final concentration of 2.0 M, adjust the pH to 9.5 with NaOH, raise the temperature to 80°C and stir for 2 hours.

[0041] 3) Centrifugation: After the homogenate is cooled naturally, centrifuge at 1000g for 10 minutes to collect the supernatant and precipitate.

[0042] During the low-salinity and high-salinity extractions, the pH change was monitored and NaOH was added to maintain the pH not lower than 9.0.

[0043] Example 3

[0044] Anserine and carnosine extract were prepared by the following steps.

[0045] 1) Low-salt water extraction: 10 L of the tuna homogenate prepared in Example 1 was put into a stirred tank, sodium acetate was added to a final concentration of 0.5 M, and the pH value was adjusted to 9.5 with NaOH. Stirring was started, the speed was set to 40 rpm, the temperature in the tank was controlled at 55° C., and stirring was performed for 8 hours.

[0046] 2) High brine extraction: Add sodium acetate to a stirred tank to a final concentration of 2.0 M, adjust the pH to 9.5 with NaOH, raise the temperature to 80°C and stir for 2 hours.

[0047] 3) Centrifugation: After the homogenate has cooled naturally, centrifuge at 1000g for 10 minutes and collect the supernatant.

[0048] During the low-salinity and high-salinity extractions, the pH change was monitored and NaOH was added to maintain the pH not lower than 9.0.

[0049] Example 4

[0050] Anserine and carnosine extracts were prepared according to the method of Example 3, except that both the low saline extraction and the high saline extraction were carried out at 55°C.

[0051] That is, step 2) is: add sodium acetate to the stirring tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, and stir at 55° C. for 2 hours.

[0052] Example 5

[0053] Anserine and carnosine extracts were prepared according to the method of Example 3, except that both the low saline extraction and the high saline extraction were carried out at 80°C.

[0054] That is, step 1 is: take 10L of the tuna meat homogenate prepared in Example 1 and put it into a stirring tank, add sodium acetate to a final concentration of 0.5 M, and adjust the pH value to 9.5 with NaOH, turn on stirring, set the speed to 40 rpm, control the temperature in the tank to 80°C, and stir for 8 hours.

[0055] Example 6

[0056] Anserine and carnosine extract were prepared according to the method of Example 2, except that sodium acetate was added instead of sodium chloride in the high saline extraction step.

[0057] That is, step 2 is: add sodium acetate to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours.

[0058] Example 7

[0059] Anserine and carnosine extract were prepared according to the method of Example 3, except that sodium chloride was added instead of sodium acetate in the high saline extraction step.

[0060] That is, step 2 is: add sodium chloride to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours.

[0061] Comparative Example 1

[0062] Anserine and carnosine extract were prepared by the following steps.

[0063] 10 L of the tuna meat homogenate prepared in Example 1 was put into a stirred tank, stirring was started, the speed was set to 40 rpm, the temperature in the tank was controlled at 55° C., and stirring was performed for 8 hours. After the homogenate was cooled naturally, it was centrifuged at 1000 g for 10 minutes to collect the supernatant.

[0064] Comparative Example 2

[0065] Anserine and carnosine extract were prepared according to the method of Example 2, except that sodium chloride was not added.

[0066] That is, step 1 is: 10 L of the tuna meat homogenate prepared in Example 1 is put into a stirring tank, the pH value is adjusted to 9.5 with NaOH, stirring is turned on, the speed is set to 40 rpm, the temperature in the tank is controlled at 55° C., and stirring is performed for 8 hours.

[0067] Step 2 is: adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours.

[0068] Comparative Example 3

[0069] Anserine and carnosine extracts were prepared according to the method of Example 2, except that the salt concentration was not increased in the high-salt water extraction step, that is, the high-salt water extraction step was as follows: adjusting the pH value to 9.5 with NaOH, heating to 80° C. and stirring for 2 hours.

[0070] Although the salt concentration is not increased here, this step is still called the "high salt water extraction" step, just for the convenience of description.

[0071] Comparative Example 4

[0072] Anserine and carnosine extracts were prepared according to the method of Example 2, except that the low-salt water extraction step was performed using high-concentration sodium chloride. That is, the low-salt water extraction step was as follows: 10 L of the tuna meat homogenate prepared in Example 1 was put into a stirred tank, sodium chloride was added to a final concentration of 2.0 M, and the pH value was adjusted to 9.5 with NaOH, stirring was turned on, the speed was set to 40 rpm, the temperature in the tank was controlled to 55°C, and stirring was performed for 8 hours. Similarly, this step is still called the "low-salt water extraction" step only for the convenience of description.

[0073] Comparative Example 5

[0074] Anserine and carnosine extracts were prepared according to the method of Example 3, except that the salt concentration was not increased in the high-salt water extraction step. That is, the high-salt water extraction step was: adjusting the pH value to 9.5 with NaOH, heating to 80° C. and stirring for 2 hours. Similarly, this step is still called the "high-salt water extraction" step only for the convenience of description.

[0075] Comparative Example 6

[0076] Anserine and carnosine extracts were prepared according to the method of Example 3, except that the low-salt water extraction step was performed with high-concentration sodium acetate. That is, the low-salt water extraction step was as follows: 10 L of the tuna meat homogenate prepared in Example 1 was put into a stirred tank, sodium acetate was added to a final concentration of 2.0 M, and the pH value was adjusted to 9.5 with NaOH, stirring was turned on, the speed was set to 40 rpm, the temperature in the tank was controlled to 55°C, and stirring was performed for 8 hours. Similarly, this step is still called the "low-salt water extraction" step only for the convenience of description.

[0077] Comparative Example 7

[0078] Anserine and carnosine extract were prepared according to the method of Example 2, except that pH adjustment was not performed.

[0079] Comparative Example 8

[0080] Anserine and carnosine extract were prepared according to the method of Example 3, except that pH adjustment was not performed.

[0081] Comparative Example 9

[0082] Anserine and carnosine extract were prepared according to the method of Example 2, except that the low-salt water extraction time was shortened to 2 hours.

[0083] Comparative Example 10

[0084] Anserine and carnosine extract were prepared according to the method of Example 2, except that both the low-salt water extraction and the high-salt water extraction were carried out at 100°C.

[0085] Comparative Example 11

[0086] Anserine and carnosine extract were prepared according to the method of Example 2, except that both the low saline extraction and the high saline extraction were carried out at 20°C. Example

[0087] 2 L of the supernatant prepared in Examples 2-6 and Comparative Examples 1-11 were filtered with a 200 nm pore size ceramic membrane, and the permeate was collected; then the permeate was filtered with a 2000 Da pore size organic membrane and a 1000 Da pore size organic membrane in turn, and the permeate was collected, and finally filtered with a 200 Da pore size organic membrane to collect the retentate. The volume of the retentate was between 60-80 mL, and after adding water to 100 mL, the concentrations of carnosine and anserine in the retentate were detected by HPLC. For each supernatant, the filtration and detection process was repeated 3 times, and the results were averaged.

[0088] The detection method of anserine and carnosine refers to NY3946-2021, using 20mM phosphate buffer solution (pH7.0) as the mobile phase at C 18 The samples were separated on a 4.6 mm × 250 mm 5 μm column at a flow rate of 1.0 mL / min and detected with a diode array detector at 210 nm.

[0089] The results are shown in Table 1. Figure 1 and Figure 2 middle.

[0090] Table 1 Test results of anserine and carnosine Sample No. Source supernatant Anserine concentration in retentate (g / L) Carnosine concentration in retentate (g / L) 1 Example 2 <![CDATA[1.452 b ]]> <![CDATA[0.587 b ]]> 2 Example 3 <![CDATA[2.043 a ]]> <![CDATA[0.698 a ]]> 3 Example 4 <![CDATA[1.315 b ]]> <![CDATA[0.425 c ]]> 4 Example 5 <![CDATA[1.024 c ]]> <![CDATA[0.332 d ]]> 5 Example 6 <![CDATA[2.322 a ]]> <![CDATA[0.683 a ]]> 6 Example 7 <![CDATA[1.545 b ]]> <![CDATA[0.527 b ]]> 7 Comparative Example 1 <![CDATA[0.277 f ]]> <![CDATA[0.135 f ]]> 8 Comparative Example 2 <![CDATA[0.305 f ]]> <![CDATA[0.127 f ]]> 9 Comparative Example 3 <![CDATA[0.588 e ]]> <![CDATA[0.237 de ]]> 10 Comparative Example 4 <![CDATA[0.512 e ]]> <![CDATA[0.195 e ]]> 11 Comparative Example 5 <![CDATA[0.729 d ]]> <![CDATA[0.233 de ]]> 12 Comparative Example 6 <![CDATA[0.715 d ]]> <![CDATA[0.216 e ]]> 13 Comparative Example 7 <![CDATA[0.546 e ]]> <![CDATA[0.281 d ]]> 14 Comparative Example 8 <![CDATA[0.695 d ]]> <![CDATA[0.248 de ]]> 15 Comparative Example 9 <![CDATA[0.833 d ]]> <![CDATA[0.317 d <!-- 6 -->]]> 16 Comparative Example 10 <![CDATA[0.809 d ]]> <![CDATA[0.273 d ]]> 17 Comparative Example 11 <![CDATA[0.648 de ]]> <![CDATA[0.189 e ]]>

[0091] Different lowercase letters in the same column indicate significant differences between groups ( p <0.05, the same lowercase letters indicate no significant difference between the groups ( p >0.05).

[0092] Some representative HPLC spectra are shown in Figure 5-8 (corresponding to the retentate of Example 2-5). Figure 3 and 4 Anserine and carnosine standard spectra are shown.

[0093] Comparing the above results, it can be seen that the two-stage water extraction with different salt concentrations will significantly promote the precipitation of anserine and carnosine (separation from other components of tuna meat). The effect of water extraction under low salt and high salt conditions is poor (see the results of Comparative Examples 3-6). Changes in pH value will also significantly affect the water extraction effect (see the results of Comparative Examples 7 and 8). In addition, water extraction time, temperature, etc. will also affect the water extraction effect (see the results of Comparative Examples 9-11).

[0094] When other salts such as sodium sulfate and disodium hydrogen phosphate are used instead of sodium chloride or sodium acetate, the separation effect of anserine and carnosine is even worse than that of comparative example 1 without adding any salt. The reason for this difference is currently unclear.

[0095] The precipitate obtained in the centrifugation step of the present invention can be subjected to repeated low-salt water extraction and high-salt water extraction steps after homogenization to increase the yield.

[0096] Since the method of the present invention uses the addition of sodium chloride and / or sodium acetate to extract anserine and carnosine, although 200Da pore size organic membrane filtration can remove most of the sodium chloride and / or sodium acetate, it is inevitable that the retentate contains a high concentration of sodium chloride and / or sodium acetate. Currently, research is underway to further remove these salts using ion exchange columns and electroosmosis methods to increase the concentration of anserine and carnosine.

Claims

1. A method for extracting anserine and / or carnosine, characterized in that: The steps include: 1) Low-salt water extraction: add the first salt solution to the tuna homogenate to a final concentration of 0.2-1.0 M of the first salt, maintain the temperature above 45° C., and stir for more than 6 hours; 2) high salt water extraction: adding a second salt solution to the tuna meat homogenate to a final second salt concentration of 1.5-2.5 M, maintaining the temperature above 45° C., and stirring for more than 1 hour; and 3) Centrifugation: After the homogenate is cooled, centrifuge and collect the supernatant. The first salt is the same as or different from the second salt; in the low-salt water extraction and high-salt water extraction steps, the pH value of the tuna meat homogenate is monitored, and the pH value is maintained at not less than 9.0 by adding an alkaline solution.

2. The method of claim 1, wherein the first salt is selected from sodium chloride, sodium acetate and a combination thereof, and the alkaline solution is sodium hydroxide solution.

3. The method according to claim 1, wherein the tuna meat homogenate is prepared by mixing chopped tuna meat and water in a volume ratio of 1:5 and then homogenizing.

4. The method of claim 1, wherein the low-salinity extraction step is performed for more than 8 hours; and the high-salinity extraction step is performed for more than 2 hours.

5. The method of claim 1, wherein the low-salinity water extraction step is carried out at 45-60°C, and the high-salinity water extraction step is carried out at 75-85°C.

6. The method according to claim 1, wherein the low-salt water extraction step comprises: adding a first salt solution to the tuna meat homogenate until the final concentration of the first salt is 0.5 M, maintaining the temperature at 55°C, and stirring for 8 hours.

7. The method according to claim 1, wherein the high salt water extraction step comprises: adding a second salt solution to the tuna meat homogenate to a final concentration of the second salt of 2.0 M, heating to 80°C, and stirring for 2 hours.

8. The method of claim 1, wherein the first salt is sodium chloride and the second salt is sodium acetate, or the first salt is sodium acetate and the second salt is sodium chloride.

9. The method according to claim 3, comprising the steps of: 1) Low-salt water extraction: put the tuna meat homogenate into a stirred tank, add sodium acetate solution to a final concentration of 0.5 M, and adjust the pH value to 9.5 with NaOH, turn on stirring, set the speed to 40 rpm, control the temperature in the tank to 55°C, and stir for 8 hours; 2) High-salt water extraction: add sodium acetate solution to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours; 3) Centrifugation: After the homogenate has cooled naturally, centrifuge at 1000g for 10 minutes and collect the supernatant. During the low-salinity extraction and high-salinity extraction processes, the pH value change was monitored and NaOH was added to maintain the pH not lower than 9.

0.

10. The method according to claim 3, comprising the steps of: 1) Low-salt water extraction: put the tuna meat homogenate into a stirred tank, add sodium chloride solution to a final concentration of 0.5 M, and adjust the pH value to 9.5 with NaOH, turn on stirring, set the speed to 40 rpm, control the temperature in the tank to 55°C, and stir for 8 hours; 2) High-salt water extraction: add sodium acetate to the stirred tank to a final concentration of 2.0 M, adjust the pH value to 9.5 with NaOH, raise the temperature to 80° C. and stir for 2 hours; 3) Centrifugation: After the homogenate has cooled naturally, centrifuge at 1000g for 10 minutes and collect the supernatant. During the low-salinity extraction and high-salinity extraction processes, the pH value change was monitored and NaOH was added to maintain the pH not lower than 9.0.

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