A method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system

Abstract

The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind of based on double aqueous phase system flaxseed cyclic peptide separation and purification method, belong to flaxseed cyclic peptide separation and purification field.The application discloses a kind

Description

TECHNICAL FIELD

[0001] The present application relates to the field of flaxseed cyclotide separation and purification, and particularly relates to a flaxseed cyclotide separation and purification method based on a two-water-phase system. BACKGROUND

[0002] In addition to the n-3 series of polyunsaturated fatty acids alpha-linolenic acid flaxseed oil, flaxseed also contains rich functional ingredients such as flaxseed lignan, flaxseed gum and flaxseed cyclotide. Among them, flaxseed cyclotide is a hydrophobic cyclic plant polypeptide naturally existing in flaxseed and rhizome, which is composed of 8-10 amino acids and is a small molecular polypeptide with a molecular weight of about 1 kDa. So far, about 38 kinds of flaxseed cyclotides have been found in flaxseed. Because the amino acid composition of flaxseed cyclotide contains methionine, it can be oxidized to form methionine sulfoxide under certain conditions, and further oxidized to form methionine sulfone, so flaxseed cyclotide has certain antioxidant activity, and cyclotide P is the precursor of cyclotide E, the main bitter source of flaxseed oil. In addition to the antioxidant activity, flaxseed cyclotide also has immunosuppressive activity, free radical scavenging, anticancer, anti-inflammatory, inhibition of osteoclast differentiation, chelation of metal ions and other physiological functions.

[0003] At present, there are mainly two extraction methods for flaxseed cyclotide in the related art, namely, extracting hydrophobic flaxseed cyclotide from flaxseed oil and extracting flaxseed cyclotide from flaxseed cake. However, both of these two extraction methods have certain disadvantages. For example, Chinese Patent Publication No. CN108329376A uses acetone to extract flaxseed oil and cyclotide from flaxseed powder, and then purifies it by using a chromatography column. This extraction method uses acetone, which is a toxic reagent, and the column chromatography purification method has a small single processing capacity and high cost. Chinese Patent Publication No. CN108853193A extracts flaxseed cyclotide from flaxseed cake. This method is beneficial to improve the economic value of flaxseed cake, but the structure of flaxseed is damaged after pressing, and the cyclotide in the cake will be oxidized. In addition, using 95% ethanol alone to extract cyclotide from flaxseed meal will have more polar components such as oil and protein remaining, and the cyclotide purity is low and needs further purification.

[0004] Therefore, it is urgent to provide a low-cost, simple-to-operate and large-scale purification process for flaxseed cyclotide. SUMMARY

[0005] In order to overcome the problems in the above-mentioned related art, the present application provides a flaxseed cyclotide separation and purification method based on a two-water-phase system, which is low in cost, non-toxic and environmentally friendly, and can realize large-scale purification production of flaxseed cyclotide.

[0006] The application provides a method for separating and purifying flaxseed cyclotide based on a two-water-phase system, which comprises the following steps:

[0007] Constructing the two-water-phase system: mixing a small-molecule alcohol and a salt solution, adjusting pH with acid and alkali, and obtaining the two-water-phase system;

[0008] Purifying flaxseed cyclotide extract with the two-water-phase system: mixing the two-water-phase system with flaxseed cyclotide extract, separating the upper phase after phase separation, removing the solvent, and obtaining a high-salt-content flaxseed cyclotide crude purification product;

[0009] Desalting the high-salt-content flaxseed cyclotide crude purification product: mixing the high-salt-content flaxseed cyclotide crude purification product with water according to a mass ratio of 1:(1-100), desalting, centrifugally separating to obtain supernatant and precipitate, removing water from the precipitate, and obtaining target purified flaxseed cyclotide powder;

[0010] Adjusting the pH of the supernatant obtained by centrifugal separation to a value at which no more precipitate and floating matter is generated, centrifugally separating to obtain precipitate, and adding the precipitate to the flaxseed cyclotide extract to perform two-water-phase purification again, i.e., step 2).

[0011] Optionally, in step 1), the small-molecule alcohol is methanol, ethanol, isopropyl alcohol or n-propyl alcohol; and the salt solution is a phosphate salt solution or a sulfate salt solution.

[0012] Optionally, in step 1), the phosphate salt solution comprises dipotassium hydrogen phosphate solution or disodium hydrogen phosphate solution; and the sulfate salt solution comprises ammonium sulfate, potassium sulfate, sodium sulfate, magnesium sulfate or zinc sulfate.

[0013] Optionally, in step 1), the small-molecule alcohol is methanol, and the salt solution is dipotassium hydrogen phosphate solution.

[0014] Optionally, in the two-water-phase system, the mass fraction of dipotassium hydrogen phosphate is 5%-30%, the mass fraction of methanol is 20%-80%, and the pH is 7-12.

[0015] Optionally, in the two-water-phase system, the mass fraction of dipotassium hydrogen phosphate is 14%-18%, the mass fraction of methanol is 44%-46%, and the pH is 8.7-9.3.

[0016] Optionally, in step 2), the flaxseed cyclotide extract and the two-water-phase system are mixed according to a mass ratio of 1:(1-120).

[0017] Optionally, in step 2), the flaxseed cyclotide extract and the two-water-phase system are mixed according to a mass ratio of 1:(30-60).

[0018] Optionally, in step 3), the water removal is performed by freeze-drying.

[0019] Optionally, the water washing step to remove salt from the crude linseed cyclotide purification product is repeated at least twice.

[0020] Optionally, in step 4), the supernatant is adjusted to a pH of 0-2.

[0021] The extraction and purification process provided by the present application mainly adopts solvent method, and has the advantages of simple and fast operation, large processing capacity, and low cost. Since the solubility of oil in the aqueous two-phase system composed of methanol and K2HPO4 is low, the present application is also applicable to purifying an oil system rich in cyclotides or a cyclotide extract system with high oil content. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a schematic diagram of the effect of the additive amount of the extract of the exemplary embodiment of the present application on the extraction rate;

[0023] Figure 2 is a picture of the aqueous two-phase multi-phase separation of the exemplary embodiment of the present application;

[0024] Figure 3 is a picture of the linseed cyclotide powder (freeze-dried) of the exemplary embodiment of the present application;

[0025] Figure 4 is a schematic diagram of the cyclotide retention in the supernatant at different pH values of the exemplary embodiment of the present application. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present application more apparent and easy to understand, the following will make a detailed description in combination with specific embodiments.

[0027] The linseed cyclotide separation and purification method based on the aqueous two-phase system in the specific embodiment of the present application comprises:

[0028] Constructing the aqueous two-phase system: mixing a small-molecule alcohol and a salt solution, adjusting the pH value with an acid and a base, and obtaining the aqueous two-phase system;

[0029] Aqueous two-phase purification of the linseed cyclotide extract: mixing the aqueous two-phase system and the linseed cyclotide extract, taking the upper phase after phase separation, removing the solvent, and obtaining the crude linseed cyclotide purification product with high salt content;

[0030] Water washing to remove salt from the crude linseed cyclotide purification product: mixing the crude linseed cyclotide purification product and water according to a mass ratio of 1:(1-100), performing desalination treatment, centrifugally separating to obtain the supernatant and the precipitate, removing water from the precipitate, and obtaining the target purified linseed cyclotide powder;

[0031] Adjust the pH of the supernatant obtained by centrifugal separation to the point where the precipitate and the float no longer increase, centrifugal separation to obtain the precipitate, and add to the flaxseed cyclic peptide extract, and perform aqueous two-phase purification again, i.e., step 2). The specific embodiment of the present application utilizes the strong hydrophobicity of flaxseed cyclic peptides, and uses the small-molecule alcohol and salt solution to form an aqueous two-phase system to purify and separate the flaxseed cyclic peptide extract, and the cyclic peptides are more distributed in the methanol phase. Considering the processing capacity, the extract addition amount is appropriately increased, and the extraction rate can reach 75% to 90%.

[0032] In the aqueous two-phase system-based flaxseed cyclic peptide separation and purification method of the specific embodiment of the present application, the small-molecule alcohol is methanol, ethanol, n-propanol or isopropanol; the salt solution is a phosphate solution or a sulfate solution; in some specific embodiments, the small-molecule alcohol is methanol, and the salt solution is dimethyl hydrogen phosphate; in some specific embodiments, the small-molecule alcohol is ethanol, and the salt solution can be dipotassium hydrogen phosphate; sodium hydrogen phosphate; ammonium sulfate; potassium sulfate; sodium sulfate; magnesium sulfate; zinc sulfate; sodium carbonate; potassium carbonate; sodium citrate; in other specific embodiments, the small-molecule alcohol is isopropanol, and the salt solution can be calcium chloride; ammonium sulfate; sodium sulfate; magnesium sulfate; in other specific embodiments, the small-molecule alcohol is n-propanol, and the salt solution can be sodium chloride; potassium chloride; barium chloride; ammonium chloride; calcium chloride; ammonium sulfate; sodium sulfate; magnesium sulfate.

[0033] In the aqueous two-phase system-based flaxseed cyclic peptide separation and purification method of the specific embodiment of the present application, the small-molecule alcohol is methanol, and the salt solution is a K2HPO4 solution. Compared with other alcohols mentioned in the present application, methanol has a low boiling point, and it is relatively easy to remove the solvent. Secondly, because methanol has a large polarity, the solubility of impurities with small polarity is low; and the dipotassium hydrogen phosphate solution can form an aqueous two-phase system with methanol, and other salt solutions are difficult to achieve.

[0034] The hydrophobic region of the protein binds with methanol, the density increases, and the protein can be suspended in the middle of the methanol phase and the water phase. At the same time, the oil in the extract has a low solubility in the methanol phase and the water phase, and the density is between the salt solution and the methanol, between the upper layer of the protein phase and the lower layer of the methanol phase. After centrifugal separation, the impurities in the extract can be effectively removed.

[0035] In the aqueous two-phase system-based flaxseed cyclic peptide separation and purification method of the specific embodiment of the present application, the mass fraction of K2HPO4 in the aqueous two-phase system is 5% to 30%, the mass fraction of methanol is 20% to 80%, and the pH is 7 to 12.

[0036] The method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application has a mass fraction of K2HPO4 of 14% to 18%, a mass fraction of methanol of 44% to 46%, and a pH of 8.7 to 9.3.

[0037] Due to the low concentration of K2HPO4, water molecules cannot be completely bound, and part of the water molecules will migrate to the methanol phase, so part of K2HPO4 will also be dissolved in the methanol phase. By using the hydrophobicity of flaxseed cyclic peptide, the upper phase of the removed solvent is washed with water to remove salt, which can effectively remove the residual impurities in the methanol phase. Among them, K2HPO4, oil, water-soluble protein, pigment, etc. will be dissolved in water or suspended in the upper layer of the water phase, and the cyclic peptide will be precipitated from the water, centrifuged, and the precipitate will be obtained. After freeze-drying, a light yellow flaxseed cyclic peptide powder with high purity can be obtained.

[0038] In step 2) of the method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application, the flaxseed cyclic peptide extract and the two-water-phase system are mixed in a mass ratio of 1:(1-120).

[0039] In step 2) of the method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application, the flaxseed cyclic peptide extract and the two-water-phase system are mixed in a mass ratio of 1:(30-60).

[0040] In step 3) of the method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application, the water is removed by freeze-drying.

[0041] In the method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application, the step of washing the flaxseed cyclic peptide crude purification product with water to remove salt is repeated at least twice. The more the number of repetitions, the higher the purity of the flaxseed cyclic peptide powder obtained.

[0042] In step 4) of the method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system in the embodiment of the present application, the supernatant is adjusted to a pH of 0 to 2. As an example, the supernatant for washing the flaxseed crude purification product is adjusted to a pH of 1.0. After the supernatant precipitates and floats, the floating and precipitated materials are added to the flaxseed cyclic peptide extract, and the two-water-phase purification is performed again. Due to the uneven distribution of cyclic peptide in the crude purification product, the loss of cyclic peptide cannot be calculated by adding the loss of two-water-phase extraction, so the loss of cyclic peptide caused by water washing is calculated according to the following formula:

[0043]

[0044] Note: m water phase: the content of cyclic peptide in water phase; m precipitate: the content of cyclic peptide in precipitate; since the floating and precipitate in supernatant can be re-extracted, it is not calculated as loss.

[0045] The flaxseed cyclic peptide extract involved in the embodiments 1-4 and comparative examples 2-9 of the present application can be obtained by the following method:

[0046] Mix 95% ethanol with flaxseed defatted meal in a percolator, and percolate at a proper temperature for 1-2 h. The percolation can be continuous or intermittent. After percolation, remove the solvent to obtain the flaxseed cyclic peptide extract. The content of flaxseed cyclic peptide is 0.4%-1%.

[0047] The target purified flaxseed cyclic peptide powder in the specific embodiments of the present application uses flaxseed cyclic peptide B (purity 95%) as a standard for external standard method quantification, and the purity can reach 80%.

[0048] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in other manners different from those described herein, and those skilled in the art can make similar generalizations without departing from the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0049] Embodiment 1:

[0050] Use methanol and K2HPO4 salt solution to form a double water phase system. The mass fraction of K2HPO4 in the double water phase system is 14%, and the pH of the salt solution is adjusted to 8.7; the mass fraction of methanol in the double water phase system is 46%.

[0051] Mix the above flaxseed cyclic peptide extract with the double water phase according to the mass ratio 1:60, shake thoroughly, and centrifuge at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptide, the middle phase of protein and oil, and the lower phase. See Figure 2 The extraction rate of cyclic peptide is 92%, see Figure 1 The distribution coefficient is 1.91. It should be noted that the higher the distribution coefficient and the extraction rate, the higher the concentration of cyclic peptide in the upper phase, or in other words, the less the residual cyclic peptide in the lower phase.

[0052] Separate the upper phase rich in cyclic peptide, remove the solvent to obtain the crude purified flaxseed cyclic peptide product with high salt content. Shake or stir with water, centrifuge to separate the precipitate, and repeat the water washing operation on the precipitate for 2-3 times. Freeze-dry the precipitate to remove water to obtain flaxseed cyclic peptide powder with a purity of 79.5%, see Figure 3The supernatant of the first water washing was adjusted to pH 1.0, and after centrifugation, the floating and precipitate were collected and added to the extract of the cyclolinopeptide of flaxseed, and the aqueous two-phase purification was performed again. By measuring the content of the cyclolinopeptide remaining in the water phase, the loss rate of the cyclolinopeptide after water washing was about 10%, see Figure 4 .

[0053] Example 2

[0054] The aqueous two-phase system was composed of methanol and K2HPO4salt solution. The mass fraction of K2HPO4in the aqueous two-phase system was 14%, and the pH of the salt solution was adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system was 46%.

[0055] The extract of the cyclolinopeptide of flaxseed was mixed with the aqueous two-phase system according to the mass ratio of 1:30, and after sufficient shaking, centrifugation was performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclolinopeptide and the intermediate phase of protein and oil and the lower phase. The extraction rate of the cyclolinopeptide was 80%, and the distribution coefficient was 0.61.

[0056] The upper phase rich in cyclolinopeptide was separated, and after removal of the solvent, the crude purified product of the cyclolinopeptide of flaxseed with high salt content was obtained. After sufficient shaking or stirring with water, the precipitate was separated by centrifugation, and the precipitate was repeatedly washed with water for 2-3 times. The precipitate was freeze-dried to remove water to obtain the powder of the cyclolinopeptide of flaxseed with a purity of 81.0%. The supernatant of the first water washing was adjusted to pH 1.5, and after centrifugation, the floating and precipitate were collected and added to the extract of the cyclolinopeptide of flaxseed, and the aqueous two-phase purification was performed again. By measuring the content of the cyclolinopeptide remaining in the water phase, the loss rate of the cyclolinopeptide after water washing was about 20%.

[0057] Example 3

[0058] The aqueous two-phase system was preferably composed of methanol and K2HPO4salt solution. The mass fraction of K2HPO4in the aqueous two-phase system was 16%, and the pH of the salt solution was adjusted to 9.3; the mass fraction of methanol in the aqueous two-phase system was 44%.

[0059] The extract of the cyclolinopeptide of flaxseed was mixed with the aqueous two-phase system according to the mass ratio of 1:60, and after sufficient shaking, centrifugation was performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclolinopeptide and the intermediate phase of protein and oil and the lower phase. The extraction rate of the cyclolinopeptide was 95%, and the distribution coefficient was 2.2.

[0060] The upper phase rich in cyclic peptide is separated and the solvent is removed to obtain the crude purified product of flaxseed cyclic peptide with high salt content. After being fully shaken or stirred with water, the precipitate is separated by centrifugation and the precipitate is washed with water for 2-3 times. The precipitate is freeze-dried to remove water to obtain the flaxseed cyclic peptide powder with a purity of 78.9%. The supernatant of the first water washing is adjusted to pH 0.5, and the floating and precipitate are collected after centrifugation and added to the flaxseed cyclic peptide extract for re-purification by the aqueous two-phase system. The loss rate of cyclic peptide after water washing is about 5% by determining the residual cyclic peptide content in the aqueous phase.

[0061] Example 4

[0062] The aqueous two-phase system is preferably composed of methanol and K2HPO4 salt solution. The mass fraction of K2HPO4 in the aqueous two-phase system is 16%, and the pH of the salt solution is adjusted to 9.3; the mass fraction of methanol in the aqueous two-phase system is 44%.

[0063] The flaxseed cyclic peptide extract is mixed with the aqueous two-phase system at a mass ratio of 1:45, fully shaken, and then centrifuged at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptide and the intermediate phase of protein and oil and the lower phase. The extraction rate of cyclic peptide is 85%, and the partition coefficient is 1.5.

[0064] The upper phase rich in cyclic peptide is separated and the solvent is removed to obtain the crude purified product of flaxseed cyclic peptide with high salt content. After being fully shaken or stirred with water, the precipitate is separated by centrifugation and the precipitate is washed with water for 2-3 times. The precipitate is freeze-dried to remove water to obtain the flaxseed cyclic peptide powder with a purity of 78.9%. The supernatant of the first water washing is adjusted to pH 0.5, and the floating and precipitate are collected after centrifugation and added to the flaxseed cyclic peptide extract for re-purification by the aqueous two-phase system. The loss rate of cyclic peptide after water washing is about 5% by determining the residual cyclic peptide content in the aqueous phase.

[0065] Example 5

[0066] The defatted flaxseed powder is mixed with 12 times the weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and then the solvent is removed to obtain the flaxseed cyclic peptide extract. The content of flaxseed cyclic peptide is 0.2%-0.5%.

[0067] The aqueous two-phase system is preferably composed of methanol and K2HPO4 salt solution. The mass fraction of K2HPO4 in the aqueous two-phase system is 5%, and the pH of the salt solution is adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system is 50%.

[0068] The flaxseed cyclic peptide extract is mixed with the aqueous two-phase system at a mass ratio of 1:60, fully shaken, and then centrifuged at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptide and the intermediate phase of protein and oil and the lower phase. The extraction rate of cyclic peptide is 80%.

[0069] The upper phase rich in cyclopeptides is separated and the solvent is removed to obtain a crude purified product of flaxseed cyclopeptides with a high salt content. After being shaken or stirred with water, the precipitate is separated by centrifugation, and the precipitate is washed with water for 2-3 times. The precipitate is freeze-dried to remove water to obtain a flaxseed cyclopeptide powder with a purity of 41.0%. The supernatant of the first water washing is adjusted to pH 1, and the floating matter and the precipitate are collected after centrifugation and added to the flaxseed cyclopeptide extract for re-purification by aqueous two-phase system. The loss rate of cyclopeptides after water washing is about 10% by determining the residual cyclopeptide content in the aqueous phase.

[0070] Example 6

[0071] The defatted flaxseed powder is mixed with 12 times the weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and the solvent is removed to obtain a flaxseed cyclopeptide extract. The flaxseed cyclopeptide content is 0.2%-0.5%.

[0072] The aqueous two-phase system is composed of methanol and K2HPO4 salt solution. The mass fraction of K2HPO4 in the aqueous two-phase system is 50%, and the pH of the salt solution is adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system is 10%.

[0073] The flaxseed cyclopeptide extract is mixed with the aqueous two-phase system at a mass ratio of 1:60, shaken thoroughly, and centrifuged at 5000 rpm for 10 min to obtain an upper phase rich in cyclopeptides and an intermediate phase of protein and oil and a lower phase. The extraction rate of cyclopeptides is 35%.

[0074] The upper phase rich in cyclopeptides is separated and the solvent is removed to obtain a crude purified product of flaxseed cyclopeptides with a high salt content. After being shaken or stirred with water, the precipitate is separated by centrifugation, and the precipitate is washed with water for 2-3 times. The precipitate is freeze-dried to remove water to obtain a flaxseed cyclopeptide powder with a purity of 41.0%. The supernatant of the first water washing is adjusted to pH 1, and the floating matter and the precipitate are collected after centrifugation and added to the flaxseed cyclopeptide extract for re-purification by aqueous two-phase system. The loss rate of cyclopeptides after water washing is about 10% by determining the residual cyclopeptide content in the aqueous phase.

[0075] Example 7

[0076] The defatted flaxseed powder is mixed with 12 times the weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and the solvent is removed to obtain a flaxseed cyclopeptide extract. The flaxseed cyclopeptide content is 0.2%-0.5%.

[0077] The aqueous two-phase system is composed of methanol and K2HPO4 salt solution. The mass fraction of K2HPO4 in the aqueous two-phase system is 50%, and the pH of the salt solution is adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system is 10%.

[0078] The above linseed cyclic peptide extract was mixed with the aqueous two-phase system according to a mass ratio of 1:15, and after sufficient shaking, centrifugation was performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptides and the middle phase of proteins and oil and the lower phase. The extraction rate of cyclic peptides was 55%.

[0079] The above upper phase rich in cyclic peptides was separated, and after removal of the solvent, a crude purified product of linseed cyclic peptides with a relatively high salt content was obtained. After sufficient shaking or stirring with water, the precipitate was separated by centrifugation, and the precipitate was subjected to water washing operation repeatedly for 2-3 times. The precipitate was freeze-dried to remove water, and a linseed cyclic peptide powder with a purity of 68.7% was obtained. The supernatant of the first water washing was adjusted to pH 1, and after centrifugation, the floating material and the precipitate were collected and added to the linseed cyclic peptide extract for re-purification by the aqueous two-phase system. By determining the residual cyclic peptide content in the aqueous phase, the loss rate of cyclic peptides after water washing was about 10%.

[0080] Example 8

[0081] The defatted linseed powder was mixed with 12 times weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and after removal of the solvent, a linseed cyclic peptide extract was obtained. The linseed cyclic peptide content was 0.2%-0.5%.

[0082] The aqueous two-phase system was preferably composed of methanol and K2HPO4 salt solution. The mass fraction of K2HPO4 in the aqueous two-phase system was 14%, and the pH of the salt solution was adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system was 46%.

[0083] The above linseed cyclic peptide extract was mixed with the aqueous two-phase system according to a mass ratio of 1:60, and after sufficient shaking, centrifugation was performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptides and the middle phase of proteins and oil and the lower phase. The extraction rate of cyclic peptides was 92%.

[0084] The above upper phase rich in cyclic peptides was separated, and after removal of the solvent, a crude purified product of linseed cyclic peptides with a relatively high salt content was obtained. After sufficient shaking or stirring with water, the precipitate was separated by centrifugation, and the precipitate was subjected to water washing operation repeatedly for 2-3 times. The precipitate was freeze-dried to remove water, and a linseed cyclic peptide powder with a purity of 79.5% was obtained. The supernatant of the first water washing was adjusted to pH 13.0, and after centrifugation, there was no obvious floating material and precipitate. By determining the residual cyclic peptide content in the aqueous phase, the loss rate of cyclic peptides after water washing was about 60%.

[0085] Example 9

[0086] The defatted linseed powder was mixed with 12 times weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and after removal of the solvent, a linseed cyclic peptide extract was obtained. The linseed cyclic peptide content was 0.2%-0.5%.

[0087] The methanol and K2HPO4 salt solution form the aqueous two-phase system. The mass fraction of K2HPO4 in the aqueous two-phase system is 10%, and the pH of the salt solution is adjusted to 9.9; the mass fraction of methanol in the aqueous two-phase system is 46%.

[0088] The above-mentioned flaxseed cyclic peptide extract is mixed with the aqueous two-phase system at a mass ratio of 1:60, and after sufficient shaking, centrifugation is performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptides and the middle phase of proteins and oils and the lower phase. The extraction rate of cyclic peptides is 75%.

[0089] The upper phase rich in cyclic peptides is separated, and after removal of the solvent, a crude purified product of flaxseed cyclic peptides with a high salt content is obtained. After sufficient shaking or stirring with water, the precipitate is separated by centrifugation, and the precipitate is repeatedly washed with water for 2-3 times. The precipitate is freeze-dried to remove water, and a flaxseed cyclic peptide powder with a purity of 80.2% is obtained. The supernatant of the first water washing is adjusted to pH 9.0, and after centrifugation, there is no obvious floating material and precipitate. By determining the residual cyclic peptide content in the aqueous phase, the loss rate of cyclic peptides after water washing is about 50%.

[0090] Example 10

[0091] The defatted flaxseed powder is mixed with 12 times the weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and after removal of the solvent, a crude flaxseed cyclic peptide extract is obtained. The flaxseed cyclic peptide content is 0.2%-0.5%.

[0092] The methanol and K2HPO4 salt solution form the aqueous two-phase system. The mass fraction of K2HPO4 in the aqueous two-phase system is 14%, and the pH of the salt solution is adjusted to 9.9; the mass fraction of methanol in the aqueous two-phase system is 46%.

[0093] The above-mentioned flaxseed cyclic peptide extract is mixed with the aqueous two-phase system at a mass ratio of 1:60, and after sufficient shaking, centrifugation is performed at 5000 rpm for 10 min to obtain the upper phase rich in cyclic peptides and the middle phase of proteins and oils and the lower phase. The extraction rate of cyclic peptides is 87%.

[0094] The upper phase rich in cyclic peptides is separated, and after removal of the solvent, a crude purified product of flaxseed cyclic peptides with a high salt content is obtained. After sufficient shaking or stirring with water, the precipitate is separated by centrifugation, and the precipitate is repeatedly washed with water for 2-3 times. The precipitate is freeze-dried to remove water, and a flaxseed cyclic peptide powder with a purity of 80.4% is obtained. The supernatant of the first water washing is adjusted to pH 3.0, and after centrifugation, there is no obvious floating material and precipitate. By determining the residual cyclic peptide content in the aqueous phase, the loss rate of cyclic peptides after water washing is about 55%.

[0095] Example 11

[0096] The defatted flaxseed powder was mixed with 12 times weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and then the solvent was removed to obtain the flaxseed cyclic peptide extract. The content of flaxseed cyclic peptide in the extract was 0.2%-0.5%.

[0097] Preferably, the methanol and K2HPO4 salt solution form the aqueous two-phase system. The mass fraction of K2HPO4 in the aqueous two-phase system is 14%, and the pH of the salt solution is adjusted to 8.7; the mass fraction of methanol in the aqueous two-phase system is 46%.

[0098] The flaxseed cyclic peptide extract was mixed with the aqueous two-phase system at a mass ratio of 1:60, and then fully shaken. After centrifugation at 5000 rpm / min for 10 min, the upper phase rich in cyclic peptides, the intermediate phase of protein and oil, and the lower phase were obtained. The extraction rate of cyclic peptides was 92%.

[0099] The upper phase rich in cyclic peptides was separated, and the solvent was removed to obtain a crude purified product of flaxseed cyclic peptides with a high salt content. After fully shaking or stirring with acidic water (pH=1.0), the precipitate was separated by centrifugation, and the precipitate was repeatedly washed with water for 2-3 times. After freeze-drying to remove water, a flaxseed cyclic peptide powder with a purity of 57.4% was obtained, which was a deep yellow granular substance.

[0100] Comparative Example 1

[0101] The defatted flaxseed powder was mixed with 12 times weight volume ratio of 95% ethanol, stirred at 50°C for 1 h, and then the solvent was removed to obtain the flaxseed cyclic peptide extract. The extract was fully shaken with acetone, and then centrifuged at 4000 rpm / min for 1 min at 4°C. The supernatant was separated, and the solvent was removed to obtain an oily purified product, in which the content of cyclic peptides was 11.9%.

[0102] Comparative Example 2

[0103] The defatted flaxseed powder was mixed with 12 times weight volume ratio of 95% ethanol, stirred at 60°C for 1 h, and then the solvent was removed to obtain the flaxseed cyclic peptide extract. The flaxseed cyclic peptide extract was mixed with the two-phase solvent (n-hexane, 60% methanol) at a ratio of 1:60, fully shaken, and then centrifuged at 10000 rpm / min for 10 min. The upper phase was separated, and the solvent was removed. The residue was freeze-dried to remove water, and a brownish paste-like purified product of flaxseed cyclic peptides with a cyclic peptide content of 57.4% was obtained.

[0104] In summary, due to the strong hydrophobicity of cyclic peptides, the methanol-K2HPO4 aqueous two-phase system is effective in purifying cyclic peptides from the extract of flaxseed cyclic peptides; it can also effectively remove most of the proteins, oils and some water-soluble impurities in the extract; water washing can effectively remove the residual salt ions, pigments, lipids and other impurities in the crude purified product, but due to the presence of a small amount of surface-active components and lipid components in the crude purified product, an oil-in-water system may be formed in water, resulting in a high loss of cyclic peptides during water washing; by adjusting the pH of the supernatant after water washing, the stability of the system can be effectively destroyed, resulting in the formation of precipitates and floaters, which can be collected by centrifugation and subjected to aqueous two-phase extraction again, thereby effectively reducing the loss of cyclic peptides during water washing.

[0105] Due to the strong hydrophobicity of cyclic peptides, the methanol-K2HPO4 aqueous two-phase system is effective in purifying cyclic peptides from the extract of flaxseed cyclic peptides; although the methanol content is too high and the salt content is low, the extraction rate of cyclic peptides is high, but the water content in the methanol phase increases, making it difficult to remove the solvent; if the salt content is too high and the methanol content is low, the extraction effect of cyclic peptides is weak. By adjusting the mass fraction of salt and methanol in the aqueous two-phase system, the extraction effect of cyclic peptides can be effectively improved, and the difficulty of solvent removal can be reduced.

[0106] Water washing for salt removal can effectively remove the residual salt ions, pigments, lipids and other impurities in the crude purified product, but the loss rate of cyclic peptides is relatively high. Although acidic water can effectively destroy the stability of the system, other impurities will also be released at the same time, therefore, it is recommended to first perform conventional water washing, precipitate part of the cyclic peptides and separate them, and then adjust the pH to precipitate the residual cyclic peptides in water, and then perform aqueous two-phase extraction again. This method can not only reduce the loss of cyclic peptides during water washing, but also increase the purity of cyclic peptides from 54.7% to 82.2%.

[0107] Compared with the n-hexane:60% methanol system, this method has low solvent cost, high purity of cyclic peptides and low loss of cyclic peptides; under the optimal conditions, the loss rate is less than 10%; compared with column chromatography, this method is simple to operate, has complex processing system, short processing time, low cost and small environmental hazards, but the purity of cyclic peptides is slightly lower; compared with the extraction of cyclic peptides from simple oil using 95% ethanol, this method has no special requirements for raw materials and is also suitable for the extraction of cyclic peptides from pressed meal, has low raw material cost and higher purity than the former 50%.

[0108] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, which should be covered by the claims of the present application.

Claims

1. A method for separating and purifying flaxseed cyclic peptide based on a two-water-phase system, characterized in that, The method comprises the following steps: 1) constructing a two-water-phase system: mixing a small-molecule alcohol and a salt solution, adjusting pH with acid and alkali, and obtaining a two-water-phase system; 2) purifying a flaxseed cyclic peptide extract with the two-water-phase system: mixing the two-water-phase system with a flaxseed cyclic peptide extract, separating the phases, taking the upper phase, removing the solvent, and obtaining a crude purified flaxseed cyclic peptide product; 3) water-washing the crude purified flaxseed cyclic peptide product to remove salt: mixing the crude purified flaxseed cyclic peptide product with water at a mass ratio of 1:(1-100), removing salt, centrifuging to obtain supernatant and precipitate, removing water from the precipitate, and obtaining a target purified flaxseed cyclic peptide powder; 4) adjusting the pH of the supernatant obtained by centrifugation to a value at which no more precipitate and floating matter is generated, centrifuging to obtain precipitate, adding the precipitate to the flaxseed cyclic peptide extract, and purifying the flaxseed cyclic peptide extract with the two-water-phase system again, i.e., step 2); In step 1), the small-molecule alcohol is methanol, and the salt solution is a potassium phosphate dibasic solution. In the two-water-phase system, the mass fraction of K2HPO4 is 14%-18%, the mass fraction of methanol is 44%-46%, and the pH is 8.7-9.

3. In step 4), the pH of the supernatant is adjusted to 0-2.

2. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to claim 1, characterized in that, In step 2), the flaxseed cyclic peptide extract and the two-water-phase system are mixed at a mass ratio of 1:(1-120).

3. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to claim 2, characterized in that, In step 2), the flaxseed cyclic peptide extract and the two-water-phase system are mixed at a mass ratio of 1:(30-60).

4. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to claim 1, characterized in that, In step 3), the desalting step is water-washing, and the water-removing step is freeze-drying.

5. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to any one of claims 1-4, characterized in that, The extraction rate of the cyclic peptide is ≥80%.

6. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to any one of claims 1-4, characterized in that, The loss rate of the cyclic peptide is ≤10%.

7. The aqueous two-phase system based separation and purification method of flaxseed cyclotripeptides according to any one of claims 1-4, characterized in that, The purity of the cyclic peptide is ≥78%.

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