Preparation method of Cyclo (Phe-Val)
Patent Information
- Application Number
- CN202510515423.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-29
AI Technical Summary
[0006]为了解决现有Cyclo(Phe-Val)制备方法存在的原料不易得到、制备工艺复杂以及白酒酿造副产物资源化利用等一系列问题，本发明提供从黄水中分离制备Cyclo(Phe-Val)的方法
[0031] 1. For the first time, the present invention isolates high-purity Cyclo(Phe-Val) from the by-product yellow water of Wuliang strong-flavor Baijiu, and develops a new source of Cyclo(Phe-Val).
Smart Images
Figure CN120383569A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of chemical analysis, and particularly relates to a preparation method of Cyclo(Phe-Val). Background Art
[0002] Cyclo(Phe-Val), also known as cyclo(phenylalanine-valine) in Chinese, CAS: 14474-71-6, is a cyclic dipeptide compound formed by cyclization of phenylalanine and valine through peptide bonds. The structural formula is as follows:
[0003]
[0004] Cyclo(Phe-Val) has a typical diketopiperazine (DKP) ring structure, with two hydrogen acceptors and two hydrogen donors, and is an important pharmacophore in medicinal chemistry, showing various biological activities such as antibacterial, antioxidant, and anticancer activities. At present, the main source of Cyclo(Phe-Val) is the microbial cells themselves and their metabolites, with the raw materials being difficult to obtain and the preparation cost being high.
[0005] Yellow water is a yellow or brownish-yellow viscous liquid produced during the process of removing the fermented grains and dripping the pits in the solid-state brewing of Baijiu. It is the fermentation product of microorganisms using grains as raw materials and is also a by-product of Baijiu brewing. Generally, about 300-400 kg of yellow water is produced for every 1000 kg of Daqu Baijiu produced, and the daily output of yellow water in a Daqu Baijiu factory with an annual output of 10,000 tons is about 10 t. The rich proteins, polysaccharides, and microbial flora in yellow water make it have a high biochemical oxygen demand (BOD) and chemical oxygen demand (COD). The pH of yellow water is about 2.5-4.2, the BOD reaches 25000-30000 mg / L, and the COD reaches 25000-40000 mg / L, far exceeding the national discharge standards for water pollutants in the fermentation alcohol and Baijiu industries. Considering the current background of "carbon neutrality", developing a resource utilization approach for the by-product yellow water in Baijiu brewing to reduce carbon emissions is an urgent problem to be solved. There are high-value small peptide compounds in yellow water. Extracting, separating, and purifying the small peptides in yellow water can increase the added value of yellow water, promote energy conservation and emission reduction in Baijiu production enterprises, and have certain economic and environmental benefits. Summary of the Invention
[0006] In order to solve a series of problems existing in the existing preparation method of Cyclo(Phe-Val), such as difficult availability of raw materials, complex preparation process, and resource utilization of Baijiu brewing by-products, the present invention provides a method for separating and preparing Cyclo(Phe-Val) from yellow water.
[0007] To achieve the above application objectives, the technical solutions adopted in this application are as follows:
[0008] In the first aspect, the present invention provides a preparation method of Cyclo(Phe-Val): It is separated from yellow water.
[0009] Among them, the said preparation method includes the following steps:
[0010] a. Take the supernatant of yellow water, dilute it with water, perform macroporous resin column chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a crude extract;
[0011] b. Perform gel column chromatography on the crude extract, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a crude product;
[0012] c. Purify the crude product by liquid chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a pure product.
[0013] Further, in step a:
[0014] The volume ratio of the supernatant of yellow water to water is 1:3 to 1:5.
[0015] The macroporous resin is HPD-450 macroporous adsorption resin.
[0016] Using ethanol-water as the elution reagent, elute in a gradient of the volume content of ethanol being 15%, 45%, 75%, and 100% in sequence.
[0017] Further, in step b:
[0018] The gel column uses Sephadex LH-20 dextran gel.
[0019] Using chloroform-methanol as the elution reagent, elute in a gradient at a ratio of v / v of 7:1, 1:1, and 1:100.
[0020] Further, in step c:
[0021] The stationary phase is a C18 column, model ZORBAX Eclipse Plus C18, 4.6×250mm, 5-Micron.
[0022] Using methanol (A) and water (D) as the elution reagent.
[0023] Preferably, gradient elution is adopted. More preferably, the gradient elution program is: 0 - 11.0min, 30 - 100% A; 11.1 - 13.0min, 100% A; 13.1 - 15min, 30% A; stop at 15min.
[0024] Collect the absorption peak at the ultraviolet absorption wavelength of 220 nm.
[0025] The flow rate is 0.8 - 1.2 mL / min, and the injection volume is 50 - 100 μL.
[0026] Further, in steps a and b, the concentration temperature is 40 - 60 °C.
[0027] Further, the yellow water is a yellow or brownish-yellow viscous liquid generated during the process of draining the pits after removing the fermented grains in the production of solid-state brewed Baijiu.
[0028] Preferably, the yellow water is a by-product generated in the production of Wuliang strong-flavor Baijiu. The "Wuliang" refers to sorghum, rice, glutinous rice, wheat, and corn.
[0029] In a second aspect, the present invention provides Cyclo(Phe-Val) prepared by the above preparation method.
[0030] Beneficial effects:
[0031] 1. For the first time, the present invention isolates high-purity Cyclo(Phe-Val) from the by-product yellow water of Wuliang strong-flavor Baijiu, and develops a new source of Cyclo(Phe-Val).
[0032] 2. The present invention prepares the bioactive peptide Cyclo(Phe-Val) with high added value from yellow water, develops a new way for the resource utilization of yellow water, improves the added value of yellow water, and also promotes energy conservation and emission reduction in Baijiu production enterprises.
[0033] 3. The present invention creatively combines separation techniques such as macroporous resin column chromatography, gel column chromatography, and liquid chromatography, and for the first time isolates and purifies Cyclo(Phe-Val) from the yellow water with complex components. The purity of Cyclo(Phe-Val) is above 98%. This method has easily available raw materials, a simple preparation method, and high product purity, providing more options for the resource utilization of yellow water and the preparation of Cyclo(Phe-Val). Description of the drawings
[0034] Figure 1 It is the secondary mass spectrometry diagram of Cyclo(Phe-Val) in the crude extract obtained in Example 1;
[0035] Figure 2 It is the total ion chromatogram (TIC) of the crude extract obtained in Example 1 and the extracted ion chromatogram (EIC) of Cyclo(Phe-Val);
[0036] Figure 3 It is the total ion chromatogram (TIC) of the crude product obtained in Example 1 and the extracted ion chromatogram (EIC) of Cyclo(Phe-Val);
[0037] Figure 4 UV absorption chromatogram at 220 nm wavelength during the purification of the crude product by liquid chromatography in Example 1;
[0038] Figure 5 Appearance diagram of the pure product of Cyclo(Phe-Val) obtained in Example 1. Detailed implementation manners
[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer and more understandable, the following further details this application in combination with implementation manners. Unless otherwise defined, all technical terms used herein have the same meanings understood by those of ordinary skill in the art.
[0040] The term "Cyclo(Phe-Val)", with the Chinese name cyclo(phenylalanine-valine), CAS: 14474-71-6, is a cyclic dipeptide compound formed by cyclizing phenylalanine and valine through a peptide bond. The structural formula is as follows:
[0041]
[0042] In one implementation manner of the present invention, a preparation method of Cyclo(Phe-Val) is first provided, which is separated from yellow water. Specifically, it includes the following steps:
[0043] a. Take the supernatant of yellow water, dilute it with water, perform macroporous resin column chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a crude extract;
[0044] b. Perform gel column chromatography on the crude extract, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a crude product;
[0045] c. Purify the crude product by liquid chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate it to obtain a pure product.
[0046] In the above implementation manner of the present invention, first, the yellow water needs to be left standing for a time ≥ 20 h to precipitate out as much starch granules and distillers' grains and other solids in the yellow water as possible to obtain the supernatant of yellow water. Since there are still a large amount of water-soluble proteins and polysaccharides in the supernatant of yellow water, which makes the yellow water highly viscous, it is difficult to effectively adsorb small molecules in the yellow water when directly loading onto the macroporous resin column chromatography. Therefore, it needs to be diluted with water, preferably deionized water, before loading onto the macroporous resin column. Macroporous adsorption resin column chromatography is used, and its principle is to adsorb organic compounds in the extraction solution and, according to the adsorption force and molecular weight of the compounds, achieve separation, purification, and impurity removal through elution with a certain solvent.
[0047] In a specific embodiment of the present invention, HPD-450 macroporous adsorption resin is selected. Because it has a certain adsorption capacity for medium-polar organic compounds, it can effectively adsorb and enrich small-molecule cyclic dipeptide compounds in yellow water, and separate them from macromolecular polysaccharides and proteins.
[0048] Then, gel column chromatography is adopted, and Sephadex LH-20 dextran gel is selected. This gel has both lipophilic and hydrophilic properties, and has the functions of gel filtration, partition chromatography and adsorption chromatography, and can separate molecules with similar structures. Using chloroform-methanol as the elution reagent, gradient elution is carried out at a ratio of v / v, 7:1, 1:1, 1:100, which can effectively separate Cyclo(Phe-Val) from weakly polar higher fatty acids and their esters in yellow water, as well as strongly polar amino acids and organic acids, and realize the enrichment and separation of Cyclo(Phe-Val) to obtain a crude product of Cyclo(Phe-Val) with a certain purity.
[0049] Then, liquid chromatography purification is adopted, and a small-particle reverse-phase C18 column is selected, which has a higher separation degree, and further elutes the components in the crude product of Cyclo(Phe-Val) in order of decreasing polarity. After investigation, the Cyclo(Phe-Val) peak (peak emergence time 6.68 min) in the crude product is close to the impurity peak (peak emergence time 6.89 min) ( Figure 3 ), using a chromatographic column (ZORBAX Eclipse Plus C18, 4.6×250 mm, 5-Micron), with methanol and water as the elution reagent, gradient elution is adopted, and the gradient elution program is: 0-11.0 min, 30-100% methanol; 11.1-13.0 min, 100% methanol; 13.1-15 min, 30% methanol; stop at 15 min, which can effectively separate the Cyclo(Phe-Val) peak from the impurity peak ( Figure 4 ). Based on the ultraviolet absorption peak at a wavelength of 220 nm and combined with the analysis results of high-performance liquid chromatography quadrupole time-of-flight mass spectrometry, the Cyclo(Phe-Val) absorption peak is collected to realize the purification of Cyclo(Phe-Val) in yellow water.
[0050] The method of the present invention is simple to operate, the organic reagents used can be recycled, the macroporous adsorption resin and dextran gel can be regenerated and reused, and the cost is low. Yellow water is rich in macromolecular polysaccharides and proteins. First, the components in yellow water need to be separated by molecular weight through macroporous resin column chromatography, and the enrichment of Cyclo(Phe-Val) is realized. Then, further enrichment and separation are carried out by gel column chromatography, and then liquid chromatography purification is carried out to realize the separation and preparation of high-purity Cyclo(Phe-Val).
[0051] Specific embodiments will be listed below to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments in terms of specific techniques or conditions, the techniques or conditions described in the literature in the art or according to the product specifications shall be followed. For the reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.
[0052] Example 1: Preparation of Cyclo(Phe-Val) from Huangshui
[0053] (1) Take 10 L of Huangshui, a by-product of Wuliangrongxiang Baijiu, let it stand for 20 h, take 8 L of the supernatant and put it into a 100 L barrel, add 40 L of deionized water for dilution, mix well and load it onto a purified macroporous resin column (HPD-450 macroporous adsorption resin) at a flow rate of 8 mL / min. Using ethanol-water as the eluent, with the volume content of ethanol being 15%, 45%, 75%, and 100% respectively, 15 L of each is used for gradient elution in turn. Collect the eluates of each section, and load the collected solutions of each section onto a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, with a UV detector at a wavelength of 220 nm and a mass spectrometry detector for simultaneous detection, to determine the 45% ethanol elution section (25 - 36# eluate) where the target compound Cyclo(Phe-Val) is located. Combine the 25 - 36# eluates, and concentrate them by vacuum distillation at 60 °C to obtain 24.36 g of the crude extract of Cyclo(Phe-Val). The total ion current chromatogram (TIC) of the crude extract and the extracted ion current chromatogram (EIC) of Cyclo(Phe-Val) are shown in Figure 2 , and the secondary mass spectrometry diagram of Cyclo(Phe-Val) is shown in Figure 1 (The molecular ion is 247.1442, the characteristic ion of valine is 72.0813, and the characteristic ion of phenylalanine is 120.0807).
[0054] (2) Take 20 g of the crude extract, dissolve it in 30 mL of chloroform-methanol (v / v, 7:1), filter and then perform gel column chromatography. The gel used is Sephadex LH-20 dextran gel. Using chloroform-methanol as the elution reagent, with the ratio of v / v being 7:1, 1:1, and 1:100 for gradient elution, and collect one tube every 30 mL. Load the collected solutions of each tube onto a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to determine the 102 - 111th collection tubes where the target compound Cyclo(Phe-Val) is located. Combine the eluates of the 102 - 111th collection tubes, and concentrate them by vacuum distillation at 40 °C to obtain 1.62 g of the crude product of Cyclo(Phe-Val). The total ion current TIC chromatogram of the crude product and the extracted ion current EIC chromatogram of Cyclo(Phe-Val) are as shown in Figure 3 shown.
[0055] (3) Dissolve the crude product obtained in step (2) in 5 mL of methanol, dilute it with 50 mL of ultrapure water, and then load it onto a liquid chromatograph. The chromatographic column is ZORBAX Eclipse Plus C18, 4.6×250 mm, 5-Micron. The flow rate is 0.8 mL / min, the injection volume is 50 μL. Use methanol (A) and ultrapure water (D) as the eluent for gradient elution: 0 - 11.0 min, 30 - 100% A; 11.1 - 13.0 min, 100% A; 13.1 - 15 min, 30% A; stop at 15 min. In the peak collection mode, collect the absorption peak at a wavelength of 220 nm. Determine the ultraviolet absorption peak of the target substance Cyclo(Phe-Val) by high performance liquid chromatography-mass spectrometry detection. Concentrate it under reduced pressure at 50 °C, recrystallize it with methanol, and dry it naturally to obtain 64 mg of pure Cyclo(Phe-Val). The ultraviolet absorption chromatogram at a wavelength of 220 nm during the purification of the crude product by liquid chromatography is as Figure 4 shown, and the pure product is in white crystalline form as Figure 5 shown.
[0056] (4) Weigh 1 mg of the pure Cyclo(Phe-Val) obtained in step (3) and dissolve it in methanol to a concentration of 0.1 mg / mL. Load it onto a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry for qualitative and quantitative analysis using the Cyclo(Phe-Val) standard. The result shows that the content of Cyclo(Phe-Val) in the pure product is 98.84%.
[0057] Example 2: Preparation of Cyclo(Phe-Val) from yellow water
[0058] (1) Take 10 L of yellow water, a by-product of Wuliangye strong-flavor liquor, let it stand for 20 h, take 8 L of the supernatant and put it into a 100 L barrel, add 24 L of deionized water for dilution. After mixing, load it onto a purified macroporous resin column (HPD-450 macroporous adsorption resin) with a flow rate of 8 mL / min. Use ethanol-water as the eluent, and perform gradient elution successively with 15 L each at the ratios of 15%, 45%, 75%, and 100% of the ethanol volume content. Collect each eluate fraction. Load each collected fraction onto a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and detect it simultaneously with a 220 nm wavelength ultraviolet detector and a mass detector to determine the 45% ethanol elution fraction (28 - 35# eluate) where the target compound Cyclo(Phe-Val) is located. Combine the 28 - 35# eluate and concentrate it by vacuum distillation at 60 °C to obtain 21.45 g of the crude extract of Cyclo(Phe-Val).
[0059] (2) Take 18 g of the crude extract, dissolve it in 30 mL of chloroform-methanol (v / v, 7:1), filter it, and then perform gel column chromatography. The gel used is Sephadex LH-20 dextran gel. Chloroform-methanol is used as the elution reagent, and gradient elution is carried out at a ratio of v / v, 7:1, 1:1, 1:100. Collect one tube every 30 mL. The collected liquid of each tube is respectively loaded onto high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to determine the 107th to 116th collection tubes where the target compound Cyclo(Phe-Val) is located. Combine the eluates of the 107th to 116th collection tubes, concentrate it by reduced pressure distillation at 40 °C to obtain 1.26 g of the crude product of Cyclo(Phe-Val).
[0060] (3) Dissolve the crude product obtained in step (2) in 5 mL of methanol, dilute it with 50 mL of ultrapure water, and then load it onto a liquid chromatograph. The chromatographic column is ZORBAX Eclipse Plus C18, 4.6×250 mm, 5-Micron, the flow rate is 1.2 mL / min, the injection volume is 100 μL, and gradient elution is carried out with methanol (A) and ultrapure water (D) as the elution agents. From 0 to 11.0 min, 30 - 100% A; from 11.1 to 13.0 min, 100% A; from 13.1 to 15 min, 30% A; stop at 15 min. In the peak collection mode, collect the absorption peak at a wavelength of 220 nm. High performance liquid chromatography-mass spectrometry is used to detect the ultraviolet absorption peak of the target substance Cyclo(Phe-Val), concentrate it by reduced pressure at 50 °C, recrystallize it with methanol, and dry it naturally to obtain 48 mg of the pure product of Cyclo(Phe-Val).
[0061] (4) Weigh 1 mg of the pure product of Cyclo(Phe-Val) obtained in step (3) and dissolve it in methanol to prepare a concentration of 0.1 mg / mL, and then load it onto high performance liquid chromatography-quadrupole time-of-flight mass spectrometry for qualitative and quantitative analysis with the Cyclo(Phe-Val) standard. The result shows that the content of Cyclo(Phe-Val) in the pure product is 98.12%.
Claims
1. A method for preparing cyclo(Phe-Val), characterized in that: Isolated from yellow water, including the following steps: a. Take the supernatant of yellow water, dilute it with water, perform macroporous resin column chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate to obtain a crude extract; b. Perform gel column chromatography on the crude extract, collect the eluate containing Cyclo(Phe-Val), and concentrate to obtain a crude product; c. Purify the crude product by liquid chromatography, collect the eluate containing Cyclo(Phe-Val), and concentrate to obtain a pure product.
2. The preparation method of Cyclo(Phe-Val) according to claim 1, characterized in that: In step a, the volume ratio of the supernatant of yellow water to water is 1:3 to 1:
5.
3. The preparation method of Cyclo(Phe-Val) according to claim 1 or 2, characterized in that: In step a, at least one of the following is satisfied: The macroporous resin is HPD-450 macroporous adsorption resin; Using ethanol-water as the elution reagent, elute in a gradient of 15%, 45%, 75%, and 100% ethanol volume content in sequence.
4. The preparation method of Cyclo(Phe-Val) according to any one of claims 1 to 3, characterized in that: In step b, at least one of the following is satisfied: The gel column uses Sephadex LH-20 dextran gel; Using chloroform-methanol as the elution reagent, v / v, elute in a gradient of 7:1, 1:1, and 1:
100.
5. The preparation method of Cyclo(Phe-Val) according to any one of claims 1 to 4, characterized in that: In step c, at least one of the following is satisfied: The liquid chromatography stationary phase is a C18 column, model ZORBAX Eclipse Plus C18, 4.6×250mm, 5-Micron; Using methanol and water as the elution reagent; Collect the absorption peak at an ultraviolet absorption wavelength of 220nm; The flow rate is 0.8 - 1.2 mL / min, and the injection volume is 50 - 100 μL.
6. The preparation method of Cyclo(Phe-Val) according to claim 5, characterized in that: Using methanol and water as the elution reagent, perform gradient elution, and the gradient elution program is: 0 - 11.0 min, 30 - 100% methanol; 11.1 - 13.0 min, 100% methanol; 13.1 - 15 min, 30% methanol; stop at 15 min.
7. The preparation method of Cyclo(Phe-Val) according to any one of claims 1 to 6, characterized in that: The concentration temperature is 40 - 60°C.
8. The preparation method of Cyclo(Phe-Val) according to any one of claims 1 to 7, characterized in that: The yellow water is a yellow or brownish-yellow viscous liquid produced during the process of draining the pits after removing the fermented grains in solid-state brewing of liquor.
9. The preparation method of Cyclo(Phe-Val) according to claim 8, characterized in that: The yellow water is a by-product produced in the brewing of Wuliangye strong-flavor liquor.
10. Cyclo(Phe-Val) prepared by the preparation method according to any one of claims 1 - 9.