A method for recovering high purity natural betaine from sugar beet molasses

Abstract

The application relates to a method for recovering high-purity natural betaine from sugar beet molasses. The method is used for solving the problems of low betaine extraction purity, complex process and high cost in the prior art, and is characterized in that: a chromatographic column is built, a strong acid cation exchange resin column is sequentially eluted with 5% inorganic salt solution and deionized water; the diluted sugar beet molasses is injected into a ligand exchange chromatography column, eluted with deionized water, and the effluent part of the betaine is collected; the concentration of the betaine is concentrated to 100-500 mg / mL; semi-preparative high performance liquid chromatography purification is carried out, the betaine solution is collected; after concentration, freeze-drying is carried out, and the betaine crystal is obtained. The orthogonality between the two chromatographies is adopted, impurities which are difficult to purify are eliminated, and the chromatographic purity of the betaine is improved to 99.5%. The application is applied to the field of separation and purification of bioactive substances in sugar beet molasses.

Description

Technical Field

[0001] This invention relates to a method for recovering high-purity natural betaine from beet molasses. Background Technology

[0002] Beet molasses is a byproduct of sugar production; it's the concentrated beet juice that can no longer be crystallized to produce sucrose. Notably, beet molasses contains 4%–8% betaine by dry weight, concentrating almost all the betaine in beets, making it one of the richest betaine resources in nature. However, currently, a large amount of molasses is only used for fermentation to produce alcohol or as livestock feed, and its deeper value has not been fully explored. If high-purity natural betaine could be extracted efficiently and economically, it would not only turn waste into treasure and improve the overall economic benefits of the sugar industry chain, but also align with the sustainable development concepts of green chemistry and a circular economy.

[0003] Betaine, chemically known as trimethylglycine, is a naturally occurring zwitterionic compound widely found in plants and animals. It was first discovered and named in beet juice in the 19th century. Betaine is an important osmotic regulator and methyl donor in living organisms. In modern industry and the health industry, betaine demonstrates enormous application potential. It plays a vital role as a highly effective feed additive in aquaculture and livestock farming, contributing to stress reduction, muscle growth promotion, and immune enhancement. Furthermore, due to its protective effects on the human liver, cardiovascular system, and nervous system, it is used in pharmaceuticals and functional foods. As a highly effective moisturizer and a relatively mild ionic surfactant, it is also highly favored in cosmetics and chemical industries.

[0004] Currently, commercial betaine is mainly produced through chemical synthesis. Although this method is mature, it has disadvantages such as high raw material costs, high environmental risks, and products that do not have a "natural" label.

[0005] Existing methods for extracting betaine from natural sources mostly employ single purification techniques such as ion exchange and crystallization, making it difficult to achieve efficient and low-cost large-scale production while ensuring high purity. In particular, beet molasses has a complex composition, containing a large amount of sugars, pigments, salts, and other impurities, which traditional methods struggle to effectively separate from betaine, resulting in low product purity and unsatisfactory recovery rates. Summary of the Invention

[0006] The purpose of this invention is to solve the problems of low purity, complex process and high cost of betaine extraction in the prior art, and to provide a method for recovering high purity natural betaine from beet molasses, so as to achieve the recovery of natural betaine with a purity of ≥99.5% from beet molasses.

[0007] The present invention discloses a method for recovering high-purity natural betaine from beet molasses:

[0008] 1. Column construction: Pack the chromatographic column with a strong acid cation exchange resin, with a column diameter-to-height ratio of 1:50 to 1:300, and then perform pretreatment to obtain the ligand exchange chromatography column;

[0009] II. Pretreatment of beet molasses: Dilute the beet molasses and filter it to obtain a pretreated solution;

[0010] III. One-dimensional ligand exchange chromatography: The pretreated solution was injected into the ligand exchange chromatography column. The column conditions were: 0.1~0.5 BV (bed volume) beet molasses injection, elution with deionized water at a flow rate of 0.5~4 BV / h (bed volume per hour) for 3~10 BV, and collection of the betaine eluent to obtain the collection solution.

[0011] IV. Preliminary Concentration: The collected liquid is concentrated by rotary evaporation to achieve a betaine concentration of 100-500 mg / mL, thus obtaining a concentrated liquid;

[0012] V. Preparative High Performance Liquid Chromatography Purification: The concentrate was purified by preparative high performance liquid chromatography to obtain a purified betaine solution.

[0013] 6. Crystallization and drying: The purified betaine solution is concentrated and then freeze-dried to obtain betaine.

[0014] The beneficial effects of this invention are:

[0015] 1. Using ligand exchange chromatography as a one-dimensional purification method with water as the eluent is low-cost and environmentally friendly, and can increase the purity of betaine from about 4% to more than 80%.

[0016] 2. Combining semi-preparative high-performance liquid chromatography (HPLC) as a two-dimensional purification method, its high resolution is used to further remove impurities such as sugars, salts, and pigments, with the final purity reaching over 99.5%.

[0017] 3. The process conditions are mild and do not require the use of toxic organic solvents, making it suitable for the green extraction of natural products.

[0018] 4. This invention achieves efficient conversion from waste molasses to high-value-added natural betaine, combining economic benefits with environmental friendliness. Attached Figure Description

[0019] Figure 1 This is a flowchart of the method for extracting high-purity betaine from beet molasses according to the present invention;

[0020] Figure 2 This is a schematic diagram of the method for extracting high-purity betaine from beet molasses according to the present invention;

[0021] Figure 3These are high-performance liquid chromatograms of beet molasses samples, samples purified by one-dimensional chromatography, and samples purified by two-dimensional chromatography.

[0022] Figure 4 This is a schematic diagram of high-purity natural betaine crystals;

[0023] Figure 5 This is the 1H NMR spectrum of high-purity betaine;

[0024] Figure 6 The product's carbon NMR spectrum. Detailed Implementation

[0025] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0026] Specific Implementation Method 1: This implementation method describes a method for recovering high-purity natural betaine from beet molasses:

[0027] 1. Column construction: Pack the chromatographic column with a strong acid cation exchange resin, with a column diameter-to-height ratio of 1:50 to 1:300, and then perform pretreatment to obtain the ligand exchange chromatography column;

[0028] II. Pretreatment of beet molasses: Dilute the beet molasses and filter it to obtain a pretreated solution;

[0029] III. One-dimensional ligand exchange chromatography: The pretreated solution was injected into the ligand exchange chromatography column. The column conditions were: 0.1~0.5 BV (bed volume) beet molasses injection, elution with deionized water at a flow rate of 0.5~4 BV / h (bed volume per hour) for 3~10 BV, and collection of the betaine eluent to obtain the collection solution.

[0030] IV. Preliminary Concentration: The collected liquid is concentrated by rotary evaporation to achieve a betaine concentration of 100-500 mg / mL, thus obtaining a concentrated liquid;

[0031] V. Preparative High Performance Liquid Chromatography Purification: The concentrate was purified by preparative high performance liquid chromatography to obtain a purified betaine solution.

[0032] 6. Crystallization and drying: The purified betaine solution is concentrated and then freeze-dried to obtain betaine.

[0033] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the strongly acidic cation exchange resin used in step one is LX1860 type strongly acidic cation exchange resin. Everything else is the same as in Specific Implementation Method One.

[0034] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the pretreatment described in step one involves sequentially rinsing the strongly acidic cation exchange resin column with a 5% (w / w) inorganic salt solution and deionized water. Everything else is the same as in Specific Implementation Method One or Two.

[0035] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the rinsing is performed by first rinsing the resin bed with an inorganic salt solution at a volume of 5-10 BV and a flow rate of 0.5-4 BV / h for 8 hours, followed by rinsing with deionized water at the same flow rate for 8 hours. Everything else is the same as in Specific Implementation Methods One to Three.

[0036] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the inorganic salt solution is a nickel chloride solution, a magnesium chloride solution, or a copper chloride solution. Everything else is the same as in Specific Implementation Methods One to Four.

[0037] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: in step two, the beet molasses is diluted by 1:1 and then filtered through quantitative filter paper. Everything else is the same as in Specific Implementation Methods One to Five.

[0038] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the concentration is carried out at a temperature of 60°C in step four. Everything else is the same as in Specific Implementation Methods One to Six.

[0039] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: in step five, 50-1000 μL of the concentrated solution is injected into a semi-preparative high-performance liquid chromatography (HPLC) column. The chromatographic column is a semi-preparative HPLC column packed with amino silica gel (250:20 mm). The eluent is a 60%-90% (v / v) acetonitrile aqueous solution, the elution flow rate is 5-30 mL / min, and the betaine peak time is 11-15 min. The purified betaine solution is collected. Everything else is the same as in Specific Implementation Methods One to Seven.

[0040] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that the concentration described in step six is ​​achieved by vacuum rotary evaporation concentration with the temperature controlled at 40–80°C and the gas pressure controlled at 10 kPa–100 kPa. Everything else is the same as in Specific Implementation Methods One to Eight.

[0041] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One to Nine in that the freeze-drying described in step six is ​​carried out under vacuum conditions, with the temperature lowered to -50~-40℃ and then heated to -20~40℃, while maintaining the vacuum degree at 10~100 Pa, and the freezing time is 1~3 days. Everything else is the same as in Specific Implementation Methods One to Nine.

[0042] The beneficial effects of the present invention are verified using the following embodiments:

[0043] Example 1: A method for recovering high-purity natural betaine from beet molasses, comprising the following steps:

[0044] 1. Column setup: Pack the column with LX1860 resin at a diameter-to-height ratio of 1:100. Elute the bed with 9-10 times bed volume (BV) of 5% nickel chloride solution at a flow rate of 2 BV / h for 8 h. Then pretreat with deionized water at the same flow rate for the same duration to obtain the ligand exchange chromatography column.

[0045] II. Pretreatment of beet molasses: The beet molasses was diluted by 1:1, and the diluted beet molasses was filtered through quantitative filter paper to obtain the pretreated solution;

[0046] III. One-dimensional ligand exchange chromatography: The pretreatment solution was injected into the ligand exchange chromatography column. The column conditions were: 0.3 BV beet molasses injection volume, elution with deionized water at a flow rate of 0.5 BV / h, elution of 5 BV, collection of the betaine eluent, and collection solution.

[0047] IV. Preliminary Concentration: Transfer the collected liquid to a rotary evaporator and concentrate it at 60°C until the betaine concentration is 300 mg / mL to obtain the concentrated liquid.

[0048] V. Two-dimensional semi-preparative high-performance liquid chromatography purification: 100 μL of the concentrate was injected into a semi-preparative high-performance liquid chromatography column (250:20 mm) with amino silica gel column packing. The concentration of acetonitrile as eluent was 75%, and the elution flow rate was 10 mL / min. The purified betaine solution was collected.

[0049] VI. Crystallization and Drying: The purified betaine solution was concentrated by rotary evaporation under reduced pressure at 60℃ and 100 Pa, followed by freeze drying. The conditions were: programmed cooling in a vacuum freeze dryer, maintaining a vacuum of 100 Pa, and lowering the temperature to -50℃. The frozen material was then reheated to -30℃ for 3 days. 20 mg of white crystalline betaine with a purity of 99.563% was obtained.

[0050] Figure 3 These are high-performance liquid chromatograms of beet molasses samples, samples purified by one-dimensional chromatography, and samples purified by two-dimensional chromatography. Figure 4 This is a schematic diagram of high-purity natural betaine crystals; Figure 5 This is the 1H NMR spectrum of high-purity betaine; Figure 6 The image shows the carbon NMR spectrum of the product, illustrating that this embodiment yielded high-purity natural betaine.

[0051] Example 2: A method for recovering high-purity natural betaine from beet molasses, comprising the following steps:

[0052] A method for recovering high-purity natural betaine from beet molasses includes the following steps:

[0053] 1. Column construction: The column was packed with LX1860 resin with a diameter-to-height ratio of 1:120. The bed was eluted with 9-10 BV of 5% magnesium chloride solution at a flow rate of 0.5 BV / h for 8 h. The column was then pretreated with deionized water at the same flow rate for the same duration to obtain the ligand exchange chromatography column.

[0054] II. Pretreatment of beet molasses: The beet molasses was diluted by 1:1, and the diluted beet molasses was filtered through quantitative filter paper to obtain the pretreated solution;

[0055] III. One-dimensional ligand exchange chromatography: The pretreatment solution was injected into the ligand exchange chromatography column. The column conditions were: 0.1 BV beet molasses injection, elution with deionized water at a flow rate of 0.5 BV / h, elution of 5 BV, collection of the betaine eluent, and collection solution.

[0056] IV. Preliminary Concentration: Transfer the collected liquid to a rotary evaporator and concentrate it at 60°C until the betaine concentration is 200 mg / mL to obtain the concentrated liquid.

[0057] V. Two-dimensional semi-preparative high-performance liquid chromatography purification: 80 μL of the concentrate was injected into a semi-preparative high-performance liquid chromatography column (250:20 mm) with amino silica gel column packing. The concentration of acetonitrile as eluent was 85%, and the elution flow rate was 5 mL / min. The purified betaine solution was collected.

[0058] VI. Crystallization and Drying: The purified betaine solution was concentrated by rotary evaporation under reduced pressure at 50℃ and 100 Pa, followed by freeze drying. The conditions were: programmed cooling in a vacuum freeze dryer, maintaining a vacuum of 100 Pa, and lowering the temperature to -50℃. The frozen material was then reheated to -30℃ for 3 days. White crystalline betaine with a purity of 99.578% was obtained.

[0059] Example 3: A method for recovering high-purity natural betaine from beet molasses, comprising the following steps:

[0060] 1. Column construction: The column was packed with LX1860 resin with a diameter-to-height ratio of 1:150. The bed was eluted with 9-10 BV of 5% copper chloride solution at a flow rate of 0.5 BV / h for 8 h. The column was then pretreated with deionized water at the same flow rate for the same duration to obtain the ligand exchange chromatography column.

[0061] II. Pretreatment of beet molasses: The beet molasses was diluted by 1:1, and the diluted beet molasses was filtered through quantitative filter paper to obtain the pretreated solution;

[0062] III. One-dimensional ligand exchange chromatography: The pretreated solution was injected into the ligand exchange chromatography column. The column conditions were: 0.1 BV beet molasses injection, elution with deionized water at a flow rate of 0.5 BV / h, elution of 5 BV, collection of the betaine eluent, and collection solution.

[0063] IV. Preliminary Concentration: Transfer the collected liquid to a rotary evaporator and concentrate it at 60°C until the betaine concentration is 200 mg / mL to obtain the concentrated liquid.

[0064] V. Two-dimensional semi-preparative high-performance liquid chromatography purification: 80 μL of the concentrate was injected into a semi-preparative high-performance liquid chromatography column (250:20 mm) with amino silica gel column packing. The concentration of acetonitrile as eluent was 85%, and the elution flow rate was 5 mL / min. The purified betaine solution was collected.

[0065] VI. Crystallization and Drying: The purified betaine solution was concentrated by rotary evaporation under reduced pressure at 50℃ and 100 Pa, followed by freeze drying. The conditions were: programmed cooling in a vacuum freeze dryer, maintaining a vacuum of 100 Pa, and lowering the temperature to -50℃. The frozen material was then reheated to -30℃ for 3 days. White crystalline betaine with a purity of 99.569% was obtained.

[0066] Example 4: A method for recovering high-purity natural betaine from beet molasses, comprising the following steps:

[0067] 1. Column setup: The column was packed with LX1860 resin with a diameter-to-height ratio of 1:200. The bed was eluted with 9-10 BV of 5% nickel chloride solution at a flow rate of 0.5 BV for 8 h. The column was then pretreated with deionized water at the same flow rate for the same duration to obtain the ligand exchange chromatography column.

[0068] II. Pretreatment of beet molasses: The beet molasses was diluted by 1:1, and the diluted beet molasses was filtered through quantitative filter paper to obtain the pretreated solution;

[0069] III. One-dimensional ligand exchange chromatography: The pretreated solution was injected into the ligand exchange chromatography column. The column conditions were: 0.1 BV beet molasses injection, elution with deionized water at a flow rate of 0.5 BV / h, elution of 5 BV, collection of the betaine eluent, and collection solution.

[0070] IV. Preliminary Concentration: Transfer the collected liquid to a rotary evaporator and concentrate it at 60°C until the betaine concentration is 300 mg / mL to obtain the concentrated liquid.

[0071] V. Two-dimensional semi-preparative high-performance liquid chromatography purification: 80 μL of the concentrate was injected into a semi-preparative high-performance liquid chromatography column (250:20 mm) with amino silica gel column packing. The concentration of acetonitrile as eluent was 75%, and the elution flow rate was 5 mL / min. The purified betaine solution was collected.

[0072] VI. Crystallization and Drying: The purified betaine solution was concentrated by rotary evaporation under reduced pressure at 50℃ and 100 Pa, followed by freeze drying. The conditions were: programmed cooling in a vacuum freeze dryer, maintaining a vacuum of 100 Pa, and lowering the temperature to -50℃. The frozen material was then reheated to -30℃ for 3 days. White crystalline betaine with a purity of 99.769% was obtained.

[0073] This invention brings significant economic benefits, has strong practicality, and can meet the market demand for natural and functional raw materials and products.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for recovering high-purity natural betaine from beet molasses, characterized in that, The method is as follows:

1. Column construction: Pack the chromatographic column with a strong acid cation exchange resin, with a column diameter-to-height ratio of 1:50 to 1:300, and then perform pretreatment to obtain the ligand exchange chromatography column; II. Pretreatment of beet molasses: Dilute the beet molasses and filter it to obtain a pretreated solution; III. One-dimensional ligand exchange chromatography: The pretreated solution was injected into the ligand exchange chromatography column. The column conditions were: injection volume of 0.1~0.5 BV beet molasses, elution with deionized water at a flow rate of 0.5~4 BV / h for 3~10 BV, and collection of the eluting portion of betaine to obtain the collection solution. IV. Preliminary Concentration: The collected liquid is concentrated by rotary evaporation to achieve a betaine concentration of 100-500 mg / mL, thus obtaining a concentrated liquid; V. Preparative High Performance Liquid Chromatography Purification: The concentrate was purified by preparative high performance liquid chromatography to obtain a purified betaine solution.

6. Crystallization and drying: The purified betaine solution is concentrated and then freeze-dried to obtain betaine.

2. The method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, The strong acid cation exchange resin used in step one is LX1860 type strong acid cation exchange resin.

3. The method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, The pretreatment described in step one involves sequentially rinsing the strongly acidic cation exchange resin column with a 5% (w / w) inorganic salt solution and deionized water.

4. The method for recovering high-purity natural betaine from beet molasses according to claim 3, characterized in that, The rinsing process involves first rinsing the resin bed with an inorganic salt solution at a volume of 5-10 BV and a flow rate of 0.5-4 BV / h for 8 hours, followed by rinsing with deionized water at the same flow rate for 8 hours.

5. A method for recovering high-purity natural betaine from beet molasses according to claim 3, characterized in that, The inorganic salt solution is a nickel chloride solution, a magnesium chloride solution, or a copper chloride solution.

6. The method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, In step two, the beet molasses is diluted by 1:1 and then filtered through quantitative filter paper.

7. The method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, In step four, concentration is carried out at a temperature of 60°C.

8. A method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, In step five, 50-1000 μL of the concentrated solution is injected into a semi-preparative high-performance liquid chromatography (HPLC) column. The HPLC column is a semi-preparative high-performance liquid chromatography column packed with amino silica gel. The eluent is acetonitrile with a volume percentage of 60%-90%. The elution flow rate is 5-30 mL / min. The peak time of betaine is 11-15 min. The purified betaine solution is then collected.

9. A method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, The concentration described in step six involves vacuum rotary evaporation concentration with the temperature controlled at 40~80℃ and the gas pressure controlled at 10~100 Pa.

10. A method for recovering high-purity natural betaine from beet molasses according to claim 1, characterized in that, The freeze-drying described in step six involves lowering the temperature to -50 to -40°C under vacuum conditions, then heating it to -20 to 40°C, maintaining the vacuum at 10 to 100 Pa, and freezing for 1 to 3 days.

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