Preparation method of high-content 3R, 3'S-dihydroxy-β-carotene

Abstract

The present application belongs to the field of 3R, 3'S-dihydroxy-β-carotene preparation, and particularly relates to a method for preparing high-content 3R, 3'S-dihydroxy-β-carotene. In order to overcome the problems of long conversion cycle, low content, low yield, and high cost of the obtained products in the prior art, the present invention provides a method for preparing high-content 3R, 3'S-dihydroxy-β-carotene, the lutein after saponification is directly isomerized, the filtering operation of the intermediate lutein is reduced, the whole process only produces mother liquor once, the operation process is simple, the present invention preferentially breaks the ester bond of lutein and turns it into lutein, and lutein is isomerized and transposed under the catalysis of malonic acid at a relatively mild temperature to produce 3R, 3'S-dihydroxy-β-carotene, reducing the loss of process lutein and final product 3R, 3'S-dihydroxy-β-carotene.

Description

Technical Field

[0001] The present application belongs to the field of 3R, 3'S-dihydroxy-β-carotene preparation, and in particular relates to a method for preparing high-content 3R, 3'S-dihydroxy-β-carotene. Background Art

[0002] 3R,3'S-dihydroxy-β-carotene is a type of dihydroxy-β-carotene. 3R,3'S-dihydroxy-β-carotene is also called zeaxanthin. It belongs to the isoprene class and is an oxygen-containing carotenoid. It is an isomer with lutein. Most of the zeaxanthin in nature is the all-trans isomer. 3R,3'S-dihydroxy-β-carotene is a new type of oil-soluble natural pigment. It is widely found in plants such as wolfberry, yellow corn, and pepper, but the content is low. In nature, it often coexists with lutein, β-carotene, cryptoxanthin, etc. to form a mixture of carotenoids.

[0003] At present, there are four ways to obtain 3R,3'S-dihydroxy-β-carotene. The first is to directly extract it from plants. However, the content of 3R,3'S-dihydroxy-β-carotene in plants is very low, the extraction steps are cumbersome, and a large amount of organic solvents are used, which has low economic value. The second is microbial fermentation. Since most microbial fermentation units are low, the content of 3R,3'S-dihydroxy-β-carotene in the fermentation broth is low, and the subsequent extraction is cumbersome and not suitable for industrial production. The third is the full chemical synthesis method. This method has many reaction steps, many harmful chemical reagents, low product yield, and the obtained 3R,3'S-dihydroxy-β-carotene is not conducive to human absorption. The fourth is to use chemical conversion to prepare 3R,3'S-dihydroxy-β-carotene, that is, to prepare 3R,3'S-dihydroxy-β-carotene through lutein diastereomerization. At present, there are some patents and literatures involving the method of separating lutein crystals from marigolds and preparing 3R,3'S-dihydroxy-β-carotene through diastereomerization.

[0004] Chinese patent CN117820184A discloses a method for efficiently preparing 3R, 3'S-dihydroxy-β-carotene from marigold oleoresin, wherein the saponified lutein is filtered and washed to obtain a lutein mixture, which is then nitrogen-displaced and pressurized at 100-130°C for 15-30 hours. The obtained lutein requires filtering, washing and purification steps, and the catalytic time is 15-30 hours. The whole process is cumbersome, the production cycle is long, and it is not conducive to industrial production.

[0005] Chinese patents CN101830841A, CN101182302A, CN107827800A and other patents all disclose different methods for preparing 3R, 3'S-dihydroxy-β-carotene from marigold oleoresin. The production methods generally have the problems of cumbersome overall operation and large amount of waste liquid, which cannot guarantee green industrial production. At the same time, the obtained product content is low and the production efficiency is low. Summary of the invention

[0006] In order to overcome the problems of long conversion cycle, low content, low yield and high cost of the obtained products in the prior art, the present invention provides an industrial method for preparing high-content 3R, 3'S-dihydroxy-β-carotene from marigold oleoresin, which solves the problem of large loss of lutein content due to long-term isomerization. The saponified lutein is directly isomerized, which reduces the filtering operation of the intermediate lutein. The whole process only produces one mother liquor, the operation process is simple, and the loss of 3R, 3'S-dihydroxy-β-carotene content is small. The solvent recovery is environmentally friendly, and the solvent is preferably ethanol, which is non-toxic and easy to recycle. In view of the degradation of lutein and 3R, 3'S-dihydroxy-β-carotene during the reaction process, the present invention uses high-pressure isomerization, that is, the ester bond of lutein is preferentially broken to become lutein. Lutein is isomerized and transposed to produce 3R, 3'S-dihydroxy-β-carotene under the catalytic action of malonic acid at a relatively mild temperature, reducing the loss of process lutein and final product 3R, 3'S-dihydroxy-β-carotene.

[0007] The present application provides a method for preparing high-content 3R, 3'S-dihydroxy-β-carotene, comprising the following steps:

[0008] S1, dissolving marigold oleoresin and phytosterol in a mass ratio of 1:0.1-1, heating to 71-80° C., and stirring to obtain a mixture;

[0009] S2, adding alkali solution to the mixture obtained in step S1 according to the weight ratio of marigold oleoresin to alkali solution of 1:0.4-0.8, and performing saponification reaction for 4.5-6 hours to obtain a mixture of lutein and higher fatty acid salts;

[0010] S3. According to the weight ratio of marigold oleoresin to malonic acid of 1:0.5-2, malonic acid is added to the product obtained in step S2 for catalytic reaction, and carbon dioxide is introduced for displacement protection at the same time. The reaction is pressurized to 0.3-1.2 MPa, and the temperature is 90-100° C. for 2-10 hours. Ethanol is added for dilution, and the reaction is carried out at 80-90° C. for high-speed stirring and crystallization for 2-10 hours. The reaction is filtered, washed, and dried by nitrogen displacement to finally obtain high-content 3R, 3'S-dihydroxy-β-carotene.

[0011] Furthermore, the alkali solution in step S2 is sodium hydroxide or potassium hydroxide with a mass fraction of 50-65%.

[0012] Furthermore, the mass fraction of ethanol in step S3 is 45%-55%.

[0013] Furthermore, the high-speed stirring in step S3 adopts a high-shear disperser with a rotation speed of 1450-2900 rpm.

[0014] Furthermore, the phytosterol in step S1 is one or more of β-sitosterol, stigmasterol, campesterol, campestanol and cholesterol.

[0015] Furthermore, the plant sterol in step S1 is stigmasterol.

[0016] Furthermore, the heating temperature in step S1 is 75°C.

[0017] Furthermore, the reaction temperature in step S3 is 90°C and the crystallization temperature is 85°C.

[0018] In the first step of the present invention, marigold oleoresin is mixed with phytosterols, and the mixture is heated and stirred to form a uniform mixture. This step utilizes the role of phytosterols as a cosolvent, which can reduce the melting point of lutein and improve its solubility, thereby promoting the effective performance of the subsequent hydrolysis reaction. In the second step, alkali solution is added to perform a saponification reaction, which is a key hydrolysis step. The ester bond is broken by the saponification reaction to release free lutein and higher fatty acid salts. The appropriate alkali solution ratio not only controls the reaction rate, but also ensures the purity of the final product, while the selected temperature range and time ensure that the reaction is completed under mild conditions to avoid product degradation caused by excessive heating.

[0019] In the third step, malonic acid was introduced as a catalyst, and carbon dioxide was introduced to protect the stability of the cis structure. The catalytic reaction lasted for 2-10 hours at 90-100°C, and then low-concentration ethanol was added for dilution and high-speed stirring for crystallization. Malonic acid played a dual role in this process: on the one hand, it could form non-covalent bonds with lutein and 3R, 3'S-dihydroxy-β-carotene, inhibit their thermal degradation, and improve the stability of these compounds at high temperatures; on the other hand, it lowered the melting point of lutein, improved its solubility, and increased the conversion rate. In addition, the use of carbon dioxide as a protective gas increased the system pressure, promoted the solubility of carbon dioxide in the system, and further stabilized the cis-lutein. Since carbon dioxide has a certain acidity, it can inhibit the occurrence of oxidation reactions to a certain extent, reduce the oxidation loss of cis-lutein during the conversion process, and thus increase the yield of the product. In addition, under the catalysis of malonic acid, carbon dioxide may form a weak interaction with the reactants or intermediates, stabilize the transition state or product, and thus improve the selectivity and yield of the target product 3R, 3'S-dihydroxy-β-carotene.

[0020] Overall, this application reduces the filtering operation of the intermediate lutein, simplifies the entire process, produces only one mother liquor, reduces the difficulty of waste liquid treatment, and meets the production requirements of green industrialization. And by using non-toxic, harmless and easily recyclable ethanol as a solvent, the environmental friendliness of the method is further enhanced. Compared with the prior art, this method not only improves the conversion efficiency of lutein to 3R, 3'S-dihydroxy-β-carotene, but also significantly reduces the production cycle, reduces costs, and makes large-scale industrial production possible.

[0021] The present application uses malonic acid to inhibit the thermal degradation of lutein and 3R, 3'S-dihydroxy-β-carotene during the isomerization process. Malonic acid can bind to lutein crystals and 3R, 3'S-dihydroxy-β-carotene through non-covalent bonds, thereby improving the stability of lutein and 3R, 3'S-dihydroxy-β-carotene at high temperatures, while reducing the melting point of lutein and improving the solubility of lutein. The present application introduces carbon dioxide as a replacement protective agent, which increases the system pressure, increases the solubility of carbon dioxide in the system, promotes the thermal motion of molecules, improves the stability of cis-lutein, reduces the oxidation of cis-lutein during the conversion process, improves the yield, and also improves production efficiency relative to the prior art. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention and its effects are further described below by specific examples. The following examples are only used to illustrate the content of the present invention and are not used to limit the protection scope of the present invention. Simple changes made to the present invention by applying the concept of the present invention are all within the scope of the protection claimed by the present invention.

[0023] The equipment used in the preparation method of the present invention can be the well-known equipment in the art. The raw materials used in the present invention are commercially available unless otherwise specified.

[0024] Example 1

[0025] Take 50g of marigold oleoresin with a content of 16.3% and 10g of β-sitosterol, mix and heat to 72°C, then add 50g of 50wt% potassium hydroxide solution, saponify for 4h, add 30g of malonic acid to the reaction solution, and pressurize to 0.9Mpa with carbon dioxide at 95°C for 6h. After dilution with ethanol solvent, use a high shear disperser at 80°C and set the speed to 1450 rpm for high-speed stirring crystallization for 3h. Filter, wash and dry at a constant temperature of 65°C to finally obtain 8.5g of 3R,3'S-dihydroxy-β-carotene crystals.

[0026] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 88.1% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 89.5% of the total carotenoids, lutein accounted for 9.3%, and the conversion rate was 82.23%.

[0027] Conversion rate = 3R, 3'S-dihydroxy-β-carotene crystal mass*UV*HPLC / marigold oleoresin mass / marigold oleoresin content*100%.

[0028] Example 2

[0029] Take 50g of marigold oleoresin with a content of 16.3% and 15g of stigmasterol, mix and heat to 80°C, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, add 25g of malonic acid to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 1.2Mpa, react at 95°C for 4h, dilute with ethanol solvent, use a high shear disperser at 65°C, set the speed to 2000 rpm for high-speed stirring crystallization for 5h, filter, wash and dry at 70°C at a constant temperature, and finally obtain 9.1g 3R,3'S-dihydroxy-β-carotene crystals.

[0030] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 92.5% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 92.2% of the total carotenoids, lutein accounted for 7.2%, and the conversion rate was 95.22%.

[0031] Example 3

[0032] Take 50g of marigold oleoresin with a content of 16.3% and 5g of campesterol, mix and heat to 75°C, add 40g of 50wt% potassium hydroxide solution, saponify for 3.5h, add 25g of malonic acid to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 0.3Mpa, react at 100°C for 8h, dilute with ethanol solvent, use a high shear disperser at 65°C, set the speed to 2900 rpm for high-speed stirring crystallization for 4h, filter, wash and dry at 72°C at a constant temperature, and finally obtain 7.4g of 3R,3'S-dihydroxy-β-carotene crystals.

[0033] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 88.3% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 88.5% of the total carotenoids, lutein accounted for 10.2%, and the conversion rate was 70.95%.

[0034] Example 4

[0035] Take 50g of marigold oleoresin with a content of 16.3% and 7.5g of rape stanol, mix and heat to 80°C, add 50g of 58.3wt% potassium hydroxide solution, saponify for 4h, add 29g of malonic acid to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 0.8Mpa, react at 93°C for 6h, dilute with ethanol solvent, use a high shear disperser at 65°C, set the speed to 1500 rpm for high-speed stirring crystallization for 6h, filter, wash and dry at 72°C at a constant temperature, and finally obtain 8.5g of 3R,3'S-dihydroxy-β-carotene crystals.

[0036] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 87.51% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 91.2% of the total carotenoids, lutein accounted for 8.5%, and the conversion rate was 83.22%.

[0037] Example 5

[0038] Take 50g of marigold oleoresin with a content of 16.3% and 20g of cholesterol, mix and heat to 78°C, add 65g of 61.5wt% potassium hydroxide solution, saponify for 6h, add 40g of malonic acid to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 1.1Mpa, react at 90°C for 10h, dilute with ethanol solvent, use a high shear disperser at 80°C, set the speed to 1450 rpm, perform high-speed stirring crystallization for 5.5h, filter, wash and dry at 72°C at a constant temperature, and finally obtain 8.2g of 3R,3'S-dihydroxy-β-carotene crystals.

[0039] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 89.2% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 90.5% of the total carotenoids, lutein accounted for 6.95%, and the conversion rate was 81.22%.

[0040] Comparative Example 1

[0041] The difference from Example 2 is that malonic acid is not added, and other conditions are the same.

[0042] Take 50g of marigold oleoresin with a content of 16.3% and 15g of stigmasterol, mix and heat to 80°C, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, and pressurize at 1.2Mpa with carbon dioxide at 95°C for 4h. After dilution with ethanol solvent, use a high shear disperser at 65°C and set the speed to 2000 rpm for high-speed stirring crystallization for 5h. Filter, wash and dry at 70°C to obtain 7.4g of 3R,3'S-dihydroxy-β-carotene crystals.

[0043] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 69.8% as determined by UV-visible spectrophotometry. High performance liquid chromatography analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 78.6% of the total carotenoids, lutein accounted for 19.5%, and the conversion rate was 49.81%.

[0044] Failure to use malonic acid as a protective agent during the translocation process resulted in a loss of pigment content and a significant decrease in the conversion rate of 3R,3'S-dihydroxy-β-carotene crystals.

[0045] Comparative Example 2

[0046] The difference from Example 2 is that stigmasterol is not added, and other conditions are the same.

[0047] Take 50g of marigold oleoresin with a content of 16.3% and heat it to 80℃, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, add 25g of malonic acid to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 1.2Mpa, react at 95℃ for 4h, dilute with ethanol solvent, use a high shear disperser at 65℃, set the speed to 2000 rpm for high-speed stirring crystallization for 5h, filter, wash and dry at 70℃, and finally obtain 6.7g of 3R,3'S-dihydroxy-β-carotene crystals.

[0048] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 78.8% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 85.12% of the total carotenoids, lutein accounted for 11.5%, and the conversion rate was 55.14%.

[0049] Not using stigmasterol as an emulsifier during the saponification process reduced the hydrolysis efficiency of marigold oleoresin and the conversion rate of 3R,3'S-dihydroxy-β-carotene crystals was significantly reduced.

[0050] Comparative Example 3

[0051] The difference from Example 2 is that carbon dioxide is not used as a replacement protective agent, and other conditions are the same.

[0052] Take 50g of marigold oleoresin with a content of 16.3% and 15g of stigmasterol, mix and heat to 80°C, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, add 25g of malonic acid to the reaction solution, react at 95°C for 4h, dilute with ethanol solvent, use a high shear disperser at 65°C, set the speed to 2000 rpm for high-speed stirring crystallization for 5h, filter, wash and dry at 70°C, and finally obtain 8.6g of 3R,3'S-dihydroxy-β-carotene crystals.

[0053] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 79.3% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 69.52% of the total carotenoids, lutein accounted for 27.4%, and the conversion rate was 58.17%.

[0054] Under normal pressure conditions, the proportion of 3R,3'S-dihydroxy-β-carotene crystal liquid phase is low, and the degree of conversion of cis-lutein to 3R,3'S-dihydroxy-β-carotene is low.

[0055] Comparative Example 4

[0056] Take 50g of marigold oleoresin with a content of 16.3% and 15g of stigmasterol, mix and heat to 80°C, add 50g of 64% potassium hydroxide solution, saponify for 6h, add 25g of malonic acid to the reaction solution, and pressurize it with nitrogen to 1.2Mpa at 95°C for 4h. After dilution with ethanol solvent, use a high shear disperser at 65°C and set the speed to 2000 rpm for high-speed stirring crystallization for 5h. Filter, wash and dry at a constant temperature of 70°C to finally obtain 8.9g of 3R,3'S-dihydroxy-β-carotene crystals.

[0057] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 89.3% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 88.82% of the total carotenoids, lutein accounted for 7.9%, and the conversion rate was 86.61%.

[0058] When nitrogen is used as a replacement protective agent, its solubility in the system is not as good as that of carbon dioxide, resulting in its protective effect being weaker than that of carbon dioxide.

[0059] Comparative Example 5

[0060] Take 50g of marigold oleoresin with a content of 16.3% and 15g of ethanol, mix and heat to 80°C, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, add 25g of malonic acid to the reaction solution, and pressurize to 1.2Mpa with carbon dioxide at 95°C for 4h. After dilution with ethanol solvent, use a high shear disperser at 65°C with a speed set to 2000 rpm for high-speed stirring crystallization for 5h, filter, wash and dry at 70°C at a constant temperature to finally obtain 8.5g of 3R,3'S-dihydroxy-β-carotene crystals.

[0061] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 87.5% as determined by UV-visible spectrophotometry. High performance liquid chromatography analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 87.94% of the total carotenoids, lutein accounted for 9.54%, and the conversion rate was 80.25%.

[0062] Comparative Example 6

[0063] Take 50g of marigold oleoresin with a content of 16.3% and 15g of stigmasterol, mix and heat to 80°C, add 50g of 64wt% potassium hydroxide solution, saponify for 6h, add 25g of sodium malonate to the reaction solution, and at the same time, charge carbon dioxide and pressurize to 1.2Mpa, react at 95°C for 4h, dilute with ethanol solvent, use a high shear disperser at 65°C, set the speed to 2000 rpm for high-speed stirring crystallization for 5h, filter, wash and dry at 70°C at a constant temperature, and finally obtain 7.2g of 3R,3'S-dihydroxy-β-carotene crystals.

[0064] The total carotenoid content of 3R,3'S-dihydroxy-β-carotene crystals was 71.25% detected by UV-visible spectrophotometry. HPLC analysis showed that 3R,3'S-dihydroxy-β-carotene accounted for 76.6% of the total carotenoids, lutein accounted for 20.2%, and the conversion rate was 48.21%.

Claims

1. A method for preparing 3R, 3'S-dihydroxy-β-carotene, characterized in that: The following steps are involved: S1, dissolving marigold oleoresin and phytosterol in a mass ratio of 1:0.1-1, heating and stirring to obtain a mixture; S2, adding alkali solution to the mixture obtained in step S1 according to a weight ratio of marigold oleoresin to alkali solution of 1:0.4-0.8 to react to obtain a mixture of lutein and higher fatty acid salts, wherein the alkali solution is sodium hydroxide or potassium hydroxide; S3. According to the weight ratio of marigold oleoresin to malonic acid of 1:0.5-2, malonic acid is added to the product obtained in step S2 for catalytic reaction, and carbon dioxide is introduced for displacement protection at the same time. The reaction is pressurized to 0.3-1.2 MPa, and the temperature is 90-100° C. for 2-10 hours. Ethanol is added for dilution, and the reaction is carried out at 80-90° C. for high-speed stirring and crystallization for 2-10 hours. The reaction is filtered, washed, and dried by nitrogen displacement to finally obtain 3R, 3'S-dihydroxy-β-carotene.

2. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: The alkali solution in step S2 is sodium hydroxide or potassium hydroxide with a mass fraction of 50-65%.

3. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: The high-speed stirring in step S3 adopts a high-shear disperser with a rotation speed of 1450-2900 rpm.

4. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: The phytosterol in step S1 is one or more of β-sitosterol, stigmasterol, campesterol, campestanol and cholesterol.

5. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 4, characterized in that: The plant sterol in step S1 is stigmasterol.

6. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: The heating temperature in step S1 is 71-80°C.

7. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 6, characterized in that: The heating temperature in step S1 is 75°C.

8. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: The reaction temperature in step S3 is 90°C and the crystallization temperature is 85°C.

9. The method for preparing 3R, 3'S-dihydroxy-β-carotene according to claim 1, characterized in that: In step S1, the marigold oleoresin and phytosterol are in a mass ratio of 1:0.1-0.4.