Method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 ferment powder
By using phospholipase A in the Coenzyme Q10 fermentation powder for enzymatic lysis reaction, combined with the steps of concentration, reflux extraction and solid-liquid separation, the high-purity Coenzyme Q10 and lysophosphatidylcholine were successfully extracted from the Coenzyme Q10 fermentation powder at the same time, solving the problem of difficulty in preparing these two substances at the same time in the prior art, achieving an efficient extraction process and improving resource utilization.
Patent Information
- Application Number
- CN202510225229.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to simultaneously prepare high contents of Coenzyme Q10 and lysophosphatidylcholine from Coenzyme Q10 fermentation bacteria powder, while using a large amount of organic solvents and producing silica gel waste.
Enzymatic reaction was performed by dissolving Coenzyme Q10 fermentation powder and adding phospholipase A, followed by concentration, reflux extraction and solid-liquid separation, and extraction was performed using anhydrous ethanol and dimethyl carbonate-petroleum ether mixture to obtain high-purity Coenzyme Q10 and lysophosphatidylcholine, respectively.
The simultaneous extraction of Coenzyme Q10 and lysophosphatidylcholine from Coenzyme Q10 fermentation bacteria powder is achieved, which improves the comprehensive utilization rate, reduces the use of organic solvents, and avoids the generation of silica gel waste.
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Figure CN120060389A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a preparation method of coenzyme Q10 and lysophosphatidylcholine, and particularly relates to a method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder. Background Art
[0002] Coenzyme Q10 has the effects of improving human immunity, antioxidation, protecting the cardiovascular and cerebrovascular systems, delaying aging, etc., and is widely used in health products, cosmetics and food additives. Lysophosphatidylcholine (LPC) has a wide range of applications in the fields of food, cosmetics, medicine, etc.
[0003] At present, the main methods for preparing coenzyme Q10 are: extraction method, semi-synthesis, synthesis method, microbial fermentation method and cell culture method; the most commonly used method is the microbial fermentation method; the preparation methods of lysophosphatidylcholine are mainly enzymatic method and synthesis method.
[0004] Coenzyme Q10 fermentation powder is a commonly used raw material for preparing coenzyme Q10, in which the content of phosphatidylcholine is greater than 10%, and the content is more than 3 times that of coenzyme Q10 therein. However, there are few reports on the technology for preparing high-content coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder. Moreover, at present, the separation and purification of coenzyme Q10 in domestic relevant literatures mainly adopt methods such as solvent extraction and silica gel chromatography, in which a large amount of organic solvents are used, and silica gel waste is generated at the same time. Summary of the Invention
[0005] The purpose of the present invention is to solve the deficiencies that the prior art cannot simultaneously prepare coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder, and the separation and purification of coenzyme Q10 usually require the use of a large amount of organic solvents and generate silica gel waste, and to provide a method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder.
[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows:
[0007] A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder, which is characterized in that it includes the following steps:
[0008] Step 1, dissolve coenzyme Q10 fermentation powder, add phospholipase A thereto, and carry out an enzymatic hydrolysis reaction;
[0009] Step 2, after the enzymatic hydrolysis reaction is completed, concentrate, then add a lower-carbon alcohol to obtain a first mixed solution, and carry out reflux extraction on the first mixed solution to obtain an extract;
[0010] Step 3: Perform the first solid-liquid separation on the extract, crystallize the supernatant obtained from the first solid-liquid separation, and then perform the second solid-liquid separation to obtain a liquid and a solid.
[0011] Step 4: Extract coenzyme Q10 and lysophosphatidylcholine separately.
[0012] Extracting coenzyme Q10: Dissolve the solid obtained from the second solid-liquid separation with absolute ethanol, perform recrystallization, perform solid-liquid separation again, and dry the separated solid to obtain coenzyme Q10.
[0013] Extracting lysophosphatidylcholine: Concentrate the liquid obtained from the second solid-liquid separation to obtain a concentrated solution, extract the concentrated solution with a dimethyl carbonate-petroleum ether mixture, let it stand for layering, discard the aqueous phase, and after concentrating and drying the organic phase, obtain lysophosphatidylcholine.
[0014] Further, step 1 is specifically: Add 2 to 10 times the weight of water to the coenzyme Q10 fermentation bacteria powder, stir, and heat to 30 to 60 °C, add phospholipase A with a weight of 1% to 30% of the coenzyme Q10 fermentation bacteria powder, and react for 1 to 8 hours.
[0015] Among them, phospholipase A plays an enzymatic hydrolysis role. In the present invention, only phospholipase A can be used. The reaction temperature is 30 to 60 °C. If the temperature is too low, the reaction proceeds slowly or does not proceed. If the temperature is too high, phospholipase A will be inactivated and the reaction cannot proceed.
[0016] Further, in step 2, the temperature of the concentration is 30 to 80 °C, and it is concentrated to a specific gravity of 1.06 to 1.3 g / mL;
[0017] In the first mixed solution, the mass fraction of the lower alcohol is 60% to 95%;
[0018] The time for the reflux extraction is 1.5 to 2.5 hours, and the extraction temperature is not lower than 60 °C;
[0019] The lower alcohol is ethanol or methanol.
[0020] Among them, if the temperature of the concentration is low, the concentration time is long, resulting in a longer production cycle. If the temperature is too high, there is a risk of scorching. If the mass fraction of the lower alcohol is low, the extraction rate is low, resulting in too many impurities being extracted. If the extraction temperature is low, the extraction rate will also be low. Therefore, it is preferably not lower than 60 °C.
[0021] In the present invention, the lower alcohol is preferably ethanol or methanol. In addition, other lower alcohols such as propanol and butanol can also be used.
[0022] Further, in step 3, before crystallizing the supernatant obtained from the first solid-liquid separation, cool the supernatant to below 40 °C; the time for the crystallization is 20 to 180 minutes.
[0023] If the crystallization time is too short, incomplete crystallization will occur; when the crystallization temperature is low, the crystallization rate is fast, and when the temperature is high, the crystallization rate is slower. Therefore, it is preferred that the crystallization temperature is below 40°C and the crystallization time is 20 - 180 minutes.
[0024] Further, in step 4, when extracting coenzyme Q10, the absolute ethanol is food-grade absolute ethanol, and the usage amount is 1 - 5 times the weight of the solid obtained from the second solid-liquid separation, and the dissolution temperature is 30 - 60°C;
[0025] The temperature of the recrystallization is below 15°C;
[0026] The drying is vacuum low-temperature drying, the drying temperature is 10 - 40°C, and the vacuum degree is -0.1 - -0.04 Mpa;
[0027] In step 4, when extracting lysophosphatidylcholine, the liquid obtained from the second solid-liquid separation is concentrated to a specific gravity of 1.01 - 1.06 g / mL, and the volume of the dimethyl carbonate - petroleum ether mixture is 1 / 10 - 1 / 2 of the volume of the concentrated liquid; the concentration temperature of the organic phase is 50 - 80°C.
[0028] Further, step 1 is specifically: adding 7 times the weight of water to the coenzyme Q10 fermentation bacteria powder, stirring, heating to 40°C, adding phospholipase A with a weight of 20% of the coenzyme Q10 fermentation bacteria powder, and reacting for 7 hours;
[0029] In step 2, the concentration temperature is 50°C, and it is concentrated to a specific gravity of 1.13 g / mL; in the first mixed solution, the lower alcohol is ethanol, and the mass fraction is 85%; the reflux extraction time is 2 hours; the extraction temperature is 80°C;
[0030] In step 3, the crystallization time is 90 minutes;
[0031] In step 4, when extracting coenzyme Q10, the usage amount of absolute ethanol is 5 times the weight of the solid obtained from the second solid-liquid separation, and the dissolution temperature is 55°C; the drying temperature is 10°C, and the vacuum degree is -0.1 Mpa;
[0032] In step 4, when extracting lysophosphatidylcholine, the liquid obtained from the second solid-liquid separation is concentrated to a specific gravity of 1.03 g / mL, and the volume of the dimethyl carbonate - petroleum ether mixture is 1 / 10 of the volume of the concentrated liquid; the concentration temperature of the organic phase is 80°C.
[0033] The beneficial effects of the present invention:
[0034] 1. The present invention makes full use of coenzyme Q10 fermented bacteria powder as a raw material for preparing lysophosphatidylcholine, simultaneously extracting coenzyme Q10 and lysophosphatidylcholine from it, avoiding waste of a large amount of coenzyme Q10 fermented bacteria powder, and improving the comprehensive utilization rate of coenzyme Q10 fermented bacteria powder.
[0035] 2. In the preparation process of coenzyme Q10 in the present invention, only alcohol solvents are used, reducing the use of other organic solvents such as petroleum ether or n-hexane, and the preparation process is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 It is a schematic diagram of the HPLC detection result of coenzyme Q10 in Example 1 of the present invention;
[0037] Figure 2 It is a schematic diagram of the HPLC detection result of lysophosphatidylcholine in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0038] Example 1
[0039] The method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermented powder in the present invention specifically includes the following steps:
[0040] Step 1: Add water in an amount twice the weight of the coenzyme Q10 fermented bacteria powder to the coenzyme Q10 fermented bacteria powder, stir, heat to 30°C, add phospholipase A with a weight of 1% of the coenzyme Q10 fermented bacteria powder, and carry out an enzymatic hydrolysis reaction for 8 hours.
[0041] Step 2: After the enzymatic hydrolysis reaction is completed, concentrate the reactant at 30°C to a specific gravity of 1.06 g / mL, add ethanol to obtain a first mixed solution, the mass fraction of ethanol in the first mixed solution is 60%, carry out reflux extraction on the first mixed solution for 2 hours, and the extraction temperature is 70°C to obtain an extract.
[0042] Step 3: Carry out the first solid-liquid separation on the extract, cool the supernatant obtained from the first solid-liquid separation to below 40°C, start crystallization, the crystallization time is 180 minutes, and carry out the second solid-liquid separation.
[0043] Step 4: Extract coenzyme Q10 and lysophosphatidylcholine respectively;
[0044] Extracting coenzyme Q10: Add edible anhydrous ethanol in an amount equal to the weight of the solid obtained from the second solid-liquid separation to the solid, heat it to 30°C to completely dissolve the crystals. After the crystal dissolution is completed, quickly place it in an environment below 15°C for recrystallization until no more crystals precipitate. After solid-liquid separation, vacuum-dry the obtained solid part at a low temperature, the drying temperature is 20°C, and the vacuum degree is -0.04 Mpa to obtain high-purity coenzyme Q10.
[0045] Extraction of lysophosphatidylcholine: The liquid obtained from the second solid-liquid separation was concentrated to a specific gravity of 1.01 g / mL, and a dimethyl carbonate - petroleum ether mixture with a volume one-fifth of the concentrated liquid volume was added thereto. The concentrated liquid was stirred and extracted, allowed to stand for layering, and the lower aqueous phase was discarded. The upper organic phase was concentrated and dried at a concentration temperature of 60 °C, and lysophosphatidylcholine was obtained after drying.
[0046] Example 2
[0047] The method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder of the present invention specifically includes the following steps:
[0048] Step 1: Add water with a weight 10 times that of the coenzyme Q10 fermentation bacterial powder to the coenzyme Q10 fermentation bacterial powder, stir, and heat to 60 °C, and add phospholipase A accounting for 30% of the weight of the coenzyme Q10 fermentation bacterial powder, and carry out an enzymatic hydrolysis reaction for 1 hour.
[0049] Step 2: After the enzymatic hydrolysis reaction is completed, the reactant is concentrated to a specific gravity of 1.3 g / mL at 80 °C, methanol is added to obtain a first mixed solution, the mass fraction of methanol in the first mixed solution is 95%, the first mixed solution is reflux-extracted for 1.5 hours at an extraction temperature of 60 °C, and an extract is obtained.
[0050] Step 3: Perform the first solid-liquid separation on the extract, quickly cool the clear liquid obtained from the first solid-liquid separation to below 40 °C, start crystallization, the crystallization time is 20 minutes, and perform the second solid-liquid separation.
[0051] Step 4: Extract coenzyme Q10 and lysophosphatidylcholine respectively;
[0052] Extraction of coenzyme Q10: Add edible anhydrous ethanol with a weight 4 times that of the solid obtained from the second solid-liquid separation thereto, heat it to 60 °C to completely dissolve the crystals. After the crystal dissolution is completed, quickly place it in an environment below 15 °C for recrystallization until no more crystals precipitate. After solid-liquid separation, the obtained solid part is vacuum-dried at a low temperature, the drying temperature is 40 °C, and the vacuum degree is -0.04 Mpa, and high-purity coenzyme Q10 is obtained.
[0053] Extraction of lysophosphatidylcholine: The liquid obtained from the second solid-liquid separation was concentrated to a specific gravity of 1.06 g / mL, and a dimethyl carbonate - petroleum ether mixture with a volume one-half of the concentrated liquid volume was added thereto. The concentrated liquid was stirred and extracted, allowed to stand for layering, and the lower aqueous phase was discarded. The upper organic phase was concentrated and dried at a concentration temperature of 50 °C, and lysophosphatidylcholine was obtained after drying.
[0054] Example 3
[0055] The method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 fermentation powder of the present invention specifically includes the following steps:
[0056] Step 1: Add water in an amount 7 times the weight of the coenzyme Q10 fermentation bacterial powder, stir, heat to 40°C, add phospholipase A in an amount 20% of the weight of the coenzyme Q10 fermentation bacterial powder, and carry out an enzymatic hydrolysis reaction for 7 hours.
[0057] Step 2: After the enzymatic hydrolysis reaction ends, concentrate the reactant at 50°C to a specific gravity of 1.13 g / mL, add ethanol to obtain a first mixed solution, where the mass fraction of ethanol in the first mixed solution is 85%, carry out reflux extraction on the first mixed solution for 2 hours, and the extraction temperature is 80°C to obtain an extract.
[0058] Step 3: Conduct the first solid-liquid separation on the extract, quickly cool the supernatant obtained from the first solid-liquid separation to below 40°C, start crystallization, with the crystallization time being 90 minutes, and conduct the second solid-liquid separation.
[0059] Step 4: Extract coenzyme Q10 and lysophosphatidylcholine respectively;
[0060] Extraction of coenzyme Q10: Add anhydrous edible ethanol in an amount 5 times the weight of the solid obtained from the second solid-liquid separation, heat it to 55°C to completely dissolve the crystals. After the crystal dissolution is completed, quickly place it in an environment below 15°C for recrystallization until no more crystals precipitate. After solid-liquid separation, vacuum-dry the obtained solid part at a low temperature, with the drying temperature being 10°C and the vacuum degree being -0.1 Mpa to obtain high-purity coenzyme Q10. Conduct HPLC detection on the obtained coenzyme Q10, and the obtained purity is 99.8%, as Figure 1 shown.
[0061] Extraction of lysophosphatidylcholine: Concentrate the liquid obtained from the second solid-liquid separation to a specific gravity of 1.03 g / mL, add a dimethyl carbonate - petroleum ether mixture with a volume 1 / 10 of the volume of the concentrated liquid, stir and extract the concentrated liquid, let it stand for layering, discard the lower aqueous phase, concentrate and dry the upper organic phase, with the concentration temperature being 80°C, and obtain lysophosphatidylcholine after drying. Conduct HPLC detection on it, and the obtained results are as Figure 2 shown.
[0062] Conduct HPLC detection on the coenzyme Q10 and lysophosphatidylcholine obtained in Example 1 and Example 2 respectively, and similarly, detection results similar to Figure 1 and Figure 2 can be obtained.
Claims
1. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder, characterized in that: The following steps are involved: Step 1, dissolving coenzyme Q10 fermentation bacteria powder, adding phospholipase A thereto, and performing enzymatic hydrolysis reaction; Step 2, after the enzymatic hydrolysis reaction is completed, the solution is concentrated, and then a low-carbon alcohol is added to obtain a first mixed solution, and the first mixed solution is subjected to reflux extraction to obtain an extract; Step 3, performing a first solid-liquid separation on the extract, crystallizing the clear liquid obtained by the first solid-liquid separation, and then performing a second solid-liquid separation to obtain a liquid and a solid; Step 4, extracting coenzyme Q10 and lysophosphatidylcholine respectively; Extraction of coenzyme Q10: using anhydrous ethanol to dissolve the solid obtained from the second solid-liquid separation, recrystallization, solid-liquid separation again, and drying the separated solid to obtain coenzyme Q10; Extraction of lysophosphatidylcholine: concentrate the liquid obtained from the second solid-liquid separation to obtain a concentrated solution, extract the concentrated solution with a dimethyl carbonate-petroleum ether mixture, allow to stand and separate, discard the aqueous phase, concentrate and dry the organic phase to obtain lysophosphatidylcholine.
2. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder according to claim 1, characterized in that: Step 1 is as follows: Add 2 to 10 times the weight of water to the coenzyme Q10 fermented bacteria powder, stir, and heat to 30 to 60° C., add phospholipase A in an amount of 1% to 30% of the weight of the coenzyme Q10 fermented bacteria powder, and react for 1 to 8 hours.
3. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder according to claim 2, characterized in that: In step 2, the concentration temperature is 30-80° C., and the concentration is performed to a specific gravity of 1.06-1.3 g / mL; In the first mixed solution, the mass fraction of low-carbon alcohol is 60% to 95%; The reflux extraction time is 1.5 to 2.5 hours, and the extraction temperature is not less than 60°C; The low-carbon alcohol is ethanol or methanol.
4. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder according to claim 3, characterized in that: In step 3, before crystallizing the clear liquid obtained from the first solid-liquid separation, the clear liquid is cooled to below 40° C.; the crystallization time is 20 to 180 minutes.
5. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder according to claim 4, characterized in that: In step 4, when extracting coenzyme Q10, the anhydrous ethanol is edible anhydrous ethanol, the amount used is 1 to 5 times the weight of the solid obtained by the second solid-liquid separation, and the dissolution temperature is 30 to 60° C.; The recrystallization temperature is lower than 15°C; The drying is vacuum low-temperature drying, the drying temperature is 10-40°C, and the vacuum degree is -0.1-0.04Mpa; In step 4, when extracting lysophosphatidylcholine, the liquid obtained by the second solid-liquid separation is concentrated to a specific gravity of 1.01-1.06 g / mL, and the volume of the dimethyl carbonate-petroleum ether mixture is 1 / 10-1 / 2 of the volume of the concentrated liquid; the concentration temperature of the organic phase is 50-80°C.
6. A method for preparing coenzyme Q10 and lysophosphatidylcholine from coenzyme Q10 baking powder according to claim 5, characterized in that: Step 1 is specifically as follows: add 7 times the weight of water to the coenzyme Q10 fermented bacterial powder, stir, and heat to 40° C., add phospholipase A in an amount of 20% by weight of the coenzyme Q10 fermented bacterial powder, and react for 7 hours; In step 2, the concentration temperature is 50°C, and the concentration is to a specific gravity of 1.13 g / mL; in the first mixed solution, the low-carbon alcohol is ethanol, and the mass fraction is 85%; the reflux extraction time is 2 hours; and the extraction temperature is 80°C; In step 3, the crystallization time is 90 minutes; In step 4, when extracting coenzyme Q10, the amount of anhydrous ethanol used is 5 times the weight of the solid obtained by the second solid-liquid separation, and the dissolution temperature is 55° C.; the drying temperature is 10° C., and the vacuum degree is -0.1 MPa; In step 4, when extracting lysophosphatidylcholine, the liquid obtained by the second solid-liquid separation is concentrated to a specific gravity of 1.03 g / mL, and the volume of the dimethyl carbonate-petroleum ether mixture is 1 / 10 of the volume of the concentrated liquid; the concentration temperature of the organic phase is 80°C.