阅读说明：本技术 一种发酵法生产辅酶q10的提取纯化方法 (Extraction and purification method for producing coenzyme Q10 by fermentation method ) 是由 石振拓 王建中 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种发酵法生产辅酶Q10的提取纯化方法,利用甲醇处理正己烷从类球红细菌发酵菌丝体提取的富含辅酶Q10的浓缩提取物,搅拌下50-65℃加热0.5-2h,甲醇提取液再经冷却析晶、过滤得到辅酶Q10；本发明通过加热下卟啉类化合物、类脂化合物与甲醇形成了一个新的溶剂体系并在冷却后析出辅酶Q10,达到初步分离及纯化辅酶Q10的目的,并且获得较高的纯度及提取率,同时大幅降低了环保压力,进一步推动了发酵法提取辅酶Q10的工业化发展。(The invention discloses an extraction and purification method for producing coenzyme Q10 by a fermentation method, which comprises the steps of treating a concentrated extract rich in coenzyme Q10 extracted from rhodobacter sphaeroides fermentation mycelium by using methanol, heating for 0.5-2h at 50-65 ℃ under stirring, cooling and crystallizing a methanol extracting solution, and filtering to obtain coenzyme Q10; according to the invention, a new solvent system is formed by the porphyrin compound, the lipoid compound and the methanol under heating, and the coenzyme Q10 is separated out after cooling, so that the purposes of primary separation and purification of the coenzyme Q10 are achieved, higher purity and extraction rate are obtained, the environmental protection pressure is greatly reduced, and the industrial development of the coenzyme Q10 extraction by a fermentation method is further promoted.)

1. An extraction and purification method for producing coenzyme Q10 by a fermentation method is characterized by comprising the following steps: the rhodobacter sphaeroides fermentation mycelium n-hexane extracting solution is subjected to solvent recovery to obtain a concentrated solution; adding methanol into the concentrated solution under stirring, heating, cooling the methanol extractive solution, crystallizing, filtering, and drying to obtain coenzyme Q10.

2. The extraction and purification method of claim 1, wherein the methanol is heated at 50-65 ℃ and stirred for 0.5-2 h.

3. The extraction and purification method according to claim 1, wherein the cooling crystallization condition is 0-25 ℃, and the standing is 4-12 h.

4. The extraction and purification method of claim 1, wherein the concentrated solution is extracted with methanol for 2-3 times, and coenzyme Q10 is combined.

5. The extraction and purification method according to claim 4, wherein the total amount of methanol is 1.75 to 2 times the volume of the extraction solution before concentration.

6. The extraction and purification method according to claim 4, wherein the repeated extraction process uses 0.25-1.5 times of the volume of the extraction solution before concentration.

7. The extraction and purification method according to claim 5, wherein in the repeated extraction step, the methanol extract after product separation is repeatedly used for the first extraction.

8. The extraction and purification method according to claim 1, wherein the coenzyme Q10 obtained by the method is purified by absolute ethanol to obtain a coenzyme Q10 pure product.

Technical Field

The invention belongs to the field of biological pharmacy, and relates to an extraction and purification method for producing coenzyme Q10 by a fermentation method.

Background

Coenzyme Q10, also known as ubiquinone 10, has the following structural formula:

the coenzyme Q10-rich concentrate extracted from fermented mycelium of rhodobacter sphaeroides by using normal hexane is rich in coenzyme Q10, and substances which are easily dissolved in normal hexane, such as fat-soluble porphyrin compounds, lipid compounds (such as stearic acid and palmitic acid), various carotenoids and glycolipid compounds, and also mixed with proteins. Because of being rich in porphyrin compounds, the coenzyme Q10-rich concentrated solution extracted by normal hexane from rhodobacter sphaeroides fermentation mycelium is purple-black tar-like. Further extraction and purification of coenzyme Q10 from purple-black tar-like substances is a key process for preparing coenzyme Q10 by a fermentation method.

Coenzyme Q10 is a fat-soluble quinone substance, is yellow or light yellow crystal, is unstable and easily degradable under alkaline conditions, and is stable to heat and acid. Has high solubility in oil and fat, and is easily dissolved in n-hexane, petroleum ether, acetone, and ethyl acetate. Chloroform, etc. Coenzyme Q10 is described as slightly soluble in ethanol and insoluble in methanol and water.

The concentrate extracted from rhodobacter sphaeroides fermentation mycelium by using n-hexane as solvent is rich in coenzyme Q10. To extract coenzyme Q10 from the concentrate, the current methods used in production include saponification, solvent extraction, ultrasonic extraction, supercritical extraction, etc. However, at present, sodium hydroxide-methanol saponification is mainly adopted at home and abroad.

Saponification is the reaction of strong base and esters, which is hydrolyzed to produce carboxylate and alcohol, and the extraction and initial purification of the extract are realized. The esters entering the extraction concentrate can be separated and removed by saponification and decomposition into carboxylate and alcohol which are insoluble in the extracting agent, and meanwhile, various impurities such as porphyrin substances and the like are removed. To achieve saponification at room temperature, the presence of a certain amount of methanol is necessary. However, emulsification tends to occur during the saponification treatment, and a large amount of sodium chloride needs to be added to suppress emulsification. The emulsification causes loss of coenzyme Q10, and the emulsification makes the production difficult. And the generated saponification liquid is black, high in alkali, high in salt and high in methanol, and cannot be treated by common environmental protection facilities and processes.

In addition, since coenzyme Q10 is unstable and easily degraded under alkaline conditions, an inhibitor such as pyrogallic acid must be added to inhibit the degradation, but this not only increases the cost, but also has the hazard of pyrogallic acid residue.

Disclosure of Invention

The invention provides an extraction and purification method for producing coenzyme Q10 by a fermentation method, which at least solves one of the problems in the prior art.

In view of this, the scheme of the invention is as follows:

an extraction and purification method for producing coenzyme Q10 by a fermentation method comprises the following steps: the rhodobacter sphaeroides fermentation mycelium n-hexane extracting solution is subjected to solvent recovery to obtain a concentrated solution; adding methanol into the concentrated solution under stirring, heating, cooling the methanol extractive solution, crystallizing, filtering, and drying to obtain coenzyme Q10.

Further, the heating condition of the methanol is 50-65 ℃, and the methanol is stirred for 0.5-2 h.

Further, the temperature of cooling crystallization is 0-25 ℃.

Further, the concentrated solution is repeatedly extracted with methanol for 2-3 times, and coenzyme Q10 is combined. Preferably, the total amount of methanol used is 1.75 to 2 times the volume of the extract before concentration. More preferably, the repeated extraction process uses 0.25 to 1.5 times the volume of the extraction solution before concentration.

Preferably, in the repeated extraction step, the methanol extract after product separation is repeatedly used for the first extraction.

Further, the coenzyme Q10 obtained by the method is purified by absolute ethyl alcohol to obtain a coenzyme Q10 pure product.

Compared with the prior art, the invention has the beneficial effects that:

1. the extraction method is economical, environment-friendly and high in feasibility. The method solves the problems of degradation of coenzyme Q10, treatment of high-alkali high-salt black waste liquid discharge after saponification, residual of a large amount of methanol in the high-alkali high-salt black waste liquid after saponification, residual of added pyrogallic acid in a coenzyme Q10 product and the like in the process of producing coenzyme Q10 by the conventional saponification method.

2. The coenzyme Q10 obtained by the extraction method has high yield and high purity, and the industrial development of coenzyme Q10 extracted by a fermentation method is further promoted.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention is further described in detail with reference to the following detailed description. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The invention aims to provide coenzyme Q10 in a concentrate extracted from rhodobacter sphaeroides fermentation mycelium by using methanol to extract and purify normal hexane as a solvent. The method for extracting and purifying the coenzyme Q10 by using methanol aims to solve the problems of degradation of the coenzyme Q10 generated in the implementation process of the saponification method in the process of producing the coenzyme Q10 by using the existing fermentation method, the treatment technical problem of discharge of high-alkali high-salt high-methanol black waste liquid after saponification, the problem of residual pyrogallic acid added in a coenzyme Q10 product and the like.

The invention provides an extraction and purification method for producing coenzyme Q10 by a fermentation method, which comprises the following steps:

s1, recovering normal hexane from an extracting solution rich in coenzyme Q10 to obtain a concentrated solution, adding methanol, stirring, extracting for 2-3 times, wherein the total amount of the methanol is 1.75-2 times of the volume of the extracting solution before concentration, heating and stirring at 50-65 ℃ for 0.5-2h, and filtering out the methanol extracting solution;

s2, respectively cooling the extracting solution to 0-25 ℃, standing for 4-12h, separating out coenzyme Q10, filtering, recovering, and merging and purifying crystals.

Preferably, the amount of methanol added in each extraction in step S1 is 0.25-1.5 times, more preferably 0.25-1 times, the volume number before concentration, heated to 60-65 deg.C under stirring, maintained for 0.5h,

preferably, the methanol extract solution of step S2 is cooled to 15 deg.C, left standing for 12h, and coenzyme Q10 is completely precipitated, filtered and recovered.

In the present invention, after the coenzyme Q10 was recovered from the first, second and third methanol extracts by filtration, methanol was recovered from the methanol solution under reduced pressure. The residue after recovery of methanol (which has been evaporated to dryness) can be used for recovery of porphyrins.

In the invention, the coenzyme Q10 recovered by cooling and filtering the methanol extracting solution of the first, second and third times is yellow, and is purified by silica gel column through dissolving n-hexane-isopropyl ether, and the absolute ethyl alcohol is recrystallized, so that the content of the obtained coenzyme 10 reaches more than 99.5 wt%.

In the invention, in order to improve the utilization rate of methanol and the yield of products, the third methanol extracting solution can be applied to the first extraction of methanol.

Prior to the practice of the present invention, those skilled in the art know or should know that coenzyme Q10 is slightly soluble in ethanol but not in methanol, and that coenzyme Q10 remains insoluble under the action of hot methanol, which alone would be an ideal solvent for purifying coenzyme Q10, on the basis of this general knowledge. After the fermentation liquor using the normal hexane as the solvent is concentrated and the normal hexane is recovered, coenzyme Q10, porphyrin compounds and lipid compounds are separated out, methanol is added under stirring, the precipitates are dissolved after heating, and the methanol, the porphyrin compounds and the lipid compounds form a new solvent system to change the polarity of the original solvent, so that the solubility of coenzyme Q10 is greatly improved; insoluble impurities are removed by filtering the methanol solution system, and then the methanol solution system is cooled, because the solubility of coenzyme Q10 is rapidly reduced and is separated out in the form of crystals, but porphyrin compounds and lipid compounds form a uniform solvent system with stable property with methanol, and therefore, the porphyrin compounds and the lipid compounds cannot be separated out. According to the principle, the coenzyme Q10 is precipitated in the form of crystals and is subjected to post-treatment to obtain a purified product.

In the invention, the extraction solution rich in coenzyme Q10 is 1000ml of n-hexane extraction solution extracted from rhodobacter sphaeroides fermentation mycelium by using n-hexane as a solvent, the content of coenzyme Q10 is 5.68mg/ml determined by a method of coenzyme Q10 in 'China pharmacopoeia 2020 edition', the n-hexane is placed in a garden bottom flask, and the n-hexane is recovered by vacuum concentration at 30 ℃ until the n-hexane is dried (no liquid drops drop out from a condenser), so that 59.43g of residue is obtained, and the content of coenzyme Q10 is 9.56%.

Example 1

59.43g of residue obtained by recovering n-hexane was subjected to extraction and purification by the following method:

1) first methanol extraction: adding 1000ml of methanol into the residue of the round-bottom flask, heating to 64 ℃, stirring for 0.5h, pouring out methanol liquid, vacuum-filtering by using a Buchner funnel while the methanol liquid is hot, and naturally cooling the methanol filtrate for 12h at 15 ℃. The crystallization of coenzyme Q10 was completed, and the crystals were filtered and dried to obtain 4.76g of coenzyme Q10 with a coenzyme Q10 content of 88.8%. The extraction rate of the first methanol extraction was 74.42% based on 100% of coenzyme Q10 in 1000ml of n-hexane extract.

Methanol solution obtained by filtering and removing coenzyme Q10 crystal is supplemented with methanol to 1000ml, the coenzyme Q10 content is determined to be 0.15mg/ml, methanol is recovered by vacuum concentration, and porphyrin compounds dissolved in methanol are recovered. The round-bottomed flask was vacuum-dried and weighed to obtain 33g of residue, and the residue obtained by adding a Buchner funnel was dissolved in n-hexane to a volume of 200ml, and the coenzyme Q10 content was found to be 6.435mg/ml, that is, 1.287g (residual rate: 22.66%).

2) And (3) second methanol extraction: adding 500ml of methanol into the residue of the round-bottom flask, heating to 60 ℃, stirring for 0.5h, pouring out methanol liquid, vacuum-filtering by using a Buchner funnel while the methanol liquid is hot, and naturally cooling the methanol filtrate after standing at 15 ℃. The crystallization was completed in 12 hours, and the residue was filtered and dried to obtain 0.99g of a content of 93.3%. The extraction rate of the second methanol extraction was 16.27% based on 100% coenzyme Q10 in 1000ml of n-hexane extract. The extraction rate was 71.79% based on the residual amount of coenzyme Q10 after the first extraction, which was 1.287g, and methanol solution obtained by filtration and from which coenzyme Q10 crystals were removed was supplemented to 500ml, and the coenzyme Q10 content was determined to be 0.14mg/ml, and methanol was recovered by vacuum concentration, and porphyrin compound dissolved in methanol was recovered. The round-bottomed flask was drained and weighed to obtain 8.05g of residue, and the residue was dissolved in n-hexane to 100ml in a Buchner funnel, whereby the content of coenzyme Q10 was measured to be 2.88 mg/ml.

3) And (3) performing third methanol extraction: adding 250ml of methanol into residues in the round-bottom flask, heating to 50 ℃, stirring for 2h, pouring out methanol solution at 20 ℃ overnight to obtain 202mg of coenzyme Q10 crystals with the content of 84.1 percent, wherein the extraction rate of the third methanol extraction is 2.99 percent based on the coenzyme Q10 content of 1000ml of n-hexane extract solution as 100 percent. Vacuum concentrating to recover 230ml of methanol, and recovering porphyrin compounds dissolved in methanol. The round bottom flask was drained and weighed to give an off-white residue, about 7.5g, which was discarded.

4) The coenzyme Q10 extracts extracted by three times of methanol are combined, and the conversion extraction rate is 93.7 percent based on the coenzyme Q10 content of 1000ml of n-hexane extract as 100 percent.

Example 2

59.43g of residue obtained by recovering n-hexane was subjected to extraction and purification by the following method:

1) first methanol extraction: adding 1500ml of methanol (containing 230ml of crystallization filtrate obtained after the third methanol extraction in the example 1) into the residue of the round-bottom flask, heating to 50 ℃, stirring for 2 hours, pouring out methanol solution, and performing vacuum filtration on a Buchner funnel while the methanol solution is hot, wherein residual liquid is used for later use; the methanol filtrate is placed at 5 ℃ and naturally cooled for 4 h. The crystallization of coenzyme Q10 was completed, and the crystals were filtered and dried to obtain 4.89g of coenzyme Q10 with a coenzyme Q10 content of 89.2%. The extraction rate of the first methanol extraction was 76.79% based on 100% of coenzyme Q10 in 1000ml of n-hexane extract.

Methanol solution obtained by filtering and removing coenzyme Q10 crystal is supplemented with methanol to 1000ml, the coenzyme Q10 content is determined to be 0.13mg/ml, methanol is recovered by vacuum concentration, and porphyrin compound dissolved in methanol is recovered. The round-bottomed flask was vacuum-dried and weighed to obtain 29g of a residue, and the residue obtained by adding a Buchner funnel was dissolved in n-hexane to a volume of 200ml, and the content of coenzyme Q10 was 5.775mg/ml, that is, the content of the residue was 1.155g (residue rate: 20.33%).

2) And (3) second methanol extraction: adding 500ml of methanol into residues in the round-bottom flask, heating to 60 ℃, stirring for 0.5h, pouring out methanol liquid, vacuum-filtering by using a Buchner funnel while the methanol liquid is hot, naturally cooling the methanol filtrate at 2 ℃, completely crystallizing and separating out after 6h, filtering and drying to obtain 1.07g with the content of 93.6%. The extraction rate of the second methanol extraction was 17.63% based on 100% coenzyme Q10 in 1000ml of n-hexane extract. The extraction rate was 86.71% based on the residual amount of coenzyme Q10 after the first extraction of 1.155g, methanol was supplemented to 300ml from the filtered methanol solution from which coenzyme Q10 crystals were removed, the coenzyme Q10 content was determined to be 0.11mg/ml, and methanol was recovered in vacuo to obtain 475 ml. The round bottom flask was drained and weighed to give an off-white residue, about 7.68g, which was discarded.

3) The coenzyme Q10 extracts extracted by two times of methanol are combined, and the conversion extraction rate is 94.4 percent based on the coenzyme Q10 content of 1000ml of n-hexane extract as 100 percent.

Example 3

59.43g of residue obtained by recovering n-hexane was subjected to extraction and purification by the following method:

1) first methanol extraction: 1200ml of methanol (containing 475ml of the crystallized filtrate obtained after the second methanol extraction in example 2) was added to the residue in the round-bottomed flask, and the same procedures as in example 2 were carried out to obtain 4.83g of coenzyme Q10, which had a coenzyme Q10 content of 89.0%.

2) And (3) second methanol extraction: 600ml of methanol was added to the residue in the round-bottom flask, and the same operation was carried out as in example 2 to obtain 1.05g of a solid having a content of 93.0%. The round bottom flask was drained and weighed to give a residue which was off white and discarded.

3) The coenzyme Q10 extracts obtained by two methanol extractions are combined, and the conversion extraction rate is 92.9 percent based on the coenzyme Q10 content of 1000ml of n-hexane extracting solution as 100 percent.

Comparative example 1

59.43g of residue obtained by recovering n-hexane was subjected to extraction and purification by the following method:

extracting with 1000ml anhydrous ethanol instead of anhydrous methanol, stirring at 78 deg.C for 0.5h, pouring out ethanol solution, vacuum filtering with Buchner funnel, standing ethanol filtrate at 15 deg.C, naturally cooling for 12h, and crystallizing out coenzyme Q10. The mixture is kept standing at 5 ℃ for 24 hours, and no coenzyme Q10 is crystallized and separated out. No coenzyme Q10 sample could be obtained.

Comparative example 2

59.43g of residue obtained by recovering n-hexane was subjected to extraction and purification by the following method:

after the first methanol extraction according to the procedure of example 1, the coenzyme Q10 is separated out after 12h at 30 ℃, filtered and dried, but the amount is only 3.52g, the coenzyme Q10 content is 85.6%, and the crystal color of the coenzyme Q10 is darker, which is not beneficial to the subsequent purification.

As can be seen from examples 1-3, in a single example, using a three-step extraction as in example 1, the amount of methanol used in the first two extractions was 1.5 times (compared to the volume of n-hexane extract before concentration), resulting in a small amount of product still contained in the residue remaining (coenzyme Q10 content greater than 2mg/ml), thus affecting the product yield. In example 2, when the amount of methanol was 2 times, the residue after the second extraction was off-white and contained a small amount, thus indicating that there was little room for increasing the yield when the amount of methanol was increased. Therefore, under the premise that the total volume of the methanol is 1.75-2 times of the volume of the n-hexane extracting solution before concentration, a two-step or three-step extraction mode is adopted, and the amount of the methanol in a single extraction step is 0.25-1.5 times of the volume of the n-hexane extracting solution before concentration, so that the comprehensive yield of about 93-94% can be obtained; in addition, the feasibility of using the recycled methanol as a solvent during the first extraction is verified, and the recycling of the methanol can be realized.

In contrast to the method of comparative example 1, the product was extracted by heating with absolute ethanol, but was not precipitated due to the slight influence of the solubility of coenzyme Q10 when the ethanol was cooled; in addition, in the comparative example 2, the coenzyme Q10 solubility is not reduced remarkably when the crystallization is carried out at a higher temperature of 30 ℃, and impurities are precipitated, so that the yield and the color of the precipitate are affected.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, and it is not intended to be limited to the specific details, representative experimental examples and examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.