Method for extracting sclareol from fermentation liquor

Abstract

The invention discloses a method for extracting sclareol from fermentation liquor. The method comprises the following operation steps: 1, treating the fermentation liquor; standing the fermentation liquor needing to be treated for 1-3 hours, putting the standing fermentation liquor into a centrifugal machine, and carrying out centrifugal treatment, so as to obtain mixed liquor of bacterial liquid and sclareol; 2, performing secondary treatment on the fermentation liquid; the centrifuged bacterial liquid and the mixed liquid are treated, the bacterial liquid is located on the upper layer of the mixed liquid, the bacterial liquid above the mixed liquid is removed, the mixed liquid is obtained, a cosolvent is added into the mixed liquid for mixing, and sclareol in the mixed liquid is dissolved by the mixed cosolvent; according to the method, the cosolvent is added into the fermentation liquor to re-dissolve the sclareol crystal part, then low-temperature crystal growing is conducted, the sclareol crystal part in the fermentation liquor is dissolved and then recrystallized into enlarged particles, the filtering difficulty is reduced, the product yield is guaranteed, and by means of subsequent drying, solvent extraction and crystallization, the yield of the sclareol is improved. The extraction process is simplified, and the product content is improved.

Description

Technical Field

[0001] The invention relates to the technical field of warehousing and logistics automation, and in particular to a method for extracting sclareol from fermentation broth. Background Art

[0002] Salvia sclareae is commonly referred to as Southern Salvia sclareae, a species of plant in the genus Salviae in the family Lamiaceae. It is an annual herb, about 1.5-2 meters tall, with an upright, quadrangular stem covered in fine hairs or green-purple hairs and many branches. Its leaves are opposite, long-stalked, and elliptical or oblong. It grows in whorls of 5-6 bisexual flowers. The nuts are oval, non-brown, and smooth. It blooms from July to September, and its seeds mature from August to October.

[0003] Sclareol is a white crystalline powder with an mp of 95-105°C. It has similar properties to other ambergris, making it an ideal raw material for the synthesis of natural ambergris substitutes such as sclareol and ambroxol. Smaller amounts are also used in the formulation of fragrances. It also exhibits antibacterial and bactericidal activity.

[0004] There are three methods for producing sclareol: one is to extract it from the sclareol plant. Due to the low content in the plant, the yield of this method is very small. The second is to rely on chemical synthesis, such as obtaining it through a series of redox reactions using sclareol as a substrate. The third is to produce it using synthetic biology methods, which is to use microbial strains to ferment and biocatalyze sclareol to produce sclareol. Currently, the most commonly used method is chemical synthesis. The chemical method has many dangerous factors in the process, low conversion yield, difficult separation and purification, and low finished product content. In addition, the chromium-containing wastewater generated during the production process pollutes the environment more. The sclareol produced by microbial fermentation catalysis can solve the above problems well. Summary of the Invention

[0005] The object of the present invention is to provide a method for extracting sclareol from a fermentation broth to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method for extracting sclareol from fermentation broth comprises the following steps:

[0008] Step 1: Fermentation Broth Treatment

[0009] The fermentation liquid to be treated is allowed to stand for 1-3 hours, and the fermentation liquid after standing is placed in a centrifuge for centrifugation to obtain a mixture of bacterial liquid and sclareol;

[0010] Step 2: Secondary treatment of fermentation broth

[0011] The bacterial liquid and the mixed liquid after centrifugation are processed, the bacterial liquid is located in the upper layer of the mixed liquid, the bacterial liquid above the mixed liquid is removed to obtain a mixed liquid, a cosolvent is added to the mixed liquid for mixing, and the mixed cosolvent dissolves sclareol in the mixed liquid;

[0012] Step 3: Obtaining sclareol

[0013] The mixed solution is placed in a centrifuge and centrifuged again to obtain a supernatant containing sclareol, a solvent is added to the supernatant, and then crystallization is performed to obtain a sclareol crystal structure;

[0014] Step 4: Purification of Sclareol Crystals

[0015] The obtained sclareol crystals are added to an ethanol solution for dissolution, the dissolved solution is frozen and diluted, and finally filtered and crystallized to obtain purified sclareol crystals;

[0016] Step 5: Sclareol Finished Product

[0017] The sclareol crystals are dried, crushed and pulverized to obtain a refined sclareol finished product.

[0018] Preferably, in the first step of processing the fermentation broth, the fermentation broth needs to be centrifuged at 135-150° C. and a speed of 500-6000 rpm.

[0019] Preferably, the cosolvent in the second step is one of petroleum ether, trichloroethylene, methanol, ethanol and petroleum ether solution of ethylene glycol.

[0020] Preferably, the addition ratio of the co-solvent to the mixed liquid is 1:6.

[0021] Preferably, the number of centrifugal treatments in the third step is 3-4 times, and the solvent added is n-hexane solvent, and the amount of n-hexane solvent added is: 1 / 6 to 1 / 8.

[0022] Preferably, the drying temperature of sclareol in the fifth step is 50-90° C., and the drying time is 2-5 hours.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of the present invention has few process steps, is simple to operate, has high yield, low production cost, and the solvent used can be recycled. It is a safe and environmentally friendly process for extracting sclareol.

[0025] The present invention adopts a method of adding a cosolvent to the fermentation broth to redissolve the crystalline part of sclareol and then growing the crystals at low temperature. The sclareol crystals in the fermentation broth are partially dissolved and then recrystallized into larger particles, which reduces the difficulty of filtration and ensures the product yield. Combined with subsequent drying, solvent extraction and crystallization methods, the extraction process is simplified and the product content is increased. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] The present invention provides a method for extracting sclareol from a fermentation broth, comprising the following steps:

[0028] Step 1: Fermentation Broth Treatment

[0029] The fermentation liquid to be treated is allowed to stand for 1-3 hours, and the fermentation liquid after standing is placed in a centrifuge for centrifugation to obtain a mixture of bacterial liquid and sclareol. The fermentation liquid needs to be centrifuged at 135-150°C and a speed of 500-6000 rpm;

[0030] Step 2: Secondary treatment of fermentation broth

[0031] The bacterial liquid and the mixed liquid after centrifugation are processed, the bacterial liquid is located in the upper layer of the mixed liquid, the bacterial liquid above the mixed liquid is removed to obtain a mixed liquid, a co-solvent is added to the mixed liquid for mixing, and the mixed co-solvent dissolves sclareol in the mixed liquid, the co-solvent being one of a petroleum ether solution of petroleum ether, trichloroethylene, methanol, ethanol, and ethylene glycol;

[0032] Step 3: Obtaining sclareol

[0033] The mixed solution is placed in a centrifuge and centrifuged again to obtain a supernatant containing sclareol. A solvent is added to the supernatant, and then crystallized to obtain a sclareol crystal structure, wherein the solvent is one of petroleum ether, trichloroethylene, methanol, ethanol, and a petroleum ether solution of ethylene glycol;

[0034] Step 4: Purification of Sclareol Crystals

[0035] The obtained sclareol crystals are added to an ethanol solution for dissolution, the dissolved solution is frozen and diluted, and finally filtered and crystallized to obtain purified sclareol crystals, wherein the number of treatments is 3-4 times, and the solvent added is n-hexane solvent, and the amount of n-hexane solvent added is: 1 / 6 to 1 / 8;

[0036] Step 5: Sclareol Finished Product

[0037] The sclareol crystals are dried, crushed and pulverized to obtain a refined sclareol finished product. The drying temperature of the sclareol is 50-90° C., and the drying time is 2-5 hours.

[0038] The present invention adopts a method for adding a cosolvent into the fermentation broth to redissolve the crystalline part of sclareol and then growing the crystals at low temperature. The sclareol crystals in the fermentation broth are partially dissolved and then recrystallized into larger particles, which reduces the difficulty of filtration and ensures the product yield. The subsequent drying, solvent extraction and crystallization methods are combined to simplify the extraction process and increase the product content. The method has the advantages of fewer process steps, simple operation, high yield and low production cost, and the solvent used can be recycled. Therefore, the method is a safe and environmentally friendly process for extracting sclareol.

[0039] Example 1:

[0040] The fermentation broth was used to extract sclareol. The corresponding fermentation broth was inoculated into a 1L glass bottle and allowed to stand for 1 hour. The fermentation broth after standing was placed in a centrifuge for centrifugation to separate the bacterial liquid in the fermentation broth and the mixed liquid containing sclareol. The centrifugation time was 5000 rpm for 15 minutes.

[0041] After 15 minutes, a precipitate containing sclareol is obtained at the bottom of the precipitate, and the precipitate is subjected to a secondary treatment by adding a cosolvent, the cosolvent being a 95% ethanol solution, adding 0.1L of the ethanol solution and stirring, each stirring time being 2 minutes, and continuously adding 4 times to dissolve it, dissolving the sclareol precipitate contained in the precipitate, and then subjecting the solution to a secondary treatment, placing the solution in a centrifuge for centrifugal treatment, and separating the liquid containing sclareol by centrifugation, the centrifugation time being 5000 rpm, and the centrifugation time being 15 minutes;

[0042] After 15 minutes, a supernatant containing sclareol was obtained, and the remaining precipitate was filtered out. The supernatant was crystallized. During the crystallization process, n-hexane solvent was added, and the amount of n-hexane solvent added was 1 / 8 of the supernatant content. Stirring was performed while adding, and the n-hexane solvent was heated and dissolved, and the insoluble precipitate was filtered out. The dissolved liquid was a sclareol solution, which was crystallized. The crystals after crystallization were sclareol crystals with a mass purity of 94.2%.

[0043] Example 2:

[0044] The fermentation broth was used to extract sclareol. The corresponding fermentation broth was inoculated into a 1L glass bottle and allowed to stand for 2 hours. The fermentation broth after standing was placed in a centrifuge for centrifugation to separate the bacterial liquid in the fermentation broth and the mixed liquid containing sclareol. The centrifugation time was 5000 rpm for 30 minutes.

[0045] After 30 minutes, a precipitate containing sclareol is obtained at the bottom of the precipitate, and the precipitate is subjected to a secondary treatment by adding a cosolvent, the cosolvent being a 95% ethanol solution, adding 0.15L of the ethanol solution and stirring, each stirring time being 2 minutes, and continuously adding 4 times to dissolve it, dissolving the sclareol precipitate contained in the precipitate, and then subjecting the solution to a secondary treatment, placing the solution in a centrifuge for centrifugal treatment, and separating the liquid containing sclareol by centrifugation, the centrifugation time being 5000 rpm, and the centrifugation time being 30 minutes;

[0046] After 30 minutes, a supernatant containing sclareol was obtained, and the remaining precipitate was filtered out. The supernatant was crystallized. During the crystallization process, n-hexane solvent was added, and the amount of n-hexane solvent added was 1 / 6 of the supernatant content. The solution was stirred while being added, and the n-hexane solvent was heated and dissolved, and the insoluble precipitate was filtered out. The dissolved liquid was a sclareol solution, which was crystallized. The crystals after crystallization were sclareol crystals with a mass purity of 96.34%.

[0047] Example 3:

[0048] The fermentation broth was used to extract sclareol. The corresponding fermentation broth was inoculated into a 1L glass bottle and allowed to stand for 1 hour. The fermentation broth after standing was placed in a centrifuge for centrifugation to separate the bacterial liquid in the fermentation broth and the mixed liquid containing sclareol. The centrifugation time was 5000 rpm for 15 minutes.

[0049] After 15 minutes, a precipitate containing sclareol is obtained at the bottom of the precipitate, and the precipitate is subjected to a secondary treatment by adding a cosolvent, the cosolvent being a 75% ethanol solution, adding 0.2L of the ethanol solution and stirring, each stirring time being 2 minutes, and continuously adding 4 times to dissolve it, dissolving the sclareol precipitate contained in the precipitate, and then subjecting the solution to a secondary treatment, placing the solution in a centrifuge for centrifugal treatment, and separating the liquid containing sclareol by centrifugation, the centrifugation time being 5000 rpm, and the centrifugation time being 15 minutes;

[0050] After 15 minutes, a supernatant containing sclareol was obtained, and the remaining precipitate was filtered out. The supernatant was crystallized. During the crystallization process, petroleum ether solvent was added, and the amount of petroleum ether solvent added was 1 / 6 of the supernatant content. Stirring was added while adding, and the solution was heated and dissolved with n-hexane solvent, and the insoluble precipitate was filtered out. The dissolved liquid was sclareol solution, which was crystallized. The crystals after crystallization were sclareol crystals with a mass purity of 95.65%.

[0051] Example 4:

[0052] The fermentation broth was used to extract sclareol. The corresponding fermentation broth was inoculated into a 1L glass bottle and allowed to stand for 1 hour. The fermentation broth after standing was placed in a centrifuge for centrifugation to separate the bacterial liquid in the fermentation broth and the mixed liquid containing sclareol. The centrifugation time was 5000 rpm for 30 minutes.

[0053] After 30 minutes, a precipitate containing sclareol is obtained at the bottom of the precipitate, and the precipitate is subjected to a secondary treatment by adding a cosolvent, the cosolvent is an ethylene glycol solution, 0.2L of ethylene glycol solution is added and stirred, each stirring time is 2 minutes, and the solution is added 4 times continuously to dissolve it, dissolve the sclareol precipitate contained in the precipitate, and then the solution is subjected to a secondary treatment, the solution is placed in a centrifuge for centrifugal treatment, and the liquid containing sclareol is separated by centrifugation, the centrifugation time is 5000 rpm, and the centrifugation time is 15 minutes;

[0054] After 15 minutes, a supernatant containing sclareol was obtained, and the remaining precipitate was filtered out. The supernatant was crystallized. During the crystallization process, trichloroethylene solvent was added, and the amount of trichloroethylene solvent added was 1 / 6 of the supernatant content. Stirring was added while adding, and the solution was dissolved by heating with n-hexane solvent, and the insoluble precipitate was filtered out. The dissolved liquid was sclareol solution, which was crystallized. The crystals after crystallization were sclareol crystals with a mass purity of 97.3%.

[0055] Example 5: Optimal Purity Extraction Conditions

[0056] The fermentation broth was used to extract sclareol. The corresponding fermentation broth was inoculated into a 1L glass bottle and allowed to stand for 3 hours. The fermentation broth after standing was placed in a centrifuge for centrifugation to separate the bacterial liquid in the fermentation broth and the mixed liquid containing sclareol. The centrifugation time was 5000 rpm for 15 minutes.

[0057] After 15 minutes, a precipitate containing sclareol is obtained at the bottom of the precipitate, and the precipitate is subjected to a secondary treatment by adding a cosolvent, wherein the cosolvent is an 85% ethanol solution, and 0.15L of the ethanol solution is added and stirred. The stirring time is 2 minutes each time, and the addition is continued for 4 times to dissolve it, dissolving the sclareol precipitate contained in the precipitate. After the solution is dissolved, the solution is subjected to a secondary treatment, and the solution is placed in a centrifuge for centrifugal treatment. By centrifugation, the liquid containing sclareol is separated, and the centrifugation time is 5000 rpm for 15 minutes.

[0058] After 15 minutes, a supernatant containing sclareol was obtained, and the remaining precipitate was filtered out. The supernatant was crystallized. During the crystallization process, n-hexane solvent was added, and the amount of n-hexane solvent added was 1 / 6 of the supernatant content. Stirring was added while heating and dissolving the n-hexane solvent, and the undissolved precipitate was filtered out. The dissolved liquid was a sclareol solution, which was crystallized. The crystals after crystallization were sclareol crystals. The crystallized sclareol crystals were then washed with cold n-hexane solvent. After washing, sclareol was obtained, and its mass purity was 98.3%.

[0059] Purification method of sclareol: purification adopts the method of dissolution filtration, and the impurities in the sclareol component are removed by repeated filtration to carry out purification treatment.

[0060] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting sclareol from a fermentation broth, characterized in that: The following steps are included: Step 1: Fermentation Broth Treatment The fermentation liquid to be treated is allowed to stand for 1-3 hours, and the fermentation liquid after standing is placed in a centrifuge for centrifugation to obtain a mixture of bacterial liquid and sclareol; Step 2: Secondary treatment of fermentation broth The bacterial liquid and the mixed liquid after centrifugation are processed, the bacterial liquid is located in the upper layer of the mixed liquid, the bacterial liquid above the mixed liquid is removed to obtain a mixed liquid, a cosolvent is added to the mixed liquid for mixing, and the mixed cosolvent dissolves sclareol in the mixed liquid; Step 3: Obtaining sclareol The mixed solution is placed in a centrifuge and centrifuged again to obtain a supernatant containing sclareol, a solvent is added to the supernatant, and then crystallization is performed to obtain a sclareol crystal structure; Step 4: Purification of Sclareol Crystals The obtained sclareol crystals are added to an ethanol solution for dissolution, the dissolved solution is frozen and diluted, and finally filtered and crystallized to obtain purified sclareol crystals; Step 5: Sclareol Finished Product The sclareol crystals are dried, crushed and pulverized to obtain a refined sclareol finished product.

2. The method for extracting sclareol from a fermentation broth according to claim 1, wherein: In the first step of processing the fermentation liquid, the fermentation liquid needs to be centrifuged at 135-150° C. and a speed of 500-6000 rpm.

3. The method for extracting sclareol from a fermentation broth according to claim 1, wherein: The cosolvent in the second step is one of petroleum ether, trichloroethylene, methanol, ethanol and ethylene glycol solution.

4. The method for extracting sclareol from a fermentation broth according to claim 1, wherein: The addition ratio of the cosolvent to the mixed liquid is 1:

6.

5. The method for extracting sclareol from a fermentation broth according to claim 1, wherein: The number of centrifugal treatments in the third step is 3-4 times, and the solvent added is n-hexane solvent, and the amount of n-hexane solvent added is: 1 / 6 to 1 / 8.

6. The method for extracting sclareol from a fermentation broth according to claim 1, wherein: In the fifth step, the drying temperature of sclareol is 50-90° C., and the drying time is 2-5 hours.