A method for separating and purifying bisabolol from bisabolol fermentation dregs

Abstract

The application provides a method for separating and purifying bisabolol from fermented residue of bisabolol, which comprises the following steps: taking the fermented residue of bisabolol, stirring with an organic solvent, filtering, taking the filtrate to decolorize, and distilling the decolorized liquid to obtain the bisabolol. The preparation method can successfully obtain high-purity bisabolol, and has the advantages of simple operation, low cost and low solvent toxicity.

Description

Technical Field

[0001] This invention belongs to the field of fermentation, and specifically relates to a method for separating and purifying bisabolol from bisabolol fermentation residue. Background Technology

[0002] Bisabolol (molecular formula C) 15 H 26 Bisabolol (O), also known as bisabolol or saposhnikovia root alcohol, is one of the most abundant sesquiterpenes in nature. It is mainly found in essential oils of chamomile, sage, and certain herbs in the genera *Salvia* and *Salvia*. Bisabolol possesses various biological activities, including reducing skin inflammation, antibacterial and anti-irritant effects, promoting blood circulation and relieving pain, healing ulcers, and dissolving gallstones. Therefore, it has wide applications in the pharmaceutical and cosmetic industries.

[0003] Currently, the main methods for producing bisabolol include extraction, chemical synthesis, and biosynthesis. Extraction primarily involves separating and extracting high-optical-purity α-bisabolol from plants rich in α-bisabolol, such as chamomile and geraniol. However, this method requires obtaining large quantities of these plants, involving transportation and storage, resulting in high costs. Furthermore, the habitat loss of these α-bisabolol-rich plants not only makes it difficult to sustainably obtain α-bisabolol from nature but also damages vegetation, contributing to desertification. Therefore, large-scale industrial production using extraction is difficult. Chemical synthesis mainly uses farnesol and other similar raw materials, involving chemical reactions with organic reagents and strong acids, followed by purification and separation to obtain racemic bisabolol.

[0004] The production of α-bisabolol using this method consumes large quantities of hazardous chemicals, causing significant environmental pollution and energy consumption. Furthermore, bisabolol contains farnesol, a skin-irritating raw material. Although chemical synthesis has enabled large-scale industrial production, difficulties remain due to the aforementioned reasons, including low product purity and content, inefficient control of racemic mixtures, and high impurity levels. Biosynthesis primarily involves microbial fermentation to obtain bisabolol fermentation broth, followed by separation and purification to obtain the final product. Biosynthesis of bisabolol offers low cost, readily available and inexpensive raw materials, and high optical purity, making it the most promising production route.

[0005] Patent CN201910333500.X discloses a method for producing α-bisabolol through microbial fermentation, achieving a yield of up to 4.15 g / L. However, extensive literature and patent searches have not yielded any research reports on extracting bisabolol from the fermentation residue. Therefore, it is necessary to develop a method for extracting high-purity bisabolol from the residue to further increase the yield of bisabolol produced by biological methods and avoid resource waste. Summary of the Invention

[0006] To address the above problems, this invention provides a method for separating and purifying bisabolol from bisabolol fermentation residue, comprising the following steps:

[0007] Take the bisabolol fermentation residue, add organic solvent and stir, filter, decolorize the filtrate, distill the decolorized solution and collect the fraction to obtain the product.

[0008] Further, the bacterial residue is mixed with 1 to 10 times the amount of v / w organic solvent, filtered, and the filter residue is then mixed with 0.1 to 1 times the amount of v / w organic solvent, and the filtrates are combined for decolorization.

[0009] Furthermore, the organic solvent is an ester solvent, an alkane solvent with more than 6 carbon atoms, an alcohol solvent, or a ketone solvent.

[0010] Furthermore, the ester solvents include ethyl acetate and butyl acetate; the alkane solvents include n-heptane, isodecane, n-dodecane, and isododecane; the alcohol solvents include methanol and ethanol; and the ketone solvents include acetone.

[0011] Furthermore, the organic solvent is butyl acetate, n-dodecane, or isododecane.

[0012] Furthermore, the organic solvent is isododecane.

[0013] Furthermore, the moisture content of the bacterial residue is 15-75%, preferably 20-40%.

[0014] Furthermore, the stirring temperature is 20℃~70℃, preferably 25℃~45℃, and the stirring time is 1~5 hours, preferably 2~3 hours.

[0015] Furthermore, the decolorization is performed by adding activated carbon; the amount of activated carbon added is 0.1-1.0% of the filtrate volume, preferably 0.3-0.6%; the decolorization time is 20-60 minutes.

[0016] Furthermore, the distillation is first carried out under reduced pressure at 25℃ to 75℃ or molecular distillation at 65℃ to 75℃ until no fraction flows out, and the crude bisabolol obtained is then distilled under reduced pressure at 140 to 150℃ or molecular distilled at 80℃ to 85℃ until no fraction flows out.

[0017] The present invention also provides a bisabolol, which is prepared according to the aforementioned method, and has a content of not less than 90% and a purity of not less than 95%.

[0018] Experimental results show that the method for separating and purifying bisabolol from bisabolol fermentation residue according to this invention can successfully obtain high-purity bisabolol from the fermentation residue. The method is simple, low-cost, and uses solvents with low toxicity. The bisabolol prepared by this method exhibits good stability, anti-inflammatory, and anti-aging properties, making it widely applicable in industries such as cosmetics. Attached Figure Description

[0019] Figure 1 This is the gas chromatogram of bisabolol standard.

[0020] Figure 2 This is the gas chromatogram of bisabolol extracted in this invention.

[0021] Figure 3 The infrared spectrum of bisabolol standard.

[0022] Figure 4 The infrared spectrum of bisabolol extracted in this invention is shown.

[0023] Figure 5 The top image shows the mass spectrum of bisabolol standard, and the bottom image shows the mass spectrum of bisabolol extracted in this invention. Detailed Implementation

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0025] Unless otherwise specified, all raw materials and equipment used in this invention can be obtained by purchasing commercially available products. The bisabolol fermentation broth used in this invention is a fermentation broth produced according to the method provided in the applicant's invention patent application (patent number: ZL2023101580889, patent name: A (-)-α-bisabolol fermentation broth culture medium and its preparation method and application), including the following steps:

[0026] (1) Preparation method of (-)-α-bisabolol fermentation medium: Weigh 20 g / L glucose, 30 g / L yeast extract, 10 g / L yeast peptone, 10 g / L soybean meal, 20 g / L corn steep liquor powder, 6 g / L dipotassium hydrogen phosphate, 8 g / L potassium dihydrogen phosphate, 0.034 g / L chloramphenicol, 0.1 g / L ampicillin, 0.1 g / L defoamer, and the remainder is water. Dissolve all raw materials except glucose in water, then add glucose and defoamer. Sterilize at 121-123℃ for 30 min to obtain the medium.

[0027] (2) A method for producing (-)-α-bisabolol: ① Take recombinant Escherichia coli and inoculate it into LB medium with pH 6.80-7.40 and a concentration of 25 g / L. Activate it by culturing it at a temperature of 35-38℃ and a rotation speed of 200-250 rpm for 6-20 h. Inoculate the activated seed liquid into the aforementioned (-)-α-bisabolol fermentation medium and ferment it for 60-100 h to obtain the fermentation broth.

[0028] Example 1: A method for separating and purifying bisabolol from fungal residue.

[0029] The bisabolol fermentation broth (10.40 kg) was filtered through a plate and frame filter to obtain bisabolol mycelial residue (2.48 kg). The bisabolol mycelial residue was dried at 45℃ (moisture content 20%–40%). 2.00 kg of the dried mycelial residue was weighed and 4 L of butyl acetate was added. The mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the filtrate and filter cake were collected. 1.6 L of butyl acetate was added to the filter cake, and the mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, w / v) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized solution. The decolorizing solution is first distilled at low temperature and reduced pressure at 50℃. When no more liquid flows out, the low temperature and reduced pressure distillation is stopped, and the liquid that does not flow out is collected, which is the crude bisabolol. Then, high temperature and reduced pressure distillation is carried out (140-150℃). When no more fractions flow out, the eluent is collected to obtain the refined bisabolol, with a yield of 80.23%, a content of 927.72 ug / mg, and a purity of 91.23%.

[0030] Example 2: A method for separating and purifying bisabolol from fungal residue.

[0031] The bisabolol fermentation broth (20.52 kg) was filtered through a plate and frame filter to obtain bisabolol bacterial residue (2.80 kg). 2.00 kg of wet bacterial residue was weighed and 4 L of butyl acetate was added. The mixture was stirred at room temperature (25℃) for 2 h. After stirring was stopped, the mixture was filtered and the filtrate and filter cake were collected. 1.6 L of butyl acetate was added to the filter cake and the mixture was stirred at room temperature (25℃) for 2 h. After stirring was stopped, the mixture was filtered and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, W / V) was added for decolorization for 30 min. The mixture was then filtered to obtain the decolorized solution. The decolorizing solution was first distilled at low temperature and reduced pressure at 50°C. When no more liquid flowed out, the low temperature and reduced pressure distillation was stopped, and the liquid that did not flow out was collected as crude bisabolol. Then, molecular distillation was performed at 80°C to 85°C. When the light phase fraction no longer flowed out, the light phase liquid was collected to obtain pure bisabolol with a yield of 70.23%, a content of 919.96 ug / mg, and a purity of 90.12%.

[0032] Example 3: A method for separating and purifying bisabolol from fungal residue.

[0033] The bisabolol fermentation broth (20.98 kg) was filtered through a plate and frame filter to obtain bisabolol mycelium residue (3.00 kg). The bisabolol mycelium residue was dried at 45℃ (moisture content 20%–40%). 2.00 kg of the dried mycelium residue was weighed and 4 L of n-heptane was added. The mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the filtrate and filter cake were collected. 1.6 L of n-heptane was added to the filter cake, and the mixture was stirred at room temperature for 2 hours. After stirring was stopped, the mixture was filtered, and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, w / v) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized solution. The decolorizing solution was first distilled at low temperature and reduced pressure at 50°C. When no more liquid flowed out, the low temperature and reduced pressure distillation was stopped, and the liquid that did not flow out was collected as crude bisabolol. Then, high temperature and reduced pressure distillation was carried out (140-150°C). When no more fraction flowed out, the eluent was collected to obtain high-quality bisabolol with a yield of 57.64%, a content of 915.95 ug / mg, and a purity of 85.235%.

[0034] Example 4: A method for separating and purifying bisabolol from fungal residue.

[0035] The bisabolol fermentation broth (26.09 kg) was filtered through a plate and frame filter to obtain bisabolol bacterial residue (2.24 kg). 2.00 kg of wet bacterial residue was weighed and 4 L of n-heptane was added. The mixture was stirred at room temperature (25℃) for 2 h. After stirring was stopped, the mixture was filtered and the filtrate and filter cake were collected. 1.6 L of n-heptane was added to the filter cake and the mixture was stirred at room temperature (25℃) for 2 h. After stirring was stopped, the mixture was filtered and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, W / V) was added for decolorization for 30 min. The mixture was then filtered to obtain the decolorized solution. The decolorizing solution was first distilled under reduced pressure at low temperature (50°C). When no more liquid flowed out, the distillation was stopped, and the undrained liquid (crude bisabolol) was collected. This crude bisabolol was then subjected to molecular distillation at 80°C–85°C. When the lighter phase fraction ceased to flow out, the lighter phase liquid was collected to obtain the refined bisabolol, with a yield of 50.67%, a content of 869.34 ug / mg, and a purity of 80.248%.

[0036] Example 5: A method for separating and purifying bisabolol from fungal residue.

[0037] The bisabolol fermentation broth (26.00 kg) was filtered through a plate and frame filter to obtain bisabolol mycelium residue (2.60 kg). The bisabolol mycelium residue was dried at 45℃ (moisture content 20%–40%). 2.00 kg of the dried mycelium residue was weighed and 4 L of isododecane was added. The mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the filtrate and filter cake were collected. 1.6 L of isododecane was added to the filter cake, and the mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, w / v) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized solution. The decolorized solution was directly subjected to molecular distillation at 65℃-75℃. When the heavy phase fraction stopped flowing out, the heavy phase liquid was collected and then subjected to molecular distillation at 80℃-85℃. When the light phase fraction stopped flowing out, the light phase liquid was collected to obtain the high-quality bisabolol, with a yield of 85%, a content of 950.56ug / mg, and a purity of 97.65%.

[0038] Example 6: A method for separating and purifying bisabolol from fungal residue.

[0039] The bisabolol fermentation broth (31.00 kg) was filtered through a plate and frame filter to obtain bisabolol bacterial residue (2.98 kg). 2.00 kg of the wet residue was weighed and 4 L of isododecane was added. The mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the filtrate and filter cake were collected. Another 1.6 L of isododecane was added to the filter cake, and the mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, V / W) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized liquid. The decolorized liquid was directly subjected to molecular distillation at 65℃-75℃. When the heavy phase fraction stopped flowing out, the heavy phase liquid was collected and then subjected to molecular distillation at 80℃-85℃. When the light phase fraction stopped flowing out, the light phase liquid was collected to obtain the purified bisabolol, with a yield of 80%, a content of 912.56 ug / mg, and a purity of 95.735%.

[0040] Example 7: A method for separating and purifying bisabolol from fungal residue.

[0041] The bisabolol fermentation broth (27.60 kg) was filtered through a plate and frame filter to obtain bisabolol mycelium residue (2.58 kg). The bisabolol mycelium residue was dried at 45℃ (moisture content 20%–40%). 2.00 kg of the dried mycelium residue was weighed and 4 L of n-dodecane was added. The mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the filtrate and filter cake were collected. 1.6 L of n-dodecane was added to the filter cake, and the mixture was stirred at room temperature (25℃) for 2 hours. After stirring was stopped, the mixture was filtered, and the two filtrates were combined. Activated carbon (0.5% of the total volume of the two filtrates, w / v) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized solution. The decolorized solution was directly subjected to molecular distillation at 65℃-75℃. When the heavy phase no longer flowed out, the heavy phase liquid was collected and then subjected to molecular distillation at 80℃-85℃. When the light phase fraction no longer flowed out, the liquid in the light phase was collected to obtain the high-quality bisabolol, with a yield of 75.46%, a content of 893.31 ug / mg, and a purity of 93.560%.

[0042] Example 8: A method for separating and purifying bisabolol from fungal residue.

[0043] The bisabolol fermentation broth (35.00 kg) was filtered through a plate and frame filter to obtain bisabolol bacterial residue (3.40 kg). 2.00 kg of the wet residue was weighed and 4 L of n-dodecane was added. The mixture was stirred at room temperature (25℃) for 2 hours. Stirring was stopped, and the mixture was filtered. The filtrate and filter cake were collected. Another 1.6 L of n-dodecane was added to the filter cake, and the mixture was stirred at room temperature (25℃) for 2 hours. Stirring was stopped, and the mixture was filtered. The two filtrates were combined, and activated carbon (0.5% of the total volume of the two filtrates, W / V) was added for decolorization for 30 minutes. The mixture was then filtered to obtain the decolorized liquid. The decolorized liquid was directly subjected to molecular distillation at 65℃-75℃. When the heavy phase fraction stopped flowing out, the heavy phase liquid was collected and then subjected to molecular distillation at 80℃-85℃. When the light phase fraction stopped flowing out, the light phase liquid was collected to obtain the pure bisabolol, with a yield of 75.23%, a content of 889.651 ug / mg, and a purity of 92.239%.

[0044] The following experimental examples further illustrate the beneficial effects of the present invention.

[0045] Experimental Example 1: Verification of the Product of the Invention

[0046] 1. Experimental Methods

[0047] The product obtained in Example 5 was subjected to gas chromatography, infrared spectroscopy, and mass spectrometry.

[0048] 2. Experimental Results

[0049] The results are as follows Figure 1-5 As shown, Figure 1 Gas chromatogram of bisabolol standard. Figure 2The gas chromatogram of bisabolol prepared in Example 5 shows that the response time of the bisabolol product and the bisabolol standard is consistent. Figure 3 The infrared spectrum of bisabolol standard. Figure 4 The infrared spectrum of bisabolol prepared in Example 5 shows a 99.02% goodness of fit between the bisabolol product and the bisabolol standard. This demonstrates the similarity between the infrared spectra of the sample and the reference standard. Figure 1 To. Figure 5 The above is the standard quality spectrum of bisabolol. Figure 5 Below is the mass spectrum of bisabolol prepared in Example 5. The molecular weight of the bisabolol product calculated from the mass spectrum is consistent with that of the bisabolol standard.

[0050] In summary, this invention provides a method for separating and purifying bisabolol from bacterial residue. The extraction method of this invention can successfully obtain high-purity bisabolol, which has good stability, anti-inflammatory and anti-aging properties, and is of wide application value in the cosmetics and other industries.

Claims

1. A method for separating and purifying bisabolol from bisabolol fermentation residue, characterized in that, It includes the following steps: Take the bisabolol fermentation residue, add organic solvent and stir, filter, decolorize the filtrate, distill the decolorized solution, and collect the distillate to obtain the product; The organic solvent is isododecane.

2. The method as described in claim 1, characterized in that, The bacterial residue is stirred with 1 to 10 times the amount of organic solvent, filtered, and the filter residue is stirred with 0.1 to 1 times the amount of organic solvent, filtered, and the filtrates are combined and decolorized.

3. The method as described in claim 1 or 2, characterized in that, The moisture content of the bacterial residue is 15-75%.

4. The method as described in claim 1 or 2, characterized in that, The stirring temperature is 20℃~70℃, and the time is 1~5 hours.

5. The method as described in claim 1, characterized in that, The decolorization is performed by adding activated carbon; the amount of activated carbon added is 0.1~1.0% of the filtrate volume; the decolorization time is 20~60 min.

6. The method as described in claim 1, characterized in that, The distillation is first carried out under reduced pressure at 25℃~75℃ or molecular distillation at 65℃-75℃ until no fraction flows out. The crude bisabolol obtained is then distilled under reduced pressure at 140-150℃ or molecular distilled at 80℃~85℃ until no fraction flows out.

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