A method for the fermentative production of (-)-alpha-bisabolol
By optimizing the culture medium and fermentation process, using ethanol as the substrate, and controlling the pH value and adjusting the feeding rate in stages, the problems of low yield and high cost in the (-)-α-bisabolol fermentation method were solved, and high-efficiency production was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HANGZHOU VIABLIFE BIOTECH CO LTD
- Filing Date
- 2026-04-20
- Publication Date
- 2026-06-05
AI Technical Summary
The existing fermentation method of (-)-α-bisabolol has low yield and high production cost due to unreasonable process, and the two-phase fermentation brings equipment damage and safety problems.
The culture medium and fermentation process were optimized by using ethanol as the substrate, controlling the pH value in stages, adding ammonia and adjusting the replenishment rate of ethanol and glucose, and optimizing stirring and aeration conditions to improve fermentation efficiency.
It significantly increases the yield of (-)-α-bisabolol, reduces production costs, and minimizes equipment wear and tear, showing promising prospects for industrial application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bio-fermentation technology, and more specifically, to a fermentation production method for (-)-α-bisabolol. Background Technology
[0002] Bisabolol, also known as sweet bisabolol or saposhnikovia root alcohol, is one of the more abundant sesquiterpenoid compounds in nature, with both α- and β-form structures. As a natural raw material, (-)-α-bisabolol has been used in pharmaceuticals and cosmetics due to its anti-inflammatory, bactericidal, antimicrobial, skin-soothing, and moisturizing properties.
[0003] Bisabolol is naturally found in Brazilian eucalyptus and German chamomile. As a secondary metabolite of plants, its yield from plant extraction is limited by various factors such as the biological growth cycle and the environment, far from meeting the needs of industrial production. In recent years, (-)-α-bisabolol has mainly relied on chemical synthesis. However, due to its complex chiral chemical structure, direct chemical synthesis is difficult, resulting in low bioactivity and purity of the synthesized product. Therefore, a method for producing (-)-α-bisabolol using engineered microorganisms has been developed, overcoming the limitations of plant extraction and chemical synthesis processes.
[0004] However, due to unreasonable processes and equipment wear and safety issues caused by biphasic fermentation, the yield of (-)-α-bisabolol is low, resulting in high production costs.
[0005] In view of this, the present invention is proposed. Summary of the Invention
[0006] The purpose of this invention is to provide a fermentation production method for (-)-α-bisabolol. By optimizing the culture medium and fermentation process, this invention can significantly increase the yield of (-)-α-bisabolol and reduce production costs.
[0007] This invention is implemented as follows: This invention provides a fermentation production method for (-)-α-bisabolol, which includes: sequentially inoculating a strain that produces (-)-α-bisabolol into a seed culture medium and a fermentation culture medium, fermenting and culturing, and then transferring the culture to a tank to obtain (-)-α-bisabolol; The strain uses ethanol as a substrate, and ethanol is added during fermentation to a concentration of 50-100 g / L. The pH is controlled in two stages: the pH of the first stage is 5.3-5.7, and the pH of the second stage is 5.8-6.2.
[0008] In some embodiments, the pH is controlled by adding ammonia during fermentation, wherein the pH is maintained at 5.3-5.7 for 0-24 h and at 5.8-6.2 for 24-96 h.
[0009] In some embodiments, the concentration of glucose fed during fermentation is 600-800 g / L.
[0010] In some embodiments, the fermentation conditions are as follows: aeration and stirring at 25-30°C and dissolved oxygen saturation of 30%-40%, with a stirring speed of 50-150 rpm and an aeration rate of 500-800 m³ / h. 3 / h.
[0011] In some embodiments, the rate of ethanol or glucose supplementation during fermentation is adjusted according to the wet weight of the cells. Specifically, the supplementation rate is 2-4 g / (L·h) when the wet weight is between 0 and 80 g / L; 4-6 g / (L·h) when the wet weight is between 80 and 120 g / L; and 6-8 g / (L·h) when the wet weight is ≥120 g / L.
[0012] In some embodiments, feeding is stopped 2 hours before discharge and discharge is carried out when the ethanol concentration is ≤50 g / L.
[0013] In some embodiments, the fermentation medium comprises the following components in the indicated mass concentrations: glucose 1%–5%, yeast extract 1%–2%, trypsin 1%–2%, magnesium sulfate heptahydrate 0.1%–0.2%, potassium dihydrogen phosphate 0.1%–0.2%, inositol 0.02%–0.05%, sodium sulfate 0.01%–0.02%, zinc sulfate heptahydrate 0.01%–0.02%, ferrous sulfate heptahydrate 0.005%–0.01%, and calcium chloride dihydrate 0.00%. 5%~0.01%, sodium molybdate dihydrate 0.001%~0.005%, cobalt chloride hexahydrate 0.001%~0.005%, thiamine hydrochloride 0.001%~0.005%, calcium pantothenate 0.001%~0.005%, nicotinic acid 0.001%~0.005%, pyridoxine hydrochloride 0.001%~0.005%, copper sulfate 0.001%~0.002%, manganese chloride tetrahydrate 0.001%~0.002%, defoamer 0.05 mL / L~0.2 mL / L.
[0014] In some embodiments, the strain is activated before being inoculated into the fermentation medium to obtain a primary seed culture, and then the primary seed culture is inoculated into the seed culture medium to obtain a secondary seed culture.
[0015] In some embodiments, the activated culture medium is yeast extract peptone glucose medium.
[0016] In some embodiments, the seed culture medium comprises the following components at mass concentrations: glucose 2%~6%, yeast extract 1%~2%, trypsin 1%~2%, magnesium sulfate heptahydrate 0.1%~0.2%, potassium dihydrogen phosphate 0.1%~0.2%, and antifoaming agent 0.05 mL / L~0.2 mL / L.
[0017] In some embodiments, the activation conditions are: aeration and stirring culture at 28~30℃ and dissolved oxygen saturation of 30%~50% for 20~25 h.
[0018] In some embodiments, the OD600 of the bacterial suspension inoculated during activation is ≥20; the inoculation amount of the bacterial suspension is 2% to 5% of the culture medium volume.
[0019] In some embodiments, the stirring speed during the activation process is 150~250 rpm, and the aeration rate is 3~6 m³ / s. 3 / h.
[0020] In some embodiments, the conditions for inoculating the primary seed culture into the seed culture medium are: aeration and stirring culture at 25-30°C and dissolved oxygen saturation of 30%-50% for 10-15 h.
[0021] In some embodiments, the stirring speed during cultivation is 100-180 rpm, and the aeration rate is 30-60 m³ / h. 3 / h.
[0022] In some embodiments, the inoculation amount of the secondary seed culture is 8% to 10% of the fermentation medium volume.
[0023] The present invention has the following beneficial effects: This invention optimizes the culture medium and fermentation process involved in the fermentation of (-)-α-bisabolol using ethanol as a substrate, and sets up optimal sugar spot segmentation control process, pH value segmentation control process, and ethanol addition process. This fermentation method can significantly improve the fermentation level of (-)-α-bisabolol and greatly reduce the production cost. At the same time, it eliminates biphasic fermentation, increases the yield of (-)-α-bisabolol, and reduces equipment wear and tear, showing good prospects for industrial application. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0025] The literature (Jiang Y, et al..Engineering Saccharomyces cerevisiae forenhanced (-)-α-bisabolol production. Synth Syst Biotechnol. 2023 Jan 20;8(2):187-195.) utilizes engineered bacteria to absorb glucose, metabolize it into acetyl-CoA, and then convert acetyl-CoA into (-)-α-bisabolol. Exogenous ethanol can also be metabolized into acetyl-CoA, thus supplementing ethanol indirectly increases the substrate. This approach uses ethanol as a substrate to synthesize (-)-α-bisabolol through yeast fermentation. However, due to unreasonable processes and equipment wear and safety issues caused by biphasic fermentation (adding organic solvents to an aqueous fermentation system, with fermentation and product extraction occurring simultaneously), the yield of (-)-α-bisabolol is low and the production cost is high. Based on the above literature, this invention optimizes the fermentation process and culture medium involved in order to improve the production efficiency of this fermentation method and reduce the production cost. Specifically, the fermentation production method of (-)-α-bisabolol provided by this invention includes the following steps: S1. The strain that produces (-)-α-bisabolol is activated to obtain a primary seed culture.
[0026] In this invention, the strain used is an engineered Saccharomyces cerevisiae that ferments ethanol to produce (-)-α-bisabolol. This engineered Saccharomyces cerevisiae is a strain with exogenously inserted (-)-α-bisabolol synthase. The starting strain and construction method used are conventional techniques in the field and can be constructed with reference to the aforementioned literature; therefore, this invention does not impose any special limitations on them.
[0027] In some embodiments, the activated culture medium is yeast extract peptone glucose medium. The preparation method of this medium is as follows: weigh 20 g peptone, 10 g yeast extract, 22 g glucose monohydrate, and 20 g agar, dissolve them in 1 L of water, and sterilize at 121°C for 20 min.
[0028] In some embodiments, the activation conditions are: aeration and stirring culture at 28~30℃ and dissolved oxygen saturation of 30%~50% for 20~25 h.
[0029] In some embodiments, the OD600 of the bacterial suspension inoculated during activation is ≥20; the inoculation amount of the bacterial suspension is 2% to 5% of the culture medium volume.
[0030] In some embodiments, the stirring speed during the activation process is 150~250 rpm, and the aeration rate is 3~6 m³ / s. 3 / h.
[0031] S2. Inoculate the primary seed culture into the seed culture medium for secondary culture to obtain the secondary seed culture.
[0032] In some embodiments, the seed culture medium comprises the following components: 2%~6% glucose, 1%~2% yeast extract, 1%~2% trypsin, 0.1%~0.2% magnesium sulfate heptahydrate, 0.1%~0.2% potassium dihydrogen phosphate, and 0.05 mL / L~0.2 mL / L defoamer.
[0033] In some embodiments, the conditions for inoculating the primary seed culture into the seed culture medium are: aeration and stirring culture at 25~30℃ and dissolved oxygen saturation of 30%~50% for 10~15 h; In some embodiments, the stirring speed during cultivation is 100-180 rpm, and the aeration rate is 30-60 m³ / h. 3 / h.
[0034] S3. Inoculate the secondary seed culture into the fermentation medium for fermentation culture and then transfer it to a container.
[0035] In some embodiments, the fermentation medium comprises the following components at concentrations: glucose 1%–5%, yeast extract 1%–2%, trypsin 1%–2%, magnesium sulfate heptahydrate 0.1%–0.2%, potassium dihydrogen phosphate 0.1%–0.2%, inositol 0.02%–0.05%, sodium sulfate 0.01%–0.02%, zinc sulfate heptahydrate 0.01%–0.02%, ferrous sulfate heptahydrate 0.005%–0.01%, and calcium chloride dihydrate 0.005%. ~0.01%, sodium molybdate dihydrate 0.001%~0.005%, cobalt chloride hexahydrate 0.001%~0.005%, thiamine hydrochloride 0.001%~0.005%, calcium pantothenate 0.001%~0.005%, nicotinic acid 0.001%~0.005%, pyridoxine hydrochloride 0.001%~0.005%, copper sulfate 0.001%~0.002%, manganese chloride tetrahydrate 0.001%~0.002%, defoamer 0.05 mL / L~0.2 mL / L.
[0036] In some embodiments, the fermentation conditions are as follows: aeration and stirring at 25-30°C and dissolved oxygen saturation of 30%-40%, with a stirring speed of 50-150 rpm and an aeration rate of 500-800 m³ / h. 3 / h.
[0037] S4. pH is controlled in stages during fermentation.
[0038] In the fermentation process of this invention, pH is controlled by adding ammonia water. The pH is maintained at 5.3-5.7 for 0-24 hours and 5.8-6.2 for 24-96 hours. Since organisms generally require a stable pH during their life activities, ammonia water is a commonly used pH regulator in fermentation, serving both as a supplementary inorganic nitrogen source and as a pH maintainer. Verification has shown that the optimal pH for the yeast used in this invention differs between the fermentation growth and synthesis stages; therefore, segmented pH control is employed.
[0039] S5. Ethanol is used to feed the fermentation process to maintain the concentration of ethanol in the fermentation broth.
[0040] In some embodiments, ethanol is added during fermentation to a concentration of 50-100 g / L; simultaneously, glucose is added at a concentration of 600-800 g / L.
[0041] The purpose of maintaining a certain ethanol concentration in the above steps is to change the permeability of the cell membrane by ethanol, thereby promoting the efflux of (-)-α-bisabolol; to maintain a certain ethanol osmotic pressure, thereby improving the efficiency of ethanol absorption by the bacteria; and to supplement with glucose in order to continuously provide a carbon source.
[0042] In some embodiments, the rate of ethanol and glucose replenishment is adjusted according to the wet weight of the cell mass during fermentation. Wet weight represents the number of cells in the fermentation broth; the greater the number of cells, the greater the demand for carbon sources (glucose and ethanol). Furthermore, the wet weight of the cell mass increases as fermentation progresses, and the demand for ethanol and glucose varies at different wet weight stages. Specifically, the replenishment rate is 2-4 g / (L·h) when the wet weight is between 0 and 80 g / L; 4-6 g / (L·h) when the wet weight is between 80 and 120 g / L; and 6-8 g / (L·h) when the wet weight is ≥120 g / L.
[0043] In some embodiments, the inoculation amount of the secondary seed culture is 8% to 10% of the fermentation medium volume.
[0044] In some embodiments, feeding is stopped 2 hours before discharge and discharge is carried out when the ethanol concentration is ≤50 g / L.
[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0046] The activation culture medium used in this embodiment of the invention is yeast extract peptone glucose medium, which is prepared as follows: weigh 20 g peptone (Angel), 10 g yeast powder (Angel), 22 g glucose monohydrate (Xiangyu), 20 g agar (Biofroxx), dissolve in 1 L of water, and sterilize at 121℃ for 20 min.
[0047] Example 1 This embodiment provides a fermentation production method for (-)-α-bisabolol, the steps of which are as follows: (a) The bacterial strain that produces (-)-α-bisabolol was activated to obtain a seed culture (OD600≥20). The activation process is as follows: Volume: Pour the prepared activation culture medium into a 9 cm round dish (25 mL / dish).
[0048] Invert plates: Cool to room temperature, solidify the culture base and store in a sealed self-sealing bag at 4°C until use.
[0049] Stirring inoculation: Take a cryopreserved glycerol tube stored at -80℃, thaw it on ice, and then streak 20 μL of bacterial culture from the glycerol tube onto a round dish in a clean bench; invert the dish and incubate at 30℃ for 60-84 h; the growth of white, round single colonies indicates that the culture is mature; pick a single colony and inoculate it into seed culture medium, and incubate at 28-30℃ and 220 rpm for 24 h to obtain the seed culture.
[0050] (b) Inoculate the seed culture medium with an inoculum of 2% at 28–30°C, dissolved oxygen saturation of 30%–50%, 200 rpm, and an aeration rate of 4 m³ / min. 3 The culture was carried out for 24 h at a pressure of 0.04~0.06 MPa to obtain the primary seed culture. Then, at an inoculum size of 2%, the primary seed culture was inoculated into seed culture medium and cultured at 28~30℃, dissolved oxygen saturation of 30%~50%, 150 rpm, and an aeration rate of 40 m³ / h. 3 The secondary seed culture was obtained by culturing for 12 h under conditions of / h and tank pressure of 0.04~0.06 MPa.
[0051] The seed culture medium contains: 6% glucose, 1% yeast extract, 2% tryptone, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.1 mL / L defoamer, and water to make up the volume.
[0052] (c) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 8%, and maintain the culture at 25–30°C, dissolved oxygen saturation of 30%–40%, 100 rpm, and an aeration rate of 800 m³ / h. 3 Fermentation was carried out under conditions of 0.04~0.05 MPa per hour and tank pressure, followed by tank discharge.
[0053] The fermentation medium contains the following components: 2% glucose, 1% yeast extract, 2% tryptone, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.02% inositol, 0.01% sodium sulfate, 0.01% zinc sulfate heptahydrate, 0.01% ferrous sulfate heptahydrate, 0.005% calcium chloride dihydrate, 0.005% sodium molybdate dihydrate, 0.005% cobalt chloride hexahydrate, 0.005% thiamine hydrochloride, 0.005% calcium pantothenate, 0.005% nicotinic acid, 0.005% pyridoxine hydrochloride, 0.002% copper sulfate, 0.002% manganese chloride tetrahydrate, and 0.05 mL / L defoamer.
[0054] Ammonia was used to control the pH during fermentation. The pH was maintained at 5.3–5.7 from 0 to 24 hours and 5.8–6.2 from 24 to 96 hours. Sufficient carbon source was provided by adding 700 g / L glucose. The feeding rate was 2.5 g / (L·h) for wet weight 0–80 g / L, 5 g / (L·h) for wet weight 80–120 g / L, and 7 g / (L·h) for wet weight ≥120 g / L. The ethanol concentration in the fermentation broth was adjusted by adding ethanol, maintaining it within 50–100 g / L. Feeding was stopped 2 hours before discharge into the fermentation tank, and discharge was only allowed when the ethanol concentration was ≤50 g / L.
[0055] Example 2 This embodiment provides a fermentation production method for (-)-α-bisabolol, the steps of which are as follows: (a) The bacterial strain that produces (-)-α-bisabolol was activated to obtain a seed culture (OD600≥20). The activation process is as follows: Dosage: Pour the prepared activation culture medium into a 9cm round dish (25 mL / dish).
[0056] Invert plates: Cool to room temperature, solidify the culture base and store in a sealed self-sealing bag at 4°C until use.
[0057] Stirring inoculation: Take a cryopreserved glycerol tube stored at -80℃, thaw it on ice, and then streak 20 μL of bacterial culture from the glycerol tube onto a round dish in a clean bench. Invert the dish and incubate at 30℃ for 60-84 h. The growth of white, round single colonies indicates that the bacteria have matured. Pick a single colony and inoculate it into seed culture medium. Incubate at 28-30℃ and 220 rpm for 24 h to obtain the seed culture.
[0058] (b) Inoculate the seed culture medium with an inoculum of 3% at 28–30°C, dissolved oxygen saturation of 30%–50%, 200 rpm, and aeration rate of 4 m³ / h. 3The culture was carried out for 24 h at a pressure of 0.04~0.06 MPa to obtain the primary seed culture. Then, at an inoculum size of 2%, the primary seed culture was inoculated into seed culture medium and cultured at 28~30℃, dissolved oxygen saturation of 30%~50%, 150 rpm, and an aeration rate of 40 m³ / h. 3 The secondary seed culture was obtained by culturing for 12 h under conditions of / h and tank pressure of 0.04~0.06 MPa.
[0059] The seed culture medium contains: 7% glucose, 1% yeast extract, 2% tryptone, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.1 mL / L defoamer, and water to make up the volume.
[0060] (c) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 9% and maintain the conditions at 25–30°C, dissolved oxygen saturation of 30%–40%, 100 rpm, and an aeration rate of 800 m³ / h. 3 Fermentation was carried out under conditions of 0.04~0.05 MPa per hour and tank pressure. The fermentation medium contained the following components: 4% glucose, 1.5% yeast extract, 1.5% trypsin, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.02% inositol, 0.01% sodium sulfate, 0.01% zinc sulfate heptahydrate, 0.01% ferrous sulfate heptahydrate, 0.005% calcium chloride dihydrate, 0.005% sodium molybdate dihydrate, 0.005% cobalt chloride hexahydrate, 0.005% thiamine hydrochloride, 0.005% calcium pantothenate, 0.005% nicotinic acid, 0.005% pyridoxine hydrochloride, 0.002% copper sulfate, 0.002% manganese chloride tetrahydrate, and 0.05 mL / L defoamer.
[0061] Ammonia was used to control the pH during fermentation. The pH was maintained at 5.4–5.6 from 0–24 h and 5.9–6.1 from 24–96 h. Sufficient carbon source was provided by adding 600 g / L glucose. The feeding rate was 2.5 g / (L·h) for wet weight 0–80 g / L, 5 g / (L·h) for wet weight 80–120 g / L, and 7 g / (L·h) for wet weight ≥120 g / L. The ethanol concentration in the fermentation broth was adjusted by adding ethanol, maintaining it within 50–100 g / L. Feeding was stopped 2 h before discharge into the fermentation tank, and discharge was only allowed when the ethanol concentration was ≤50 g / L.
[0062] Example 3 This embodiment provides a fermentation production method for (-)-α-bisabolol, the steps of which are as follows: (a) The bacterial strain that produces (-)-α-bisabolol was activated to obtain a seed culture (OD600≥20). The activation process is as follows: Volume: Pour the prepared activation culture medium into a 9 cm round dish (25 mL / dish).
[0063] Invert plates: Cool to room temperature, solidify the culture base and store in a sealed self-sealing bag at 4°C until use.
[0064] Stirring inoculation: Take a cryopreserved glycerol tube stored at -80℃, thaw it on ice, and then streak 20 μL of bacterial culture from the glycerol tube onto a round dish in a clean bench. Invert the dish and incubate at 30℃ for 60-84 h. The growth of white, round single colonies indicates that the bacteria have matured. Pick a single colony and inoculate it into seed culture medium. Incubate at 28-30℃ and 220 rpm for 24 h to obtain the seed culture.
[0065] (b) Inoculate the seed culture medium with an inoculum of 4% at 28–30°C, dissolved oxygen saturation of 30%–50%, 200 rpm, and an aeration rate of 4 m³ / min. 3 The culture was carried out for 24 h at a pressure of 0.04~0.06 MPa to obtain the primary seed culture. Then, at an inoculum size of 2%, the primary seed culture was inoculated into seed culture medium and cultured at 28~30℃, dissolved oxygen saturation of 30%~50%, 150 rpm, and an aeration rate of 40 m³ / h. 3 The secondary seed culture was obtained by culturing for 12 h under conditions of / h and tank pressure of 0.04~0.06 MPa.
[0066] The seed culture medium includes: 8% glucose, 1% yeast extract, 2% tryptone, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.1 mL / L defoamer, and water to make up the volume.
[0067] (c) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 10%, and maintain the culture at 25–30°C, dissolved oxygen saturation of 30%–40%, 100 rpm, and an aeration rate of 800 m³ / h. 3 Fermentation was carried out under conditions of 0.04~0.05 MPa / h and tank pressure, and the culture medium contained the following components: Glucose 3%, yeast extract 1.2%, tryptone 2%, magnesium sulfate heptahydrate 0.1%, potassium dihydrogen phosphate 0.1%, inositol 0.02%, sodium sulfate 0.01%, zinc sulfate heptahydrate 0.01%, ferrous sulfate heptahydrate 0.01%, calcium chloride dihydrate 0.005%, sodium molybdate dihydrate 0.005%, cobalt chloride hexahydrate 0.005%, thiamine hydrochloride 0.005%, calcium pantothenate 0.005%, nicotinic acid 0.005%, pyridoxine hydrochloride 0.005%, copper sulfate 0.002%, manganese chloride tetrahydrate 0.002%, defoamer 0.05 mL / L.
[0068] Ammonia was used to control the pH during fermentation, maintaining a pH of 5.4–5.6 from 0 to 24 hours and 5.9–6.1 from 24 to 96 hours. Sufficient carbon source was provided by adding 800 g / L glucose, with a feeding rate of 2.5 g / (L·h) for wet weights of 0–80 g / L, 5 g / (L·h) for wet weights of 80–120 g / L, and 7 g / (L·h) for wet weights ≥120 g / L. The ethanol concentration in the fermentation broth was adjusted by adding ethanol, maintaining it within 50–100 g / L. Feeding was stopped 2 hours before discharge into the fermentation tank, and discharge was only allowed when the ethanol concentration was ≤50 g / L.
[0069] Comparative Example 1 The only difference between this comparative example and Example 2 is that no ethanol is added to adjust the ethanol concentration during the fermentation process in step (c).
[0070] Comparative Example 2 The only difference between this comparative example and Example 2 is that in step (c), ammonia water was used to control the pH to 5.8-6.2 during the fermentation process from 0 to 96 hours.
[0071] Comparative Example 3 The only difference between this comparative example and Example 2 is that the dissolved oxygen saturation during the fermentation process in step (c) is 40%~50%.
[0072] Comparative Example 4 The only difference between this comparative example and Example 2 is that the glucose supplementation rate is fixed at 5 g / (L·h) throughout the process.
[0073] Comparative Example 5 The only difference between this comparative example and Example 2 is the composition of the fermentation medium. The composition of the culture medium in this comparative example is as follows: 4% glucose, 1.5% yeast extract, 1.5% tryptone, 0.1% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.01% sodium sulfate, and 0.05 mL / L defoamer.
[0074] Comparative Example 6 The only difference between this comparative example and Example 2 is the composition of the seed culture medium. The composition of the culture medium in this comparative example is as follows: 7% glucose, 1% yeast extract, 1% tryptic peptone, and 0.1 mL / L defoamer.
[0075] Experimental Example In this experiment, yeast strain BMK6 was used as the experimental strain (BMK6 is an engineered brewing yeast, modified according to the reference: Jiang Y, et al..Engineering Saccharomyces cerevisiae for enhanced (-)-α-bisabolol production.Synth Syst Biotechnol.2023 Jan 20;8(2):187-195). The fermentation production of (-)-α-bisabolol was carried out according to the fermentation production methods in Example 2 and Comparative Examples 1 to 6.
[0076] The yield of (-)-α-bisabolol in different experimental groups was detected by GC method.
[0077] Reference solution: Weigh approximately 100 mg of (-)-α-bisabolol reference standard accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol to obtain the reference solution.
[0078] Test solution: Take an appropriate amount of (-)-α-bisabolol fermentation broth, place it in a 25 mL volumetric flask, dissolve and dilute it to the mark with methanol to obtain the test solution.
[0079] Instrument: Shimadzu GC-2010Pro Gas phase conditions: Injection volume: 1 μL; Vaporization chamber temperature: 250℃; Pressure: 87.5 kPa; Total flow rate: 17 mL / min; Column flow rate: 1 mL / min; Linear velocity: 27.4 cm / sec; Purging flow rate: 1 mL / min; End time: 20 min; The temperature program for the column oven is shown in Table 1: Table 1. Column Oven Temperature Program
[0080] The test results are shown in Table 2: Table 2 Test Results
[0081] As shown in Table 2, compared with Comparative Examples 1-6, Examples 1-3, through a specific fermentation process, can better improve the fermentation yield of (-)-α-bisabolol.
[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A fermentation method for producing (-)-α-bisabolol, characterized in that, include: The strain that produces (-)-α-bisabolol was sequentially inoculated into seed culture medium and fermentation culture medium, and after fermentation and culturing, (-)-α-bisabolol was obtained. The strain uses ethanol as a substrate, and ethanol is added during fermentation to a concentration of 50-100 g / L; the pH is controlled in two stages, with the first stage having a pH of 5.3-5.7 and the second stage having a pH of 5.8-6.
2.
2. The fermentation production method according to claim 1, characterized in that, During fermentation, the pH was controlled by adding ammonia water. The pH was maintained at 5.3-5.7 from 0 to 24 h and at 5.8-6.2 from 24 to 96 h.
3. The fermentation production method according to claim 2, characterized in that, The concentration of glucose added during fermentation is 600~800 g / L.
4. The fermentation production method according to claim 3, characterized in that, The fermentation conditions were as follows: aeration and stirring at 25-30℃ and dissolved oxygen saturation of 30%-40%, with a stirring speed of 50-150 rpm and an aeration rate of 500-800 m³ / h. 3 / h.
5. The fermentation production method according to claim 4, characterized in that, During fermentation, the rate of ethanol and glucose replenishment is adjusted according to the wet weight of the cells. Specifically, the replenishment rate is 2-4 g / (L·h) when the wet weight is 0-80 g / L; 4-6 g / (L·h) when the wet weight is 80-120 g / L; and 6-8 g / (L·h) when the wet weight is ≥120 g / L. Preferably, feeding is stopped 2 hours before discharge and discharge is carried out when the ethanol concentration is ≤50 g / L.
6. The fermentation production method according to claim 1, characterized in that, The fermentation medium comprises the following components at the following mass concentrations: Glucose 1%~5%, yeast extract 1%~2%, trypsin 1%~2%, magnesium sulfate heptahydrate 0.1%~0.2%, potassium dihydrogen phosphate 0.1%~0.2%, inositol 0.02%~0.05%, sodium sulfate 0.01%~0.02%, zinc sulfate heptahydrate 0.01%~0.02%, ferrous sulfate heptahydrate 0.005%~0.01%, calcium chloride dihydrate 0.005%~0.01%, molybdic acid dihydrate Sodium 0.001%~0.005%, cobalt chloride hexahydrate 0.001%~0.005%, thiamine hydrochloride 0.001%~0.005%, calcium pantothenate 0.001%~0.005%, nicotinic acid 0.001%~0.005%, pyridoxine hydrochloride 0.001%~0.005%, copper sulfate 0.001%~0.002%, manganese chloride tetrahydrate 0.001%~0.002%, defoamer 0.05 mL / L~0.2 mL / L.
7. The fermentation production method according to claim 1, characterized in that, Before the strain is inoculated into the fermentation medium, it is first activated to obtain a primary seed liquid, and then the primary seed liquid is inoculated into the seed medium to obtain a secondary seed liquid. Preferably, the activated culture medium is a yeast extract peptone glucose medium; Preferably, the seed culture medium contains the following components at the following mass concentrations: glucose 2%~6%, yeast extract 1%~2%, trypsin 1%~2%, magnesium sulfate heptahydrate 0.1%~0.2%, potassium dihydrogen phosphate 0.1%~0.2%, and defoamer 0.05 mL / L~0.2 mL / L.
8. The fermentation production method according to claim 7, characterized in that, The activation conditions are: aeration and stirring culture at 28~30℃ and dissolved oxygen saturation of 30%~50% for 20~25 h; Preferably, the OD600 of the bacterial solution used for activation is ≥20; the inoculation amount of the bacterial solution is 2%~5% of the culture medium volume; Preferably, the stirring speed during the activation process is 150~250 rpm, and the aeration rate is 3~6 m³ / s. 3 / h.
9. The fermentation production method according to claim 7, characterized in that, The conditions for inoculating the primary seed culture into the seed culture medium are: aeration and stirring at 25-30℃ and dissolved oxygen saturation of 30%-50% for 10-15 h. Preferably, the stirring speed during cultivation is 100~180 rpm, and the aeration rate is 30~60 m³ / h. 3 / h.
10. The fermentation production method according to claim 7, characterized in that, The inoculation amount of the secondary seed liquid is 8% to 10% of the fermentation medium volume.