A process for the production of resveratrol by fermentation using a strain of yarrowia lipolytica

Abstract

The application discloses a process for producing resveratrol by fermentation of Yarrowia lipolytica, and belongs to the technical field of microorganisms. Through a series of fermentation condition optimization, including adjustment of the basic medium composition, concentration, feeding composition, pH and feeding mode. The fermentation yield after adjustment is increased from 6g / L at the beginning to 22g / L, and the purity of the extracted resveratrol is greater than 80%, which solves the problems of insufficient yield, difficult extraction and low purity compared with resveratrol from plants.

Description

Technical Field

[0001] This invention relates to a process for producing resveratrol by fermentation using a strain of Yeastra lipolytica, belonging to the field of microbial technology. Background Technology

[0002] Resveratrol is a natural antitoxin widely distributed in grapes, berries, peanuts, green ferns, and other plants. Clinical studies have shown that resveratrol can effectively improve diabetes, cardiovascular disease, and neurological disorders, and also possesses anti-tumor properties. Due to its diverse physiological activities, resveratrol is marketed as a raw material for food and cosmetics. Resveratrol is primarily obtained from plant extraction through complex processes, but this process is hampered by high production costs and low potency, making it difficult to meet demand. Therefore, using microbial cell factories offers an attractive and sustainable alternative for the biosynthesis of resveratrol, as it offers higher productivity, lower production costs, and green economic sustainability compared to plant extraction processes.

[0003] Y. lipolytica is widely recognized as a safe bioengineering strain. Due to its abundant acetyl-CoA and malonyl-CoA, Y. lipolytica is extensively used to synthesize many high-value compounds. Furthermore, Y. lipolytica possesses a high-throughput pentose phosphate (PP) pathway, which can also provide efficient precursors for phenylpropane compounds. Because of these properties, many flavonoids, fatty acids, and terpenoids are beginning to utilize Y. lipolytica as their production host.

[0004] In conclusion, while vigorously developing the extraction of resveratrol from plants, we should gradually shift our focus to microbial fermentation technology to achieve an economical and sustainable production route for resveratrol. Summary of the Invention

[0005] Based on the problems existing in the background technology, the present invention overcomes the disadvantages of low yield, low potency, and cumbersome steps in extracting resveratrol from plants, and provides a scheme for efficient production and extraction of resveratrol by microbial fermentation, including the following:

[0006] This invention provides a method for preparing resveratrol by fermentation. The method involves inoculating a seed culture prepared by activating a resveratrol-producing strain into a fermenter containing a basal culture medium for fermentation. During the fermentation process:

[0007] In the small-scale fermentation, dissolved oxygen was controlled at 15-20% and multiple feedings were made. During the fermentation period of 0-12 hours, dissolved oxygen was controlled by adjusting the rotation speed between 200-500 rpm. After fermentation for 13 hours, dissolved oxygen was controlled by alternating increases in rotation speed and aeration.

[0008] Alternatively, in pilot-scale fermentation, dissolved oxygen can be controlled at 8–12% by alternating increases in rotation speed and aeration, and multiple feeding cycles can be performed.

[0009] In one embodiment, during the small-scale fermentation, the feeding is as follows: at 9 hours of fermentation, feeding 1 is started at a feeding rate of 30-40 mL / h; at 11 hours, the rate is adjusted to 50-60 mL / h; at 12 hours, the rate is adjusted to 80-100 mL / h; at 13 hours, the rate is adjusted to 140-160 mL / h; at 14 hours, the rate is adjusted to 170-190 mL / h; thereafter, the feeding rate of feeding 1 is increased by 20 mL every hour until it reaches a maximum feeding rate of 380-420 mL / h at 25 hours; at 26 hours, the feeding rate of feeding 1 is maintained at 380-420 mL / h; from 27 hours onwards, the feeding rate of feeding 1 is decreased by 50-60 mL / h every hour until it reaches a feeding rate of 180-220 mL / h at 30 hours and is maintained until feeding 1 is exhausted; at 18 hours of fermentation, feeding 2 is started, and the DE value is adjusted between 0.1 and 0.6.

[0010] Alternatively, in pilot-scale fermentation, the feeding is initiated at a rate of 18-22 L / h when fermentation reaches 8 hours. This rate is maintained for 3 hours, and then the feeding rate is gradually increased every hour until it reaches a maximum of 42-48 L / h at 18 hours, which is maintained for 6 hours. After fermentation reaches 24 hours, the feeding rate of feeding 1 is gradually decreased—the amount of feeding is reduced by 5 L every five hours until the feeding rate of feeding 1 returns to 18-22 L / h at 44 hours. This rate is maintained until feeding 1 is exhausted.

[0011] In one implementation, when fermentation has reached 18-19 hours, feeding 2 is started, and the DE value is adjusted between 0.1 and 0.6.

[0012] In one implementation, during small-scale fermentation, the maximum rotation speed is 1000 rpm and the maximum aeration rate is 3.6 M. 3 / h.

[0013] In one implementation, during pilot-scale fermentation, the maximum rotation speed was 45 Hz and the maximum aeration rate was 2600 m³ / h. 3 / h.

[0014] In one embodiment, the temperature is 27–29°C; pH is 4.8–5; and the tank pressure is 0.02–0.03 MPa.

[0015] In one embodiment, during a small-scale fermentation, the initial rotation speed was 200 rpm and the initial aeration rate was 1 M. 3 / h.

[0016] In one embodiment, during pilot-scale fermentation, the initial rotation speed was 10 Hz and the initial aeration rate was 260 M³ / h. 3 / h.

[0017] In one embodiment, the basal culture medium comprises 30-50 g / L glucose, 3.5 g / L KH2PO4, 6.7-10 g / L (NH4)2SO4, 0.4-0.6 g / L MgSO4·7H2O, 8-12 g / L peptone, 2 mL / L trace elements, and 1 mL / L vitamin solution.

[0018] In one embodiment, the trace elements include CaCl2·2H2O 4-5 g / L, ZnSO4·7H2O 4-5 g / L, FeSO4·7H2O 2-4 g / L, CuCl·2H2O 1 g / L, H3BO3 1 g / L, Na2MoO4·2H2O 0.3-0.5 g / L, CoCl·6H2O 0.3-0.5 g / L, CuSO4·5H2O 0.1 g / L, KI 0.1 g / L, and EDTA 13-17 g / L.

[0019] In one embodiment, the vitamin solution comprises 45-55 mg / L biotin, 150-250 mg / L para-aminobenzoic acid, 1 g / L nicotinic acid, 1 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 1 g / L thiamine hydrochloride, and 20-30 g / L inositol.

[0020] In one embodiment, feed 1 consists of 130-150 g / L KH2PO4, 180-220 g / L (NH4)2SO4, and 18-22 g / L MgSO4·7H2O.

[0021] In one embodiment, feed 2 is 500-800 g / L of glucose.

[0022] In one embodiment, the resveratrol-producing strain is the one disclosed in the literature *Remodellingmetabolism for high-level resveratrol production in Yarrowia lipolytica*.

[0023] Beneficial effects:

[0024] The lipolytic yeast ST890 of this invention can metabolize resveratrol, which has anti-aging, anti-tumor, cardiovascular disease prevention, and neuroprotective effects. Microbial fermentation is low-cost and not limited by time or season, expanding the production pathways of resveratrol and reducing its production cost. Through the optimization of the resveratrol fermentation method in this invention, the fermenter yield reaches 24.29–28.2 g / L, significantly increasing the resveratrol production.

[0025] This invention optimizes the culture medium by adjusting the nitrogen source, carbon source, and feed composition. This ensures that the yeast maintains a round yeast morphology during fermentation and effectively improves product purity, reducing the content of the precursor paracoumaric acid to 0.01-0.03 g / L, which helps simplify the subsequent resveratrol extraction steps. Furthermore, this invention optimizes the feed feeding method, changing from a constant feed rate to varying feed rates per hour, increasing the yield to a maximum of 28.2 g / L. Attached Figure Description

[0026] Figure 1. HPLC detection of resveratrol; 1-1. Standard curve; 1-2. HPLC detection;

[0027] Figure 2. Effects of different feeds on yeast cells; 2-150% glucose concentration; 2-2100% glycerol concentration. Detailed Implementation

[0028] The invention will be further explained below with specific examples.

[0029] The detection methods involved in the following embodiments:

[0030] Detection of resveratrol: The content of the extracted fermentation broth was determined by HPLC.

[0031] Standard preparation: Weigh an appropriate amount of standard and dissolve it in methanol (greater than 95%) to prepare a standard solution for testing.

[0032] The determination was performed using Agilent high-performance liquid chromatography.

[0033] HPLC conditions:

[0034] Chromatographic column: InertSustain C18 250 mm × 4.6 mm column (particle size 5 μm);

[0035] Mobile phase A: ultrapure water containing 1‰ trifluoroacetic acid; Mobile phase B: acetonitrile containing 1‰ trifluoroacetic acid; Column temperature: 40℃; Injection volume: 10μL; Detector wavelength: 305nm.

[0036] The HPLC elution program is shown in the table below:

[0037]

[0038] Peak elution time: Resveratrol approximately 12.8 min

[0039] The strains involved in the following examples:

[0040] The Yarrowia lipolytica strain ST890 used in this invention was obtained from the research group of Zhou Jingwen at the School of Bioengineering, Jiangnan University. The article is titled "Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica".

[0041] Extraction method of resveratrol: After microbial fermentation, the fermentation broth is heated at 70℃ for 30 min to inactivate the bacteria. Anhydrous ethanol is added at a ratio of (v / v) 1:2 (fermentation broth:ethanol), and the mixture is thoroughly mixed and extracted for 4 h, stirring every 20 min during extraction. After removing the upper extract, the same volume of anhydrous ethanol is added to the precipitate, and the previous steps are repeated. The two extracts are combined and filtered through a 0.22 μm filter membrane to remove bacteria and solid impurities. After vacuum rotary drying, crude resveratrol with a purity ≥80% is obtained.

[0042] Example 1

[0043] A process for producing resveratrol using *Yamylostella lipolytica* strain through fermentation includes the following:

[0044] The specific plan is as follows:

[0045] (1) Basic culture medium formula: glucose 40 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 6.7 g / L, MgSO4·7H2O 0.5 g / L, peptone 10 g / L, trace elements 2 mL / L, vitamin solution 1 mL / L. Glucose was sterilized separately at 115℃ for 20 min; KH2PO4, (NH4)2SO4, and MgSO4·7H2O were dissolved separately and then transferred to the fermenter for sterilization at 115-118℃ for 30 min; vitamin solution was sterilized by filtration using a syringe filter.

[0046] Trace element formula: CaCl2·2H2O 4.5g / L, ZnSO4·7H2O 4.5g / L, FeSO4·7H2O 3g / L, CuCl·2H2O 1g / L, H3BO3 1g / L, Na2MoO4·2H2O 0.4g / L, CoCl·6H2O 0.3g / L, CuSO4·5H2O 0.1g / L, KI 0.1g / L, EDTA 15g / L; adjust pH to 4.0.

[0047] Vitamin solution formula: Biotin 50mg / L, para-aminobenzoic acid 200mg / L, niacin 1g / L, calcium pantothenate 1g / L, pyridoxine hydrochloride 1g / L, thiamine hydrochloride 1g / L, inositol 25g / L.

[0048] Feed culture medium formula: Feed 1: KH2PO4 140 g / L, (NH4)2SO4 200 g / L, MgSO4·7H2O 20 g / L, the feed volume is about 40% of the fermentation volume; Feed 2: glucose 700 g / L.

[0049] The formula for primary stock culture medium is: glucose 20g / L, yeast extract 20g / L, and peptone 10g / L.

[0050] Secondary seed culture medium formula: glucose 20 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 5 g / L, MgSO4·7H2O 0.5 g / L.

[0051] (2) Seed culture: 50L tank small-scale fermentation: The preserved Yeast strain ST890 was taken out of the -80℃ freezer, thawed naturally, streaked on YPD plates, activated and cultured in a 30℃ incubator for 48h, and then 5 strong and fresh colonies were picked and placed in the primary seed mother liquor culture medium. After that, it was placed in a shaker at 28℃ and 200rpm for 22h, and then transferred to the secondary seed mother liquor culture medium at a 5% (v / v) inoculation rate. After culturing in a shaker at 28℃ and 200rpm for 24h, it was transferred to the tank at a 10% (v / v) inoculation rate.

[0052] (3) Small-scale fermentation: The volume of basic culture medium in the 50L small-scale fermenter was 20L; the temperature was controlled at 28±0.5℃ throughout the process; the pH was 4.9±0.1 (adjusted with ammonia); the tank pressure was 0.02~0.03Mpa; the initial rotation speed was 200rpm; and the initial aeration rate was 1M. 3 / h; During fermentation 0-12h, adjust the turbine speed to 200-500 rpm. As the dissolved oxygen decreases with cell growth, adjust the turbine speed to maintain dissolved oxygen between 15-20%. After 13h, continue to control dissolved oxygen between 15-20% by alternately increasing the turbine speed and aeration, until the maximum turbine speed of 1000 rpm and the maximum aeration rate of 3.6M are reached. 3 / h. When fermentation reaches 9h, feed 1 is started at a feed rate of 30-40mL / h. At 11h, the rate is adjusted to 50-60mL / h, at 12h to 80-100mL / h, at 13h to 140-160mL / h, and at 14h to 170-190mL / h. Thereafter, the feed 1 rate is increased by 20mL every hour until it reaches the maximum feed 1 rate of 380-420mL / h at 25h. At 26h, the feed 1 rate is maintained at 380-420mL / h. From 27h onwards, the feed 1 rate is decreased by 50mL every hour until it reaches a feed 1 rate of 180-220mL / h at 30h and is maintained until feed 1 is exhausted. When fermentation reaches 18h, feed 2 is started according to the measured DE value, and the DE value is controlled between 0.1-0.6. The total fermentation time is 162h. When the fermentation liquid volume is 41L, the resveratrol yield in the fermentation liquid is 26.9g / L, then the total yield of the 50L tank is 1102.9g, and the sugar conversion rate can reach 6.35%.

[0053] Example 2

[0054] A process for producing resveratrol using *Yamylostella lipolytica* strain through fermentation includes the following:

[0055] (1) Basic culture medium formula: glucose 40 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 6.7 g / L, MgSO4·7H2O 0.5 g / L, peptone 10 g / L, trace elements 2 mL / L, vitamin solution 1 mL / L. Glucose was sterilized separately at 115℃ for 20 min; KH2PO4, (NH4)2SO4, and MgSO4·7H2O were dissolved separately and then transferred to the fermenter for sterilization at 115-118℃ for 30 min; vitamin solution was sterilized by filtration using a syringe filter.

[0056] Trace element formula: CaCl2·2H2O 4.5g / L, ZnSO4·7H2O 4.5g / L, FeSO4·7H2O 3g / L, CuCl·2H2O 1g / L, H3BO3 1g / L, Na2MoO4·2H2O 0.4g / L, CoCl·6H2O 0.3g / L, CuSO4·5H2O 0.1g / L, KI 0.1g / L, EDTA 15g / L; adjust pH to 4.0.

[0057] Vitamin solution formula: Biotin 50mg / L, para-aminobenzoic acid 200mg / L, niacin 1g / L, calcium pantothenate 1g / L, pyridoxine hydrochloride 1g / L, thiamine hydrochloride 1g / L, inositol 25g / L.

[0058] Feed culture medium formula: Feed 1: KH2PO4 140 g / L, (NH4)2SO4 200 g / L, MgSO4·7H2O 20 g / L, the feed volume is about 40% of the fermentation volume; Feed 2: glucose 700 g / L.

[0059] The formula for primary stock culture medium is: glucose 20g / L, yeast extract 20g / L, and peptone 10g / L.

[0060] The culture medium formulas for the secondary and tertiary seed mother liquors are as follows: glucose 20 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 5 g / L, MgSO4·7H2O 0.5 g / L.

[0061] (2) Seed culture: Pilot fermentation in a 30T tank: The preserved *Yarrowia lipolytica* strain ST890 was taken out of the -80℃ freezer, thawed naturally, and streaked onto YPD plates. After activation culture in a 30℃ incubator for 48h, 5 robust and fresh colonies were picked and placed in the primary seed stock culture medium. Then, it was placed in a shaker at 28℃ and 200rpm for 24h. After that, it was transferred to the secondary seed stock culture medium at a 5% (v / v) inoculation rate and cultured in a shaker at 28℃ and 200rpm for 25h. Then, it was transferred to a 1T seed tank at a 10% (v / v) inoculation rate. The culture conditions in the seed tank were: temperature 28±0.5℃; rotation speed 10HZ; tank pressure 0.02~0.03Mpa; ventilation ratio 1:1.2; culture time 10h.

[0062] (3) Fermentation: The volume of the basal culture medium in the 30T pilot-scale fermenter was 10T; the seed tank was inoculated into the pilot-scale fermenter at a transfer rate of 6% (v / v), and the temperature was controlled at 28±0.5℃ throughout the process; pH was 4.9±0.1 (adjusted with ammonia); tank pressure was 0.02~0.03Mpa; initial rotation speed was 10HZ; initial aeration rate was 260M 3 / h; As the dissolved oxygen level decreases with bacterial growth, it is controlled at around 10% by alternating increases in rotation speed and ventilation until the maximum rotation speed of 45Hz and the maximum ventilation volume of 2600 m³ / h are reached. 3 / h; When fermentation reaches 8h, feed 1 is started at a rate of 18-22L / h and maintained at this rate for 3h. Then, the feed rate is gradually increased every hour until it reaches a maximum of 42-48L / h at 18h, and maintained for 6h. After fermentation reaches 24h, the feed rate of feed 1 is gradually decreased—reduced by 5L every five hours, until at 44h the feed rate of feed 1 returns to 18-22L / h, and this rate is maintained until feed 1 is exhausted. When fermentation reaches 19h, feed 2 is started based on the measured DE value, and the DE value is controlled between 0.1-0.6. The total fermentation time is 202h. The fermentation liquid volume at the time of discharge is 25T, and the resveratrol yield in the fermentation liquid is 24.29g / L. Therefore, the total yield of the 30T tank is 607.25kg.

[0063] Weigh resveratrol standard and prepare concentrations of 50 μg / L, 100 μg / L, 200 μg / L, 300 μg / L, and 400 μg / L. Analyze the concentrations using high-performance liquid chromatography (HPLC). Plot a standard curve based on peak area. Figure 1-1 As shown: R 2 ≥0.99; y=2E -5 *x+9.2889. Calculate the resveratrol concentration based on the standard curve and the peak area obtained from subsequent measurements.

[0064] like Figure 1-2 As shown, after 192 hours of fermentation broth from a 30T tank was fully extracted with 50 times the amount of methanol using ultrasonic extraction, the extract was analyzed by HPLC. The resveratrol peak area reached 16,869,145, which was calculated to be 24.29 g / L. The peak area ratio at a detection wavelength of 305 nm reached 95.74%, indicating high purity.

[0065] Example 3

[0066] A process for producing resveratrol using *Yamylostella lipolytica* strain through fermentation includes the following:

[0067] (1) Basic culture medium formula: glucose 40 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 6.7 g / L, MgSO4·7H2O 0.5 g / L, peptone 10 g / L, trace elements 2 mL / L, vitamin solution 1 mL / L. Glucose was sterilized separately at 115℃ for 20 min; KH2PO4, (NH4)2SO4, and MgSO4·7H2O were dissolved separately and then transferred to the fermenter for sterilization at 115-118℃ for 30 min; vitamin solution was sterilized by filtration using a syringe filter.

[0068] Trace element formula: CaCl2·2H2O 4.5g / L, ZnSO4·7H2O 4.5g / L, FeSO4·7H2O 3g / L, CuCl·2H2O 1g / L, H3BO3 1g / L, Na2MoO4·2H2O 0.4g / L, CoCl·6H2O 0.3g / L, CuSO4·5H2O 0.1g / L, KI 0.1g / L, EDTA 15g / L; adjust pH to 4.0.

[0069] Vitamin solution formula: Biotin 50mg / L, para-aminobenzoic acid 200mg / L, niacin 1g / L, calcium pantothenate 1g / L, pyridoxine hydrochloride 1g / L, thiamine hydrochloride 1g / L, inositol 25g / L.

[0070] Feed culture medium formula: Feed 1: KH2PO4 140 g / L, (NH4)2SO4 200 g / L, MgSO4·7H2O 20 g / L, the feed volume is about 40% of the fermentation volume; Feed 2: glucose 700 g / L.

[0071] The formula for primary stock culture medium is: glucose 20g / L, yeast extract 20g / L, and peptone 10g / L.

[0072] Secondary seed culture medium formula: glucose 20 g / L, KH2PO4 3.5 g / L, (NH4)2SO4 5 g / L, MgSO4·7H2O 0.5 g / L.

[0073] (2) Seed culture: 50L tank small-scale fermentation: The preserved Yeast strain ST890 was taken out of the -80℃ freezer, thawed naturally, streaked on YPD plates, activated and cultured in a 30℃ incubator for 48h, and then 4 strong and fresh colonies were picked and placed in the primary seed mother liquor culture medium. After that, it was placed in a shaker at 28℃ and 200rpm for 24h, and then transferred to the secondary seed mother liquor culture medium at a 5% (v / v) inoculation rate. After culturing in a shaker at 28℃ and 200rpm for 26h, it was transferred to the tank at a 10% (v / v) inoculation rate.

[0074] (3) Fermentation: The volume of the basic culture medium in the 50L pilot fermenter was 20L; the temperature was controlled at 28±0.5℃ throughout the process; the pH was 4.9±0.1 (adjusted with ammonia); the tank pressure was 0.02~0.03Mpa; the initial rotation speed was 200rpm; and the initial aeration rate was 1M. 3 / h; During fermentation 0-12h, adjust the turbine speed to 200-500 rpm. As the dissolved oxygen decreases with cell growth, adjust the turbine speed to maintain dissolved oxygen between 15-20%. After 13h, continue to control dissolved oxygen between 15-20% by alternately increasing the turbine speed and aeration, until the maximum turbine speed of 1000 rpm and the maximum aeration rate of 3.6M are reached. 3 / h; When fermentation reaches 9h, start feeding 1 at a feeding rate of 30-40mL / h. At 11h, adjust the rate to 50-60mL / h, at 12h to 80-100mL / h, at 13h to 140-160mL / h, and at 14h to 170-190mL / h. Then, increase the feeding 1 rate by 20mL every hour until reaching the maximum feeding 1 rate of 380-420mL / h at 25h. At 26h, maintain the feeding 1 rate of 380-420mL / h. From 27h onwards, decrease the feeding 1 rate by 50mL every hour until reaching the feeding 1 rate of 180-220mL / h at 30h and maintain it until feeding 1 is exhausted. When fermentation reaches 18h, start feeding 2, and adjust the DE value between 0.1-0.6. The total fermentation time is 168h. When the fermentation liquid volume was 43L, the resveratrol yield in the fermentation liquid was 27.3g / L, so the total yield of the 50L tank was 1173.9g.

[0075] Comparative Example 1

[0076] The specific implementation method is the same as in Example 1, except that the basic culture medium formula is adjusted, and experiments are conducted under small-scale conditions:

[0077] (1) Through small-scale optimization of the types and concentrations of nitrogen sources in the basal culture medium, a series of different types and concentrations of nitrogen sources, including (NH4)2SO4, yeast extract, peptone, and soybean meal, were tried. After comprehensively considering production costs, the optimal nitrogen source was determined to be the combination of (NH4)2SO4 and peptone, with the optimal concentration being 6.7 g / L of (NH4)2SO4 and 10 g / L of peptone. Although yeast extract can increase yield to a certain extent, from a cost control perspective, the price of yeast extract is relatively high, and the increase in yield is limited, making the combination of (NH4)2SO4 and peptone far more cost-effective.

[0078] Table 1 Nitrogen source optimization

[0079] nitrogen source Yield (g / L) <![CDATA[(NH4)2SO4(6.7g / L)]]> 8.6 <![CDATA[(NH4)2SO4(10g / L)]]> 8.8 Yeast powder (10g / L) 6.5 Peptone (10g / L) 9.4 Soy flour (10g / L) 5.3 <![CDATA[(NH4)2SO4 (6.7 g / L) + Peptone (10 g / L)]]> 16.3 <![CDATA[(NH4)2SO4 (6.7 g / L) + Peptone (20 g / L)]]> 15.1 <![CDATA[(NH4)2SO4 (6.7 g / L) + peptone (10 g / L) + yeast extract (10 g / L)]]> 16.8 <![CDATA[(NH4)2SO4 (6.7 g / L) + yeast powder (10 g / L)]]> 11.6

[0080] (2) By optimizing the types and concentrations of carbon sources in the basal culture medium in small-scale trials, and after trying different types and concentrations of carbon sources including glucose and glycerol, the optimal carbon source was determined to be glucose, with an optimal concentration of 40 g / L.

[0081] Table 2 Carbon Source Optimization

[0082] carbon source Yield (g / L) Glucose (50g / L) 16.8 Glucose (40g / L) 17.5 Glucose (30g / L) 17.3 Glucose (20g / L) 16 Glucose (10g / L) 12.4 Glycerin (30g / L) 8.8 Glycerin (20g / L) 10.4 Glycerin (10g / L) 10.1

[0083] The results showed that the fermentation liquid volume was 35L when it was added to the tank, and the resveratrol yield in the fermentation liquid was 17.5g / L. Therefore, the total yield of the 50L tank was 612.5g.

[0084] Comparative Example 2

[0085] The specific implementation method is the same as in Example 1, except that the basic culture medium formula is adjusted, and the formula of supplement 2 in the supplemental culture medium is adjusted as follows:

[0086] After trying 100% glycerol, it was found that the yeast cells exhibited significant elongation and deformation when supplemented with glycerol, such as... Figure 2-2 As shown. The fermentation liquid volume was only 23L when it was added to the tank, and the yield was 4.5g / L. The total yield of the 50L tank was 103.5g.

[0087] Setting glucose feed concentrations of 50%, 60%, 70%, and 80% had no significant impact on the total yield of the 50L tank, only affecting the resveratrol concentration in the fermentation broth. Throughout the fermentation process, the yeast cells maintained a round morphology, such as... Figure 2-1 As shown.

[0088] However, the residual sugar concentration in the fermentation broth after 18 hours of fermentation has a significant impact on the yield and product purity. When the basal sugar in the culture medium is depleted, and the sugar replenishment rate is controlled with high sugar (residual sugar concentration > 1), the precursor of resveratrol, p-coumaric acid, accumulates and cannot be converted into the target product resveratrol. This results in a final resveratrol yield of 15-18 g / L, while p-coumaric acid concentration is found to be as high as 1-3 g / L. When the basal sugar in the culture medium is depleted and controlled with low sugar (0.1 ≥ residual sugar concentration < 0.6), the resveratrol yield in the fermentation broth can reach 18-22.5 g / L, while the precursor p-coumaric acid concentration is only 0.01-0.03 g / L, greatly improving product purity and simplifying the subsequent extraction steps. In this case, the fermentation broth volume in the lower tank is 30-40 L, so the total yield of a 50 L tank is approximately 700 g.

[0089] Comparative Example 3

[0090] The specific implementation method is the same as in Example 1. Preliminary experiments showed that the strain yielded higher output under acidic conditions. The pH values ​​for fermentation in a 50L fermenter were adjusted to 4, 4.5, 5, 5.5, 6, and 6.5. KOH, ammonia, and NaOH were used to adjust the pH respectively. The results showed that when the pH was 4.5 and 5, the resveratrol yield in the fermentation broth was significantly increased compared to other conditions, reaching 18.9 g / L. The yeast cells exhibited good morphology. With a lower tank volume of approximately 35L, the total yield in a 50L tank was 661.5 g.

[0091] Comparative Example 4

[0092] The specific implementation method is the same as in Example 1, only the feeding method of feed 1 is adjusted:

[0093] The 50L pilot-scale fermentation tank was filled with 20L of liquid; the temperature was controlled at 28±0.5℃ throughout the process; the pH was 4.9±0.1 (adjusted with ammonia); the tank pressure was 0.02~0.03Mpa; the initial rotation speed was 200rpm; and the initial aeration rate was 1M. 3 / h; During fermentation 0-12h, adjust the turbine speed to 200-500 rpm. As the dissolved oxygen decreases with cell growth, adjust the turbine speed to maintain dissolved oxygen between 15-20%. After 13h, continue to control dissolved oxygen between 15-20% by alternately increasing the turbine speed and aeration, until the maximum turbine speed of 1000 rpm and the maximum aeration rate of 3.6M are reached. 3 / h.

[0094] During fermentation, a feeding method was first tested, starting at 9 hours with a constant feeding rate of 150, 200, 250, 300, and 350 mL / h. It was found that when the feeding rate was ≤200 mL / h, fermentation easily resulted in a large amount of foam, cell death due to nitrogen deficiency, and lysis between 15-25 hours. OD 600 The maximum value can only reach around 150; while when the feeding rate is ≥250mL / h, the addition of a large amount of inorganic salts in the early logarithmic phase of the bacteria causes short-term growth stagnation, affects metabolic pathways, and inhibits product production.

[0095] Therefore, based on the growth characteristics of this strain and after continuous optimization of the feeding rate, the feeding rate was adjusted as follows: Feeding 1 was started at a rate of 30-40 mL / h at 9 hours of fermentation; at 11 hours, the rate was adjusted to 50-60 mL / h; at 12 hours, it was adjusted to 80-100 mL / h; at 13 hours, it was adjusted to 140-160 mL / h; at 14 hours, it was adjusted to 170-190 mL / h; thereafter, the feeding rate was increased by 20 mL every hour until it reached a maximum of 380-420 mL / h at 25 hours; at 26 hours, the feeding rate was maintained at 380-420 mL / h; from 27 hours onwards, the feeding rate was decreased by 50 mL per hour until it reached 180-220 mL / h at 30 hours and maintained until feeding 1 was exhausted. The adjusted feeding 1 had a very significant positive effect on cell growth and product accumulation, reducing the initial OD... 600 =150 upgraded to OD 600At approximately 360, the bacterial cells achieved high-density fermentation, and the fermentation process was more stable and controllable without excessive foaming. The product concentration increased from 21 g / L to 28.2 g / L. Feeding was initiated at 18 hours of fermentation based on the measured DE value. The DE value was maintained between 0.1 and 0.5 during the later stages of fermentation. The total fermentation time was 162 hours.

[0096] When the fermentation liquid volume is 38L, the resveratrol yield in the fermentation liquid is 28.2g / L, so the total yield of the 50L tank is 1071.6g.

[0097] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method of fermentatively producing resveratrol, characterized in that, The method is to inoculate the seed liquid prepared by activating the resveratrol production strain into a fermenter containing a basic medium for fermentation production, the resveratrol production strain is Yarrowia lipolytica strain ST890, the basic medium comprises 30-50 g / L of glucose, 3.5 g / L of KH2PO4, 6.7-10 g / L of (NH4)2SO4, 0.4-0.6 g / L of MgSO4·7H2O, 8-12 g / L of proteose peptone, 2 mL / L of trace elements, and 1 mL / L of vitamin solution, and in the fermentation process: In the small-scale fermentation, the dissolved oxygen is controlled to be 15-20% and multiple feedings are performed, wherein, the rotation speed is controlled to be between 200-500 rpm to realize the dissolved oxygen control in the fermentation for 0-12 h, and the rotation speed and aeration are alternately increased to realize the dissolved oxygen control after 13 h of fermentation; the feeding is started at a feeding speed of 30-40 mL / h at 9 h of fermentation, the speed is adjusted to be 50-60 mL / h at 11 h, the speed is adjusted to be 80-100 mL / h at 12 h, the speed is adjusted to be 140-160 mL / h at 13 h, the speed is adjusted to be 170-190 mL / h at 14 h, then the speed of the feeding 1 is increased by 20 mL every hour until the highest speed of 380-420 mL / h of the feeding 1 is reached at 25 h, the speed of the feeding 1 is kept at 380-420 mL / h at 26 h, the speed of the feeding 1 is decreased by 50 mL every hour from 27 h until the speed of 180-220 mL / h of the feeding 1 is reached at 30 h and kept until the feeding 1 is exhausted; the feeding 2 is started at 18 h of fermentation, and the DE value is controlled to be between 0.1-0.6; Or, in the pilot-scale fermentation, the dissolved oxygen is controlled to be 8-12% by alternately increasing the rotation speed and aeration, and multiple feedings are performed; the feeding is started at a speed of 18-22 L / h at 8 h of fermentation, the speed is kept for 3 h, then the speed of the feeding is uniformly increased every hour until the highest speed of 42-48 L / h is reached at 18 h and kept for 6 h, the speed of the feeding 1 is uniformly decreased after 24 h of fermentation--the feeding amount is decreased by 5 L every five hours until the speed of the feeding 1 returns to 18-22 L / h at 44 h, and the speed is kept until the feeding 1 is exhausted; the feeding 2 is started at 19 h of fermentation according to the measured DE value, and the DE value is controlled to be between 0.1-0.6; The feeding 1 is 130-150 g / L of KH2PO4, 180-220 g / L of (NH4)2SO4, and 18-22 g / L of MgSO4·7H2O, and the feeding 2 is 500-800 g / L of glucose.

2. The method of claim 1, wherein, The feeding 2 is started at 18-19 h of fermentation, and the DE value is controlled to be between 0.1-0.

6.

3. The method of claim 1, wherein, In the small-scale fermentation, the rotation speed is up to 1000 rpm and the ventilation is up to 3.6 M 3 / h.

4. The method of claim 1, wherein, In the pilot fermentation, the highest rotation speed was 45HZ, and the highest ventilation volume was 2600M 3 / h.

5. The method of claim 1, wherein, The temperature is 27-29 ℃, the pH is 4.8-5, and the tank pressure is 0.02-0.03 MPa.

6. The method of claim 1, wherein, In the small-scale fermentation, the initial rotation speed was 200 rpm; the initial aeration amount was 1 M / h 3 In the pilot-scale fermentation, the initial rotation speed was 10 HZ; the initial aeration amount was 260 M / h 3 / h.

7. The method of claim 1, wherein, The trace elements include CaCl2·2H2O 4-5 g / L, ZnSO4·7H2O 4-5 g / L, FeSO4·7H2O 2-4 g / L, CuCl·2H2O 1 g / L, H3BO3 1 g / L, Na2MoO4·2H2O 0.3-0.5 g / L, CoCl·6H2O 0.3-0.5 g / L, CuSO4·5H2O 0.1 g / L, KI 0.1 g / L, EDTA 13-17 g / L; The vitamin solution includes biotin 45-55 mg / L, p-aminobenzoic acid 150-250 mg / L, nicotinic acid 1 g / L, calcium pantothenate 1 g / L, pyridoxine hydrochloride 1 g / L, thiamine hydrochloride 1 g / L, myo-inositol 20-30 g / L.

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