A method for biosynthesis of sclareolide

Abstract

The present application relates to a kind of biosynthesis methods of sclareolide, comprising the following steps: 1) in the fermentation process, every interval one sampling period t1, the content of sclareol in fermentation liquor is sampled and detected, and according to the content of sclareol in fermentation liquor detection value, the feeding speed of sclareol is adjusted;2) the volatilization amount of sclareol in fermentation tail gas is continuously detected, every interval one metering period t2, the volatilization total amount of sclareol is counted once, and the total amount of sclareol feeding in metering period is counted, the ratio of the volatilization total amount of sclareol and the total amount of sclareol feeding is calculated;3) when the ratio is less than threshold value, increase the feeding speed of sclareol.The present application judges the consumption variation of sclareol by continuously monitoring the ratio of the volatilization amount of sclareol in fermentation tail gas and the feeding amount of sclareol, can improve the timeliness of the concentration control of sclareol in fermentation liquor.The conversion rate and yield of biosynthesis sclareolide can be further improved by using the method.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a method for biosynthesizing perilla lactone. Background Technology

[0002] Perilla lactone, as a key intermediate in the synthesis of ambergris substitute—ambergris ether—has broad application prospects and extremely high economic value in the fields of fragrances, food, medicine, and cosmetics.

[0003] The synthesis of perillaldehyde using perillyl alcohol as a substrate has become one of the most feasible and economical synthetic routes. Chemical synthesis methods require the use of heavy metal oxidants such as chromium and manganese, or toxic catalysts, limiting their application in food, cosmetics, and other fields. Biosynthesis, due to its unique advantages such as environmental friendliness, mild reaction conditions, high product purity, and low energy consumption, has gradually become a research hotspot and development trend in perillaldehyde synthesis technology. The biosynthesis of perillaldehyde from perillyl alcohol essentially utilizes the metabolic system of microorganisms or their produced enzymes to convert perillyl alcohol into perillaldehyde through a specific catalytic reaction during fermentation. The entire process does not require the use of toxic or harmful chemical reagents, meeting the requirements of green and environmentally friendly industries.

[0004] During fermentation, perillyl alcohol exhibits toxic side effects on microbial cells; excessively high concentrations inhibit cell activity and affect conversion rates. Slow feed flow rates result in low perillyl alcohol concentrations, impacting the yield of perillyl lactones. Therefore, strict control of perillyl alcohol concentration in the fermentation broth is necessary. Current methods involve periodically sampling the fermentation broth to detect perillyl alcohol concentration and controlling the continuous feed flow rate based on the detected values. However, sampling and testing are time-consuming, typically occurring at 6-hour intervals, resulting in poor timeliness. Existing technologies disclose continuous online detection of the effective component concentration in fermentation broth using HPLC, but the equipment is expensive, and the complex composition of the fermentation broth leads to numerous interferences, significantly affecting the accuracy of the results. These factors make perillyl alcohol concentration control extremely challenging. Summary of the Invention

[0005] To address the above problems, this invention provides a device for the biosynthesis of perilla lactone, the specific solution of which is as follows:

[0006] A method for biosynthesizing perilla lactone includes the following steps: 1) During the fermentation process, the fermentation liquid is sampled and the content of perillaldehyde is detected at each sampling cycle t1. The perillaldehyde feeding rate is adjusted according to the detected value of perillaldehyde content in the fermentation liquid. 2) Continuously monitor the amount of perillaldehyde volatilized in the fermentation tail gas. At each metering cycle t2, count the total amount of perillaldehyde volatilized and the total amount of perillaldehyde added within the metering cycle. Calculate the ratio of the total amount of perillaldehyde volatilized to the total amount of perillaldehyde added. 3) When the ratio is less than the threshold, increase the feeding rate of perillaldehyde.

[0007] The ratio of the total volatilization of perillaldehyde to the total amount of perillaldehyde fed in the fermentation broth can be used to reflect the consumption rate of perillaldehyde in the fermentation broth. The smaller the ratio, the faster the consumption rate. Under the condition that the bacterial concentration and other process conditions do not fluctuate significantly, the perillaldehyde feeding rate can be appropriately increased, which is beneficial to increasing the final yield of perillaldehyde lactone.

[0008] Furthermore, in step 1), the sampling cycle is 6 hours and the metering cycle is 1 hour. The sampling cycle is close to or consistent with the current sampling and testing cycle of the fermentation broth. Multiple metering cycles can be set within one sampling cycle. By jointly controlling the feeding rate of perillaldehyde by the metering statistical value and the sampling and testing value, the timeliness of perillaldehyde monitoring and adjustment can be improved, and the accuracy of perillaldehyde concentration control in the fermentation broth can be improved.

[0009] Furthermore, in step 3), when the ratio is greater than the threshold, additional samples are taken from the fermentation broth to test the perillaldehyde content. If the perillaldehyde content is higher than the previous test value, the perillaldehyde feeding rate is reduced. A higher ratio of total perillaldehyde volatilization to total perillaldehyde feeding indicates a decreased perillaldehyde consumption rate, suggesting that excessively high perillaldehyde concentrations may be inhibiting strain activity. Sampling and testing are used to verify whether the perillaldehyde feeding rate should be reduced. If the perillaldehyde concentration detected in the samples does not increase, the perillaldehyde feeding rate remains unchanged, while simultaneously analyzing whether changes in other process conditions caused the decrease in perillaldehyde consumption rate.

[0010] Furthermore, in step 3), when the ratio is greater than the threshold, the fermentation broth is sampled again to detect the perillaldehyde content. When the perillaldehyde content is higher than the previous detection value, the perillaldehyde feeding rate is reduced while the sugar feeding rate is increased. When the strain activity decreases, the perillaldehyde feeding rate is reduced and the sugar feeding rate is increased to help restore the strain activity.

[0011] Furthermore, in step 2), the oxygen supply and residual oxygen in the fermentation exhaust gas are simultaneously measured within a metering cycle, and the ratio of residual oxygen to oxygen supply is calculated. When this ratio exceeds a threshold, the feeding rate of perillaldehyde is reduced, while the feeding rate of sugar is increased. Since sampling and testing of the fermentation broth is time-consuming, this scheme employs another auxiliary method to verify whether an increase in perillaldehyde concentration inhibits strain activity. Specifically, the oxygen consumption rate is indirectly monitored by the ratio of residual oxygen in the exhaust gas to oxygen supply; a decrease in the oxygen consumption rate further demonstrates a decrease in strain activity.

[0012] Furthermore, the perillaldehyde content in the fermentation broth was controlled at 15-25 g / L. A perillaldehyde concentration of 30 g / L in the fermentation broth would have a significant toxic inhibitory effect on the fermenting bacteria. Conversely, too low a perillaldehyde concentration would affect the final yield of perillaldehyde lactone. Therefore, the perillaldehyde concentration was controlled at 15-25 g / L.

[0013] Furthermore, the content of perillaldehyde and residual oxygen in the fermentation tail gas were detected online using gas chromatography. Gas chromatography systems are simple to operate and maintain, significantly less expensive than liquid chromatography systems, and provide more reliable results.

[0014] This invention uses the ratio of continuously monitored online perillaldehyde supplementation to the amount of perillaldehyde volatilized in the fermentation tail gas to determine the changes in perillaldehyde consumption, thereby improving the timeliness of perillaldehyde concentration control in the fermentation broth. This method can further improve the conversion rate and yield of perillaldehyde in biosynthetic products. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments and comparative examples, but the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the following embodiments are all under conventional experimental conditions.

[0016] I. Formulation of basal culture medium and feed solution 1. Seed culture medium (g / L) 25g glucose, 10g peptone, 5g yeast extract, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, sterilized at 121°C for 20min.

[0017] 2. Fermentation basal medium (g / L) 15g glucose, 8g corn steep liquor powder, 3g dipotassium hydrogen phosphate, 0.8g magnesium sulfate, 0.3g calcium chloride, pH 6.5-7.0, sterilized at 121℃ for 20min.

[0018] 3. Perilla frutescens alcohol replenishment solution Perillyl alcohol with a purity ≥98% was prepared into a feed solution with a mass concentration of 200 g / L using anhydrous ethanol as a dissolving agent, and then aseptically filtered for later use.

[0019] 4. Sugar supplement solution (g / L) 400 mg of glucose was sterilized at 115°C for 15 minutes and used to regulate bacterial activity.

[0020] II. Detection Methods 1. Detection of perillaldehyde content: Fermentation broth was extracted with ethyl acetate, the supernatant was collected by centrifugation, and high performance liquid chromatography (HPLC) was used for detection. Quantification was performed using the external standard method at a detection wavelength of 210 nm.

[0021] 2. Gas chromatography was used to continuously detect the volatilization of perillaldehyde and residual oxygen in the fermentation tail gas online.

[0022] 3. Detection of perilla lactone yield: After fermentation, the fermentation broth was extracted and treated, and the product content was quantitatively detected by HPLC. The substrate conversion rate and product yield were calculated.

[0023] 4. The oxygen supply and perillaldehyde feed amount are determined by flow rate statistics.

[0024] III. Examples and Comparative Examples Example 1: 1. Fermentation culture: The activated seed liquid was inoculated into a 50L fermenter at a volume fraction of 8%, with a liquid volume of 30L, a fermentation temperature of 30℃, an aeration rate of 1.5vvm, a stirring speed of 300r / min, and a fermentation cycle of 72h.

[0025] 2. Feeding control: After fermentation begins, start the feeding of perillaldehyde at an initial rate of 3 g / (L·h). Once it reaches 20 g / L, use a feeding rate of 0.3 g / (L·h). ① Offline sampling: The sampling cycle is 6 hours, and the content of perillol in the fermentation broth is measured every 6 hours, with a target control concentration of 15-25 g / L; ② Exhaust gas monitoring: Continuously monitor the amount of volatile perillaldehyde in the exhaust gas. The measurement cycle is t2=1 hour. Every hour, the total amount of volatile perillaldehyde and the total amount added are counted within the cycle, and the ratio is calculated. The threshold is set at 1‰-5‰. ③ Rate control: When the calculated ratio is <1‰, it is determined that the consumption rate of perillaldehyde substrate is relatively fast, and the feeding rate is increased by 0.05 g / (L·h); the perillaldehyde concentration in the fermentation broth is kept stable at 18-22 g / L throughout the process; when the ratio of total volatiles to total added is >5‰, the fermentation broth is immediately sampled again to test the perillaldehyde content; if the test value is higher than the previous routine sampling value, it is determined that the consumption rate of perillaldehyde is reduced, and the feeding rate is reduced by 0.05 g / (L·h), while the sugar feeding rate is increased by 0.2 g / (L·h) to improve the carbon source supply to the cells and restore cell activity.

[0026] 3. After fermentation, the conversion rate of perillaldehyde was 62%, and the concentration of perillaldehyde lactone in the fermentation broth was 22 g / L.

[0027] Example 2: The basic fermentation conditions, culture medium, initial feeding rate, and cycle parameters are the same as in Example 1. The threshold for the volatility ratio is 1‰-5‰, and the threshold for the ratio of residual oxygen to oxygen supply is 0.70-0.80.

[0028] Regulation and optimization: ① Count the ratio of total volatilized styraxol to total added styraxol every hour, and simultaneously count the ratio of residual oxygen in the exhaust gas to oxygen supply. When the ratio of total volatilized styraxol to total added styraxol is greater than 5‰ and the ratio of residual oxygen to oxygen supply is greater than 0.80, reduce the styraxol feeding rate by 0.05 g / (L·h), while increasing the sugar feeding rate by 0.2 g / (L·h) to improve the carbon source supply for the microorganisms and restore their activity. Control the styraxol concentration at 18-22 g / L throughout the process.

[0029] The remaining operations are the same as in Example 1.

[0030] Results: After fermentation, the conversion rate of perillaldehyde was 65%, and the concentration of perillaldehyde lactone in the fermentation broth was 25 g / L.

[0031] Comparative Example 1: The fermentation conditions, culture medium, and initial feeding rate were the same as in Example 1. The only difference was that the concentration of perillaldehyde was detected by routine 6-hour offline sampling. The feeding rate was adjusted solely based on the detection results. No monitoring or real-time control of tail gas volatilization was performed.

[0032] During fermentation, the concentration of perillyl alcohol fluctuated between 12-28 g / L. When the concentration exceeded the standard, the cell activity decreased significantly, the substrate conversion rate was 51%, and the concentration of perillyl lactone in the fermentation broth was 18 g / L. The regulation lag was obvious, and the overall efficiency was far lower than that of the embodiment of the present invention.

Claims

1. A method for biosynthesizing perilla lactone, characterized in that, Includes the following steps: 1) During the fermentation process, the fermentation liquid is sampled and the content of perillaldehyde is detected at each sampling cycle t1. The perillaldehyde feeding rate is adjusted according to the detected value of perillaldehyde content in the fermentation liquid. 2) Continuously monitor the amount of perillaldehyde volatilized in the fermentation tail gas. At each metering cycle t2, count the total amount of perillaldehyde volatilized and the total amount of perillaldehyde added within the metering cycle. Calculate the ratio of the total amount of perillaldehyde volatilized to the total amount of perillaldehyde added. 3) When the ratio is less than the threshold, increase the feeding rate of perillaldehyde.

2. The method for biosynthesizing perilla lactone according to claim 1, characterized in that, In step 1), the sampling period is 6 hours and the measurement period is 1 hour.

3. The method for biosynthesizing perilla lactone according to claim 1, characterized in that, In step 3), when the ratio is greater than the threshold, the fermentation broth is sampled again to detect the content of perillaldehyde. When the detected value of perillaldehyde content is higher than the previous detection value, the feeding rate of perillaldehyde is reduced.

4. The method for biosynthesizing perilla lactone according to claim 3, characterized in that, In step 3), when the ratio is greater than the threshold, the fermentation broth is sampled again to detect the perillaldehyde content. When the perillaldehyde content is higher than the previous detection value, the perillaldehyde feeding rate is reduced while the sugar feeding rate is increased.

5. The method for biosynthesizing perillaldehyde according to claim 3, characterized in that, In step 2), the oxygen supply and residual oxygen in the fermentation tail gas are counted simultaneously within a metering cycle. The ratio of residual oxygen to oxygen supply is calculated. When the ratio is greater than the threshold, the feeding rate of perillaldehyde is reduced, while the feeding rate of sugar is increased.

6. The method for biosynthesizing perilla lactone according to claim 1, characterized in that, The content of perillaldehyde in the fermentation broth should be controlled at 15-25 g / L.

7. The method for biosynthesizing perilla lactone according to claim 5, characterized in that, The content of perillol and residual oxygen in the fermentation tail gas were detected online using a gas chromatograph.