Preparation method of noottanone

Abstract

The invention relates to a noottanone preparation method which comprises the following steps: 1) setting a first batch of culture medium and a second batch of culture medium, and the culture medium contains a substrate, namely valenvaccinene; (2) firstly adding an enzyme concentrated solution into the first batch of culture medium, reacting for a period of time under controlled reaction conditions, then adding a second batch of culture medium into the enzyme concentrated solution, and reacting for a period of time under controlled reaction conditions; 3) supplementing the first batch of culture medium into the second batch of culture medium, and controlling reaction conditions to continue reaction to prepare a conversion solution containing noottanone, and 4) purifying the conversion solution to obtain noottanone. The method can improve the conversion yield of noottanone.

Description

Technical Field

[0001] The invention relates to a preparation method of grapefruit ketone, belonging to the technical field of biosynthesis. Background Art

[0002] Nootone is a terpenoid compound with a unique aroma and significant application value in food additives, cosmetics, and other fields. The synthesis of nootone using valenciaene as a raw material through biotransformation and oxidation has been widely studied. Compared to oxidation methods using metal catalysts, the product contains no heavy metal residues and is therefore safer. Prior art methods using Yarrowia lipolytica to convert valenciaene to nootone have a conversion rate of 27%. By optimizing the culture medium, the conversion rate can be increased to 49%, with a yield of approximately 500 mg / L. However, for commercial applications, the conversion yield needs to be increased. In this reaction, P450 enzymes produced within the yeast Yarrowia lipolytica convert valenciaene to nootone. In typical enzymatic reactions, the conversion yield can be increased by increasing the initial substrate concentration before saturation or by adding substrate intermittently or continuously during the reaction. However, this approach has not achieved the desired effect when used to produce nootone. Summary of the Invention

[0003] In view of the above problems, the present invention provides a method for preparing grapefruit ketone, comprising the following steps: 1) setting up a first batch of culture medium and a second batch of culture medium containing the substrate valencene; 2) Add the enzyme concentrate to the first batch of culture medium, control the reaction conditions and react for a period of time, then add the enzyme concentrate to the second batch of culture medium and control the reaction conditions and react for a period of time; 3) Adding the first batch of culture medium to the second batch of culture medium, controlling the reaction conditions and continuing the reaction to prepare a conversion solution containing oleic acid; 4) Purify the conversion solution to obtain grapefruit ketone.

[0004] When converting valenciadene to naringenone, the conversion yield is 400 mg / L at a substrate concentration of 900 mg / L, resulting in a conversion rate of less than 50%. Increasing the substrate concentration decreases the conversion rate, but the naringenone yield does not increase. The conversion of valenciadene to naringenone begins with oxygenation to produce naringenol, which is then dehydrogenated to produce naringenone. The accumulation of intermediates during the reaction limits the conversion of the substrate to naringenol, which is the primary cause of the decreased conversion rate and yield.

[0005] The present invention provides first batch culture medium and second batch culture medium, and two batch culture mediums are reacted in steps. After the first batch culture medium reacts for a long time, the substrate concentration descends, and the intermediate product and target product concentrations rise, and the intermediate product is restricted to the conversion of the target product. After the intermediate product concentration approximately rises to the limit value, the restriction substrate continues to convert. The second batch culture medium reacts for a shorter time, and the substrate concentration is in the descending stage, and the intermediate product concentration is in the ascending stage. The intermediate product concentration is lower, and the efficiency that the intermediate product converts to the target product is lower. Now, the first batch culture medium is supplemented in the second batch culture medium of the shorter reaction time, improve the second batch culture medium intermediate product concentration, improve the conversion efficiency of the intermediate product to grapefruit ketone, promote the conversion of substrate to the intermediate product simultaneously, finally realize the further conversion of substrate to the target product, improve the conversion yield of grapefruit ketone.

[0006] Preferably, in step 1), the concentration of the substrate valencene in the culture medium is 2 g / L.

[0007] Preferably, in step 2), when the conversion rate of the substrate in the first batch of culture medium reaches 40% and the conversion rate of the substrate in the second batch of culture medium reaches 20%, the first batch of culture medium is added to the second batch of culture medium.

[0008] Preferably, in step 2), after the first batch of culture medium reacts for 10-12 hours, the enzyme concentrate is added to the second batch of culture medium for reaction. After the first batch of culture medium reacts for 20-24 hours, the first batch of culture medium is added to the second batch of culture medium.

[0009] Preferably, the weight ratio of the first batch of culture medium to the second batch of culture medium is 1:1-2.

[0010] Preferably, in step 2), the first batch of culture medium is added to the second batch of culture medium in batches, with a single feeding weight ratio of 1:5 and a feeding interval of 1 hour.

[0011] Preferably, in step 2), the first batch of culture medium is added to the second batch of culture medium at one time.

[0012] Preferably, the enzyme concentrate used is a concentrate extracted from Yarrowia lipolytica fermentation.

[0013] The present invention achieves the effect of adjusting the concentration ratio of substrate, intermediate product and target product in the culture medium by arranging two batches of culture medium for step-by-step conversion and then mixing the two batches of culture medium. On this basis, the conversion yield of grapefruit ketone is improved by increasing the substrate concentration. DETAILED DESCRIPTION

[0014] The following describes the solution of the present invention in detail with reference to specific examples.

[0015] Before carrying out the following examples or comparative examples, an enzyme concentrate was prepared according to the following method.

[0016] 1. P450 enzyme fermentation 1. Bacteria cultivation The culture room is connected to the shake flask for fermentation and culture of an appropriate amount of bacteria, and the bacteria used is Yarrowia lipolytica.

[0017] 2. Seed tank expansion Sterilize the seed culture medium at 121°C for 30 minutes. The medium contains 40 g / L α-lactose, 15 g / L peptone, and 0.6 g / L FeSO₄·7H₂O. Inoculate the culture from the shake flask in the culture chamber. Cultivate the culture for an appropriate time under conditions of pH 5.0-5.5, temperature 33-35°C, pressure 0.05-0.08 MPa, and air flow at 50 m³ / h to prepare the seed solution for the fermenter.

[0018] 3. Fermentation The culture medium was sterilized at 121°C for 30 min, and the culture was inoculated into the shake flask culture medium in the culture room. The culture was cultured for an appropriate time under the conditions of pH 5.0-5.5, temperature 35-37°C, pressure 0.05-0.08 MPa, stirring speed 130 rpm, and air flow 300 m³ / h to prepare the fermentation broth containing P450 enzyme.

[0019] 2. Isolation and Extraction of P450 Enzymes 1. Cell disruption The prepared fermentation broth and the diluent are stirred and mixed evenly, and then pumped into a homogenizer to break the cells.

[0020] 2. Centrifugal separation Add flocculants to the broken cell solution and mix thoroughly. Place the solution in a high-speed centrifuge to separate the broken cell mycelium and the clear liquid. After a second centrifugation, the mycelium is dried and recovered. The clear liquid is temporarily stored in a buffer tank.

[0021] 3. Enzyme solution concentration The supernatant after centrifugation is concentrated through reverse osmosis and ultrafiltration membranes to remove part of the water and small molecular impurities to obtain an enzyme concentrate.

[0022] 4. Purification and filtration The enzyme concentrate is filtered to remove the remaining macromolecular insoluble matter in the liquid, and the refined enzyme liquid is obtained and temporarily stored for use.

[0023] Example 1 1. Prepare enzymatic hydrolysis tank #1 and enzymatic hydrolysis tank #2. Add 1 kg of culture medium into each of the two tanks. The concentration of the substrate valencene in the culture medium is 2 g / L. The composition of the culture medium is citrate solution, and 0.6 g / L of FeSO4·7H2O is added.

[0024] 2. Add 20g of enzyme concentrate to the 1# enzymolysis tank, control the pH at 5.5-6.0, temperature at 35-37°C, pressure at 0.08-0.1MPa, stirring speed at 60rpm, and incubate for 12 hours under the conditions of 300m³ / h air flow. Add 20g of enzyme concentrate to the 2# enzymolysis tank, control the pH at 5.5-6.0, temperature at 35-37°C, pressure at 0.08-0.1MPa, stirring speed at 60rpm, and incubate for 12 hours under the conditions of 300m³ / h air flow. At this time, the reaction in the 1# enzymolysis tank should be carried out for 24 hours.

[0025] 3. Sample testing showed that the substrate concentration in culture medium #1 was 1.2 g / L, and the substrate conversion rate reached 40%. At this time, the substrate concentration in enzymatic hydrolysis tank #2 was 1.6 g / L, and the substrate conversion rate reached 20%.

[0026] 4. Add the culture medium in the 1# enzymolysis tank to the 2# enzymolysis tank at one time, control the pH at 5.5-6.0, temperature at 35-37°C, pressure at 0.08-0.1 MPa, stirring speed at 60 rpm, and air flow at 300 m³ / h for 12 hours to obtain the conversion solution containing grapefruit ketone.

[0027] 5. Add ethyl acetate to the conversion solution and mix thoroughly. Separate the organic and aqueous phases, recover the aqueous phase, and purify the organic phase by distillation. The organic phase containing the product is pumped into a high-vacuum distillation apparatus, where the organic solvent and organic impurities are separated at high temperature to obtain a concentrated crude product of grapefruit ketone. The resulting concentrated crude product of grapefruit ketone is then subjected to precision filtration to remove insoluble impurities, yielding the refined grapefruit ketone product.

[0028] Example 2 The difference from Example 1 is that after the 1# enzymolysis tank reacted for 10 hours, the enzyme concentrate was added to the 2# enzymolysis tank to start the reaction. After the 2# enzymolysis tank reacted for 10 hours, the culture medium in the 1# enzymolysis tank was added to the 2# enzymolysis tank.

[0029] Example 3 The difference from Example 1 is that after the reaction in the 2# enzymolysis tank for 12 hours, 200 g of the culture medium in the 1# enzymolysis tank was added to the 2# enzymolysis tank every 1 hour.

[0030] Example 4 The difference from Example 1 is that the weight of the material fed into the 2# enzymolysis tank is 2 kg.

[0031] Comparative Example 1 1. Add 1 kg of culture medium to the enzymatic hydrolysis tank #1. The concentration of the substrate valencene in the culture medium is 1 g / L. The composition of the culture medium is citrate, and 0.6 g / L of FeSO4·7H2O is added.

[0032] 2. Add 25 g of enzyme concentrate to the 2.1# enzymolysis tank, control the pH at 5.5-6.0, temperature at 35-37°C, pressure at 0.08-0.1 MPa, stirring speed at 60 rpm, and incubate for 36 hours under the conditions of air flow at 300 m³ / h to obtain a conversion solution containing grapefruit ketone.

[0033] 3. Add an appropriate proportion of organic solvent (ethyl acetate) to the conversion solution and mix thoroughly. Separate the organic and aqueous phases, recover the aqueous phase, and purify the organic phase by distillation. The organic phase containing the product is pumped into a high-vacuum distillation apparatus, where the organic solvent and organic impurities are separated at high temperature to obtain a concentrated crude product of grapefruit ketone. The resulting concentrated crude product of grapefruit ketone is then subjected to precision filtration to remove insoluble impurities, yielding the refined grapefruit ketone product.

[0034] Comparative Example 2 1. Add 1 kg of culture medium to the enzymatic hydrolysis tank #1. The concentration of the substrate valencene in the culture medium is 2 g / L. The composition of the culture medium is citrate, and 0.6 g / L of FeSO4·7H2O is added.

[0035] 2. Add 25 g of enzyme concentrate to the 2.1# enzymolysis tank, control the pH at 5.5-6.0, temperature at 35-37°C, pressure at 0.08-0.1 MPa, stirring speed at 60 rpm, and incubate for 36 hours under the conditions of air flow at 300 m³ / h to obtain a conversion solution containing grapefruit ketone.

[0036] 3. Add an appropriate proportion of organic solvent (ethyl acetate) to the conversion solution and mix thoroughly. Separate the organic and aqueous phases, recover the aqueous phase, and purify the organic phase by distillation. The organic phase containing the product is pumped into a high-vacuum distillation apparatus, where the organic solvent and organic impurities are separated at high temperature to obtain a concentrated crude product of grapefruit ketone. The resulting concentrated crude product of grapefruit ketone is then subjected to precision filtration to remove insoluble impurities, yielding the refined grapefruit ketone product.

[0037] The conversion solutions in Examples 1-4 and Comparative Examples 1-2 were sampled to detect the concentration of oleic acid ketone and the conversion rate of oleic acid ketone was calculated. The results are compared as follows:

[0038] The above comparison shows that the conversion yield did not increase after increasing the substrate concentration in Comparative Example 2. Examples 1-4 used two batches of step-by-step reactions followed by mixed reactions, which significantly increased the conversion yield, with Example 3 having the highest conversion yield.

Claims

1. A method for preparing grapefruit ketone, characterized in that, The following steps are involved: 1) setting up a first batch of culture medium and a second batch of culture medium containing the substrate valencene; 2) Add the enzyme concentrate to the first batch of culture medium, control the reaction conditions and react for a period of time, then add the enzyme concentrate to the second batch of culture medium and control the reaction conditions and react for a period of time; 3) Adding the first batch of culture medium to the second batch of culture medium, controlling the reaction conditions and continuing the reaction to prepare a conversion solution containing oleic acid; 4) Purify the conversion solution to obtain grapefruit ketone.

2. The method for preparing grapefruit ketone according to claim 1, wherein In step 1), the concentration of the substrate valencene in the culture medium is 2 g / L.

3. The method for preparing grapefruit ketone according to claim 1, wherein In step 2), when the conversion rate of the substrate in the first batch of culture medium reaches 40% and the conversion rate of the substrate in the second batch of culture medium reaches 20%, the first batch of culture medium is added to the second batch of culture medium.

4. The method for preparing grapefruit ketone according to claim 1, wherein In step 2), after the first batch of culture medium has reacted for 10-12 hours, the enzyme concentrate is added to the second batch of culture medium for reaction. After the first batch of culture medium has reacted for 20-24 hours, the first batch of culture medium is added to the second batch of culture medium.

5. The method for preparing grapefruit ketone according to claim 1, wherein The weight ratio of the first batch of culture medium to the second batch of culture medium is 1:1-2.

6. The method for preparing grapefruit ketone according to claim 1, wherein In step 2), the first batch of culture medium is added to the second batch of culture medium in batches, with a single feeding weight ratio of 1:5 and a feeding interval of 1 hour.

7. The method for preparing grapefruit ketone according to claim 1, wherein In step 2), the first batch of culture medium is added to the second batch of culture medium at one time.

8. The method for preparing grapefruit ketone according to claim 1, wherein The enzyme concentrate used is the concentrate extracted from Yarrowia lipolytica fermentation.

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