Fermentation method of strain for synthesizing beta-carotene
By enhancing the antioxidant capacity of the strain with sodium selenite and monitoring with qPCR, combined with dynamic regulation of strain ratio and dissolved oxygen, the metabolic degradation and product loss of *Blancium trispora* strain during subculturing were solved, achieving efficient and safe β-carotene production.
Patent Information
- Application Number
- CN202510943176.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2025-11-21
AI Technical Summary
In the existing technology, *Blancium trispora* strains are prone to metabolic degradation during continuous subculturing, resulting in a decrease in the production of β-carotene. Differences in the growth rate of strains lead to an imbalance in the ratio, and they are highly sensitive to dissolved oxygen environment, resulting in a high product loss rate. Furthermore, additive residues may affect safety.
By enhancing the antioxidant capacity of the strain with sodium selenite, combined with qPCR monitoring of genetic stability, dynamic regulation of strain ratio and dissolved oxygen, precise addition of precursor substances, and segmented control of carbon source and dissolved oxygen, fermentation process parameters were optimized.
It significantly improved the genetic stability and yield of the strain, reduced yield volatility, decreased product loss rate, increased unit biomass yield and product purity, extended strain lifespan, and reduced production costs.
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Figure CN120989201A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of β-carotene synthesis, and particularly relates to a fermentation method of a β-carotene synthetic strain. BACKGROUND
[0002] The fermentation method of the β-carotene synthetic strain is mainly based on microbial metabolic regulation, and common strains include Blakeslea trispora and Yarrowia lipolytica. During the fermentation process, the β-carotene yield is significantly improved by dynamically regulating dissolved oxygen, feeding strategies (such as adding soybean oil in batches) and precursor substances (such as β-ionone), and the content in dry mycelium can reach 5.97%-7.18%. For Yarrowia lipolytica, by means of synthetic biology to overexpress key genes (such as CarPR and CarB) and optimize the promoter combination, combined with lipid metabolism regulation, the β-carotene yield can reach 24.0 mg / g DCW.
[0003] However, the fermentation method of the conventional β-carotene synthetic strain has substantial deficiencies, mainly manifested as follows: the natural Blakeslea trispora is prone to metabolic degradation during continuous subculture, and the β-carotene yield decreases by 35%-40% after 5 times of continuous subculture. The main reason is that the genetic stability of the strain for synthesizing pigments is poor, and the positive and negative strains (ATCC14271 / ATCC14272) need to be cooperatively fermented at a strict ratio (the ideal ratio is 1:10-1:100), and in actual operation, the difference in growth rate of the strains easily leads to imbalance in the ratio, reduces the accumulation efficiency of the β-carotene, and further affects the synthesis of β-carotene. The β-carotene is highly sensitive to light and oxygen during the fermentation process. The dissolved oxygen (DO) in the traditional open reactor is >10%, and there is no light shielding design, which leads to a product loss rate of more than 20%. In addition, a high dissolved oxygen environment accelerates the oxidative degradation of β-carotene, and reducing the dissolved oxygen inhibits the aerobic metabolism of the mycelium, forming a process contradiction. The traditional mechanical cell wall disruption method damages the crystal structure of β-carotene, and subsequent extraction relies on organic solvents such as n-hexane, and the solvent residue is often >50 ppm, which is difficult to meet the infant food standards. Although subcritical extraction (such as butane) can be used as an alternative, the equipment investment cost increases dramatically, and small and medium-sized enterprises are difficult to bear. In order to improve the yield, precursors or blocking agents such as β-ionone and imidazole derivatives are often added, but the residues of some additives (such as antibiotics) may induce human drug resistance. Although the alternative scheme (such as adding VB1 and glutamic acid) has improved safety, the yield fluctuates significantly (±30%) when the optimization is insufficient. SUMMARY
[0004] The present application aims to provide a fermentation method of a β-carotene synthetic strain to solve the problems in the background art.
[0005] In order to achieve the above object, the present application provides the following technical scheme: a strain fermentation method for synthesizing beta-carotene, comprising the following steps:
[0006] S1: strain preparation and stability enhancement;
[0007] S2: seed liquid preparation and proportion pre-optimization;
[0008] S3: dynamic proportion regulation in fermentation process;
[0009] S4: feeding and process optimization;
[0010] S5: termination and harvesting;
[0011] The strain preparation and stability enhancement includes strain selection and activation, genetic stability monitoring, the seed liquid preparation and proportion pre-optimization includes seed culture medium formula, culture parameters, the dynamic proportion regulation in fermentation process includes fermentation culture medium formula, initial inoculation and conditions, dynamic proportion regulation strategy, the feeding and process optimization includes carbon source feeding strategy, dissolved oxygen segmented control, precursor addition.
[0012] As a further technical scheme of the present application, the strain selection and activation includes using Blakeslea trispora positive strain ATCC14271 and negative strain ATCC14272, adding sodium selenite with a concentration of 0.1 millimole per liter to the positive strain pre-culture to enhance the antioxidant capacity and reduce the degradation of passage, taking the negative 80 degrees Celsius glycerol preserved strain to inoculate in PDA slant containing 20 grams per liter of glucose and 0.1 grams per liter of vitamin B1, and culturing at 28 degrees Celsius for 5 days, collecting the spore suspension and controlling the positive strain concentration to be 1 times 10 to the 8th power CFU per milliliter, and the negative strain concentration to be 8 times 10 to the 8th power CFU per milliliter, the genetic stability monitoring includes detecting the copy number of sex hormone synthesis key genes tsp1 and carRA carB by qPCR after each generation of strain passage, and screening the strain with a copy number fluctuation of less than 5 percent for fermentation.
[0013] As a further technical scheme of the present application, the seed culture medium formula includes 35 grams of corn starch, 45 grams of corn syrup, 15 grams of glucose, 1.0 gram of potassium dihydrogen phosphate, 0.015 gram of vitamin B1, and 3 grams of soybean oil per liter of culture medium, and sodium selenite with a concentration of 0.1 millimole per liter is added at the same time, the culture parameters include inoculating the spore suspension into the seed tank according to the volume ratio of positive strain to negative strain being 1 to 8, culturing at 28 degrees Celsius with a rotation speed of 200 revolutions per minute and a ventilation volume of 1.0 volume per liter of culture medium per minute for 48 hours, and controlling the mycelium wet weight at the end point to be 25 plus or minus 2 grams per liter.
[0014] As a further technical scheme of the present application, the fermentation medium formula comprises 240 grams of corn starch, 200 grams of corn syrup, 400 grams of soybean powder, 100 grams of glucose, 10 grams of potassium dihydrogen phosphate, 0.15 grams of vitamin B1, and 500 grams of soybean oil per liter.
[0015] As a further technical scheme of the present application, the initial inoculation and conditions comprise inoculating 10% of the seed liquid into the fermenter by volume percentage, the initial ratio of positive bacteria to negative bacteria being 1:8, the temperature being controlled at 28 degrees Celsius, the dissolved oxygen being greater than 25%, the pH being 7.0, the stirring rate being 150 revolutions per minute, and the aeration volume being 0.8 volume per liter of medium per minute.
[0016] As a further technical scheme of the present application, the dynamic ratio control strategy comprises sampling every 6 hours and detecting the genomic abundance of positive bacteria-specific gene matA and negative bacteria-specific gene matB by qPCR, triggering control when the respiratory entropy first drops to 0.95 plus or minus 0.05, adding negative bacteria seed liquid if the proportion of positive bacteria is greater than 10%, the spore number being 1.5 times that of positive bacteria, adding positive bacteria seed liquid if the proportion of negative bacteria is greater than 90%, the spore number being 0.1 times that of negative bacteria, and the target positive to negative bacteria ratio being maintained at 1:10 plus or minus 0.5.
[0017] As a further technical scheme of the present application, the carbon source flow addition strategy comprises a glucose solution with a concentration of 500 grams per liter as the feed solution, with a constant flow rate of 12 grams per hour, and maintaining the residual sugar concentration at 8 plus or minus 1 grams per liter.
[0018] As a further technical scheme of the present application, the dissolved oxygen control comprises controlling the dissolved oxygen at 50% during the growth period from 0 to 24 hours, with a stirring rate of 200 revolutions per minute, and controlling the dissolved oxygen at 20% during the factor production period from 24 to 120 hours, with the stirring rate increased to 400 revolutions per minute.
[0019] As a further technical scheme of the present application, the precursor addition comprises adding β-ionone at a concentration of 0.3% (w / v) at 48 hours, and adding malic acid twice at 24 hours and 72 hours, with a concentration of 0.2% (w / v) each time.
[0020] As a further technical scheme of the present application, the termination and harvesting comprise terminating the fermentation at 120 hours, and discharging the tank when the mycelium β-carotene content is greater than or equal to 7.5% (w / w).
[0021] The beneficial effects of the present application are as follows:
[0022] 1、The present application ensures the stability of key synthetic genes by integrating sodium selenite protection mechanism and qPCR real-time gene monitoring, reduces the influence of genetic drift and growth rate difference systematically by combining dynamic regulation strategy, the design reduces the yield fluctuation rate to less than 5% in the process of subculture, the accumulation efficiency of tricarboxylic acid is increased by 18%-25%, thereby effectively avoiding the interruption of synthesis caused by proportion imbalance, this effect is due to the synergistic effect of stabilization of metabolic pathways in the strain and external regulation, not simply repeating technical steps, significantly prolongs the industrial life of the production strain, and reduces the batch quality control cost.
[0023] 2、The present application solves the problem by segmenting the dissolved oxygen control, that is, 50% dissolved oxygen in the growth early stage promotes the growth of bacteria, 20% dissolved oxygen in the production period reduces the oxidation loss, and the timing of accurate precursor addition is solved, the product loss rate caused by light oxygen sensitivity is compressed from more than 20% to less than 8% by this optimization, at the same time, by maintaining the stability of residual sugar concentration ±1g / L and target proportion control, the unit biomass yield is increased to more than 7.5%, in addition, the accurate timing of precursor addition reduces the risk of excessive accumulation, reduces the dependence on high-toxicity solvents in the extraction stage, indirectly improves the product purity, thanks to the timing synergistic strategy of process parameters, in essence, it is to combine metabolic kinetics and engineering control to achieve the balance of high yield, low consumption and safety. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 It is the whole fermentation process schematic diagram of the present application;
[0025] Figure 2 It is the process schematic diagram of strain preparation and stability enhancement of the present application;
[0026] Figure 3 It is the process schematic diagram of seed liquid preparation and proportion pre-optimization of the present application;
[0027] Figure 4 It is the process schematic diagram of dynamic proportion regulation of the present application fermentation process;
[0028] Figure 5 It is the process schematic diagram of feeding and process optimization of the present application;
[0029] Figure 6 It is the test data comparison table of the present application. DETAILED DESCRIPTION
[0030] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.
[0031] As Figures 1 to 6 shown in the present application, a strain fermentation method for synthesizing β-carotene comprises the following steps:
[0032] S1: strain preparation and stability enhancement;
[0033] S2: seed liquid preparation and proportion pre-optimization;
[0034] S3: dynamic proportion control during fermentation process;
[0035] S4: feeding and process optimization;
[0036] S5: termination and harvesting;
[0037] Strain preparation and stability enhancement includes strain selection and activation, genetic stability monitoring, seed liquid preparation and proportion pre-optimization includes seed medium formula, culture parameters, dynamic proportion control during fermentation process includes fermentation medium formula, initial inoculation and conditions, dynamic proportion control strategy, feeding and process optimization includes carbon source feeding strategy, dissolved oxygen control in sections, precursor addition.
[0038] As Figure 2 shown, strain selection and activation includes using Blakeslea trispora positive strain ATCC14271 and negative strain ATCC14272, adding sodium selenite with a concentration of 0.1 millimoles per liter to the positive strain during pre-culture to enhance antioxidant capacity and reduce passage degradation, taking negative 80 degrees Celsius glycerol preserved strains and inoculating them on PDA slants containing 20 grams per liter of glucose and 0.1 grams per liter of vitamin B1, culturing at 28 degrees Celsius for 5 days, collecting spore suspension and controlling the positive strain concentration to be 1 times 10 to the 8th power CFU per milliliter, the negative strain concentration to be 8 times 10 to the 8th power CFU per milliliter, genetic stability monitoring includes detecting the copy number of sex hormone synthesis key genes tsp1 and carRA carB after each generation of strain passage by qPCR, and screening strains with a copy number fluctuation of less than 5 percent for fermentation.
[0039] As Figure 3 shown, seed medium formula includes 35 grams of corn starch, 45 grams of corn syrup, 15 grams of glucose, 1.0 gram of potassium dihydrogen phosphate, 0.015 gram of vitamin B1, and 3 grams of soybean oil per liter of medium, while adding sodium selenite with a concentration of 0.1 millimoles per liter, culture parameters include spore suspension inoculation into seed tank at a positive to negative strain volume ratio of 1 to 8, mixing at a speed of 200 revolutions per minute and aeration of 1.0 volume per liter of medium per minute at 28 degrees Celsius for 48 hours, and controlling the mycelium wet weight at the end point to be 25 plus or minus 2 grams per liter.
[0040] As Figure 4As shown, the fermentation medium formula includes 240g corn starch, 200g corn steep liquor, 400g soybean flour, 100g glucose, 10g potassium dihydrogen phosphate, 0.15g vitamin B1, and 500g soybean oil per liter. Initial inoculation and conditions include adding 10% (by volume) of seed culture to the fermenter, an initial positive-to-negative bacteria ratio of 1:8, and controlling the temperature at 28°C, dissolved oxygen greater than 25%, and pH... 7.0, stirring rate 150 rpm, aeration rate 0.8 volumes per liter of culture medium, volume mixing ratio, dynamic ratio control strategy includes sampling every 6 hours and detecting the genomic abundance of positive bacteria-specific gene matA and negative bacteria-specific gene matB by qPCR. When the respiratory entropy first drops to 0.95 plus or minus 0.05, regulation is triggered. If the proportion of positive bacteria is greater than 10%, negative bacteria seed culture is added, with its spore count being 1.5 times that of positive bacteria. If the proportion of negative bacteria is greater than 90%, positive bacteria seed culture is added, with its spore count being 0.1 times that of negative bacteria. The target positive to negative bacteria ratio is maintained at 1:10 plus or minus 0.5.
[0041] like Figure 5 As shown, the carbon source feeding strategy includes feeding a glucose solution with a concentration of 500 g / L at a constant rate of 12 g / L, maintaining a residual sugar concentration of 8 ± 1 g / L. Segmented dissolved oxygen control includes controlling dissolved oxygen at 50% and stirring at 200 rpm during the early growth stage (0-24 hours) of fermentation, and controlling dissolved oxygen at 20% and stirring at 400 rpm during the nutrient production stage (24-120 hours). Precursor addition includes adding β-ionone at a concentration of 0.3% by weight / volume at 48 hours of fermentation, and adding malic acid twice at 24 hours and 72 hours, each time at a concentration of 0.2% by weight / volume.
[0042] The termination and harvesting process includes stopping fermentation after 120 hours and releasing the mycelium into the tank when the mycelial β-carotene content is greater than or equal to 7.5% by weight.
[0043] Preferred embodiment:
[0044] A fermentation method for synthesizing β-carotene by a strain includes the following steps:
[0045] S1: Strain preparation and stability enhancement
[0046] Strain preparation and stability enhancement include strain selection and activation, and genetic stability monitoring.
[0047] Strain selection and activation includes using positive strain ATCC14271 and negative strain ATCC14272 of Blakeslea trispora, adding sodium selenite with a concentration of 0.15 millimoles per liter to the pre-culture of the positive strain to enhance the antioxidant capacity and reduce the degradation of the passage, and taking the negative strain preserved at 80 degrees Celsius in glycerol to inoculate PDA slant containing 25 grams per liter of glucose and 0.12 grams per liter of vitamin B10, and culturing at 28 degrees Celsius for 4 days, collecting the spore suspension, and controlling the concentration of the positive strain to be 2 times 10 to the 8th power CFU per milliliter and the concentration of the negative strain to be 1 times 10 to the 9th power CFU per milliliter.
[0048] Genetic stability monitoring includes detecting the copy number of the key genes tsp1 and carRP in the synthesis of sex hormones by qPCR after each generation of strain passage, and screening strains with a copy number fluctuation of less than 3% for fermentation.
[0049] S2: Seed liquid preparation and proportion pre-optimization
[0050] Seed liquid preparation and proportion pre-optimization includes seed medium formula and culture parameters.
[0051] The seed medium formula includes 40 grams of corn starch, 50 grams of corn syrup, 20 grams of glucose, 1.2 grams of potassium dihydrogen phosphate, 0.018 grams of vitamin B10, and 4 grams of soybean oil per liter of medium, and sodium selenite with a concentration of 0.15 millimoles per liter is added.
[0052] The culture parameters include inoculating the seed tank with a spore suspension in a volume ratio of 1:9 of positive strain to negative strain, culturing at 28 degrees Celsius with a rotation speed of 220 revolutions per minute and a ventilation rate of 1.2 volume per liter of medium per minute for 42 hours, and controlling the mycelium wet weight at the end to be 28 plus or minus 1 grams per liter.
[0053] S3: Dynamic proportion control in the fermentation process
[0054] Dynamic proportion control in the fermentation process includes fermentation medium formula, initial inoculation and conditions, and dynamic proportion control strategy.
[0055] The fermentation medium formula includes 260 grams of corn starch, 220 grams of corn syrup, 450 grams of soybean meal, 120 grams of glucose, 12 grams of potassium dihydrogen phosphate, 0.18 grams of vitamin B10, and 550 grams of soybean oil per liter.
[0056] The initial inoculation and conditions include inoculating the fermentation tank with seed liquid at a volume percentage of 12%, with the initial proportion of positive strain to negative strain being 1:9, controlling the temperature at 29 degrees Celsius, the dissolved oxygen being greater than 30%, the pH being 6.8, the stirring rate being 180 revolutions per minute, and the ventilation rate being 1.0 volume per liter of medium per minute.
[0057] Dynamic proportion regulation strategy includes sampling every 5 hours and detecting the genome abundance of positive bacteria specific gene matA and negative bacteria specific gene matB by qPCR, when the respiratory entropy first drops to 0.92 plus or minus 0.03, the regulation is triggered, if the proportion of positive bacteria is greater than 12%, the negative bacteria seed solution is added, the spore number is 1.8 times of that of positive bacteria, if the proportion of negative bacteria is greater than 88%, the positive bacteria seed solution is added, the spore number is 0.08 times of that of negative bacteria, the target positive and negative bacteria ratio is maintained at 1 to 11 plus or minus 0.3.
[0058] S4: feeding and process optimization
[0059] Feeding and process optimization includes carbon source flow addition strategy, dissolved oxygen segmented control, precursor addition.
[0060] Carbon source flow addition strategy includes that the feeding liquid is glucose solution with a concentration of 550 grams per liter, and is added at a constant speed of 15 grams per hour to maintain the residual sugar concentration at 9 plus or minus 0.8 grams per liter.
[0061] Dissolved oxygen segmented control includes controlling the dissolved oxygen at 55% during the growth period from 0 to 26 hours of fermentation, and the stirring rate is 220 revolutions per minute; controlling the dissolved oxygen at 25% during the enzyme production period from 26 to 110 hours, and the stirring rate is increased to 420 revolutions per minute.
[0062] Precursor addition includes adding β-ionone at a concentration of 3.5 grams per liter at 44 hours of fermentation, and adding malic acid twice at 26 hours and 68 hours, each time at a concentration of 2.2 grams per liter.
[0063] S5: termination and harvesting
[0064] Termination and harvesting includes terminating fermentation at 110 hours, and when the mycelium β-carotene content is greater than or equal to 8.2% by weight, the tank is discharged
[0065] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, the scope of the present application being defined by the appended claims and their equivalents.
Claims
1. A fermentation process for a strain synthesizing β-carotene, characterized by: Comprising the following steps: S1: strain preparation and stability enhancement; S2: seed broth preparation and scale pre-optimization; S3: dynamic scale regulation during fermentation process; S4: feeding and process optimization; S5: termination and harvest; The strain preparation and stability enhancement includes strain selection and activation, genetic stability monitoring, the seed broth preparation and scale pre-optimization includes seed medium formulation, culture parameters, the dynamic scale regulation during fermentation process includes fermentation medium formulation, initial inoculation and conditions, dynamic scale regulation strategy, the feeding and process optimization includes carbon source feeding strategy, dissolved oxygen segmented control, precursor addition.
2. The fermentation method of a β-carotene-producing strain according to claim 1, characterized by: The strain selection and activation includes using positive strain Trichoderma harzianum ATCC14271 and negative strain ATCC14272, adding sodium selenite with concentration of 0.1 millimole per liter to pre-culture positive strain to enhance antioxidant capacity and reduce subculture degradation, taking negative strain glycerol preservation strain at 80 degrees Celsius to inoculate PDA slant containing glucose 20 grams per liter and vitamin B1 0.1 grams per liter, culturing at 28 degrees Celsius for 5 days, collecting spore suspension and controlling positive strain concentration to be 1 times 10 to the 8th power CFU per milliliter, negative strain concentration to be 8 times 10 to the 8th power CFU per milliliter, the genetic stability monitoring includes detecting copy number of sex hormone synthesis key genes tsp1 and carRA carB through qPCR after each generation of strain subculture, screening strain with copy number fluctuation less than 5 percent for fermentation.
3. The fermentation method of a β-carotene-producing strain according to claim 1, characterized by: The seed medium formulation includes corn starch 35 grams, corn syrup 45 grams, glucose 15 grams, potassium dihydrogen phosphate 1.0 gram, vitamin B1 0.015 gram, soybean oil 3 grams per liter of medium, and adding sodium selenite with concentration of 0.1 millimole per liter, the culture parameters include inoculating seed tank with spore suspension at positive to negative strain volume ratio of 1 to 8, culturing at 28 degrees Celsius with speed of 200 revolutions per minute and aeration amount of 1.0 volume to volume mixing ratio per liter of medium per minute for 48 hours, and controlling mycelium wet weight at 25 plus or minus 2 grams per liter at the end.
4. The fermentation method of a β-carotene-producing strain according to claim 1, characterized by: The fermentation medium formulation includes corn starch 240 grams, corn syrup 200 grams, soybean powder 400 grams, glucose 100 grams, potassium dihydrogen phosphate 10 grams, vitamin B1 0.15 gram, soybean oil 500 grams per liter.
5. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The initial inoculation and conditions include inoculating fermentation tank with seed broth at volume percentage of 10 percent, initial positive to negative strain ratio of 1 to 8, controlling temperature at 28 degrees Celsius, dissolved oxygen greater than 25 percent, pH 7.0, stirring speed of 150 revolutions per minute, and aeration amount of 0.8 volume to volume mixing ratio per liter of medium per minute.
6. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The dynamic scale regulation strategy includes sampling every 6 hours and detecting genome abundance of positive strain specific gene matA and negative strain specific gene matB through qPCR, triggering regulation when respiratory entropy first decreases to 0.95 plus or minus 0.05, if positive strain proportion is greater than 10 percent, adding negative strain seed broth with spore number being 1.5 times of positive strain, if negative strain proportion is greater than 90 percent, adding positive strain seed broth with spore number being 0.1 times of negative strain, and target positive to negative strain ratio is maintained at 1 to 10 plus or minus 0.
5.
7. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The carbon source feeding strategy includes a glucose solution concentration of 500 grams per liter fed at a constant rate of 12 grams per hour to maintain a residual sugar concentration of 8 plus or minus 1 gram per liter.
8. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The dissolved oxygen fractionation control includes a dissolved oxygen fraction of 50 percent for the first 24 hours of growth, with an agitation rate of 200 revolutions per minute, and a dissolved oxygen fraction of 20 percent for the next 96 hours of production, with an agitation rate of 400 revolutions per minute.
9. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The precursor addition includes the addition of beta-ionone at a concentration of 0.3 percent weight by volume at 48 hours, and the addition of malic acid at a concentration of 0.2 percent weight by volume at 24 hours and 72 hours.
10. The fermentation method for synthesizing β-carotene according to claim 1, characterized in that: The termination and harvest includes termination at 120 hours when the beta-carotene content of the mycelium is greater than or equal to 7.5 percent by weight.