A culture medium for producing lycopene by fermentation using blakeslea trispora
By using lauryl phosphate and potassium humate in the fermentation medium of *Blancium trispora*, the carbon-nitrogen ratio was optimized, the problem of insufficient oxygen supply was solved, the yield of lycopene was increased, and the production cost was reduced, thus achieving stability and safety in the fermentation process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NINGXIA KINGVIT PHARMA
- Filing Date
- 2021-08-23
- Publication Date
- 2026-06-19
AI Technical Summary
In the current process of fermenting lycopene with Bacillus trispora to produce lycopene, insufficient oxygen supply leads to reduced biomass and energy metabolism, which affects lycopene yield. Furthermore, existing oxygen carrier methods pose safety risks or quality issues.
Lauryl phosphate was used as a component of the fermentation medium, combined with potassium humate, to optimize the carbon and nitrogen source ratio of the seed and fermentation medium, ensuring oxygen transfer efficiency and stable cell growth environment during fermentation. The heat-resistant lauryl phosphate was used to replace the nonionic surfactant Span20.
It increased fermentation biomass, increased lycopene production, reduced production costs, and maintained the quality stability of fermentation products.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fermentation technology, and in particular relates to a culture medium for the fermentation of lycopene using Bacillus trispora. Background Technology
[0002] Lycopene, also known as ψ-carotene, is a straight-chain polyunsaturated olefin composed of 11 conjugated double bonds and 2 non-conjugated double bonds. It belongs to the isoprene class of compounds and is a type of carotenoid, an isomer of β-carotene. It was first isolated from tomatoes, hence the name lycopene. Lycopene possesses superior physiological functions. It not only has anti-cancer and cancer-suppressing effects but also plays an important role in preventing cardiovascular diseases, arteriosclerosis, and various adult-onset diseases, enhancing the human immune system, and delaying aging. It is a promising new type of functional natural pigment, used in health foods, food coloring additives, and cosmetics.
[0003] Lycopene is an intermediate in the biosynthetic pathways of β-carotene and lutein.
[0004] The molecular formula is C 40 H 56 Molecular weight 536.85
[0005] Structural formula:
[0006]
[0007] Lycopene production methods include: chemical synthesis, natural plant extraction, and bio-fermentation.
[0008] The natural plant extraction method primarily uses tomatoes as raw materials, resulting in a completely natural product with naturally occurring isomers that are easily accepted by the general public. However, the availability of raw materials is greatly affected by the season, preservation is difficult, yields are low, and production costs are high.
[0009] Chemical synthesis is low-cost and simple to produce, but it is not suitable for long-term use and is mainly used in cosmetics and food coloring additives.
[0010] Bio-fermentation is a safe and simple production technology with high output, low production cost, and good product quality. The quality of the product is similar to that of lycopene extracted from natural plants. It can be used in health foods, cosmetics, and food coloring additives, and is gradually becoming the mainstream method for lycopene production.
[0011] Currently, the process of producing lycopene using the bio-fermentation method of Blakeslea trispora (B trispora) is relatively mature and can be carried out on a large scale.
[0012] The fermentation process of *B. trispora* is aerobic, and *B. trispora* is highly aerobic, therefore, the oxygen consumption during fermentation is relatively large. As the biomass of *B. trispora* increases during fermentation, the fermentation broth becomes viscous. Furthermore, the presence of soybean oil-like substances in the culture medium easily adheres to the surface of *B. trispora*, hindering oxygen mass transfer and inhibiting aerobic growth. This insufficient oxygen supply severely affects the growth of *B. trispora*, resulting in reduced biomass, lower energy metabolism levels, and ultimately, reduced lycopene production. Therefore, ensuring a sufficient oxygen supply is crucial during fermentation.
[0013] Regarding methods to improve oxygen supply during fermentation, in addition to changing the liquid volume, stirring speed, aeration rate, and fermenter structure, Reference 1 (Study on the pigment synthesis regulation mechanism of high-yield lycopene mutant strain induced by B. trispora; Fu Jinju; Master's thesis, Henan Normal University; 2018, pp. 41-48) discloses that adding oxygen carriers during fermentation can effectively improve the dissolved oxygen status of the fermentation broth, increase the dissolved oxygen concentration, ensure the normal growth and metabolism of B. trispora, and thus increase lycopene yield. Common oxygen carriers include hemoglobin, n-hexane, and n-dodecane. References 2 (A Unique Pattern of Mycelial Elongation of Blakeslea trispora, and Its Effect on Morphological Characteristics and β-Carotene Synthesis; Jeong JC, Lee J, Park YH; Curr Microbiol; 2001, 42(3):225-228) and 3 (Study on the Interaction Mode of Blakeslea trispora Producing β-Carotene and Optimization of Fermentation Conditions; Chen Guangqian; Master's Thesis, Huazhong University of Science and Technology; 2017, P9) disclose that adding the nonionic surfactant Span20 can adsorb on the surface of B. trispora cells, forming an oil-water interface, reducing the tension of the solid-liquid interface, and forming a certain steric hindrance, effectively limiting the aggregation of hyphae, making the hyphae more uniformly dispersed, improving oxygen transfer efficiency, and increasing lycopene production.
[0014] In the above methods involving the addition of oxygen carriers, hemoglobin is a biochemical substance, requiring high technical standards for collection, storage, and use, resulting in high production and application costs. Furthermore, population differences can affect its effectiveness. The fermentation medium requires high-temperature sterilization before entering the fermentation tank, which can easily denature the protein and cause loss of physiological activity. Hexane and dodecane are flammable and explosive substances, posing significant safety risks in production and application. The nonionic surfactant Span20 has poor heat resistance and is prone to quality changes during high-temperature sterilization of the medium before entering the tank, affecting the emulsification effect. Summary of the Invention
[0015] The purpose of this invention is to overcome the shortcomings of the prior art and provide a culture medium for the fermentation of lycopene using Bacillus trispora to effectively improve oxygen supply, increase fermentation biomass, increase lycopene yield, and reduce production costs.
[0016] The technical solution adopted to achieve the above objectives is as follows:
[0017] A culture medium for producing lycopene using *Bacillus trispora* fermentation is characterized by comprising: *B. trispora*(+) seed medium, *B. trispora*(-) primary seed medium, secondary seed medium, and fermentation medium, wherein:
[0018] The B. trispora(+) seed culture medium consisted of: corn starch 40-60 g / L, soybean meal 20-26 g / L, potassium humate 1.2-1.6 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, and foaming agent 0.2-0.6 g / L.
[0019] The primary seed culture medium for B. trispora(-) consisted of: corn starch 40-60 g / L, soybean meal 20-26 g / L, potassium humate 1.2-1.6 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, and bubbly spores 0.2-0.6 g / L.
[0020] The secondary seed culture medium for B. trispora(-) consisted of: corn starch 40-60 g / L, soybean meal 20-26 g / L, potassium humate 1.2-1.6 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, and foaming agent 0.2-0.6 g / L.
[0021] The fermentation medium consists of: corn starch 15-22 g / L, soybean meal 26-36 g / L, soybean oil 35-55 g / L, 2,6-di-tert-butyl-4-methylphenol (BHT) 0.3-0.9 g / L, potassium humate 1.2-1.6 g / L, corn steep liquor 15-20 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-1.3 g / L, foaming agent 0.2-0.6 g / L, and lauryl phosphate 0.2-0.6 g / L.
[0022] The quality requirements for the sterilized B. trispora(+) seed culture medium are: amino nitrogen 100-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.5.
[0023] The quality requirements for the sterilized primary seed culture medium of B. trispora(-) are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.5.
[0024] The quality requirements for the sterilized B. trispora(-) secondary seed culture medium are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.5.
[0025] The quality requirements for the sterilized fermentation medium are: amino nitrogen 150-300 mg / 100 ml, total sugar 2.0-3.6 g / 100 ml, pH 6.8-7.5.
[0026] The technical advantages of this invention are reflected in:
[0027] 1. The fermentation medium of this invention uses lauryl phosphate, which has strong heat resistance. It is sterilized at high temperature before being put into the tank without any quality change. It replaces the nonionic surfactant Span20, has a good emulsification effect, and helps to increase the yield of lycopene, a fermentation product.
[0028] 2. Potassium humate is used in the fermentation culture medium of this invention. The quinone groups contained in potassium humate participate in the redox process of bacterial cells, promote cell proliferation, alleviate oxygen deficiency and increase cell activity, enhance the adaptability of the strain to the fermentation system environment, facilitate the propagation of fermentation seed culture, and increase the yield of the fermentation product lycopene.
[0029] 3. This invention confirms the optimal ratio of carbon and nitrogen sources in B. trispora(+) seed culture medium, B. trispora(-) primary seed culture medium, secondary seed culture medium, and fermentation medium for the fermentation production of lycopene using B. trispora. The strain growth and metabolic environment is stable, which is beneficial to the stable fermentation yield and quality. Detailed Implementation
[0030] The invention is illustrated below with examples. It should be understood that these examples are for illustrative purposes only and not for limiting the invention. The scope and core content of the invention are defined by the claims.
[0031] The strain processing procedure in the following embodiments is as follows:
[0032] B. trispora(+) seed culture process:
[0033] Under flame protection, the mycelium of the pre-cultured B. trispora(+) mother bottle was inoculated at a rate of about 0.8L into a sterilized B. trispora(+) seed culture medium that was maintained under sterile air pressure at a temperature of 28±1℃ for B. trispora(+) seed culture.
[0034] The seed culture conditions for B. trispora(+) are as follows: tank pressure 0.03~0.05MPa; tank temperature 28±1℃; air flow rate 50-100m3 / h; stirring speed 120-200r / min; culture time approximately 48h before transferring the seed to a fermenter for further culture.
[0035] B. trispora (-) Primary seed culture process:
[0036] Under flame protection, the mycelia of the pre-cultured B. trispora(-) mother bottle were inoculated at a rate of about 0.6L into a sterilized, pressurized B. trispora(-) primary seed culture medium at 28±1℃ for B. trispora(-) primary seed culture.
[0037] The primary seed culture conditions for B. trispora(-) are as follows: tank pressure 0.03~0.05MPa; tank temperature 28±1℃; air flow rate 50-100m3 / h; stirring speed 120-200r / min; culture time approximately 48h, then transfer to a secondary seed tank for B. trispora(-) culture.
[0038] B. trispora (-) secondary seed culture process:
[0039] The secondary seed culture conditions for B. trispora(-) are as follows: tank pressure 0.03~0.05MPa; tank temperature 28±1℃; air flow rate 1500-1000m3 / h; stirring speed 100-200r / min; culture time approximately 24h before transferring to the fermentation tank.
[0040] Transplanted into fermentation tank
[0041] Fermentation tank: Seed ratio 1:0.08~0.12 (V / V), including: B. trispora(-) secondary seed and B. trispora(+) seed, in a ratio of B. trispora(+) seed : B. trispora(-) secondary seed = 1:8~12. Transfer to sterilized fermentation medium, pressurized with sterile air, at a temperature of 28±1℃, for fermentation culture. When transferring, first transfer the B. trispora(-) secondary seed, then transfer the B. trispora(+) seed.
[0042] Fermentation culture process:
[0043] The fermentation conditions are as follows: fermentation temperature: 28±1℃; tank pressure: 0.03~0.05MPa; air flow rate: 1:0.6~1.2V / V / min (fermentation liquid volume m3: sterile air volume m3 introduced per minute); stirring speed: 110-130r / min; pH controlled at 6.0-7.0.
[0044] Fermentation feeding process:
[0045] Feeding begins 10 hours after the inoculum is transferred to the fermenter and stops approximately 30 hours before the end of fermentation. A fed-batch method is used, with the feed solution being sterile filtered nicotine at a rate of 0.5 g / L.
[0046] Fermentation process monitoring and control: sugar concentration 1.0–3.0 g / 100 ml, amino nitrogen 100–200 mg / 100 ml
[0047] Requirements for completion of fermentation culture:
[0048] The fermentation cycle is controlled at around 120 hours.
[0049] Example 1
[0050] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0051] B. trispora(+) seed culture medium: corn starch 44kg, soybean meal 22kg, potassium humate 1.32kg, light calcium carbonate 6.6kg, dipotassium hydrogen phosphate 0.33kg, and bubbly agent 0.22kg.
[0052] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 100 mg / 100 ml, total sugar 3.0 g / 100 ml, pH 6.5.
[0053] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0054] The primary seed culture medium for B. trispora(-) consists of: 36 kg corn starch, 18 kg soybean meal, 1.08 kg potassium humate, 5.4 kg light calcium carbonate, 0.27 kg dipotassium hydrogen phosphate, and 0.18 kg of bufotoxin.
[0055] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 110 mg / 100 ml, total sugar 3.0 g / 100 ml, and pH 6.5.
[0056] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0057] The secondary seed culture medium for B. trispora(-) consists of: 440 kg corn starch, 220 kg soybean meal, 13.2 kg potassium humate, 66 kg light calcium carbonate, 3.3 kg dipotassium hydrogen phosphate, and 2.2 kg of bubbly spores.
[0058] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 110 mg / 100 ml, total sugar 3.0 g / 100 ml, pH 6.8.
[0059] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0060] 1650 kg corn starch, 2860 kg soybean meal, 3850 kg soybean oil, 33 kg 2,6-di-tert-butyl-4-methylphenol (BHT), 132 kg potassium humate, 1650 kg corn steep liquor, 660 kg light calcium carbonate, 33 kg dipotassium hydrogen phosphate, 22 kg foaming agent, and 22 kg lauryl phosphate.
[0061] The sterilized fermentation medium had the following composition: amino nitrogen 150 mg / 100 ml, total sugar 2.0 g / 100 ml, and pH 6.8.
[0062] Fermentation culture was completed, with a fermentation titer of 2.39 g / L and a fermentation cycle of 116 h.
[0063] Example 2
[0064] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0065] B. trispora(+) seed culture medium: corn starch 49.5 kg, soybean meal 24.2 kg, potassium humate 1.43 kg, light calcium carbonate 7.7 kg, dipotassium hydrogen phosphate 0.44 kg, and bubbly agent 0.33 kg.
[0066] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 122 mg / 100 ml, total sugar 3.8 g / 100 ml, and pH 7.1.
[0067] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0068] The primary seed culture medium for B. trispora(-) consists of: 40.5 kg corn starch, 19.8 kg soybean meal, 1.17 kg potassium humate, 6.3 kg light calcium carbonate, 0.36 kg dipotassium hydrogen phosphate, and 0.27 kg of bufotoxin.
[0069] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 126 mg / 100 ml, total sugar 3.9 g / 100 ml, and pH 6.9.
[0070] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0071] The secondary seed culture medium for B. trispora(-) consists of: 495 kg corn starch, 242 kg soybean meal, 14.3 kg potassium humate, 77 kg light calcium carbonate, 4.4 kg dipotassium hydrogen phosphate, and 3.3 kg of bufotoxin.
[0072] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 125 mg / 100 ml, total sugar 4.2 g / 100 ml, pH 7.2.
[0073] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0074] 1870 kg corn starch, 3190 kg soybean meal, 4400 kg soybean oil, 55 kg 2,6-di-tert-butyl-4-methylphenol (BHT), 143 kg potassium humate, 1870 kg corn steep liquor, 770 kg light calcium carbonate, 66 kg dipotassium hydrogen phosphate, 33 kg bufotoxin, and 33 kg lauryl phosphate.
[0075] The sterilized fermentation medium had the following composition: amino nitrogen 175 mg / 100 ml, total sugar 2.8 g / 100 ml, and pH 7.3.
[0076] Fermentation culture was completed, with a fermentation titer of 2.46 g / L and a fermentation cycle of 119 h.
[0077] Example 3
[0078] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0079] B. trispora(+) seed culture medium: corn starch 55kg, soybean meal 26.4kg, potassium humate 1.54kg, light calcium carbonate 8.8kg, dipotassium hydrogen phosphate 0.55kg, and bubbly agent 0.44kg.
[0080] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 138 mg / 100 ml, total sugar 4.2 g / 100 ml, and pH 7.3.
[0081] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0082] The primary seed culture medium for B. trispora(-) consists of: 45 kg corn starch, 21.6 kg soybean meal, 1.26 kg potassium humate, 7.2 kg light calcium carbonate, 0.45 kg dipotassium hydrogen phosphate, and 0.36 kg of bufotoxin.
[0083] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 142 mg / 100 ml, total sugar 4.1 g / 100 ml, and pH 7.2.
[0084] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0085] The secondary seed culture medium for B. trispora(-) consists of: 550 kg corn starch, 264 kg soybean meal, 15.4 kg potassium humate, 88 kg light calcium carbonate, 5.5 kg dipotassium hydrogen phosphate, and 4.4 kg of bubbly spores.
[0086] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 136 mg / 100 ml, total sugar 4.4 g / 100 ml, pH 7.3.
[0087] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0088] 2090 kg of corn starch, 3520 kg of soybean meal, 4950 kg of soybean oil, 77 kg of 2,6-di-tert-butyl-4-methylphenol (BHT), 154 kg of potassium humate, 2090 kg of corn steep liquor, 880 kg of light calcium carbonate, 99 kg of dipotassium hydrogen phosphate, 44 kg of foaming agent, and 44 kg of lauryl phosphate.
[0089] The sterilized fermentation medium had the following composition: amino nitrogen 230 mg / 100 ml, total sugar 3.2 g / 100 ml, and pH 7.4.
[0090] Fermentation culture was completed, with a fermentation titer of 2.53 g / L and a fermentation cycle of 118 hours.
[0091] Example 4
[0092] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0093] B. trispora(+) seed culture medium: corn starch 60.5 kg, soybean meal 28.6 kg, potassium humate 1.65 kg, light calcium carbonate 9.9 kg, dipotassium hydrogen phosphate 0.88 kg, and bubbly agent 0.55 kg.
[0094] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 150 mg / 100 ml, total sugar 4.6 g / 100 ml, and pH 7.5.
[0095] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0096] The primary seed culture medium for B. trispora(-) consists of: 49.5 kg corn starch, 23.4 kg soybean meal, 1.35 kg potassium humate, 8.1 kg light calcium carbonate, 0.72 kg dipotassium hydrogen phosphate, and 0.45 kg of bufotoxin.
[0097] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 150 mg / 100 ml, total sugar 4.5 g / 100 ml, and pH 7.5.
[0098] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0099] The secondary seed culture medium for B. trispora(-) consists of: 605 kg corn starch, 286 kg soybean meal, 16.5 kg potassium humate, 99 kg light calcium carbonate, 8.8 kg dipotassium hydrogen phosphate, and 5.5 kg of bubbly spores.
[0100] The sterilized secondary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 150 mg / 100 ml, total sugar 4.6 g / 100 ml, and pH 7.5.
[0101] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0102] 2420 kg of corn starch, 3960 kg of soybean meal, 5500 kg of soybean oil, 99 kg of 2,6-di-tert-butyl-4-methylphenol (BHT), 165 kg of potassium humate, 2200 kg of corn steep liquor, 990 kg of light calcium carbonate, 121 kg of dipotassium hydrogen phosphate, 55 kg of foaming agent, and 55 kg of lauryl phosphate.
[0103] The sterilized fermentation medium had the following composition: amino nitrogen 300 mg / 100 ml, total sugar 3.6 g / 100 ml, and pH 7.5.
[0104] Fermentation culture was completed, with a fermentation titer of 2.68 g / L and a fermentation cycle of 121 h.
[0105] Example 5
[0106] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0107] B. trispora(+) seed culture medium: corn starch 66kg, soybean meal 25.3kg, potassium humate 1.76kg, light calcium carbonate 8.25kg, dipotassium hydrogen phosphate 0.605kg, and bubbly agent 0.66kg.
[0108] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 129 mg / 100 ml, total sugar 5.0 g / 100 ml, and pH 7.3.
[0109] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0110] The primary seed culture medium for B. trispora(-) consists of: 54 kg corn starch, 20.7 kg soybean meal, 1.44 kg potassium humate, 6.75 kg light calcium carbonate, 0.495 kg dipotassium hydrogen phosphate, and 0.54 kg of bufotoxin.
[0111] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 132 mg / 100 ml, total sugar 5.0 g / 100 ml, and pH 7.1.
[0112] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0113] The secondary seed culture medium for B. trispora(-) consists of: 660 kg corn starch, 253 kg soybean meal, 17.6 kg potassium humate, 82.5 kg light calcium carbonate, 6.05 kg dipotassium hydrogen phosphate, and 6.6 kg of bubbly spores.
[0114] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 130 mg / 100 ml, total sugar 5.0 g / 100 ml, pH 6.9.
[0115] Fermentation culture: Effective volume of fermentation medium 110m³3 .
[0116] 2035 kg of corn starch, 3410 kg of soybean meal, 6050 kg of soybean oil, 66 kg of 2,6-di-tert-butyl-4-methylphenol (BHT), 176 kg of potassium humate, 1925 kg of corn steep liquor, 825 kg of light calcium carbonate, 143 kg of dipotassium hydrogen phosphate, 66 kg of foaming agent, and 66 kg of lauryl phosphate.
[0117] The sterilized fermentation medium had the following composition: amino nitrogen 220 mg / 100 ml, total sugar 3.0 g / 100 ml, and pH 7.2.
[0118] Fermentation culture was completed, with a fermentation titer of 2.55 g / L and a fermentation cycle of 122 h.
[0119] Comparison Case 1
[0120] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0121] B. trispora(+) seed culture medium: corn starch 55kg, soybean meal 26.4kg, light calcium carbonate 8.8kg, dipotassium hydrogen phosphate 0.55kg, and bubbly agent 0.44kg.
[0122] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 136 mg / 100 ml, total sugar 4.1 g / 100 ml, and pH 7.2.
[0123] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0124] The primary seed culture medium for B. trispora(-) consists of: 45 kg corn starch, 21.6 kg soybean meal, 7.2 kg light calcium carbonate, 0.45 kg dipotassium hydrogen phosphate, and 0.36 kg of bubbly spores.
[0125] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 142 mg / 100 ml, total sugar 4.2 g / 100 ml, and pH 7.1.
[0126] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0127] The secondary seed culture medium for B. trispora(-) consists of: 550 kg corn starch, 264 kg soybean meal, 88 kg light calcium carbonate, 5.5 kg dipotassium hydrogen phosphate, and 4.4 kg of bubbly agent.
[0128] The sterilized secondary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 138 mg / 100 ml, total sugar 4.4 g / 100 ml, and pH 7.2.
[0129] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0130] 2090 kg of corn starch, 3520 kg of soybean meal, 4950 kg of soybean oil, 77 kg of 2,6-di-tert-butyl-4-methylphenol (BHT), 2090 kg of corn steep liquor, 880 kg of light calcium carbonate, 99 kg of dipotassium hydrogen phosphate, 44 kg of foaming agent, and 55 kg of Span20.
[0131] The sterilized fermentation medium had the following composition: amino nitrogen 235 mg / 100 ml, total sugar 3.3 g / 100 ml, and pH 7.2.
[0132] Fermentation culture was completed, with a fermentation titer of 2.16 g / L and a fermentation cycle of 117 h.
[0133] Comparison Case 2
[0134] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0135] B. trispora(+) seed culture medium: corn starch 55kg, soybean meal 26.4kg, light calcium carbonate 8.8kg, dipotassium hydrogen phosphate 0.55kg, and bubbly agent 0.44kg.
[0136] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 134 mg / 100 ml, total sugar 4.3 g / 100 ml, and pH 7.3.
[0137] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0138] The primary seed culture medium for B. trispora(-) consists of: 45 kg corn starch, 21.6 kg soybean meal, 7.2 kg light calcium carbonate, 0.45 kg dipotassium hydrogen phosphate, and 0.36 kg of bubbly spores.
[0139] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 145 mg / 100 ml, total sugar 4.1 g / 100 ml, and pH 7.2.
[0140] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0141] The secondary seed culture medium for B. trispora(-) consists of: 550 kg corn starch, 264 kg soybean meal, 88 kg light calcium carbonate, 5.5 kg dipotassium hydrogen phosphate, and 4.4 kg of bubbly agent.
[0142] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 135 mg / 100 ml, total sugar 4.2 g / 100 ml, pH 7.0.
[0143] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0144] 2090 kg of corn starch, 3520 kg of soybean meal, 4950 kg of soybean oil, 77 kg of 2,6-di-tert-butyl-4-methylphenol (BHT), 2090 kg of corn steep liquor, 880 kg of light calcium carbonate, 99 kg of dipotassium hydrogen phosphate, 44 kg of foaming agent, and 44 kg of lauryl phosphate.
[0145] The sterilized fermentation medium had the following composition: amino nitrogen 232 mg / 100 ml, total sugar 3.0 g / 100 ml, and pH 7.3.
[0146] Fermentation culture was completed, with a fermentation titer of 2.29 g / L and a fermentation cycle of 118 h.
[0147] Comparison Case 3
[0148] B. trispora(+) seed culture: effective volume of seed culture medium 1.1 ml 3 .
[0149] B. trispora(+) seed culture medium: corn starch 55kg, soybean meal 26.4kg, light calcium carbonate 8.8kg, potassium humate 1.54kg, dipotassium hydrogen phosphate 0.55kg, and bubbly agent 0.44kg.
[0150] The sterilized B. trispora(+) seed culture medium had the following composition: amino nitrogen 135 mg / 100 ml, total sugar 4.2 g / 100 ml, and pH 7.2.
[0151] B. trispora (-) Primary seed culture: Effective volume of seed culture medium 0.9 ml 3 .
[0152] The primary seed culture medium for B. trispora(-) consists of: 45 kg corn starch, 21.6 kg soybean meal, 7.2 kg light calcium carbonate, 1.26 kg potassium humate, 0.45 kg dipotassium hydrogen phosphate, and 0.36 kg of bufotoxin.
[0153] The sterilized primary seed culture medium for B. trispora(-) had the following composition: amino nitrogen 142 mg / 100 ml, total sugar 4.3 g / 100 ml, and pH 7.1.
[0154] B. trispora (-) Secondary seed culture: Seed culture medium effective volume 11 ml 3 .
[0155] The secondary seed culture medium for B. trispora(-) consists of: 550 kg corn starch, 264 kg soybean meal, 88 kg light calcium carbonate, 15.4 kg potassium humate, 5.5 kg dipotassium hydrogen phosphate, and 4.4 kg of bubbly spores.
[0156] The sterilized secondary seed culture medium for B. trispora(-) had the following composition after sterilization: amino nitrogen 140 mg / 100 ml, total sugar 4.5 g / 100 ml, pH 7.2.
[0157] Fermentation culture: Effective volume of fermentation medium 110m³ 3 .
[0158] 2090 kg corn starch, 3520 kg soybean meal, 4950 kg soybean oil, 77 kg 2,6-di-tert-butyl-4-methylphenol (BHT), 2090 kg corn steep liquor, 880 kg light calcium carbonate, 154 kg potassium humate, 99 kg dipotassium hydrogen phosphate, 44 kg foaming agent, and 55 kg Span20.
[0159] The sterilized fermentation medium had the following composition: amino nitrogen 239 mg / 100 ml, total sugar 3.2 g / 100 ml, and pH 7.4.
[0160] Fermentation culture was completed, with a fermentation titer of 2.36 g / L and a fermentation cycle of 121 h.
Claims
1. A culture medium for producing lycopene by fermentation using Blakeslea trispora (B trispora), characterized in that... This includes B. trispora(+) seed culture medium, B. trispora(-) primary seed culture medium, secondary seed culture medium, and fermentation medium, wherein: The B. trispora(+) seed culture medium consists of: corn starch 40-60 g / L, soybean meal 20-26 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, bufotenol 0.2-0.6 g / L, and potassium humate 1.2-1.6 g / L. The sterilized B. trispora(+) seed culture medium has the following quality requirements: amino nitrogen 100-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, and pH 6.5-7.
5. The primary seed culture medium for B. trispora(-) consists of: corn starch 40-60 g / L, soybean meal 20-26 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, bufotenol 0.2-0.6 g / L, and potassium humate 1.2-1.6 g / L. The sterilized quality requirements for the primary seed culture medium for B. trispora(-) are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, and pH 6.5-7.
5. The secondary seed culture medium for B. trispora(-) consists of: corn starch 40-60 g / L, soybean meal 20-26 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-0.8 g / L, bufotenol 0.2-0.6 g / L, and potassium humate 1.2-1.6 g / L. The sterilized quality requirements for the secondary seed culture medium for B. trispora(-) are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, and pH 6.5-7.
5. The fermentation medium consists of: corn starch 15-22 g / L, soybean meal 26-36 g / L, soybean oil 35-55 g / L, 2,6-di-tert-butyl-4-methylphenol (BHT) 0.3-0.9 g / L, corn steep liquor 15-20 g / L, light calcium carbonate 6-9 g / L, dipotassium hydrogen phosphate 0.3-1.3 g / L, foaming agent 0.2-0.6 g / L, potassium humate 1.2-1.6 g / L, and lauryl phosphate 0.2-0.6 g / L. The sterilized fermentation medium has the following quality requirements: amino nitrogen 150-300 mg / 100 ml, total sugar 2.0-3.6 g / 100 ml, and pH 6.8-7.
5.
2. The culture medium according to claim 1, wherein the potassium humate is 1.4 to 1.6 g / L.
3. The culture medium according to claim 2, wherein the potassium humate is 1.5 g / L.
4. Use of the culture medium according to any one of claims 1-3 for fermentation production of lycopene.