Bacteria enrichment nutrient solution for fermentation and preparation method thereof

Abstract

The invention relates to the technical field of bacterium enrichment nutrient solutions, in particular to a bacterium enrichment nutrient solution for fermentation and a preparation method of the bacterium enrichment nutrient solution. The culture medium is prepared from the following raw materials in parts by weight: maltodextrin, crystalline glucose, low-pigment yeast extract powder, refined soy peptone, dipotassium phosphate, monopotassium phosphate, magnesium sulfate heptahydrate, sodium chloride, phytic acid, L-ascorbic acid, manganese sulfate monohydrate, biotin, thiamine hydrochloride and choline chloride. Through a double-carbon-source structure, maltodextrin and crystalline glucose cooperate, metabolic oscillation is avoided, and the energy utilization rate is increased; the low-pigment yeast extract powder and the refined soy peptone are complementary, so that the rapid growth requirement of thalli is met; phytic acid is chelated with metal ions to prevent precipitation, L-ascorbic acid is used for resisting oxidation, and biotin and thiamine activate a coenzyme pathway to realize accurate regulation and control of a metabolic network.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial enrichment nutrient solution, in particular to a bacterial enrichment nutrient solution for fermentation and a preparation method thereof. Background Art

[0002] Nutrient solution is a liquid culture medium that provides comprehensive nutritional components for microbial growth and metabolism. It contains core substances such as carbon source, nitrogen source, inorganic salts, vitamins and growth factors. It is specially used in the field of industrial fermentation and is suitable for high-density culture of engineered bacteria such as Escherichia coli and Bacillus subtilis. It can efficiently produce recombinant proteins, amino acids, vaccines and other biological products, and has wide application value in biopharmaceuticals, food additives, biofuels and other fields.

[0003] Generally, traditional nutrient solutions mostly use simple carbon sources, crude nitrogen sources and basic inorganic salts, which have the following defects: an imbalance in the carbon-nitrogen ratio can easily lead to metabolic inhibition, resulting in residual sugar accumulation or premature aging of the bacteria; crude raw materials contain impurities such as pigments and endotoxins, which inhibit bacterial activity and increase the difficulty of purification; high-temperature sterilization destroys the activity of heat-sensitive substances such as vitamins and growth factors, reducing bioavailability.

[0004] Based on this, the present invention provides a bacterial enrichment nutrient solution for fermentation and a preparation method thereof to solve the above-mentioned technical problems. Summary of the Invention

[0005] The object of the present invention is to provide a bacterial enrichment nutrient solution for fermentation and a preparation method thereof, so as to solve the problems mentioned in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a bacterial enrichment nutrient solution for fermentation. The nutrient solution is composed of the following raw materials in parts by weight: 120.0-180.0 parts of maltodextrin; 20.0-40.0 parts of crystalline glucose; 25.0-45.0 parts of low-pigment yeast extract powder; 15.0-30.0 parts of refined soy peptone; 2.0-5.0 parts of dipotassium hydrogen phosphate; 0.5-2.0 parts of potassium dihydrogen phosphate; 0.8-1.5 parts of magnesium sulfate heptahydrate; 0.5-2.0 parts of sodium chloride; 0.1-0.3 parts of phytic acid; 0.05-0.15 parts of L-ascorbic acid; 0.005-0.02 parts of manganese sulfate monohydrate; 0.0001-0.0005 parts of biotin; 0.001-0.005 parts of thiamine hydrochloride; and 0.1-0.4 parts of choline chloride.

[0008] Preferably, the low-pigment yeast extract is prepared by subjecting baker's yeast to CRISPR-engineered bacteria-assisted enzymatic hydrolysis coupled with nanofiltration under an anaerobic micro-pressure environment.

[0009] Preferably, the refined soy peptone is obtained by purifying defatted soybean meal by directional enzymatic decomposition coupled with electrodialysis in a low-temperature plasma environment, and the purity is greater than 96.0%.

[0010] Preferably, the phytic acid is prepared by coupling rice bran fermentation with ionic liquid extraction under a supercritical carbon dioxide environment.

[0011] Preferably, the L-ascorbic acid is produced by crystallizing glucose through a genetically engineered cyanobacteria photosynthetic conversion coupled membrane in a photobioreactor environment.

[0012] Preferably, the biotin is prepared by enzyme immobilization catalysis coupled with ultracentrifugation of fumaric acid in a constant temperature magnetic nanoparticle environment.

[0013] Preferably, the thiamine hydrochloride is prepared by electrochemical synthesis of pyrimidine derivatives in a microfluidic chip environment and coupled with real-time monitoring of a biosensor, and has a purity greater than 97.0%.

[0014] Preferably, the choline chloride is prepared by coupling trimethylamine and epichlorohydrin with ion exchange resin for purification under supercritical water environment through microwave-assisted reaction, and the purity is greater than 96.0%.

[0015] The present invention also provides a method for preparing a bacterial enrichment nutrient solution for fermentation, comprising the following steps:

[0016] S1. Pretreatment and accurate weighing of raw materials: 120.0-180.0 parts of maltodextrin and 20.0-40.0 parts of crystallized glucose were placed in a vacuum drying oven and dehydrated at 45±5℃ and vacuum degree -0.08MPa for 2 hours to ensure that the water content was ≤0.5%. 2.0-5.0 parts of dipotassium hydrogen phosphate, 0.5-2.0 parts of potassium dihydrogen phosphate, 0.8-1.5 parts of magnesium sulfate heptahydrate, and 0.5-1.5 parts of chlorinated starch were added. 0.5-2.0 parts of sodium and 0.005-0.02 parts of manganese sulfate monohydrate are pre-crushed by a jet mill, and 0.1-0.3 parts of phytic acid, 0.05-0.15 parts of L-ascorbic acid, 0.0001-0.0005 parts of biotin, 0.001-0.005 parts of thiamine hydrochloride, and 0.1-0.4 parts of choline chloride are weighed according to the ratio using a high-precision microbalance with an accuracy of 0.0001g for later use;

[0017] S2. Synthesis of the main nutrient solution: 400 L of ultrapure water was added to a bidirectional shear dispersion tank, and the temperature was raised to 60 ± 2 ° C. After high-speed stirring, pretreated maltodextrin, crystalline glucose, phosphate, magnesium salt, sodium chloride and manganese sulfate monohydrate were added in sequence. Nitrogen was introduced to maintain an inert environment. The temperature was raised to 85 ± 1 ° C at a rate of 0.5 ° C / min. The reaction was stirred at a constant speed for 30 minutes until completely dissolved. The solution was rapidly cooled to 45 ± 1 ° C within 15 seconds through a plate heat exchanger and temporarily stored in a sterile buffer tank;

[0018] S3. Integration of thermosensitive bioactive components: 25.0-45.0 parts low-pigment yeast extract powder and 15.0-30.0 parts purified soy peptone were added to an ultrasonic dispersing vessel and ultrasonically dispersed at 35±1°C and pH 6.5±0.2 for 20 minutes to form a homogeneous colloid. Phytic acid, L-ascorbic acid, biotin, thiamine hydrochloride, and choline chloride were then sequentially added to the mixture in a low-temperature laminar flow mixer. The mixture was stirred at 200 rpm for 10 minutes. The main nutrient solution was then added in a 1:1 volume ratio gradient. The mixing rate was controlled at ≤2 L / min to avoid excessive local concentration.

[0019] S4. Dynamic sterilization and nano-homogenization: The mixed solution was treated with a continuous flow UV-hydrogen peroxide synergistic sterilization system at a flow rate of 5 L / min to ensure a microbial kill rate of ≥99.99%. The mixture was then broken into particles ≤100 nm by a high-pressure nano-homogenizer and the dissolved oxygen was removed to ≤0.5 ppm in a low-temperature vacuum degassing tower.

[0020] S5. Final product shaping and filling: Use an online pH-conductivity monitor to adjust the solution to pH 6.8±0.1, sterilize by filtration through a membrane filtration system with a 0.22μm PVDF filter element and a pressure difference of ≤0.3MPa, and fill the solution into light-proof containers on a sterile filling line and seal them with nitrogen.

[0021] Preferably, the parameters of the continuous flow UV-hydrogen peroxide synergistic sterilization system in step S4 are: UV wavelength 254 nm, intensity 80 mJ / cm², and H2O2 concentration 50 ppm.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses a dual-carbon source structure, maltodextrin and crystallized glucose to cooperate, avoid metabolic shock and improve energy utilization; low-pigment yeast extract powder and refined soy peptone complement each other to meet the rapid growth needs of bacteria; phytic acid chelates metal ions to prevent precipitation, L-ascorbic acid is antioxidant, biotin and thiamine activate coenzyme pathways, and achieve precise regulation of metabolic networks. At the same time, innovative processes such as genetically engineered cyanobacteria to synthesize L-ascorbic acid and plasma-activated enzymatic hydrolysis of soy peptone are adopted to ensure high activity and low pigmentation of key components; supercritical CO2 extraction of phytic acid, microfluidic synthesis of thiamine and other processes avoid chemical solvent residues and improve biocompatibility; low-temperature laminar mixing and ultraviolet-hydrogen peroxide non-thermal sterilization make the vitamin activity retention rate higher; high-pressure nano-homogenization makes nutrients close to the bacterial membrane pore size, with high absorption efficiency. At the same time, the multi-stage deoxygenation design from dehydration to filling blocks the oxidation chain reaction and ensures stability. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0025] 1. Materials:

[0026] The present invention provides a bacterial enrichment nutrient solution for fermentation, and the materials are all commercially available unless otherwise specified.

[0027] The invention is composed of the following raw materials in parts by weight: 120.0-180.0 parts of maltodextrin; 20.0-40.0 parts of crystalline glucose; 25.0-45.0 parts of low-pigment yeast extract powder; 15.0-30.0 parts of refined soy peptone; 2.0-5.0 parts of dipotassium hydrogen phosphate; 0.5-2.0 parts of potassium dihydrogen phosphate; 0.8-1.5 parts of magnesium sulfate heptahydrate; 0.5-2.0 parts of sodium chloride; 0.1-0.3 parts of phytic acid; 0.05-0.15 parts of L-ascorbic acid; 0.005-0.02 parts of manganese sulfate monohydrate; 0.0001-0.0005 parts of biotin; 0.001-0.005 parts of thiamine hydrochloride; and 0.1-0.4 parts of choline chloride.

[0028] It should also be noted that the low-pigment yeast extract powder is made by coupling baker's yeast with CRISPR-engineered bacteria-assisted enzymatic hydrolysis and nanofiltration under an anaerobic micro-pressure environment.

[0029] It should also be noted that refined soy peptone is obtained by purifying defatted soybean meal through directional enzymatic hydrolysis coupled with electrodialysis in a low-temperature plasma environment, with a purity of greater than 96.0%.

[0030] It should also be noted that phytic acid is produced by coupling rice bran with ionic liquid extraction through microbial fermentation in a supercritical carbon dioxide environment.

[0031] It should also be noted that L-ascorbic acid is produced by crystallizing glucose through genetically engineered cyanobacteria photosynthetic conversion coupled membrane in a photobioreactor environment.

[0032] It should also be noted that biotin is prepared by enzyme-immobilized catalytic coupling and ultracentrifugation of fumaric acid in a constant temperature magnetic nanoparticle environment.

[0033] It should also be noted that thiamine hydrochloride is prepared by electrochemical synthesis of pyrimidine derivatives in a microfluidic chip environment and real-time monitoring by a biosensor, with a purity greater than 97.0%.

[0034] It should also be noted that choline chloride is prepared by coupling trimethylamine and epichlorohydrin with ion exchange resin through microwave-assisted reaction in a supercritical water environment, with a purity greater than 96.0%.

[0035] 2. Process:

[0036] Based on the above bacterial enrichment nutrient solution formula, the present invention also proposes a method for preparing a bacterial enrichment nutrient solution for fermentation, comprising the following steps:

[0037] S1. Pretreatment and accurate weighing of raw materials: 120.0-180.0 parts of maltodextrin and 20.0-40.0 parts of crystallized glucose were placed in a vacuum drying oven and dehydrated at 45±5℃ and vacuum degree -0.08MPa for 2 hours to ensure that the water content was ≤0.5%. 2.0-5.0 parts of dipotassium hydrogen phosphate, 0.5-2.0 parts of potassium dihydrogen phosphate, 0.8-1.5 parts of magnesium sulfate heptahydrate, and 0.5-1.5 parts of chlorinated starch were added. 0.5-2.0 parts of sodium and 0.005-0.02 parts of manganese sulfate monohydrate are pre-crushed by a jet mill, and 0.1-0.3 parts of phytic acid, 0.05-0.15 parts of L-ascorbic acid, 0.0001-0.0005 parts of biotin, 0.001-0.005 parts of thiamine hydrochloride, and 0.1-0.4 parts of choline chloride are weighed according to the ratio using a high-precision microbalance with an accuracy of 0.0001g for later use;

[0038] S2. Synthesis of the main nutrient solution: 400 L of ultrapure water was added to a bidirectional shear dispersion tank, and the temperature was raised to 60 ± 2 ° C. After high-speed stirring, pretreated maltodextrin, crystalline glucose, phosphate, magnesium salt, sodium chloride and manganese sulfate monohydrate were added in sequence. Nitrogen was introduced to maintain an inert environment. The temperature was raised to 85 ± 1 ° C at a rate of 0.5 ° C / min. The reaction was stirred at a constant speed for 30 minutes until completely dissolved. The solution was rapidly cooled to 45 ± 1 ° C within 15 seconds through a plate heat exchanger and temporarily stored in a sterile buffer tank;

[0039] S3. Integration of thermosensitive bioactive components: 25.0-45.0 parts low-pigment yeast extract powder and 15.0-30.0 parts purified soy peptone were added to an ultrasonic dispersing vessel and ultrasonically dispersed at 35±1°C and pH 6.5±0.2 for 20 minutes to form a homogeneous colloid. Phytic acid, L-ascorbic acid, biotin, thiamine hydrochloride, and choline chloride were then sequentially added to the mixture in a low-temperature laminar flow mixer. The mixture was stirred at 200 rpm for 10 minutes. The main nutrient solution was then added in a 1:1 volume ratio gradient. The mixing rate was controlled at ≤2 L / min to avoid excessive local concentration.

[0040] S4. Dynamic sterilization and nano-homogenization: The mixed solution was treated with a continuous flow UV-hydrogen peroxide synergistic sterilization system at a flow rate of 5 L / min to ensure a microbial kill rate of ≥99.99%. The mixture was then broken into particles ≤100 nm by a high-pressure nano-homogenizer and the dissolved oxygen was removed to ≤0.5 ppm in a low-temperature vacuum degassing tower.

[0041] S5. Final product shaping and filling: Use an online pH-conductivity monitor to adjust the solution to pH 6.8±0.1, sterilize by filtration through a membrane filtration system with a 0.22μm PVDF filter element and a pressure difference of ≤0.3MPa, and fill the solution into light-proof containers on a sterile filling line and seal them with nitrogen.

[0042] It should also be noted that the parameters of the continuous flow UV-hydrogen peroxide synergistic sterilization system in step S4 are: UV wavelength 254nm, intensity 80mJ / cm², and H2O2 concentration 50ppm.

[0043] In order to verify the scientific nature of the components of the bacterial enrichment nutrient solution formula of the present invention and the synergistic performance of the preparation process, examples within the component range of the present invention and comparative examples outside the range were designed for analysis, wherein:

[0044] The following fixed parameters are included:

[0045] S1. Dehydration temperature 45°C, vacuum -0.08MPa;

[0046] S2. Dispersion tank water temperature 60℃, dissolution temperature 85℃;

[0047] S3. Ultrasonic dispersion pH 6.5, laminar mixing temperature 4°C;

[0048] S4. Sterilization UV intensity 80mJ / cm², H2O250ppm, homogenization pressure 180MPa;

[0049] S5. Filling temperature 25℃;

[0050] The following fermentation tests are included:

[0051] Bacterial strain: E. coli K12 (ATCC29425);

[0052] Fermenter: 5L automatic bioreactor, 2L liquid volume, 5% inoculum;

[0053] Parameters: temperature 37°C, pH 7.0±0.1, dissolved oxygen 30%, stirring speed 300 rpm;

[0054] Test time: 24 hours;

[0055] Includes the following performance indicators:

[0056] Bacterial density (OD600): Samples were taken every hour to measure absorbance;

[0057] Product concentration (g / L): HPLC was used to measure the expression of recombinant protein (target product: green fluorescent protein);

[0058] Specific growth rate (μ,h⁻¹): calculated by fitting OD600 in the exponential phase;

[0059] Residual sugar (g / L): residual glucose at the end of fermentation;

[0060] Example 1: In this example, a method for preparing a bacterial enrichment nutrient solution for fermentation comprises the following steps:

[0061] S1 Raw material pretreatment:

[0062] 150.0 parts of maltodextrin and 30.0 parts of crystalline glucose were placed in a vacuum drying oven and dehydrated at 45°C and -0.08 MPa for 2 hours to a water content of 0.4%; 3.5 parts of dipotassium hydrogen phosphate, 1.25 parts of potassium dihydrogen phosphate, 1.15 parts of magnesium sulfate heptahydrate, 1.25 parts of sodium chloride, and 0.012 parts of manganese sulfate monohydrate were ground using a jet mill (12000 rpm) to a D90 of 8 μm; 0.2 parts of phytic acid, 0.10 parts of L-ascorbic acid, 0.0003 parts of biotin, 0.003 parts of thiamine hydrochloride, and 0.25 parts of choline chloride were weighed using a microbalance (0.0001 g accuracy);

[0063] S2 main nutrient solution synthesis:

[0064] Add 400 L of ultrapure water (18.2 MΩ·cm) to a 500 L bidirectional shear dispersion tank, heat to 60°C, and stir at 800 rpm. Then, add all the raw materials in S1 except for trace components in sequence. Flow nitrogen (5 L / min) for protection, heat to 85°C at 0.5°C / min, and stir at 400 rpm for 30 minutes to dissolve. Cool to 45°C within 15 seconds using a plate heat exchanger and transfer to a sterile buffer tank.

[0065] S3 heat-sensitive component integration:

[0066] 35.0 parts of low-pigment yeast extract powder and 22.5 parts of refined soy peptone were added to an ultrasonic dispersing kettle (10 kW, 28 kHz) and ultrasonically dispersed at 35°C and pH 6.5 for 20 minutes to form a colloid. The mixture was transferred to a low-temperature laminar flow mixer (4°C, wind speed 0.2 m / s), and phytic acid, L-ascorbic acid, biotin, thiamine hydrochloride, and choline chloride were added in sequence. The mixture was stirred at 200 rpm for 10 minutes. The main nutrient solution was then added in a gradient at a rate of 2 L / min.

[0067] S4 sterilization and homogenization:

[0068] The mixed solution was passed through a UV-H2O2 sterilization system (254nm, 80mJ / cm², 50ppmH2O2) at a speed of 5L / min, achieving a kill rate of 99.995%. It was then circulated three times in a high-pressure nano-homogenizer (180MPa) until the particle size reached 95nm. Finally, it was deoxygenated to 0.4ppm in a low-temperature vacuum degassing tower (30°C, -0.095MPa).

[0069] S5 shaping and filling:

[0070] The pH was adjusted to 6.8 using an online pH meter and filtered through a 0.22 μm PVDF membrane (pressure difference 0.25 MPa). The solution was then dispensed into brown bottles using an aseptic filling line (ISO 5 grade) and sealed with nitrogen.

[0071] Example 2: In this example, maltodextrin is 120.0 parts, crystalline glucose is 20.0 parts, low-pigment yeast extract powder is 25.0 parts, and purified soy peptone is 15.0 parts. Other components are the same as in Example 1.

[0072] Example 3: In this example, maltodextrin is 180.0 parts, crystalline glucose is 40.0 parts, low-pigment yeast extract powder is 45.0 parts, and purified soy peptone is 30.0 parts. Other components are the same as in Example 1.

[0073] The component parameters in the embodiment are shown in Table 1:

[0074] Table 1: Material ratio table of the examples

[0075] raw material Example 1 Example 2 Example 3 Maltodextrin 150.0 120.0 180.0 Crystallized glucose 30.0 20.0 40.0 Low-pigment yeast extract powder 35.0 25.0 45.0 Refined soy peptone 22.5 15.0 30.0 Dipotassium hydrogen phosphate 3.5 3.5 3.5 Potassium dihydrogen phosphate 1.25 1.25 1.25 Magnesium sulfate heptahydrate 1.15 1.15 1.15 Sodium chloride 1.25 1.25 1.25 Phytic acid 0.2 0.2 0.2 L-ascorbic acid 0.10 0.10 0.10 Manganese sulfate monohydrate 0.012 0.012 0.012 Biotin 0.0003 0.0003 0.0003 Thiamine hydrochloride 0.003 0.003 0.003 Choline chloride 0.25 0.25 0.25

[0076] Comparative Example 1: In this example, maltodextrin is 100.0 parts (lower than the range), and the other components are the same as in Example 1;

[0077] Comparative Example 2: In this example, the amount of crystalline glucose is 50.0 parts (higher than the range), the amount of low-pigment yeast extract powder is 50.0 parts (higher than the range), and the other components are the same as those in Example 1;

[0078] Comparative Example 3: In this example, the amount of purified soy peptone was 0 (missing), the amount of biotin was 0 (missing), and the other components were the same as in Example 1;

[0079] The component parameters in the comparative example are shown in Table 2:

[0080] Table 2: Comparative Example Material Ratio

[0081] raw material Comparative Example 1 Comparative Example 2 Comparative Example 3 Maltodextrin 100.0↓ 150.0 150.0 Crystallized glucose 30.0 50.0↑ 30.0 Low-pigment yeast extract powder 35.0 50.0↑ 35.0 Refined soy peptone 22.5 22.5 0.0↓ Biotin 0.0003 0.0003 0.0↓ Other components Same as Example 1 Same as Example 1 Same as Example 1

[0082] 3. Performance test:

[0083] Nutrient solution samples were prepared according to the examples and comparative examples, and the following performance tests were performed. The performance data of the nutrient solution prepared in the examples are shown in Table 3:

[0084] Table 3: Performance comparison of examples

[0085] index Example 1 Example 2 Example 3 Maximum OD600 18.5 15.2 17.8 Specific growth rate μ(h⁻¹) 0.62 0.51 0.59 Product concentration (g / L) 3.85 2.90 3.55 Residual sugar content (g / L) 0.8 2.1 1.2

[0086] The performance data of the nutrient solution prepared in the comparative example are shown in Table 4:

[0087] Table 4: Comparative Example Performance Comparison

[0088] index Comparative Example 1 Comparative Example 2 Comparative Example 3 Maximum OD600 12.3 14.1 9.8 Specific growth rate μ(h⁻¹) 0.41 0.38 0.29 Product concentration (g / L) 1.95 2.10 0.75 Residual sugar content (g / L) 5.6 8.3 3.2

[0089] 4. Data Analysis

[0090] According to Tables 1 to 4, the residual sugar content of Examples 1-3 of the present invention (0.8-2.1 g / L) is significantly lower than that of Comparative Example 1 (5.6 g / L) and Comparative Example 2 (8.3 g / L), proving that the carbon source ratio (maltodextrin + glucose) within this range can be fully utilized; while Comparative Example 3 lacks peptone and biotin, resulting in severe restriction of bacterial growth (OD600 of only 9.8) and a decrease in product concentration by 80%;

[0091] Example 1 had the highest bacterial density (OD600 18.5) and product (3.85 g / L) due to the optimal C / N ratio (maltodextrin:peptone = 6.7:1); whereas Comparative Example 2 had the lowest specific growth rate (0.38 h⁻¹) due to the inhibition of carbon metabolism caused by excess glucose (50 parts);

[0092] Therefore, the component formula setting interval of the present invention is reasonable;

[0093] As can be seen from Tables 1 and 2, in Example 1 of the present invention, maltodextrin (150 parts) provides a continuous carbon source, avoiding metabolic stress caused by excessive glucose consumption (compared to 8.3 g / L residual sugar in Comparative Example 2); the combined nitrogen source ratio of yeast extract (35 parts) to soy peptone (22.5 parts) (1.56:1) meets the amino acid requirements of the bacteria, and the OD600 is 21.7% higher than that of Example 2; trace components (such as 0.0003 parts of biotin) accurately support coenzyme synthesis, and when they are absent (Comparative Example 3), the product concentration decreases by 80.5%; therefore, Example 1 is the best embodiment of the present invention.

[0094] At the same time, the median parameters used in Example 1 of the present invention enable the activity of heat-sensitive components (such as L-ascorbic acid) to be retained by 98% in S3 low-temperature laminar mixing; the homogeneous particle size of 95 nm (close to the bacterial membrane pore size) improves the nutrient absorption efficiency.

[0095] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0096] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A bacterial enrichment nutrient solution for fermentation, characterized in that: The invention is composed of the following raw materials in parts by weight: 120.0-180.0 parts of maltodextrin; 20.0-40.0 parts of crystalline glucose; 25.0-45.0 parts of low-pigment yeast extract powder; 15.0-30.0 parts of refined soy peptone; 2.0-5.0 parts of dipotassium hydrogen phosphate; 0.5-2.0 parts of potassium dihydrogen phosphate; 0.8-1.5 parts of magnesium sulfate heptahydrate; 0.5-2.0 parts of sodium chloride; 0.1-0.3 parts of phytic acid; 0.05-0.15 parts of L-ascorbic acid; 0.005-0.02 parts of manganese sulfate monohydrate; 0.0001-0.0005 parts of biotin; 0.001-0.005 parts of thiamine hydrochloride; and 0.1-0.4 parts of choline chloride.

2. The bacterial enrichment nutrient solution for fermentation according to claim 1, characterized in that: The low-pigment yeast extract is prepared by subjecting baker's yeast to CRISPR-engineered bacteria-assisted enzymatic hydrolysis coupled with nanofiltration under an anaerobic micro-pressure environment.

3. The bacterial enrichment nutrient solution for fermentation according to claim 2, characterized in that: The refined soy peptone is prepared by purifying defatted soybean meal through directional enzymatic decomposition coupled with electrodialysis in a low-temperature plasma environment, and has a purity of more than 96.0%.

4. The bacterial enrichment nutrient solution for fermentation according to claim 1, characterized in that: The phytic acid is prepared by subjecting rice bran to microbial fermentation coupled with ionic liquid extraction under a supercritical carbon dioxide environment.

5. The bacterial enrichment nutrient solution for fermentation according to claim 4, characterized in that: The L-ascorbic acid is prepared by crystallizing glucose through photosynthetic conversion coupling membrane of genetically engineered cyanobacteria in a photobioreactor environment.

6. The bacterial enrichment nutrient solution for fermentation according to claim 4, characterized in that: The biotin is prepared by subjecting fumaric acid to enzyme immobilization catalysis coupling ultracentrifugation in a constant temperature magnetic nanoparticle environment.

7. The bacterial enrichment nutrient solution for fermentation according to claim 1, characterized in that: The thiamine hydrochloride is prepared by electrochemical synthesis of pyrimidine derivatives in a microfluidic chip environment and then coupled with a biosensor for real-time monitoring, with a purity greater than 97.0%.

8. The bacterial enrichment nutrient solution for fermentation according to claim 1, characterized in that: The choline chloride is prepared by coupling trimethylamine and epichlorohydrin through microwave-assisted reaction in a supercritical water environment and then purified by ion exchange resin, with a purity greater than 96.0%.

9. The method for preparing a bacterial enrichment nutrient solution for fermentation according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Pretreatment and accurate weighing of raw materials: 120.0-180.0 parts of maltodextrin and 20.0-40.0 parts of crystallized glucose were placed in a vacuum drying oven and dehydrated at 45±5℃ and vacuum degree -0.08MPa for 2 hours to ensure that the water content was ≤0.5%. 2.0-5.0 parts of dipotassium hydrogen phosphate, 0.5-2.0 parts of potassium dihydrogen phosphate, 0.8-1.5 parts of magnesium sulfate heptahydrate, and 0.5-1.5 parts of chlorinated starch were added. 0.5-2.0 parts of sodium and 0.005-0.02 parts of manganese sulfate monohydrate are pre-crushed by a jet mill, and 0.1-0.3 parts of phytic acid, 0.05-0.15 parts of L-ascorbic acid, 0.0001-0.0005 parts of biotin, 0.001-0.005 parts of thiamine hydrochloride, and 0.1-0.4 parts of choline chloride are weighed according to the ratio using a high-precision microbalance with an accuracy of 0.0001g for later use; S2. Synthesis of the main nutrient solution: 400 L of ultrapure water was added to a bidirectional shear dispersion tank, and the temperature was raised to 60 ± 2 ° C. After high-speed stirring, pretreated maltodextrin, crystalline glucose, phosphate, magnesium salt, sodium chloride and manganese sulfate monohydrate were added in sequence. Nitrogen was introduced to maintain an inert environment. The temperature was raised to 85 ± 1 ° C at a rate of 0.5 ° C / min. The reaction was stirred at a constant speed for 30 minutes until completely dissolved. The solution was rapidly cooled to 45 ± 1 ° C within 15 seconds through a plate heat exchanger and temporarily stored in a sterile buffer tank; S3. Integration of thermosensitive bioactive components: 25.0-45.0 parts low-pigment yeast extract powder and 15.0-30.0 parts purified soy peptone were added to an ultrasonic dispersing vessel and ultrasonically dispersed at 35±1°C and pH 6.5±0.2 for 20 minutes to form a homogeneous colloid. Phytic acid, L-ascorbic acid, biotin, thiamine hydrochloride, and choline chloride were then sequentially added to the mixture in a low-temperature laminar flow mixer. The mixture was stirred at 200 rpm for 10 minutes. The main nutrient solution was then added in a 1:1 volume ratio gradient. The mixing rate was controlled at ≤2 L / min to avoid excessive local concentration. S4. Dynamic sterilization and nano-homogenization: The mixed solution was treated with a continuous flow UV-hydrogen peroxide synergistic sterilization system at a flow rate of 5 L / min to ensure a microbial kill rate of ≥99.99%. The mixture was then broken into particles ≤100 nm by a high-pressure nano-homogenizer and the dissolved oxygen was removed to ≤0.5 ppm in a low-temperature vacuum degassing tower. S5. Final product shaping and filling: Use an online pH-conductivity monitor to adjust the solution to pH 6.8±0.1, sterilize by filtration through a membrane filtration system with a 0.22μm PVDF filter element and a pressure difference of ≤0.3MPa, and fill the solution into light-proof containers on a sterile filling line and seal them with nitrogen.

10. The method for preparing a bacterial enrichment nutrient solution for fermentation according to claim 9, characterized in that: The parameters of the continuous flow UV-hydrogen peroxide synergistic sterilization system in step S4 are: UV wavelength 254nm, intensity 80mJ / cm², and H2O2 concentration 50ppm.