A method for reducing the lincomycin fermentation group b component

Abstract

The application discloses a method for reducing lincomycin B component in a lincomycin fermentation process, which comprises adding soybean protein hydrolysate A in the lincomycin fermentation process, so that the lincomycin B component can be effectively reduced, and the A component titer can be improved. The soybean protein hydrolysate A is a complex amino acid mixture obtained by enzymolysis of soybean raw materials, and the components include peptide segment A, free amino acid solution A, water or others, wherein the mass percentage of the peptide segment A is 30-35%, the mass percentage of the free amino acid solution A is 55-65%, and the water and the others account for 5-15%. After optimization, the production level of the B component in a 50 L fermenter is at least 166 mu g / mL, which is reduced by more than 75% compared with the yield of the B component in the original fermentation culture method, the product quality and the production efficiency are improved, and the method is suitable for industrialized scale production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for reducing the B component in lincomycin fermentation. Background Technology

[0002] Lincomycin is a lincosamide antibiotic produced by Streptomyces lincosamide, with the molecular formula C64. 18 H 34 N₂O₆S, with a relative molecular weight of 406.56, is primarily used clinically as lincomycin A. However, during fermentation, the bacteria simultaneously synthesize lincomycin B, which has a similar chemical structure to A but lower antibacterial activity and higher toxicity. The structural difference between the two components is that component A has a n-propyl group at the 4-position, while component B has an ethyl group. Pharmacopoes of various countries impose strict limits on the content of lincomycin B in the finished product (e.g., the Chinese Pharmacopoeia stipulates that it must not exceed 5%), and since lincomycin A and B are homologues, their separation and purification are relatively difficult. Therefore, effectively reducing the amount of component B in the fermentation broth is a crucial quality control challenge in the industrial production of lincomycin.

[0003] Currently, industrial production mainly relies on subsequent solvent extraction and multiple crystallization to remove component B. This method has a long process, large yield loss, high solvent consumption, and extremely high requirements for crystallization process control, which increases production costs and environmental burden.

[0004] Existing technologies also include some attempts to control fermentation at its source. For example, strains that produce low levels of B components are selected through mutagenesis, but this method is labor-intensive, time-consuming, and may be accompanied by the risk of decreased potency. Other technologies propose adding certain precursors to fermentation, but the effects are often unstable, or they fail to form effective synergies with other key process parameters, making it impossible to stably control B components at extremely low levels in industrial-scale production.

[0005] Therefore, there is an urgent need in this field to develop an efficient, stable, and easily industrially applicable fermentation control method that can inhibit the generation of lincomycin B at the source, simplify post-processing procedures, and enhance product competitiveness. Summary of the Invention

[0006] In view of this, the present invention aims to propose a method for reducing lincomycin B fraction during fermentation, focusing on precise nutritional regulation of secondary metabolism in the mid-to-late stages of fermentation. Unexpectedly, it was discovered that not all organic nitrogen supplementation can effectively guide metabolic flow. Only by supplementing soybean protein hydrolysate with specific peptide-amino acid composition and molecular morphology during a specific metabolic window can extremely precise regulation of the lincomycin A / B synthesis ratio be achieved without introducing exogenous inorganic salts, while maintaining or even enhancing the potency of the main product.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] A method for reducing component B during lincomycin fermentation involves adding soybean protein hydrolysate A during fermentation to control the formation of component B. Component B is (2S,4R)-4-ethyl-N-((1R,2R)-2-hydroxy-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(methylthio)tetrahydro-2H-pyran-2-yl)propyl)-1-methylpyrrolidine-2-carboxamide, with the chemical formula C. 17 H 32 N2O6S.

[0009] The soybean protein hydrolysate A is a complex amino acid mixture obtained from soybean raw materials through enzymatic hydrolysis technology. The components include peptide A and free amino acid solution A, wherein the mass percentage of peptide A is 30-35%, the mass percentage of free amino acid solution A is 55-65%, and the proportion of moisture and other components is 5-15%.

[0010] Preferably, the small molecule peptides with a molecular weight of less than or equal to 1000 Da account for ≥96% of the total mass of peptide A; the free amino acid solution A includes glutamic acid, aspartic acid, valine and other amino acids, and the mass percentages of glutamic acid, aspartic acid, valine and tyrosine in the total mass of free amino acid solution A are 10-12%, 7-8.5%, 3-3.5% and 1.6-1.8%, respectively.

[0011] More preferably, in peptide A, peptides with a molecular weight less than 150 Da account for 21-25% of the total mass of peptide A, and peptides with a molecular weight of 150-400 Da account for 45-55% of the total mass of peptide A.

[0012] More preferably, the peptides with a molecular weight less than 150 Da account for 23.68% of the total mass of peptide A, and the peptides with a molecular weight of 150-400 Da account for 49.87% of the total mass of peptide A; the percentages of glutamic acid, aspartic acid, valine, and tyrosine in the total mass of free amino acid solution A are 11.89%, 7.40%, 3.28%, and 1.73%, respectively.

[0013] The amount of soybean protein hydrolysate A added is 0.05-0.10% of the fermentation liquid volume, and the soybean protein hydrolysate A is added when the fermentation has been going on for 130-190 hours.

[0014] Preferably, the amount of soybean protein hydrolysate A added is 0.05-0.07% of the fermentation liquid volume, and the soybean protein hydrolysate A is added 150-170 hours after fermentation.

[0015] More preferably, the amount of soybean protein hydrolysate A added is 0.05% of the fermentation liquid volume, and the soybean protein hydrolysate A is added at 162 h of fermentation.

[0016] The fermentation strain is *Streptomyces lincosinate*, with an inoculum size of 20-40%. The fermentation medium used in the method comprises the following components by mass fraction: starch 0.5-1.0%, soybean flour 2.0-3.0%, sodium citrate 0.2-0.8%, ammonium sulfate 0.2-0.5%, potassium dihydrogen phosphate 0.01-0.03%, sodium nitrate 0.7-0.8%, calcium chloride 0.6-0.8%, glucose 5.0-6.0%, corn steep liquor powder 1.0-2.0%, and defoamer 0.02-0.1%.

[0017] The fermentation method is used to produce lincomycin, and the culture medium used in the fermentation method comprises the following components in the following mass fractions:

[0018] Seed culture medium (30 L fermenter): starch 0.2-0.5%, cold-pressed soybean flour 1.0-2.0%, ammonium sulfate 0.1-0.4%, ammonium nitrate 0.1-0.3%, potassium dihydrogen phosphate 0.01-0.03%, sodium nitrate 0.2-0.5%, calcium chloride 0.4-0.7%, glucose 3.0-6.0%, corn steep liquor powder 1.0-2.0%, defoamer 0.02-0.1%, sterilized at 121℃ for 30 min, inoculation amount is one slant.

[0019] Fermentation medium (50 L fermenter): starch 0.5-1.0%, cold-pressed soybean flour 2.0-3.0%, sodium citrate 0.2-0.8%, ammonium sulfate 0.2-0.5%, potassium dihydrogen phosphate 0.01-0.03%, sodium nitrate 0.7-0.8%, calcium chloride 0.6-0.8%, glucose 5.0-6.0%, corn steep liquor powder 1.0-2.0%, defoamer 0.02-0.1%, sterilized at 121℃ for 30 min. The cold-pressed soybean flour has a protein content ≥50% and has not undergone high-temperature denaturation, making it more easily absorbed by the microorganisms.

[0020] Preferably, the fermentation process is carried out under the following conditions: fermentation cycle of 200-240 h, culture temperature of 30±1℃, pH value of 6.6±0.1, stirring speed of 150-700 rpm, and air flow rate of 1500-2400 L / h.

[0021] Preferably, the fermentation method uses *Streptomyces lincosum* strain, and the seeds cultured in the seed tank are inoculated into a 50 L fermenter using the pressure difference method. The inoculation amount is 20-40%, the fermentation cycle is 200-240 h, the culture temperature is 30±1℃, the pH value is 6.6±0.1, the stirring speed is 150-700 rpm, and the air flow rate is 1500-2400 L / h.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) In the process of lincomycin fermentation, the addition of exogenous soybean protein hydrolysate A at different fermentation cycles can effectively reduce the content of lincomycin B component, which helps to reduce production costs and improve equipment utilization.

[0024] (2) All control parameters of this invention are conventional fermentation control parameters, requiring no special equipment or complex modifications, making them easy to implement and scale up on existing production lines with good reproducibility. While achieving excellent control levels of component B, this invention completely avoids the introduction of high-concentration inorganic salts, significantly reducing the environmental burden of subsequent wastewater treatment, and causing less corrosion to the fermenter, thus exhibiting greater industrial friendliness and environmental advantages.

[0025] (3) The content of lincomycin B component in this invention is reduced to a minimum of 166 μg / mL, which is more than 75% lower than the yield of B component (679 μg / mL) of the original fermentation culture method. The proportion of B component in total lincomycin at the fermentation endpoint is stably reduced to below 1.6%, which is far superior to the pharmacopoeia standard. Attached Figure Description

[0026] Figure 1 Example 3: Detection results of lincomycin B component;

[0027] Figure 2 Comparative Example 1: Detection results of lincomycin B component. Detailed Implementation

[0028] Unless otherwise defined, the technical terms used in the following embodiments and comparative examples have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments and comparative examples are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0029] B ratio = B content / (A content + B content) 100%;

[0030] The detection methods for lincomycin A and B are as follows:

[0031] High performance liquid chromatograph, syringe filter (organic type, pore size 0.45μm);

[0032] Chromatographic system column: XBridge-C18 or equivalent packing material, 4.6 mm × 250 mm, 5 μm;

[0033] Detector: Ultraviolet detector, detection wavelength: 210 nm, injection volume: 20 μl, column temperature: 50 °C;

[0034] Mobile phase: pH 6.5 buffer: acetonitrile: methanol = 100: 170: 80, flow rate: 1.0 ml / min; pH 6.5 buffer: 34 g phosphate dissolved in 900 mL water, pH adjusted to 6.5 with concentrated ammonia, and water added to 1000 mL.

[0035] Control solution a: Accurately weigh approximately 62.5 mg of lincomycin hydrochloride reference standard, dissolve it in the mobile phase and dilute to 25 ml, then shake well.

[0036] Preparation of test solution: Shake the fermentation broth or extraction intermediate to be tested well. Accurately measure an appropriate amount of the test solution by mass or volume and place it in a 25 ml volumetric flask (controlling the sample concentration after dilution between 50 and 200 μg / ml). Add methanol to 3 / 4 of the volumetric flask to dissolve the sample (note that the sample should be shaken while adding methanol to ensure complete dissolution). Let it stand until the solution temperature reaches room temperature. Then, dilute to the mark with the mobile phase, shake well, and immediately transfer 8–9 ml of the above solution to a 10 ml centrifuge tube. Centrifuge at 4000 r / min for 5 min, filter through a 0.45 μm microporous membrane, and inject the sample. Take 20 μl of the control solution and the test solution respectively, inject one syringe into the chromatograph, and record the chromatograms of the control solution and the test solution for 40 min. Record the peak areas of lincomycin A and lincomycin B respectively, and calculate their contents.

[0037] In the embodiments of the present invention, unless otherwise specified, all proportions used are mass percentages.

[0038] In an embodiment of the present invention, the fermentation medium is formulated as follows: starch 1.0%, cold-pressed soybean flour 2.5%, sodium citrate 0.6%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.02%, sodium nitrate 0.8%, calcium chloride 0.8%, glucose 6.0%, corn steep liquor powder 1.5%, defoamer 0.05%, and the remainder is water.

[0039] The strain used in the examples and comparative examples was *Streptomyces lincosum*. The seeds cultured in the seed tank were inoculated into a 50 L fermenter using the pressure difference method. The inoculation amount was 30%, the fermentation period was 220 h, the culture temperature was 30±1℃, the pH value was 6.6±0.1, the stirring speed was 600 rpm, and the air flow rate was 2000 L / h.

[0040] Soybean protein hydrolysate A is added during the fermentation process. The addition time is in the middle and late stages of fermentation (130-180 h), and the amount added is 0.05-0.10% of the fermentation liquid volume.

[0041] Preparation process of soybean protein hydrolysate A:

[0042] Mix soybean meal powder and deionized water at a ratio of 1:10 (w / v), stir well to form a suspension;

[0043] Pretreatment: The suspension was preheated to 50±2°C and the pH was adjusted to 8.5±0.2 with sodium hydroxide solution. The pH was maintained for 15 minutes to moderately denature the protein and improve the enzymatic hydrolysis efficiency.

[0044] Enzymatic hydrolysis: Add 0.8% alkaline protease by weight of substrate, react at 55±1°C and pH 8.5 with continuous stirring for 3 hours; quickly adjust the pH of the solution to 6.5 with hydrochloric acid solution and cool to 50°C; then add 0.5% flavor protease by weight of substrate, react at 50±1°C and pH 6.5 with continuous stirring for 1 hour.

[0045] After enzymatic hydrolysis, rapidly heat the solution to 85°C and maintain this temperature for 15 minutes to completely inactivate the enzyme. Use a plate and frame filter or centrifuge (≥5000 rpm) to remove unhydrolyzed fine soybean residue particles and large molecular impurities to obtain a clear enzymatic hydrolysate.

[0046] Membrane system fractionation, including:

[0047] Ultrafiltration: An ultrafiltration membrane with a molecular weight cutoff of 1000 Da is used, operating at a pressure of 0.1 MPa and a temperature of 26°C. This removes any residual large peptides (molecular weight greater than 1000 Da) and trace amounts of thermally denatured polymers from the permeate, ensuring that the proportion of small peptides with a molecular weight of ≤1000 Da in the product is ≥96%. Nanofiltration fractionation: A nanofiltration membrane with a molecular weight cutoff of 150 Da is used, operating at a pressure of 1.8 MPa and a temperature of 26°C. The retentate with a molecular weight between 150 and 1000 Da is collected; this fraction is rich in peptides between 150 and 400 Da.

[0048] The permeate from nanofiltration (mainly containing free amino acids and very short peptides with a molecular weight less than 150 Da) and the retentate (150-400 Da peptides) are mixed at a volume ratio of (6-7):(2-4). If the content of a specific amino acid is low according to the amino acid test results, it is appropriately supplemented. The mixture is then filtered and sterilized to achieve the composition of soybean protein hydrolysate A. This invention does not simply use soybean hydrolysate, but rather employs an enzymatic hydrolysis process to prepare product A with a unique peptide-amino acid complex form, and supplements it during the critical metabolic window.

[0049] The soybean protein hydrolysate A described in Examples 1-5 is a complex obtained using the above-described process. The complex comprises peptide A and free amino acid solution A, wherein peptide A comprises 30-35% by mass, free amino acid solution A comprises 55-65% by mass, and water and other components comprise 5-15%. Peptide A encompasses peptides of all molecular weights, and free amino acid solution A refers to a single amino acid molecule without peptide bonds.

[0050] Preferably, the small molecule peptides with a molecular weight of less than or equal to 1000 Da account for ≥96% of the total mass of peptide A; the free amino acid solution A includes glutamic acid, aspartic acid, valine or other amino acids, and the mass percentages of glutamic acid, aspartic acid and valine in the total mass of free amino acid solution A are 10-12%, 7-8.5% and 3-3.5%, respectively.

[0051] More preferably, in peptide A, peptides with a molecular weight less than 150 Da account for 21-25% of the total mass of peptide A, and peptides with a molecular weight of 150-400 Da account for 45-55% of the total mass of peptide A.

[0052] More preferably, the peptides with a molecular weight less than 150 Da account for 23.68% of the total mass of peptide A, and the peptides with a molecular weight of 150-400 Da account for 49.87% of the total mass of peptide A; the percentages of glutamic acid, aspartic acid, and valine in the total mass of free amino acid solution A are 11.89%, 7.40%, and 3.28%, respectively.

[0053] In specific embodiments and comparative examples:

[0054] The permeate and retentate from the 150 Da nanofiltration process were mixed at a volume ratio of 7:3. The free amino acid composition was analyzed. If insufficient, specific components such as glutamic acid, aspartic acid, and valine were added to reach the target range, followed by filtration and sterilization. In the soybean protein hydrolysate A described in Examples 1-5, peptide A accounted for 32% by mass, free amino acid solution A accounted for 60% by mass, and water and other specific components accounted for 8%.

[0055] Preferably, in peptide A, the molecular weight of small molecule peptides less than or equal to 1000 Da accounts for 96% of the total mass of peptide A;

[0056] In peptide A, peptides with a molecular weight less than 150 Da accounted for 23.68% of the total mass of peptide A, and peptides with a molecular weight between 150 and 400 Da accounted for 49.87% of the total mass of peptide A. The percentages of glutamic acid, aspartic acid, valine, and tyrosine in the total mass of free amino acid solution A were 11.89%, 7.40%, 3.28%, and 1.73%, respectively. Other amino acids in the free amino acid solution A included threonine (2.67%), serine (2.62%), glycine (6.19%), alanine (4.27%), cysteine ​​(0.35%), methionine (1.13%), isoleucine (2.86%), leucine (4.26%), phenylalanine (2.56%), lysine (4.09%), histidine (1.55%), arginine (4.75%), proline (5.02%), with the remainder being water.

[0057] The specific implementation examples and comparative examples have the following basic parameters:

[0058] The following examples and comparative examples are all based on a 50L fermenter system, with consistent core fermentation parameters: 30% *Streptomyces lincosinate* inoculum, 220 h fermentation period, 30 ± 1℃ temperature, pH 6.6 ± 0.1, stirring speed 600 rpm, and air flow rate 2000 L / h. The fermentation medium formula is standardized.

[0059] Starch 0.6%, cold-pressed soybean flour 2.7%, sodium citrate 0.4%, ammonium sulfate 0.4%, potassium dihydrogen phosphate 0.02%, sodium nitrate 0.72%, calcium chloride 0.6%, glucose 5.6%, corn steep liquor powder 1.4%, defoamer 0.05%.

[0060] Example 1

[0061] Hydrolysate A replenishment parameters: 0.08% soybean protein hydrolysate A was added at 138 h during the experiment. At 220 h, the concentration of component A was 13296 μg / mL, and the concentration of component B was 188 μg / mL, with the proportion of component B being 1.39%.

[0062] Example 2

[0063] Hydrolysate A replenishment parameters: 0.07% soybean protein hydrolysate A was added at 154 h during the experiment. At 220 h, the concentration of component A was 12114 μg / mL, and the concentration of component B was 182 μg / mL, with the proportion of component B being 1.48%.

[0064] Example 3

[0065] Hydrolysate A replenishment parameters: 0.05% soybean protein hydrolysate A was added at 162 h during the experiment. At 220 h, the concentration of component A was 13343 μg / mL, and the concentration of component B was 166 μg / mL, with the proportion of component B being 1.23%.

[0066] Example 4

[0067] Hydrolysate A replenishment parameters: 0.06% soybean protein hydrolysate A was added at 170 h during the experiment. At 220 h, the concentration of component A was 11969 μg / mL, and the concentration of component B was 187 μg / mL, with the proportion of component B being 1.53%.

[0068] Example 5

[0069] Hydrolysate A replenishment parameters: 0.10% soybean protein hydrolysate A was added at 180 h during the experiment. At 220 h, the concentration of component A was 12569 μg / mL, and the concentration of component B was 195 μg / mL, with the proportion of component B being 1.52%.

[0070] Comparative Example 1

[0071] Based on Example 3, no soybean protein hydrolysate A was added throughout the experiment. During fermentation, the concentration of component B continuously increased. At 220 h, component A was 12286 μg / mL and component B was 679 μg / mL, with a component B ratio of 5.24%. The results indicate that, without any adjustments, component B significantly exceeded the pharmacopoeia standard.

[0072] Comparative Example 2

[0073] Based on Example 3, 0.05% yeast powder solution was added at the same time point. After 220 h, the concentration of component A in the tank was 12783 μg / mL, and that of component B was 513 μg / mL, with a proportion of 3.86%. Due to the disordered amino acid composition of the yeast powder, the effect was poor.

[0074] Comparative Example 3

[0075] Based on Example 3, at the same time point, 0.05% fish peptone solution was added. After 220 h, the concentration of component A was 13011 μg / mL, and the concentration of component B was 369 μg / mL, with the proportion of component B being 2.76%.

[0076] Comparative Example 4

[0077] Based on Example 3, 0.05% of hydrolyzed soybean protein solution from Zhongguang Biotechnology Co., Ltd. was added at the same time point. After 220 h, the concentration of component A was 12627 μg / mL, and the concentration of component B was 343 μg / mL, with the proportion of component B being 2.64%.

[0078] Comparing Comparative Example 4 with Example 3, it can be seen that not all soybean-derived hydrolysates can achieve the same effect.

[0079] Comparative Examples 2-4, which used yeast powder, fish peptone, and commercially available hydrolyzed soybean protein liquid, all showed significantly inferior results compared to this invention. This demonstrates that not all organic nitrogen sources are effective.

[0080] Comparative Example 5

[0081] Based on Example 3, 0.05% soybean protein hydrolysate A was added 100 h before (90 h). At 220 h, the concentration of component A in the tank was 11736 μg / mL, and the concentration of component B was 297 μg / mL, with the proportion of component B being 2.47%.

[0082] Comparative Example 6

[0083] Based on Example 3, 0.05% soybean protein hydrolysate A was added after 200 h (210 h). At 220 h, the concentration of component A in the tank was 12783 μg / mL, and the concentration of component B was 466 μg / mL, with the proportion of component B being 3.52%.

[0084] Comparative Examples 5-6 showed significantly reduced effects when nutrients were added either too early (90h) or too late (210h) during fermentation. This confirms that precise nutrient regulation is crucial during the critical window of 130-190h for the synthesis of this primary metabolite; adding nutrients too early or too late will cause the optimal regulatory opportunity to be missed.

[0085] Comparative Example 7

[0086] Based on Example 3, at the same time point (162 h), a corresponding proportion (0.05%) of soybean protein hydrolysate B was added. Soybean protein hydrolysate B contained 20% peptides, 40% free amino acids, and 40% water. The peptide and amino acid composition was the same as soybean protein hydrolysate A. At 220 h, the concentration of component A was 12786 μg / mL, and component B was 357 μg / mL, with a proportion of 2.72%. This indicates that even with the same amino acid and peptide composition, insufficient total μg / mL content of peptides and amino acids cannot achieve the desired effect.

[0087] Preparation of hydrolysate B: Enzymatic hydrolysis, ultrafiltration, and 150Da nanofiltration fractionation are performed using the same standard process. Permeate (amino acid-short peptide fraction) and retentate (150-1000Da peptides) are separated. The permeate and retentate are mixed at a ratio of 7:3 and diluted with deionized water to reduce the peptide concentration in the mixture from 32% to 20% and the free amino acid concentration from 60% to 40% (the relative ratio of glutamic acid, aspartic acid, valine, and tyrosine remains 11.89:7.40:3.28:1.73). No additional amino acids are required.

[0088] Comparative Example 8

[0089] Based on Example 3, soybean protein hydrolysate D in the corresponding proportion (0.05%) was added at the same time point (162 h). After 220 h, the concentration of component A was 12458 μg / mL, and the concentration of component B was 252 μg / mL, with the proportion of component B being 1.98%.

[0090] The specific preparation steps for hydrolysate D are exactly the same as those for hydrolysate A. The difference is that a 300Da nanofiltration membrane (2.0MPa / 26℃) is used instead of a 150Da membrane for fractionation. The amino acid loss in the permeate is 30%, and the peptide retention rate in the retentate is 95%. After mixing, an additional 1.2kg / 100L of glutamic acid needs to be added to reach the target ratio because the 300Da membrane retains less than 150Da short peptides, resulting in insufficient amino acid-short peptide components.

[0091] Comparative Example 9

[0092] Based on Example 3, soybean protein hydrolysate E in the corresponding proportion (0.05%) was added at the same time point (162 h). After 220 h, the concentration of component A was 12915 μg / mL, and the concentration of component B was 248 μg / mL, with the proportion of component B being 1.89%.

[0093] A low percentage of segments weakens the synergistic effect.

[0094] The specific preparation steps of hydrolysate F are completely consistent with the standard process of hydrolysate A, including enzymatic hydrolysis and ultrafiltration fractionation. The difference from hydrolysate A is that the 150Da nanofiltration permeate and retentate are mixed at an 8:2 ratio (the standard is 7:3). The test revealed that valine was only 2.9%. After adding 0.05kg / 100L, the content was increased to 3.28%, resulting in a final peptide content of 28% and a free amino acid content of 62%. The peptide content was too low and the amino acid content was excessive, which weakened the synergistic effect between the two.

[0095] Comparative Example 10

[0096] Based on Example 3, soybean protein hydrolysate F was added at the same time point (162 h) in a corresponding proportion (0.05%). After 220 h, the concentration of component A was 12678 μg / mL, and the concentration of component B was 264 μg / mL, with the proportion of component B being 2.04%. Valine did not reach the threshold and could not inhibit the synthesis of component B.

[0097] The specific preparation steps of hydrolysate G are completely consistent with the standard process of hydrolysate A. The difference from hydrolysate A is that after fractionation and mixing at 150 Da, only glutamic acid (11.92%) and aspartic acid (7.45%) were detected, while valine (2.9%) and tyrosine (1.3%) were not supplemented.

[0098] Comparative Example 11

[0099] Based on Example 3, 0.05% of free amino acid solution B was added at the same time point (162 h). Free amino acid solution B was a mixture of glutamic acid, aspartic acid, valine, and tyrosine, with a mass percentage of 60%, the remainder being water. The mass ratio of glutamic acid, aspartic acid, and valine was 11.89:7.4:3.28:1.73, containing only these three amino acids, without peptides or other substances. At 220 h, the concentration of component A was 12580 μg / mL, and component B was 312 μg / mL, with a proportion of 2.42%. The system containing only fast-acting amino acids lacked the sustained-release effect of peptides, resulting in poor regulation of release duration.

[0100] Comparative Example 12

[0101] Based on Example 3, a corresponding proportion (0.05%) of small molecule peptide protein solution A was added at the same time point (162 h). After 220 h, the concentration of component A was 13028 μg / mL, and the concentration of component B was 225 μg / mL, with a proportion of 1.70%. Due to the lack of amino acid initiation signals, the onset of action was slow.

[0102] The specific preparation procedure for small molecule peptide protein solution A is as follows: First, obtain a 150Da nanofiltration retentate (150-1000Da peptides) according to the standard process of hydrolysate A, and then deacidify it by circulating it through a 300Da nanofiltration system at 2.0MPa / 26℃ (solution:water = 1:5) until the amino acid content of the permeate is ≤0.1g / L, and then concentrate it to 32% peptides and ≤0.5% free amino acids.

[0103] Comparative Example 13

[0104] Based on Example 3, soybean protein hydrolysate H was added at the same time point (162 h) in a corresponding proportion (0.05%). After 220 h, the concentration of component A was 12583 μg / mL, and the concentration of component B was 216 μg / mL, with the proportion of component B being 1.69%.

[0105] Soybean protein hydrolysate H: Small molecule peptide protein solution A (32%) and free amino acid solution B (60%) were mixed, and water (8%) was added to prepare a system consistent with component A. The mixture was stirred at room temperature for 10 min (300 rpm) to obtain soybean protein hydrolysate H. Physical mixing disrupted the peptide-amino acid complex formed in situ during enzymatic hydrolysis, resulting in decreased cell absorption efficiency. Even though the final peptide and amino acid composition and content were completely consistent with hydrolysate A, the product obtained through physical mixing still showed significantly worse regulatory effects than that of Example 3. This demonstrates that the specific peptide-amino acid complex formed in situ during enzymatic hydrolysis is crucial for efficient cell absorption and metabolic regulation, and cannot be replaced by simple composition alone.

[0106] Comparative Example 14

[0107] Based on Example 3, a corresponding proportion (0.05%) of soybean protein hydrolysate C was added at the same time point (162 h).

[0108] Hydrolysate C contains 5% glutamic acid, 3% aspartic acid, 2% valine, and 1.5% tyrosine, with a peptide distribution consistent with hydrolysate A. After 220 h, the concentration of component A was 12762 μg / mL, and component B was 209 μg / mL, representing 1.61% of the total. The lack of key amino acids hinders the guidance of metabolic pathways.

[0109] Preparation of hydrolysate C: After diluting the reference mixture of hydrolysate A, alanine was added as a filler amino acid to make the total free amino acid 60% (alanine accounts for 48.5%), and the peptide distribution was the same as that of A.

[0110] As can be seen from the above examples and comparative examples, compared with no nitrogen source added, or adding other nitrogen sources or other soybean protein hydrolysates, the addition of a specific concentration of soybean protein hydrolysate at a specific time in this invention can significantly reduce the content of component B in the lincomycin fermentation process and increase the potency of lincomycin A. Soybean protein hydrolysate is rich in amino acids, among which glutamic acid, as a readily available nitrogen source, can be rapidly absorbed and utilized by the cells, participating in protein synthesis and purine and pyrimidine metabolism; simultaneously, the transamination reaction of glutamic acid can regulate the pH of the fermentation broth, maintaining the nitrogen source required for cell growth. Valine degradation produces isobutyric acid, which participates in fatty acid synthesis and affects the cell's nitrogen metabolism balance by regulating branched-chain amino acid dehydrogenases. Although tyrosine content is low, it is also a key substance affecting component B synthesis. The specific ratio of small molecule peptides and amino acid composition affects the rapid absorption and metabolic regulation of the hydrolysate, competitively inhibiting the key enzymes in component B synthesis, thereby reducing component B to extremely low levels during fermentation, effectively inhibiting the synthesis of by-products and improving the purity of the main product. This method is worthy of widespread application.

[0111] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for reducing component B in the lincomycin fermentation process, characterized in that, Component B is (2S,4R)-4-ethyl-N-((1R,2R)-2-hydroxy-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(methylthio)tetrahydro-2H-pyran-2-yl)propyl)-1-methylpyrrolidine-2-carboxamide, with the chemical formula C 17 H 32 N2O6S, the method involves adding soybean protein hydrolysate A during fermentation to control the formation of component B; the soybean protein hydrolysate A is a complex obtained by enzymatic hydrolysis of soybean raw materials, the complex includes peptide A and free amino acid solution A; wherein, the mass percentage of peptide A is 30-35%, the mass percentage of free amino acid solution A is 55-65%, and the proportion of moisture and other components is 5-15%; The percentage of small molecule peptides with a molecular weight of less than or equal to 1000 Da in peptide A is ≥96% of the total mass of peptide A. In the free amino acid solution A, the mass percentages of glutamic acid, aspartic acid, valine, and tyrosine in the total mass of free amino acid solution A are 10-12%, 7-8.5%, 3-3.5%, and 1.6-1.8%, respectively. In peptide A, peptides with a molecular weight less than 150 Da account for 21-25% of the total mass of peptide A; in peptide A, peptides with a molecular weight of 150-400 Da account for 45-55% of the total mass of peptide A. The preparation process of soybean protein hydrolysate A includes the following steps: (1) Mix soybean meal powder and deionized water at a ratio of 1:10 w / v, stir evenly to form a suspension; Pretreatment: The suspension was preheated to 50±2°C and the pH was adjusted to 8.5±0.2 with sodium hydroxide solution and maintained for 15 minutes; (2) Enzymatic hydrolysis: Add 0.8% alkaline protease by weight of substrate, and stir continuously for 3 hours at 55±1°C and pH 8.5; adjust the pH of the solution to 6.5 with hydrochloric acid solution and cool to 50°C; then add 0.5% flavor protease by weight of substrate, and stir continuously for 1 hour at 50±1°C and pH 6.

5. (3) After the enzymatic hydrolysis is completed, the liquid is heated to 85°C and kept for 15 minutes to completely deactivate the enzyme; use a plate and frame filter or centrifuge to remove the unhydrolyzed fine soybean residue particles and macromolecular impurities to obtain a clear enzymatic hydrolysate. (4) Membrane system fractionation, including: Ultrafiltration: An ultrafiltration membrane with a molecular weight cutoff of 1000 Da is used to obtain the permeate at an operating pressure of 0.1 MPa and a temperature of 26°C. This removes residual macromolecular peptides with a molecular weight greater than 1000 Da and trace amounts of thermally denatured polymerized proteins, ensuring that the proportion of small molecule peptides with a molecular weight of less than or equal to 1000 Da in the product is ≥96%. Nanofiltration fractionation: A nanofiltration membrane with a molecular weight cutoff of 150 Da was used. The operating pressure was 1.8 MPa and the temperature was 26°C. The retentate with a molecular weight between 150 and 1000 Da was collected. This fraction is rich in peptides with a molecular weight of 150-400 Da. (5) Mix the permeate and retentate of nanofiltration in a volume ratio of (6-7):(2-4), adjust the amino acid content according to the amino acid test results, filter and sterilize to obtain soybean protein hydrolysate A.

2. The method for reducing component B in the lincomycin fermentation process according to claim 1, characterized in that, The peptides with a molecular weight less than 150 Da accounted for 23.68% of the total mass of peptide A, and the peptides with a molecular weight of 150-400 Da accounted for 49.87% of the total mass of peptide A; the percentages of glutamic acid, aspartic acid, valine, and tyrosine in the total mass of free amino acid solution A were 11.89%, 7.40%, 3.28%, and 1.73%, respectively.

3. The method for reducing component B in the lincomycin fermentation process according to claim 1, characterized in that, The amount of soybean protein hydrolysate A added is 0.05-0.10% of the fermentation liquid volume, and the soybean protein hydrolysate A is added when the fermentation has been going on for 130-190 hours.

4. The method for reducing component B in the lincomycin fermentation process according to claim 3, characterized in that, The amount of soybean protein hydrolysate A added is 0.05-0.07% of the fermentation liquid volume, and the soybean protein hydrolysate A is added 150-170 hours after fermentation.

5. The method for reducing component B in the lincomycin fermentation process according to claim 4, characterized in that, The amount of soybean protein hydrolysate A added is 0.05% of the fermentation liquid volume, and the soybean protein hydrolysate A is added when the fermentation has been going on for 162 hours.

6. The method for reducing component B in the lincomycin fermentation process according to claim 1, characterized in that, The fermentation strain is *Streptomyces lincosae*, with an inoculum size of 20-40%. The fermentation medium used in the method comprises the following components by mass fraction: starch 0.5-1.0%, soybean flour 2.0-3.0%, sodium citrate 0.2-0.8%, ammonium sulfate 0.2-0.5%, potassium dihydrogen phosphate 0.01-0.03%, sodium nitrate 0.7-0.8%, calcium chloride 0.6-0.8%, glucose 5.0-6.0%, corn steep liquor powder 1.0-2.0%, defoamer 0.02-0.1%, and the balance being water.

7. The method for reducing component B in the lincomycin fermentation process according to claim 1, characterized in that, The fermentation process was carried out under the following conditions: fermentation cycle of 200-240 h, culture temperature of 30±1℃, pH value of 6.6±0.1, stirring speed of 150-700 rpm, and air flow rate of 1500-2400 L / h.

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