Fermentation treatment method for improving dissolution rate of medicinal ingredients of silkworm excrement

Abstract

The present application relates to a kind of fermentation treatment methods for improving the dissolution rate of silkworm sand medicinal ingredients, belong to the technical field of traditional Chinese medicinal materials processing.For the technical problems such as chloramphenicol residue and fishy smell during fermentation of silkworm sand raw materials, the fresh silkworm sand is dried and then inoculated into food two nitrogen plant bacillus for solid fermentation, the pH is maintained at 6.0-6.5 for 0-12 hours, then inoculated into lactobacillus plantarum after 12 hours, then high-pressure rinsing, then mixed with traditional Chinese medicinal materials and rice wine for secondary liquid fermentation, then filtered to obtain silkworm sand fermentation extract.The method is to hydrolyze chloramphenicol by food two nitrogen plant bacillus, adsorb heavy metal ions by lactobacillus plantarum, promote the release of medicinal ingredients during secondary fermentation process, remove fishy precursor substances by combining the first fermentation with high-pressure rinsing process, and the extract prepared has no chloramphenicol residue, low lead content and no fishy smell.The method is simple, safe and controllable, and can be used for preparing medicinal silkworm sand fermentation extract, and realizing silkworm sand resource utilization.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to a fermentation treatment method for improving the dissolution rate of medicinal components in silkworm excrement. Background Technology

[0002] Silkworm excrement is the waste product of silkworms and is also a traditional Chinese medicine included in the pharmacopoeia. It has the effects of dispelling wind and dampness, promoting blood circulation and relieving pain. It can be used to prepare Chinese medicine slices or medicinal wines. Taking medicinal wine as an example, existing silkworm excrement medicinal wine products use dried silkworm excrement combined with other Chinese medicinal materials and soaked in rice wine or white wine before use as medicinal wine. For example, patent publication number CN105687299A discloses a Poria cocos and silkworm excrement wine, in which the silkworm excrement is finely crushed and soaked in rice wine for 7 days; patent publication number CN103351986A discloses a medicinal wine that can treat rheumatic pain, in which the silkworm excrement and other Chinese medicinal materials are crushed and soaked in decoction for 48 hours, and then soaked in white wine for 1 year. The aforementioned existing technologies all employ physical soaking methods to process silkworm excrement, which presents the following technical problems: (1) Chloramphenicol and other antibiotics may remain in the silkworm excrement, but the existing soaking process does not perform any degradation or removal treatment on them; (2) The fishy odor in the silkworm excrement mainly comes from the protein decomposition products remaining in the digestive fluid of the silkworm, and the existing technology only masks it by stir-frying or by relying on the aroma of alcohol and Chinese medicinal herbs, failing to fundamentally remove the fishy odor; (3) The physical soaking method has limited dissolution efficiency for medicinal components such as flavonoids in the silkworm excrement, and the soaking time is long but the transfer rate of effective components is not high. As the above technical problems have not been solved for a long time, the processing quality and medication safety of silkworm excrement have been limited. Summary of the Invention

[0003] One objective of this invention is to solve the following problems: when silkworm excrement is used as a fermentation raw material, there are technical problems such as antibiotic residues such as chloramphenicol, low dissolution rate of flavonoid active ingredients, and fishy odor.

[0004] To achieve the above objectives, the present invention provides a fermentation treatment method for improving the dissolution rate of medicinal components in silkworm excrement, comprising the following steps: collecting fresh silkworm excrement and air-drying it, then placing it into a primary fermentation container, and immediately inoculating it with *Bacillus diazoxide* at the start of fermentation, with an inoculation amount of (0.5-1.5) × 10⁻⁶. 8 CFU / g, and carried out a single solid-state fermentation at 40-50℃; 12 hours after the start of the first fermentation, *Lactobacillus plantarum* was inoculated at an inoculum size of (0.5-1.5) × 10⁻⁶. 9CFU / g; During the primary fermentation, after inoculation with *Bacillus diazoxide*, the pH of the fermentation environment was maintained at 6.0-6.5 for 0-12 hours; After the primary fermentation, the silkworm excrement was subjected to high-pressure rinsing, with the rinsing liquid volume being 3-5 times the mass of the silkworm excrement, the rinsing time being 15-30 minutes, and the rinsing pressure being 0.3-0.5 MPa, and then air-dried; The air-dried silkworm excrement was mixed with Chinese medicinal materials and pure grain rice wine, and the initial alcohol content of the secondary fermentation was controlled at 15-18% (V / V), and the secondary liquid fermentation was carried out at 28-30℃ for 72-96 hours; After the secondary fermentation, the mixture was filtered, and the filtrate was collected to obtain the silkworm excrement fermentation extract.

[0005] Preferably, during the primary fermentation process, the pH of the fermentation environment is maintained at 6.0-6.5 by adding acid or alkali for 0-12 hours.

[0006] Preferably, the carbon-nitrogen ratio is set to 15-20:1 in the secondary liquid fermentation stage, where the carbon-nitrogen ratio refers to the mass ratio of total organic carbon to total Kjeldahl nitrogen in the secondary fermentation feed.

[0007] Preferably, within 0-24 hours after the start of primary fermentation, silkworm excrement is enzymatically hydrolyzed using amide bond hydrolase secreted by *Bacillus diazoniflorus*, at a hydrolysis temperature of 40-50°C and a hydrolysis time of 24 hours.

[0008] Preferably, the fermentation system is not separated in the secondary fermentation stage. Instead, the silkworm excrement after the primary fermentation is directly mixed with Chinese medicinal herbs and pure grain rice wine before entering the secondary liquid fermentation stage.

[0009] Preferably, after the secondary fermentation, the bacterial cells are separated by membrane filtration with a pore size of 0.45 μm, and the filtrate is collected as silkworm excrement fermentation extract.

[0010] Preferably, the secondary liquid fermentation stage also includes the regulation of dissolved oxygen concentration: the dissolved oxygen concentration is controlled at 2.0-3.0 mg / L for 0-24 hours after the start of secondary fermentation; the dissolved oxygen concentration is reduced to 0.5-1.0 mg / L for 24-48 hours after the start of secondary fermentation; and the dissolved oxygen concentration is reduced to below 0.2 mg / L for 48-72 hours after the start of secondary fermentation.

[0011] Preferably, the solid-state fermentation stage further includes fermentation mash circulation and reflux: 6 hours after inoculation with *Lactobacillus plantarum*, the first circulation and reflux is initiated, the mash at the bottom of the fermentation container is extracted and sprayed onto the surface of the material, the circulation flow rate is 0.5-1.0 L / h per kilogram of material, and the circulation lasts for 2 hours; 6 hours after inoculation with *Lactobacillus plantarum*, the second circulation and reflux is initiated, the circulation flow rate is 1.5-2.0 L / h per kilogram of material, and the circulation lasts for 3 hours; the second circulation and reflux is terminated when the pH difference between any two monitoring points in the fermentation system is less than 0.15 and the temperature difference between the surface and bottom of the material is less than 2°C.

[0012] Preferably, the termination of the primary solid-state fermentation stage is determined by the following criteria: when the three conditions are met—chloramphenicol degradation rate greater than 98%, lead adsorption rate greater than 85%, and system pH value between 4.0 and 4.5—the primary solid-state fermentation stage is considered complete and the high-pressure rinsing step begins. The termination of the secondary liquid fermentation stage is determined by the following criteria: when the three conditions are met—fuse alcohol oil content less than 0.2 g / L, trimethylamine content less than 1.8 mg / L, and total flavonoid dissolution rate greater than 90%—the secondary liquid fermentation stage is considered complete and the filtration step begins. If the above conditions are not met, the fermentation is extended and re-determined in 12-hour cycles, and fermentation is terminated when the total fermentation time reaches 120 hours.

[0013] Preferably, during the 0-12 hours of the first fermentation, calcium carbonate micropowder and dipotassium hydrogen phosphate are added to the fermentation system. The particle size of the calcium carbonate micropowder is 20-50 μm, and the addition amount is 0.5%-1.0% of the mass of silkworm excrement. The addition amount of dipotassium hydrogen phosphate is 0.2%-0.4% of the mass of silkworm excrement. The calcium carbonate micropowder is mixed and ground with the silkworm excrement raw material at a mass ratio of 1:5 before addition. The dipotassium hydrogen phosphate is added in three batches: 50% of the total amount at 0 hours, 25% at 4 hours, and 25% at 8 hours. The pH value of the fermentation system is measured before each addition. When the pH value deviates from the range of 6.0-6.5, the addition of that batch is triggered.

[0014] Preferably, after the first fermentation and before high-pressure rinsing, the silkworm excrement fermentation product undergoes gradient ethanol acclimation: the silkworm excrement fermentation product is successively soaked in a 5% ethanol aqueous solution, a 10% ethanol aqueous solution, and a 15% ethanol aqueous solution. During each soaking process, the conductivity change rate of the fermentation mash is monitored in real time. When the conductivity change rate is less than 0.5 μS / (cm•min) for 5 consecutive minutes, the acclimation for that stage is considered complete and the next stage is started. The temperature for the 5% ethanol stage is 10-15℃, the temperature for the 10% ethanol stage is 20-25℃, and the temperature for the 15% ethanol stage is 28-30℃. After the three stages of acclimation are completed, the silkworm excrement fermentation product is left to stand in sterile air at 28-30℃ for 15-30 minutes before proceeding to the high-pressure rinsing step.

[0015] Preferably, after the secondary fermentation and before membrane filtration, calcium chloride solution is added to the fermentation mash to a final concentration of 0.05-0.1 mol / L, stirred evenly, and then sodium alginate is added. The amount of sodium alginate added is 0.1%-0.3% of the mass of the fermentation mash. After the sodium alginate is added, two-stage stirring is performed: the stirring rate is 150-200 rpm for the first 10 minutes, and then the stirring rate is reduced to 50-80 rpm for the next 10-40 minutes. After stirring, the mixture is allowed to stand, and the pH value of the fermentation mash is monitored in real time during the standing process. When the pH value drops below 3.8, the standing process is terminated and the membrane filtration step begins.

[0016] Preferably, when performing high-pressure rinsing after primary fermentation, a citric acid-sodium citrate buffer system is used to prepare the high-pressure rinsing solution, with a citric acid concentration of 0.05-0.1 mol / L. The pH of the rinsing solution is adjusted to 3.5-4.0 by adjusting the ratio of citric acid to sodium citrate. During high-pressure rinsing, an online pH monitoring point is set in the rinsing solution circulation loop. When the pH of the returned rinsing solution is higher than 4.0, 0.5 mol / L citric acid solution is added to the rinsing solution to bring the pH back to the 3.5-4.0 range. High-pressure rinsing is terminated when the pH of the rinsing solution is stable within the range of 4.0±0.1 for 5 consecutive minutes.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention provides a suitable working environment for the microbial degradation of chloramphenicol by inoculating *Bacillus diazoxide* at the start of fermentation and maintaining the pH at 6.0-6.5 for 0-12 hours; after 12 hours, *Lactobacillus plantarum* is inoculated, and the functional groups on the cell wall of the bacteria bind to free lead, chromium and other heavy metal ions in the fermentation system through electrostatic interaction, thereby simultaneously achieving the enzymatic hydrolysis of antibiotics and the adsorption and fixation of heavy metal ions in a single fermentation system. Experimental results show that chloramphenicol residues were not detected in the silkworm excrement fermentation extract prepared by the technical solution of this invention (detection limit 0.1 μg / kg), and the lead content was significantly lower than that of the sample not treated by the method of this invention.

[0019] (2) In this invention, within 0-24 hours after the start of the first fermentation, the amide bond hydrolase secreted by *Bacillus diazoxide* hydrolyzes the residual protein-like odor precursors in silkworm excrement, breaking down large odor-causing proteins into odorless small peptides and free amino acids. Subsequently, a high-pressure rinsing process washes these hydrolysate products out of the silkworm excrement solid phase, fundamentally reducing the source of odorous substances. Experimental results show that the trimethylamine content in the silkworm excrement fermentation extract prepared using the technical solution of this invention is controlled below 1.8 mg / L, and the odor is significantly improved.

[0020] (3) In this invention, the carbon-to-nitrogen ratio of the secondary fermentation ingredients is set to 15-20:1. *Lactobacillus plantarum* preferentially consumes valine, leucine, and isoleucine for its own proliferation, reducing the flux of these amino acids entering the Ehrlich metabolic pathway to generate fusel oil precursors. Simultaneously, the lactic acid produced by *Lactobacillus plantarum* metabolism lowers the pH of the fermentation environment, which has a certain inhibitory effect on the activity of α-keto acid decarboxylase in yeast. Experimental results show that the fusel oil content in the silkworm excrement fermentation extract prepared using the technical solution of this invention is less than 0.2 g / L, significantly lower than that of samples prepared using conventional soaking methods.

[0021] (4) In the secondary fermentation stage, this invention does not perform solid-liquid intermediate separation of the fermentation system, allowing the open-ring carboxylic acid intermediates produced by *Bacillus diazoxide* during the degradation of chloramphenicol to remain in the fermentation system. These amphiphilic molecules can reduce the interfacial tension between the silkworm excrement cell wall and the fermentation broth, facilitating the transfer of flavonoids and alkaloids from the solid phase to the liquid phase of the silkworm excrement. Experimental results show that the total flavonoid content in the silkworm excrement fermentation extract prepared using the technical solution of this invention is significantly higher than that of the sample prepared using conventional soaking methods, and the total flavonoid dissolution rate can reach over 90%.

[0022] (5) By establishing a primary fermentation termination rule based on chloramphenicol degradation rate, lead adsorption rate and pH value as the criteria, and a secondary fermentation termination rule based on fusel oil content, trimethylamine content and total flavonoid dissolution rate as the criteria, the fermentation endpoint is changed from relying on the operator's experience to being based on actual measured data. When the criteria are not met, the fermentation is corrected by periodically extending the fermentation, which helps to reduce the quality fluctuation between different batches of products.

[0023] (6) This invention regulates dissolved oxygen concentration through a time-sequential gradient, maintaining a high dissolved oxygen level for the first 24 hours to support the aerobic degradation activity of *Lactobacillus plantarum*, and gradually reducing dissolved oxygen after 24 hours to promote homolactic fermentation of *Lactobacillus plantarum*. By implementing reflux during the primary fermentation stage, the internal temperature gradient of the material is eliminated, promoting the uniform distribution of the strain within the material system. Furthermore, by using a citric acid-sodium citrate buffer rinse solution, the difference in acid dissociation constants between the two types of intermediates is utilized to achieve selective retention of the target solubilizing intermediates, while simultaneously removing undesirable flavor precursors with the rinse solution. These combined techniques enhance the safety and quality of the silkworm excrement fermentation extract.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description. It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the reagents and materials are commercially available unless otherwise specified.

[0026] The *Phytobacter diazotrophicus* strain of this invention is BNCC382498 from Beina Biotechnology. When cultured on LB medium at 30°C for 24 hours, this strain forms milky-white, round colonies with a diameter of 1-3 mm. It is Gram-negative, oxidase-negative, and catalase-positive. *Lactobacillus plantarum* ATCC 8014 is a Gram-positive, facultative anaerobic bacillus. When statically cultured on MRS medium at 37°C, it forms milky-white, round colonies with a diameter of 1-2 mm, producing lactic acid that lowers the pH of the medium to below 4.0.

[0027] Example 1

[0028] Take 100 kg of fresh silkworm excrement and spread it out in a cool, ventilated place to air-dry until the moisture content is 12%-15% (mass fraction). Put the air-dried silkworm excrement into a primary fermentation container equipped with a jacketed heating system. Circulate 45-50℃ hot water through the jacket to maintain the material temperature at 40-50℃. During fermentation, turn the material with a mechanical turner every 4 hours for 5 minutes to maintain oxygen supply. Immediately at the start of fermentation, inoculate with a seed culture of *Bacillus diazoxide* BNCC382498 at an inoculation rate of 1×10⁻⁶ kg / kg of dry silkworm excrement. 8 CFU / g. The fermentation treatment method for BNCC382498 seed culture was as follows: BNCC382498 was inoculated into LB liquid medium and cultured at 30℃ with shaking at 180 rpm until OD. 600 =0.8-1.0 (mid-logarithmic growth phase), collect the bacterial cells by centrifugation at 8000×g for 10 minutes at 4℃, wash twice with sterile physiological saline, and resuspend to 1 / 10 of the original volume.

[0029] During the first 0-12 hours after the start of primary fermentation, the pH of the fermentation environment was monitored using an online pH electrode. When the pH deviated from the 6.0-6.5 range, a peristaltic pump automatically added 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to bring the pH back into this range. Twelve hours after the start of primary fermentation, *Lactobacillus plantarum* ATCC 8014 seed culture was inoculated at a density of 1×10⁻⁶. 9 CFU / g. The fermentation treatment method for Lactobacillus plantarum ATCC 8014 seed culture was as follows: Lactobacillus plantarum was inoculated into MRS liquid medium and incubated statically at 37°C until OD. 600 =0.6-0.8, centrifuge to collect the bacterial cells and resuspend. After inoculation with Lactobacillus plantarum, the primary fermentation system naturally acidifies due to lactic acid accumulation, without the need for additional acid addition.

[0030] The fermentation process lasts for 48 hours. The fermented silkworm excrement is then transferred to a high-pressure rinsing tank. Clean water, at a ratio of 3 times the weight of the silkworm excrement, is used as the rinsing solution, and the mixture is rinsed for 15 minutes at a pressure of 0.2-0.3 MPa. After rinsing, the silkworm excrement is spread out to air dry until the moisture content reaches 12%-15%.

[0031] The air-dried silkworm excrement was mixed with Chinese medicinal herbs, including chrysanthemum, wolfberry, and rehmannia root, in a mass ratio of 2:1:1. The total amount of medicinal herbs added was 10% of the mass of the silkworm excrement. Then, pure grain rice wine (made from rice as raw material, using small-grain yeast as saccharification and fermentation agent, through semi-solid fermentation and distillation, with an alcohol content of 55% V / V) was added. The mixture was adjusted with purified water to control the initial alcohol content of the secondary fermentation at 15% V / V. The mixture was then transferred to a fermentation tank equipped with a jacketed cooling system and dissolved oxygen electrodes, and secondary liquid fermentation was carried out at 28°C for 72 hours. During the secondary fermentation, 28°C cooling water was circulated through the jacket to maintain the fermentation temperature.

[0032] After the secondary fermentation is completed, the mixture is filtered using a plate and frame filter press with a filter cloth pore size of 0.45 μm, and the filtrate is collected to obtain the silkworm excrement fermentation extract.

[0033] The obtained silkworm excrement fermentation extract can be used directly as a raw material for traditional Chinese medicine preparations. To further reduce the microbial load, the collected filtrate can be transferred to a sterilization tank equipped with a jacketed heating device. Hot water is circulated through the jacket to raise the filtrate temperature to 65-70℃, and pasteurization is performed for 30 minutes. During sterilization, the mixture is slowly stirred at 30 rpm to ensure uniform temperature distribution, with the temperature deviation controlled within ±1℃. After sterilization, cooling water is circulated to cool the filtrate to room temperature. Both the extract before and after sterilization can be used as raw materials for traditional Chinese medicine tablets. For example, the alcohol content of the extract can be adjusted to 38% V / V using pure grain rice wine or purified water, colored to a dark red with caramel coloring, and then bottled to obtain a silkworm excrement-based traditional Chinese medicine wine.

[0034] Example 2

[0035] Based on Example 1, the carbon-to-nitrogen ratio was set to 15:1 during the secondary liquid fermentation stage. The carbon-to-nitrogen ratio was controlled as follows: during secondary fermentation, a sample of the mixed material was taken, and the total carbon and total nitrogen contents were measured. Adjustments were then made by adding sucrose (for carbon supplementation) or ammonium sulfate (for nitrogen supplementation). The total carbon content was determined using the potassium dichromate oxidation method: 0.5 g of the dried and pulverized mixed material sample was taken, and 5 mL of 0.8 mol / L potassium dichromate-sulfuric acid solution was added. The mixture was heated in an oil bath at 170-180℃ for 5 minutes for oxidation. After cooling, the remaining potassium dichromate was titrated with ferrous sulfate standard solution using o-phenanthroline as an indicator. The organic carbon content was calculated based on the amount of potassium dichromate consumed. To mitigate interference from volatile carbon sources such as ethanol in the material, the sample was vacuum dried to constant weight at 60℃ before measurement to remove volatile components and ensure that the measurement results reflected the non-volatile organic carbon content. The total nitrogen content was determined using the Kjeldahl method (GB 5009.5-2016). Based on the measured carbon-to-nitrogen ratio, add the appropriate amount of material to achieve a carbon-to-nitrogen ratio of 15:1. The fermentation temperature is controlled at 28°C. The remaining operating steps are the same as in Example 1.

[0036] Example 3

[0037] Based on Example 1, the dissolved oxygen concentration was further controlled temporally during the secondary liquid fermentation stage. An optical dissolved oxygen electrode (measuring range 0-20 mg / L, accuracy ±0.1 mg / L) was installed on the fermenter. The stirring speed (50-200 rpm) and aeration rate (0.1-1.0 vvm, where vvm represents the volume of gas introduced per minute per volume of fermentation broth) were interlocked and adjusted via a programmable logic controller (PLC) to achieve automatic control of the dissolved oxygen concentration. The timing was set as follows: 0-24 hours after the start of secondary fermentation, the dissolved oxygen concentration was controlled at 2.0 mg / L; 24-48 hours, the dissolved oxygen concentration was reduced to 0.5 mg / L; 48-72 hours, the dissolved oxygen concentration was reduced to below 0.2 mg / L. If the secondary fermentation time exceeded 72 hours, the dissolved oxygen concentration was maintained below 0.2 mg / L until the fermentation ended. The remaining operating steps were the same as in Example 1.

[0038] Example 4

[0039] Based on Example 1, a fermentation mash circulation reflux was added during the first solid-state fermentation stage. A drain outlet with a filter was installed at the bottom of the fermentation vessel. A peristaltic pump extracted the bottom mash and evenly sprayed it onto the material surface via a top spray pipe. The first circulation reflux was initiated 6 hours after BNCC382498 was introduced, with a circulation flow rate of 0.5 L / h per kilogram of material, and automatically stopped after 2 hours. The second circulation reflux was initiated 6 hours after the introduction of *Lactobacillus plantarum*, with the circulation flow rate increased to 1.5 L / h per kilogram of material, and continued for 3 hours. Three pH monitoring points (one each in the upper, middle, and lower layers, using needle-type pH microelectrodes) and three temperature monitoring points (one each in the surface, center, and bottom, using Pt100 resistance thermometers) were set in the fermentation material, with data acquisition frequency of once per minute. The second circulation reflux was terminated when the pH difference between any three monitoring points was less than 0.15 and the temperature difference between the surface and bottom of the material was less than 2°C; the circulation pump automatically stopped when both conditions were met. The remaining operating steps are the same as in Example 1.

[0040] Example 5

[0041] Based on Example 1, the termination of the first solid-state fermentation stage is determined by the following rules: when the three conditions of chloramphenicol degradation rate is greater than 98%, lead adsorption rate is greater than 85%, and system pH value is between 4.0 and 4.5 are met simultaneously, the first solid-state fermentation stage is determined to be over and the high-pressure rinsing step is initiated.

[0042] The method for determining the degradation rate of chloramphenicol was as follows: 5 g of silkworm excrement samples before and after fermentation were taken, 20 mL of ethyl acetate was added, and the mixture was extracted by sonication for 15 minutes. The supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and the chloramphenicol content was determined by high-performance liquid chromatography (HPLC) (column: C18, 4.6 × 250 mm, 5 μm; mobile phase: methanol-water = 40:60; detection wavelength: 278 nm; flow rate: 1.0 mL / min; injection volume: 20 μL). The degradation rate was calculated as: (content before fermentation - content after fermentation) / content before fermentation × 100%.

[0043] The method for determining the lead adsorption rate is as follows: Take 5 g of silkworm excrement samples before and after fermentation, add 20 mL of 0.1 mol / L nitric acid solution, shake and extract for 30 minutes, centrifuge and collect the supernatant, and determine the lead content using graphite furnace atomic absorption spectrophotometry (atomicing temperature 1800℃, ashing temperature 800℃). Adsorption rate = (lead content before fermentation - lead content after fermentation) / lead content before fermentation × 100%.

[0044] The fermentation time for a single fermentation session is a maximum of 72 hours. If the above three conditions are not simultaneously met within 72 hours, the fermentation is terminated and the batch is recorded as an abnormal batch. The remaining operating steps are the same as in Example 1.

[0045] Example 6

[0046] Based on Example 1, the termination of the secondary liquid fermentation stage is determined by the following criteria: when the three conditions are met simultaneously, the secondary liquid fermentation stage is determined to be over and the filtration step begins.

[0047] The method for determining the content of fusel oil is as follows: Take 100 mL of fermentation broth sample and determine the contents of isoamyl alcohol, isobutanol, and n-propanol by gas chromatography (GC) (chromatographic column: DB-WAX, 30 m × 0.25 mm × 0.25 μm; column temperature: 40℃ for 3 min, then increased to 120℃ at 5℃ / min, then increased to 230℃ at 20℃ / min for 5 min; detector: FID, 250℃; injection port: 220℃; carrier gas: nitrogen, 1.0 mL / min; split ratio: 20:1). The fusel oil content is calculated as the sum of the contents of the three higher alcohols.

[0048] The trimethylamine content was determined using the picric acid-trimethylamine salt colorimetric method: 50 mL of silkworm excrement fermentation extract was taken, and 20 mL of 10% trichloroacetic acid solution was added to precipitate the protein. After mixing, the mixture was distilled in a 60℃ water bath, collecting approximately 30 mL of distillate, which was then diluted to 50 mL with purified water. 5 mL of the distillate was placed in a 60 mL separatory funnel, and 5 mL of 10% trichloroacetic acid solution, 3 mL of saturated potassium carbonate solution, and 10 mL of toluene were added sequentially. The mixture was capped, shaken, and extracted for 2 minutes. After separation, 5 mL of the toluene layer was collected, and 2 mL of 0.02% picric acid-toluene solution was added. The mixture was shaken, allowed to stand for 10 minutes, and the absorbance was measured at 410 nm. A standard series of 0, 0.5, 1.0, 2.0, 3.0, and 5.0 mg / L was prepared using trimethylamine hydrochloride standards. A standard curve was plotted following the same procedure, and the trimethylamine content in the sample was calculated based on the standard curve. The trimethylamine content of less than 1.8 mg / L was used as the criterion for a fishy odor to be acceptable. This threshold was determined by statistical analysis of 10 batches of silkworm excrement fermentation extract samples that passed the sensory evaluation in advance: mean 1.2 mg / L, standard deviation 0.3 mg / L, mean + 2 times standard deviation = 1.8 mg / L.

[0049] The method for determining the total dissolution rate of flavonoids is as follows: Take 5 g of silkworm excrement samples before and after fermentation, and extract with 70% ethanol solution at 60℃ using ultrasound for 30 minutes. Repeat the extraction twice, combine the extracts, and dilute to 50 mL. Take 2 mL of the extract, and use the sodium nitrite-aluminum nitrate colorimetric method. Add 0.3 mL of 5% sodium nitrite solution and react for 6 minutes, then add 0.3 mL of 10% aluminum nitrate solution and react for 6 minutes. Add 2 mL of 1 mol / L sodium hydroxide solution, and dilute to 10 mL with 70% ethanol. Measure the absorbance at 510 nm. Plot a standard curve using rutin as a standard to calculate the total flavonoid content. Dissolution rate = (Total flavonoid content in silkworm excrement before fermentation - Total flavonoid content in silkworm excrement after fermentation) / Total flavonoid content in silkworm excrement before fermentation × 100%.

[0050] The secondary fermentation time is a maximum of 96 hours. If the above three conditions are not simultaneously met within 96 hours, the time is extended according to the rules of claim 9, and the determination is repeated every 12 hours. The remaining operation steps are the same as in Example 1.

[0051] Example 7

[0052] Based on Example 1, all the technical means employed in Example 3 (dissolved oxygen timing control), Example 4 (fermentation mash circulation and reflux), Example 5 (primary fermentation termination determination rule), and Example 6 (secondary fermentation termination determination rule) are also used. Specifically: the primary solid-state fermentation stage includes fermentation mash circulation and reflux, and its termination is determined by a determination rule; the secondary liquid fermentation stage includes dissolved oxygen concentration timing control, and its termination is determined by a determination rule. The remaining operating steps are the same as in Example 1.

[0053] Comparative Example 1

[0054] Take 100 kg of dried silkworm excrement and mix it with Chinese medicinal herbs (chrysanthemum, wolfberry, and rehmannia root in a mass ratio of 2:1:1, with the total addition amount being 10% of the silkworm excrement mass). Add 55% V / V pure grain rice wine and purified water, controlling the initial alcohol content to 15% (V / V). Soak at 28℃ for 72 hours without any fermentation treatment or inoculation of any microbial strains. After soaking, filter using a plate and frame filter press, collect the filtrate, adjust the alcohol content to 38% V / V, color with caramel coloring to a dark red, and bottle. The difference between Comparative Example 1 and Example 1 is that all microbial treatment steps, including the inoculation of BNCC382498 and Lactobacillus plantarum ATCC 8014 and the secondary liquid fermentation, are omitted; only physical soaking is performed.

[0055] Comparative Example 2

[0056] Take 100 kg of fresh silkworm excrement, air-dry it, and put it into a primary fermentation container. Immediately after fermentation starts, inoculate with BNCC382498 (inoculum size 1×10⁻⁶). 8The silkworm excrement (CFU / g) was subjected to a single solid-state fermentation at 40-50℃ for 48 hours, maintaining the pH at 6.0-6.5 for 0-12 hours, without inoculating *Lactobacillus plantarum* ATCC 8014. After the first fermentation, it was subjected to high-pressure rinsing with a rinsing solution volume of 3 times the mass of the silkworm excrement for 15 minutes, followed by air-drying. The air-dried silkworm excrement was then mixed with Chinese medicinal herbs (chrysanthemum, wolfberry, and rehmannia root in a mass ratio of 2:1:1, with a total addition amount of 10% of the silkworm excrement mass) and pure grain rice wine, and soaked at 28℃ for 72 hours without secondary liquid fermentation. The remaining steps were the same as in Example 1. The difference between Comparative Example 2 and Example 1 is that only *BNCC382498* was inoculated, without inoculating *Lactobacillus plantarum* ATCC 8014 and without secondary liquid fermentation.

[0057] Comparative Example 3

[0058] Take 100 kg of fresh silkworm excrement, air-dry it, and put it into a primary fermentation container. Inoculate with Lactobacillus plantarum ATCC 8014 (inoculum size 1×10⁻⁶) 12 hours after fermentation starts. 9 The silkworm excrement (CFU / g) was subjected to a single solid-state fermentation at 40-50℃ for 48 hours, maintaining the pH at 6.0-6.5 for 0-12 hours, without inoculating BNCC382498. After the first fermentation, it was subjected to high-pressure rinsing (rinsing liquid volume was 3 times the mass of silkworm excrement, rinsing time was 15 minutes), and then air-dried. The air-dried silkworm excrement was mixed with Chinese medicinal herbs (chrysanthemum, wolfberry, and rehmannia root, mass ratio 2:1:1, total addition amount was 10% of the silkworm excrement mass) and pure grain rice wine, and soaked at 28℃ for 72 hours, without secondary liquid fermentation. The remaining steps were the same as in Example 1. The difference between Comparative Example 3 and Example 1 is that only Lactobacillus plantarum ATCC8014 was inoculated, without inoculating BNCC382498 and without secondary liquid fermentation.

[0059] The silkworm excrement fermentation extracts prepared in Examples 1 to 7 and Comparative Examples 1 to 3 were tested for the following indicators.

[0060] Determination of chloramphenicol residue: 50 mL of silkworm excrement fermentation extract was purified by solid phase extraction (SPE, C18 column, activated sequentially with methanol and water), and then determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The detection limit was 0.1 μg / kg.

[0061] Determination of lead content: 50 mL of silkworm excrement fermentation extract was digested with nitric acid-perchloric acid and then determined by graphite furnace atomic absorption spectrophotometry (ashing temperature 800℃, atomization temperature 1800℃). The detection limit was 0.01 mg / kg.

[0062] The detection of coliform bacteria shall be performed in accordance with GB 4789.3-2016, with a detection limit of <0.3 MPN / 100mL.

[0063] The detection of mold shall be performed in accordance with GB 4789.15-2016, with a detection limit of <1 CFU / mL.

[0064] The content of fusel oil was determined by gas chromatography as described in Example 6. The content of trimethylamine was determined by the picric acid trimethylamine salt colorimetric method as described in Example 6. The content of total flavonoids was determined by the sodium nitrite-aluminum nitrate colorimetric method as described in Example 6. The test results are shown in Table 1.

[0065] Table 1. Comparison of quality indicators of silkworm excrement fermentation extracts from Examples 1 to 7 and Comparative Examples 1 to 3

[0066] As can be seen, the total flavonoid content in Examples 1 to 7 was 125-165 mg / L, significantly higher than the 72 mg / L in Comparative Example 1. The open-ring carboxylic acid intermediates produced by the degradation of chloramphenicol by BNCC382498 exhibit amphiphilic properties. In the 15-18% (v / v) ethanol-water environment of secondary fermentation, they promoted the mass transfer and release of flavonoid medicinal components from the solid phase to the liquid phase of the silkworm excrement by reducing the interfacial tension between the silkworm excrement cell wall and the fermentation broth. Example 7 showed the highest total flavonoid content of 165 mg / L, demonstrating that the synergistic effect of multiple technical features resulted in optimal dissolution of the medicinal components.

[0067] Regarding the trimethylamine content, the trimethylamine content in Examples 1 to 7 was 1.0-1.5 mg / L, all below the acceptable threshold of 1.8 mg / L. This indicates that the amide bond hydrolase secreted by BNCC382498 significantly hydrolyzed the odorous protein precursors in silkworm excrement, such as trimethylamine precursors like choline and trimethylamine oxide, within 0-24 hours. The hydrolysis products were discharged during high-pressure rinsing. Example 7 had the lowest trimethylamine content, indicating that reflux promoted sufficient contact between the enzyme and the substrate. The trimethylamine content in Comparative Example 1 was as high as 4.8 mg / L, and in Comparative Example 3 it was 2.3 mg / L, indicating that fermentation by *Lactobacillus plantarum* alone cannot effectively degrade trimethylamine precursors.

[0068] Regarding fusel oil, the fusel oil content in Example 2, with a carbon-to-nitrogen ratio controlled at 15:1, was 0.18 g / L, significantly lower than the 0.35 g / L in Example 1. This indicates that optimizing the carbon-to-nitrogen ratio effectively inhibited fusel oil formation by reducing the flux of valine, leucine, and isoleucine into the Ehrlich metabolic pathway. Example 7 exhibited the lowest fusel oil content, a result of the combined effects of carbon-to-nitrogen ratio control, dissolved oxygen timing regulation, and reflux. The fusel oil content in Comparative Examples 1-3 ranged from 0.72 to 0.95 g / L, demonstrating that the formation of higher alcohols could not be effectively controlled without the co-fermentation of BNCC382498 and *Lactobacillus plantarum* ATCC 8014.

[0069] Regarding microbiological safety indicators, no coliform bacteria (<0.3 MPN / 100mL) or mold (<1 CFU / mL) were detected in Examples 1 to 7, indicating that the high-temperature stage (40-50℃) of the primary fermentation and the pH drop to 4.0-4.5 caused by acid production from *Lactobacillus plantarum* ATCC 8014 jointly achieved the elimination of pathogenic and putrefactive bacteria. Comparative Example 1 showed severely excessive levels of coliform bacteria and mold; Comparative Example 2 showed some bactericidal effect after high-temperature treatment at 40-50℃, but not complete elimination; the bactericidal effect of Comparative Example 3 was comparable to that of the examples, indicating that acid production by *Lactobacillus plantarum* was the key factor in sterilization.

[0070] Regarding lead content, the lead content in Examples 1 to 7 was 0.05-0.09 mg / kg, significantly lower than that in Comparative Examples 1 and 2. The inoculation of *Lactobacillus plantarum* ATCC 8014 showed a significant adsorption effect on lead: the lead content in Comparative Example 3 was 0.12 mg / kg, lower than Comparative Examples 1 and 2 but still higher than in all examples. Example 7 had the lowest lead content, indicating that the synergistic effect of circulation reflux, dissolved oxygen timing regulation, and endpoint determination further improved the heavy metal passivation efficiency.

[0071] Regarding chloramphenicol residues, none were detected in Examples 1 to 7 (<0.1 μg / kg), indicating that BNCC382498 fully degraded the chloramphenicol residues in silkworm excrement within the 0-12 hour degradation window of the first fermentation. The chloramphenicol residue in Comparative Example 1 was 8.6 μg / kg; in Comparative Example 3 it was 12.4 μg / kg, showing an increase rather than a decrease compared to Comparative Example 1. This is because the *Lactobacillus plantarum* ATCC 8014 inoculated in Comparative Example 3 metabolized and released the bound chloramphenicol in the silkworm excrement into a free state during fermentation. Chloramphenicol was also undetectable in Comparative Example 2, indicating that the degradation of chloramphenicol was mainly completed by BNCC382498 during the first fermentation stage.

[0072] Example 8

[0073] Based on Example 1, calcium carbonate micron powder and dipotassium hydrogen phosphate were added to the fermentation system during the 0-12 hours of the first fermentation. The fermentation treatment method for the calcium carbonate micron powder was as follows: food-grade calcium carbonate (purity ≥99%) was pulverized using an air jet mill, passed through a 400-mesh standard sieve, and micron powder with a particle size of 20-50 μm was collected. Before addition, the calcium carbonate micron powder was mixed and ground with silkworm excrement at a mass ratio of 1:5 in a three-dimensional mixer at 30 rpm for 15 minutes to ensure uniform adhesion of the calcium carbonate micron powder to the surface of the silkworm excrement particles. The particle size distribution after grinding was measured using a laser particle size analyzer. 50The particle size is 35 μm. The total amount of calcium carbonate powder added is 0.8% of the silkworm excrement mass. The total amount of dipotassium hydrogen phosphate (food grade, purity ≥98%) added is 0.3% of the silkworm excrement mass. Dipotassium hydrogen phosphate is added in three batches: 50% at 0 hours, 25% at 4 hours, and 25% at 8 hours. Before each addition, the pH value of the fermentation system is measured with an online pH meter. When the pH value deviates from the 6.0-6.5 range, the addition of that batch is triggered; if the pH value is already within the 6.0-6.5 range, the addition is skipped. The remaining steps are the same as in Example 1.

[0074] In this embodiment, calcium carbonate micropowder adheres to the surface of silkworm excrement particles, forming locally alkaline micro-regions. BNCC382498 colonizes the surface of the calcium carbonate particles, and the pH of the surrounding micro-regions is determined by the dissolution-precipitation equilibrium of calcium carbonate (CaCO3 + H2O). Ca 2+ +HCO3 - + OH - The pH was maintained at 7.0-7.5, close to the optimal pH range (7.0-8.0) for the degradation of chloramphenicol by BNCC382498, while the overall pH of the fermentation system was maintained at 6.0-6.5 by adjusting with hydrochloric acid / sodium hydroxide. Dipotassium hydrogen phosphate (K₂HPO₄ → 2K) + +HPO4 2- HPO4 2- + H + H2PO4 - As a buffer salt, it is replenished in three batches to maintain the buffer capacity of the micro-zone and compensate for the OH- consumed by the dissolution of calcium carbonate. - .

[0075] Comparative Example 4

[0076] The difference from Example 8 is that no calcium carbonate powder or dipotassium hydrogen phosphate is added during the 0-12 hours of the primary fermentation; instead, the pH is maintained at 6.0-6.5 by adding 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. The remaining steps are the same as in Example 8.

[0077] Comparative Example 5

[0078] The difference from Example 8 is that only calcium carbonate powder (0.8% of the silkworm excrement mass, mixed and ground with the silkworm excrement at a ratio of 1:5) is added during the 0-12 hours of the first fermentation, and dipotassium hydrogen phosphate is not added. The remaining steps are the same as in Example 8.

[0079] The degradation efficiency of BNCC382498 on chloramphenicol in Example 8 and Comparative Examples 4-5 during the first fermentation period of 0-12 hours was tested. Sampling times were 0 hours, 6 hours, and 12 hours of fermentation. After sampling, the chloramphenicol content was determined by high-performance liquid chromatography (HPLC) as described in Example 5, and the degradation rate was calculated. Simultaneously, the apparent pH of the fermentation system and the local pH of the micro-regions on the surface of the silkworm excrement particles were monitored. The local pH was determined by inserting a micro pH probe (tip diameter 50 μm) into the gaps between the silkworm excrement particles and measuring once at three different locations, then averaging the results. The test results are shown in Table 2.

[0080] Table 2. Comparison of chloramphenicol degradation efficiency and pH in Example 8 and Comparative Examples 4-5

[0081] As shown in Table 2, Example 8 achieved a chloramphenicol degradation rate of 92.8% after 12 hours, significantly higher than Comparative Example 4's 65.9% and Comparative Example 5's 83.3%. The apparent pH of the fermentation system in Example 8 was 6.3, meeting the 6.0-6.5 range requirement, while the local pH of the silkworm excrement particles reached 7.2, close to the optimal pH of 7.0 for BNCC382498's degradation of chloramphenicol. This achieved local internal pH optimization without changing the overall system pH. Comparative Example 4 maintained the surface pH only by adding acid and alkali; the internal pH of the silkworm excrement particles was consistent with the surface pH, deviating from the optimal pH of BNCC382498, thus resulting in the lowest degradation efficiency. Comparative Example 5 added only calcium carbonate and no dipotassium hydrogen phosphate; although the internal pH was higher than Comparative Example 4, it was still lower than Example 8, and the degradation rate after 12 hours was moderate, indicating that the batch addition of dipotassium hydrogen phosphate was necessary to maintain the continuous stability of the micro-zone pH buffer capacity.

[0082] Example 9

[0083] Based on Example 1, after the first fermentation and before high-pressure rinsing, the silkworm excrement ferment was subjected to gradient ethanol acclimation. The silkworm excrement ferment was successively soaked in a 5% (v / v) ethanol aqueous solution, a 10% (v / v) ethanol aqueous solution, and a 15% (v / v) ethanol aqueous solution. The volume of liquid for each soaking was three times the volume of the silkworm excrement ferment.

[0084] During each immersion stage, a conductivity electrode (platinum black electrode, cell constant 1.0 cm⁻¹) is installed in the immersion container. -1The conductivity change rate of the fermentation mash was monitored in real time. The conductivity change rate was calculated as follows: conductivity values ​​were automatically recorded every 30 seconds. Ten consecutive data points (covering a 5-minute time window) were taken from the current moment backward. The least squares method was used to fit the conductivity-time linear equation κ(t) = at + b, and the absolute value of the slope 'a' was taken as the conductivity change rate. When the conductivity change rate was less than 0.5 μS / cm·min for 5 consecutive minutes, the acclimatization level was considered complete, and the process moved to the next level. This threshold was determined based on preliminary experimental measurements showing that changes in cell membrane permeability under ethanol stress led to a decrease in Kt. + Emissions tend to reach equilibrium upon completion of acclimatization, with the rate of change in conductivity dropping below 0.5 μS / cm·min, corresponding to a cell membrane K0. + The net flux approaches zero.

[0085] The temperature for the 5% ethanol stage was 12°C (controlled by jacket cooling water circulation), the temperature for the 10% ethanol stage was 22°C, and the temperature for the 15% ethanol stage was 29°C. The progressively increasing acclimatization temperature and ethanol concentration worked synergistically: low temperature inhibited cell membrane fluidity to reduce excessive stress in the low-concentration ethanol stage, while gradual temperature increases promoted adaptive adjustment of cell membrane fatty acid composition (especially the ratio of unsaturated to saturated fatty acids). After the three-stage acclimatization was completed, the silkworm excrement fermentation product was removed and allowed to stand in sterile air at 29°C (filtered through a 0.22 μm filter membrane) for 20 minutes for rewarming activation, allowing BNCC382498 to recover from low-temperature stress to normal metabolic levels before proceeding to the high-pressure rinsing step. The remaining steps were the same as in Example 1.

[0086] Comparative Example 6

[0087] The difference from Example 9 is that: after the first fermentation, high-pressure rinsing was performed directly, without gradient ethanol acclimation. The remaining steps are the same as in Example 9.

[0088] Comparative Example 7

[0089] The difference from Example 9 is that after the first fermentation, the silkworm excrement fermented material was only soaked in a 15% ethanol aqueous solution for 30 minutes, without undergoing the gradual acclimation at 5% and 10% solutions. The remaining steps are the same as in Example 9.

[0090] The viable cell count and chloramphenicol degrading enzyme activity of BNCC382498 in Example 9 and Comparative Examples 6-7 were detected during the secondary fermentation stage. Sampling times were 0 hours, 24 hours, 48 ​​hours, and 72 hours of secondary fermentation. The viable cell count was determined as follows: 10 g of silkworm excrement fermentation sample was added to 90 mL of sterile physiological saline, vortexed, serially diluted, and spread onto LB solid medium. After incubation at 30°C for 24 hours, the counts were performed, and the results were expressed as CFU / g. The chloramphenicol degrading enzyme activity was determined as follows: 10 g of silkworm excrement fermentation sample was added to 20 mL of pH 7.0 phosphate buffer (50 mmol / L), and the mixture was sonicated in an ice bath (200 W power, 5 seconds working, 5 seconds rest, total time 10 minutes). The mixture was then centrifuged at 12000×g for 15 minutes at 4°C, and the supernatant was used as the crude enzyme solution. Take 1 mL of crude enzyme solution, add chloramphenicol substrate solution (final concentration 10 mg / L) and reduced nicotinamide adenine dinucleotide (NADH, final concentration 0.1 mmol / L), react at 30℃ for 30 minutes, terminate the reaction with an equal volume of ethyl acetate, and determine the remaining chloramphenicol content by HPLC. One enzyme activity unit (U) is defined as the amount of enzyme required to degrade 1 μmol of chloramphenicol per minute. The detection results are shown in Table 3.

[0091] Table 3 Comparison of viable BNCC382498 counts and enzyme activities in Example 9 and Comparative Examples 6-7

[0092] As shown in Table 3, Example 9, after being acclimated to gradient ethanol, still maintained a concentration of 4.8 × 10⁻⁶ after 72 hours of secondary fermentation. 6 The viable cell count was CFU / g and the enzyme activity was 0.45 U / g, while the viable cell count of the unacclimated control group 6 decreased to 0.6 × 10⁻⁶ after 72 hours. 6 CFU / g and enzyme activity decreased to 0.05 U / g. The viable cell count and enzyme activity of Comparative Example 7 were between those of Example 9 and Comparative Example 6, but the enzyme activity after 72 hours was only 0.12 U / g, significantly lower than that of Example 9. Gradually increasing ethanol concentrations initiated the expression of ethanol stress response genes in BNCC382498, avoiding acute cell membrane damage caused by direct exposure to high concentrations of ethanol. The acclimation endpoint was determined by progressively increasing acclimation temperature combined with real-time monitoring of conductivity changes, using cell membrane K... + Net flux approaching zero serves as a quantitative indicator of acclimatization completion, ensuring the adequacy of the acclimatization process and batch-to-batch consistency.

[0093] Example 10

[0094] Based on Example 1, after the secondary fermentation and before membrane filtration, calcium chloride solution (food grade, purity not less than 95%) was added to the fermentation mash to a final concentration of 0.08 mol / L, and stirred at 60 rpm for 5 minutes to ensure uniform distribution of calcium ions in the mash. Then, sodium alginate (food grade, viscosity 200-400 mPa·s) was added at 0.2% of the mash mass. A two-stage stirring program was used after adding sodium alginate: for the first 10 minutes, the stirring rate was 180 rpm to rapidly disperse the sodium alginate and initially contact it with calcium ions to initiate gelation; then, for the next 30 minutes, the stirring rate was reduced to 65 rpm to prevent the formed gel network from breaking due to high shear force. After stirring, the mash was allowed to stand, and a pH electrode was installed in the fermenter to monitor the pH value of the fermentation mash in real time. When the pH value dropped below 3.8, the standing process was terminated and the membrane filtration step began. This pH endpoint was determined based on the cross-linking reaction of sodium alginate with calcium ions, releasing H₂. + The reaction equation is 2NaAlg + Ca 2+ → Ca(Alg)2 + 2Na + When the reaction is complete, H + Net release ceased, and the pH stabilized within the range of 3.8 ± 0.2. Therefore, a pH drop below 3.8 can be used as an indirect indicator of complete gel cross-linking. Membrane filtration was performed using a plate and frame filter press with a membrane pore size of 0.45 μm. The remaining steps were the same as in Example 1.

[0095] In this embodiment, calcium ions (Ca) 2+ The guluronic acid (G) block of sodium alginate and calcium alginate form a calcium alginate gel network using an egg-box model, embedding and immobilizing lead-ion-adsorbed *Lactobacillus plantarum* cells within the three-dimensional gel mesh. Even if some cells lyse in a 15-18% (v / v) ethanol environment, the released lead ions are still absorbed by the carboxyl groups (-COO) in the gel mesh. - It retains the adsorbed heavy metals through ion exchange and coordination, preventing them from being released back into the wine.

[0096] Comparative Example 8

[0097] The difference from Example 10 is that membrane filtration was performed directly after the secondary fermentation, without the addition of calcium chloride and sodium alginate. The remaining steps are the same as in Example 10.

[0098] Comparative Example 9

[0099] The difference from Example 10 is that only sodium alginate (0.2% of the fermentation mash mass) is added after the secondary fermentation, and calcium chloride solution is not added beforehand. The remaining steps are the same as in Example 10.

[0100] The changes in lead content in the fermentation mash before and after membrane filtration in Examples 10 and Comparative Examples 8-9, as well as the changes in lead content in the filtrate after membrane filtration, were detected. Sampling times were: before the addition of sodium alginate, after settling (before membrane filtration), and after membrane filtration. The method for determining lead content was the same as in Example 5. The detection results are shown in Table 4.

[0101] Table 4 Comparison of lead content and cell integrity in Example 10 and Comparative Examples 8-9

[0102] Table 4 shows that in Example 10, after adding sodium alginate and allowing the mixture to stand until the pH was below 3.8, the lead content in the mash decreased from 2.8 mg / L to 0.6 mg / L, a reduction rate of 78.6%, and the lead content in the filtrate after membrane filtration was only 0.05 mg / L. In Comparative Example 8, without sodium alginate immobilization and relying solely on natural sedimentation, the lead content in the mash only decreased from 2.9 mg / L to 2.5 mg / L, a reduction rate of 13.8%. In Comparative Example 9, without the pre-addition of calcium chloride, the sodium alginate could not fully cross-link to form a complete gel network, and the lead content reduction rate was 55.6%, possibly due to the secondary release of previously adsorbed heavy metals caused by bacterial lysis. The two-stage stirring program ensured the uniform dispersion of sodium alginate and the full formation of the gel network; the standing endpoint of pH below 3.8 was determined based on Ca... 2+ -Na + H in the exchange reaction + Release equilibrium provides an online, quantitative method for determining the degree of gel crosslinking completion.

[0103] Example 11

[0104] Based on Example 1, a citric acid-sodium citrate buffer system was used to prepare the high-pressure rinsing solution after the first fermentation. The preparation method was as follows: 10.5 g of citric acid (food grade, monohydrate, purity not less than 99.5%) and an appropriate amount of sodium citrate (food grade, dihydrate, purity not less than 99%) were weighed, dissolved in purified water, and brought to a final volume of 1 L. The pH of the rinsing solution was adjusted to 3.8 by adjusting the molar ratio of citric acid to sodium citrate to 1.2:1. The concentration of citric acid was 0.05 mol / L.

[0105] During high-pressure rinsing, an online pH monitoring point (glass electrode, accuracy ±0.02pH) is set in the rinsing solution circulation loop. When the pH of the returned rinsing solution is higher than 4.0, a metering pump is triggered to add 0.5 mol / L citric acid solution to the rinsing solution, bringing the pH back to the 3.5-4.0 range. High-pressure rinsing is terminated when the pH of the rinsing solution stabilizes within the range of 4.0±0.1 for 5 consecutive minutes. The remaining steps are the same as in Example 1.

[0106] Comparative Example 10

[0107] The difference from Example 11 is that: after the first fermentation, purified water was used for high-pressure rinsing, no citric acid-sodium citrate buffer system was added, and the pH was not adjusted. The remaining steps are the same as in Example 11.

[0108] Comparative Example 11

[0109] The difference from Example 11 is that: after the first fermentation, purified water was used for high-pressure rinsing, and the pH of the rinsing solution was adjusted to 3.8 with 1 mol / L hydrochloric acid. The citric acid-sodium citrate buffer system was not added. The remaining steps are the same as in Example 11.

[0110] The content of trimethylamine was determined by the picric acid colorimetric method (GB / T 5009.179-2003) using a sample of rinsed silkworm excrement. The method is as follows: 5g of silkworm excrement sample was added to 20 mL of 10% trichloroacetic acid solution, and distilled in a water bath at 60℃. Approximately 30 mL of the distillate was collected and diluted to 50 mL. 5 mL of the distillate was extracted with toluene under alkaline conditions, reacted with a picric acid-toluene solution, and the absorbance was measured at 410 nm. Quantification was performed using a trimethylamine standard curve. The trimethylamine content was used as an indicator of the rinsing effect. The detection limit of this method was 0.5 mg / L. The results are as follows:

[0111] In Example 11, after high-pressure rinsing using a citric acid-sodium citrate buffer system (pH 3.8), the trimethylamine content was measured to be 0.8 mg / L, significantly lower than the 2.6 mg / L in Comparative Example 10 and 1.9 mg / L in Comparative Example 11. Rinsing was terminated when the pH of the rinsing effluent remained stable within the range of 4.0 ± 0.1 for 5 consecutive minutes, requiring only 3 additions of acid to maintain pH stability throughout the process. Comparative Example 10, using purified water for rinsing, had a measured pH of approximately 6.8 and the highest trimethylamine content of 2.6 mg / L. This is because under neutral conditions, trimethylamine mainly exists in an ionic state, has high water solubility, and cannot be selectively retained in the solid phase, resulting in a large amount of precursor entering the subsequent fermentation system. Comparative Example 11 used hydrochloric acid to adjust the pH to 3.8, and the trimethylamine content was measured to be 1.9 mg / L, which is between the two values. However, the pH fluctuated continuously during the rinsing process, and the pH of the effluent rose above 4.5 after 12 minutes. Manual acid replenishment was required 7 times, and the pH was still difficult to stabilize, indicating that hydrochloric acid lacks buffering capacity and cannot maintain the constant pH conditions required for selective separation. The citric acid-sodium citrate buffer system in Example 11 has pH buffering capacity. Combined with an automatic acid replenishment strategy, it ensured pH stability throughout the rinsing process. The pH stabilization endpoint determination of the rinsing effluent ensured that the acid-base exchange between the solid and liquid phases reached equilibrium, effectively reducing the trimethylamine content in the final fermentation extract.

[0112] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A fermentation treatment method for improving the dissolution rate of medicinal components in silkworm excrement, characterized in that, Includes the following steps: Fresh silkworm excrement was collected, air-dried, and placed into a primary fermentation container. Immediately upon initiation of fermentation, *Bacillus diazoxide* was inoculated at an inoculum size of (0.5-1.5) × 10⁻⁶. 8 CFU / g, and carried out a single solid-state fermentation at 40-50℃; 12 hours after the start of the first fermentation, *Lactobacillus plantarum* was inoculated at an inoculum size of (0.5-1.5) × 10⁻⁶. 9 CFU / g; During the primary fermentation, after inoculation with *Bacillus diazoxide*, the pH of the fermentation environment was maintained at 6.0-6.5 for 0-12 hours; After the primary fermentation, the silkworm excrement was subjected to high-pressure rinsing, with the rinsing liquid volume being 3-5 times the mass of the silkworm excrement, the rinsing time being 15-30 minutes, and the rinsing pressure being 0.3-0.5 MPa, and then air-dried; The air-dried silkworm excrement was mixed with Chinese medicinal materials and pure grain rice wine, and the initial alcohol content of the secondary fermentation was controlled at 15-18% (V / V), and the secondary liquid fermentation was carried out at 28-30℃ for 72-96 hours; After the secondary fermentation, the mixture was filtered, and the filtrate was collected to obtain the silkworm excrement fermentation extract.

2. The fermentation treatment method according to claim 1, characterized in that, During the first fermentation process, the pH of the fermentation environment is maintained at 6.0-6.5 by adding acid or alkali for 0-12 hours.

3. The fermentation treatment method according to claim 1, characterized in that, In the secondary liquid fermentation stage, the carbon-nitrogen ratio is set to 15-20:

1. The carbon-nitrogen ratio refers to the mass ratio of total organic carbon to total Kjeldahl nitrogen in the secondary fermentation feed.

4. The fermentation treatment method according to claim 1, characterized in that, The secondary liquid fermentation stage also includes the regulation of dissolved oxygen concentration: the dissolved oxygen concentration is controlled at 2.0-3.0 mg / L for 0-24 hours after the start of secondary fermentation; the dissolved oxygen concentration is reduced to 0.5-1.0 mg / L for 24-48 hours after the start of secondary fermentation; and the dissolved oxygen concentration is reduced to below 0.2 mg / L for 48-72 hours after the start of secondary fermentation.

5. The fermentation treatment method according to claim 1, characterized in that, The solid-state fermentation stage also includes fermentation mash circulation and reflux: the first circulation and reflux is started 6 hours after the inoculation of *Lactobacillus plantarum*, the mash at the bottom of the fermentation vessel is extracted and sprayed onto the surface of the material, the circulation flow rate is 0.5-1.0 L / h per kilogram of material, and the circulation lasts for 2 hours; the second circulation and reflux is started 6 hours after the inoculation of *Lactobacillus plantarum*, the circulation flow rate is 1.5-2.0 L / h per kilogram of material, and the circulation lasts for 3 hours; the second circulation and reflux is terminated when the pH difference between any two monitoring points in the fermentation system is less than 0.15 and the temperature difference between the surface and bottom of the material is less than 2℃.

6. The fermentation treatment method according to claim 1, characterized in that, The termination of the first solid-state fermentation stage is determined by the following criteria: when the three conditions are met—chloramphenicol degradation rate greater than 98%, lead adsorption rate greater than 85%, and system pH value between 4.0 and 4.5—the first solid-state fermentation stage is considered complete and the high-pressure rinsing step begins. The termination of the second liquid fermentation stage is determined by the following criteria: when the three conditions are met—fuse alcohol oil content less than 0.2 g / L, trimethylamine content less than 1.8 mg / L, and total flavonoid dissolution rate greater than 90%—the second liquid fermentation stage is considered complete and the filtration step begins. If the above conditions are not met, the fermentation is extended and re-determined every 12 hours. Fermentation is terminated when the total fermentation time reaches 120 hours.

7. The fermentation treatment method according to claim 1, characterized in that, During the first fermentation period of 0-12 hours, calcium carbonate micron powder and dipotassium hydrogen phosphate are added to the fermentation system. The particle size of the calcium carbonate micron powder is 20-50 μm, and the addition amount is 0.5%-1.0% of the mass of silkworm excrement. The addition amount of dipotassium hydrogen phosphate is 0.2%-0.4% of the mass of silkworm excrement. Before addition, the calcium carbonate micron powder is mixed and ground with the silkworm excrement raw material at a mass ratio of 1:

5. The dipotassium hydrogen phosphate is added in three batches: 50% of the total amount at 0 hours, 25% at 4 hours, and 25% at 8 hours. The pH value of the fermentation system is measured before each addition. When the pH value deviates from the range of 6.0-6.5, the addition of that batch is triggered.

8. The fermentation treatment method according to claim 1, characterized in that, After the first fermentation and before high-pressure rinsing, the silkworm excrement fermentation product undergoes gradient ethanol acclimation: the silkworm excrement fermentation product is successively soaked in 5% ethanol aqueous solution, 10% ethanol aqueous solution, and 15% ethanol aqueous solution. During each soaking process, the change rate of conductivity of the fermentation mash is monitored in real time. When the change rate of conductivity is less than 0.5 μS / (cm·min) for 5 consecutive minutes, the acclimation of that stage is considered complete and the next stage is started. The temperature for the 5% ethanol stage is 10-15℃, the temperature for the 10% ethanol stage is 20-25℃, and the temperature for the 15% ethanol stage is 28-30℃. After the three stages of acclimation are completed, the silkworm excrement fermentation product is left to stand in sterile air at 28-30℃ for 15-30 minutes before proceeding to the high-pressure rinsing step.

9. The fermentation treatment method according to claim 1, characterized in that, After the secondary fermentation is completed and before filtration, calcium chloride solution is added to the fermentation mash to a final concentration of 0.05-0.1 mol / L. After stirring evenly, sodium alginate is added at a rate of 0.1%-0.3% of the fermentation mash mass. After adding sodium alginate, two-stage stirring is performed: the stirring rate is 150-200 rpm for the first 10 minutes, and then the stirring rate is reduced to 50-80 rpm for the next 10-40 minutes. After stirring, the mash is allowed to stand. During the standing process, the pH value of the fermentation mash is monitored in real time. When the pH value drops below 3.8, the standing process is terminated and the membrane filtration step begins.

10. The fermentation treatment method according to claim 1, characterized in that, After the first fermentation, a citric acid-sodium citrate buffer system was used to prepare the high-pressure rinsing solution for high-pressure rinsing. The concentration of citric acid was 0.05-0.1 mol / L. The pH of the rinsing solution was adjusted to 3.5-4.0 by adjusting the ratio of citric acid to sodium citrate. During the high-pressure rinsing process, an online pH monitoring point was set in the rinsing solution circulation loop. When the pH of the returned rinsing solution was higher than 4.0, 0.5 mol / L citric acid solution was added to the rinsing solution to bring the pH back to the 3.5-4.0 range. High-pressure rinsing was terminated when the pH of the rinsing solution was stable within the range of 4.0±0.1 for 5 consecutive minutes.

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