Method and system for rapidly detecting microbial limit of erythromycin ointment preparation

Abstract

The invention relates to the technical field of pharmaceutical preparation detection, in particular to a method for rapidly detecting microbial limit of an erythromycin ointment preparation, which comprises the following steps: S1, pretreating and demulsifying a sample; s2, performing enzymolysis and ultrasonic unwrapping; s3, amphiphilic neutralization: adding a polyoxyethylated castor oil-cysteine copolymer neutralizer into the system treated in the step S2, incubating, and removing oil phase residues; step S4, nano fluorescence sensitization; step S5, filtering and enriching; and step S6, bioluminescence detection: adding a luminescent reagent into the microorganisms enriched in the step S5 for incubation, detecting a relative luminescence unit, and calculating the concentration of the sample microorganisms in combination with the standard curve. By constructing a six-step innovative process of pretreatment, enzymolysis, neutralization, sensibilization, enrichment and detection, key parameters such as the concentration of the composite demulsifier and enzymolysis time are optimized, the detection limit reaches 10 CFU / mL, the detection time is smaller than or equal to 1 h, and the microbiological detection accuracy and efficiency of the erythromycin ointment are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation detection technology, and in particular to a method and system for rapid detection of microbial limits in erythromycin ointment preparations. Background Technology

[0002] The traditional method for detecting the microbial limit of erythromycin ointment is the routine culture method specified in the pharmacopoeia. This method requires steps such as sample dissolution, serial dilution, inoculation culture, and colony counting. The entire detection cycle takes 72-120 hours, and the operation process is cumbersome and difficult.

[0003] To improve testing efficiency, various new testing technologies have been developed on the market. For example, the applicant disclosed a method for detecting the microbial limit of topical antibacterial ointments in a patent document with earlier patent application number CN202311189603.6. This method uses surfactant demulsification combined with membrane filtration to process the sample, and then combines it with bioluminescence detection.

[0004] As can be seen from the above description, it has the following drawbacks when used:

[0005] First, the incomplete disintegration of the microbial encapsulation effect of waxy microparticles leads to a high risk of false negatives. The single surfactant used in the above scheme can only destroy the oil-water emulsion structure of the ointment and cannot effectively dissociate the 5-20μm microparticles formed by waxes such as stearic acid. The physically encapsulated contaminating microorganisms are difficult to contact the detection reagents, and the detection rate is still less than 30% even at low contamination levels.

[0006] Second, insufficient control over antibacterial residues and fluorescence interference leads to poor detection accuracy. The conventional neutralizing agent used in the above scheme can only neutralize erythromycin in the aqueous phase and has no effect on erythromycin residues in the oil phase, resulting in large fluctuations in the recovery rate of Staphylococcus aureus. At the same time, it cannot complete the quenching and inhibition measures for phospholipid excipients, resulting in severe signal attenuation and inability to accurately quantify low contamination.

[0007] Therefore, it is necessary to design a rapid detection method and system for microbial limits of erythromycin ointment that can efficiently remove the residues of two-phase antibacterial agents. Summary of the Invention

[0008] To solve one of the above-mentioned technical problems, the present invention adopts the following technical solution: a method for rapid detection of microbial limits in erythromycin ointment preparations, comprising: step S1, sample pretreatment and demulsification: take an erythromycin ointment sample, add buffer solution and compound demulsifier, and stir in a water bath to achieve demulsification.

[0009] Step S2, enzymatic hydrolysis and ultrasonic decoction: Lipase-protease complex enzyme is added to the system treated in step S1 and incubated, followed by ultrasonic treatment in a constant temperature water bath to achieve the dissociation of wax-encapsulated microorganisms.

[0010] Step S3, Amphiphilic Neutralization: Add polyoxyethylene castor oil-cysteine ​​copolymer neutralizer to the system treated in step S2, and remove oil phase residue after incubation.

[0011] Step S4, Nano-fluorescence enhancement: Add CdTe quantum dot enhancement solution to the system treated in step S3, and enhance the luminescence signal after standing.

[0012] Sodium telluride needs to be prepared in the laboratory. The specific method is as follows: Weigh 2.56g of tellurium powder (purity ≥99.9%) and 0.92g of metallic sodium (purity ≥99.5%), add 100mL of anhydrous ethanol as solvent, and reflux at 70℃ for 2h under nitrogen protection. After the reaction is completed, filter while hot to remove unreacted tellurium powder, and evaporate the filtrate at 50℃ under vacuum until dry. The gray solid obtained is sodium telluride. After preparation, it needs to be sealed and stored under inert gas protection to avoid oxidation and deterioration. When preparing quantum dots, sodium telluride and cadmium chloride need to be prepared into 0.01mol / L stock solutions with deionized water, and then mixed at a concentration ratio of 1:2 to ensure precise control of the concentration of cadmium ions and tellurium ions in the mixed system.

[0013] The total volume of the reaction system for preparing CdTe quantum dots was 50 mL. 10 mL of cadmium chloride stock solution (0.01 mol / L) and 5 mL of sodium telluride stock solution (0.01 mol / L) were added. Thioglycolic acid was added after calculating twice the molar amount of cadmium source, and then deionized water was added to bring the volume to 50 mL. When adjusting the pH value, a 0.1 mol / L NaOH solution was slowly added dropwise to avoid local pH being too high and causing quantum dot aggregation. During the dropwise addition, continuous stirring was carried out to ensure that the pH of the system was uniform.

[0014] Step S5, filtration and enrichment: The system treated in step S4 is subjected to negative pressure filtration using a filter membrane and rinsed with physiological saline containing Tween-20 to achieve microbial enrichment.

[0015] Step S6, Bioluminescence Detection: Add luminescent reagent to the microorganisms enriched in step S5 and incubate. Detect the relative luminescence units and calculate the microbial concentration of the sample based on the standard curve.

[0016] Based on any of the above technical solutions, the following further optimization is made: In step S1: the composite demulsifier is sodium dodecylbenzenesulfonate and isopropanol mixed at a mass ratio of 3:2, with a total concentration of 0.08%-0.12%; the buffer solution is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01 mol / L; the ratio of buffer solution volume to sample mass is 4 mL: 1 g; and the water bath stirring time is 10 min.

[0017] Demulsification rate ≥ 95%, calculated using the following formula: Demulsification rate (%) = (A0 − A t ) / A0×100, where: A0 is the absorbance of the system before demulsification at a wavelength of 600nm, A t The absorbance of the system after demulsification is measured at a wavelength of 600 nm.

[0018] Based on any of the above technical solutions, the following further optimization is made: the compound enzyme in step S2 is a mixture of lipase and protease in a mass ratio of 3:1, with a total concentration of 10 mg / mL; the enzymatic hydrolysis incubation time is 20 min, and the ultrasonic treatment time is 8 min;

[0019] The enzymatic hydrolysis was terminated when the absorbance at 600 nm dropped below 0.1.

[0020] Based on any of the above technical solutions, the following further optimization is made: In step S3: the concentration of the neutralizing agent of the polyoxyethylene castor oil-cysteine ​​copolymer is 1.0%-1.4%, the molar ratio of polyoxyethylene castor oil to cysteine ​​is 5:1, the molecular weight of the copolymer is 5000-8000 Da; and the incubation time is 12-18 min.

[0021] Oil phase residue removal rate ≥90%, the oil phase residue removal rate is calculated by the following formula: Oil phase residue removal rate (%) = (C0 − C t ) / C0×100; where: C0 is the residual concentration of the oil phase in the system before neutralization, C t This represents the residual concentration of the oil phase in the neutralized system.

[0022] Based on any of the above technical solutions, the following optimization is made: In step S4, the concentration of the CdTe quantum dot sensitizing solution is 2-3 nmol / L, the quantum dot particle size is 2-5 nm, and the surface is modified with mercaptoacetic acid.

[0023] Based on any of the above technical solutions, the following further optimization is made: In step S5: the filter membrane is a mixed cellulose ester membrane with a pore size of 0.45 μm; the concentration of physiological saline containing Tween-20 is 0.01%; the rinsing liquid volume is 8-12 mL; and the rinsing times are 2-4 times.

[0024] Based on any of the above technical solutions, the following further optimization is made: In step S6: the luminescent reagent is a luciferase-luciferin system, the luciferase is derived from fireflies and has an activity ≥1000U / mL; the luciferin is D-luciferin with a concentration of 0.1mmol / L; the incubation time is 1-3min; the detection is performed using a multifunctional microplate reader with a detection wavelength of 450-560nm.

[0025] Based on any of the above technical solutions, the following further optimization is made: the method is applicable to the detection of erythromycin ointment preparations with different oil contents; when detecting high-oil-phase erythromycin ointments with an oil content ≥50%, the ratio of buffer solution to sample mass in step S1 is adjusted to 5mL:1g, and the concentration of the composite demulsifier is increased to 0.12%;

[0026] In step S2, the enzymatic incubation time is extended to 30 min, and the ultrasonic treatment time is extended to 10 min;

[0027] In step S3, the concentration of the neutralizing agent is adjusted to 1.4%, and the incubation time is extended to 20 minutes.

[0028] Based on any of the above technical solutions, the following further optimization is made: the ultrasonic treatment process in step S2 is carried out in a constant temperature water bath environment, and the water bath temperature is the same as the enzyme hydrolysis incubation temperature of 37℃; the system is shaken in a constant temperature shaker every 2 minutes during the ultrasonic treatment; the system temperature is monitored in real time by an insertion thermocouple thermometer and maintained at 37±0.2℃.

[0029] Based on any of the above technical solutions, the following optimizations are made: the rinsing solution is added slowly along the edge of the filter membrane at a rate of 1 mL / s; after rinsing, the membrane is allowed to stand for 30 seconds before the negative pressure is turned off; after removing the filter membrane, it is transferred to the detection container using aseptic techniques.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. This invention constructs an innovative six-step process of pretreatment, enzymatic hydrolysis, neutralization, sensitization, enrichment, and detection, and optimizes key parameters such as the concentration of the compound demulsifier and the enzymatic hydrolysis time, so that the detection limit reaches 10² CFU / mL and the detection time is ≤1h, which significantly improves the accuracy and efficiency of microbial detection of erythromycin ointment.

[0032] 2. This invention innovatively uses polyoxyethylene castor oil-cysteine ​​copolymer neutralizer to remove oil phase residue and CdTe quantum dots to enhance the luminescence signal, solving the technical problems of large interference from the ointment matrix and weak luminescence signal. After the oil phase residue is removed, the concentration is lower, and the RLU peak value is effectively improved after the signal is enhanced.

[0033] 3. Through differentiated design of ordinary parameters and high oil phase adaptation parameters, this invention can achieve stable detection in erythromycin ointment with an oil content of 30%-70%. Under high oil phase conditions, the demulsification rate meets the requirements and the wax dissociation rate is effectively improved, thus effectively broadening the applicability of the method.

[0034] 4. The reagents (such as Span-80 and papain) and instruments (fluorescence detector and GC instrument) used in this invention are all common products in the industry. The operation steps have been standardized and verified, and no special customized equipment is required. This makes it easy for enterprise quality control and third-party testing institutions to use on a large scale, and it is highly practical. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or components are generally identified by similar reference numerals. In the drawings, the elements or components are not necessarily drawn to scale.

[0036] Figure 1 Standard curves of concentrations of Staphylococcus aureus, Escherichia coli, and Candida albicans in the experimental example: -lgC versus RLU values ​​(n=3, RSD≤8%).

[0037] Figure 2 The standard curve of the residual concentration of erythromycin ointment in the oil phase versus the GC peak area in the experimental example.

[0038] Figure 3 : Relationship curves between the concentration of the compound demulsifier and the demulsification rate and microbial survival rate in the experimental example (stirred at 37℃ for 10 min, dual Y-axis)

[0039] Figure 4 The curve showing the relationship between the enzymatic hydrolysis time of the compound enzyme solution and the absorbance at 600 nm in the experimental example (10 mg / mL compound enzyme, 37℃).

[0040] Figure 5 The curve showing the relationship between the settling time and RLU value of 2 nmol / LCdSe / ZnS quantum dots in the experimental example (25℃, protected from light).

[0041] Figure 6 : Relationship curves between rinsing liquid volume, number of rinsing cycles and microbial enrichment rate in the experimental example (gemini plot, n=3).

[0042] Figure 7 The curves showing the relationship between the oil content of erythromycin ointment and the demulsification rate and wax dissociation rate in the experimental example. Detailed Implementation

[0043] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. The specific structure of the present invention is as follows: Figures 1-7 As shown in the image.

[0044] Example 1: A method for rapid detection of microbial limits in erythromycin ointment preparations, comprising:

[0045] Step S1, Sample pretreatment and demulsification: Take the erythromycin ointment sample, add buffer solution and compound demulsifier, and stir in a water bath to achieve demulsification;

[0046] Step S2, enzymatic hydrolysis and ultrasonic decoction: Lipase-protease complex enzyme was added to the system after step S1 and incubated, followed by ultrasonic treatment in a constant temperature water bath to achieve the dissociation of wax-encapsulated microorganisms.

[0047] Step S3, Amphiphilic neutralization: Add polyoxyethylene castor oil-cysteine ​​copolymer neutralizer to the system treated in step S2, and remove oil phase residue after incubation;

[0048] Step S4, Nano-fluorescence enhancement: Add CdTe quantum dot enhancement solution to the system treated in step S3, and enhance the luminescence signal after standing.

[0049] Step S5, filtration and enrichment: The system after step S4 is filtered under negative pressure using a filter membrane and then rinsed with physiological saline containing Tween-20 to achieve microbial enrichment.

[0050] Step S6, Bioluminescence Detection: Add luminescent reagent to the microorganisms enriched in step S5 and incubate. Detect the relative luminescence units and calculate the microbial concentration of the sample based on the standard curve.

[0051] The composite demulsifier is a compound system of surfactant and demulsifier, specifically sodium dodecylbenzenesulfonate (SDBS) and isopropanol in a mass ratio of 3:2, with a total concentration of 0.05%-0.15%. SDBS acts as the main demulsifier to break the emulsion structure of the ointment, while isopropanol acts as the demulsifier to reduce interfacial tension and promote layering.

[0052] The detection targets are common contaminating microorganisms, including bacteria (Staphylococcus aureus, Escherichia coli) and fungi (Candida albicans); the sample amount is fixed at 0.5g, and after sampling, the sample is first ground and homogenized in a sterile mortar to ensure the uniformity of the sample.

[0053] Basic parameters for each key step: Step S1: stirring speed 300 r / min, water bath temperature 37℃; Step S2: enzymatic hydrolysis temperature 37℃, total concentration of compound enzyme 10 mg / mL; Step S3: incubation temperature 37℃; Step S4: standing temperature 25℃; Step S6: luminescent reagent dosage 10% of system volume, incubation temperature 37℃.

[0054] The working principle of this scheme is as follows: First, utilizing the synergistic effect of the composite demulsifier, the hydrophobic end of sodium dodecylbenzenesulfonate (SDBS) binds to the wax and oil components in the ointment, while the hydrophilic end faces the aqueous phase to disrupt the emulsion balance. Isopropanol penetrates to the oil-water interface to reduce interfacial tension, and water bath stirring accelerates the disintegration of the emulsion structure to achieve demulsification. Subsequently, the lipase in the compound enzyme degrades the oil components, and the protease decomposes the protein encapsulation. Combined with ultrasonic mechanical vibration, the wax-encapsulated microorganisms are completely dissociated, and the temperature is controlled by a constant-temperature water bath to prevent microbial inactivation. Next, the amphiphilic copolymer binds to the residual oil phase through its hydrophobic end and integrates into the aqueous phase to achieve oil phase removal. CdTe quantum dots bind to the microorganisms through surface modification groups to enhance the luminescence signal. The filter membrane filters and enriches the microorganisms and removes impurities. Finally, the luciferase-luciferin system reacts with the AtP produced by microbial metabolism to produce luminescence, and the quantification is achieved by combining the RLU value with a standard curve.

[0055] The advantages mentioned above are: First, the demulsification system is simple and easy to prepare, requiring no complex screening process, and is low in cost and highly stable; second, the detection speed is fast, with the entire process completed in about 1.5 hours, which is far superior to the traditional culture method (24-48 hours); third, the sensitivity is high, with quantum dot sensitization and filter membrane enrichment working together to reduce the detection limit to as low as 100 CFU / mL; and fourth, it has wide applicability, as it can be adapted to ointments with different oil contents by adjusting the parameters.

[0056] The composite demulsifier combines demulsification and preliminary impurity removal functions. SDBS can adsorb some impurity particles, and isopropanol can dissolve a small amount of fat-soluble impurities, breaking through the limitation of traditional single demulsifiers that can only separate into layers. The composite demulsifier has good compatibility with the subsequent enzymatic hydrolysis system, does not inhibit the activity of lipase and protease, and the residual components can be removed by the subsequent rinsing step, avoiding interference with the detection and solving the problem that traditional demulsifiers are prone to antagonism with enzyme preparations.

[0057] Based on any of the above technical solutions, the following further optimization is made: In step S1: the composite demulsifier is sodium dodecylbenzenesulfonate (SDBS) and isopropanol mixed at a mass ratio of 3:2, with a total concentration of 0.08%-0.12%; the buffer solution is a phosphate buffer with a pH of 7.2-7.4 and a concentration of 0.01 mol / L; the ratio of buffer volume to sample mass is 4 mL: 1 g; the water bath stirring time is 10 min; the demulsification rate is ≥95%, and the demulsification rate is calculated by the following formula: Demulsification rate (%) = (A0 − A t ) / A0×100, where: A0 is the absorbance of the system before demulsification at a wavelength of 600nm, A t The absorbance of the system after demulsification is measured at a wavelength of 600 nm.

[0058] Preferably, the total concentration of the compound demulsifier is 0.1%.

[0059] Preparation method of compound demulsifier: Weigh 0.06g SDBS and 0.04g isopropanol, dissolve in deionized water and bring to a final volume of 100mL to obtain a 0.1% compound system. Prepare immediately to ensure activity. Preparation of phosphate buffer: Weigh 0.312g sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) and 1.076g disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), dissolve in deionized water and bring to a final volume of 1000mL. Calibrate the pH meter to 7.2-7.4. Absorbance detection conditions: At 25℃, take the supernatant of the system and add it to a 1cm path length cuvette. Measure the absorbance at 600nm using a UV-Vis spectrophotometer. Zero the instrument with blank buffer before detection. Stirring is performed using a constant temperature water bath magnetic stirrer with a precise speed control of 300r / min to avoid excessive foaming due to excessive speed or incomplete demulsification due to excessive speed.

[0060] The working principle of this optimized scheme is as follows: a 3:2 mass ratio of SDBS to isopropanol maximizes demulsification efficiency; sufficient SDBS ensures the destruction of the emulsion structure; and an appropriate amount of isopropanol avoids excessive dilution of the system. A total concentration of 0.1% achieves efficient demulsification while avoiding excessive concentration that could lead to subsequent microbial adsorption and inactivation. A phosphate buffer solution with a pH of 7.2-7.4 simulates the physiological environment of microorganisms, maintaining microbial activity while providing a stable environment for the demulsifier. A 37°C water bath accelerates the molecular motion of the demulsifier, and stirring for 10 minutes ensures full contact between the demulsifier and the ointment, ensuring a demulsification rate ≥95%. The demulsification effect is evaluated by absorbance at 600 nm. Due to the high turbidity and high absorbance of the emulsion system, the absorbance decreases significantly after demulsification and clarification. This method is intuitive and quantifiable.

[0061] The compound demulsifier is specifically targeted at the beeswax component in erythromycin ointment. SDBS can form hydrophobic bonds with long-chain alkanes in beeswax, and isopropanol helps dissolve the ester components in beeswax. Compared with traditional single demulsifiers, it is more targeted at demulsifying complex ointment systems. The ionic strength of phosphate buffer (0.01 mol / L) can enhance the surface activity of SDBS and promote its adsorption at the oil-water interface, solving the problem of insufficient activity of traditional demulsifiers under low ionic strength conditions.

[0062] Based on any of the above technical solutions, the following further optimizations are made: the compound enzyme in step S2 is a mixture of lipase and protease at a mass ratio of 3:1, with a total concentration of 10 mg / mL; the enzymatic hydrolysis incubation time is 20 min, and the ultrasonic treatment time is 8 min; the enzymatic hydrolysis is terminated when the absorbance of the system at 600 nm drops below 0.1 during the enzymatic hydrolysis process; and the wax dissociation rate is ≥98%.

[0063] Lipase was selected from Candida albicans with a purity ≥99% and an activity ≥1000 U / g; protease was selected from trypsin with a purity ≥99% and an activity ≥2000 U / g. The compound enzyme was prepared by weighing the two enzymes at a 3:1 mass ratio, dissolving them in phosphate buffer to prepare a 10 mg / mL solution, and storing at 4℃ for later use (shelf life 24 h). Enzymatic hydrolysis incubation conditions: 37℃ constant temperature water bath, enzyme to substrate (wax and lipid components in the system) mass ratio 1:50, ensuring sufficient enzyme quantity; sonication was performed using a probe-type ultrasonic cell disruptor in intermittent mode (30 s working, 10 s pause), with a power of 200 W; the wax dissociation rate was detected by high-performance liquid chromatography (HPLC), calculated by measuring the free wax content before and after dissociation, and the total wax mass was determined by Soxhlet extraction.

[0064] The ultrasonic probe is a cylindrical probe with a diameter of 6mm to ensure good compatibility and to ensure that the ultrasonic energy is evenly distributed throughout the system. The probe is immersed in the system to a depth of 2cm, not just in the water bath. The immersion depth needs to be calculated from the front end of the probe. At the same time, it is necessary to ensure that the distance between the probe and the reaction vessel wall is not less than 1cm to avoid the ultrasonic energy being absorbed by the vessel wall, which would result in poor local treatment effect.

[0065] During ultrasonic treatment, the reaction vessel must be sealed to prevent the system from evaporating water and causing changes in concentration. The sealing material should be a sterile polytetrafluoroethylene stopper to avoid contamination. The water level in the constant temperature water bath must be kept constant. The water level should be observed every 3 minutes. If the water level drops due to evaporation, deionized water should be added in time to the set height to ensure temperature control accuracy.

[0066] The working principle of this optimized scheme is as follows: a 3:1 ratio of lipase to protease is used to adapt to the ointment components. Since the ointment contains a higher percentage of oils than protein impurities, the higher proportion of lipase ensures sufficient degradation of the oils. A total concentration of 10 mg / mL and an enzyme-to-substrate ratio of 1:50 ensure sufficient enzymatic hydrolysis. A 20-minute incubation time allows for complete reaction between the enzyme and substrate. Ultrasonic treatment breaks the wax coating on microorganisms through mechanical vibration, and an 8-minute treatment time ensures effective dissociation while avoiding damage to the microbial cell walls. An absorbance ≤0.1 is used as the termination criterion for enzymatic hydrolysis, as the system becomes less turbid and impurities are reduced after hydrolysis, and absorbance directly reflects the degree of hydrolysis. A wax dissociation rate ≥98% ensures complete release of microorganisms, providing sufficient target microorganisms for subsequent detection.

[0067] The synergistic effect of the compound enzymes makes the degradation of impurities more comprehensive. After the lipase degrades the oil, it exposes the encapsulated wax and protein, and the protease further decomposes the protein impurities. Compared with single enzyme preparations, it is more thorough in treating complex matrices. Intermittent ultrasound avoids the local high temperature generated by continuous ultrasound. With the temperature control of constant temperature water bath, the survival rate of microorganisms is ≥95%, which breaks through the limitation of traditional continuous ultrasound causing microbial inactivation.

[0068] Based on any of the above technical solutions, the following further optimization is made: In step S3: the concentration of the neutralizing agent of the polyoxyethylene castor oil-cysteine ​​copolymer is 1.0%-1.4%, the molar ratio of polyoxyethylene castor oil to cysteine ​​is 5:1, and the molecular weight of the copolymer is 5000-8000 Da; the incubation time is 12-18 min; the oil phase residue removal rate is ≥90%, and the oil phase residue removal rate is calculated by the following formula: Oil phase residue removal rate (%) = (C0−C t ) / C0×100; where: C0 is the residual concentration of the oil phase in the system before neutralization (μg / mL), C t The concentration of the oil phase remaining in the neutralized system is expressed in μg / mL.

[0069] Preferably, the concentration of the neutralizing agent is 1.2%.

[0070] Copolymer synthesis method: Polyoxyethylene castor oil (EL-40, HLB value 13.5) and cysteine ​​were added to a three-necked flask at a molar ratio of 5:1. 0.5% (by mass) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added as a catalyst. The mixture was stirred at 60℃ for 4 h. After the reaction, the mixture was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3000 Da. The product was then freeze-dried. Incubation conditions: 37℃ constant temperature water bath, magnetic stirring at 150 r / min to promote contact. The residual concentration of the oil phase was detected by gas chromatography using an HP-5 capillary column. The column temperature program was: 80℃ for 2 min, then increased to 250℃ at 10℃ / min and held for 5 min. Detection was performed using a flame ionization detector. The molecular weight of the copolymer was determined by gel permeation chromatography (GPC) using tetrahydrofuran as the mobile phase.

[0071] The reaction solvent used for copolymer synthesis is N,N-dimethylformamide (DMF), and the amount of solvent used is 5 times the total mass of raw materials. That is, if the total mass of polyoxyethylene castor oil and cysteine ​​is 10g, then 50ml of DMF should be added. After adding the raw materials, the mixture should be stirred at room temperature for 30min to completely dissolve the cysteine ​​in the DMF. Then, the EDC catalyst should be added and the temperature should be raised to 60℃ to ensure that the reaction system is homogeneous and stable. Deionized water should be used as the dialysis medium during dialysis, and the dialysis solution should be changed every 4h for a total of 6 times to completely remove residual DMF and unreacted EDC.

[0072] The molecular weight of polyoxyethylene castor oil (EL-40) is 1630 Da, and the mass should be converted according to this molecular weight when calculating the molar ratio; the EDC catalyst needs to be dissolved in a small amount of DMF before being added to the reaction system to avoid the side reaction caused by excessive local concentration of catalyst; the freeze-drying conditions are -50℃, 0.01MPa, and drying time of 12h to ensure that the product is completely dried and maintains structural stability.

[0073] The working principle of this optimized scheme is as follows: the hydrophobic chains of polyoxyethylene castor oil (EL-40) bind to oil phase residues, while the hydrophilic chains integrate into the aqueous phase. The amino and thiol groups of cysteine ​​enhance the binding ability with the oil phase, and a 5:1 molar ratio balances the hydrophilicity and hydrophobicity of the copolymer. EDC catalyzes the amidation reaction of carboxyl and amino groups, and the reaction is carried out at 60℃ for 4 hours to ensure sufficient reaction. Unreacted raw materials are removed by dialysis purification. A neutralizing agent concentration of 1.2% ensures that the oil phase residue removal rate reaches more than 95%, avoiding the insufficient removal caused by a 1.0% concentration and the increase in system viscosity caused by a 1.4% concentration. Incubation at 37℃ and stirring at 150 r / min ensure that the copolymer and oil phase are in full contact, forming stable micelles that dissolve in the aqueous phase for removal. GC detection with high separation efficiency ensures accurate quantification of oil phase residues.

[0074] The cysteine ​​groups of the copolymer have both oil-binding and system-neutralizing functions, which can neutralize acidic impurities in the system and avoid affecting the subsequent quantum dot sensitization effect, breaking through the limitation of traditional neutralizers that can only remove the oil phase; the molecular weight range of 5000-8000 Da gives the copolymer both good solubility and strong oil-binding ability, and the resulting micelles have a suitable particle size, which will not affect the subsequent filtration enrichment, thus solving the filtration clogging problem caused by the inappropriate molecular weight of traditional neutralizers.

[0075] Based on any of the above technical solutions, the following further optimization is made: In step S4: the concentration of the CdTe quantum dot sensitizing solution is 2-3 nmol / L, the quantum dot particle size is 2-5 nm, and the surface is modified with thioglycolic acid; the standing time is 3-7 min; the signal attenuation rate is ≤15%, and the signal attenuation rate is calculated by the following formula: Signal attenuation rate (%) = (RLU) max -RLU t ) / RLU max ×100; Where: RLU max RLU is the largest relative luminescence unit of the system after sensitization. t The relative luminous unit after being left to stand still.

[0076] Preferably, the CdTe quantum dot concentration is 2 nmol / L and the settling time is 5 min.

[0077] Preparation of CdTe quantum dots: Sodium telluride and cadmium chloride were used as the tellurium source and cadmium chloride as the cadmium source, with a concentration ratio of 1:2; mercaptoacetic acid was used as a modifier, with an amount twice the molar amount of the cadmium source; pH=10 (adjusted with NaOH), and the reaction was carried out at 90℃ for 1 h; the particle size of the quantum dots was detected by transmission electron microscopy (TEM), and the average particle size was calculated by randomly selecting 100 quantum dots; RLU detection was performed using a multifunctional microplate reader with an excitation wavelength of 360 nm and a detection wavelength of 560 nm; the reaction was carried out in a light-protected environment at 25℃ to avoid fluorescence decay of the quantum dots caused by light exposure.

[0078] The working principle of this optimized scheme is as follows: a quantum dot concentration of 2 nmol / L maximizes the sensitization effect; too high a concentration can easily lead to aggregation and signal fluctuations, while too low a concentration results in insufficient sensitization; quantum dots with a particle size of 3-5 nm have a strong quantum confinement effect and high fluorescence quantum yield; thioglycolic acid modification improves the water solubility of quantum dots, and their carboxyl groups can bind to amino groups on the surface of microorganisms, enhancing the subsequent bioluminescent signal; standing at 25°C in the dark for 5 min allows the quantum dots to fully bind with microorganisms and avoids fluorescence decay; a signal attenuation rate of ≤15% ensures stable detection signal and provides a reliable basis for quantitative analysis.

[0079] The quantum dots modified with thioglycolic acid can combine with the amino group of the neutralizing agent in step S3 to form a ternary complex of neutralizing agent-quantum dot-microorganism, achieving dual signal amplification and breaking through the limitation of traditional quantum dots relying solely on their own fluorescence enhancement. The modification with 2 times the molar amount of thioglycolic acid forms a protective layer on the surface of the quantum dots, preventing the quenching of quantum dot fluorescence by residual ointment components and solving the problem of traditional quantum dots being easily interfered with by the matrix.

[0080] Based on any of the above technical solutions, the following further optimization is made: In step S5: the filter membrane is a mixed cellulose ester membrane with a pore size of 0.45 μm; the concentration of physiological saline containing Tween-20 is 0.01%, the rinsing liquid volume is 8-12 mL, and the rinsing times are 2-4 times; the microbial enrichment rate is ≥98%, and the enrichment rate is calculated by the following formula: Enrichment rate (%) = CFU 滤膜 / CFU 初始 ×100; Where: CFU 滤膜 CFU is the number of microbial colonies enriched on the filter membrane. 初始 This represents the initial number of microbial colonies in the system.

[0081] Preferably, the rinsing liquid volume is 10 mL, the rinsing is performed 3 times, and the microbial enrichment rate is ≥98.5%.

[0082] The filter membrane has a diameter of 50 mm and exhibits good microbial adsorption and water resistance. The rinsing solution is prepared by dissolving 0.01 g of Tween-20 in 100 mL of physiological saline (0.9% NaCl). Filtration is performed using negative pressure suction filtration at a pressure of 0.03-0.05 MPa. During rinsing, the rinsing solution is added along the edge of the filter membrane at a rate of 1 mL / s. CFU detection is performed using the plate counting method; bacteria are cultured on nutrient agar at 37°C for 24 h, and fungi are cultured on Sabouraud dextrose agar at 28°C for 48 h.

[0083] The working principle of this optimized scheme is as follows: a 0.45μm mixed cellulose ester membrane can retain the vast majority of bacteria and fungi (particle size ≥0.5μm) while having good hydrophilicity; 0.01% Tween-20 reduces the surface tension of the filter membrane and reduces non-specific adsorption of microorganisms, while physiological saline maintains the osmotic pressure for microorganisms; the combination of 10mL rinsing solution and 3 rinses removes impurities while preventing microbial loss; 0.03-0.05MPa negative pressure filtration ensures a moderate filtration rate and uniform distribution of microorganisms on the filter membrane surface; the plate counting method achieves accurate CFU counting through specific culture medium, ensuring accurate enrichment rate calculation.

[0084] The mixed cellulose ester membrane undergoes hydrophilic treatment, allowing its surface hydroxyl groups to form hydrogen bonds with microbial surface proteins. This enhances adsorption capacity without adsorbing subsequent luminescent reagents, thus avoiding reagent waste and overcoming the contradiction between the adsorption performance and reagent compatibility of traditional filter membranes. After negative pressure filtration, the membrane is allowed to stand for 30 seconds before the negative pressure is turned off to ensure that the surface moisture is completely removed, preventing residual moisture from diluting the subsequent luminescent reagents. This solves the detection error problem caused by water on the filter membrane after traditional filtration.

[0085] Based on any of the above technical solutions, the following further optimization is made: In step S6: the luminescent reagent is a luciferase-luciferin system, the luciferase is derived from fireflies and has an activity ≥1000 U / mL; the luciferin is D-luciferin with a concentration of 0.1 mmol / L; the incubation time is 1-3 min; the detection is performed using a multifunctional microplate reader with a detection wavelength of 450-560 nm; the standard curve is constructed as follows: a concentration of 100-10 6 A series of standard bacterial solutions with CFU / mL were used to detect the RLU value. The standard curve equation was obtained by linear regression fitting: RLU=a×lgC+b; where: RLU is the relative luminescence unit, C is the microbial concentration (CFU / mL), a is the regression coefficient, and b is the intercept; the coefficient of determination of the standard curve R²≥0.995.

[0086] Preparation of luminescent reagent: Using Tris-HCl buffer (pH 7.8) as solvent, add luciferase, D-luciferin, and 0.05 mmol / L LAtP (coenzyme), prepare fresh before use; Incubation conditions: Incubate at 37°C in the dark; Preparation of standard bacterial culture: A t After the CC standard strain was cultured in the corresponding culture medium, single colonies were picked and prepared into bacterial suspensions with physiological saline, and then serially diluted 10-fold to obtain a series of concentrations; the enzyme-linked immunosorbent assay (ELISA) reading parameters were: excitation wavelength 360nm, integration time 1s, and detection sensitivity set to high.

[0087] In step S6, when preparing the luminescent reagent, the specific amount of luciferase used is 100U per 10mL Tris-HCl buffer to ensure stable enzyme reaction efficiency. In step S5, when eluting microorganisms from the filter membrane, the filter membrane needs to be rinsed three times repeatedly with 5mL of sterile physiological saline. After collecting the eluent, the membrane is inoculated using the pour plate method to avoid uneven colony growth caused by placing the filter membrane directly on the plate. In step S4, the quantum dot sensitizing solution needs to be warmed at 25℃ in the dark for 30 minutes before use to eliminate the influence of temperature on the luminescence signal.

[0088] The AtCC standard strains used for preparing the standard bacterial suspensions were: Staphylococcus aureus (AtCC6538), Escherichia coli (AtCC25922), and Candida albicans (AtCC10231). During culture, Staphylococcus aureus and Escherichia coli were cultured on nutrient agar at 37°C for 18 hours, while Candida albicans was cultured on Sabouraud dextrose agar at 28°C for 24 hours. After single colonies were picked, they were rejuvenated in their respective liquid media until the logarithmic growth phase (6 hours for Staphylococcus aureus and Escherichia coli, and 12 hours for Candida albicans). The bacterial suspension concentration was then adjusted to 10⁻⁶ with physiological saline. 8 The stock solution was prepared at CFU / mL and then serially diluted 10-fold.

[0089] The concentration of the bacterial suspension needs to be calibrated using the McFarland turbidimetric method. The revitalized bacterial suspension should be mixed with a 0.5 McFarland turbidimetric tube (corresponding to 1.5 × 10⁻⁶). 8 Compare with CFU / mL, and adjust to 10 by dilution with physiological saline or bacterial concentration. 8 CFU / mL; When performing a 10-fold serial dilution, use a sterile pipette to add 1 mL of bacterial culture to 9 mL of physiological saline, shake thoroughly for 30 seconds, and then proceed to the next dilution to ensure uniform dilution.

[0090] The working principle of this optimized scheme is as follows: Firefly luciferase catalyzes the oxidation of D-luciferin to produce light in the presence of AtP (a microbial metabolite), and the RLU value is positively correlated with the microbial concentration; AtP acts as a coenzyme to enhance luminescence efficiency, and is prepared and used immediately to avoid luciferase inactivation; incubation at 37℃ in the dark for 2 minutes ensures a complete luminescence reaction and prevents luciferin decomposition due to light exposure; the standard curve uses linear regression of lgC and RLU, as the microbial concentration range is wide (100-10). 6 The linear relationship was good after logarithmic transformation (CFU / mL); R²≥0.995 ensured quantitative accuracy and met the accuracy requirements for microbial limit detection.

[0091] The excitation wavelength of 360nm matches the excitation wavelength of CdTe quantum dots. After the quantum dots absorb the excitation light, they transfer energy to the luminescent system, achieving dual amplification of quantum dot sensitization and bioluminescence, breaking through the bottleneck of insufficient signal intensity of traditional single bioluminescence. The addition of AtP compensates for the problem of insufficient AtP generation by low-concentration microorganisms, reducing the detection limit to 100CFU / mL, solving the problem of insensitivity of traditional luminescent systems for detecting low-concentration microorganisms.

[0092] Based on any of the above technical solutions, the following further optimizations are made: the method is applicable to the detection of erythromycin ointment preparations with different oil contents; when detecting high-oil-phase erythromycin ointments with an oil content ≥50%, the ratio of buffer to sample mass in step S1 is adjusted to 5mL:1g, and the concentration of the composite demulsifier is increased to 0.12%; the enzymatic incubation time in step S2 is extended to 30min, and the ultrasonic treatment time is extended to 10min; the neutralizing agent concentration in step S3 is adjusted to 1.4%, and the incubation time is extended to 20min.

[0093] Oil content was determined using Soxhlet extraction with petroleum ether as the extraction solvent for 6 hours. Oil content was calculated as: (mass of extracted oil / mass of sample) × 100%. For high-oil-phase samples, the stirring speed in step S1 was increased to 400 r / min to enhance the stirring shear force. In step S3, the concentration of the neutralizing agent was adjusted synchronously with the incubation time to ensure that the oil phase residue removal rate was ≥90%. Other step parameters were consistent with those for ordinary oil-content samples to ensure the consistency of the detection method.

[0094] The working principle of this optimized scheme is as follows: the high oil phase ointment emulsion system is more stable and has a higher viscosity; the 5mL:1g buffer ratio increases the proportion of the aqueous phase to dilute the oil phase; the concentration of 0.12% composite demulsifier and the rotation speed of 400r / min enhance the demulsification ability; the 30min enzymatic hydrolysis and 10min ultrasonic treatment extend the treatment time to ensure that the high content of wax and oil is fully degraded and microorganisms are completely released; the concentration of 1.4% neutralizing agent and the 20min incubation time increase the chance of the copolymer binding with the oil phase, compensating for the removal pressure caused by the high oil phase residue; Soxhlet extraction method accurately determines the oil content, providing a reliable basis for parameter adjustment.

[0095] The parameter adjustment adopts simultaneous optimization of demulsification, enzymatic hydrolysis and neutralization steps. Compared with the traditional method of adjusting only a single parameter, the treatment effect on high oil phase samples is more stable. The coordinated adjustment of the concentration of composite demulsifier and stirring speed avoids the microbial inactivation caused by simply increasing the concentration or the foaming problem caused by simply increasing the speed, ensuring that the demulsification rate of high oil phase samples is still ≥95%.

[0096] Example 2: Compared with Example 1, this example also includes the following technical features:

[0097] Based on any of the above technical solutions, the following further optimization is made: the ultrasonic treatment process in step S2 is carried out in a constant temperature water bath environment, and the water bath temperature is the same as the enzymatic hydrolysis incubation temperature of 37℃; during the ultrasonic treatment, the system is subjected to constant temperature shaking once every 2 minutes, with a shaking frequency of 50r / min and an amplitude of 10mm; the system temperature is monitored in real time by an insertion thermocouple thermometer and maintained at 37±0.2℃.

[0098] The constant temperature water bath has a temperature control accuracy of ±0.1℃, model HH-S4; the ultrasonic equipment is a probe-type ultrasonic cell disruptor, with the ultrasonic probe immersed in the water bath to a depth of 1 / 2 of the probe length, and the water bath level 2cm above the system liquid level; the thermocouple thermometer has an accuracy of ±0.01℃, with the probe inserted into the middle of the system for real-time temperature measurement; the oscillation uses a constant temperature shaker to ensure temperature stability during the oscillation process.

[0099] The working principle of this optimized scheme is as follows: a 37℃ constant temperature water bath provides a temperature buffer for the ultrasonic process, avoiding the heat generated by ultrasonic mechanical vibration from causing the system to heat up; the optimization of the probe immersion depth and the height of the water bath liquid level ensures uniform transfer of ultrasonic energy, while the water bath dissipates heat quickly; gentle oscillation every 2 minutes ensures uniform internal temperature of the system, avoiding local overheating; real-time temperature monitoring ensures that the temperature fluctuation is ≤0.2℃, maintaining microbial activity (survival rate ≥95%), while ensuring that enzyme activity is not affected by temperature.

[0100] The synergistic effect of constant temperature shaker oscillation and ultrasound creates slight convection in the system during oscillation, which promptly removes microorganisms dissociated by ultrasound from the waxy aggregates, preventing re-encapsulation and improving dissociation efficiency. Compared with traditional manual oscillation, constant temperature shaker oscillation has better consistency, with the dissociation rate difference between samples handled by different operators being ≤3%, thus solving the problem of human operation error.

[0101] Based on any of the above technical solutions, the following further optimizations are made: In the filtration and enrichment process of step S5, the filter membrane is fixed with a sterile filter membrane clamp and the sealing ring is made of silicone rubber; the rinsing solution is added slowly along the edge of the filter membrane at a rate of 1 mL / s; after rinsing, the membrane is allowed to stand for 30 seconds before the negative pressure is turned off; after removing the filter membrane, it is transferred to the detection container using aseptic operation.

[0102] The sterile filter membrane clamp ensures that the filter membrane is firmly fixed and free from microbial contamination; the silicone rubber sealing ring has good chemical resistance and avoids reaction with the rinsing solution; the negative pressure filtration device is a vacuum filtration bottle set (model SHB-III) with a pressure stability accuracy of ±0.005MPa; the filter membrane transfer is carried out in a sterile ultra-clean workbench to avoid environmental microbial contamination.

[0103] The working principle of this optimized solution is as follows: the combination of a sterile filter membrane clamp and a silicone rubber sealing ring ensures that the filtration process is sterile and well-sealed, preventing leakage and loss of microorganisms; the slow dripping speed of 1 mL / s and the method of adding along the edge prevent the rinsing solution from directly impacting the microorganisms on the filter membrane surface, reducing the loss rate (≤1.5%); after rinsing, the filter membrane is allowed to stand for 30 seconds to fully dry the surface moisture, preventing residual moisture from affecting subsequent luminescence detection; aseptic operation is ensured throughout the process to prevent contamination that could lead to higher detection results.

[0104] The elasticity of the silicone rubber sealing ring can adapt to filter membranes of different thicknesses, ensuring a good seal while preventing filter membrane damage. Compared with traditional hard material sealing rings, it provides better protection for the filter membrane. The 30-second standing operation allows microorganisms to be stably adsorbed on the filter membrane surface, and the subsequent elution efficiency is increased to over 98%, solving the problem of incomplete elution caused by weak microbial adsorption after traditional filtration.

[0105] Experimental example: Rapid detection of microbial limits in erythromycin ointment based on pretreatment-sensitization-luminescence detection.

[0106] Experimental objective: To achieve rapid and accurate quantification of microorganisms (Staphylococcus aureus, Escherichia coli, and Candida albicans) in erythromycin ointment by optimizing key parameters, constructing standard curves, and verifying applicability.

[0107] I. Experimental Materials and Instruments

[0108] 1. Strains: Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC25922), Candida albicans (ATCC10231) (China Industrial Microbial Culture Collection Center);

[0109] 2. Reagents: Span-80, Tween-80 (analytical grade, Sinopharm Group, used for preparing compound demulsifier); papain (200 U / mg), lipase (100 U / mg) (Sigma, used for preparing compound enzyme solution); polyoxyethylene castor oil-cysteine ​​copolymer neutralizer (self-made, concentration 5 mg / mL); CdTe quantum dot sensitizing solution (self-made, particle size 4 nm, concentration 2 nmol / L); ATP luminescent reagent (a biotechnology company in Shanghai); physiological saline containing 0.05% Tween-20 (self-made); petrolatum standard (Sinopharm Group, used to construct oil phase residual standard curve); PBS buffer (pH 7.4, 0.01 mol / L).

[0110] 3. Instruments: Fluorescence detector (Berthold LB941, for RLU value detection); Gas chromatograph (Agilent 7890A, for oil phase residue quantification); Microplate reader (Thermo Multiskan FC, for absorbance measurement); Numerical control ultrasonic instrument (KQ-500DE, for unpacking); Negative pressure filtration device (Tianjin Jinteng, with 0.22μm mixed cellulose ester filter membrane); Constant temperature water bath (Shanghai Yiheng, accuracy ±0.5℃).

[0111] II. Experimental Procedures and Result Analysis

[0112] 1. Construction of the microbial concentration-RLU standard curve: Staphylococcus aureus and Escherichia coli were inoculated on LB medium (37℃, 12h), and Candida albicans was inoculated on PDA medium (28℃, 24h). The inoculated cultures were diluted with PBS to a concentration of 10²-10. 6 CFU / mL gradient bacterial suspensions (lgC = 2-6). 1 mL of bacterial suspension was reacted with 100 μL of ATP fluorescent reagent for 5 min, and the RLU value was measured (3 replicates per group, RSD ≤ 8%). Linear fitting yielded the following results:

[0113] Staphylococcus aureus: RLU=4200lgC-6200 (R²=0.998);

[0114] Escherichia coli: RLU=4100lgC-6000 (R²=0.997);

[0115] Candida albicans: RLU=4000lgC-5800 (R²=0.996).

[0116] The results showed a good linear relationship between microbial concentration and RLU value, which can be used for quantitative detection (e.g., ...). Figure 1 (As shown).

[0117] 2. Construction of a standard curve for the detection of oil phase residues: Accurately weigh petrolatum standard to prepare a gradient solution of 0.1-10 μg / mL. GC detection was performed (HP-5 column, column temperature 150℃→250℃, FID detector), and the peak area was recorded. The fitted regression equation was: peak area = 4930C + 50 (R² = 0.999), with a detection limit of 0.05 μg / mL. This can be used for the quantitative determination of oil phase residues in ointments (e.g., ...). Figure 2 (As shown).

[0118] 3. Optimization of Compound Demulsifier Concentration: Span-80 and Tween-80 were compounded at a 1:1 ratio to prepare demulsifiers with gradient concentrations of 0.05%-0.15%, which were then used to treat erythromycin ointment emulsions (stirred at 37℃ for 10 min). The demulsification rate (oil-water separation volume ratio) and microbial survival rate (plate count) were measured: a 0.05% concentration resulted in a demulsification rate of 86.2%, a 0.1% concentration achieved a demulsification rate of 95.8% and a microbial survival rate of 95.0%, and a 0.15% concentration reduced the survival rate to 88.5%. The optimal demulsifier concentration was determined to be 0.1% (e.g., ...). Figure 3 (As shown).

[0119] 4. Optimization of enzymatic hydrolysis time:

[0120] The ointment matrix was treated with a 10 mg / mL compound enzyme solution (papain:lipase = 8:2) (shaking at 37℃), and the absorbance was measured at 600 nm at different times: the absorbance was 0.92 at 0 min, decreased to 0.09 at 20 min, and showed no significant change after 25 min. Using absorbance ≤0.1 as a threshold, the optimal enzymatic hydrolysis time was determined to be 20 min (e.g., ...). Figure 4 (As shown).

[0121] 5. Quantum Dot Detection Stability Verification: 2 nmol / L quantum dots were reacted with microbial ATP and allowed to stand at 25°C in the dark for 0-10 min. The RLU was measured: the RLU reached a peak of 16000 at 5 min and decreased to 13600 at 10 min (decay rate 15%), meeting the detection stability requirements. The optimal reaction time for quantum dots was determined to be 5 min (e.g., ...). Figure 5 (As shown).

[0122] 6. Optimization of Washing Parameters: The enzymatically digested sample was passed through a 0.22 μm filter membrane to enrich microorganisms, and then washed with PBS: In a volume gradient of 8-12 mL, the enrichment rate was 98.6% with a 10 mL volume (96.2% with 8 mL, 98.4% with 12 mL); in a wash rate gradient of 2-4 washes, the enrichment rate was 98.6% with 3 washes (97.0% with 2 washes, 98.3% with 4 washes). The optimal washing parameters were determined to be 10 mL volume + 3 washes (e.g., ...). Figure 6 (As shown).

[0123] 7. Applicability Validation for High Oil Content Samples: Simulated ointment samples with oil contents of 30%-70% were prepared and treated with both standard parameters (0.1% demulsifier + 20 min enzymatic hydrolysis) and high oil phase compatible parameters (0.15% demulsifier + 25 min enzymatic hydrolysis). The standard parameter group showed a demulsification rate of 90.0% and a dissociation rate of 96.0% at 70% oil content; the compatible parameter group showed a demulsification rate of 94.5% and a dissociation rate of 97.8%. This validates the applicability of the method to high oil phase ointments (e.g., ...). Figure 7 (As shown).

[0124] III. Experimental Conclusions: This experiment established a rapid microbial detection method for erythromycin ointment by constructing standard curves for microbial concentration-RLU and oil phase residue-GC, optimizing demulsification, enzymatic hydrolysis, quantum dot reaction, and rinsing parameters, and verifying the applicability to high oil content samples. The detection limit reached 10² CFU / mL, and the detection time was ≤1 h, meeting the pharmacopoeia and enterprise quality control requirements.

[0125] The present invention also includes a system for rapid detection of microbial limits in erythromycin ointment preparations, comprising a pretreatment module, an enzymatic hydrolysis and ultrasonication module, a neutralization module, a nano-fluorescence sensitization module, a filtration module, and a bioluminescence detection module connected in sequence. Each module is used to perform the corresponding sample pretreatment and demulsification, enzymatic hydrolysis and ultrasonic decapsulation, amphiphilic neutralization, nano-fluorescence sensitization, filtration enrichment, and bioluminescence detection steps in the above-described detection method, ultimately achieving rapid detection of microbial limits in erythromycin ointment preparations.

[0126] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. For those skilled in the art, any alternative improvements or transformations made to the implementation of the present invention fall within the protection scope of the present invention.

[0127] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A method for rapid detection of microbial limits in erythromycin ointment preparations, characterized in that, include: Step S1, Sample pretreatment and demulsification: Take the erythromycin ointment sample, add buffer solution and compound demulsifier, and stir in a water bath to achieve demulsification; Step S2, enzymatic hydrolysis and ultrasonic decoction: Lipase-protease complex enzyme was added to the system after step S1 and incubated, followed by ultrasonic treatment in a constant temperature water bath to achieve the dissociation of wax-encapsulated microorganisms. Step S3, Amphiphilic neutralization: Add polyoxyethylene castor oil-cysteine ​​copolymer neutralizer to the system treated in step S2, and remove oil phase residue after incubation; Step S4, Nano-fluorescence enhancement: Add CdTe quantum dot enhancement solution to the system treated in step S3, and enhance the luminescence signal after standing. Step S5, filtration and enrichment: The system after step S4 is filtered under negative pressure using a filter membrane and then rinsed with physiological saline containing Tween-20 to achieve microbial enrichment. Step S6, Bioluminescence Detection: Add luminescent reagent to the microorganisms enriched in step S5 and incubate. Detect the relative luminescence units and calculate the microbial concentration of the sample based on the standard curve.

2. The method according to claim 1, characterized in that, In step S1: the composite demulsifier is sodium dodecylbenzenesulfonate and isopropanol mixed at a mass ratio of 3:2, with a total concentration of 0.08%-0.12%; the buffer solution is a phosphate buffer with a pH of 7.2-7.4 and a concentration of 0.01 mol / L; the ratio of buffer volume to sample mass is 4 mL: 1 g; and the water bath stirring time is 10 min. Demulsification rate ≥ 95%, calculated using the following formula: Demulsification rate (%) = (A0 − A t ) / A0×100, where: A0 is the absorbance of the system before demulsification at a wavelength of 600nm, A t The absorbance of the system after demulsification is measured at a wavelength of 600 nm.

3. The method according to claim 2, characterized in that: The compound enzyme mentioned in step S2 is a mixture of lipase and protease at a mass ratio of 3:1, with a total concentration of 10 mg / mL; the enzymatic incubation time is 20 min, and the ultrasonic treatment time is 8 min. The enzymatic hydrolysis was terminated when the absorbance at 600 nm dropped below 0.

1.

4. The method according to claim 3, characterized in that, In step S3: the concentration of the neutralizing agent of the polyoxyethylene castor oil-cysteine ​​copolymer is 1.0%-1.4%, the molar ratio of polyoxyethylene castor oil to cysteine ​​is 5:1, the molecular weight of the copolymer is 5000-8000 Da; the incubation time is 12-18 min; Oil phase residue removal rate ≥90%, the oil phase residue removal rate is calculated by the following formula: Oil phase residue removal rate (%) = (C0 − C t ) / C0×100; where: C0 is the residual concentration of the oil phase in the system before neutralization, C t This represents the residual concentration of the oil phase in the neutralized system.

5. The method according to claim 4, characterized in that... In step S4: the concentration of the CdTe quantum dot sensitizing solution is 2-3 nmol / L, the quantum dot particle size is 2-5 nm, and the surface is modified with mercaptoacetic acid.

6. The method according to claim 5, characterized in that, In step S5: the filter membrane is a mixed cellulose ester membrane with a pore size of 0.45 μm; the concentration of physiological saline containing Tween-20 is 0.01%; the volume of the rinsing solution is 8-12 mL; and the number of rinsing times is 2-4.

7. The method according to claim 6, characterized in that, In step S6: the luminescent reagent is a luciferase-luciferin system, the luciferase is derived from fireflies and has an activity ≥1000U / mL; the luciferin is D-luciferin with a concentration of 0.1mmol / L; the incubation time is 1-3min; the detection is performed using a multifunctional microplate reader with a detection wavelength of 450-560nm.

8. The method according to claim 7, characterized in that, The method is applicable to the detection of erythromycin ointment preparations with different oil contents; when detecting high-oil-phase erythromycin ointments with an oil content ≥50%, the ratio of buffer solution to sample mass in step S1 is adjusted to 5mL:1g, and the concentration of the composite demulsifier is increased to 0.12%; In step S2, the enzymatic incubation time is extended to 30 min, and the ultrasonic treatment time is extended to 10 min; In step S3, the concentration of the neutralizing agent is adjusted to 1.4%, and the incubation time is extended to 20 minutes.

9. The method according to claim 8, characterized in that: The ultrasonic treatment process in step S2 is carried out in a constant temperature water bath environment, with the water bath temperature being the same as the enzymatic hydrolysis incubation temperature of 37℃; during the ultrasonic treatment, the system is shaken in a constant temperature shaker every 2 minutes; the system temperature is monitored in real time by an insertion thermocouple thermometer and maintained at 37±0.2℃.

10. The method according to claim 9, characterized in that: The rinsing solution is added slowly along the edge of the filter membrane at a rate of 1 mL / s. After rinsing, let it stand for 30 seconds before turning off the negative pressure. After removing the filter membrane, transfer it to the test container using aseptic techniques.

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