Sodium pyrosulfite microbial limit detection method

Abstract

The invention discloses a sodium pyrosulfite microbial limit detection method, and relates to the technical field of microbial detection. Aiming at the characteristics of strong reducibility and acidity of sodium metabisulfite, the method realizes accurate detection through pH regulation to neutralize acidity and sodium sulfite oxidase to eliminate the reducibility, and then through microorganism enrichment, membrane filtration and selective culture medium culture. The method solves the problems of microorganism survival inhibition and inaccurate detection result in the traditional method, has the advantages of high detection precision, good repeatability and simplicity and convenience in operation, and is suitable for microbial limit detection of the sodium pyrosulfite raw material as well as food additives, medicine auxiliary materials and the like containing the sodium pyrosulfite raw material.

Description

Technical Field

[0001] This application relates to the field of microbial detection technology, specifically to a method for testing the microbial limits of sodium metabisulfite. Background Technology

[0002] Sodium metabisulfite (Na₂S₂O₂) is a commonly used food additive and pharmaceutical excipient with preservative, antioxidant, and bleaching properties, and is widely used in food processing, pharmaceutical manufacturing, and other industries. However, sodium metabisulfite is susceptible to microbial contamination during production and storage. If microbial limits are exceeded, product quality will be affected, and even human health may be harmed. Therefore, establishing accurate and reliable methods for testing the microbial limits of sodium metabisulfite is crucial.

[0003] Traditional methods for microbial limit testing mainly include plate counting and membrane filtration. However, directly applying these methods to sodium metabisulfite presents several problems: First, sodium metabisulfite has strong reducing properties, which can destroy nutrients in the culture medium and inhibit microbial growth and reproduction, leading to lower counts. Second, sodium metabisulfite dissolves in water to form an acidic solution (pH approximately 3.5-4.5), and this acidic environment adversely affects the survival of most microorganisms, further reducing the accuracy of the test. Furthermore, sodium metabisulfite readily decomposes in water to produce sulfur dioxide, and the resulting bubbles can interfere with filtration and colony observation, affecting the stability of the testing process.

[0004] In existing technologies, some studies have attempted to reduce interference by lowering the concentration of sodium metabisulfite through dilution. However, excessive dilution can lead to undetectable low concentrations of microorganisms, resulting in insufficient detection sensitivity. Other studies have used neutralizing agents to neutralize acidity, but these studies have not considered the impact of reducing properties on microbial growth, leading to significant errors in the detection results. Therefore, there is an urgent need for a microbial limit testing method that can effectively eliminate the reducing and acidic interference of sodium metabisulfite while ensuring both detection sensitivity and accuracy. Summary of the Invention

[0005] To address the problems of reducing and acidic interference, low detection accuracy, and insufficient sensitivity in existing methods for testing the microbial limits of sodium metabisulfite, this application provides a method for testing the microbial limits of sodium metabisulfite. Through targeted pretreatment steps and optimized detection procedures, it achieves accurate detection of microorganisms in sodium metabisulfite.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] A method for testing the microbial limits of sodium metabisulfite includes steps such as sample preparation, pretreatment, microbial enrichment, filtration, culture, and counting, as detailed below:

[0008] Weigh out the sodium metabisulfite sample and add it to a pH 7.0 sterile sodium chloride-peptone buffer solution. Dissolve the sample by shaking to obtain the initial sample solution. The mass-to-volume ratio of the sample to the sterile diluent is 1 g: 10 mL. Choosing a pH 7.0 sterile sodium chloride-peptone buffer solution as the diluent can initially maintain the stability of the system and reduce the initial impact of acidity on microorganisms.

[0009] Add 1 mol / L sterile sodium hydroxide solution to the initial sample solution, and adjust the pH to 6.8-7.2 using a sterile pH meter for real-time monitoring. Then add sterile sodium sulfite oxidase solution with an enzyme activity of 100 U / mL (the amount added is 1%-2% of the initial sample volume), and incubate at 37℃ for 10-15 min. pH adjustment can effectively neutralize the acidity of sodium metabisulfite, and sodium sulfite oxidase can oxidize the sulfite ions produced by the decomposition of sodium metabisulfite to sulfate ions, eliminating reducing interference and providing a suitable acid-base and nutrient environment for microbial growth.

[0010] The pretreatment solution was transferred to sterile nutrient broth and incubated at 37°C with shaking at 150-180 rpm for 4-6 hours. Shaking incubation promotes sufficient contact between microorganisms and nutrients, enabling rapid enrichment of microorganisms, improving detection sensitivity, and avoiding missed detection of low-concentration microorganisms.

[0011] A sterile filter membrane with a pore size of 0.45 μm was used to filter the enriched solution under a negative pressure of 0.03-0.05 MPa. After filtration, the filter membrane was rinsed three times with 10 mL of sterile physiological saline each time. Membrane filtration can retain microorganisms on the filter membrane, and the rinsing step can remove residual sodium metabisulfite and culture medium components, avoiding interference with subsequent culture.

[0012] The filter membrane was attached to tryptic soy agar medium (for aerobic bacteria counting) and Sabouraud dextrose agar medium (for mold and yeast counting), respectively. Aerobic bacteria were incubated at 37°C for 48-72 h, while molds and yeasts were incubated at 25-28°C for 72-96 h. After the incubation period, the number of colonies was counted to obtain the microbial limit results.

[0013] Furthermore, the conditions for oscillation dissolution are: oscillation at 200-250 r / min for 10-15 min.

[0014] Furthermore, a sterile pH meter is used for real-time monitoring during pH adjustment to ensure pH adjustment accuracy of ±0.1.

[0015] Furthermore, the sodium sulfite oxidase solution must be prepared within 30 minutes before use and temporarily stored in a refrigerated environment at 4°C after preparation.

[0016] Furthermore, the oscillation rate for the oscillation culture is 150-180 r / min.

[0017] Furthermore, the medium-nutrient broth culture medium needs to be sterilized by high-pressure steam at 121℃ for 20 minutes before use, and then cooled to 37℃±1℃ before adding the pretreatment solution.

[0018] Furthermore, the membrane filtration uses a negative pressure filtration device, with the negative pressure controlled at 0.03-0.05 MPa.

[0019] Furthermore, the aerobic bacteria count medium was tryptic soy agar, and the mold and yeast count medium was Sabouraud dextrose agar.

[0020] Furthermore, the volume ratio of the pretreatment solution to the nutrient broth culture medium was 1:5.

[0021] Furthermore, when counting colonies, filter membranes with colony counts in the range of 30-300 CFU should be selected for counting. If the colony counts of all filter membranes exceed this range, the sample dilution factor should be readjusted and the test should be repeated.

[0022] Beneficial technical effects:

[0023] This application addresses the problem of sodium metabisulfite's inhibition of microbial growth at its root by neutralizing its acidity through pH adjustment and eliminating its reducing properties through sodium sulfite oxidase in the pretreatment step, thus ensuring detection accuracy. Combined with the microbial enrichment step, it enables rapid proliferation of low-concentration microorganisms, effectively improving detection sensitivity. Parameter optimization in key steps such as filtration and culturing ensures stable and highly repeatable detection processes. The entire process is clearly defined, requires no complex equipment, and is easy to routinely apply in laboratories. Attached Figure Description

[0024] Figure 1 Flowchart of a method for testing the microbial limit of sodium metabisulfite Detailed Implementation

[0025] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application will be provided below.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of this application. However, this application may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0027] Example 1

[0028] like Figure 1 As shown in the figure, this embodiment provides a method for testing the microbial limit of sodium metabisulfite, including the following steps:

[0029] Weigh 10.0g of food-grade sodium metabisulfite sample (production batch: 20250601, purity ≥97%) and add it to 100mL of pH7.0 sterile sodium chloride-peptone buffer (pre-sterilized at 121℃ for 20min and cooled to room temperature). Place the buffer in a constant temperature shaker and shake at 220r / min for 12min to ensure that the sample is completely dissolved and no bubbles are generated, thus obtaining the initial sample solution. The entire operation is carried out in a Class 100 sterile clean bench to avoid external microbial contamination.

[0030] In a Class 100 clean bench, 1 mol / L sterile sodium hydroxide solution (filtered through a 0.22 μm filter membrane for sterilization) was slowly added dropwise to the initial sample solution using a sterile pipette. At the same time, the pH of the solution was monitored in real time using a sterile pH meter (accuracy ±0.1) and the pH was adjusted to 7.0. Then, 1.5 mL of sterile sodium sulfite oxidase solution (enzyme activity 100 U / mL, prepared within 30 min before use and stored at 4℃) was added. The solution was transferred to a sterile conical flask and incubated in a 37℃ incubator for 12 min to obtain the pretreatment solution.

[0031] Take 20 mL of pretreatment solution and transfer it into 100 mL of sterile nutrient broth (sterilized by autoclaving at 121℃ for 20 min and cooled to 37℃±1℃). The volume ratio of pretreatment solution to nutrient broth is 1:5. Place the conical flask in a constant temperature shaking incubator and shake at 37℃ and 160 r / min for 5 h. During this period, ensure that the shaking is uniform so that the microorganisms can fully contact the nutrients to obtain the enrichment solution.

[0032] In a Class 100 clean bench, a negative pressure filtration device was assembled. A sterile filter membrane with a pore size of 0.45 μm (pre-sterilized by dry heat at 121℃ for 2 hours) was installed in the filter cup. The enrichment solution was slowly poured into the filter cup, and the negative pressure pump was turned on to control the negative pressure at 0.04 MPa for filtration. After filtration, the filter membrane was rinsed 3 times with 10 mL of sterile physiological saline (sterilized by high-pressure steam at 121℃ for 20 minutes). After each rinse, the filter membrane was allowed to dry before the next rinse was performed to remove residual sodium metabisulfite and culture medium components.

[0033] Using sterile forceps, pick up the rinsed filter membranes and affix them to tryptic soy agar medium (for aerobic bacteria counting, after...). The filter membrane was sterilized by autoclaving for 20 minutes and by autoclaving at 121°C for 20 minutes on Sabouraud dextrose agar (for mold and yeast counting). The surface of the filter membrane was ensured to be tightly adhered to the agar and free of air bubbles. Tryptic soy peptone agar was incubated at 37°C for 60 hours and Sabouraud dextrose agar was incubated at 26°C for 84 hours. After incubation, filter membranes with colony counts in the range of 30-300 CFU were selected and counted using a colony counter. Three parallel experiments were performed for each sample.

[0034] The average total aerobic bacteria count was 85 CFU / g, and the relative standard deviation (RSD) of the three parallel experiments was 3.2%. The average total mold and yeast count was 11 CFU / g, with an RSD of 3.8%. The colony morphology was regular, with no shrinkage or abnormal growth. The test results met the microbial limit requirements in GB1886.7-2015 "National Food Safety Standard for Food Additives Sodium Metabisulfite" (total aerobic bacteria count ≤100 CFU / g, total mold and yeast count ≤20 CFU / g).

[0035] Example 2

[0036] like Figure 1 As shown in the figure, this embodiment provides a method for testing the microbial limit of sodium metabisulfite, including the following steps:

[0037] Weigh 10.0 g of pharmaceutical-grade sodium metabisulfite sample (production batch: 20250608, conforming to the Chinese Pharmacopoeia 2020 edition, Part IV standard), add it to 100 mL of pH 7.0 sterile sodium chloride-peptone buffer, and shake at 250 r / min for 10 min to ensure complete dissolution of the sample to obtain the initial sample solution; the buffer used has been sterilely validated in advance to confirm that there is no microbial contamination.

[0038] Add 1 mol / L sterile sodium hydroxide solution to the initial sample solution to adjust the pH to 6.8; add 2.0 mL of sterile sodium sulfite oxidase solution (enzyme activity 100 U / mL), and incubate at 37℃ for 15 min to obtain the pretreatment solution; gently shake the conical flask every 5 min during incubation to ensure the enzyme reaction is complete.

[0039] Transfer 20 mL of the pretreatment solution into 100 mL of sterile nutrient broth and incubate at 37 °C and 180 r / min for 6 h to obtain the enrichment solution. Observe the state of the solution during the incubation process to ensure that there is no turbidity or precipitation (if any abnormality occurs, prepare the sample again).

[0040] A sterile filter membrane with a pore size of 0.45 μm was used to filter the enriched solution under a negative pressure of 0.03 MPa. During rinsing, the surface of the filter membrane was slowly rinsed with sterile physiological saline to avoid dispersing the trapped microorganisms. The rinsing was performed 3 times, with 10 mL each time.

[0041] The filter membranes were attached to tryptic soy agar and Sabouraud dextrose agar, respectively. Aerobic bacteria were incubated at 37°C for 72 hours, while molds and yeasts were incubated at 25°C for 96 hours. Because the expected microbial content of the samples was low, a magnifying glass was used to assist in the observation of the filter membrane during counting to avoid missing areas with low colony counts. Three sets of parallel experiments were performed for each sample.

[0042] The average total aerobic bacteria count was 16 CFU / g, with an RSD of 4.1%; total mold and yeast counts were not detected. It meets the microbial limits for pharmaceutical excipient sodium metabisulfite as specified in the 2020 edition of the Chinese Pharmacopoeia, Part IV (total aerobic bacteria ≤100 CFU / g, total mold and yeast ≤10 CFU / g); parallel experiments showed no cross-contamination, and the test results were stable and reliable.

[0043] Example 3

[0044] like Figure 1 As shown in the figure, this embodiment provides a method for testing the microbial limit of sodium metabisulfite, including the following steps:

[0045] For the compound food additive sample (production batch: 20250615, containing 30% sodium metabisulfite, 20% citric acid, and 50% sucrose), weigh 33.3g of the sample (equivalent to 10.0g sodium metabisulfite) and add it to 100mL of pH 7.0 sterile sodium chloride-peptone buffer. Shake at 200r / min for 15min, gently stirring with a sterile glass rod during the process to help the citric acid and sucrose completely dissolve, to obtain the initial sample solution. After dissolution, observe the solution to ensure that there are no undissolved particles remaining.

[0046] Add 1 mol / L sterile sodium hydroxide solution to the initial sample solution to adjust the pH to 7.2 (because the sample contains citric acid, the amount of sodium hydroxide solution needs to be increased appropriately, and the pH should be monitored in real time to avoid over-adjustment); add 1.0 mL of sterile sodium sulfite oxidase solution, and incubate at 37℃ for 10 min to obtain the pretreatment solution; after incubation, take a small amount of the pretreatment solution and use pH test paper to quickly verify whether the pH is within the range of 6.8-7.2.

[0047] Take 20 mL of pretreatment solution and transfer it into 100 mL of sterile nutrient broth medium. Incubate at 37 °C and 150 r / min for 4 h with shaking to obtain enrichment solution. Because the matrix of the compound sample is complex, gently blow the pretreatment solution with a sterile pipette before incubation to avoid matrix components from agglomerating and affecting microbial enrichment.

[0048] Filter the enriched solution under a negative pressure of 0.05 MPa, and control the filtration rate at 5-10 mL / min to avoid clogging of the filter membrane due to excessive flow rate. When rinsing the filter membrane, ensure that physiological saline covers the entire area of ​​the filter membrane, paying particular attention to the edges of the filter membrane to remove residual matrix components such as citric acid and sucrose.

[0049] After attaching the filter membrane to the corresponding culture medium, observe the colony growth regularly during the culture period to distinguish between target colonies (aerobic bacteria are milky white or pale yellow, molds are fluffy, and yeasts are round and raised) and mottling formed by matrix interference (irregular shape, no proliferation); count after culture, perform 3 parallel experiments for each sample, and convert the results to sodium metabisulfite content.

[0050] The average total aerobic bacteria count after conversion was 92 CFU / g (calculated as sodium metabisulfite), with an RSD of 3.7%; the average total mold and yeast count was 12 CFU / g (calculated as sodium metabisulfite), with an RSD of 4.2%. Interference from citric acid and sucrose on colony observation was successfully eliminated, and the test results met the microbiological quality control standards for compound food additives (total aerobic bacteria count). The total number of molds and yeasts is ≤20 CFU / g.

[0051] Comparative Example 1

[0052] This embodiment provides a method for testing the microbial limit of sodium metabisulfite, including the following steps:

[0053] Weigh 10.0g of food-grade sodium metabisulfite sample (production batch: 20250601, purity ≥97%), add it to 100mL of sterile physiological saline (replacing pH 7.0 sterile sodium chloride-peptone buffer), place it in a constant temperature shaker, and shake at 220r / min for 12min to obtain the sample solution; the entire operation is carried out in a Class 100 sterile clean bench to avoid external microbial contamination.

[0054] In a Class 100 clean bench, 100 mL of sample solution was directly poured into a negative pressure filtration device without pH adjustment or sodium sulfite oxidase treatment. A sterile filter membrane with a pore size of 0.45 μm (pre-sterilized by dry heat at 121°C for 2 hours) was used for filtration under a negative pressure of 0.04 MPa. After filtration, the filter membrane was rinsed twice with 10 mL of sterile physiological saline (sterilized by high-pressure steam at 121°C for 20 minutes). After each rinse, the filter membrane was allowed to dry before the next rinse was performed.

[0055] Using sterile forceps, pick up the rinsed filter membranes and affix them to tryptic soy agar medium (for aerobic bacteria counting, after...). The surface of the filter membrane was sterilized by autoclaving for 20 min and by autoclaving at 121℃ for 20 min on Sabouraud dextrose agar (for mold and yeast counting). Ensure that the filter membrane is in close contact with the culture medium without air bubbles. Tryptic soy peptone agar was incubated at 37℃ for 60 h and Sabouraud dextrose agar was incubated at 26℃ for 84 h. After incubation, colony counts were performed using a colony counter, and three parallel experiments were conducted for each sample.

[0056] The average total number of aerobic bacteria was 38 CFU / g, with an RSD of 12.5% ​​for the three parallel experiments; the average total number of molds and yeasts was 4 CFU / g, with an RSD of 15.2%; during filtration, a large number of tiny bubbles were generated due to the decomposition of sodium metabisulfite, leading to local blockage of the filter membrane; after cultivation, the colony morphology was irregular, and some colonies were shrunken due to acid and reducing inhibition, resulting in significantly lower test results and poor stability, which could not reflect the true microbial contamination of the sample.

[0057] Comparative Example 2

[0058] This embodiment provides a method for testing the microbial limit of sodium metabisulfite, including the following steps:

[0059] Weigh 10.0g of food-grade sodium metabisulfite sample (production batch: 20250601, purity ≥97%) and add it to 900mL of sterile physiological saline (sterilized by autoclaving at 121℃ for 20min and cooled to room temperature). Place the saline in a constant temperature shaker and shake at 220r / min for 12min to obtain the sample dilution. The entire operation is carried out in a Class 100 sterile clean bench to avoid external microbial contamination.

[0060] In a Class 100 clean bench, 1 mol / L sterile sodium hydroxide solution (filtered through a 0.22 μm filter membrane for sterilization) was slowly added dropwise to the sample dilution using a sterile pipette. At the same time, the pH of the solution was monitored in real time using a sterile pH meter (accuracy ±0.1) and the pH was adjusted to 7.0 to obtain a 10-fold dilution. Without adding sodium sulfite oxidase solution, 100 mL of the dilution was directly transferred to 100 mL of sterile nutrient broth culture medium (sterilized by autoclaving at 121℃ for 20 min and cooled to 37℃±1℃).

[0061] The conical flask was placed in a constant temperature shaking incubator and shaken at 37℃ and 160r / min for 5h to obtain the enriched solution. In a Class 100 clean bench, a negative pressure filtration device was assembled. A sterile filter membrane with a pore size of 0.45μm (pre-sterilized by dry heat at 121℃ for 2h) was installed in the filter cup. The enriched solution was slowly poured into the filter cup, and the negative pressure pump was turned on to control the negative pressure at 0.04MPa for filtration. After filtration, the filter membrane was rinsed 3 times with 10mL of sterile physiological saline (sterilized by high pressure steam at 121℃ for 20min). After each rinse, the filter membrane was allowed to dry before the next rinse was performed.

[0062] Using sterile forceps, pick up the rinsed filter membranes and affix them to tryptic soy agar medium (for aerobic bacteria counting, after...). The surface of the filter membrane was sterilized by autoclaving for 20 min and by autoclaving at 121℃ for 20 min on Sabouraud dextrose agar (for mold and yeast counting). Ensure that the filter membrane is in close contact with the culture medium without air bubbles. Tryptic soy peptone agar was incubated at 37℃ for 60 h and Sabouraud dextrose agar was incubated at 26℃ for 84 h. After incubation, colony counts were performed using a colony counter, and three parallel experiments were conducted for each sample.

[0063] The average total aerobic bacteria count was 45 CFU / g, with an RSD of 10.8% for the three parallel experiments; the average total mold and yeast count was 5 CFU / g, with an RSD of 13.6%. Due to the absence of sodium sulfite oxidase to eliminate reducing properties, proteins in the culture medium were oxidized and degraded, resulting in slow colony growth and a small number of colonies. In addition, the 10-fold dilution caused the low concentration of microorganisms in the original sample to be diluted below the detection limit, posing a risk of missed detection. The accuracy and sensitivity of the detection results were both insufficient.

[0064] Examples 1-3 respectively target food-grade sodium metabisulfite raw materials, pharmaceutical-grade sodium metabisulfite excipients, and compound food additives containing sodium metabisulfite. The sample preparation, pretreatment, microbial enrichment, filtration, culture, and counting steps of the method in this application were strictly followed. Interference was eliminated through dual methods of pH adjustment and sodium sulfite oxidase treatment. Combined with optimized enrichment, filtration, and culture parameters, accurate detection was achieved. The RSD of the detection results was ≤4.2%, and they met the microbial limit standards for the corresponding products, demonstrating the accuracy, stability, and wide applicability of the method.

[0065] Comparative Example 1 did not undergo pH adjustment and reducing agent elimination treatment. It was directly filtered and cultured after dilution with sterile physiological saline. Due to the acidity and reducing properties of sodium metabisulfite inhibiting microbial growth, and the bubbles generated during filtration interfering with the operation, the test results were significantly lower, with an RSD ≥ 12.5%, and extremely poor stability.

[0066] Comparative Example 2 only neutralized the acidity but did not eliminate the reducing properties. Furthermore, the sodium metabisulfite concentration was reduced by a 10-fold dilution, which led to the destruction of nutrients in the culture medium and the missed detection of low-concentration microorganisms. The accuracy and sensitivity of the detection results were insufficient, with an RSD ≥ 10.8%, and it could not reflect the true contamination of the sample.

[0067] It should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the spirit and scope of the technical solutions of this application, and all such modifications and substitutions should be covered within the scope of the claims of this application.

Claims

1. A method for sodium metabisulfite microbial limit test, characterized by, It comprises the following steps: A sodium pyrosulfite sample is weighed, added to a sterile diluent, and shaken to dissolve, to obtain a sample initial solution; the sterile diluent is a pH 7.0 sterile sodium chloride-peptone buffer, and the mass-volume ratio of the sample to the sterile diluent is 1 g:10 mL; A sterile sodium hydroxide solution is added to the sample initial solution obtained above, to adjust the pH to 6.8-7.2, and then a sterile sodium sulfite oxidase solution is added, and the mixture is incubated at 37°C for 10-15 min, to obtain a pretreatment solution; the concentration of the sterile sodium hydroxide solution is 1 mol / L, the enzyme activity of the sterile sodium sulfite oxidase solution is 100 U / mL, and the addition amount is 1%-2% of the volume of the sample initial solution; The pretreatment solution obtained above is transferred to a sterile nutrient broth culture medium, and shaken to culture at 37°C for 4-6 h, to obtain an enrichment solution; The enrichment solution obtained above is subjected to membrane filtration using a sterile filter membrane with a pore size of 0.45 μm, and after the filtration is completed, the filter membrane is washed with sterile normal saline for 3 times, with a washing volume of 10 mL each time; The washed filter membrane is attached to an aerobic bacteria total count culture medium and a mold and yeast total count culture medium, respectively, the aerobic bacteria culture condition is 37°C for 48-72 h, the mold and yeast culture condition is 25-28°C for 72-96 h, and after the culture is completed, colony counting is performed, to obtain the microbial limit results of the sodium pyrosulfite.

2. A method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The shaking dissolution condition is shaking at 200-250 r / min for 10-15 min.

3. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, When the pH is adjusted, a sterile pH meter is used for real-time monitoring, to ensure that the pH adjustment accuracy is ±0.

1.

4. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The sodium sulfite oxidase solution needs to be prepared within 30 min before use, and is temporarily stored in a 4°C cold storage environment after preparation.

5. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The shaking culture rate is 150-180 r / min.

6. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The nutrient broth culture medium needs to be sterilized by 121°C high-pressure steam for 20 min before use, and after sterilization, the medium is cooled to 37°C±1°C before the pretreatment solution is added.

7. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The membrane filtration is performed using a negative pressure filtration device, and the negative pressure is controlled at 0.03-0.05 MPa.

8. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The aerobic bacteria total count culture medium is a tryptone soy peptone agar culture medium, and the mold and yeast total count culture medium is a sabouraud dextrose agar culture medium.

9. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, The volume ratio of the pretreatment solution to the nutrient broth culture medium is 1:

5.

10. The method for sodium metabisulfite microbial limit test according to claim 1, characterized in that, When the colony counting is performed, the filter membrane with a number of colonies in the range of 30-300 CFU is selected for counting, and if the number of colonies on all filter membranes exceeds the range, the sample dilution factor needs to be adjusted and the detection is performed again.

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