Quantitative fresh culture bacterial liquid of staphylococcus aureus and preparation method of quantitative fresh culture bacterial liquid

Abstract

The invention provides a quantifiable staphylococcus aureus fresh culture bacterial liquid which is different from a traditional culture medium and is obtained by limiting growth of staphylococcus aureus through vitamin C and a preparation method of the quantifiable staphylococcus aureus fresh culture bacterial liquid. The staphylococcus aureus fresh culture bacterial liquid comprises a bacterial strain, a nitrogen source, a carbon source, inorganic salt and a limiting factor. On the basis of reducing components of a carbon source, a nitrogen source and an energy source, a limiting factor vitamin C is added into the culture medium, and the growth trend of the staphylococcus aureus can be accurately controlled by controlling the concentration of the vitamin C, so that a staphylococcus aureus fresh culture bacterium solution with the strain concentration of less than or equal to 1000cfu / 100mu L is accurately and efficiently obtained; therefore, the requirements of Chinese pharmacopoeia are met, the requirement that the generations of working strains do not exceed 5 generations is met, and a fresh culture with the colony count meeting the specification of the pharmacopoeia can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and in particular relates to a quantifiable fresh culture solution of Staphylococcus aureus and a preparation method thereof. Background Art

[0002] When pharmaceutical companies conduct new product testing and method suitability checks, there are two methods for medium suitability checks and method suitability checks:

[0003] One method is to use commercial freeze-dried bacterial pellets. Although the quality characteristics of the strain and the requirements for stable colony count (not more than 100 cfu) are met during the suitability check of the new product method, it cannot 100% meet the requirements of the Chinese Pharmacopoeia for the use of strains: that is, "preparation of bacterial suspension using fresh culture obtained after cultivation."

[0004] Another method is to use the 0-generation standard strain to perform medium suitability checks and method suitability checks, but this method is cumbersome to operate and has the risk of failure of the suitability check and method suitability check.

[0005] Based on the above considerations, how to efficiently prepare fresh cultures that meet specific quality characteristics and colony counts in the bacterial suspension preparation process has become increasingly urgent. Summary of the invention

[0006] The object of the present invention is to provide a quantifiable fresh culture solution of Staphylococcus aureus and a preparation method thereof.

[0007] In order to achieve the above application purpose, the technical solution adopted by the present invention is as follows:

[0008] The invention provides a quantifiable fresh culture bacterial liquid of Staphylococcus aureus, the components of which include bacterial strains, nitrogen sources, carbon sources, inorganic salts and limiting factors.

[0009] Furthermore, the strain is Staphylococcus aureus, and the limiting factor is vitamin C. For the specific quality control bacteria Staphylococcus aureus in a low-nutrient specific culture medium environment, by controlling the concentration of vitamin C, the growth trend of Staphylococcus aureus can be accurately controlled, thereby efficiently obtaining a fresh culture solution of Staphylococcus aureus with a strain concentration of ≤1000 cfu / 100 μL.

[0010] Furthermore, every 100 ml of the quantifiable fresh culture solution of Staphylococcus aureus contains 5-100 cfu / 100 μL of the strain, 1-8 g of a nitrogen source, 1-2 g of a carbon source, 0.1-0.5 g of an inorganic salt, and 0.05-1.5 g of a limiting factor.

[0011] The Staphylococcus aureus quantitative fresh culture liquid of the present invention has a carbon source comprising sucrose and / or glucose; the nitrogen source comprising one or more of gelatin, tryptone and skimmed milk powder; and the inorganic salt comprising one or more of dipotassium hydrogen phosphate, magnesium chloride and sodium chloride.

[0012] The Staphylococcus aureus quantitative fresh culture liquid of the present invention comprises the following steps:

[0013] S1: Preparation of freeze-dried pellets of Staphylococcus aureus;

[0014] S2: Preparation of culture medium suitable for quantitative fresh culture of Staphylococcus aureus;

[0015] S3: Add one freeze-dried pellet of Staphylococcus aureus with a colony count of no more than 100 cfu / pellet into a culture medium suitable for quantitative fresh culture of Staphylococcus aureus for culture at a temperature of 30-35°C for a period of 20h±1h.

[0016] Furthermore, the preparation method of the freeze-dried Staphylococcus aureus pellets is as follows:

[0017] S1: Inoculate the standard strain of Staphylococcus aureus into TSB medium and culture it at 30-35°C for 18-24 hours for bacterial enrichment;

[0018] S2: After the bacterial enrichment culture, a fresh culture with a high colony count is obtained. The grown bacteria are the second-generation standard stock bacteria. After the fresh culture is shaken by an oscillator until it is uniform, 0.9% NaCl is used as a diluent to dilute the fresh culture in a 1:10 manner;

[0019] S3: If the turbidity of the fresh culture stock solution is greater than 4.0McF, then conduct a turbidity test on the diluted 1:10 bacterial suspension and control the turbidity of the 1:10 bacterial suspension to 0.9-1.5McF; then perform subsequent dilutions until the dilution reaches 1:10 -6 . ;

[0020] S4: Select dilution to 10 -4 , 10 -5 and 10 -6 The bacterial suspension was inoculated on TSA solid medium plates, cultured at 30-35°C for 3 days, and the colonies were counted;

[0021] S5: Determine the dilution gradient corresponding to the colony count of the dilution solution ≤ 100 cfu by observing the number of TSA growing colonies;

[0022] S6: Based on the results of S4 and S5, determine the dilution gradient corresponding to the accurate turbidity value of the 1:10 dilution;

[0023] S7: Repeat steps S1-S6, and according to the dilution gradient of the colony count less than 100 cfu, calculate the corresponding dilution concentration of the bacterial suspension and add it to the stabilizer, and the ratio of the diluted bacterial suspension to the stabilizer is 1:99;

[0024] S8: freeze-dry the mixed solution in step 7 using a microsphere forming apparatus and a freeze-drying apparatus to prepare freeze-dried bacterial pellets, with a colony count of ≤100 cfu / bacterial pellet.

[0025] Furthermore, the stabilizer composition of the present invention includes a protective agent, a filler, an antioxidant and an osmotic pressure regulator, wherein the protective agent is composed of glucose, trehalose and (2-hydroxypropyl)-β-cyclodextrin, the filler is composed of mannitol, the antioxidant is composed of skim milk powder, and the osmotic pressure regulator is composed of dipotassium hydrogen phosphate trihydrate. The formula of the specific components of the stabilizer is as follows:

[0026] Table 1

[0027]

[0028]

[0029] The components and proportions of the culture medium suitable for quantitative fresh culture of Staphylococcus aureus of the present invention are as follows: 2-6g of nitrogen source, 1-2g of carbon source, 0.2-0.4g of inorganic salt, 0.1-1g of limiting factor, and the balance is water per 100 ml of the culture medium. The carbon source of the present invention is glucose, the nitrogen source is tryptone, and the inorganic salt is dipotassium hydrogen phosphate trihydrate.

[0030] The beneficial effects of the present invention are as follows:

[0031] The present invention provides a method for preparing a quantifiable fresh culture solution of Staphylococcus aureus. On the basis of reducing carbon source, nitrogen source and energy components, a limiting factor vitamin C is added to a culture medium. By controlling the concentration of vitamin C, the growth trend of Staphylococcus aureus can be accurately controlled, so that a fresh culture solution of Staphylococcus aureus with a strain concentration of ≤1000 cfu / 100 μL can be accurately and efficiently obtained, so as to meet the requirements of the Chinese Pharmacopoeia, achieve the requirement that the generation number of the working strain shall not exceed 5 generations, and obtain a fresh culture with a colony count that meets the requirements of the Pharmacopoeia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is a graph showing the effect of limiting factor vitamin C on the growth of Staphylococcus aureus. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0035] Example 1: Preparation of a culture medium suitable for quantitative fresh culture of Staphylococcus aureus

[0036] As shown in Table 2, each 100 ml culture medium contains 2 g of gelatin, 0.5 g of tryptone, 0.15 g of skimmed milk powder, 0.5 g of sucrose, 0.5 g of glucose, 0.2 g of dipotassium hydrogen phosphate trihydrate, and the balance is water. The concentration of the limiting factor vitamin C in experimental groups 1-4 is different, which are 0.2%, 0.3%, 0.4%, and 0.5%, respectively.

[0037] Table 2

[0038]

[0039]

[0040] Gelatin, tryptone, skim milk powder, sucrose, glucose, potassium hydrogen phosphate trihydrate, vitamin C and water were mixed at 25° C. for 3 minutes to obtain a culture medium suitable for quantitative fresh culture of Staphylococcus aureus.

[0041] Example 2: Preparation of freeze-dried Staphylococcus aureus pellets

[0042] 1. Produce a single dose of freeze-dried bacterial pellets (0.10x10 2 ~1.0x10 2 / a ball), the freeze-dried ball size was controlled at 20 μL. The bacteria used in this example were Staphylococcus aureus.

[0043] 2. Preparation of bacterial suspension: Prepare working standard according to the requirements of the Chinese Pharmacopoeia. First, inoculate the 0th generation standard bacteria into the liquid culture medium TSB according to the Chinese Pharmacopoeia, obtain fresh bacterial suspension under the specified culture temperature and time conditions, and then use 0.9% sterile NaCl as a diluent to dilute the bacterial suspension step by step, adjust the concentration of the bacterial suspension to control the turbidity at 1.0±0.1McF (McFurter turbidity unit), and then dilute it step by step at 1:10. After testing, obtain 10 -5 The number of colonies in the dilution solution can be controlled to be less than or equal to 150 cfu / 100 μL (when the turbidity is 0.95: 10 -5 The average number of colonies in each 100 μL dilution is 40 cfu-70 cfu; when the turbidity is 1.05: 10 -5 The average number of colonies in each 100 μL dilution was 80 cfu-120 cfu).

[0044] 3. The freeze-dried bacterial pellets are composed of the test bacteria and the stabilizer. If the diluent and the stabilizer are mixed at a ratio of 1:99, then according to step (2), the reverse calculation is that a single dose of freeze-dried bacterial pellets (0.25x10 2 ~1.0x10 2 The dilution level and volume of the test bacteria (per pellet) were added to the stabilizer, and then freeze-dried pellets were prepared using a microsphere former and a freeze dryer.

[0045] Example 3: Control of Staphylococcus aureus growth by limiting factor vitamin C

[0046] The freeze-dried bacterial pellets prepared in Example 2 were added to the four culture media suitable for quantitative fresh culture of Staphylococcus aureus in Example 1, and cultured at a temperature of 30°C-35°C for 20h±1h; 100 μL was taken out from the obtained cultured liquid culture medium and added in parallel to the surface of tryptic soy agar medium TSA, spread and dried, and cultured at a temperature of 30°C-35°C for no more than 3 days. After the culture was completed, the colonies were counted. The results are shown in Table 3.

[0047] Table 3

[0048]

[0049] The relationship between the logarithmic values ​​of the colony count changes of Staphylococcus aureus in experimental groups 1-4 after 20 hours of culture is shown in Figure 1 , from Table 3 and Figure 1The data trend shows that: in the culture medium formulas of experimental groups 1 to 44, when the addition amount of carbon source, nitrogen source, and inorganic salt remains unchanged, although vitamin C has the effect of promoting microbial growth, as the addition amount continues to increase, it has a certain inhibitory effect on the growth of microorganisms. The data in Table 3 show that when the logarithm of the change in the number of colonies of microorganisms before and after 20 hours of culture is low (for example, 0.74), it just meets the requirement of the culture medium required for obtaining a quantifiable fresh culture after culture (100 cfu / 100 μL), and this culture medium is a specific culture medium for quantifiable fresh culture liquid of Staphylococcus aureus.

[0050] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A quantifiable fresh culture solution of Staphylococcus aureus, characterized in that: The invention comprises the following components: bacterial strain, nitrogen source, carbon source, inorganic salt and limiting factor.

2. The quantifiable fresh culture solution of Staphylococcus aureus according to claim 1, characterized in that: The strain is Staphylococcus aureus.

3. The quantifiable fresh culture solution of Staphylococcus aureus according to claim 1, characterized in that: The limiting factor is vitamin C. For the specific quality control bacteria Staphylococcus aureus in a low-nutrient specific culture medium environment, by controlling the concentration of vitamin C, the growth trend of Staphylococcus aureus can be accurately controlled, thereby efficiently obtaining a fresh culture solution of Staphylococcus aureus with a strain concentration of ≤1000cfu / 100μL.

4. The quantifiable fresh culture solution of Staphylococcus aureus according to claim 1, characterized in that: Each 100 ml of the culture solution contains 5-100 cfu of the strain, 1-8 g of a nitrogen source, 1-2 g of a carbon source, 0.1-0.5 g of an inorganic salt, and 0.05-1.5 g of a limiting factor.

5. The quantifiable fresh culture solution of Staphylococcus aureus according to any one of claims 1 to 4, characterized in that: The carbon source includes sucrose and / or glucose; and / or, the nitrogen source comprises one or more of gelatin, tryptone and skimmed milk powder; And / or, the inorganic salt includes one or more of dipotassium hydrogen phosphate trihydrate, magnesium chloride and sodium chloride.

6. A method for preparing a quantitative fresh culture solution of Staphylococcus aureus, characterized in that: The following steps are involved: S1: Preparation of freeze-dried pellets of Staphylococcus aureus; S2: Preparation of culture medium suitable for quantitative fresh culture of Staphylococcus aureus; S3: Add one freeze-dried pellet of Staphylococcus aureus with a colony count of no more than 100 cfu / pellet into a culture medium suitable for quantitative fresh culture of Staphylococcus aureus for culture at a temperature of 30-35°C for a period of 20h±1h.

7. The method for preparing a quantifiable fresh culture solution of Staphylococcus aureus according to claim 6, characterized in that: The preparation method of the freeze-dried Staphylococcus aureus pellets is as follows: S1: Inoculate the standard strain of Staphylococcus aureus into TSB medium and culture it at 30-35°C for 18-24 hours for bacterial enrichment; S2: After the bacterial enrichment culture, a fresh culture with a high colony count is obtained. The grown bacteria are the second-generation standard stock bacteria. After the fresh culture is shaken by an oscillator until it is uniform, 0.9% NaCl is used as a diluent to dilute the fresh culture in a 1:10 manner; S3: If the turbidity of the fresh culture stock solution is greater than 4.0McF, then conduct a turbidity test on the diluted 1:10 bacterial suspension and control the turbidity of the 1:10 bacterial suspension to 0.9-1.5McF; then perform subsequent dilutions until the dilution reaches 1:10 -6 . ; S4: Select dilution to 10 -4 , 10 -5 and 10 -6 The bacterial suspension was inoculated on TSA solid medium plates, cultured at 30-35°C for 3 days, and the colonies were counted; S5: Determine the dilution gradient corresponding to the colony count of the dilution solution ≤ 100 cfu by observing the number of TSA growing colonies; S6: Based on the results of S4 and S5, determine the dilution gradient corresponding to the accurate turbidity value of the 1:10 dilution; S7: Repeat steps S1-S6, and according to the dilution gradient of the colony count less than 100 cfu, calculate the corresponding dilution concentration of the bacterial suspension and add it to the stabilizer, and the ratio of the diluted bacterial suspension to the stabilizer is 1:99; S8: freeze-dry the mixed solution in step 7 using a microsphere forming apparatus and a freeze-drying apparatus to prepare freeze-dried bacterial pellets, with a colony count of ≤100 cfu / bacterial pellet.

8. The method for preparing a quantitative fresh culture solution of Staphylococcus aureus according to claim 7, characterized in that: The stabilizer component comprises a protective agent, a filler and an antioxidant, wherein the protective agent is composed of glucose and trehalose, the filler is composed of mannitol, and the antioxidant is composed of serum and skim milk powder.

9. The method for preparing a quantifiable fresh culture solution of Staphylococcus aureus according to claim 6, characterized in that: The components and proportions of the culture medium suitable for quantitative fresh culture of Staphylococcus aureus are as follows: 2-6g of nitrogen source, 1-2g of carbon source, 0.2-0.4g of inorganic salt, 0.1-1g of limiting factor, and the balance is water per 100 ml of the culture medium.

10. The method for preparing a quantifiable fresh culture solution of Staphylococcus aureus according to claim 9, characterized in that: The carbon source is glucose; the nitrogen source is trypsin; and the inorganic salt is dipotassium hydrogen phosphate trihydrate.

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