Preparation method of bacterial standard substance capable of being stored for long time and stable in content

Abstract

The invention relates to a preparation method of a bacterial standard substance capable of being stored for a long time and stable in content, which sequentially comprises the following steps: step 1, preparing a protective solution, adding a bacterial solution, and uniformly mixing; step 2, loading the mixture uniformly mixed in the step 1 into a tray, and performing pre-freezing treatment; step 3, carrying out freeze-drying treatment on the bacteria liquid sample pre-frozen in the step 2; and 4, quantitatively sub-packaging the freeze-dried bacterial liquid sample in the step 3, and carrying out vacuum refrigeration. According to the present invention, the number and the characteristics of the microorganisms of the freeze-dried coliform bacterial powder can be maintained stable for a long time through the compounded protective agent formula and the combination of the specific storage conditions so as to prepare the coliform bacterial standard product for quantitative detection.

Description

Technical Field

[0001] The present invention relates to the field of microorganism-related technologies, and in particular to a method for preparing a bacterial standard product that can be stored for a long time and has a stable content. Background Art

[0002] Excessive microbial content is one of the main hidden dangers of food safety. Microbial testing can evaluate the sanitary quality and safety of products, determine whether products meet relevant standards and regulations, and protect the health rights and interests of consumers. As a limit indicator for drinking water, food, and medicine, coliform bacteria are almost a must-test item for all foods. Microbial proficiency testing refers to a verification activity that uses inter-laboratory comparisons to determine laboratory capabilities and ensure that the laboratory maintains a high level of testing to assess, supervise, and confirm its capabilities. The preparation of microbial standards is a technology that must be possessed by proficiency testing organizers. For molds, yeasts, and most Gram-positive bacteria, existing methods can meet the needs of standard preparation. However, coliform bacteria are Gram-negative rods. The use of freeze-drying will destroy protein molecules and inactivate cells, killing a large number of cells; the moisture content of samples obtained by the dry inoculation method is unstable, and the uniformity of inoculated microorganisms cannot be guaranteed. At present, there is a lack of information on the preparation of coliform bacteria standards. Therefore, a reliable method is needed to prepare coliform bacteria quantitative standards to organize microbial proficiency testing activities.

[0003] Vacuum freeze drying is one of the most common methods for preserving microbial materials. During the freeze drying process, cells are subjected to the effects of two intense factors, freezing and drying, and cell damage is inevitable. There are many factors that affect the quality of surviving cells, including strains, growth status, pH value, pre-freezing temperature, freeze drying conditions, protective agents, etc. For most bacteria, the key to the success of freeze drying lies in the use of protective agents. Protective agents can change the physical conditions and chemical environment of the bacteria in the freeze-dried state, reduce the damage to the cells during the freeze drying process, and maintain the original physiological and biochemical characteristics and biological activity of the microorganisms as much as possible. According to the properties of the protective agent, it can be divided into: polyols: such as glycerol, mannitol, ethylene glycol; sugars: such as dextran, maltodextrin, lactose, sucrose; amino acids: such as proline, lysine, alanine; inorganic salts: such as phosphates, calcium carbonate, manganese sulfate; proteins: such as casein, bovine serum albumin, etc.

[0004] There are many types of freeze-drying protective agents, and their freeze-drying protective effects on different bacteria are not the same, especially for Gram-negative bacteria represented by coliform bacteria. Due to the unique cell wall structure of Gram-negative bacteria, their resistance to freeze-drying conditions is not good. After adding a certain protective agent, the number of surviving cells after freeze-drying may not be low, but they cannot be preserved for a long time. Under refrigerated conditions for seven days, their number can drop sharply by 1,000 times or more. However, the coliform standards used for quantitative detection require that their quantity and characteristics must be stable.

[0005] In view of the above-mentioned defects, the designers have actively carried out research and innovation in order to create a method for preparing bacterial standards that can be preserved for a long time and have stable content, so as to make it more valuable for industrial use. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a method for preparing a bacterial standard that can be stored for a long time and has a stable content.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing a bacterial standard substance that can be stored for a long time and has a stable content, comprising the following steps in sequence:

[0009] Step 1, prepare protective solution, add bacterial solution and mix well;

[0010] Step 2, placing the mixture mixed in the above step 1 on a plate for pre-freezing;

[0011] Step 3, freeze-drying the pre-frozen bacterial liquid sample in the above step 2;

[0012] Step 4: quantitatively package the freeze-dried bacterial liquid sample in the above step 3 and refrigerate it in a vacuum.

[0013] As a further improvement of the present invention, the protective solution in step 1 is composed of a protective agent and sterile water, and the protective agent includes skimmed milk powder, trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate.

[0014] As a further improvement of the present invention, the amount of skim milk powder in the protective agent is 10% (w / v), and the amounts of trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate in the protective agent are all ≤3% (w / v).

[0015] As a further improvement of the present invention, the volume proportion of the bacterial liquid in step 1 is ≤1%.

[0016] As a further improvement of the present invention, the pre-freezing treatment condition in step 2 is freezing at -80°C for 24 hours.

[0017] As a further improvement of the present invention, in step 3, the bacterial liquid sample is freeze-dried in a freeze dryer.

[0018] As a further improvement of the present invention, the freeze-drying treatment time in step 3 is ≤48h, and the moisture content of the freeze-dried bacterial liquid sample is ≤3%.

[0019] As a further improvement of the present invention, in step 4, the freeze-dried bacterial liquid sample is quantitatively packaged into sterile sealed bags, and the air in the sterile sealed bags is removed and then sealed.

[0020] As a further improvement of the present invention, in step 4, the mixture is refrigerated at 2-8°C.

[0021] As a further improvement of the present invention, the bacterial solution in step 1 is Escherichia coli and the concentration is 10 5 ~10 9 CFU / mL.

[0022] By means of the above scheme, the present invention has at least the following advantages:

[0023] The invention uses a compounded protective agent formula combined with specific storage conditions to keep the microbial quantity and characteristics of the freeze-dried coliform bacteria powder stable for a long time, thereby being able to prepare a coliform bacteria standard for quantitative detection.

[0024] The coliform group standard prepared by the present invention can maintain the stability of microbial quantity and characteristics for up to 6 months under refrigerated conditions.

[0025] Conventional quantitative microbial standards are packaged in modified atmosphere, and if leakage occurs after the production of the present invention, it can be discovered in time.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 It is a schematic diagram of a method for preparing a bacterial standard substance that can be stored for a long time and has a stable content according to the present invention. DETAILED DESCRIPTION

[0029] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0030] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. The components of the embodiment of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiment of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiment of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0031] Example

[0032] like Figure 1 As shown,

[0033] A method for preparing a bacterial standard substance that can be stored for a long time and has a stable content, comprising the following steps in sequence:

[0034] Step 1, prepare protective solution, add bacterial solution and mix well;

[0035] Step 2, placing the mixture mixed in the above step 1 on a plate for pre-freezing;

[0036] Step 3, freeze-drying the pre-frozen bacterial liquid sample in the above step 2;

[0037] Step 4: quantitatively package the freeze-dried bacterial liquid sample in the above step 3 and refrigerate it in a vacuum.

[0038] Preferably, the protective solution in step 1 is composed of a protective agent and sterile water, and the protective agent includes skimmed milk powder, trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate.

[0039] Preferably, the amount of skim milk powder in the protective agent is 10% (w / v), and the amounts of trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate in the protective agent are all ≤3% (w / v).

[0040] Preferably, the volume proportion of the bacterial liquid in step 1 is ≤1%.

[0041] Preferably, the pre-freezing treatment condition in step 2 is freezing at -80°C for 24 hours.

[0042] Preferably, in step 3, the bacterial liquid sample is freeze-dried in a freeze dryer.

[0043] Preferably, the freeze-drying treatment time in step 3 is ≤48h, and the moisture content of the bacterial liquid sample after freeze-drying is ≤3%.

[0044] Preferably, the freeze-dried bacterial liquid sample in step 4 is quantitatively packaged into sterile sealed bags, and the air in the sterile sealed bags is removed and then sealed.

[0045] Preferably, in step 4, the mixture is refrigerated at 2-8°C.

[0046] Preferably, the bacterial solution in step 1 is Escherichia coli and has a concentration of 105 to 109 CFU / mL.

[0047] The first embodiment of the present invention:

[0048] Preparation of microbial standards for quantitative coliform bacteria

[0049] Bacterial solution concentration: 10 5 ~10 9 CFU / mL;

[0050] Protective agent composition: skim milk powder, trehalose, sucrose, sodium glutamate, dextrin, sodium ascorbate;

[0051] The amount of skim milk powder is 10% (w / v), and the other components are ≤ 3% (w / v);

[0052] Step 1: Prepare protective solution with sterile water according to the protective agent formula, add bacterial solution and mix well (the volume proportion of bacterial solution is ≤1%).

[0053] Step 2: The above mixture is pre-frozen in a plate and frozen at -80°C for 24 hours.

[0054] Step 3: After pre-freezing, the sample enters the freeze dryer for freeze drying. The freeze drying parameters and duration are determined by the differences in the freeze dryer. The freeze drying time should be ≤48h, and the moisture content of the sample after freeze drying should be ≤3%.

[0055] Step 4: Quantitatively package the lyophilized material into sterile sealed bags, remove all air from the bags and seal them, and store them at 2-8°C.

[0056] The invention uses a compounded protective agent formula combined with specific storage conditions to keep the microbial quantity and characteristics of the freeze-dried coliform bacteria powder stable for a long time, thereby being able to prepare a coliform bacteria standard for quantitative detection.

[0057] The coliform group standard prepared by the present invention can maintain the stability of microbial quantity and characteristics for up to 6 months under refrigerated conditions.

[0058] Conventional quantitative microbial standards are packaged in modified atmosphere, and if leakage occurs after the production of the present invention, it can be discovered in time.

[0059] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and the like are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly referring to the number of technical features indicated. Thus, features defined as "first", "second", and the like may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0060] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a bacterial standard substance that can be stored for a long time and has a stable content, characterized in that: The following steps are included in sequence: Step 1, prepare protective solution, add bacterial solution and mix well; Step 2, placing the mixture mixed in the above step 1 on a plate for pre-freezing; Step 3, freeze-drying the pre-frozen bacterial liquid sample in the above step 2; Step 4: quantitatively package the freeze-dried bacterial liquid sample in the above step 3 and refrigerate it in a vacuum.

2. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: The protective solution in step 1 is composed of a protective agent and sterile water, wherein the protective agent includes skimmed milk powder, trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate.

3. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 2, characterized in that: The amount of skimmed milk powder in the protective agent is 10% (w / v), and the amounts of trehalose, sucrose, sodium glutamate, dextrin and sodium ascorbate in the protective agent are all less than 3% (w / v).

4. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: The volume proportion of the bacterial liquid in step 1 is ≤1%.

5. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: The pre-freezing treatment condition in step 2 is freezing at -80°C for 24 hours.

6. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: In step 3, the bacterial liquid sample is freeze-dried in a freeze dryer.

7. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: The freeze-drying treatment time in step 3 is ≤48h, and the moisture content of the bacterial liquid sample after freeze-drying is ≤3%.

8. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: In step 4, the freeze-dried bacterial liquid samples are quantitatively packaged into sterile sealed bags, and the air in the sterile sealed bags is removed and then sealed.

9. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: In step 4, the mixture is refrigerated at 2-8°C.

10. The method for preparing a bacterial standard substance that can be stored for a long time and has a stable content as claimed in claim 1, characterized in that: In step 1, the bacterial solution is Escherichia coli and the concentration is 10 5 ~10 9 CFU / mL.

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