Strict anaerobic bacteria detection method and application thereof
By using shallow liquid protection methods in the surface hygiene detection of brewery equipment, the problem of strict anaerobic bacteria prone to death in an oxygen environment is solved, and efficient and low-cost strict anaerobic bacteria detection is achieved.
Patent Information
- Application Number
- CN202510640822.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-08-08
AI Technical Summary
The existing surface hygiene detection methods for brewery equipment are difficult to detect strict anaerobic bacteria, mainly because oxygen has a toxic effect on it. Conventional methods fail to provide an effective oxygen-avoiding environment, resulting in strict anaerobic bacteria death during the detection process.
By keeping strict anaerobic bacteria in shallow liquid for protection during the detection process, sampling and culture with deoxygenation transfer liquid, avoiding direct exposure to air, ensuring their activity, and improving survival rate by protecting the shallow liquid.
It realizes efficient detection of strictly anaerobic bacteria, improves the accuracy and simplicity of detection, and reduces the detection cost.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial detection, in particular to a detection method for strictly anaerobic bacteria and application thereof. Background Art
[0002] Currently, breweries often use cotton swab culture methods to test the surface hygiene of production equipment. Common target microorganisms and their culture conditions are shown in the table below.
[0003] Table 1 Detection target microorganisms and their culture conditions
[0004] Microbial species Growth type Culture conditions Lactobacillus Anaerobic <![CDATA[CO2 environment]]> Pediococcus Facultative anaerobic Microaerobic environment Pectobacterium, Megasphaera Strict anaerobic type (obligate anaerobic type) oxygen-free environment Acetobacter Aerobic type oxygen-rich environment
[0005] The existing beer equipment surface hygiene detection method (cotton swab wiping culture method) is as follows:
[0006] (1) Sterile cotton swabs should be moistened with sterile saline on a clean bench in advance. The moistened sterile cotton swabs can be stored in the refrigerator for up to 2 days.
[0007] (2) Use a sterile cotton swab to vigorously wipe the designated test surface. Note: Each point should be sampled in crevices, rough or uneven areas, corners, lower surfaces, and other places that are difficult to clean. The area sampled by the cotton swab should be as large as 15cm*15cm. When sampling, rotate the cotton swab to ensure that the dirt adheres to the largest possible area.
[0008] (3) Place the cotton swab after sampling in a sterile test tube and bring it back to the laboratory, then pour it into NBB culture medium for culture as soon as possible.
[0009] However, although the above detection methods can detect aerobic bacteria, facultative anaerobic bacteria, and general anaerobic bacteria, they can rarely detect strict anaerobic bacteria. Summary of the Invention
[0010] In response to the above problems, the present invention provides a method for detecting strict anaerobic bacteria. This detection method prevents the strict anaerobic bacteria from being directly exposed to the air, but instead protects them in a shallow layer of liquid. Even if the liquid layer is very shallow, the strict anaerobic bacteria can still maintain a high activity for a period of time, thereby enabling the strict anaerobic bacteria obtained by sampling to support the completion of subsequent culture and detection steps, thereby achieving the detection of strict anaerobic bacteria.
[0011] The present invention provides a method for detecting strict anaerobic bacteria, comprising the following steps:
[0012] Pre-deoxygenation: deoxygenating the transfer liquid and culture medium to obtain deoxygenated transfer liquid and deoxygenated culture medium;
[0013] Sampling and shallow liquid protection: immerse the sampling tool in the deoxygenated transfer liquid, and use the sampling tool soaked in the deoxygenated transfer liquid to wipe the object to be tested or the environment to be tested for sampling, so that the microorganisms are immersed in the deoxygenated transfer liquid and shallow liquid protection is performed; or spray the deoxygenated transfer liquid on the surface of the object to be tested or the environment to be tested, so that the microorganisms are immersed in the deoxygenated transfer liquid and shallow liquid protection is performed, and sampling is performed using the sampling tool;
[0014] Transfer, culture, and detection: Transfer the deoxygenated transfer liquid containing microorganisms to a deoxygenated culture medium for deep liquid culture, and detect the culture results of the deep liquid culture.
[0015] During the research on the problems existing in the detection of strict anaerobic bacteria, the inventors found that the main reasons why strict anaerobic bacteria are difficult to detect are the following factors: (1) The equipment surface hygiene detection method is mainly used to detect aerobic bacteria, facultative anaerobic bacteria, and general anaerobic bacteria, and rarely detects strict anaerobic bacteria. The reason is that oxygen in the air is toxic to strict anaerobic bacteria, and strict anaerobic bacteria will die quickly when exposed to the air. In the detection process, strict anaerobic bacteria must be strictly avoided in order to detect strict anaerobic bacteria. However, the conventional equipment surface hygiene detection method does not pay attention to strict avoidance of oxygen. Strict anaerobic bacteria that survive on the surface of the equipment will not be directly exposed to the air. They are usually tightly wrapped by the viscous liquid and beer residue produced by the growth of other types of microorganisms on the surface of the equipment, isolating the air to form an oxygen-deficient environment so that they can survive and reproduce. When using the cotton swab wiping culture method to perform beer equipment surface hygiene detection, it is difficult to detect strict anaerobic bacteria because the cotton swab will destroy this oxygen-deficient environment when wiping dirt on the surface of the equipment. (2) In other industries, strict anaerobic bacteria detection methods for equipment surface hygiene mostly require the use of pre-reducing agents, and the detection steps are relatively cumbersome and costly, and the final detection results are not necessarily better. In other industries, strict anaerobic bacteria detection on equipment surfaces mainly relies on oxygen avoidance methods such as the use of pre-reducing agents and deep liquid culture in test tubes. Pre-reducing agents can consume oxygen in liquids.
[0016] In the classic liquid test tube culture experiment of strict anaerobic bacteria, the liquid at the bottom of the test tube has the lowest oxygen content, so strict anaerobic bacteria can grow; the liquid near the top of the test tube has a high oxygen content because oxygen from the air dissolves into the liquid, so strict anaerobic bacteria cannot grow. Figure 1As shown in Figure 1, the bacteria cultured in test tube 1 are obligate aerobes, which require oxygen to grow. Since oxygen only penetrates shallowly into the test tube, they grow in the upper layer, where the oxygen content is highest. The bacteria cultured in test tube 2 are strict anaerobes, which can only grow in the absence of oxygen and therefore grow in the lower layer, where the oxygen content is lowest. As can be seen, the strict anaerobes in test tube 2 die when exposed to oxygen, as oxygen is toxic to them. Oxygen acts as a sterilizing agent for strict anaerobes. The lower the oxygen content, the easier it is for strict anaerobes to survive. The bacteria cultured in test tube 3 are facultative anaerobes, which can grow in both aerobic and anaerobic conditions. Therefore, they are distributed throughout the test tube culture medium. However, they grow better in aerobic conditions, resulting in a higher concentration of bacteria in the upper layer. Therefore, the facultative anaerobes in test tube 3 are not killed when exposed to oxygen and even require oxygen for growth and metabolism. The bacteria cultured in test tube 4 are microaerophiles, which can only grow in lower oxygen levels and therefore grow in the upper layer of the test tube. The bacteria cultured in test tube 5 are oxygen-tolerant anaerobic bacteria that can grow anaerobically in the presence of oxygen, so they are evenly distributed throughout the test tube culture medium.
[0017] The inventors have conducted in-depth research on the detection method of strict anaerobic bacteria using pectin bacteria (strict anaerobic bacteria) as the research object, and found that pectin bacteria will die quickly when completely exposed to the air (for more than 1 minute). However, when pectin bacteria is in a shallow pre-deoxygenated liquid protection (the experimental conditions are that the liquid layer depth is about 0.1mm and the liquid is exposed to the air), it can maintain a high survival rate for a long time. After 20 minutes under the experimental conditions, the survival rate of pectin bacteria in shallow liquid culture is still more than 80%. As the oxygen in the air gradually dissolves into the liquid layer, the survival rate of pectin bacteria is also gradually decreasing. The shallow liquid culture study shows that as long as pectin bacteria is not directly exposed to the air, but is in a liquid, even if the liquid depth is very shallow (0.1mm), pectin bacteria can still maintain a high activity within a certain period of time, and the activity and quantity of pectin bacteria obtained by sampling are sufficient to support the completion of the equipment surface hygiene detection step. In the past classic strict anaerobic bacteria culture theory, only the deep layer (about 10 cm or above) culture theory of test tube liquid was mentioned, and no research on the survival of strict anaerobic bacteria in shallow liquid was involved.
[0018] In one embodiment, the shallow liquid protection time is ≥20 minutes.
[0019] In one embodiment, the shallow liquid protection has a liquid layer depth of 0.05-2 mm.
[0020] During the research on shallow pre-deoxygenation liquid protection, the inventors found that when the liquid layer depth was 0.1 mm, the survival rate of pectin bacteria was still above 80% after 20 minutes of shallow liquid protection; when the liquid layer depth was 1.5 mm, the survival rate of pectin bacteria was maintained at above 85% after 30 minutes of shallow liquid protection. Therefore, when the liquid layer depth in the present case is within the above range, it is convenient to operate, so that the operator only needs to perform shallow liquid protection to achieve the sampling of strict anaerobic bacteria, and can also maintain a high survival rate for a longer period of time.
[0021] In one embodiment, the deoxygenated transfer liquid is exposed to air during the shallow liquid protection process.
[0022] In one embodiment, the transfer liquid includes at least one of water and physiological saline.
[0023] In one embodiment, the sampling tool includes at least one of a cotton swab, a sponge smear stick, a sampling swab, and sterile gauze.
[0024] In one embodiment, the strict anaerobic bacteria include at least one of Pectobacterium, Macrosphaera, and Bifidobacterium.
[0025] The present invention also provides a microbial monitoring method for beer production, comprising the following steps: using the detection method to detect surface microorganisms of beer production equipment and beer production environment.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] The present invention provides a detection method for strict anaerobic bacteria and its application. The detection method prevents the strict anaerobic bacteria from being directly exposed to the air, but instead protects them in a shallow layer of liquid. Even if the liquid layer is very shallow, the strict anaerobic bacteria can still maintain a high activity (i.e., maintain a high survival rate) for a period of time. The strict anaerobic bacteria obtained by sampling can then be supported until the completion of subsequent culture and detection steps, thereby achieving the detection of strict anaerobic bacteria and improving the detection rate of strict anaerobic bacteria. In addition, the detection method is simpler, easier to implement, and has lower cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of test tube culture of microorganisms with different oxygen requirements;
[0029] Figure 2 Schematic diagram of the process flow of the detection method in Example 1;
[0030] Figure 3 This is the peak graph of the PCR melting curve of sequence number 5, with the horizontal axis representing temperature;
[0031] Figure 4 This is the peak graph of the PCR melting curve of sequence number 6, with the horizontal axis representing temperature;
[0032] Figure 5 This is the PCR standard bacteria library reference table (Pectobacterium part) and the reference table that comes with the PCR identification instrument system. DETAILED DESCRIPTION
[0033] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.
[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0035] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.
[0036] Example 1
[0037] In the detection method of the present invention, as long as a trace amount of liquid is provided for immersion of strict anaerobic bacteria, good oxygen protection can be provided to them during the detection time, thereby increasing their survival rate and the positive detection rate. Compared with the principle of deep culture of strict anaerobic bacteria in liquid test tubes, the inventors found in the shallow liquid protection research that the liquid layer depth is about 0.1mm, and when exposed to the atmosphere, the pectin bacteria (strict anaerobic bacteria) immersed in the liquid still has 80% activity after 20 minutes. Based on the conclusion of this study, the detection method of the present invention provides liquid protection for strict anaerobic bacteria before, during, and after the detection. The process flow of the detection method is as follows: Figure 2 As shown, the details are as follows:
[0038] 1. Pre-deoxidation.
[0039] The prepared sterile saline and NBB culture medium test tubes were placed in an anaerobic workstation at room temperature. The sterile saline was deoxygenated for 24 hours, and the NBB culture medium test tubes were deoxygenated for 48 hours to remove oxygen from the liquid for later use.
[0040] 2. Sampling and shallow liquid protection.
[0041] Bring deoxygenated saline, a tube of deoxygenated NBB culture medium, and a cotton swab to the testing site. Before use, dip the cotton swab tip (length x width: 10mm x 5mm) into the saline and shake for 5 seconds to moisten it. Wetting the cotton swab at the sampling site helps reduce the amount of oxygen that dissolves into the moistened tip and reduces the amount of water that evaporates from the tip over time. A fully moistened cotton swab tip helps protect the strictly anaerobic bacteria being tested.
[0042] Wipe quickly, using each cotton swab within one minute. Wipe until the dirt and microorganisms on the cotton swab head are soaked with liquid.
[0043] Note: Each point should be sampled from crevices, rough or uneven areas, corners, lower surfaces, and other places that are difficult to clean. When sampling, rotate the cotton swab to ensure that the dirt adheres to the largest possible area.
[0044] 3. Transfer, culture, and detection.
[0045] Transfer and culture: Immediately break the cotton swab head after sampling and place it in a deoxygenated NBB culture medium test tube for culture (deep liquid culture), plugged with a breathable rubber stopper; within 2 hours, bring the NBB test tube with the wipe cotton swab head back to the laboratory and place it in an anaerobic workstation for culture for 10 days.
[0046] Detection: The culture results are identified by PCR to determine whether they are strictly anaerobic bacteria (e.g., Pectobacterium, Macrosphaera).
[0047] Example 2
[0048] The detection method of Example 1 is compared with the detection method of strict anaerobic bacteria in the prior art:
[0049] Table 2 Comparison of the detection method of Example 1 and the detection method of strict anaerobic bacteria in the prior art
[0050]
[0051]
[0052] A comparison of the above technical solutions reveals that the key point of the present invention is that strict anaerobic bacteria can maintain a high survival rate as long as they are surrounded by a deoxygenated liquid, even if the liquid is very thin (0.1mm) and exposed to air. The detection method of the present invention is simple, low-cost, and effective in detecting strict anaerobic bacteria.
[0053] Example 3
[0054] The detection method of Example 1 was used to perform microbial detection on the surfaces of different beer production equipment. The PCR identification results are shown in the following table.
[0055] Table 3 Microbial detection results on the surfaces of different beer production equipment
[0056]
[0057] The peak values of PCR melting curves for numbers 5 and 6 in the table above are as follows: Figure 3 、 4 As shown in the table, the PCR standard bacteria library reference table (Pectobacterium part) is as follows Figure 5 As shown, Figure 3 、 Figure 4 The horizontal axis is temperature. Figure 3 The PCR melting curve analysis shows that the melting temperature (TM) value of the positive sample No. 5 is 70.17℃. With reference to the TM value of the PCR standard bacteria library (Pectobacterium part), it is confirmed that it is a clear peak melting temperature of Pectobacterium. This curve can be used to identify the presence of Pectobacterium in the sample. Figure 4 The PCR melting curve analysis showed that the melting temperature (TM) value of the positive sample No. 6 was 70.22℃. Referring to the TM value of the PCR standard bacteria library control table (Pectobacterium part), it was confirmed that it was an obvious Pectobacterium melting peak temperature. This curve can be used to identify the presence of Pectobacterium in the sample.
[0058] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0059] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.
Claims
1. A method for detecting strict anaerobic bacteria, characterized in that: The following steps are involved: Pre-deoxygenation: deoxygenating the transfer liquid and culture medium to obtain deoxygenated transfer liquid and deoxygenated culture medium; Sampling and shallow liquid protection: immerse the sampling tool in the deoxygenated transfer liquid, and use the sampling tool soaked in the deoxygenated transfer liquid to wipe the object to be tested or the environment to be tested for sampling, so that the microorganisms are immersed in the deoxygenated transfer liquid and shallow liquid protection is performed; or spray the deoxygenated transfer liquid on the surface of the object to be tested or the environment to be tested, so that the microorganisms are immersed in the deoxygenated transfer liquid and shallow liquid protection is performed, and sampling is performed using the sampling tool; Transfer, culture, and detection: Transfer the deoxygenated transfer liquid containing microorganisms to a deoxygenated culture medium for deep liquid culture, and detect the culture results of the deep liquid culture.
2. The detection method according to claim 1, wherein The shallow liquid protection time is ≥20 minutes.
3. The detection method according to claim 1, wherein The depth of the liquid layer of the shallow liquid protection is 0.05-2 mm.
4. The detection method according to claim 1, wherein During the shallow liquid protection process, the deoxygenated transfer liquid is exposed to air.
5. The detection method according to claim 1, wherein The transfer liquid includes at least one of water and physiological saline.
6. The detection method according to claim 1, characterized in that The sampling tool comprises at least one of a cotton swab, a sponge smear stick, a sampling swab, and sterile gauze.
7. The detection method according to any one of claims 1 to 6, characterized in that The strict anaerobic bacteria include at least one of pectinobacterium, macrosphaeria, and bifidobacteria.
8. A microbial monitoring method for beer production, characterized in that: The method comprises the following steps: using the detection method according to any one of claims 1 to 7 to detect surface microorganisms of beer production equipment and beer production environment.