Rapid detection method of streptomyces specific bacteriophage for fermentation production
Through the mycelial germination culture and double-layer detection plate methods, the problem of difficulty in quickly detecting Streptomyces's specific phages during fermentation is solved, and the rapid and effective detection and observation of phages are achieved to prevent phage infection and outbreaks.
Patent Information
- Application Number
- CN202411888916.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-25
AI Technical Summary
During the fermentation process, it is difficult for the prior art to detect Streptomyces' specialized phages quickly and effectively, resulting in rapid infection and outbreaks and economic losses.
The growth of plaques was observed to detect phages by culturing Streptomyces mycelium under specific conditions and contacting them with possible phages in the sample or environment using Streptomyces mycelium to detect phages.
It realizes rapid and effective detection of Streptomyces' specialized phages, can intuitively observe the existence of phages, and detect their number and sedimentation rate to prevent phage infection and outbreak and ensure the safety of the production environment.
Smart Images
Figure CN119685529A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological detection, and in particular to a rapid detection method for a Streptomyces-specific bacteriophage used in fermentation production. Background Art
[0002] Bacteriophage is a general term for a class of bacterial viruses that can infect microorganisms, including bacteria, fungi, actinomycetes or spirochetes. As a type of virus, bacteriophage has some common characteristics of viruses: individuals are tiny and do not have a complete cell structure, only contain a single nucleic acid, and have a simple genetic structure; they rely on host cells to replicate and grow, and once they leave the host cell, they cannot grow and replicate. After the bacteriophage individual infects the host cell, through simple replication, it can quickly generate hundreds or thousands of progeny bacteriophage individuals, and each progeny individual can quickly infect other bacterial cells and generate countless progeny bacteriophage individuals again. Therefore, once a bacteriophage breaks out in a fermentation enterprise, its development speed is alarming, causing a reduction in production at the least, or even causing a shutdown, and the economic losses brought about are incalculable.
[0003] If bacterial contamination occurs during the fermentation process, the type and condition of the contaminated bacteria can be directly confirmed under a microscope, but bacteriophages are too small to be observed with the naked eye, so an effective, fast, and easy-to-observe detection method must be sought. Summary of the invention
[0004] The purpose of the present application is to provide a rapid detection method for Streptomyces-specific phages used in fermentation production, so as to achieve effective and rapid detection of Streptomyces-specific phages and easy observation of phages.
[0005] In order to solve the above technical problems, the present application provides a rapid detection method for Streptomyces specific phage for fermentation production, comprising:
[0006] Streptomyces mycelium germination culture: prepare a Streptomyces spore suspension, add the Streptomyces spore suspension to a mycelium culture medium, and culture at 30-32° C. for 22-24 hours. When the appearance is yellow and thick and the mycelium is intact and moderate, the culture is terminated to obtain Streptomyces mycelium;
[0007] Preparation of a double-layer detection plate: prepare a lower culture medium and an upper culture medium, and spread the lower culture medium on a 90 cm plate, adding 15-20 mL / plate and waiting for solidification; adding the Streptomyces mycelium to the upper culture medium at 45-50° C. at a concentration of 10-15%, shaking well, and spreading it on the solidified lower culture medium, adding 10-15 mL / plate, and forming a double-layer detection plate after solidification;
[0008] Sample detection: dilute the seed liquid or fermentation liquid to obtain the test liquid, and add the test liquid to the double-layer test plate, add 100 μL to each plate, evenly spread it, and culture it at a constant temperature of 32-34°C and a humidity of 40-45% for 24-28 hours, then observe the growth of the plaques and record the infection status;
[0009] Environmental testing: Place the double-layer test plate in the environmental area to be tested for a set time and then take it back. Place the taken back double-layer test plate at a temperature of 32-34°C and a humidity of 40-45% for constant temperature culture for 24-28 hours. After the culture is completed, observe the growth of plaques and record the infection status.
[0010] As a preferred embodiment, a rapid detection method for a specific bacteriophage of Streptomyces used in fermentation production is provided, wherein the mycelium culture medium has a ratio of: 5.0% starch, 1.0% soy protein powder, 0.4% peptone, 0.3% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.2% magnesium sulfate, pH 7.0, and the percentages are measured in g / 100mL purified water.
[0011] What needs to be explained in detail in the scheme is a rapid detection method for a specific bacteriophage of Streptomyces used in fermentation production, wherein the components of the upper culture medium are: 1.0% starch, 0.2% plant peptone, 0.3% ammonium sulfate, 0.2% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and 0.1% magnesium sulfate, and the percentages are calculated in g / 100mL purified water, adjusted to pH 7.0 with 20-40% sodium hydroxide solution, and sterilized at 118-122°C for 30 minutes.
[0012] What needs to be further explained in the scheme is a rapid detection method for a specific bacteriophage of Streptomyces used in fermentation production. The components of the lower culture medium are specifically: 2.2% soluble starch, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, the percentage content is calculated in g / 100mL purified water, adjusted to pH 7.0 with 20-40% sodium hydroxide solution, and sterilized at 118-122°C for 30 minutes.
[0013] As a preferred embodiment, a method for rapid detection of Streptomyces specific phage for fermentation production, wherein the test solution obtained by diluting the seed solution or the fermentation solution comprises:
[0014] In the early stage of infection, the seed solution or the fermentation solution is diluted by 10 - 3-10 -6 times;
[0015] In the late infection period, the seed solution or the fermentation solution was diluted by 10 -5 ~10 -8 times.
[0016] As a preferred embodiment, a method for rapid detection of Streptomyces-specific bacteriophage for fermentation production is provided, wherein the environmental areas to be detected include the factory environment, the factory building environment, the clean area and the inoculation room, the set time corresponding to the factory environment is 15 minutes, the set time corresponding to the factory building environment is 20 minutes, the set time corresponding to the clean area is 30 minutes, and the dynamic monitoring during the inoculation process is 60 minutes.
[0017] Compared with the prior art, the present invention provides a method for rapid detection of a specific bacteriophage of Streptomyces for fermentation production. The cultured Streptomyces mycelium is mixed into the upper culture medium of a double-layer detection plate, and then laid on the lower culture medium of the solidified double-layer detection plate. After solidification, it is placed in an environment that needs to be detected for fixed-point and timed detection. After being retrieved, it is cultured at a constant temperature of 32 to 34°C and a humidity of 40 to 45% for 24 to 28 hours, and the results are observed and recorded, so that the detection of bacteriophages in the environment can be realized. At the same time, by adding the test liquid to the double-layer detection plate, evenly coating it, and then culturing it at a constant temperature of 32 to 34°C and a humidity of 40 to 45% for 24 to 28 hours, observing the growth of plaques, and recording the infection situation, the detection of bacteriophages infected with seed liquid or fermentation liquid can be realized. The presence of bacteriophages can be observed visually, and the number and sedimentation rate of bacteriophages can also be detected. It is an effective, fast and simple method for detecting Streptomyces phages, which is suitable for the detection of ambient air and infected bacterial fluids. It can effectively prevent the infection and outbreak of Streptomyces phages, and can also monitor bacteriophages in the production and research environments of enterprises, so as to detect them very early and take disinfection and other measures in time to prevent the pollution of the factory environment and laboratory, and completely eliminate the possibility of outbreaks. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solution of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without inventiveness.
[0019] Figure 1 A schematic diagram of a rapid detection method for a Streptomyces-specific bacteriophage used in fermentation production provided in an embodiment of the present application. DETAILED DESCRIPTION
[0020] In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.
[0021] The core of the present application is to provide a rapid detection method for Streptomyces-specific phage used in fermentation production, so as to achieve effective and rapid detection of Streptomyces-specific phage and easy observation of phage.
[0022] Figure 1 A schematic diagram of a rapid detection method for a Streptomyces-specific phage for fermentation production provided in the present application example, see Figure 1 shown.
[0023] Example 1
[0024] A rapid detection method for Streptomyces specific bacteriophage used in fermentation production, comprising:
[0025] Streptomyces mycelium germination culture: prepare a Streptomyces spore suspension, add the Streptomyces spore suspension to the mycelium culture medium, culture at 30-32°C for 22-24 hours, observe the appearance of yellow and thick, and terminate the culture when the mycelium is intact and moderate, and obtain Streptomyces mycelium. The growth process of Streptomyces spore buds has the following physiological characteristics: after spore germination, it grows into mycelium, its cell wall becomes thinner and the structure changes, and it is easy to be infected by specific bacteriophages. It has been confirmed by experiments that the mycelium growth stage belongs to the susceptible stage. Therefore, when selecting Streptomyces for detection, it is necessary to use mycelium in the susceptible stage, that is, to use the bacterial solution cultured to this stage for detection, so as to achieve the effect of quickly obtaining results.
[0026] Preparation of double-layer test plate: prepare lower culture medium and upper culture medium, and spread the lower culture medium on a 90cm plate, add 15-20mL / dish and wait for solidification; add the Streptomyces mycelium to the upper culture medium at 45-50°C at a concentration of 10-15%, shake well, and spread on the solidified lower culture medium, add 10-15mL / dish, and form a double-layer test plate after solidification. Requirements for double-layer test plates: the lower culture medium must contain basic nutrients for microbial growth, and the material of the upper culture medium should be easy for spores to germinate and grow into mycelium, so as to achieve the effect of forming plaques after infection.
[0027] Sample detection: dilute the seed liquid or fermentation liquid to obtain the test liquid, and add the test liquid to the double-layer test plate, add 100 μL to each plate, evenly spread it, and culture it at a constant temperature of 32-34°C and a humidity of 40-45% for 24-28 hours, then observe the growth of the plaques and record the infection status.
[0028] For samples that may be infected with bacteriophages, the test concentration is determined based on the time of possible bacteriophage infection and then tested.
[0029] (1) Early stage of infection, that is, the viscosity of the seed solution or fermentation solution is lower than that of the normal seed solution or fermentation solution, and the solution adheres to the wall, but the time of adherence to the wall is significantly shortened. During this period, the seed solution or fermentation solution that may be infected with phage is diluted, and the dilution multiple can be controlled at 10 -3 ~10 -6 Add the diluted test solution to the double-layer test plate, 100 μL per plate, evenly spread, and culture at a constant temperature of 32-34°C and 40-45% humidity for 24-28 hours. After the culture is completed, observe the growth of plaques and record the infection status.
[0030] (2) Late infection stage, when the viscosity of the seed solution or fermentation solution is much lower than that of the normal seed solution or fermentation solution, and the solution does not stick to the wall. During this stage, the seed solution or fermentation solution that may be infected with phages is diluted, and the dilution factor can be controlled at 10 -5 ~10 -8 Add the diluted test solution to the double-layer test plate, 100 μL per plate, evenly spread, and culture at a constant temperature of 32-34°C and 40-45% humidity for 24-28 hours. After the culture is completed, observe the growth of plaques and record the infection status.
[0031] Environmental testing: Place the double-layer test plate in the environmental area to be tested for a set time and then take it back. Place the taken back double-layer test plate at a temperature of 32-34°C and a humidity of 40-45% for constant temperature culture for 24-28 hours. After the culture is completed, observe the growth of plaques and record the infection status.
[0032] Environmental testing that may be infected with bacteriophages, confirmation of environmental testing location: According to the company's need to test and the environment where infection may occur, the testing environment is divided into factory environment, factory environment, clean area (regardless of level) and inoculation room. Placement: The location that needs to be tested in the factory, the four corners and center of the room, the top and table of the clean bench.
[0033] Confirmation of testing time and frequency: After verification, the testing time is determined to be 15 minutes for factory environment, 20 minutes for workshop environment, 30 minutes for clean area, and 60 minutes for dynamic monitoring during the inoculation process; the testing frequency is determined to be once a week, and timely testing is required if there are changes in conditions such as equipment replacement, air disinfection, etc.
[0034] Confirmation of test results: After the double-layer test plate is placed for a certain period of time, it will be collected and placed in a constant temperature incubation at 32-34°C and 40-45% humidity for 24-28 hours. After the incubation, observe the growth of plaques and record the infection situation. If plaques are found, disinfect them immediately and re-verify.
[0035] On the basis of Example 1, a rapid detection method for a specific bacteriophage of Streptomyces for fermentation production is provided, wherein the mycelium culture medium is formulated as follows: 5.0% starch, 1.0% soy protein powder, 0.4% peptone, 0.3% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.2% magnesium sulfate, pH 7.0, and the percentages are measured in g / 100mL purified water.
[0036] In this embodiment, a method for rapid detection of a specific bacteriophage of Streptomyces for fermentation production is provided, wherein the upper culture medium comprises 1.0% starch, 0.2% phytokinin, 0.3% ammonium sulfate, 0.2% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and 0.1% magnesium sulfate, and the percentages are calculated in g / 100mL purified water, and the pH is adjusted to 7.0 with 20-40% sodium hydroxide solution, and sterilized at 118-122°C for 30 minutes.
[0037] In this embodiment, a method for rapid detection of a specific bacteriophage of Streptomyces for fermentation production is provided, wherein the components of the lower culture medium are specifically as follows: 2.2% soluble starch, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, the percentages being calculated in g / 100mL of purified water, adjusted to pH 7.0 with 20-40% sodium hydroxide solution, and sterilized at 118-122°C for 30 minutes.
[0038] Determination of prevention methods
[0039] There are corresponding disinfectants for the prevention and treatment of microbial contamination. As a special microorganism, the prevention method of bacteriophage mainly requires the selection of suitable disinfectants for regular disinfection.
[0040] Verification Method
[0041] The detected plaques of Streptomyces specific phages for fermentation production are frozen and stored, and the concentration is tested. Different disinfectants are reacted with a certain concentration of phage liquid at a certain concentration ratio, and different action times are set. The liquid after the action is quantitatively diluted and separated for culture, and the count is detected using the solid double-layer plate method, and the control count is set at the same time. The mortality rate is calculated according to the dilution multiple. The disinfection effects of several disinfectants or physical sterilization methods are compared, and at least 3 to 5 verifications are carried out to finally determine the disinfectant or physical disinfection method with better killing effect.
[0042] Validated disinfection methods
[0043] (1) Disinfectants: A. 0.2% chlorhexidine solution; B. 5% Lysol; C. 75% ethanol solution; D. 3% hydrogen peroxide; E. 1% hydrogen peroxide; F. Formaldehyde fumigation.
[0044] (2) Physical sterilization method: ultraviolet disinfection.
[0045] Verify the results
[0046] Using 0.2% Sanisol solution for air spraying or wiping can achieve a killing rate of more than 99% for Streptomyces specific phages used in fermentation production, and can be used as the main means of wiping prevention. The killing rate of formaldehyde is also above 99%, which can be used as the main means of prevention in inoculation rooms and microbial culture rooms. The killing rate of ultraviolet irradiation disinfection is above 80%, but its use has certain limitations, so ultraviolet irradiation can be used to regularly disinfect the air in non-clean areas, such as bottle storage rooms, raw material mixing rooms, etc.
[0047] The present invention provides a method for rapid detection of Streptomyces specific phage for fermentation production, wherein the cultured Streptomyces mycelium is mixed into the upper culture medium of a double-layer detection plate, and then laid on the lower culture medium of the solidified double-layer detection plate, and after solidification, it is placed in an environment that needs to be detected for fixed-point and timed detection, and after retrieval, it is cultured at a constant temperature of 32 to 34°C and a humidity of 40 to 45% for 24 to 28 hours, and the results are observed and recorded, so as to detect phages in the environment. At the same time, by adding the test liquid to the double-layer detection plate, evenly coating it, and then culturing it at a constant temperature of 32 to 34°C and a humidity of 40 to 45% for 24 to 28 hours, observing the growth of the plaque, and recording the infection situation, the detection of phages infected with the seed liquid or fermentation liquid is realized. The presence of bacteriophages can be observed visually, and the number and sedimentation rate of bacteriophages can also be detected. It is an effective, fast and simple method for detecting Streptomyces phages, which is suitable for the detection of ambient air and infected bacterial fluids. It can effectively prevent the infection and outbreak of Streptomyces phages, and can also monitor bacteriophages in the production and research environments of enterprises, so as to detect them very early and take disinfection and other measures in time to prevent the pollution of the factory environment and laboratory, and completely eliminate the possibility of outbreaks.
[0048] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the application disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art disclosed in the present application. The specification and examples are to be regarded as exemplary only, and the true scope of the present application is indicated by the claims.
[0049] It should be understood that the present application is not limited to the precise structure described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The above-described embodiments of the present application do not constitute a limitation on the scope of protection of the present application.
Claims
1. A rapid detection method for Streptomyces specific bacteriophage used in fermentation production, characterized in that: include: Streptomyces mycelium germination culture: prepare a Streptomyces spore suspension, add the Streptomyces spore suspension to a mycelium culture medium, and culture at 30-32° C. for 22-24 hours. When the appearance is yellow and thick and the mycelium is intact and moderate, the culture is terminated to obtain Streptomyces mycelium; Preparation of a double-layer detection plate: prepare a lower culture medium and an upper culture medium, and spread the lower culture medium on a 90 cm plate, adding 15-20 mL / plate and waiting for solidification; adding the Streptomyces mycelium to the upper culture medium at 45-50° C. at a concentration of 10-15%, shaking well, and spreading it on the solidified lower culture medium, adding 10-15 mL / plate, and forming a double-layer detection plate after solidification; Sample detection: dilute the seed liquid or fermentation liquid to obtain the test liquid, and add the test liquid to the double-layer test plate, add 100 μL to each plate, evenly spread it, and culture it at a constant temperature of 32-34°C and a humidity of 40-45% for 24-28 hours, then observe the growth of the plaques and record the infection status; Environmental testing: Place the double-layer test plate in the environmental area to be tested for a set time and then take it back. Place the taken back double-layer test plate at a temperature of 32-34°C and a humidity of 40-45% for constant temperature culture for 24-28 hours. After the culture is completed, observe the growth of plaques and record the infection status.
2. The rapid detection method for Streptomyces-specific phage for fermentation production according to claim 1, characterized in that: The mycelium culture medium has a ratio of: 5.0% starch, 1.0% soybean protein powder, 0.4% peptone, 0.3% ammonium sulfate, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.2% magnesium sulfate, pH 7.0, and the percentages are calculated in g / 100mL purified water.
3. The rapid detection method for Streptomyces-specific phage for fermentation production according to claim 2, characterized in that: The upper culture medium comprises: 1.0% starch, 0.2% vegetable peptone, 0.3% ammonium sulfate, 0.2% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and 0.1% magnesium sulfate, and the percentages are calculated in g / 100mL purified water. The pH value is adjusted to 7.0 with 20-40% sodium hydroxide solution, and sterilized at 118-122°C for 30 minutes.
4. The rapid detection method for Streptomyces-specific phage for fermentation production according to claim 3, characterized in that: The lower culture medium has the following components: 2.2% soluble starch, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the percentages being calculated in g / 100 mL of purified water. The pH is adjusted to 7.0 with 20-40% sodium hydroxide solution and sterilized at 118-122° C. for 30 minutes.
5. The rapid detection method for Streptomyces-specific phage for fermentation production according to claim 1, characterized in that: The test solution obtained by diluting the seed solution or the fermentation solution comprises: In the early stage of infection, the seed solution or the fermentation solution is diluted by 10 - 3~10 -6 times; In the late infection period, the seed solution or the fermentation solution was diluted by 10 -5 ~10 -8 times.
6. The rapid detection method for Streptomyces-specific phage for fermentation production according to claim 1, characterized in that: The environmental areas to be tested include the factory environment, factory environment, clean area and inoculation room. The setting time corresponding to the factory environment is 15 minutes, the setting time corresponding to the factory environment is 20 minutes, and the setting time corresponding to the clean area is 30 minutes. The dynamic monitoring during the inoculation process is 60 minutes.