CN111718976A - Method for detecting thermophilic bacteria in CIP cleaning residual water - Google Patents
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Abstract
The invention discloses a method for detecting thermophilic bacteria in CIP cleaning residual water, which comprises the following steps: (1) measuring 50-150 mL of residual water, filtering the residual water by using a filtering device under aseptic operation, and collecting microorganisms in the residual water on a filter membrane; (2) enrichment culture: culturing the filter membrane with a first culture medium at 35-37 ℃ for 36-60 h; (3) diluting the colony cultured in the step (2) in a diluent, and treating for 10-20min at 85-100 ℃; (4) respectively measuring 1mL of the thermally treated diluent as a first sample and a second sample, respectively culturing for 36-60 h at 32-38 ℃ and 50-60 ℃ by using a second culture medium, and counting; (5) gram staining and microscopic examination. The method expands the sample amount of CIP cleaning residual water, comprehensively and effectively detects the colony number and the morphology of the thermophilic bacteria under different growth conditions, so as to judge the CIP cleaning effect and the operation condition of equipment in each production link, and has a guiding function in the production of food.
Description
Technical Field
The invention relates to the technical field of food analysis and detection, in particular to a method for detecting thermotolerant bacteria in CIP cleaning residual water.
Background
The CIP system (in-situ cleaning system) is a cleaning system which can achieve the purpose of cleaning by using water and different cleaning solutions according to a certain program through circulation without disassembling and assembling equipment, and is widely applied to the food, beverage and pharmaceutical industries. The detection of the microorganisms after CIP cleaning is also a health key control point for food processing, and if the microorganisms remained in the CIP system flushing water after cleaning are too high, the hidden danger of microorganism pollution is brought to the food processing in the later period. Coli and the like are easily killed in the CIP process, most of residual bacteria are thermotolerant bacteria, the thermotolerant bacteria generate spores, the thermotolerant bacteria have extremely strong resistance to heat, can survive in the pasteurization process in the production at the later stage of food processing, and even some thermotolerant bacteria are difficult to completely kill in UHT (ultra high temperature) sterilization and autoclaving measures. If the thermophilic bacteria are left in the food, the nutrient substances in the food can be decomposed and utilized, the food is rotten and deteriorated, and even the human health is harmed. Therefore, the detection of the thermophilic bacteria in the cleaning residual water of the CIP system has great guiding significance for the sanitary management and control of food production and processing, and must be strictly controlled. Common thermophilic bacteria include heat-resistant spores of rackets, acinetobacter lofoenii, enterococcus and the like, the most suitable growth temperature of common thermophilic bacteria is 28-42 ℃, thermophilic bacteria in some thermophilic bacteria need to be above 40 ℃ for normal development, the most suitable growth temperature is generally 50-60 ℃, and therefore, the growth environments of different kinds of thermophilic bacteria are different.
Chinese patent document CN106906304A discloses a method for detecting thermotolerant bacteria in dairy products, wherein the thermotolerant bacteria are separated from the dairy products, then bacterial colonies are cultured and counted, and bacterial colonies with typical morphology are selected and then streaked for separate culture; DNA is extracted from the cultured thermotolerant bacteria respectively, and PCR (polymerase chain reaction) sequencing is adopted to detect the types of the thermotolerant bacteria. The method can detect the variety and the quantity of the thermophilic bacteria and has strong qualitative capability, but the molecular biology detection method based on the PCR technology needs to detect the thermophilic bacteria in different forms one by one, and has high detection cost, time consumption and labor consumption. Chinese patent document CN108676841A discloses a method for inspecting spores and heat-resistant spores, which comprises measuring a first sample and a second sample, respectively performing heat treatment at different temperatures on the first sample and the second sample, diluting, performing plate culture at different temperatures, counting the number of thermophilic bacteria and mesophilic bacteria in the samples, and determining whether UHT sterilization is qualified or not according to the counting result. The method has the advantages that the thermophilic bacteria with different growth conditions can be detected, but because the first sample and the second sample are respectively taken during sampling, the method for detecting the thermophilic bacteria in the residual water after CIP cleaning can cause inaccuracy of the detection result due to the sampling difference, the number of the thermophilic bacteria in the residual water after CIP cleaning is less, and the number of the thermophilic bacteria in the residual water can not be effectively detected by using the method. Therefore, there is still a need to provide a method for efficiently and accurately detecting thermophilic bacteria in CIP cleaning residual water.
Disclosure of Invention
The invention aims to solve the problems that the number of thermophilic bacteria in residual water after CIP cleaning is small and thermophilic bacteria under different growth conditions cannot be accurately detected, and provides a method for detecting thermophilic bacteria in CIP cleaning residual water.
In order to achieve the above object, the present invention provides a method for detecting thermophilic bacteria in CIP cleaning residual water, comprising the steps of:
(1) filtering and enriching: measuring 50-150 mL of CIP cleaning residual water, filtering the residual water by using a filtering device under aseptic operation, and collecting microorganisms in the residual water on a filter membrane;
(2) enrichment culture: culturing the filter membrane collected with the microorganisms obtained in the step (1) for 36-60 h at 35-37 ℃ by using a first culture medium;
(3) and (3) heat treatment: placing the bacterial colony cultured in the step (2) in a sterilization container under aseptic operation, diluting with a diluent to obtain a diluent, plugging an outlet, placing the container at 85-100 ℃, preserving heat for 10-20min, and cooling;
(4) culturing and counting: taking the diluent subjected to the heat treatment in the step (3) as a first sample and a second sample respectively, culturing the first sample at 32-38 ℃ by using a second culture medium, culturing the second sample at 50-60 ℃ by using the second culture medium, and counting the thermophilic bacteria after culturing for 36-60 h respectively to obtain the colony number of the thermophilic bacteria in the residual water at two culture temperatures;
(5) gram staining and microscopic examination, and observing colony morphology.
In the invention, the sampling amount of CIP cleaning residual water is enlarged, microorganisms are collected on a filter membrane through filtration, the filter membrane with the microorganisms is cultured by a culture medium, the microorganisms in the residual water are enriched, thermophilic bacteria are left after heat treatment, the thermophilic bacteria are cultured at two different temperatures respectively and then counted and subjected to dyeing microscopic examination, the colony number and the colony morphology in the residual water at the two culture temperatures are finally detected, the thermophilic bacteria with different growth conditions can be detected at the different culture temperatures, so that whether the process flows such as equipment cleaning and disinfection and the like and the execution parameters meet the production requirements can be judged, and the method has a guiding function in the production of food.
In a preferred embodiment of the present invention, in the step (2), the first medium is a PCA medium (plate count agar medium) or a BHI medium (brain heart infusion broth medium).
As a preferred embodiment of the invention, the PCA culture medium is prepared by the following steps: weighing 23.5g of commercially available plate count agar particles, adding into distilled water, stirring, heating, boiling to dissolve completely, adjusting pH to 7.0 + -0.2, diluting to 1000mL, autoclaving at 121 deg.C for 15min, and keeping the temperature at 45-50 deg.C.
As a preferred embodiment of the invention, the BHI culture medium preparation process comprises the following steps: weighing 38.5g of commercially available brain heart extract broth particles, adding into distilled water, stirring, heating, boiling to dissolve completely, adjusting pH to 7.0 + -0.2, diluting to 1000mL, autoclaving at 121 deg.C for 15min, and keeping the temperature to 45-50 deg.C.
In a preferred embodiment of the present invention, in the step (2), the filter membrane with the microorganisms is cultured with the first culture medium by first transferring the filter membrane with the microorganisms to the surface of the culture medium, and this process is required to ensure that no air bubbles remain in the contact between the filter membrane and the culture medium, and the microorganisms are transferred to the surface of the culture medium along with the filter membrane.
In a preferred embodiment of the present invention, in the step (3), the diluent is sterile physiological saline, and the volume of the diluent is 8 to 20 mL.
As a preferred embodiment of the present invention, the preparation method of the sterile physiological saline comprises: 8.5g of sodium chloride is weighed and dissolved in 1000mL of distilled water, and autoclaved for 15min at 121 ℃ for standby.
As a preferable embodiment of the present invention, in the step (3), the cooling process is: and taking out the heat-treated container, and placing the container in flowing water for cooling to room temperature for 5-12 min, wherein the process needs rapid cooling to prevent the propagation of residual heat-resistant spores in the slow cooling process to cause high results.
In a preferred embodiment of the present invention, the heat-treated diluted solution obtained in the step (3) is diluted 10 to 100 times with a diluent. After the heat treatment, if the magnitude of the microorganism is too large, the heat-treated dilution solution needs to be diluted and then cultured.
In a preferred embodiment of the present invention, the volume of the diluent in the step (4) is 0.5 to 3 mL.
In a preferred embodiment of the present invention, in the step (4), the second culture medium is a PCA culture medium or a BHI culture medium, and the first culture medium and the second culture medium are independently selected.
In a preferred embodiment of the present invention, in the step (4), the first sample and the second sample are each provided with two parallel samples.
In a preferred embodiment of the present invention, in the step (4), the first sample is cultured at 32 to 38 ℃, and the preferred culture temperature is 35 to 37 ℃.
In a preferred embodiment of the present invention, in the step (4), the second sample is cultured at 50 to 60 ℃, and the preferred culture temperature is 54 to 56 ℃.
In a preferred embodiment of the present invention, in the step (4), the method for calculating the number of colonies of the thermotolerant bacteria in the residual water comprises: and (3) multiplying the number of colonies after counting the plates by the volume of the diluent in the step (3), and dividing by the volume of the diluent in the step (4) and the volume of the residual water measured in the step (1) to obtain the number of colonies of the thermotolerant bacteria in each 1mL of the residual water. And judging whether the number of the heat-resisting bacteria in the detected residual water deviates from a normal level value or not so as to judge whether equipment cleaning and running conditions of each link in the production process meet the production quality requirement or not.
As a preferred embodiment of the invention, the gram staining comprises four steps of primary staining, mordant staining, decoloration and counterstaining. The specific operation is as follows: smearing with inoculating bacteria, primarily staining with crystal violet, mordanting with iodine solution, decolorizing with ethanol, and counterstaining with red stain.
Compared with the prior art, the invention has the beneficial effects that:
1. the detection method provided by the invention enlarges the sampling amount of CIP cleaning residual water, and can accurately detect the thermophilic bacteria when the number of the thermophilic bacteria in the residual water is small by culturing after the microbes are collected by filtering.
2. The detection method provided by the invention cultures microorganisms at two different temperatures, and can comprehensively and effectively detect the colony number and the morphology of the thermophilic bacteria under different growth conditions in the residual water.
3. According to the invention, the morphology of the thermophilic bacteria can be obviously observed through dyeing microscopic examination, the thermophilic bacteria with different morphologies can be detected, the CIP cleaning effect and the operation condition of equipment in each production link can be judged, the method has a guiding effect on production, and the quality of products is ensured.
Drawings
FIG. 1 shows the results of microscopic examination of thermotolerant bacteria cultured at 36 ℃ in example 1;
FIG. 2 shows the results of microscopic examination of thermotolerant bacteria cultured at 55 ℃ in example 1;
FIG. 3 shows the results of microscopic examination of thermotolerant bacteria cultured at 36 ℃ in example 2;
FIG. 4 shows the results of microscopic examination of thermotolerant bacteria cultured at 55 ℃ in example 2.
Detailed Description
The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
Example 1
Measuring 50mL of residual water after certain equipment is cleaned by CIP, loading a sterile filter membrane with the diameter of 5cm and the pore diameter of 0.45 mu m into a split type funnel, filtering the residual water by using the funnel through vacuum filtration on a sterile operating platform, and collecting microorganisms in the residual water on the filter membrane; 20mL of PCA medium incubated at about 46 ℃ was added to a 100mm dish, after solidification of the PCA medium, the filter was transferred to the surface of the medium with sterile forceps to obtain a culture plate, which was inverted and cultured at 36 ℃ for 48 hours.
Picking all the cultured colonies into a sterilized test tube filled with 10mL of sterile normal saline on a sterile operation table to obtain a diluent, plugging a bottle mouth with a rubber plug, performing spiral rotation for 2-3min, uniformly mixing, placing the test tube in a water bath at 100 ℃ for heat preservation treatment for 10min, taking out the test tube after the treatment is finished, placing the test tube in flowing water, and cooling to room temperature for 8 min; respectively measuring 1mL of the thermally treated diluent as a first sample and a second sample, respectively adding the first sample and the second sample into two groups of 100mm dishes, respectively arranging 2 parallel samples in each group, measuring 1mL of sterile physiological saline as a blank control, adding 20mL of PCA culture medium which is kept at about 46 ℃ into the dishes, and shaking and mixing uniformly; solidifying the culture medium to obtain a culture plate, inverting the culture plate, culturing the first sample at 36 ℃, culturing the second sample at 55 ℃, and culturing for 48h respectively; and counting the heat-resistant bacteria in the first sample plate and the second sample plate after the culture is finished, and calculating to obtain the colony numbers of the heat-resistant bacteria in the residual water under the culture at 36 ℃ and 55 ℃. Picking bacterial colonies from the counted flat plates by using an inoculating loop, uniformly coating the bacterial colonies on a glass slide, quickly passing the smear above flame for multiple times, fixing, dropwise adding a crystal violet staining solution, staining for 1min, timely washing with water, dropwise adding an iodine solution, re-staining for 1min, and timely washing with water; and (3) dropwise adding 95% ethanol for decolorization for about 15-30 s, washing with water, dropwise adding a Czar dyeing solution for re-dyeing for 1min, washing with water, drying, and performing microscopic examination. The low power is used firstly and then the high power is used, and finally the staining condition and the colony morphology are observed by an oil lens. FIG. 1 and FIG. 2 show the colony morphology observed by microscopy.
The colony number of the thermotolerant bacteria in the residual water cultured at 36 ℃ is 68CFU/mL, the colony number of the thermotolerant bacteria in the residual water cultured at 55 ℃ is 11CFU/mL, and the thermotolerant bacteria cultured and grown at 36 ℃ is shown in figure 1, and the thermotolerant bacteria are purple after being dyed, are gram-positive (G +) bacteria and comprise G + bacillus, G + bacillus and G + capsule-shaped spore; FIG. 2 shows thermophilic bacteria grown in culture at 55 ℃ and stained purple, gram-positive bacteria with G + capsuloid spores.
Example 2
Measuring 100mL of residual water after certain equipment is cleaned by CIP, loading a sterile filter membrane with the diameter of 5cm and the pore diameter of 0.45 mu m into a split type funnel, filtering the residual water by using the funnel through vacuum filtration on a sterile operating platform, and collecting microorganisms in the residual water on the filter membrane; adding 15mL of BHI culture medium which is kept at about 46 ℃ into a 100mm dish, transferring the filter membrane to the surface of the culture medium by using sterile forceps after the BHI culture medium is solidified to obtain a culture plate, inverting the culture plate, and culturing for 48h at 36 ℃.
Picking all the cultured colonies into a sterilized test tube filled with 15mL of sterile normal saline on a sterile operation table to obtain a diluent, plugging a bottle mouth with a rubber plug, performing spiral rotation for 2-3min, uniformly mixing, placing the test tube in a water bath at 100 ℃ for heat preservation treatment for 10min, taking out the test tube after the treatment is finished, placing the test tube in flowing water, and cooling to room temperature for 10 min; respectively measuring 1mL of the diluent after heat treatment as a first sample and a second sample, respectively adding the samples into two groups of 100mm plates, wherein each group is provided with 2 parallel samples, measuring 1mL of sterile physiological saline as a blank control, adding 15mL of BHI culture medium which is kept at about 46 ℃ into the plates, and uniformly mixing by shaking; solidifying the culture medium to obtain a culture plate, inverting the culture plate, culturing the first sample at 36 ℃, culturing the second sample at 55 ℃, and culturing for 48h respectively; and counting the heat-resistant bacteria in the first sample plate and the second sample plate after the culture is finished, and calculating to obtain the colony numbers of the heat-resistant bacteria in the residual water under the culture at 36 ℃ and 55 ℃. Picking bacterial colonies from the counted flat plates by using an inoculating loop, uniformly coating the bacterial colonies on a glass slide, quickly passing the smear above flame for multiple times, fixing, dropwise adding a crystal violet staining solution, staining for 1min, timely washing with water, dropwise adding an iodine solution, re-staining for 1min, and timely washing with water; and (3) dropwise adding 95% ethanol for decolorization for about 15-30 s, washing with water, dropwise adding a Czar dyeing solution for re-dyeing for 1min, washing with water, drying, and performing microscopic examination. The low power is used firstly and then the high power is used, and finally the staining condition and the colony morphology are observed by an oil lens. FIG. 3 and FIG. 4 show the colony morphology observed by microscopy.
The colony number of the thermotolerant bacteria in the residual water cultured at 36 ℃ of the sample is 186CFU/mL, the colony number of the thermotolerant bacteria in the residual water cultured at 55 ℃ is 11CFU/mL, and FIG. 3 shows that the thermotolerant bacteria cultured and grown at 36 ℃ have G + bacillus, G + bacillus and G + capsule-shaped spores and G + racket-shaped spores; FIG. 4 shows thermophilic bacteria grown by culturing at 55 ℃ and stained G + filamentous bacteria and gram negative (G-) filamentous bacteria, which are red, as long-rod bacteria with coexisting yin and yang, and thermophilic bacteria.
Example 3
The difference between example 3 and example 1, which is the same as the detection method of example 1, is that the volumes of CIP cleaning residual water measured were 20mL, 50mL, 100mL, 150mL and 200mL, respectively, in order to test the influence of different sample amounts of residual water on the colony detection results. 2 kinds of equipment CIP washs back residual water has been got to this embodiment, is residual water 1 and residual water 2 respectively, through detecting, and the testing result sees table 1.
TABLE 1 Effect of different sample volumes on colony assay results
As can be seen from Table 1, when the content of thermophilic bacteria in the residual water is small, such as residual water 1, if 20mL of samples are taken for enrichment detection, particularly thermophilic spores, the deviation of the detection result is large due to the small volume of the residual water, which is not favorable for the judgment of the result; when the content of the thermophilic bacteria in the residual water is large, such as the residual water 2, if more than 200mL of the thermophilic bacteria is extracted for enrichment detection, the selection of the dilution during re-inoculation after heat screening is not facilitated, and errors caused by multiple dilutions or the plate which is not selected to have the optimal dilution cannot be counted. Therefore, the volume of the residual water measured in the detection of the thermophilic bacteria of the CIP cleaning residual water is 50-150 mL.
Example 4
The same test method as that of example 1, and example 4 is different from example 1 in that the heat treatment conditions of the diluent are 75 ℃/10min, 85 ℃/10min, 90 ℃/10min, 100 ℃/10min and 100 ℃/20min, respectively, in order to test the influence of different heat treatment conditions on the detection result of the microorganisms in the residual water. The same residual water is selected, different treatment temperatures and different treatment times are adopted in the detection, and the detection results are shown in table 2.
TABLE 2 Effect of different Heat treatment conditions on the results of colony detection
As is clear from Table 2, after the diluent was treated at 75 ℃ for 10 minutes, some of the heat-labile bacteria were not killed, and after the diluent was treated at 85 ℃, 90 ℃ or 100 ℃ for 10 minutes, only the heat-labile bacteria survived, and most of the microorganisms remained after the diluent was treated at 100 ℃ for 20 minutes were spores of variable types, such as Racket spores, capsulated spores and Bacillus lentus, and most of them were thermophilic spores. Therefore, the temperature of the heat treatment is 85-100 ℃ and the time is 10-20 min.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A detection method for thermophilic bacteria in CIP cleaning residual water is characterized by comprising the following steps:
(1) filtering and enriching: measuring 50-150 mL of CIP cleaning residual water, filtering the residual water by using a filtering device under aseptic operation, and collecting microorganisms in the residual water on a filter membrane;
(2) enrichment culture: culturing the filter membrane collected with the microorganisms obtained in the step (1) for 36-60 h at 35-37 ℃ by using a first culture medium;
(3) and (3) heat treatment: placing the bacterial colony cultured in the step (2) in a sterilization container under aseptic operation, diluting with a diluent to obtain a diluent, plugging an outlet, placing the container at 85-100 ℃, preserving heat for 10-20min, and cooling;
(4) culturing and counting: taking the diluent subjected to the heat treatment in the step (3) as a first sample and a second sample respectively, culturing the first sample at 32-38 ℃ by using a second culture medium, culturing the second sample at 50-60 ℃ by using the second culture medium, and counting the thermophilic bacteria after culturing for 36-60 h respectively to obtain the colony number of the thermophilic bacteria in the residual water at two culture temperatures;
(5) gram staining and microscopic examination, and observing colony morphology.
2. The method for detecting Thermus according to claim 1, wherein in the step (2), the first medium is a PCA medium or a BHI medium.
3. The method for detecting Thermus according to claim 1, wherein in the step (3), the diluent is sterile physiological saline.
4. The method for detecting thermotolerant bacteria according to claim 3, wherein the volume of the diluent is 8 to 20 mL.
5. The method for detecting thermotolerant bacteria according to claim 1, wherein in the step (3), the cooling step is: and taking out the container after the heat treatment, and placing the container in flowing water to cool to room temperature for 5-12 min.
6. The method for detecting thermotolerant bacteria according to claim 1, wherein the diluent obtained in the step (3) after the heat treatment is diluted 10 to 100 times with a diluent.
7. The method for detecting Thermus-resistant bacterium according to claim 1, wherein the volume of the diluent in step (4) is 0.5 to 3 mL.
8. The method for detecting Thermus according to claim 1, wherein in the step (4), the second medium is a PCA medium or a BHI medium.
9. The method for detecting Thermus according to claim 1, wherein in step (4), the first sample and the second sample are each provided with two parallel samples.
10. The method for detecting thermotolerant bacteria according to claim 1, wherein in the step (4), the number of colonies of the thermotolerant bacteria in the residual water is calculated by: and (3) multiplying the number of colonies after counting the plates by the volume of the diluent in the step (3), and dividing by the volume of the diluent in the step (4) and the volume of the residual water measured in the step (1) to obtain the number of colonies of the thermotolerant bacteria in each 1mL of the residual water.
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