Rapid detection method and test kit for bacterial count in food

Abstract

The present invention provides a rapid detection method and a detection kit for the number of bacteria in food. The detection kit includes a sterile liquid culture medium, a bacteria enricher, a bacteria color developer and a detection reaction blank container; the bacteria enricher includes a fiber separation membrane and a detachable filter head. The detection process is: first, the fiber separation membrane is used to separate and enrich the bacteria in the liquid food or the solid food dispersed with the liquid; then the fiber separation membrane is placed in the detection reaction container, and the bacteria color developer is added to the reaction container for a color reaction of 2-4 hours, and the absorbance value of the color developer is measured by a spectrophotometer to obtain the bacterial concentration. The present invention significantly improves the efficiency of bacterial separation and enrichment by improving the fiber separation membrane, and can also realize the separation of pigments in food, thereby improving the detection speed and accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection in food, and in particular to a rapid detection method and a detection kit for the number of bacteria in food. Background Art

[0002] Food bacteria refers to bacteria that often exist in food. There are many kinds of bacteria in nature, but due to the limitations of environmental conditions and processing factors of the physical and chemical properties of food, the bacteria that exist in food are only a small part of the bacteria in nature. Food bacteria include pathogenic bacteria and non-pathogenic bacteria: Pathogenic bacteria refer to bacteria that can cause foodborne diseases after entering the human body with food. Common ones include Salmonella and Shigella, which are directly related to the occurrence of diseases and are generally not allowed to be detected in food; non-pathogenic bacteria generally do not cause human diseases, but some of them are spoilage bacteria, such as Pseudomonas, which are closely related to food spoilage and are important indicators for evaluating food hygiene quality.

[0003] In conventional food hygiene testing methods, determining the number of bacteria in food involves operating under strictly specified culture methods and conditions. This method ensures that under specific conditions, each living bacterial cell can generate a colony visible to the naked eye, and the total number of colonies generated is the total number of bacteria in the food. There are many types of bacteria in food, and their physiological characteristics and required culture conditions are different. If the culture method is used to count all the types and numbers of bacteria in food, different culture media and culture conditions must be used, which is a lot of work. Usually, the method of detecting indicator bacteria is used instead.

[0004] The method of testing the bacterial content in food through bacterial culture has a high accuracy rate, but it takes too long and cannot meet the needs of rapid testing in the food field. In addition, in actual production, the shelf life of many foods is only 7 days. If the detection time is too long, the production cost will increase, the production cycle will also increase accordingly, and it is easy to cause the goods to be unsalable.

[0005] It can be seen that the current food bacteria detection methods cannot meet the needs of simple, rapid and accurate detection. For food-borne pathogens that are difficult to culture and identify, more efficient detection methods are needed.

[0006] In view of this, it is necessary to design an improved rapid detection method and detection kit for the number of bacteria in food to solve the above problems. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a method and a test kit for rapid detection of the number of bacteria in food. The present invention enriches the bacteria in the food on a separation membrane, destroys the bacterial cell structure at the same time, so as to release the dehydrogenase in the cell, and then uses the rapid color reaction of the CCK-8 detection reagent and the dehydrogenase to judge the bacterial content in the food. The separation membrane has high separation and enrichment efficiency and fast color reaction speed, so the detection speed is fast, and the sensitivity and accuracy are high.

[0008] To achieve the above-mentioned object of the invention, the present invention provides a method for rapid detection of the number of bacteria in food, comprising the following steps:

[0009] S1. Enriching bacteria in liquid food or solid food dispersion using fiber separation membrane;

[0010] S2. The fiber separation membrane enriched with the microorganism to be detected is placed in a blank sterile reactor, and CCK-8 colorimetric reagent is added immediately or after a certain period of incubation to perform a color reaction, and the absorbance value is tested after the reaction is completed;

[0011] S3. The concentration of microorganisms in the bacterial solution is obtained according to the absorbance value, and then the content of bacteria in the food to be tested is obtained.

[0012] As a further improvement of the present invention, the fiber separation membrane described in step S2 is formed by hot pressing two layers of fiber membranes, the surface of the first layer of fiber membrane is cation-modified, and the surface of the second layer of fiber membrane is super-hydrophobic modified.

[0013] As a further improvement of the present invention, the first layer is a cation-modified nanofiber membrane with a pore size of 200-400 nm, and the surface modification molecules of the cation-modified fiber separation membrane are:

[0014]

[0015] Wherein, n is a positive integer of 4-8.

[0016] As a further improvement of the present invention, the second layer of fiber membrane is a super hydrophobic micron fiber membrane with a pore size of 15-20 um and surface modified with fluorosilane, and the contact angle is greater than 150°.

[0017] As a further improvement of the present invention, in step S2, the color development reaction time is 2-4 h and the temperature is 30-40°C.

[0018] As a further improvement of the present invention, the detection method is used for detecting the content of yeast in food during the production process of fermented food or for detecting the content of bacteria in food before it leaves the factory.

[0019] As a further improvement of the present invention, in step S1, the liquid food or solid food dispersion is an original food sample or a sample obtained by culturing the original food in a sterile culture medium for a certain period of time.

[0020] As a further improvement of the present invention, the preparation method of the sterile culture medium comprises: dissolving yeast extract and peptone in deionized water, treating at high pressure and 121° C. for 20 minutes, and then adding a glucose solution; wherein the content of the yeast extract is (0.5-1.5) wt%, the content of the peptone is (1.5-2.5) wt%, and the content of the glucose is (1.5-2.5) wt%; and the volume ratio of the original food to the sterile culture medium is 1:(50-150).

[0021] A rapid detection kit for the number of bacteria in food comprises a sterile liquid culture medium, a bacteria enricher, a bacteria color developer and a detection reaction blank container; the bacteria enricher comprises the fiber separation membrane and a detachable filter head as described in any one of the above items.

[0022] The beneficial effects of the present invention are:

[0023] 1. The rapid detection method of the number of bacteria in food provided by the present invention uses an improved fiber separation membrane to filter and enrich the bacteria in the food, and then uses CCK-8 to perform a rapid color development reaction and absorbance value detection to quickly obtain the bacterial concentration. In this way, the test results can be quickly obtained directly through the CCK-8 detection reagent. The detection process is simple and fast, and the time consumption is short. The process of colony counting is omitted, and the error caused by colony counting is reduced, which provides a quick method for the rapid detection of bacteria in the food industry.

[0024] 2. Most foods contain pigments, which can interfere with detection. The present invention performs cation modification on the fiber separation membrane and designs the super-hydrophobic property. With this arrangement, on the one hand, the surface of microorganisms is negatively charged, and the cation-modified nanofiber membrane can actively capture and adsorb microorganisms through the interaction between positive and negative charges; on the other hand, the super-hydrophobic fiber separation membrane can directly filter out the pigment, avoiding the interference of the pigment and improving the accuracy of detection.

[0025] 3. The design of the fiber separation membrane of the present invention, the surface cation-modified nanofiber membrane can achieve bacterial filtration, but the nanofiber membrane also has defects such as fragility. Therefore, the second layer of super-hydrophobic micron fiber membrane can not only filter pigments but also play a supporting role, effectively solving the problems of pigment interference and fragility of nanofiber membrane.

[0026] 4. The design of the pore size of the fiber separation membrane in the present invention well combines the interception effect of the microporous structure with the adsorption and killing effect of cationic modification on bacteria, which can significantly improve the bacterial interception rate and accelerate the dissolution rate of dehydrogenase, thereby improving the detection rate and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a schematic flow chart of the method for rapid detection of bacteria quantity in food of the present invention.

[0028] Figure 2 Schematic diagram of the structure of the bacteria enricher.

[0029] Figure 3 Schematic diagram of the top view structure of the fiber separation membrane.

[0030] Figure 4 Schematic diagram of the layer structure of the fiber separation membrane. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below in conjunction with specific embodiments.

[0032] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present invention are shown in the specific embodiments, while other details that are not closely related to the present invention are omitted.

[0033] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0034] See also Figure 1-4 As shown, the method for rapid detection of bacteria count in food provided by the present invention comprises the following steps:

[0035] S1. Use fiber separation membrane to enrich bacteria in liquid food or solid food dispersion, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, etc.; it can be used to detect the yeast content in food during the production process of fermented food or the bacterial content of food before leaving the factory, so as to evaluate food safety.

[0036] In step S1, the liquid food or solid food dispersion is the original food sample or the sample after the original food is cultured in a sterile culture medium for 9-12 hours. When it is the original food sample, the measured bacterial concentration is the actual concentration in the food sample. When it is a sample after culture, the bacterial concentration can be used to qualitatively determine whether the food contains bacteria and the relative size of the bacterial content. The role of culture is to increase the bacterial content in the sample with a low bacterial content in the food through culture, thereby improving the detection sensitivity. This method can also infer the original bacterial content by comparing the concentration of the test sample with the detection concentration of the standard culture sample.

[0037] The sterile culture medium is preferably a yeast extract peptone glucose culture medium. The yeast extract peptone glucose culture medium comprises: dissolving yeast extract and peptone in deionized water, treating at high pressure and 121°C for 20 minutes, and then adding glucose solution; wherein the content of the yeast extract is (0.5-1.5) wt%, preferably 1 wt%; the content of the peptone is (1.5-2.5) wt%, preferably 2 wt%; the content of the glucose is (1.5-2.5) wt%, preferably 2 wt%. The volume ratio of the original food to the sterile culture medium is 1: (50-150).

[0038] S2. Place the fiber separation membrane enriched with bacteria in a blank sterile reactor, and then add CCK-8 colorimetric reagent (cell counting reagent, containing WST-8: chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt, which is reduced to a highly water-soluble yellow formazan product by dehydrogenase in the cell mitochondria under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate. The amount of formazan generated is proportional to the number of living cells. The light absorption value is measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells) for a color reaction of 2-4h at a temperature of 30-40℃. After the reaction is completed, the absorbance value at a wavelength of 450nm is tested. The bacterial concentration is proportional to the absorbance value, and the bacterial concentration can be determined by the high or low photometric value. The detection principle is: after enriching the bacteria with a filter membrane, adding CCK-8 solution, in the presence of an electron coupling reagent, the bacteria can be reduced to a water-soluble yellow formazan product. The more living cells there are, the more formazan is produced, and the darker the color will be. After reacting for 2 hours, the absorbance value at 450nm is measured to calculate the number of bacteria. The detection method of the present invention is simple to operate, takes a short time, and provides a quick method for rapid detection of bacteria in the food industry.

[0039] The fiber separation membrane described in step S2 is formed by hot pressing two layers of fiber membranes, the surface of the first layer of fiber membrane is modified by cations, and the surface of the second layer of fiber membrane is modified by superhydrophobicity.

[0040] The first layer is a cation-modified nanofiber membrane with a pore size of 200-400 nm. The surface modification molecules of the cation-modified fiber separation membrane are:

[0041]

[0042] In the formula, n is a positive integer of 4 to 8. The length of the alkyl chain of the present invention has a significant influence on the enrichment effect. If the alkyl chain is too short, the reaction with bacteria is not sufficient; if the alkyl chain is too long, it is not conducive to rapid detection. Therefore, the optimal alkyl chain length is 4 to 8.

[0043] The second layer of fiber membrane is a super hydrophobic micron fiber membrane with a pore size of 15-20um and surface modified with fluorosilane, with a contact angle greater than 150°. Figure 3 and 4 As shown, the filtration and enrichment effect is better through the combination of two layers of micron and nanometer fiber membranes.

[0044] S3. Obtain the bacterial concentration in the food according to the absorbance value.

[0045] By adopting the above technical scheme, the bacterial separation and enrichment rate of the present invention is fast, the enrichment effect is good, and the color development reaction only takes about 2 hours. Compared with the existing technology, it does not require long-term cultivation and has a fast detection rate, which significantly shortens the detection time of food bacterial content and provides good guidance for the food industry to achieve rapid detection of bacteria.

[0046] A rapid detection kit for the number of bacteria in food, comprising a sterile liquid culture medium, a bacteria enricher, a bacteria colorimetric agent and a blank detection reaction container; the bacteria enricher comprises a fiber separation membrane and a detachable filter head as described in any of the above items. The filter head structure, which is easily detachable, is provided with a pull ring, which can be directly pulled open. The detection process of the rapid detection kit is as follows: 1) using a bacteria enricher to separate and enrich the sample to be tested; 2) breaking the outer shell of the separator; 3) placing the separation membrane enriched with bacteria in a blank sterile reaction container; 4) adding a bacteria colorimetric agent to the reaction container and continuing to culture for 2 hours; 5) detecting the absorbance of the liquid in step 4, and obtaining the bacterial concentration by the absorbance value. Prior to this, a standard curve of absorbance value and bacterial concentration can be prepared.

[0047] Example 1

[0048] A method for rapid detection of bacterial content in liquid food comprises the following steps:

[0049] S1. Enriching bacteria in liquid food, beer, etc. by using fiber separation membrane; the fiber separation membrane is formed by hot pressing two layers of fiber membranes, namely, a cationic-modified nanofiber membrane and a fluorosilane-surface-modified super-hydrophobic microfiber membrane; the cationic-modified molecules are as follows:

[0050]

[0051] Among them, n is 6.

[0052] S2. Take out the fiber separation membrane enriched with bacteria, put it into a centrifuge tube, make three groups of the same concentration in parallel, add 4mL (to ensure that the filter membrane is completely immersed in the solution) CCK-8 solution (diluted with PBS at a ratio of 1:3), react in a 35.5°C constant temperature incubator for 2h, and then measure the absorbance value at 450nm;

[0053] S3. Obtain the bacterial content in food based on the absorbance value.

[0054] Example 2

[0055] A method for rapid detection of bacterial content in solid food, compared with Example 1, is different in that step S1 includes: crushing and dispersing the solid food in water, and then enriching the bacteria in the solid food with a fiber separation membrane. The rest is substantially the same as Example 1 and will not be repeated here.

[0056] Example 3

[0057] A method for rapid detection of bacterial content in liquid food, compared with Example 1, the difference is that step S1 includes: the first layer is a cationic modified nanofiber membrane, and the second layer is a micron fiber membrane that has not been modified with a super-hydrophobic structure and is formed by hot pressing. The rest is substantially the same as Example 1 and will not be repeated here.

[0058] The absorbance value of this embodiment can be used to determine whether liquid food contains bacteria, and can qualitatively determine the bacterial content. If the liquid food contains a small amount of bacteria, the number of bacteria increases after cultivation, and when enrichment detection is performed, the sensitivity is higher and the judgment accuracy is higher. This overcomes the problem of not being able to detect when the bacterial content is too small without cultivation and direct detection.

[0059] Table 1 Test results of Examples 1-3

[0060]

[0061] As can be seen from Table 1, the design of the physical structure of the double-layer membrane and the modified design of the chemical structure are conducive to improving the effective enrichment of bacteria in the sample to be tested and the effective filtration of pigments, avoiding the interference of pigments on the experiment, and improving the accuracy of detection. The bottom layer is not super-hydrophobic, the bacterial interception rate is reduced, and the residual pigment is as high as 40%, so the bacterial detection deviation rate is very high, reaching 12.6%, which has a great interference with the test results. In addition, the double quaternary ammonium salt modified fiber membrane with a special structure can effectively intercept bacteria, thereby improving the enrichment rate and thus improving the detection accuracy.

[0062] In summary, the present invention uses an improved fiber separation membrane to filter and enrich bacteria in food, and then quickly obtains the bacterial concentration by performing a rapid color development reaction and absorbance value detection by CCK-8. In this way, the test results can be quickly obtained directly through the CCK-8 detection reagent. The detection process is simple and fast, and the time consumption is short. The process of colony counting is omitted, and the error caused by colony counting is reduced, which provides a quick method for the food industry to quickly detect bacteria. The interception effect of the microporous structure is well combined with the adsorption and killing effect of cations on bacteria and the separation of pigments by hydrophobicity, which can significantly improve the bacterial interception rate and accelerate the dissolution rate of dehydrogenase, thereby improving the detection rate and accuracy.

[0063] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for rapid detection of bacterial counts in food, characterized in that: The following steps are involved: S1. Enriching bacteria in liquid food or solid food dispersion using fiber separation membrane; S2. Place the fiber separation membrane enriched with the microorganisms to be detected in a blank sterile reactor, add CCK-8 colorimetric reagent for color development, and test the absorbance value after the reaction is completed; The fiber separation membrane is formed by hot pressing two layers of fiber membranes, the surface of the first layer of fiber membrane is modified by cations, and the surface of the second layer of fiber membrane is modified by super hydrophobicity; The first layer is a cation-modified nanofiber membrane with a pore size of 200-400 nm. The surface modification molecules of the cation-modified fiber separation membrane are: Wherein, n is a positive integer of 4-8; The second layer of fiber membrane is a super hydrophobic micron fiber membrane with a pore size of 15-20 um and surface modified with fluorosilane, and the contact angle is greater than 150°; S3. The concentration of microorganisms in the bacterial solution is obtained based on the absorbance value, and then the content of bacteria in the food to be tested is obtained.

2. The method for rapid detection of bacterial counts in food according to claim 1, characterized in that: In step S2, the color development reaction takes 2-4 hours at a temperature of 30-40°C.

3. The method for rapid detection of bacteria in food according to claim 1, characterized in that: The detection method is used for detecting the yeast content in food during the production process of fermented food or for detecting the bacteria content in food before it leaves the factory.

4. The method for rapid detection of bacteria in food according to claim 1, characterized in that: In step S1, the liquid food or solid food dispersion is an original food sample or a sample obtained by culturing the original food in a sterile culture medium for a certain period of time.

5. The method for rapid detection of bacterial counts in food according to claim 4, characterized in that: The preparation method of the sterile culture medium comprises: dissolving yeast extract and peptone in deionized water, treating under high pressure and 121° C. for 20 minutes, and then adding a glucose solution; wherein the content of the yeast extract is (0.5-1.5) wt%, the content of the peptone is (1.5-2.5) wt%, and the content of the glucose is (1.5-2.5) wt%; and the volume ratio of the original food to the sterile culture medium is 1:(50-150).

6. A rapid detection kit for the number of bacteria in food, characterized in that: The invention comprises a sterile liquid culture medium, a bacteria enricher, a bacteria color developer and a detection reaction blank container, wherein the bacteria enricher comprises the fiber separation membrane and a detachable filter head according to any one of claims 1 to 5; and the bacteria color developer is CCK-8.

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