A device and method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy

Abstract

The application discloses a device and method for rapidly determining total number of bacteria based on multi-wavelength reflection spectrum, which comprises: a light emitting element for emitting a continuous light beam; a monochromator arranged in the emission direction of the light beam for separating the light beam into monochromatic light of different wavelengths; an integrating sphere for receiving the monochromatic light separated by the monochromator and converging the diffused monochromatic light; a constant temperature element arranged in the integrating sphere for keeping the standard sample at constant temperature, the diffused reflection being generated after the monochromatic light irradiates the constant temperature standard sample; a photoelectric conversion element connected with the integrating sphere for converting the optical signal into an electrical signal; and an oscilloscope connected with the photoelectric conversion element for reading, calculating and recording the electrical signal conducted by the photoelectric conversion element. The application solves the technical problem that the existing detection method has a large error in detection result due to the size of bacteria individuals, the state of the sample and the color difference, so as to achieve the purpose of rapidly and accurately determining the total number of bacteria.

Description

Technical Field

[0001] This invention relates to the field of bacterial detection technology, and specifically to a device and method for rapidly determining the total bacterial count based on multi-wavelength reflectance spectroscopy. Background Technology

[0002] Total bacterial count refers to the number of bacterial colonies that grow on a sample after it has been cultured aerobically on a standard nutrient agar plate at 37°C for 48 hours. The total bacterial count is calculated and converted to the total number of bacterial colonies per gram (per milliliter) of sample. Total bacterial count is a crucial parameter for determining the quality of many tested samples. Exposure to food and other samples with severely excessive total bacterial counts can easily lead to various diseases, endangering health and even life.

[0003] With the significant improvement in living standards, people have increasingly higher demands for food quality, and safe, high-quality food is becoming increasingly popular among consumers. As a sunrise industry, my country's food industry has seen rapid growth in total output value in recent years, creating an urgent need for rapid and efficient quality control methods and testing equipment. Total bacterial count, as an important biological indicator of food, is measured throughout the entire production process of milk, meat, and eggs. It is a crucial reference for evaluating food quality and a mandatory national hygiene indicator. Microbial contamination of my country's water resources is a serious problem; therefore, developing rapid and accurate bacterial detection technologies is urgently needed. Rapidly and accurately evaluating the effectiveness of cleaning and disinfection of repeatedly used medical devices is a key factor in effectively controlling cross-infection within hospitals. Through bacterial sampling and culture monitoring, and rapid detection, cross-contamination within hospitals can be effectively controlled. Furthermore, daily chemical products are also highly susceptible to microbial contamination, especially when the total bacterial count exceeds the standard, which can seriously damage the product's quality and economic benefits.

[0004] Nationally mandated standards for total bacterial count include those for food, drinking water, and daily chemical products. Furthermore, in enclosed environments such as nuclear submarines and astronaut living quarters, excessive bacterial counts can easily occur. Therefore, developing rapid bacterial count detection instruments that are fast, accurate, and widely applicable to monitor changes in total bacterial counts in food, solid samples, and drinking water is of significant and practical importance for protecting the health of soldiers and civilians.

[0005] To determine the total bacterial count in samples, researchers have developed various detection methods. Current methods for total bacterial count detection mainly fall into two categories: traditional standard plate counting methods and a range of new rapid detection methods, such as flow cytometry, electrical impedance analysis, ELISA (enzyme-linked immunosorbent assay), ATP (adenosine triphosphate) bioluminescence assay, and turbidimetry. Although many rapid methods exist for total bacterial count detection, none currently simultaneously meets the requirements of accurate results, low operating costs, and ease of operation; each method has certain limitations and applicability.

[0006] 1. The standard plate count (SPC) method is the most widely used method for detecting total bacterial count. The total bacterial count is determined by counting the colonies that grow on a standard nutrient agar plate at 37°C for 48 hours under aerobic conditions. The SPC method involves counting 30-300 dilutions on each plate under strictly defined national standards (incubation temperature, time, sample processing, etc.), and then multiplying the count by the dilution factor to obtain the bacterial content per milliliter of sample. As a national standard method, the SPC method measures the number of viable bacteria, providing a relatively accurate reflection of bacterial contamination in the sample. It has the advantage of good repeatability and is suitable for samples with both high and low total bacterial counts. However, the main disadvantages are: (1) the operator needs to have skilled techniques; (2) the results are delayed and generally take 2 days, while in production practice, the test results should be real-time; (3) the obtained results are less than the actual values. During the dilution process, bacteria are usually not single, but several or more aggregated together to form clumps or chains. If the sample is not diluted evenly, the test results will be lower.

[0007] 2. To address the problems of time-consuming and complex operation in traditional bacterial count detection methods, developing rapid bacterial detection methods that are simple to operate, accurate, and suitable for large-scale sample testing has become a research hotspot. The accuracy of flow cytometry is affected by the bacterial species, the dye staining process, and the degree of damage to the bacteria during the detection process. ATP fluorescence methods cannot distinguish between microbial and non-microbial ATP, and certain ions in the sample itself and ATP extraction reagents can interfere with ATP measurement and inhibit luminescence, resulting in insufficient accuracy. Furthermore, the reagents are expensive, increasing the detection cost.

[0008] 3. Based on the shortcomings of the above detection methods, researchers further developed turbidimetry. Turbidimetry, under 600nm monochromatic light, utilizes the inverse relationship between the concentration of a bacterial suspension and its transmittance within a certain range, using transmittance (T value) or absorbance (A value) to reflect the concentration of the sample bacterial suspension. This method is simple to operate, fast in detection, requires no complex pretreatment process, and has relatively high accuracy. However, turbidimetry still has the following drawbacks:

[0009] (1) The turbidimetric method uses monochromatic light of one wavelength to reflect the absorbance value of all bacterial samples, without taking into account the influence of the size difference of different types of bacteria on absorbance (the size difference of different types of bacteria leads to different maximum absorption wavelengths), resulting in reduced accuracy.

[0010] (2) The turbidimetric method is only for measuring the absorbance of transmissive samples that conform to the Lambert-Beer law. It does not take into account that many actual biological samples are in the state of emulsion or suspension (such as raw milk). Directly applying the transmissivity method to measure their absorbance will bring a large error and cause deviation in the bacterial detection results.

[0011] (3) The turbidimetric method fails to eliminate the influence of the sample’s color intensity on absorbance. For samples with darker colors, the detection results are more erroneous. Summary of the Invention

[0012] To overcome the shortcomings of existing technologies, this invention provides a device and method for rapidly determining the total bacterial count based on multi-wavelength reflectance spectroscopy. This solves the technical problem that existing detection methods have large errors in detection results due to differences in bacterial size, sample state, and color, thereby achieving the goal of rapidly and accurately determining the total bacterial count.

[0013] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0014] A device for rapidly determining the total bacterial count based on multi-wavelength reflectance spectroscopy, comprising:

[0015] Light-emitting elements are used to emit continuous beams of light;

[0016] A monochromator, located in the direction of the beam's emission, is used to separate the beam into monochromatic light of different wavelengths;

[0017] An integrating sphere receives the monochromatic light separated by the monochromator and converges the diffusely reflected monochromatic light;

[0018] A thermostatic element is located inside the integrating sphere to keep the standard sample at a constant temperature. When monochromatic light is irradiated onto the thermostatic standard sample, diffuse reflection occurs.

[0019] A photoelectric conversion element, connected to the integrating sphere, is used to convert optical signals into electrical signals;

[0020] An oscilloscope, connected to the photoelectric conversion element, is used to read, calculate, and record the electrical signals transmitted by the photoelectric conversion element.

[0021] In a preferred embodiment of the present invention, the photoelectric conversion element includes:

[0022] An optical fiber, connected to the integrating sphere, is used to receive the monochromatic light converged by the integrating sphere;

[0023] A photomultiplier tube, connected to the optical fiber, is used to convert the optical signal transmitted through the optical fiber into an electrical signal.

[0024] In a preferred embodiment of the present invention, the monochromator is a filter or a grating.

[0025] In a preferred embodiment of the present invention, the light-emitting element is a halogen lamp.

[0026] In a preferred embodiment of the present invention, the temperature-regulating element is a semiconductor temperature controller.

[0027] A method for rapidly determining total bacterial count based on multi-wavelength reflectance spectroscopy includes the following steps:

[0028] The sample to be tested is sampled and prepared into a standard sample;

[0029] After heating the standard sample, the first average reflected light intensity value of the standard sample at different wavelengths was measured under monochromatic light of different wavelengths.

[0030] After the first measurement was completed, the sample was heated and incubated to measure the average reflected light intensity value of the standard sample a second time.

[0031] Calculate the difference between the first and second average reflected light intensity values;

[0032] The difference in average reflected light intensity values ​​was converted into the total bacterial count of the sample using a standard curve.

[0033] In a preferred embodiment of the present invention, the conditions for the heat treatment are: heating at 45-47°C for 3-5 minutes, and the conditions for the heat culture are: heating at 45-47°C for 15-17 minutes, and the total time for the heat treatment and heat culture is 20 minutes.

[0034] In a preferred embodiment of the present invention, the preparation of the standard sample includes:

[0035] If the sample to be tested is water, emulsion, or water sample, the sample to be tested is directly mixed with sterilized skim milk at a volume ratio of 1:1~2 to obtain a standard sample.

[0036] If the sample to be tested is any one of solid, semi-solid, semi-liquid, gel, liquid, or suspension, the sample to be tested is mixed with physiological saline to obtain a sample homogenate. The content of the sample to be tested in the sample homogenate is 5-20% by weight. The sample homogenate is then mixed with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0037] In a preferred embodiment of the present invention, the process of obtaining the standard curve is as follows:

[0038] The sample to be tested is sampled multiple times, and any sample is randomly selected to prepare a standard sample.

[0039] After heating the standard sample, the first average reflected light intensity value of the standard sample at different wavelengths was measured under monochromatic light of different wavelengths.

[0040] After the first measurement was completed, the sample was heated and incubated to measure the average reflected light intensity value of the standard sample a second time.

[0041] Calculate the difference between the first and second average reflected light intensity values;

[0042] Continue to select any sample and repeat the above steps until the difference in the average reflected light intensity values ​​of all samples is obtained.

[0043] By using the difference in the average reflected light intensity values ​​of all collected samples, a standard curve was established between the difference in reflected light intensity values ​​and the true value of the total bacterial count.

[0044] In a preferred embodiment of the present invention, the true value of the total bacterial count is obtained by detecting the total bacterial count of the standard sample after heating and culturing according to the national standard GB4789.2—2016.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] (1) This invention uses monochromatic light of different wavelengths to simultaneously measure the absorbance of the sample at different wavelengths and take the average value, thereby solving the influence of individual bacterial differences on absorbance and improving the accuracy of detection.

[0047] (2) The present invention includes a process of preparing the sample to be tested into a standard sample before bacterial determination, thereby solving the problem of deviation in bacterial detection results caused by different states of the sample to be tested, and making the detection results more accurate;

[0048] (3) This invention utilizes the direct proportionality between the intensity of reflected light and the total number of bacteria, effectively overcoming the problem of absorbance error caused by direct transmissivity measurement, and further making the detection results more accurate;

[0049] (4) Based on the differential method, this invention overcomes the problem that the variation in the color of the sample itself affects the absorbance, and effectively improves the accuracy of detection.

[0050] (5) The present invention does not require a complicated pretreatment process, is easy to operate, and has a fast detection speed.

[0051] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0052] Figure 1 - is a schematic diagram of the device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to an embodiment of the present invention;

[0053] Figure 2 - This is a flowchart illustrating the method steps for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to an embodiment of the present invention;

[0054] Figure 3 - is a standard curve diagram of Embodiment 1 of the present invention;

[0055] Figure 4 - is the standard curve graph of Embodiment 2 of the present invention.

[0056] Explanation of reference numerals in the attached diagram: 1. Light-emitting element; 2. Monochromator; 3. Integrating sphere; 4. Temperature control element; 5. Optical fiber; 6. Photomultiplier tube; 7. Oscilloscope. Detailed Implementation

[0057] The device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy provided by this invention, such as... Figure 1 As shown, the system includes: a light-emitting element 1, a monochromator 2, an integrating sphere 3, a thermostatic element 4, a photoelectric conversion element, and an oscilloscope 7. The light-emitting element 1 emits a continuous light beam; the monochromator 2 is positioned in the direction of the beam's emission and separates the beam into monochromatic lights of different wavelengths; the integrating sphere 3 receives the monochromatic light separated by the monochromator 2 and converges the diffusely reflected monochromatic light; the thermostatic element 3 is located inside the integrating sphere 3 and maintains the standard sample at a constant temperature, causing diffuse reflection when monochromatic light irradiates the thermostatic standard sample; the photoelectric conversion element is connected to the integrating sphere 3 and converts the optical signal into an electrical signal; the oscilloscope 7 is connected to the photoelectric conversion element and reads, calculates, and records the electrical signal transmitted by the photoelectric conversion element. Preferably, the oscilloscope 7 is a digital oscilloscope, which has an internal microprocessor and an external digital display. It can perform operations such as addition, subtraction, multiplication, division, averaging, square root calculation, and root mean square calculation on the captured waveform parameters and display the results. Using a digital oscilloscope, the total bacterial count can be directly calculated from the established standard curve and displayed.

[0058] The integrating sphere 3 comprises a hollow inner surface, forming a spherical cavity with extremely high diffuse reflectivity. Its inner surface can be considered a Lambertian emitter. It is a photometric instrument commonly used for measuring laser power and energy, and material reflectivity. When a laser beam enters the integrating sphere, it forms a uniform, isotropic diffuse reflection field within the sphere. Using the integrating sphere 3 can reduce and eliminate measurement errors caused by differences in light beam shape, divergence angle, and responsivity at different locations on the measuring device.

[0059] In view of the fact that many actual biological samples are in different states such as emulsions or suspensions, a thermostatic element 4 is designed. After monochromatic light passes through the standard sample in the thermostatic element 4, diffuse reflection is generated in the integrating sphere 3. The diffuse reflectance spectrum absorption of the standard sample is measured. By utilizing the relationship between the intensity of reflected light and the total number of bacteria, the problem of absorbance error caused by direct transmittance measurement is effectively overcome.

[0060] The detection process of this invention is as follows:

[0061] (1) Connect the power cord to the device on the clean bench and turn on the device power. At this time, the device will start self-testing. After the self-test is completed, it will indicate that the test is completed. The self-test takes about 10 minutes. After the self-test, it needs to continue to preheat for 30 minutes before the device can be used to test the sample.

[0062] (2) Using BaSO4 as a standard white substance as a reflection reference, its reflected light intensity value was measured and automatically set to 100%;

[0063] (3) Take out the reagent cup, add 1.0 mL of liquid sample to the reagent cup, add 1.0 mL of skim milk, mix thoroughly and it becomes the standard sample. Place the standard sample in the thermostat 4, set the temperature of the thermostat 4 to 47℃, and preheat the standard sample at 47℃ for 5 minutes.

[0064] (4) After preheating for 5 minutes, a continuous light beam is emitted through the light-emitting element 1. The monochromator 2 separates the light beam into monochromatic light of different wavelengths. Preferably, the light beam is separated into monochromatic light of four wavelengths: 440nm, 520nm, 600nm, and 640nm. The monochromatic light of different wavelengths irradiates the isothermal standard sample in the isothermal element 4, producing diffuse reflection. The integrating sphere 3 converges the diffusely reflected monochromatic light, and after the light signal is converted into an electrical signal by the photoelectric conversion element, the initial reflected light intensity value of the test sample is read by the oscilloscope 7 and automatically set to the value "0".

[0065] (5) The reagent cup is placed in the thermostatic element 4 at 47°C and incubated at a constant temperature for 15 minutes. The intensity of reflected light after the liquid sample is incubated is then measured using the measuring device. After 20 minutes of incubation, the intensity of reflected light increases due to bacterial growth. The instrument records the intensity of reflected light of the tested liquid sample at this time. After a large number of samples are tested, the intensity of reflected light of the samples that have been incubated for 20 minutes is fitted with the true value of the total number of bacteria to establish a standard curve, which is then input into the oscilloscope 7.

[0066] For subsequent testing of liquid samples, the reflected light intensity values ​​at 5 min and 20 min were measured according to the above steps. The oscilloscope 7 was used to convert the total bacterial count of the sample into a standard curve and then displayed.

[0067] Furthermore, the measuring device is also equipped with a "view" button, which can be pressed to display the test results of the sample.

[0068] Furthermore, the measuring device is also equipped with a "print" button. By pressing the "print" button, the measurement results can be printed out.

[0069] Furthermore, this measuring device can also be used in conjunction with the sscom 3.2 software. The data is recorded by the software and converted into a TXT text file, which facilitates the subsequent analysis, comparison and tracking of the measured data, and storage in the flash memory medium.

[0070] Furthermore, the photoelectric conversion element includes an optical fiber 5 and a photomultiplier tube 6; the optical fiber 5 is connected to the integrating sphere 3 and is used to receive the monochromatic light converged by the integrating sphere 3; the photomultiplier tube 6 is connected to the optical fiber 5 and is used to convert the optical signal transmitted by the optical fiber 5 into an electrical signal.

[0071] A photomultiplier tube (PMT) is a photoelectric sensing element based on the external photoelectric effect, secondary electron emission, and electron optics theories. In practical applications, it is generally used in the ultraviolet, visible, and near-infrared spectral regions. Besides the photocathode and anode, the PMT's internal structure includes multiple tile-shaped dynodes. During operation, a voltage is generated between adjacent dynodes to accelerate electrons. When the photocathode is irradiated by a light source, it releases photoelectrons, which are then directed towards the first dynode under the influence of an electric field, resulting in secondary and tertiary electron emissions, ultimately multiplying the number of electrons within the tube. Ultimately, the number of electrons collected at the anode of the PMT can increase by 10-1. 4 ~10 8 Times, to achieve

[0072] Detection of weak light signals within the system.

[0073] Furthermore, the monochromator 2 is a filter or a grating. The filter is a Fabry-Perot filter, which uses liquid crystal as the cavity material and has advantages such as narrow bandwidth, low power consumption, wide tuning range, low driving voltage, and simple structure.

[0074] A grating is formed by etching numerous parallel, equally wide, and equally spaced slits onto a transparent glass plate, with the slits themselves being opaque. A grating typically has dozens or even thousands of slits per millimeter. When light waves are transmitted or reflected through the grating, diffraction occurs, creating a specific diffraction pattern. Because different wavelengths of light diffract at different angles, the grating can separate different wavelengths of incident light.

[0075] Furthermore, the light-emitting element 1 is a halogen lamp. The principle of a halogen lamp is to inject halogen gas such as iodine or bromine into the bulb. At high temperature, the sublimated tungsten filament reacts chemically with the halogen. After cooling, the tungsten will re-solidify on the filament, forming a balanced cycle and preventing the filament from breaking prematurely. Therefore, halogen lamps have a longer lifespan than incandescent lamps.

[0076] Furthermore, the constant temperature element 4 is a semiconductor temperature controller. The semiconductor temperature controller has the following advantages: (1) simple structure, no refrigerant, no wear, long life, high reliability, and low requirements for working environment; (2) heating temperature and speed can be controlled by working current, which is flexible and quick to start; (3) small size, light weight, and easy maintenance; (4) high control accuracy; (5) large control temperature range; therefore, the use of a semiconductor temperature controller can further ensure the accuracy of the measurement results.

[0077] The method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy provided by this invention, such as... Figure 2 As shown, it includes the following steps:

[0078] S1: Sample the sample to be tested and prepare it into a standard sample;

[0079] S2: After heating the standard sample, measure the first average reflected light intensity value of the standard sample at different wavelengths under monochromatic light irradiation.

[0080] S3: After completing the first measurement, perform heating and incubation, and measure the average reflected light intensity value of the standard sample for the second time;

[0081] S4: Calculate the difference between the first average reflected light intensity value and the second average reflected light intensity value;

[0082] S5: Convert the difference in average reflected light intensity values ​​into the total bacterial count of the sample using a standard curve.

[0083] In step S2 above, the heat treatment conditions are: heating at 45-47℃ for 3-5 min, and the heat culture conditions are: heating at 45-47℃ for 15-17 min, with a total heat treatment and heat culture time of 20 min.

[0084] Furthermore, the standard sample was heated at 47°C for 5 min and used as a blank to measure the reflected light intensity. After further incubation at 47°C for 15 min, its reflected light intensity was measured again. Since the change in the reflected light intensity of the mixture is caused by bacterial reproduction, the difference between the two intensities can eliminate the interference of sample color on the measurement results. Simultaneously, bacteria reproduce by binary fission, and after 20 min, the total bacterial count in the standard sample doubled. The total bacterial count of the original sample can be obtained from the total bacterial count of the standard sample.

[0085] In step S2 above, the monochromatic light of different wavelengths is 440nm, 520nm, 600nm, and 640nm, respectively. At the same time, the absorbance of the sample at different wavelengths is measured and the average value is taken. The maximum absorption wavelength of the largest bacteria is 440nm, the maximum absorption wavelength of the large bacteria is 520nm, the maximum absorption wavelength of the small bacteria is 640nm, and the maximum absorption wavelength of the medium-sized bacteria is 580-600nm. By using the above four wavelengths of monochromatic light, bacteria of different sizes can be included, thereby overcoming the technical problem that the maximum absorption wavelength is different due to the difference in the size of different types of bacteria.

[0086] Furthermore, the preparation of standard samples includes:

[0087] If the sample to be tested is water, emulsion, or water sample, mix the sample to be tested directly with sterilized skim milk at a volume ratio of 1:1~2 to obtain a standard sample.

[0088] If the sample to be tested is any one of the following: solid, semi-solid, semi-liquid, gel, liquid, or suspension, the sample to be tested is mixed with physiological saline to obtain a sample homogenate. The content of the sample to be tested in the sample homogenate is 5-20% by weight. The sample homogenate is then mixed with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0089] Specifically as follows:

[0090] If the sample to be tested is an emulsion or water, mix the sample to be tested with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0091] If the sample to be tested is solid or semi-liquid, first mix the sample to be tested with physiological saline to obtain a sample homogenate containing 10% by weight of the sample to be tested. Then mix the sample homogenate with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0092] If the sample to be tested is a semi-solid, first mix the sample to be tested with physiological saline to obtain a sample homogenate containing 15% by weight of the sample to be tested. Then mix the sample homogenate with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0093] If the sample to be tested is in gel form, first mix the sample to be tested with physiological saline to obtain a sample homogenate containing 20% ​​by weight of the sample to be tested. Then mix the sample homogenate with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample.

[0094] If the sample to be tested is a liquid, first mix the sample to be tested with physiological saline to obtain a sample homogenate containing 5% by weight of the sample to be tested. Then mix the sample homogenate with sterilized skim milk at a volume ratio of 1:1~2 to obtain a standard sample.

[0095] If the sample to be tested is a suspension, first mix the sample to be tested with physiological saline to obtain a sample homogenate containing 10% by weight of the sample to be tested. Centrifuge the sample and take the supernatant and mix it with sterile skim milk at a volume ratio of 1:1~2 to obtain a standard sample.

[0096] Furthermore, the process of obtaining the standard curve is as follows:

[0097] The sample to be tested is sampled multiple times, and any sample is randomly selected to prepare a standard sample.

[0098] After heating the standard sample, the first average reflected light intensity value of the standard sample at different wavelengths was measured under monochromatic light of different wavelengths.

[0099] After the first measurement was completed, the sample was heated and incubated to measure the average reflected light intensity value of the standard sample a second time.

[0100] Calculate the difference between the first and second average reflected light intensity values;

[0101] Continue to select any sample and repeat the above steps until the difference in the average reflected light intensity values ​​of all samples is obtained.

[0102] By using the difference in the average reflected light intensity values ​​of all collected samples, a standard curve was established between the difference in reflected light intensity values ​​and the true value of the total bacterial count.

[0103] Furthermore, the true value of the total bacterial count is obtained by measuring the total bacterial count of a standard sample after heating and culturing, according to the national standard GB4789.2—2016 "Determination of Total Microbial Colony Count in Food". This standard specifies the method for determining the total bacterial count in food.

[0104] The present invention will be further illustrated by specific embodiments below.

[0105] Examples 1-15

[0106] Detection of emulsion samples (raw milk, milk beverages, etc.):

[0107] The standard plate count method (GB4789.2—2016) was used to detect the true value of total bacterial count in dairy products. 1.0 mL of dairy product was added to 1.0 mL of sterilized skim milk. The absorbance measured by the instrument was used as a reference value. The initial absorbance of the test emulsion sample was set to 0. Using BaSO4 as a white standard as a reflectance reference, the initial reflected light intensity of the instrument was set to 100%. After heating the mixture at 47℃ for 5 min, its reflected light intensity was measured as a blank. After further incubation at 47℃ for 15 min, its reflected light intensity was measured again. After 20 min of incubation, due to bacterial growth, the reflected light intensity increased, and the instrument recorded the reflected light intensity value of the tested dairy product at this time. After large-scale sampling and testing, the reflected light intensity value of the dairy products incubated for 20 min was fitted with the true value of total bacterial count to establish a standard curve.

[0108] Take 15 groups of emulsion samples (including raw milk, milk beverages, etc.), and measure their reflected light intensity values ​​according to the above steps. Then, use a standard curve to convert the total bacterial count of the samples.

[0109] Examples 16-30

[0110] Water sample testing:

[0111] Take 10 mL of water sample and first perform aseptic filtration using a 10 μm pore size filter membrane to remove solid particles from the water and eliminate their interference with the experiment. Take 1.0 mL of the aseptically filtered water sample and add it to 1.0 mL of sterile skim milk. The absorbance value measured by the instrument is the reference value. Set the initial absorbance value of the water sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its absorbance. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, the reflected light intensity value increases. The instrument records the reflected light intensity value of the test water sample at this time. After large-scale sampling and testing, fit the reflected light intensity value of the water sample after 20 min of incubation with the true value of the total bacterial count to establish a standard curve.

[0112] Take 15 groups of water samples (including bottled water, river water, etc.), and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert the total bacterial count of the samples.

[0113] Examples 31-45

[0114] Detection of solid samples

[0115] Take 25.00g of solid sample (bread, candy, dried fruit, sausage, etc.) and place it in a sterile homogenizing cup containing 225.00 mL of physiological saline. Homogenize at 8000 r / min to 10000 r / min for 1 min to 2 min, or place it in a sterile homogenizing bag containing 225 mL of diluent and beat it with a tapping homogenizer for 1 min to 2 min to obtain a sample homogenate with a solid sample weight ratio of 10%, and set aside for later use.

[0116] Take 1.0 mL of sample homogenate and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflected light intensity value. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, its reflected light intensity value increases. Record the reflected light intensity value of the tested solid sample at this time. After a large number of samples are tested, fit the reflected light intensity value of the solid sample that has been incubated for 20 min with the true value of the total bacterial count to establish a standard curve.

[0117] Take 15 groups of solid samples (including candy, sausage, etc.), and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert the total bacterial count of the samples.

[0118] Examples 46-60

[0119] Detection of semi-solid samples

[0120] Take 15.00g of semi-solid sample (vegetable puree, fruit puree, etc.) and place it in an Erlenmeyer flask containing 85.00 mL of physiological saline. Mix it evenly under a vortex mixer to obtain a sample homogenate with a semi-solid sample weight ratio of 15%, and set it aside for later use.

[0121] Take 1.0 mL of the pretreated semi-solid sample solution and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflected light intensity value. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, its reflected light intensity value increases. Record the reflected light intensity value of the tested solid sample at this time. After a large number of samples are tested, fit the reflected light intensity value of the semi-solid sample after 20 min of incubation with the true value of the total bacterial count to establish a standard curve.

[0122] Take 15 groups of semi-solid samples (including rice noodles, etc.), and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert the total bacterial count of the samples.

[0123] Examples 61-75

[0124] Detection of semi-liquid samples

[0125] Take 10.00g of semi-liquid sample (daily chemical products, eight-treasure porridge, etc.) and place it in an Erlenmeyer flask containing 90.00 mL of physiological saline. Mix it evenly under a vortex mixer to obtain a sample homogenate with a semi-liquid sample weight ratio of 10%, and set it aside for later use.

[0126] Take 1.0 mL of sample homogenate and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflection intensity value. Continue to incubate at 47℃ for 15 min and measure its reflection intensity value again. After incubation for 20 min, due to bacterial growth, its reflection intensity value increases. Record the reflection intensity value of the tested solid sample at this time. After a large number of samples are tested, fit the reflection intensity value of the semi-liquid sample that has been incubated for 20 min with the true value of the total bacterial count to establish a standard curve.

[0127] Take 15 groups of semi-liquid samples (including rice noodles, etc.), and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert the total bacterial count of the samples.

[0128] Examples 76-90

[0129] Detection of gel-like samples

[0130] Take 20.00g of gel-like sample (such as tofu) and place it in a sterile homogenizing cup containing 80.00 mL of physiological saline. Homogenize at 8000 r / min to 10000 r / min for 1 min to 2 min to obtain a sample homogenate with a gel-like sample weight ratio of 20% for later use.

[0131] Take 1.0 mL of the pretreated gel sample solution and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflected light intensity value. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, its reflected light intensity value increases. Record the reflected light intensity value of the gel sample at this time. After a large number of samples are tested, fit the reflected light intensity value of the gel sample after 20 min of incubation with the true value of the total bacterial count to establish a standard curve.

[0132] Take 15 groups of gel-like samples (jelly, etc.) and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert them into the total bacterial count value of the samples.

[0133] Examples 91-105

[0134] Detection of liquid samples

[0135] Take 10.00g of liquid sample (fruit juice, soy milk, etc.) and place it in an Erlenmeyer flask containing 190.00 mL of physiological saline. Mix it evenly under a vortex mixer to obtain a sample homogenate with a liquid sample weight ratio of 5%, and set it aside for later use.

[0136] Take 1.0 mL of the pretreated liquid sample solution and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflected light intensity value. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, its reflected light intensity value increases. Record the reflected light intensity value of the solid sample at this time. After a large number of samples are tested, fit the reflected light intensity value of the liquid sample that has been incubated for 20 min with the true value of the total bacterial count to establish a standard curve.

[0137] Take 15 groups of liquid samples (broth, etc.) and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert the total bacterial count of the samples.

[0138] Examples 105-120

[0139] Suspension samples

[0140] Take 20.00g of suspension sample (such as blood) and place it in an Erlenmeyer flask containing 800.00 mL of physiological saline. Mix it evenly under a vortex mixer to obtain a sample homogenate with a suspension sample weight ratio of 10%. Centrifuge at 7000-10000 speed for 15 minutes and take the supernatant for later use.

[0141] Take 1.0 mL of the pretreated suspension sample solution and add it to 1.0 mL of sterile skim milk. The absorbance measured by the instrument is the reference value. Set the initial absorbance value of the solid sample to be tested to 0. Use BaSO4 standard white substance as the reflection reference and set the initial reflected light intensity of the instrument to 100%. After heating the mixture at 47℃ for 5 min, use it as a blank to measure its reflected light intensity value. Continue to incubate at 47℃ for 15 min and measure its reflected light intensity value again. After incubation for 20 min, due to bacterial growth, its reflected light intensity value increases. Record the reflected light intensity value of the tested solid sample at this time. After a large number of samples are tested, fit the reflected light intensity value of the suspension samples that have been incubated for 20 min with the true value of the total bacterial count to establish a standard curve.

[0142] Take 15 groups of suspension samples (barium sulfate suspension), and measure their reflected light intensity values ​​according to the above steps. Then, use the standard curve to convert them into the total bacterial count value of the samples.

[0143] The total bacterial count of 15 groups (Examples 1-15) of emulsion samples was determined using the apparatus and method of the present invention (hereinafter referred to as the instrumental method), and the accuracy was analyzed. The specific measurement results and analysis results are shown in Tables 1 and 2:

[0144] Table 1. Results of total bacterial count determination for emulsion samples using instrumental and national standard methods (unit: μg / L) 4 cfu / mL)

[0145]

[0146] Table 2. Comparative Analysis of T-tests for Instrumental Methods in Emulsion Samples

[0147]

[0148] As shown in Table 2, within the 95% confidence interval, the P-value (two-tailed value) obtained by the paired T-test is 0.335, P > 0.05. The P-value is greater than 0.05 (i.e., the difference between samples is not significant), indicating that there is no significant difference between the results of the instrumental method for detecting the total bacterial count of emulsion samples and the results of the standard method (standard plate colony count method). The accuracy of the instrumental method for detecting emulsion samples is high.

[0149] The total bacterial count of 15 water samples (Examples 16-30) was determined using an instrumental method, and the accuracy was analyzed. The specific measurement and analysis results are shown in Tables 3 and 4.

[0150] Table 3. Results of total bacterial count determination in water samples using instrumental and national standard methods (unit: μg / L) 4 cfu / mL)

[0151]

[0152] Table 4. Comparative Analysis of T-tests for Instrumental Methods in Water Samples

[0153]

[0154] As shown in Table 4, within the 95% confidence interval, the P-value (two-tailed) obtained by the paired T-test is 0.721, P > 0.05, P > 0.05 (i.e., the difference between samples is not significant), indicating that there is no significant difference between the results of the instrument method for detecting the total number of bacteria in water samples and the results of the standard method, and the accuracy of the instrument method for detecting the total number of bacteria in water samples is high.

[0155] Solid, semi-solid, semi-liquid, gel, liquid, and suspension samples (Examples 30-120) were all compared using a T-test. The P-values ​​were all greater than 0.05, indicating that there was no significant difference between the results of the instrumental method for detecting the total bacterial count in the above samples and the results of the standard method. The accuracy of the instrumental method for detecting the total bacterial count in the above samples is high.

[0156] Standard curves established for emulsion samples, such as Figure 3 As shown, by Figure 3 It can be seen that R 2 =0.994, R 2 Ri is the correlation coefficient of the standard curve, used to evaluate the goodness of fit between the experimental data and the fitted function. 2 The closer the value is to 1, the higher the degree of agreement. Therefore, the standard curve established for emulsion samples is in high agreement with the measured data.

[0157] The standard curve established for the water sample is as follows: Figure 4 As shown, by Figure 4 It can be seen that R 2 =0.9931, therefore the standard curve established for the water sample is in high agreement with the measured data.

[0158] Standard curves were established for samples including solids, semi-solids, semi-liquids, gels, liquids, and suspensions. R0 2 All values ​​are greater than 99%, therefore the standard curves established for solid, semi-solid, semi-liquid, gel, liquid, and suspension samples are in high agreement with the measured data.

[0159] As can be seen from the above, the p-values ​​of the T-test comparison analysis results of each embodiment are all greater than 0.05, and the correlation coefficients R of the standard curves established for each type of sample are also high. 2 The values ​​are all greater than 99%, which fully demonstrates that the device and method of the present invention can accurately measure the total bacterial count of emulsions, water, solids, semi-solids, semi-liquids, gels, liquids, and suspensions, overcoming the technical problem of large measurement errors in existing technologies.

[0160] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy, characterized in that, Includes the following steps: The sample to be tested is sampled and prepared into a standard sample. If the sample to be tested is water, emulsion, or a water sample, the sample to be tested is directly mixed with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain a standard sample. If the sample to be tested is any one of solid, semi-solid, semi-liquid, gel, liquid, or suspension, the sample to be tested is mixed with physiological saline to obtain a sample homogenate. The content of the sample to be tested in the sample homogenate is 5% by weight. 20%, mix the sample homogenate with sterilized skim milk at a volume ratio of 1:1 to 2 to obtain the standard sample; After heating the standard sample, the first average reflected light intensity value of the standard sample at different wavelengths was measured under monochromatic light of different wavelengths. After the first measurement was completed, the standard sample was heated and cultured at the same temperature as the first heating temperature, and the average reflected light intensity value was measured a second time. Calculate the difference between the first and second average reflected light intensity values; The difference in average reflected light intensity values ​​was converted into the total bacterial count of the sample using a standard curve.

2. The method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 1, characterized in that, The heat treatment conditions are: at 45°C Heating at 47℃ for 3 days The heating and incubation conditions were as follows: 5 min at 45°C. Heating at 47℃ for 15 minutes The total time for the heat treatment and heat culture was 20 minutes, which was 17 minutes.

3. The method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 1, characterized in that, The process of obtaining the standard curve is as follows: The sample to be tested is sampled multiple times, and any sample is randomly selected to prepare a standard sample. After heating the standard sample, the first average reflected light intensity value of the standard sample at different wavelengths was measured under monochromatic light of different wavelengths. After the first measurement was completed, the sample was heated and incubated to measure the average reflected light intensity value of the standard sample a second time. Calculate the difference between the first and second average reflected light intensity values; Continue to select any sample and repeat the above steps until the difference in the average reflected light intensity values ​​of all samples is obtained. By using the difference in the average reflected light intensity values ​​of all collected samples, a standard curve was established between the difference in reflected light intensity values ​​and the true value of the total bacterial count.

4. The method for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 3, characterized in that, The true value of the total bacterial count is obtained by measuring the total bacterial count of the standard sample after heating and culturing according to the national standard GB4789.2—2016.

5. A device for rapidly determining the total bacterial count based on multi-wavelength reflectance spectroscopy, characterized in that, The apparatus is used to perform the measurement step of the method as described in any one of claims 1 to 4, the apparatus comprising: Light-emitting elements, used to emit continuous beams of light; A monochromator, located in the direction of the beam's emission, is used to separate the beam into monochromatic light of different wavelengths; An integrating sphere receives the monochromatic light separated by the monochromator and converges the diffusely reflected monochromatic light; A thermostatic element is located inside the integrating sphere to keep the standard sample at a constant temperature. When monochromatic light is irradiated onto the thermostatic standard sample, diffuse reflection occurs. A photoelectric conversion element, connected to the integrating sphere, is used to convert optical signals into electrical signals; An oscilloscope, connected to the photoelectric conversion element, is used to read, calculate, and record the electrical signals transmitted by the photoelectric conversion element.

6. The device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 5, characterized in that, The photoelectric conversion element includes: An optical fiber, connected to the integrating sphere, is used to receive the monochromatic light converged by the integrating sphere; A photomultiplier tube, connected to the optical fiber, is used to convert the optical signal transmitted through the optical fiber into an electrical signal.

7. The device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 5, characterized in that, The monochromator is a filter or a grating.

8. The device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 5, characterized in that, The light-emitting element is a halogen lamp.

9. The device for rapid determination of total bacterial count based on multi-wavelength reflectance spectroscopy according to claim 5, characterized in that, The temperature control element is a semiconductor temperature controller.

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