A method for visually monitoring the spoilage of milk

Abstract

The present invention discloses a method for visually monitoring the spoilage of milk, which comprises: providing an environment simulating the spoilage period to simulate the spoilage of milk; constructing a milk spoilage-fluorescence model to determine a luminescence change color band or a linear graph of quantum yield change reflecting the degree of milk spoilage; and determining the degree of spoilage of the milk to be tested based on the milk spoilage-fluorescence model. The present invention evaluates the quality of milk by monitoring the change in the photoluminescence characteristics of milk, and has the characteristics of being able to immediately and quickly judge the freshness of milk, with visible results, high accuracy, simple operation and short operation time.

Description

Technical Field

[0001] The present invention belongs to the technical field of milk detection, and particularly relates to a method for visually monitoring the spoilage of milk. Background Art

[0002] Dairy products, especially milk, are an important natural food, which contains rich nutrients essential for the human body, such as proteins, fats, carbohydrates, vitamins, etc. However, the rich nutrients make milk extremely prone to spoilage, seriously affecting the taste and nutritional value. Although milk undergoes strict sterilization treatment during the production and processing process, there is a risk of secondary contamination during filling, storage, and transportation. Especially for pasteurized fresh milk, its sterilization treatment is usually carried out in the range of 60 - 82°C, which is much lower than the temperature of ultra-high temperature sterilization. Its bacterial content is much higher than that of ultra-high temperature sterilized pure milk, so fresh milk is more likely to be affected by the environment and deteriorate. Therefore, simply judging the spoilage of milk by the shelf life may be inaccurate. And in recent years, food safety problems have occurred frequently, making consumers more concerned about the freshness and quality of milk.

[0003] At present, there are many mature methods for detecting the freshness and quality of milk. Its basic principle is to test or analyze materials by measuring the chemical or physical properties of the materials, mainly including chemical detection methods, ATP bioluminescence technology, electronic nose technology, infrared spectroscopy, ultrasonic detection methods, etc. Many of these methods have problems such as destroying samples, long detection cycles, complex operations, and high costs. For example, chemical detection methods need to detect the pH, nutrients, etc. of milk, which is not only cumbersome to operate but also has a risk of secondary contamination; infrared spectroscopy is limited by the use of instruments and is easily interfered by environmental factors in the near-infrared band, so it is difficult to monitor the quality changes of milk in real time and accurately. Due to the above problems, there is an urgent need to develop a detection technology for the freshness and quality of milk that can be monitored in real time, is non-destructive, efficient, and convenient. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for visually monitoring the spoilage of milk in view of the deficiencies of the above-mentioned prior art.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: providing an environment simulating the spoilage period to simulate the spoilage of milk;

[0006] Constructing a milk spoilage - fluorescence model to determine the emission change color band or quantum yield change linear diagram reflecting the degree of milk spoilage;

[0007] Determining the degree of spoilage of the milk to be tested.

[0008] The present invention has the following advantages compared with the prior art:

[0009] 1. Based on the correspondence between the photoluminescence characteristics of milk and its degree of spoilage, the present invention provides a method for evaluating the quality of milk by monitoring the changes in the photoluminescence characteristics of milk. This method includes simulating milk spoilage, constructing a milk spoilage-fluorescence model, determining the luminescence change color spectrum band or the linear graph of quantum yield change that reflects the degree of milk spoilage, and judging the degree of milk spoilage based on the above model. It has the characteristics of being able to instantly and quickly evaluate the freshness of milk, with visible results, high accuracy, simple operation, and short operation time.

[0010] 2. Preferably, the milk spoilage-fluorescence model constructed by the present invention includes testing the photophysical properties and physicochemical properties of milk during the spoilage simulation period. The physicochemical properties include pH, lactic acid content, free amino acid content, and / or protein aggregation state. Using the above multi-dimensional integrated physicochemical properties to determine the freshness and quality of milk is more comprehensive and accurate than judging based on a single acidity value.

[0011] 3. Preferably, the present invention can judge the degree of milk spoilage only by obtaining the luminescence color or quantum yield of the milk to be tested under ultraviolet light irradiation, without the need to process the milk to be tested.

[0012] The following combines the drawings and embodiments to make a further detailed description of the technical solution of the present invention. Description of the Drawings

[0013] Figure 1 are the luminescence photos of different brand milks under ultraviolet excitation at 254, 312, and 365 nm and the spectrograms at different excitation wavelengths;

[0014] Figure 2 are the emission spectra, CIE chromaticity diagrams, and lifetime diagrams of fresh milk at different excitation wavelengths;

[0015] Figure 3 are the emission spectra of casein, whey protein, and riboflavin at different excitation wavelengths and the luminescence photos under 365 nm ultraviolet excitation;

[0016] Figure 4 are the luminescence photos of milk placed for different times under 312 and 365 nm ultraviolet excitation;

[0017] Figure 5 are the luminescence photos of casein and riboflavin before and after being placed for three days under 254, 312, and 365 nm ultraviolet excitation;

[0018] Figure 6 are the graphs of the changes in lactic acid content, pH, free amino acid concentration, and solution particle size of milk over time;

[0019] Figure 7Particle size distribution and TEM images of protein aggregates in milk after standing for different times;

[0020] Figure 8 Emission spectra and CIE chromaticity diagrams of milk standing for different times;

[0021] Figure 9 Luminescence change color bands reflecting the degree of milk spoilage;

[0022] Figure 10 Schematic diagram of the determination results of the degree of spoilage of milk sample A and milk sample B to be tested;

[0023] Figure 11 Linear diagram of the change in quantum yield reflecting the degree of milk spoilage;

[0024] Figure 12 Quantum yield diagram of milk sample C to be tested;

[0025] Figure 13 Schematic diagram of the determination results of the degree of spoilage of milk sample D to be tested. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0027] In the following description, the term "and / or" is used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, B exists alone, and both A and B exist simultaneously. Where A and B may be singular or plural.

[0028] In the following description, the terms "including", "comprising", "having", "containing", etc. are all open-ended terms, that is, they are meant to include but not limited to.

[0029] Those skilled in the art should understand that in the following description of the embodiments of the present application, the sequence numbers do not mean the order of execution, and some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0030] Those skilled in the art should understand that the numerical ranges in the embodiments of the present application should be understood as specifically disclosing each intermediate value between the upper and lower limits of the range. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, the technical / scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] The technical principle adopted by the present invention:

[0033] The spoilage of milk is mainly caused by the reproduction and metabolism of microorganisms. For example, the growth or reproduction of microorganisms such as Lactobacillus, Pseudomonas, and coliforms. During the metabolism of organic compounds by the above microorganisms, milk will show phenomena such as sour flavor, abnormal smell, and flocculent coagulation. These biochemical processes cause changes in the physical and chemical properties of milk, such as protein denaturation, lactic acid generation, and riboflavin oxidation. Milk has the photoluminescence property under the excitation of ultraviolet light with a specific wavelength. This property stems from the photoluminescence properties of the main nutrients in milk, including proteins and riboflavin (vitamin B2). That is, the photoluminescence phenomenon of milk is the result of the luminescence effect generated by the main nutrients contained therein under the irradiation of an excitation light source. Spoilage will cause changes in the structure, morphology, and aggregation state of the main nutrient components in milk, resulting in significant changes in the luminescence properties. Based on this, the present invention provides a method for evaluating the quality of milk by monitoring the changes in the photoluminescence properties of milk, including:

[0034] Providing an environment simulating the spoilage period to simulate the spoilage of milk;

[0035] Constructing a milk spoilage-fluorescence model to determine the luminescence change color band or the linear diagram of quantum yield change reflecting the degree of milk spoilage;

[0036] Based on the milk spoilage-fluorescence model, determining the degree of spoilage of the milk to be tested.

[0037] In the present invention, there are no restrictions on the brands of milk used to construct the milk spoilage-fluorescence model and the milk to be tested. Different brands have little effect on the luminescence of milk and have compositional consistency. For example, they are all commercially available pasteurized fresh milk or ultra-high temperature sterilized pure milk with only raw milk as the ingredient. To further improve the judgment accuracy, it is specified that the milk to be tested and the milk used to construct the milk spoilage-fluorescence model are of the same brand and the same batch.

[0038] Commercially available pure milk or fresh milk has a universal intrinsic luminescence phenomenon. Through measuring the luminescence photos of milk of different brands under ultraviolet excitation at 254 nm, 312 nm, and 365 nm, the present invention finds that, as Figure 1 shown in a of Figure 1 , milk produces weak blue-violet light under 254 nm ultraviolet light excitation, bright blue-white light and yellow-green light under 312 nm and 365 nm ultraviolet light excitation respectively, and the immediate emission spectra ( Figure 2 and Figure 3 ) of milk of different brands are very similar, indicating that milk has a universal intrinsic luminescence phenomenon. The main components of milk are water and nutritional components. The nutritional components include proteins, fats, lactose, vitamins, etc. The present invention measures the emission spectra of milk and its main nutritional components, and the test results are as Figure 2 and Figure 2 shown. Among them, Figure 2 a of Figure 2 is the emission spectrum, Figure 3 b of Figure 3 is the CIE chromaticity diagram, Figure 3 c of Figure 3 is the lifetime diagram, which are the emission spectra of fresh milk at different excitation wavelengths, the CIE chromaticity diagram, and the lifetime diagram. Among them, Figures 1 - 3 , the emission spectrum peaks of milk in the 300 - 500 nm band are mainly based on casein and whey protein, the emission spectrum peak at 340 nm mainly comes from the tryptophan residues of proteins, the emission peaks in the 390 - 480 nm range are based on aggregates of proteins and their derivatives with strong emission, and the emission peak around 530 nm comes from riboflavin. It can be seen that proteins and riboflavin (vitamin B2) make important contributions to the photoluminescence of milk. By testing the photoluminescence performance parameters of milk and its main components, the present invention determines that the luminescence effect of milk mainly depends on the proteins and riboflavin in it, which is consistent with the existing research conclusions. Spoilage will cause changes in the physical and chemical properties and photoluminescence characteristics of milk, and these changes are highly correlated with the changes in proteins and riboflavin. Through measuring the ultraviolet luminescence of milk and its main nutritional components during the spoilage period, the present invention shows that the results are as Figure 4and Figure 5 as shown, where Figure 4 are ultraviolet luminescence photos of milk at different storage times, Figure 5 are shown under ultraviolet excitation light at 254, 312, and 365 nm after casein and riboflavin are placed for three days. Combining Figure 4 and Figure 5 it can be seen that deterioration will cause changes in the physical properties and luminescence characteristics of milk, casein, and riboflavin. The luminescence display of milk deterioration shows a consistent change trend with the luminescence display of casein and riboflavin deterioration.

[0039] The described simulated deterioration period environment is used to provide a place close to the general milk storage scenario to obtain milk quality change sampling samples required for modeling. This environment can be an ideal environment or an experimental environment. The present invention preferably uses a constant temperature ideal environment to ensure the repeatability of the experiment. In some preferred embodiments, the simulation of milk deterioration is specifically as follows: under a sterile environment, fresh milk is dispensed into multiple centrifuge tubes, sealed, and placed under a preset constant temperature condition for static settlement; the constant temperature is 4 - 25°C.

[0040] The construction of the milk deterioration - fluorescence model to determine the luminescence change color band or quantum yield change linear graph reflecting the degree of milk deterioration includes:

[0041] Performing photophysical property tests and physicochemical property tests on milk during the simulated deterioration period; the photophysical properties include luminescence display, emission spectrum, and / or quantum yield; the physicochemical properties include pH, lactic acid content, free amino acid content, and / or protein aggregation state; in some embodiments, the photophysical properties also include fluorescence lifetime; the tests are continuous tests or intermittent tests. The denser the test points, even continuous tests, the more accurately the obtained photophysical properties and physicochemical properties can reflect the milk deterioration process; in some embodiments, based on the operability of the test, the photophysical property tests and physicochemical property tests are carried out in an intermittent test manner, and the sampling time interval of the intermittent test is 1 - 24 h;

[0042] Analyzing the results of the photophysical property tests and the results of the physicochemical property tests, determining the corresponding relationship between the degree of deterioration and the luminescence display or quantum yield, and establishing a luminescence change color band or quantum yield change linear graph reflecting the degree of milk deterioration.

[0043] As described above, the spoilage of milk is mainly caused by the reproduction and metabolism of microorganisms including Lactobacillus, Pseudomonas, and coliforms. At the same time, the spoilage of milk will cause changes in the luminescence characteristics of milk, such as fluorescence color and quantum yield. The inventors found that by monitoring the biochemical activities of microorganisms by judging pH, lactic acid content, free amino acid content, and protein aggregation state, the spoilage degree of milk can be determined. Milks with different spoilage degrees have different luminescence characteristics. Based on this, the inventors established the corresponding relationship between the luminescence characteristics of milk and the changes in its physical and chemical properties, and obtained a luminescence change color spectrum band or a linear graph of quantum yield change reflecting the spoilage degree of milk.

[0044] Among them, the above-mentioned photophysical properties and physical and chemical properties can be detected and obtained by common detection methods and / or detection equipment in the art. In some embodiments, the luminescence display includes a luminescence state photo and / or a luminescence state chromaticity diagram. The luminescence state photo is obtained by shooting, and the luminescence state chromaticity diagram is determined by the luminescence state chromaticity coordinates; the luminescence state is the luminescence state under natural light or ultraviolet excitation, and the ultraviolet light wavelength is 254 nm, 312 nm, or 365 nm. In some preferred embodiments, the ultraviolet light wavelength for obtaining the luminescence display is 365 nm;

[0045] In some embodiments, the emission spectrum, fluorescence lifetime, and quantum yield are all detected and obtained by a steady-state transient fluorescence spectrometer (FLS1000).

[0046] In some preferred embodiments, the excitation wavelength for obtaining the emission spectrum is 254 - 395 nm, the signal intensity is 500 - 1000 kcps, the scanning step is 1 - 2 nm, and the residence time is 0.1 - 0.2 s. Multiple spoilage color spectrum bands can be obtained within the above wavelength range, which is convenient for selecting a spectrum band with a high contrast of color change; by restricting the above signal intensity, scanning step, and residence time, more accurate test results can be obtained more efficiently. If the residence time is less than 0.1 s, the obtained data fluctuates greatly.

[0047] The excitation wavelength for obtaining the emission spectrum, fluorescence lifetime, and quantum yield is equal to the excitation light wavelength used for obtaining the luminescence photo.

[0048] In some embodiments, the signal intensity for obtaining the quantum yield is 500 - 1000 kcps, the step is 0.1 - 0.5 nm, and the residence time is 0.2 - 0.5 s.

[0049] In some embodiments, the pH is obtained by testing with a pH meter; the lactic acid content is obtained by detection with a 1260 / UV high performance liquid chromatograph; the free amino acid content is obtained by detection with a fully automatic amino acid analyzer (L-8900-1); the protein aggregation state is determined by combining particle size analysis and protein transmission electron microscope pictures, wherein the particle size analysis is based on dynamic light scattering (DLS) testing, and the transmission electron microscope is a biological field emission transmission electron microscope (Talos F200C G2).

[0050] Currently, the reference basis for routinely confirming the degree of milk spoilage is acidity. For example, in the patent application document 201510941205.4, a method for detecting the freshness of milk based on multi-source spectral data fusion discloses that when the acidity value ≥ 25°T, it is determined that the milk sample to be tested is not fresh; when the acidity value < 25°T, it is determined that the milk sample to be tested is fresh. Some other ways of distinguishing freshness believe that fresh milk has a pH of 6.6 - 6.8, milk in the process of spoiling has a pH of 5.5 - 6.0, and milk that is completely spoiled has a pH of 4.5 - 5.5 (less than 5.5). The acidity or pH value is affected by various factors, and using it alone as an evaluation index for milk freshness lacks certain reliability and accuracy. The inventor found that lactic acid produced by lactose fermentation causes protein denaturation, and the metabolism of microorganisms produces various enzymes that promote protein decomposition. By combining the pH, lactic acid content, free amino acid content, and protein aggregation state, the changes in nutrients and microorganisms in milk can be more accurately determined, and the milk freshness can be determined.

[0051] Based on the above analysis, it can be seen that the current classification of milk freshness is usually the dichotomy or trichotomy. This determination standard is usually based on the ease of operation of the test to meet the rapid detection requirements, and is also related to the test accuracy. For example, it is difficult to obtain high-precision test results by the traditional method of determining acidity by titration or pH meter. There are obvious drawbacks in classifying the degree of milk spoilage in this way. Based on the above comprehensive physical indicators and referring to the traditional standard for classifying the degree of milk spoilage, the inventor determines the four-stage degree of milk spoilage and the corresponding physical parameters; among them, the degree of spoilage is successively the fresh stage, the mild acidification stage, the partial spoilage stage, and the complete spoilage stage, where:

[0052] The pH, lactic acid content, free amino acid content, and protein aggregation state in the fresh stage are: the pH is 6.6 - 6.8, the lactic acid content is less than 0.05%, the contents of free proline, phosphoserine, and alanine are less than 3, 15, and 32 nmol / mL respectively, the protein size is relatively uniform, the PDI is less than 0.2, and the average particle diameter of the protein micelles is about 200 nm; the lactic acid content less than 0.05 means that the mass of lactic acid in 100 mL of milk is less than 0.05 g;

[0053] In the mild acidification stage, the pH, lactic acid content, free amino acid content, and protein aggregation state are as follows: the pH is 6.51 - 6.66, the lactic acid content is 0.05 - 0.2%, the contents of free proline, phosphoserine, and alanine are 3 - 30, 15 - 70, and 32 - 40 nmol / mL respectively, the protein size is relatively uniform, the PDI slightly increases but is still less than 0.2, and the average particle size of the protein micelles is about 200 nm;

[0054] In the partial deterioration stage, the pH, lactic acid content, free amino acid content, and protein aggregation state are as follows: the pH is 5.5 - 6.0, the lactic acid content is 0.2 - 0.4%, the contents of free proline, phosphoserine, and alanine are 30 - 60, 70 - 100, and 40 - 55 nmol / mL respectively, the protein shows non - uniformity, with significantly larger and smaller particle size distributions, the PDI is greater than 0.2, and the average micelle particle size is greater than 300 nm;

[0055] In the complete deterioration stage, the pH, lactic acid content, free amino acid content, and protein aggregation state are as follows: the pH is less than 5.5, the lactic acid content is greater than 0.4%, the contents of free proline, phosphoserine, and alanine are greater than 60, 100, and 55 nmol / mL respectively, the protein particle size distribution is more extreme, a large number of protein micelles with particle sizes less than 100 nm appear, and protein aggregates with particle sizes greater than 1000 nm gradually appear, and the PDI continues to increase to approach 1.

[0056] In some embodiments, the luminescence change color band is a band formed by connecting the luminescence colors of milk under ultraviolet excitation during the simulated deterioration period. In some preferred embodiments, the luminescence change color band includes the color of the region formed by connecting the chromaticity coordinates of the luminescence colors of each milk during the simulated deterioration period on the chromaticity diagram. In some specific embodiments, the chromaticity coordinates corresponding to the luminescence color in the fresh stage, the starting luminescence color in the mild acidification stage, the starting luminescence color in the partial deterioration stage, and the starting luminescence color in the complete deterioration stage are connected on the chromaticity diagram, and the color of the formed region is the full - stage color band. According to the fresh stage, mild acidification stage, partial deterioration stage, and complete deterioration stage, the deterioration degree of the full - stage color band is marked to obtain the luminescence change color band reflecting the deterioration degree of milk, and the transition regions between each stage are gradually filled based on the changing trend of the chromaticity coordinates. In some preferred embodiments, the ultraviolet excitation wavelength for obtaining the luminescence change color band is 365 nm.

[0057] In some embodiments, the quantum yield change linear graph is the statistics of the luminescence change quantum yield of milk under ultraviolet excitation during the simulated deterioration period. In some preferred embodiments, the ultraviolet excitation wavelength for obtaining the quantum yield change linear graph is 365 nm.

[0058] Based on the color spectrum of luminescence change, determining the spoilage degree of the milk to be tested specifically includes:

[0059] Obtain the luminescence color of the milk to be tested under ultraviolet light irradiation, compare this luminescence color with the color spectrum of luminescence change. If the luminescence color of the milk to be tested falls within the spectral band segment corresponding to a certain stage in the color spectrum of luminescence change, determine the spoilage degree of the milk to be tested based on the spoilage degree of this stage spectral band segment. In some embodiments, to further improve the accuracy of determination, the method for determining the spoilage degree of the milk to be tested is to mark the chromaticity coordinates of the luminescence photo of the above-mentioned milk to be tested in the color spectrum of luminescence change, and determine the spoilage degree of the milk according to the area it falls into. Chromaticity is a physical quantity that objectively represents the nature of color, which is only related to the dominant wavelength and purity, and has nothing to do with brightness. The purpose of chromaticizing the luminescence color is to remove the influence of different sampling times, shooting environments, and camera parameters on color recording, without changing the color display.

[0060] Among them, obtaining the luminescence color of the milk to be tested under ultraviolet light irradiation specifically includes:

[0061] Use ultraviolet light with the same wavelength as that used to obtain the color spectrum of luminescence change to excite the milk to be tested, and obtain the luminescence color of the milk to be tested.

[0062] Based on the linear graph of quantum yield change, determining the spoilage degree of the milk to be tested specifically includes:

[0063] Obtain the quantum yield of the milk to be tested under ultraviolet light irradiation, compare this quantum yield with the linear graph of quantum yield change. If the quantum yield of the milk to be tested falls within the internal interval of the linear graph of quantum yield change, determine the spoilage degree of the milk to be tested according to the spoilage degree to which this interval belongs.

[0064] Furthermore, as a feasible implementation method, the operations for obtaining the above-mentioned photophysical property test results and physicochemical property test results provided by the present invention are as follows:

[0065] 1. The method for determining the luminescence display by taking a luminescence state photo includes:

[0066] Step 101: Fix the ultraviolet lamp and the cuvette, install the test tube clamp, place two pieces of black cardboard respectively below and behind the test tube clamp to remove background interference. Then adjust the position of the iron stand and the test tube clamp so that the lower test tube clamp is parallel to the tabletop and at the 1 / 3 position of the cuvette, tighten the clamp to fix the position of the cuvette, and adjust the upper test tube clamp so that the ultraviolet lamp hangs 3 - 6 cm above the cuvette and is slightly forward as a whole;

[0067] Step 102: Adjust and fix the camera position and shooting angle to ensure consistent shooting conditions each time, specifically including: adjusting the tripod and camera position to make the lens face and be parallel to the cuvette surface. Then, slowly add 2.5 - 3.5 mL of milk sample into the cuvette using a pipette, and adjust the camera focus to make the sample appear clearest within the lens.

[0068] Step 103: Adjust the shooting parameters and take photos, specifically including: adjusting and fixing the ISO value according to the brightness of the object, setting a delay of 3 seconds to ensure stable shooting, and then taking photos under natural light, 254 nm, 312 nm, and / or 365 nm ultraviolet light. After each group of samples is photographed, loosen the screw on one side of the clamping device to ensure clamping at the same position each time. Remove the cuvette, suck out the internal sample with a dropper, rinse the cuvette 2 - 3 times alternately with water and ethanol, and then dry the cuvette. Repeat the above shooting process and select clear photos as the luminescent photos for determining luminescence display.

[0069] 2. The emission spectrum test method includes:

[0070] Step 201: Wash the quartz cuvette with water and dry it. Use a pipette to add 2.5 - 3.5 mL of milk sample into the cuvette, place it in the sample chamber of a steady-state transient fluorescence spectrometer (FLS1000), set the excitation wavelength to 254 - 395 nm and insert the corresponding filter, adjust the appropriate slit width to make the signal intensity between 500 - 1000 kcps, set the scanning step size to 1 - 2 nm, and the residence time to 0.1 - 0.2 s.

[0071] Step 202: After fixing the above test parameters, conduct the emission spectrum test and normalize some of the results through conventional means in the art.

[0072] 3. The fluorescence lifetime test method includes:

[0073] After determining the excitation and emission wavelengths through the emission spectrum, switch the steady-state transient fluorescence spectrometer to the lifetime mode, replace the light source with a laser emitter, and conduct the lifetime test.

[0074] 4. The quantum yield test method includes:

[0075] Step 301: Adjust the integrating sphere to the liquid measurement mode. Use a pipette to add 2.5 - 3.5 mL of PBS solution with pH = 6.8 into the cuvette and place it in the integrating sphere. Switch the steady-state transient fluorescence spectrometer to the quantum yield test page, adjust the appropriate slit width to make the signal intensity between 500 - 1000 kcps, set the step size to 0.1 - 0.5 nm, and the residence time to 0.2 - 0.5 s, and conduct the spectral test.

[0076] Step 302: After the test, take out the cuvette, clean and dry it, add a milk sample equal in amount to PBS, transfer the liquid according to the method in Step 301 for spectral testing, and obtain the quantum yield of the sample through integral fitting.

[0077] 5. The pH measurement method includes:

[0078] First, calibrate the portable pH meter (YHBJ - 262) with two buffer solutions. Rinse the test electrode 2 - 3 times with clear water and dry it. Use a pipette to take 4 - 8 mL of the sample into a 15 mL centrifuge tube, immerse the electrode probe into the sample, and let it stand for 1 - 3 min. After the data stabilizes, read the pH value. The two buffer solutions are a phosphate buffer solution with a concentration of 0.025 M and a pH of 6.86, and a potassium hydrogen phthalate buffer solution with a concentration of 0.05 M and a pH of 4.00.

[0079] 6. The lactic acid content test method includes:

[0080] Step 401: Dissolve pure lactic acid in deionized water to obtain lactic acid standard samples with different concentration gradients, and use HPLC to test and plot the standard curve of lactic acid concentration.

[0081] Step 402: Extract lactic acid from the milk sample, specifically including: Mix 2 mL of the milk sample with 408 mg of trichloroacetic acid and stir for 2 - 4 min, incubate at 37°C for 1 h, centrifuge for 25 min, take the supernatant, adjust the pH to 7.5 with 50 g / L KOH solution, then dilute it 2.5 times with PBS buffer solution with a pH of 6.8, centrifuge, and take the supernatant.

[0082] Step 403: Use HPLC for determination, compare the determination result with the standard curve of lactic acid concentration, and determine the lactic acid content.

[0083] 7. The free amino acid content test method includes:

[0084] Centrifuge 1 - 3 mL of the milk sample to remove the upper layer of milk fat. Add the pretreatment solution and sonicate for 5 - 10 min, then centrifuge for 20 - 30 min to remove proteins. Take the supernatant and use an automatic amino acid analyzer (L - 8900 - 1) to determine the content of free amino acids. The pretreatment solution is a mixed aqueous solution of hydrochloric acid and trichloroacetic acid, where the HCl concentration is 0.1 mol / L and the mass percentage of trichloroacetic acid is 10%.

[0085] 8. The dynamic light scattering (DLS) test method includes:

[0086] Take 5 - 10 mL of milk sample and centrifuge it to remove the upper cream part. Dilute it 100 times with PBS solution at pH = 6.8. Take 2.5 mL of the diluted system and place it in a clean cuvette, and measure it with a nano particle size and Zeta potential analyzer (ZS90).

[0087] 9. Transmission electron microscopy (TEM) tests include:

[0088] Take 3 - 5 mL of milk sample and centrifuge it to remove the upper cream part. Dilute it to 100 times with PBS solution at pH = 6.8. Drop one drop on a 600 - mesh copper grid, let it stand for 5 - 10 min, then suck off the liquid with filter paper. Then, perform negative staining with 2% phosphotungstic acid solution by mass. After 1 min, suck off the staining solution and observe the sample and record it under a biological field - emission transmission electron microscope (Talos F200C G2) at 25°C.

[0089] A series of experiments were carried out before the application of this invention. Now, some test results are cited to further describe the invention in detail. The following is a detailed description in combination with the embodiments.

[0090] Example 1

[0091] Step 1: Provide an environment simulating the deterioration period and simulate the deterioration of milk, specifically including:

[0092] Under a sterile environment, dispense fresh milk into 7 sterilized 50 - mL centrifuge tubes, with 30 mL of milk in each tube. Set the temperature at 25°C to simulate the deterioration process of milk at room temperature. During the simulated deterioration period, take out one tube of sample at 0, 1, 6, 12, 24, 48, and 72 h respectively, divide it equally into three parts and put them into 15 - mL sterilized centrifuge tubes, and seal them to obtain the samples to be tested; the fresh milk is taken from brand A pure milk.

[0093] Step 2: Construct a milk deterioration - fluorescence model and determine the emission color spectrum band reflecting the degree of milk deterioration, specifically including:

[0094] Perform both photophysical property tests and physicochemical property tests on the milk that has been left standing for the preset time; the photophysical properties include luminescence display and emission spectrum; the physicochemical properties include pH, lactic acid content, free amino acid content, and protein aggregation state.

[0095] The photo shooting is carried out according to the above - mentioned method for taking luminescence photos. Among them, in step 101, adjust the upper test tube clamp to make the ultraviolet lamp hang 5 cm above the cuvette; in step 102, slowly add 2.8 mL of the sample to the cuvette with a pipette; in step 103, rinse the cuvette 3 times alternately with water and ethanol.

[0096] The emission spectrum test is carried out according to the above-mentioned emission spectrum test method. Among them, in step 201, 3 mL of the sample is added to the cuvette with a pipette. The excitation wavelength is set to 312 or 365 nm and the corresponding filter with 330 nm or 395 nm is placed. The slit width is adjusted to ExBW = 5 nm and EmBW = 4 nm. The scanning step is 1 nm and the residence time is 0.1 s.

[0097] The pH measurement is carried out according to the above-mentioned pH measurement method. The test electrode is rinsed with clean water 3 times. 6 mL of the sample is taken with a pipette into a 15 mL centrifuge tube and left standing for 2 min.

[0098] The lactic acid content is determined according to the above-mentioned lactic acid content test method. Among them, in step 402, the time for adding trichloroacetic acid and mixing is 3 min.

[0099] The determination of the free amino acid content is carried out according to the above-mentioned free amino acid content test method. Among them, the volume of the centrifuged milk sample is 2 mL and the volume of the pretreatment solution is 2 mL; the ultrasonic time is 8 min and the time for centrifuging to remove proteins is 25 min.

[0100] The dynamic light scattering (DLS) test is carried out according to the above-mentioned dynamic light scattering (DLS) test method. Among them, the volume of the milk sample is 6 mL.

[0101] The transmission electron microscopy (TEM) test is carried out according to the above-mentioned transmission electron microscopy (TEM) test method. The standing time is 9 min.

[0102] Analyze the test results of the photophysical properties and the physicochemical properties corresponding to the preset time, determine the corresponding relationship between the degree of deterioration and the fluorescence display, and establish a luminescence change color band reflecting the degree of milk deterioration;

[0103] Figure 6 a is the graph of the lactic acid content and pH changes at different stages. Figure 6 b is the graph of the free amino acid concentration changes at different stages. Figure 6 c is the graph of the particle size changes at different stages. At 72 h, the milk has stratified and large protein aggregates have appeared, and there is no need for DLS to show the dispersion uniformity. Figure 7 is the particle size distribution of protein aggregates in milk after being placed for different times and its TEM image. According to Figure 6 and Figure 7 , it is determined that the physicochemical properties of the milk change as follows:

[0104] From 0 to 6 h, the pH decreased from 6.69 to 6.68, the lactic acid content was < 0.015% (0.015 g / 100 mL milk), the contents of free proline, phosphoserine, and alanine were less than 3, 15, and 32 nmol / mL respectively, and the aggregation degree of free amino acids and proteins remained almost unchanged during the whole period. The protein size was relatively uniform, the PDI was all less than 0.2, and the DLS test results showed that the average particle size of protein micelles was about 200 nm, indicating that the milk was very fresh. From 6 to 12 h, the pH decreased from 6.68 to 6.66, the lactic acid increased slightly, and the content was between 0.015% and 0.05%. The aggregation degree of free amino acids and proteins did not change. It was determined that from 0 to 12 h, the milk was in the fresh stage;

[0105] From 12 to 24 h, the pH continued to decrease, ranging from 6.51 to 6.66. The lactic acid content increased significantly but did not exceed 0.2%. The free amino acids increased. Among them, the contents of free proline, phosphoserine, and alanine were all < 30, 70, and 40 nmol / mL respectively. The protein size was relatively uniform, the PDI increased slightly but was still less than 0.2 overall, and the average particle size of protein micelles was still about 200 nm. The physicochemical properties of the milk changed slightly from 12 to 24 h. It was determined that 12 h was the starting point of the mild acidification stage of the milk;

[0106] From 24 to 48 h, the pH continued to decrease, ranging from 6.51 to 5.96. The lactic acid content continued to increase and was between 0.1% and 0.25%. The contents of free proline, phosphoserine, and alanine were between 16 - 86, 39 - 128, and 40 - 63 nmol / mL respectively. The protein homogeneity deteriorated, and larger and smaller particle size distributions began to appear. The PDI increased significantly and was greater than 0.2, and the average particle size of protein micelles gradually increased to more than 300 nm. It was determined that 48 h was the starting point of the partial deterioration stage of the milk;

[0107] From 48 to 72 h, the pH decreased significantly and finally was less than 5.5. The lactic acid content continued to increase, ranging from 0.25% to 0.5%. The contents of free proline, phosphoserine, and alanine increased rapidly and finally were greater than 60, 100, and 55 nmol / mL respectively. The protein particle size distribution became more extreme, and a large number of protein micelles with particle sizes less than 100 nm and greater than 1000 nm gradually appeared. The PDI continued to increase and even reached 1. Analysis showed that at 72 h, it was in the early stage of the complete deterioration stage. Referring to the time point with a pH of 5.5, the starting point of the complete deterioration stage was determined to be 56 h;

[0108] Based on the above data, the corresponding physicochemical parameters for the fresh stage, mild acidification stage, partial deterioration stage, and complete deterioration stage were determined as follows:

[0109] The pH, lactic acid content, free amino acid content, and protein aggregation state in the fresh stage are as follows: pH is 6.6 - 6.8, the lactic acid content is less than 0.05% (0.05 g / 100 mL milk), the contents of free proline, phosphoserine, and alanine are less than 3, 15, and 32 nmol / mL respectively, the protein size is relatively uniform, the PDI is less than 0.2, and the average particle size of protein micelles is about 200 nm;

[0110] The pH, lactic acid content, free amino acid content, and protein aggregation state in the slightly acidified stage are as follows: pH is 6.51 - 6.66, the lactic acid content is 0.05 - 0.2% (0.05 - 0.2 g / 100 mL milk), the contents of free proline, phosphoserine, and alanine are 3 - 30, 15 - 70, and 32 - 40 nmol / mL respectively, the protein size is relatively uniform, the PDI increases slightly but is still less than 0.2, and the average particle size of protein micelles is about 200 nm;

[0111] The pH, lactic acid content, free amino acid content, and protein aggregation state in the partially deteriorated stage are as follows: pH is 5.5 - 6.0, the lactic acid content is 0.2 - 0.4%, the contents of free proline, phosphoserine, and alanine are 30 - 60, 70 - 100, and 40 - 55 nmol / mL respectively, the protein shows non - uniformity, with significantly larger and smaller particle size distributions, the PDI is greater than 0.2, and the average micelle particle size is greater than 300 nm;

[0112] The pH, lactic acid content, free amino acid content, and protein aggregation state in the completely deteriorated stage are as follows: pH is less than 5.5, the lactic acid content is greater than 0.4%, the contents of free proline, phosphoserine, and alanine are greater than 60, 100, and 55 nmol / mL respectively, the protein particle size distribution is more extreme, with a large number of protein micelles with particle sizes less than 100 nm appearing, and protein aggregates with particle sizes greater than 1000 nm gradually emerging, and the PDI continues to increase to approach 1.

[0113] Figure 4 Figures are the luminescence photos of milk at different stages under 365 nm and 312 nm ultraviolet excitation, Figure 8 a in is the test result of the corresponding emission spectrum under 365 nm ultraviolet excitation, Figure 8 b in is Figure 8 the normalized intensity of a in, Figure 8 c in is the CIE chromaticity diagram under 365 nm ultraviolet excitation. According to Figure 4 and Figure 8It can be seen that fresh milk shows bright yellow-green light under 365 nm ultraviolet excitation, and bright blue-white light under 312 nm ultraviolet excitation. The luminescence of milk at different stages shows obvious changes under the same wavelength ultraviolet excitation. Under 312 nm ultraviolet excitation, the color of milk during the deterioration simulation period changes from bright blue to blue-purple. Under 365 nm ultraviolet excitation, the color of milk during the deterioration simulation period changes from bright yellow-green to blue. Specifically, from 0 to 6 h, the luminescence shows bright yellow-green under the excitation of 365 nm ultraviolet light. From 6 to 12 h, the emission blue-shifts to green light. From 12 to 24 h, the emission continues to blue-shift and gradually dims. From 24 to 48 h, the emission is blue-green light, and from 48 to 72 h, it becomes a weak blue light. The overall change is from yellow-green light to blue light.

[0114] The chromaticity coordinates of the luminescent colors under 365 nm ultraviolet excitation are connected in sequence on the chromaticity diagram, with the 12 h chromaticity coordinates as the starting point of the mild acidification stage, the 48 h chromaticity coordinates as the starting point of the partial deterioration stage, and the 56 h chromaticity coordinates as the starting point of the complete deterioration stage. The transition area is gradually filled based on the CIE coordinate change trend to obtain a full-stage color band including the fresh stage, mild acidification stage, partial deterioration stage, and complete deterioration stage. The degree of deterioration corresponding to each starting point is marked on the full-stage color band to obtain a luminescent change color band reflecting the degree of milk deterioration, as shown in Figure 2. Figure 9 shown.

[0115] Step 3: Determine the degree of deterioration of the milk to be tested based on the color spectrum of the luminescent change;

[0116] According to the above-mentioned photo shooting method, the milk sample to be tested is irradiated with 365 nm ultraviolet light and a luminescent photo is obtained. The obtained luminescent photo is matched with the above-mentioned luminescent change color spectrum band. The result is as follows Figure 10 As shown, the milk A to be tested is fresh milk that has been placed at room temperature for 3 hours, and the milk B to be tested is fresh milk that has been placed at room temperature for 60 hours. The results show that the milk A to be tested is yellow-green, and the milk B to be tested is a weaker blue-green color, indicating that the milk A to be tested is relatively fresh and still edible, while the milk B to be tested has partially deteriorated.

[0117] Example 2

[0118] This embodiment is the same as embodiment 1, except that step 2 is to construct a linear model of milk deterioration-quantum yield, wherein:

[0119] The quantum yield test was performed according to the above-mentioned quantum yield test method. In step 301, 3 mL of PBS solution was added to the cuvette using a pipette and placed in an integrating sphere, with a signal intensity of 600 kcps, a step size of 0.4 nm, and a dwell time of 0.2 s;

[0120] The remaining tests were the same as those in Example 1;

[0121] Analyze the test results of photophysical properties and physicochemical properties corresponding to the preset time, determine the corresponding relationship between the degree of deterioration and the quantum yield, and establish a linear model of milk deterioration - quantum yield reflecting the degree of milk deterioration;

[0122] The degree of milk deterioration over time at room temperature was the same as that in Example 1;

[0123] Figure 11 It is a linear graph of the quantum yield and the change in quantum yield of milk at different stages under ultraviolet excitation, where Figure 11 a is the quantum yield graph under ultraviolet excitation at 365 nm and 312 nm, Figure 11 b is the linear graph of the change in quantum yield under ultraviolet excitation at 365 nm. According to Figure 11 It can be seen that the quantum yield decreases as the degree of deterioration deepens.

[0124] Step 3: Based on the linear graph of the change in quantum yield, determine the degree of deterioration of the milk to be tested;

[0125] According to the above method for testing the quantum yield, the emission spectrum and quantum yield of the milk to be tested were obtained by a steady-state transient fluorescence spectrometer. Among them, the milk to be tested C was fresh milk placed at room temperature for 5 days. The obtained results were compared with the linear graph of the change in quantum yield. The results were as Figure 12 shown. The results showed that under ultraviolet excitation at 365 nm, the quantum yield ln Ф was 1.99. Compared with Figure 11 b, it was in the stage of complete deterioration, indicating that the milk to be tested C had completely deteriorated.

[0126] Example 3

[0127] This example was the same as Example 1, except that the milk to be tested D was fresh milk taken out after being placed in the refrigerator's chilled area for 7 days. The milk to be tested was irradiated with ultraviolet light at 365 nm and a luminescence photo was obtained according to the above photo-taking method. The obtained luminescence photo was corresponded to the luminescence change color band in Example 1. The results were as Figure 13 shown. The results showed that the milk to be tested D presented bright yellow-green light as a whole and was in the fresh stage.

Claims

1. A method for visually monitoring milk spoilage, characterized in that: include: Provide a simulated spoilage environment to simulate the spoilage of milk; Construct a milk deterioration-fluorescence model to determine the color spectrum of luminescence changes or the linear graph of quantum yield changes that reflect the degree of milk deterioration; Determining the degree of deterioration of the milk to be tested, wherein, based on the luminescence change color spectrum band, determining the degree of deterioration of the milk to be tested specifically comprises: obtaining the luminescence color of the milk to be tested under ultraviolet light, comparing the luminescence color with the luminescence change color spectrum band, the luminescence color of the milk to be tested falls into the stage spectrum band segment corresponding to the luminescence change color spectrum band, and determining the degree of deterioration of the milk to be tested based on the deterioration degree of the stage spectrum band segment; Based on the linear graph of quantum yield change, determining the degree of deterioration of the milk to be tested specifically includes: obtaining the quantum yield of the milk to be tested under ultraviolet light, comparing the quantum yield with the linear graph of quantum yield change, and determining the degree of deterioration of the milk to be tested according to the degree of deterioration to which the interval belongs if the quantum yield of the milk to be tested falls into the linear graph of quantum yield change.

2. The method for visually monitoring milk deterioration according to claim 1, characterized in that: The method of constructing a milk deterioration-fluorescence model and determining a luminescence change color spectrum band or a quantum yield change linear graph reflecting the degree of milk deterioration comprises: Conduct photophysical and physicochemical property tests on milk during the simulated spoilage period; Analyze the test results of photophysical properties and physicochemical properties, determine the corresponding relationship between the degree of deterioration and the luminescence display or quantum yield, and establish a linear graph of luminescence change color spectrum band or quantum yield change reflecting the degree of milk deterioration; The photophysical properties include luminescence display, emission spectrum and / or quantum yield; the physicochemical properties include pH, lactic acid content, free amino acid content and / or protein aggregation state.

3. The method for visually monitoring milk deterioration according to claim 2, characterized in that: The luminous display includes a luminous state photograph and / or a luminous state chromaticity diagram, and the luminous state is a luminous state under natural light or ultraviolet excitation.

4. The method for visually monitoring milk deterioration according to claim 3, characterized in that: The wavelength of ultraviolet light is 365nm.

5. The method for visually monitoring milk deterioration according to claim 2, characterized in that: The emission spectrum was obtained at an excitation wavelength of 254-395 nm, a signal intensity of 500-1000 kcps, a scanning step of 1-2 nm, and a dwell time of 0.1-0.2 s.

6. The method for visually monitoring milk deterioration according to claim 2, characterized in that: The signal intensity for obtaining the quantum yield is 500-1000 kcps, the step size is 0.1-0.5 nm, and the dwell time is 0.2-0.5 s.

7. The method for visually monitoring milk deterioration according to claim 2, characterized in that: The luminescence change color spectrum band or quantum yield change linear graph were obtained under ultraviolet excitation light with a wavelength of 365 nm.

8. The method for visually monitoring milk deterioration according to claim 2, characterized in that: Establishing a luminescence change color spectrum band reflecting the degree of milk spoilage includes: connecting the chromaticity coordinates corresponding to the luminescence color of the fresh stage, the luminescence color at the starting point of the slightly acidified stage, the luminescence color at the starting point of the partially deteriorated stage and the luminescence color at the starting point of the completely deteriorated stage on a chromaticity diagram.

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