Method for visually monitoring milk deterioration

Abstract

The invention discloses a method for visually monitoring milk deterioration, which comprises the following steps: providing a deterioration period simulation environment, and carrying out deterioration simulation on milk; constructing a milk deterioration-fluorescence model, and determining a luminescence change color band or a quantum yield change linear diagram reflecting the milk deterioration degree; and based on the milk deterioration-fluorescence model, determining the deterioration degree of the to-be-detected milk. The milk quality is evaluated by monitoring the change of the photoluminescence characteristics of the milk, and the method has the characteristics of capability of immediately and quickly judging the freshness of the milk, visual result, high accuracy, simplicity and convenience in operation and short operation time.

Description

Technical Field

[0001] This invention belongs to the field of milk detection technology, specifically relating to a method for visually monitoring milk spoilage. Background Technology

[0002] Dairy products, especially milk, are an important natural food, rich in essential nutrients such as protein, fat, carbohydrates, and vitamins. However, this abundance of nutrients makes milk highly susceptible to spoilage, severely impacting its taste and nutritional value. Although milk undergoes rigorous sterilization during production and processing, secondary contamination risks exist during bottling, storage, and transportation. Pasteurized milk, in particular, is typically sterilized at temperatures between 60-82℃, much lower than ultra-high temperature (UHT) sterilization. Its bacterial content is significantly higher than that of UHT milk, making it more vulnerable to environmental influences and spoilage. Therefore, judging milk spoilage solely by its expiration date may be inaccurate. Furthermore, the frequent food safety issues in recent years have heightened consumer awareness of milk freshness and quality.

[0003] Currently, there are many mature methods for testing milk freshness and quality. Their basic principle lies in testing or analyzing materials by measuring their chemical or physical properties. These methods mainly include chemical detection methods, ATP biofluorescence technology, electronic nose technology, infrared spectroscopy, and ultrasonic detection. However, many of these methods suffer from drawbacks such as sample destruction, long testing cycles, complex operation, and high costs. For example, chemical detection methods require testing milk pH and nutrients, which is not only cumbersome but also carries the risk of secondary contamination. Infrared spectroscopy, limited by instrument availability and easily interfered with by environmental factors in the near-infrared band, makes it difficult to accurately monitor changes in milk quality in real time. Due to these problems, there is an urgent need to develop a real-time, non-destructive, efficient, and convenient technology for testing milk freshness and quality. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for visually monitoring milk spoilage, addressing the shortcomings of the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: to provide a simulated spoilage environment to simulate the spoilage of milk;

[0006] Construct a milk spoilage-fluorescence model to determine the color bands of luminescence changes or the linear graph of quantum yield changes that reflect the degree of milk spoilage;

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

[0008] Compared with the prior art, the present invention has the following advantages:

[0009] 1. Based on the correlation between the photoluminescence properties of milk and the degree of spoilage, this invention provides a method for evaluating milk quality by monitoring changes in the photoluminescence properties of milk. This method simulates milk spoilage, constructs a milk spoilage-fluorescence model, determines the color spectrum of luminescence changes or the linear graph of quantum yield changes that reflect the degree of milk spoilage, and determines the degree of milk spoilage based on the above model. It has the characteristics of being able to quickly and instantly assess the freshness of milk, providing visible results, high accuracy, simple operation, and short operation time.

[0010] 2. Preferably, the present invention constructs a milk spoilage-fluorescence model by performing photophysical and physicochemical property tests on milk during the spoilage simulation period. The physicochemical properties include pH, lactic acid content, free amino acid content and / or protein aggregation state. The freshness and quality of milk are determined by using the above-mentioned multi-dimensional integrated physicochemical properties, which is more comprehensive and accurate than the determination by a single acidity value.

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

[0012] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 Photographs of the luminescence of different brands of milk under ultraviolet excitation at 254, 312 and 365 nm and spectra at different excitation wavelengths;

[0014] Figure 2 Emission spectra, CIE chromaticity diagrams, and lifetime diagrams of fresh milk under different excitation wavelengths;

[0015] Figure 3 Emission spectra of casein, whey protein, and riboflavin at different excitation wavelengths and luminescence images under 365 nm UV excitation;

[0016] Figure 4 Photographs of the luminescence of milk stored for different times under UV excitation at 312 and 365 nm;

[0017] Figure 5 Luminescence images of casein and riboflavin under UV excitation at 254, 312, and 365 nm before and after three days of storage;

[0018] Figure 6 The graph shows 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 after milk has been stored for different times;

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

[0021] Figure 9 The color bands representing changes in light emission reflect the degree of milk spoilage;

[0022] Figure 10 This is a schematic diagram showing the results of determining the degree of spoilage of milk A and milk B to be tested;

[0023] Figure 11 A linear graph of quantum yield variation reflecting the degree of milk spoilage;

[0024] Figure 12 The quantum yield diagram of milk C to be tested;

[0025] Figure 13 This is a schematic diagram showing the results of determining the degree of spoilage of the milk D to be tested. Detailed Implementation

[0026] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0027] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0028] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0029] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. 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 on the implementation process of the embodiments of this application.

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

[0031] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] The technical principles employed in this invention are as follows:

[0033] Milk spoilage is mainly caused by microbial reproduction and metabolism, such as the growth or reproduction of microorganisms like Lactobacillus, Pseudomonas, and Escherichia coli. During the metabolism of organic compounds, these microorganisms cause milk to exhibit sour flavors, off-flavors, and flocculent coagulation. These biochemical processes lead to changes in the physicochemical properties of milk, such as protein denaturation, lactic acid formation, and riboflavin oxidation. Milk possesses photoluminescence properties under specific wavelengths of ultraviolet light excitation. This property originates from the photoluminescence properties of the main nutrients in milk, including proteins and riboflavin (vitamin B2). In other words, the photoluminescence phenomenon of milk is the result of the luminescent effect produced by its main nutrients under the illumination of an excitation light source. Spoilage causes changes in the structure, morphology, and aggregation state of the main nutrients in milk, resulting in significant changes in the photoluminescence properties. Based on this, the present invention provides a method for assessing milk quality by monitoring changes in the photoluminescence properties of milk, including:

[0034] It provides a simulated spoilage environment to simulate the spoilage of milk;

[0035] Construct a milk spoilage-fluorescence model to determine the color bands of luminescence changes or the linear graph of quantum yield changes that reflect the degree of milk spoilage;

[0036] The degree of spoilage of the milk under test is determined based on the milk spoilage-fluorescence model.

[0037] In this invention, the brands of milk used to construct the milk spoilage-fluorescence model and the milk to be tested are not limited. Different brands have little impact on the milk's luminescence and are consistent in composition. For example, they are all commercially available pasteurized fresh milk or UHT pure milk with raw milk as the sole ingredient. To further improve the accuracy of the determination, the milk to be tested and the milk used to construct the milk spoilage-fluorescence model are limited to the same brand and batch.

[0038] Commercially available pure milk or fresh milk exhibits a universal intrinsic luminescence phenomenon. This invention, through measuring the luminescence of different brands of milk under ultraviolet excitation at 254 nm, 312 nm, and 365 nm, reveals that... Figure 1 As shown in Figure a, milk produces weak blue-violet light under 254 nm ultraviolet light excitation, and bright blue-white and yellow-green light under 312 nm and 365 nm ultraviolet light excitation, respectively. The instantaneous emission spectra of different brands of milk ( Figure 1 The results are very similar to those in b), indicating that milk exhibits a universal intrinsic luminescence phenomenon. Milk's main components are water and nutrients, including protein, fat, lactose, and vitamins. This invention measures the emission spectra of milk and its main nutrients, and the test results are as follows: Figure 2 and Figure 3 As shown, where Figure 2 The emission spectra, CIE chromaticity diagrams, and lifetime diagrams of fresh milk at different excitation wavelengths are shown below. Figure 2 'a' represents the emission spectrum. Figure 2 b is the CIE chromaticity diagram. Figure 2 c represents the lifetime diagram. Figure 3 The images show the emission spectra of casein, whey protein, and riboflavin at different excitation wavelengths, as well as their luminescence under 365 nm UV excitation. Figure 3 The a-value corresponds to casein. Figure 3 The 'b' corresponds to whey protein. Figure 3 The 'c' corresponds to riboflavin, and the luminescence image is the small image in the corresponding picture. Combined with... Figures 1-3 The emission spectral peaks of milk in the 300-500 nm wavelength range are mainly based on casein and whey protein; the emission peak at 340 nm mainly originates from tryptophan residues in proteins; the emission peak at 390-480 nm is based on aggregates of strongly emitting proteins and their derivatives; and the emission peak around 530 nm originates from riboflavin. It is evident that proteins and riboflavin (vitamin B2) make significant contributions to the photoluminescence of milk. This invention, by testing the photoluminescence performance parameters of milk and its main components, determines that the luminescence effect of milk mainly depends on its proteins and riboflavin, which is consistent with existing research conclusions. Spoilage leads to changes in the physicochemical properties and photoluminescence characteristics of milk, and these changes are highly correlated with changes in proteins and riboflavin. This invention, by measuring the ultraviolet emission of milk and its main nutrients during the spoilage period, shows the results as follows: Figure 4and Figure 5 As shown, where Figure 4 Ultraviolet emission photographs of milk at different storage times. Figure 5 Casein and riboflavin were observed under UV excitation at 254, 312, and 365 nm after being stored for three days, showing their binding properties. Figure 4 and Figure 5 It is known that spoilage will cause changes in the physical properties and luminescent characteristics of milk, casein, and riboflavin. The luminescence display of spoiled milk shows a consistent trend with that of spoiled casein and riboflavin.

[0039] The simulated spoilage environment is used to provide a location that closely resembles the general storage scenario for milk, in order to obtain milk quality change 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, simulating milk spoilage specifically involves, under aseptic conditions, dispensing fresh milk into multiple centrifuge tubes, sealing them, and placing them under a preset constant-temperature condition; the constant temperature is 4–25°C.

[0040] The construction of the milk spoilage-fluorescence model, and the determination of the luminescence color bands or quantum yield change linear graphs reflecting the degree of milk spoilage, include:

[0041] Photophysical and physicochemical properties of milk during a simulated spoilage period are tested. 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 can be continuous or intermittent; the denser the test points, or even continuous the testing, the more accurately the obtained photophysical and physicochemical properties reflect the milk spoilage process. In some embodiments, based on the operability of the tests, photophysical and physicochemical property tests are performed intermittently, with a sampling time interval of 1–24 hours.

[0042] Analyze the results of photophysical and physicochemical property tests to determine the correlation between the degree of spoilage and luminescence display or quantum yield, and establish a linear graph of luminescence change color bands or quantum yield change that reflects the degree of milk spoilage.

[0043] As mentioned above, milk spoilage is mainly caused by the reproduction and metabolism of microorganisms, including lactic acid bacteria, pseudomonas, and coliform bacteria. Simultaneously, milk spoilage leads to changes in its luminescent properties, such as fluorescence color and quantum yield. The inventors discovered that by monitoring pH, lactic acid content, free amino acid content, and protein aggregation state to determine the biochemical activity of microorganisms, the degree of milk spoilage can be identified. Milk with different degrees of spoilage exhibits different luminescent properties. Based on this, the inventors established a correlation between the luminescent properties of milk and changes in its physicochemical properties, obtaining luminescent color bands or linear graphs of quantum yield changes reflecting the degree of milk spoilage.

[0044] The aforementioned photophysical and physicochemical properties can be obtained by detection using commonly used detection methods and / or detection equipment in the field. In some embodiments, the luminescence display includes a luminescence state photograph and / or a luminescence state chromaticity diagram. The luminescence state photograph is obtained by taking a picture, 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, emission spectra, fluorescence lifetimes, and quantum yields are all obtained by detection using 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 scan step size is 1–2 nm, and the dwell time is 0.1–0.2 s. Multiple modified color bands can be obtained within this wavelength range, facilitating the selection of bands with high contrast in color change. Limiting the signal intensity, scan step size, and dwell time allows for more efficient and accurate test results; however, dwell times below 0.1 s result in large data fluctuations.

[0047] The excitation wavelength used to obtain the emission spectrum, fluorescence lifetime, and quantum yield is equal to the excitation wavelength used to obtain the luminescent photograph.

[0048] In some embodiments, the signal strength for obtaining 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.

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

[0050] Currently, acidity is the conventional reference for confirming the degree of milk spoilage. For example, patent application 201510941205.4, "A Method for Detecting Milk Freshness Based on Multi-Source Spectral Data Fusion," discloses that when the acidity value is ≥25°T, the milk sample is considered not fresh; when the acidity value is <25°T, the milk sample is considered fresh. Other methods of distinguishing freshness consider a pH of 6.6-6.8 as fresh milk, a pH of 5.5-6.0 as milk that is spoiling, and a pH of 4.5-5.5 (less than 5.5) as completely spoiled milk. However, acidity or pH value is affected by various factors, and using it alone as an evaluation indicator of milk freshness lacks reliability and accuracy. The inventors discovered that lactic acid produced by lactose fermentation leads to protein denaturation, and microbial metabolism produces various enzymes that promote protein decomposition. By combining 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, thus determining milk freshness.

[0051] Based on the above analysis, it is known that current classifications of milk freshness typically use a binary or triadic method. This standard is usually based on ease of testing to meet rapid testing needs, but it is also related to testing accuracy. For example, traditional methods of determining acidity using titration or pH meters are difficult to obtain highly accurate test results, making this method of classifying milk spoilage a significant drawback. Based on the aforementioned comprehensive physical indicators and referring to traditional standards for classifying milk spoilage, the inventors have determined four stages of milk spoilage and their corresponding physical parameters; wherein the spoilage stages are, in order, the freshness stage, the slightly acidified stage, the partially spoiled stage, and the completely spoiled stage, wherein:

[0052] The fresh stage of milk exhibits the following characteristics: pH 6.6–6.8, lactic acid content less than 0.05%, free proline, phosphoserine, and alanine contents less than 3, 15, and 32 nmol / mL, respectively; relatively uniform protein size, PDI less than 0.2, and average protein micelle diameter around 200 nm. The lactic acid content of less than 0.05% refers to a lactic acid mass of less than 0.05 g per 100 mL of milk.

[0053] During the mild acidification stage, the pH, lactic acid content, free amino acid content, and protein aggregation state were as follows: pH 6.51–6.66, lactic acid content 0.05–0.2%, free proline, phosphoserine, and alanine contents 3–30, 15–70, and 32–40 nmol / mL, respectively. The protein size was relatively uniform, the PDI increased slightly but remained less than 0.2, and the average particle size of the protein micelles was around 200 nm.

[0054] The pH, lactic acid content, free amino acid content, and protein aggregation state during the partial deterioration stage were as follows: pH 5.5-6.0, lactic acid content 0.2-0.4%, free proline, phosphoserine, and alanine contents 30-60, 70-100, and 40-55 nmol / mL, respectively. The protein exhibited heterogeneity, with significant distributions of larger and smaller particle sizes, a PDI greater than 0.2, and an average micelle size greater than 300 nm.

[0055] During the complete deterioration stage, the pH, lactic acid content, free amino acid content, and protein aggregation state were as follows: pH less than 5.5, lactic acid content greater than 0.4%, free proline, phosphoserine, and alanine content greater than 60, 100, and 55 nmol / mL, respectively. The protein particle size distribution became more extreme, with a large number of protein micelles with a particle size less than 100 nm appearing, and protein aggregates with a particle size greater than 1000 nm gradually appearing. The PDI continued to increase to close to 1.

[0056] In some embodiments, the luminescence color change bands are bands formed by connecting the luminescence colors of milk under ultraviolet excitation during the simulated spoilage period. In some preferred embodiments, the luminescence color change bands include the colors of the regions formed by connecting the chromaticity coordinates of each milk luminescence color during the simulated spoilage period on a chromaticity diagram. In some specific embodiments, the chromaticity coordinates corresponding to the luminescence colors of the fresh stage, the starting luminescence color of the mild acidification stage, the starting luminescence color of the partial spoilage stage, and the starting luminescence color of the complete spoilage stage are connected on a chromaticity diagram, and the resulting region color is the full-stage color band. The full-stage color bands are then labeled with the degree of spoilage according to the fresh stage, mild acidification stage, partial spoilage stage, and complete spoilage stage to obtain luminescence color change bands reflecting the degree of milk spoilage. The transition regions between each stage are gradually filled based on the trend of chromaticity coordinate changes. In some preferred embodiments, the ultraviolet excitation wavelength for obtaining the luminescence color change bands is 365 nm.

[0057] In some embodiments, the quantum yield change linear graph is a statistical representation of the quantum yield change in 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] Determining the degree of spoilage of milk based on luminescence color band changes specifically includes:

[0059] The emission color of the milk under ultraviolet light is obtained, and this emission color is compared with the emission color change spectrum. If the emission color of the milk falls into the corresponding segment of the emission color change spectrum, the degree of spoilage of the milk is determined based on the degree of deterioration in that segment. In some embodiments, to further improve the accuracy of the determination, the method for determining the degree of spoilage of the milk is to plot the chromaticity coordinates of the emission image of the milk on the emission color change spectrum, and determine the degree of spoilage based on the region in which the coordinates fall. Chromaticity is a physical quantity that objectively represents the properties of color. It is only related to the dominant wavelength and purity, and is independent of brightness. The purpose of colorizing the emission color is to remove the influence of different sampling times, shooting environment, and camera parameters on color recording, without changing the color display.

[0060] Specifically, obtaining the luminescence color of the milk under ultraviolet light irradiation includes:

[0061] The milk to be tested is excited with ultraviolet light of the same wavelength as that used to obtain the color band of the emission change, and the emission color of the milk to be tested is obtained.

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

[0063] The quantum yield of the milk to be tested under ultraviolet light irradiation is obtained. The quantum yield is compared with the quantum yield change line graph. If the quantum yield of the milk to be tested falls within the interval of the quantum yield change line graph, the degree of spoilage of the milk to be tested is determined according to the degree of spoilage to which the interval belongs.

[0064] Furthermore, as a feasible implementation method, the present invention provides the following operation for obtaining the above-mentioned photophysical property test results and physicochemical property test results:

[0065] 1. Methods for determining luminous display by taking photographs of the luminous state include:

[0066] Step 101: Fix the UV lamp and cuvette, install the test tube clamp, place two pieces of black cardstock below and behind the test tube clamp respectively 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 table surface and at 1 / 3 of the position of the cuvette, tighten the clamp to fix the position of the cuvette, and adjust the upper test tube clamp so that the UV lamp is suspended 3-6 cm above the cuvette and slightly forward.

[0067] Step 102: Adjust and fix the camera position and shooting angle to ensure that the conditions are consistent for each shooting. Specifically, this includes: adjusting the tripod and camera position so that the lens is facing and parallel to the cuvette surface, then slowly adding 2.5~3.5mL of milk sample to the cuvette with a pipette, and adjusting the camera focus to make the sample appear as clear as possible in the lens.

[0068] Step 103: Adjust the shooting parameters and take photos. Specifically, this includes: adjusting and fixing the ISO value according to the actual brightness of the object, setting a 3-second delay 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 that it is clamped in the same position each time. Remove the cuvette, use a dropper to remove the sample inside, rinse the cuvette with water and ethanol alternately 2-3 times, and then blow dry the cuvette. Repeat the above shooting process and select a clear photo as the photo for determining the luminescence display.

[0069] 2. Emission spectroscopy testing methods include:

[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 and place it in the sample chamber of the steady-state transient fluorescence spectrometer (FLS1000). Set the excitation wavelength to 254~395 nm and place the corresponding filter. Adjust the appropriate slit width to make the signal intensity 500~1000 kcps. Set the scanning step size to 1~2 nm and the dwell time to 0.1~0.2 s.

[0071] Step 202: After fixing the above test parameters, perform emission spectrum testing, and normalize some of the results according to conventional methods in this field.

[0072] 3. Fluorescence lifetime testing methods include:

[0073] After determining the excitation and emission wavelengths using emission spectroscopy, the steady-state transient fluorescence spectrometer was switched to lifetime mode, and the light source was replaced with a laser emitter for lifetime testing.

[0074] 4. Quantum yield testing methods include:

[0075] Step 301: Adjust the integrating sphere to liquid measurement mode. Use a pipette to add 2.5~3.5 mL of pH=6.8 PBS solution to a cuvette and place it inside 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 500~1000 kcps, set the step size to 0.1~0.5 nm, and the dwell time to 0.2~0.5 s, and perform spectral testing.

[0076] Step 302: After the test is completed, remove the cuvette, clean and dry it, add an equal amount of milk sample to PBS, pipette the sample according to the method in step 301, and perform spectral testing. Obtain the quantum yield of the sample by integral fitting.

[0077] 5. pH measurement methods include:

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

[0079] 6. Methods for testing lactic acid content include:

[0080] Step 401: Dissolve pure lactic acid in deionized water to obtain lactic acid standards 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 milk sample, specifically including: mixing 2 mL of milk sample with 408 mg of trichloroacetic acid and stirring for 2-4 min, incubating at 37℃ for 1 h, centrifuging for 25 min, taking the supernatant and adjusting the pH to 7.5 with 50 g / L KOH solution, then diluting 2.5 times with PBS buffer solution at pH=6.8, centrifuging, and taking the supernatant;

[0082] Step 403: Use HPLC to determine the lactic acid content by comparing the results with the standard curve of lactic acid concentration.

[0083] 7. Methods for testing free amino acid content include:

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

[0085] 8. Dynamic light scattering (DLS) testing methods include:

[0086] Centrifuge 5-10 mL of milk sample to remove the upper layer of milk fat. Dilute 100 times with PBS solution at pH 6.8. Take 2.5 mL of the diluted system and place it in a clean cuvette for determination in a nanoparticle size and zeta potential analyzer (ZS90).

[0087] 9. Transmission electron microscopy (TEM) testing includes:

[0088] Centrifuge 3-5 mL of milk sample to remove the upper layer of milk fat. Dilute the sample to 100 times with PBS solution at pH 6.8. Place one drop on a 600-mesh copper grid and let it stand for 5-10 minutes. Then, blot the liquid with filter paper. Next, perform negative staining with 2% phosphotungstic acid solution. After 1 minute, remove the staining solution and observe and record the sample under a bio-type field emission transmission electron microscope (Talos F200C G2) at 25°C.

[0089] Prior to the application for this invention, a series of experiments were conducted. Some of the test results are presented below to further describe the invention in detail. The following is a detailed description in conjunction with the embodiments.

[0090] Example 1

[0091] Step 1: Provide a simulated spoilage environment to simulate spoilage of the milk, specifically including:

[0092] Under aseptic conditions, fresh milk was dispensed into seven 50 mL sterile centrifuge tubes, each containing 30 mL of milk. The temperature was set to 25°C to simulate the spoilage process of milk at room temperature. During the simulated spoilage period, at 0, 1, 6, 12, 24, 48, and 72 hours, one sample tube was taken out, divided into three equal portions, and placed into three 15 mL sterile centrifuge tubes, which were then sealed to obtain the test samples. The fresh milk was taken from brand A pure milk.

[0093] Step 2: Construct a milk spoilage-fluorescence model to determine the luminescence color bands reflecting the degree of milk spoilage, specifically including:

[0094] Milk that has been left to stand for a preset time is subjected to both photophysical and physicochemical property tests. 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 photographs were taken according to the above-described method for taking luminescent photographs. In step 101, the upper test tube clamp was adjusted so that the UV lamp was suspended 5 cm above the cuvette. In step 102, 2.8 mL of sample was slowly added to the cuvette using a pipette. In step 103, the cuvette was rinsed three times with alternating water and ethanol.

[0096] The emission spectrum test was performed according to the emission spectrum test method described above. In step 201, 3 mL of sample was added to a cuvette using a pipette. The excitation wavelength was set to 312 or 365 nm and a filter of 330 nm or 395 nm was placed accordingly. The slit width ExBW = 5 nm and EmBW = 4 nm were adjusted, the scanning step size was 1 nm, and the dwell time was 0.1 s.

[0097] pH measurement was performed according to the pH measurement method described above. The test electrode was rinsed with water 3 times. 6 mL of sample was taken with a pipette and placed in a 15 mL centrifuge tube and allowed to stand for 2 min.

[0098] The lactic acid content was determined according to the above-mentioned lactic acid content test method, wherein in step 402, trichloroacetic acid was added and mixed for 3 minutes.

[0099] The free amino acid content was determined according to the above-mentioned free amino acid content test method, wherein the volume of the centrifuged milk sample was 2 mL, the volume of the pretreatment solution was 2 mL, the ultrasonic time was 8 min, and the centrifugation time to remove protein was 25 min.

[0100] Dynamic light scattering (DLS) testing was performed according to the above-described dynamic light scattering (DLS) testing method, with a milk sample volume of 6 mL.

[0101] Transmission electron microscopy (TEM) testing was performed according to the above-described TEM testing method, with a settling time of 9 minutes.

[0102] Analyze the photophysical and physicochemical property test results at the preset time, determine the correspondence between the degree of deterioration and fluorescence display, and establish a color spectrum of luminescence changes reflecting the degree of milk deterioration;

[0103] Figure 6 Figure 'a' shows the changes in lactic acid content and pH at different stages. Figure 6 Figure b shows the changes in free amino acid concentration at different stages. Figure 6 Figure c shows the particle size changes at different stages. At 72 h, the milk had already separated into layers, with large protein aggregates appearing, so DLS was not needed to show the dispersion uniformity. Figure 7 Particle size distribution and TEM images of protein aggregates in milk after different storage times. Figure 6 and Figure 7 The changes in the physicochemical properties of milk were determined as follows:

[0104] From 0 to 6 h, the pH decreased from 6.69 to 6.68, and 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. Throughout this period, the degree of aggregation of free amino acids and proteins remained almost unchanged, and the protein size was relatively uniform, with a PDI of less than 0.2. DLS testing showed that the average particle size of the protein micelles was around 200 nm, indicating very fresh milk. From 6 to 12 h, the pH decreased from 6.68 to 6.66, and the lactic acid content increased slightly, ranging from 0.015% to 0.05%. The degree of aggregation of free amino acids and proteins remained unchanged. Therefore, from 0 to 12 h, the milk was considered to be in the fresh stage.

[0105] Between 12 and 24 hours, the pH continued to decrease, ranging from 6.51 to 6.66. The lactic acid content increased significantly but did not exceed 0.2%. Free amino acids increased, with free proline, phosphoserine, and alanine all <30, 70, and 40 nmol / mL, respectively. The protein size was relatively uniform, and the PDI increased slightly but remained below 0.2 overall. The average particle size of the protein micelles remained around 200 nm. The physicochemical properties of the milk changed slightly between 12 and 24 hours; 12 hours was determined to be the starting point of the mild acidification stage of the milk.

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

[0107] Between 48 and 72 h, the pH decreased significantly and eventually fell below 5.5. Lactic acid content continued to increase, ranging from 0.25% to 0.5%. The contents of free proline, phosphoserine, and alanine increased rapidly and eventually exceeded 60, 100, and 55 nmol / mL, respectively. The protein particle size distribution became more extreme, with a large number of protein micelles with diameters less than 100 nm and greater than 1000 nm gradually appearing. The PDI continued to increase and even reached 1. Analysis showed that 72 h was in the early stage of complete deterioration. Referring to the time point when the pH was 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, slightly acidified stage, partially deteriorated stage, and completely deteriorated stage are 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 6.6~6.8, lactic acid content less than 0.05% (0.05 g / 100 mL milk), free proline, phosphoserine and alanine content less than 3, 15 and 32 nmol / mL respectively, protein size is relatively uniform, PDI is less than 0.2, and the average particle size of protein micelles is about 200 nm.

[0110] During the mild acidification stage, the pH, lactic acid content, free amino acid content, and protein aggregation state were as follows: pH 6.51–6.66, lactic acid content 0.05–0.2% (0.05–0.2 g / 100 mL milk), free proline, phosphoserine, and alanine contents 3–30, 15–70, and 32–40 nmol / mL, respectively. The protein size was relatively uniform, the PDI increased slightly but remained less than 0.2, and the average particle size of the protein micelles was around 200 nm.

[0111] The pH, lactic acid content, free amino acid content, and protein aggregation state during the partial deterioration stage were as follows: pH 5.5-6.0, lactic acid content 0.2-0.4%, free proline, phosphoserine, and alanine contents 30-60, 70-100, and 40-55 nmol / mL, respectively. The protein exhibited heterogeneity, with significant distributions of larger and smaller particle sizes, a PDI greater than 0.2, and an average micelle size greater than 300 nm.

[0112] During the complete deterioration stage, the pH, lactic acid content, free amino acid content, and protein aggregation state were as follows: pH less than 5.5, lactic acid content greater than 0.4%, free proline, phosphoserine, and alanine content greater than 60, 100, and 55 nmol / mL, respectively. The protein particle size distribution became more extreme, with a large number of protein micelles with a particle size less than 100 nm appearing, and protein aggregates with a particle size greater than 1000 nm gradually appearing. The PDI continued to increase to close to 1.

[0113] Figure 4 Images of milk luminescence at different stages under UV excitation at 365 nm and 312 nm. Figure 8 'a' represents the emission spectrum measured under 365 nm ultraviolet excitation. Figure 8 b is Figure 8 The normalized strength of a, Figure 8 c is the CIE chromaticity diagram under 365 nm UV excitation. According to... Figure 4 and Figure 8It is evident that fresh milk exhibits a bright yellow-green light under 365 nm UV excitation and a bright bluish-white light under 312 nm UV excitation. The luminescence of milk shows significant changes at different stages under the same wavelength of UV excitation. Under 312 nm UV excitation, the milk color changes from bright blue to bluish-purple during the spoilage simulation period. Under 365 nm UV excitation, the milk color gradually changes from bright yellow-green to blue during the spoilage simulation period. Specifically, from 0 to 6 hours, the overall luminescence under 365 nm UV excitation is bright yellow-green; from 6 to 12 hours, the emission shifts to green; from 12 to 24 hours, the emission continues to shift to blue and gradually dims; from 24 to 48 hours, the emission is bluish-green; and from 48 to 72 hours, it becomes a weak blue light. Overall, it exhibits a change from yellow-green to blue light.

[0114] The chromaticity coordinates of the emission colors under 365 nm ultraviolet excitation were sequentially connected on the chromaticity diagram. The 12-hour chromaticity coordinate was used as the starting point for the mild acidification stage, the 48-hour chromaticity coordinate as the starting point for the partial spoilage stage, and the 56-hour chromaticity coordinate as the starting point for the complete spoilage stage. The transition region was gradually filled based on the CIE coordinate trend, resulting in a full-stage color spectrum encompassing the fresh, mild acidification, partial spoilage, and complete spoilage stages. The degree of spoilage corresponding to each starting point was marked on the full-stage color spectrum, yielding the luminescence change color spectrum reflecting the degree of milk spoilage, as shown below. Figure 9 As shown.

[0115] Step 3: Determine the degree of spoilage of the milk to be tested based on the color bands of the emission changes;

[0116] Following the photographic method described above, the milk sample to be tested was irradiated with 365 nm ultraviolet light to obtain a luminescence photograph. The obtained luminescence photograph was then compared with the aforementioned luminescence color bands, and the results are as follows: Figure 10 As shown, test milk A was fresh milk left at room temperature for 3 hours, and test milk B was fresh milk left at room temperature for 60 hours. The results showed that test milk A was yellowish-green, and test milk B was a weaker bluish-green, indicating that test milk A was relatively fresh and still edible, while test milk B had partially spoiled.

[0117] Example 2

[0118] This embodiment is the same as Embodiment 1, except that step two involves constructing a linear model of milk spoilage-quantum yield.

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

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

[0121] Analyze the photophysical and physicochemical property test results at the corresponding preset time, determine the correspondence between the degree of deterioration and quantum yield, and establish a linear model of milk deterioration-quantum yield that reflects the degree of milk deterioration;

[0122] The milk deteriorated to the same extent over time at room temperature as in Example 1;

[0123] Figure 11 This is a linear graph showing the changes in milk quantum yield and quantum yield at different stages under ultraviolet excitation. Figure 11 'a' represents the quantum yield diagram under ultraviolet excitation at 365 nm and 312 nm. Figure 11 b is a linear graph of the quantum yield change under 365 nm ultraviolet excitation. According to... Figure 11 It is evident that the quantum yield decreases as the degree of alteration increases.

[0124] Step 3: Determine the degree of spoilage of the milk to be tested based on the linear graph of quantum yield change;

[0125] Following the method described above for testing quantum yield, the emission spectrum and quantum yield of the milk under test were obtained using a steady-state transient fluorescence spectrometer. The milk under test, C, was fresh milk stored at room temperature for 5 days. The results were compared with a linear graph of quantum yield change, as shown below. Figure 12 As shown, the results indicate that under 365 nm ultraviolet excitation, the quantum yield ln Ф is 1.99, which is consistent with... Figure 11 As can be seen from the comparison with b, it is in the stage of complete spoilage, indicating that the milk C tested has completely spoiled.

[0126] Example 3

[0127] This embodiment is the same as Embodiment 1, except that the milk to be tested, D, is fresh milk that has been stored in the refrigerator for 7 days. The milk to be tested is irradiated with 365 nm ultraviolet light according to the photographic method described above to obtain a luminescence photograph. The obtained luminescence photograph is compared with the luminescence color bands of Embodiment 1, and the results are as follows: Figure 13 As shown in the figure, the test milk D exhibits a bright yellow-green color, indicating that it is in a 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; Determine the degree of deterioration of the milk to be tested.

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: Photophysical and physicochemical properties of milk during the simulated spoilage period were tested; 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 photo 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.

9. The method for visually monitoring milk deterioration according to claim 8, characterized in that: Based on the luminescence color spectrum, the degree of deterioration of the milk to be tested is determined by: The luminescent color of the milk to be tested under ultraviolet light is obtained, and the luminescent color is compared with the luminescent change color spectrum band. The luminescent color of the milk to be tested falls into the corresponding stage spectrum band segment of the luminescent change color spectrum band. The deterioration degree of the milk to be tested is determined based on the deterioration degree of the stage spectrum band segment.

10. The method for visually monitoring milk deterioration according to claim 2, characterized in that: Based on the linear graph of quantum yield changes, the degree of deterioration of the milk to be tested is determined by: The quantum yield of the milk to be tested under ultraviolet light is obtained, and the quantum yield is compared with a linear graph of quantum yield changes. The quantum yield of the milk to be tested falls into an interval within the linear graph of quantum yield changes. The degree of deterioration of the milk to be tested is determined according to the degree of deterioration to which the interval belongs.

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