Method for testing for blood in milk, and device for testing for blood in milk

Abstract

The present invention makes it easy to detect blood in milk regardless of whether the milk has been homogenized.　A method according to the present invention includes: a methemoglobin formation step S1 for changing hemoglobin contained in blood in a target sample derived from milk, which is a food product, into methemoglobin; a target exposure step for exposing the target sample to light; a target transmitted light detection step for detecting transmitted light from the target sample through a diffuser that diffuses and transmits the light; and a quantitative evaluation step for quantitatively evaluating the blood in the target sample on the basis of the intensity of light of a predetermined wavelength included in the transmitted light detected in the target transmitted light detection step.

Description

Bloody milk testing method and bloody milk testing device

[0001] The present invention relates to a bloody milk testing method and a bloody milk testing device used therefor.

[0002] Conventionally, devices have been developed for testing blood (bloody milk) contaminated in milk. One example is the device described in Non-Patent Document 1. Meanwhile, Non-Patent Document 2 describes the relationship between milk component analysis and homogenization processing.

[0003] Hokkaido Research Organization, "Development of a Portable Raw Milk Testing Device," 2016 Technical Support Results, URL: https: / / www.hro.or.jp / industrial / research / iri / koho / publications / casebook / example-9693.html; Ben Aernouts, Evgeny Polshin, Wouter Saeys, Jeroen Lammertyn, "Mid-infrared spectrometry of milk for dairy metabolomics: A comparison of two sampling techniques and the effect of homogenization," Analytica Chimica Acta, Volume 705, Issues 1-2, pp. 88-97, 31 October 2011.

[0004] As described in Non-Patent Document 1, the results of bloody milk tests may be affected by the amount of milk fat and other components. Non-Patent Document 2 describes that homogenization improves the accuracy of milk component analysis. Therefore, to make it easier to detect bloody milk, it may be possible to homogenize the milk beforehand.

[0005] On the other hand, homogenization requires the use of specialized equipment and is time-consuming. Therefore, assuming homogenization as a pre-treatment for testing, the testing environment must be well-prepared to a certain extent. However, such an environment is not always available at testing sites.

[0006] An object of the present invention is to provide a bloody milk testing method that can easily detect bloody milk regardless of whether homogenization has been performed or not, and a bloody milk testing device used therefor.

[0007] The blood and milk testing method of the present invention comprises a methemoglobination step of converting hemoglobin contained in blood in a target sample derived from milk, which is a food product, to methemoglobin; a target irradiation step of irradiating light onto the target sample after the methemoglobination step; a target transmitted light detection step of detecting transmitted light from the target sample irradiated with light in the target irradiation step through a diffuser that diffuses and transmits light; and a quantitative evaluation step of quantitatively evaluating the blood in the target sample based on the intensity of light of a predetermined wavelength contained in the transmitted light detected in the target transmitted light detection step.

[0008] As a result of extensive research, the inventors have found that the use of methemoglobin and a diffusing agent makes it possible to quantitatively evaluate blood regardless of the components of milk. The fact that the quantitative evaluation is independent of the components of milk means that the quantitative evaluation is not dependent on the state of milk fat due to homogenization. Therefore, the present invention makes it possible to provide a test method that can easily detect blood in milk regardless of whether homogenization has been performed.

[0009] In the present invention, the target sample may be obtained from milk that has not been subjected to homogenization, which allows quantitative evaluation of blood in bloody milk for a target sample using milk that has not been subjected to homogenization.

[0010] In the present invention, it is also preferable that sodium lauryl sulfate is mixed with the target sample in the methemoglobination step, since the use of sodium lauryl sulfate enables safe methemoglobination.

[0011] In the present invention, the diffuser is preferably an opal glass, whereby the use of opal glass as the diffuser makes it possible to appropriately diffuse transmitted light from the target sample.

[0012] Furthermore, the present invention further includes a control illumination step of irradiating a control sample with light and a control transmitted light detection step of detecting, through the diffuser, transmitted light from the control sample irradiated with light in the control illumination step, and it is preferable that, in the quantitative evaluation step, the degree of absorbance of the target sample is obtained based on the intensity of light of a predetermined wavelength contained in the transmitted light detected in both the control transmitted light detection step and the target transmitted light detection step. This allows the degree of absorbance of the target sample to be appropriately obtained from the detection results of transmitted light from both the control sample and the target sample.

[0013] In addition, the blood milk testing device of the present invention is an apparatus used in the above method, and comprises a light irradiation unit that irradiates light onto the control sample or the target sample in each of the control transmitted light detection step and the target transmitted light detection step, a container for containing the target sample, a passage unit that supports the control sample so that it can slide between an irradiation position where light from the light irradiation unit is irradiated and a retracted position retracted from the irradiation position, and a restoring force generating unit that applies a restoring force to the control sample to maintain the control sample at the irradiation position, and the passage unit has an insertion opening through which the container is inserted, and when the container is inserted into the passage unit, the container pushes the control sample from the irradiation position to the retracted position against the restoring force of the restoring force generating unit, and the container is configured to be positioned at a position where light from the light irradiation unit is irradiated onto the target sample.

[0014] The blood milk testing device of the present invention can detect transmitted light from a control sample when a container containing a target sample is not inserted into the passage, and can detect transmitted light from the target sample when the container is inserted into the passage. This realizes a testing device that can perform detection of both the target sample and the control sample in a simple procedure.

[0015] 1 is a block diagram showing the schematic configuration of a blood milk testing device according to one embodiment of the present invention. It is a cross-sectional view of the sample holder of FIG. 1. It is a cross-sectional view of the sample holder of FIG. 1, showing a state different from that of FIG. 2. It is a conceptual diagram showing how light transmitted through the sample is scattered by the diffuser of FIG. 2. It is a flowchart showing the flow of a blood milk testing method using the blood milk testing device of FIG. 1. It is a graph showing the measurement results of the absorbance of a sample in Example 1, which is an embodiment of the present invention. It is a graph extracting the absorbance of a specific wavelength from the measurement results shown in FIG. 6. It is a graph showing the result of obtaining a regression line when the absorbance obtained in Example 1 is y and the hemoglobin concentration is x. FIG. 9(a) is a graph showing the result of predicting the hemoglobin concentration of a sample obtained in a different region from Example 2 using the calibration curve obtained in Example 2. FIG. 9(b) is a table showing the result of classifying samples into normal milk and bloody milk using a predetermined standard. It is a graph showing the distribution of the residuals for the regression line obtained in Example 2 versus the solids-non-fat content. It is a graph showing the distribution of the residuals for the regression line obtained in Example 2 versus the milk fat content.

[0016] A blood milk testing method and device according to one embodiment of the present invention will be described with reference to Figures 1 to 5. The blood milk testing device 1 according to this embodiment quantitatively evaluates the blood in blood milk by utilizing the light absorption properties of hemoglobin. As shown in Figure 1, the blood milk testing device 1 comprises a light source 10, a sample holder 100, a measurement unit 20, and an evaluation unit 200. The light source 10 is composed of a halogen lamp, a light-emitting diode, or the like, and emits measurement light.

[0017] The sample holder 100 is a device that holds a sample container S onto which measurement light from the light source 10 is irradiated. As shown in Figures 2 and 3, the sample container S is a plastic container that is long in the vertical direction of the figure. A target sample, a milk specimen, is sealed inside the sample container S. The container wall of the sample container S is transparent and allows light to pass through. As shown in Figure 2, the measurement light from the light source 10 enters the sample holder 100 along an optical path α that is parallel to the horizontal direction of the figure, passes through the sample container S inside, and then exits the sample holder 100 along an optical path β.

[0018] The sample holder 100 includes a housing 101, a lid 103, lenses 110 and 120, a filter 130, a diffuser 140, a control sample 150, and an elastic body 160. The housing 101 is a box-shaped member that houses the main components. The lens 110 and the filter 130 are arranged along the optical path α of the incident light. Of these, the lens 110 focuses the measurement light from the light source 10. The filter 130 removes components unnecessary for measurement from the light focused by the lens 110. The light that passes through the filter 130 further passes through the sample and becomes output light. Note that, depending on the type of light source 10, the measurement light incident on the sample holder 110 may not originally contain unnecessary components. In such cases, the filter 130 does not need to be installed in the sample holder 110.

[0019] The diffuser 140 and the lens 120 are arranged along the optical path β of the emitted light. Of these, the diffuser 140 is a flat plate-shaped member made of opal glass or frosted glass that diffuses and transmits light incident thereon. When the diffuser 140 is made of opal glass, it may be made of two layers: a normal transparent glass substrate layer and a layer containing a light-diffusing component, or the entire diffuser may be made of a layer containing a light-diffusing component. The diffuser 140 uniformly scatters the light incident thereon (see FIG. 4 ). The light scattered by the diffuser 140 is collected by the lens 120 and emitted from the sample holder 100 along the optical path β.

[0020] A passage 102 extending linearly in the vertical direction of the figure is formed within the housing 101. A hollow passage is formed within the passage 102. An opening 102a is formed at the upper end of the passage 102, connecting the internal passage with the outside. A lid 103 is attached to the opening 102a. The lid 103 selectively takes a state in which the opening 102a is closed, as shown in FIG. 2, or a state in which the opening 102a is open, as shown in FIG. 3. A control sample 150 is accommodated below the passage of the passage 102. The control sample 150 is made of a standard white board made of polytetrafluoroethylene. A protrusion 102b is formed in the passage of the passage 102 to restrict upward movement of the control sample 150. The control sample 150 is supported by the passage of the passage 102 and can move up and down between the retracted position shown in FIG. 2 and the irradiation position shown in FIG. 3. The irradiation position is a position where the measurement light from the light source 10 is irradiated onto the control sample 150 along the optical path α. The retracted position is a position retracted downward from the measurement position. An elastic body 160 is installed at the bottom of the passage 102. The elastic body 160 (corresponding to the "restoring force generating unit" of the present invention) is made of, for example, a spring, and applies an upward elastic force to the control sample 150. This elastic force functions as a restoring force that maintains the control sample 150 at the irradiation position shown in FIG. 3.

[0021] In the state shown in FIG. 3 , measurement light from the light source 10 is irradiated onto the control sample 150. Therefore, light transmitted through the control sample 150 travels toward the measurement unit 20 via the diffuser 140 and the lens 120. Meanwhile, when setting the sample container S in the sample holder 100, in the state shown in FIG. 3 , the sample container S is inserted from above through the opening of the opening 102a (corresponding to the "insertion port" of the present invention). The sample container S is inserted to the back of the passage 102 until its bottom end contacts the top end of the control sample 150. Then, by further moving the sample container S to the back of the passage 102, the control sample 150 can be pushed to the retracted position against the elastic force of the elastic body 160. After the control sample 150 reaches the retracted position, the lid 103 is operated to transition to the state shown in FIG. 2 . In the state shown in FIG. 2 , measurement light from the light source 10 is irradiated onto the sample container S. Therefore, light transmitted through the target sample in the sample container S travels through the diffuser 140 and the lens 120 toward the measurement unit 20. When removing the sample container S from the sample holder 100 after measurement, the lid 103 is operated to transition to the state shown in Figure 3, and the elastic force of the elastic body 160 causes the control sample 150 to move to the measurement position while pushing the sample container S outward from the opening 102a. When the sample container S pushed out from the opening 102a is removed from the sample holder 100, the sample holder 100 returns to the state shown in Figure 3.

[0022] However, milk-derived components contained in the target sample strongly scatter light incident on the target sample. Therefore, if the diffuser 140 is not used, the intensity of the light emitted from the target sample and incident on the measurement unit 20 through the lens 120 varies depending on the scattering pattern in the target sample. The scattering pattern differs depending on the condition of the components in the target sample. Therefore, if the condition of the milk-derived components contained in the target sample differs, this may cause fluctuations in the measurement results obtained by the measurement unit 20.

[0023] In contrast, the sample holder 100 uses a diffuser 140. As shown in FIG. 4, the diffuser 140 uniformly scatters the light incident on it. Therefore, the light incident on the measurement unit 20 is less affected by the scattering pattern in the target sample. In other words, the measurement results by the measurement unit 20 are less affected by the state of the components in the target sample. Similarly, the diffuser 140 also uniformly scatters the light emitted from the control sample 150. Therefore, if the absorbance A in this embodiment is defined as follows, the evaluation based on the absorption characteristics is less affected by fluctuations in the components in the target sample.

[0024] A = log(I0 / I) (Formula 1) I0: Intensity of light incident from the control sample 150 to the measurement unit 20 I: Intensity of light incident from the target sample to the measurement unit 20

[0025] The measurement unit 20 measures the intensity of light emitted from the sample holder 100. The measurement unit 20 has a spectrometer that acquires the spectrum of the light, and an AD converter that converts an analog signal representing the spectrum output by the spectrometer into a digital signal. The digital signal from the measurement unit 20 is input to the evaluation unit 200.

[0026] The evaluation unit 200 includes a computer having software and hardware including a CPU (Central Processing Unit), memory, etc. The software causes the hardware to function so as to execute the following processes.

[0027] The evaluation unit 200 evaluates the blood milk concentration of the target sample based on the spectrum represented by the digital signal transmitted from the measurement unit 20, as follows. First, the evaluation unit 200 obtains the light intensity of a specific wavelength (corresponding to 531.531 nm in the examples described below) in the transmitted light spectra measured for each of the target sample and the control sample 150. Next, the evaluation unit 200 calculates the absorbance A based on Equation 1. Here, the light intensity in the control sample 150 is substituted for I0 in Equation 1, and the light intensity in the target sample is substituted for I in Equation 1. Next, the evaluation unit 200 derives the blood milk concentration using a calibration curve (corresponding to the regression line in the examples described below) that shows the relationship between the blood milk concentration and the absorbance A. The derived blood milk concentration is output from the evaluation unit 200 using appropriate means. Suitable means include displaying on a display, printing on printing paper using a printer, recording on a recording medium such as a USB memory, transmitting to another device via a communication line such as the Internet, etc.

[0028] Hereinafter, the bloody milk testing method according to this embodiment using the bloody milk testing device 1 will be described with reference to FIG.

[0029] First, milk is prepared as a target sample. This milk may or may not have been homogenized. A reagent is added to the milk to methemoglobinize the hemoglobin contained in the milk (S1). Methemoglobinization corresponds to oxidizing the iron ions in the hemoglobin, changing its ionic valence from 2 to 3. For safety reasons, sodium lauryl sulfate is preferably used as the reagent, but potassium ferricyanide, potassium cyanide, sodium nitrite, or sodium azide may also be used. Step S1 corresponds to the "methemoglobinization step" of the present invention.

[0030] Next, the target sample prepared in S1 is placed in a sample container S and set in the blood milk testing device 1. The target sample is irradiated with measurement light from the light source 10 and the spectrum of the transmitted light is measured (S2). This step corresponds to the "target irradiation step" and "target transmitted light detection step" of the present invention.

[0031] Next, the sample container S is removed from the blood milk testing device 1, and the control sample 150 is irradiated with measurement light from the light source 10 to measure the spectrum of the transmitted light (S3). This step corresponds to the "control irradiation step" and "control transmitted light detection step" of the present invention.

[0032] Next, the concentration of the bloody milk is evaluated by the evaluation unit 200 based on the spectra measured in S2 and S3 (S4). This step corresponds to the "quantitative evaluation step" of the present invention.

[0033] [Examples] Examples of the present invention will be described below.

[0034] (First Example) In the first example, sample I of milk with known blood concentration was prepared, and the calibration curve was derived as follows. First, the combined milk used for sample I was collected from unhomogenized bulk milk stored in multiple bulk tanks in the Tokachi region in August, collected separately from each tank, and samples that showed no erythrocytic sedimentation when centrifuged were extracted. Nine types of sample I with different hemoglobin concentrations were prepared by mixing defibrinated cow's blood with the combined milk from each bulk tank. Specifically, sample I with hemoglobin concentrations of 0, 0.0075, 0.01, 0.025, 0.05, 0.075, 0.1, 0.125, and 0.15 (g / L) were prepared and placed in cells, respectively.

[0035] Next, sodium lauryl sulfate was dissolved in 500 ml of 10-fold diluted phosphate buffer (1 / 15 mol / L, pH 7.2) to a concentration of 35 mmol / L. This solution was added to the cells containing sample A at 200 μl per cell. Each cell of sample A was then placed in a sample container S, and the spectrum of light transmitted through the sample container S was acquired using the light source 10, sample holder 100, and measurement unit 20 of the blood milk testing device 1. The spectrum of light transmitted through the control sample 150 was also acquired. The absorbance A shown in Equation 1 was then calculated from the acquired spectrum, and noise was removed from the calculated value by appropriate smoothing. The graph in Figure 6 shows one of the results. Figure 7 is a graph plotting the absorbance values ​​at a wavelength of 531.531 nm, extracted for each of different concentrations of sample A, against the concentration, calculated as shown in Figure 6. The wavelength of 531.531 nm corresponds to the position where a weak peak appears in absorbance due to methemoglobinized blood.

[0036] As described above, a regression line was derived where y represents the absorbance obtained for the multiple samples prepared for each concentration, and x represents the hemoglobin concentration. The regression line was y = -0.656 * x + 0.054 (coefficient of determination R^2: 0.979) as shown in FIG. 8. The regression line obtained in this example can be used as a calibration curve according to this embodiment. The same applies to the following examples.

[0037] (Example 2) A regression line was derived in the same manner as in Example 1 for Sample B, which was obtained from milk mixtures obtained in the same region as Sample A at four different times (September to December) from Sample A used in Example 1. The results, including those for Example 1, are as follows: (August) y = -0.656 * x + 0.054 R^2: 0.979 (Example 1) (September) y = -0.653 * x + 0.055 R^2: 0.982 (October) y = -0.672 * x + 0.055 R^2: 0.983 (November) y = -0.675 * x + 0.055 R^2: 0.982 (December) y = -0.698 * x + 0.055 R^2: 0.971

[0038] (Example 3) Using the regression line obtained from the November samples in Example 2 as a calibration curve, Sample C, which used synthetic milk obtained in a region (Tokachi district) different from the region where Sample A in Example 1 and Sample B in Example 2 were obtained, was evaluated. Specifically, similar to Example 1, multiple Samples C with known but different hemoglobin concentrations were prepared, and smoothed absorbance values ​​were obtained for each Sample C. The hemoglobin concentration for each Sample C was then predicted by comparing the obtained absorbance values ​​with the calibration curve. Figure 9(a) shows the results. The horizontal axis of the graph in Figure 9(a) represents the predicted hemoglobin concentration, and the vertical axis represents the correct hemoglobin concentration. The regression line, with the former as x and the latter as y, was y = 0.929x + 0.008 (R^2: 0.952, root mean square error (RMSE): 0.011). Furthermore, when milk with a hemoglobin concentration below 2.5*10e^-2 [g / L] was classified as normal milk and milk with a hemoglobin concentration above the standard as bloody milk, the relationship between the predicted classification and the correct classification was as shown in Figure 9(b). The numbers in Figure 9(b) represent the number of samples that fell into each classification.

[0039] (Summary of the Examples) The residuals for each sample were calculated for each regression line obtained in Example 2. The percentage of solids-not-fat and the percentage of milk fat in the combined milk from which each sample was derived were also obtained. Based on this, the distribution of residuals for solids-not-fat is shown in the graph of FIG. 10 . The distribution of residuals for milk fat is shown in the graph of FIG. 11 . As shown in FIGS. 10 and 11 , the residuals vary in both positive and negative ranges around 0, regardless of the sample components. This indicates that the regression line obtained in Example 2 is highly generalizable, regardless of the sample components.

[0040] [Effects of the Embodiment] According to the present embodiment described above, a test method that easily detects blood in milk is possible, regardless of whether homogenization has been performed. The reasons for this are as follows. As shown in the above examples, the calibration curve obtained using the test method of this embodiment is highly generalizable regardless of the milk components. On the other hand, the test method has two features: methemoglobinization of hemoglobin in the target sample and scattering of light transmitted through the target sample by the diffuser 140. These two features enable stable test results to be obtained regardless of the state of hemoglobin in the target sample or the components that scatter light. Therefore, the calibration curve obtained using the test method of this embodiment is not affected by differences in components. In other words, by employing methemoglobinization and the diffuser 140, this embodiment enables quantitative evaluation of blood in milk regardless of the milk components. The fact that the quantitative evaluation is independent of the milk components means that the quantitative evaluation is not dependent on the state of milk fat caused by the homogenization process. This fact is also shown in the above example in which unhomogenized milk was used as the sample. That is, according to this embodiment, quantitative evaluation of blood in bloody milk can be appropriately performed not only when homogenized milk is used, but also when non-homogenized milk is used as the target sample.

[0041] Furthermore, according to this embodiment, safe methemoglobinization is possible by using sodium lauryl sulfate.

[0042] Furthermore, according to this embodiment, by using opal glass for the diffuser 140, it is possible to appropriately diffuse the transmitted light from the target sample.

[0043] Furthermore, according to this embodiment, the absorbance of the target sample can be appropriately obtained from the detection results of the transmitted light in both the control sample 150 and the target sample.

[0044] Furthermore, according to this embodiment, when the sample container S is not inserted into the passage 102 (see FIG. 3), it is possible to detect transmitted light from the control sample 150, and when the sample container S is inserted into the passage 102 (see FIG. 2), it is possible to detect transmitted light from the target sample in the sample container S. Therefore, detection of both the target sample and the control sample 150 can be performed by a simple procedure.

[0045] [Modifications] Although embodiments of the present invention have been described above with reference to the drawings, it should be understood that the specific configurations are not limited to these embodiments. The scope of the present invention is indicated not only by the description of the above-mentioned embodiments but also by the claims, and further includes all modifications within the meaning and scope equivalent to the claims. Below, modifications of the above-mentioned embodiments will be described. Furthermore, parts common to the above-mentioned embodiments will be designated by the same reference numerals as above, and descriptions will be omitted as appropriate.

[0046] In the above-described embodiment, the measurement unit 20 is configured as a spectroscope. Alternatively, a filter that extracts light of a specific wavelength from transmitted light and a detector that detects the intensity of the transmitted light that has passed through the filter and is now light of only the specific wavelength may be used.

[0047] In the above embodiment, the light source 10 is a halogen lamp or the like. Alternatively, the light source 10 may be a light-emitting diode.

[0048] S: Sample container 1: Blood milk testing device 10: Light source 20: Measurement section 100: Sample holding section 102: Passage section 102a: Opening 140: Diffuser 150: Control sample 160: Elastic body 200: Evaluation section

Claims

1. A blood and milk testing method comprising: a methemoglobination step of converting hemoglobin contained in blood in a target sample derived from milk, which is a food product, to methemoglobin; a target illumination step of irradiating light onto the target sample after the methemoglobination step; a target transmitted light detection step of detecting transmitted light from the target sample irradiated with light in the target illumination step through a diffuser that diffuses and transmits light; and a quantitative evaluation step of quantitatively evaluating the blood in the target sample based on the intensity of light of a predetermined wavelength contained in the transmitted light detected in the target transmitted light detection step.

2. The blood milk testing method according to claim 1, characterized in that the target sample is obtained from milk that has not been subjected to homogenization.

3. The blood milk testing method according to claim 1, characterized in that in the methemoglobinization step, sodium lauryl sulfate is mixed with the target sample.

4. The method for testing blood in milk according to claim 1, wherein the diffusing material is opal glass.

5. A blood milk testing method as described in claim 1, further comprising a control illumination step of irradiating a control sample with light, and a control transmitted light detection step of detecting, through the diffuser, transmitted light from the control sample irradiated with light in the control illumination step, wherein, in the quantitative evaluation step, the degree of light absorption of the target sample is obtained based on the intensity of light of a predetermined wavelength contained in the transmitted light detected in both the control transmitted light detection step and the target transmitted light detection step.

6. A blood milk testing device for use in the method described in claim 5, comprising: a light irradiation unit that irradiates light onto the control sample or the target sample in each of the control transmitted light detection step and the target transmitted light detection step; a container for containing the target sample; a passage unit that supports the control sample so as to be slidable between an irradiation position where light from the light irradiation unit is irradiated and a retracted position retracted from the irradiation position; and a restoring force generating unit that applies a restoring force to the control sample so as to maintain the control sample at the irradiation position, wherein the passage unit has an insertion opening through which the container is inserted, and when the container is inserted into the passage unit, the container pushes the control sample from the irradiation position to the retracted position against the restoring force of the restoring force generating unit, and the container is positioned at a position where light from the light irradiation unit is irradiated onto the target sample.

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