Method and system for evaluating somatic cells in raw milk

Abstract

To provide an evaluation method of a somatic cell in raw milk and an evaluation system of a somatic cell in raw milk in which a somatic cell of raw milk to be inspected can be stably evaluated simply and securely.SOLUTION: An evaluation method of a somatic cell in raw milk is a method for evaluating the number of somatic cells in raw milk extracted from a mammal, mainly cow. In the method, a centrifugal separation process, a collagen gel-forming process, an immobilized chip manufacturing process, an oxygen reduction current measuring process, and a somatic cell number evaluating process are executed. Further, in executing the oxygen reduction current measuring process, an electrode refreshing process may be executed.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a raw milk somatic cell evaluation method and a raw milk somatic cell evaluation system for evaluating the somatic cell count in raw milk extracted from mammals.

Background Art

[0002] It is known that in the initial stage of mastitis, the somatic cell count in raw milk mainly composed of increased white blood cells to detect and sterilize bacteria invading the mammary gland increases, and it is effective to perform somatic cell measurement for early detection of mastitis. Various methods have been developed and studied for measuring the somatic cell count.

[0003] For example, as shown in Patent Document 1 below, as this type of raw milk inspection device, a stimulation chamber is provided on a flow path into which the raw milk to be inspected is poured, and an electromagnetic wave or an electric field is irradiated or applied as a stimulation means to activate somatic cells in the raw milk. Next, while the raw milk containing the activated somatic cells passes through the measurement chamber, it is adhered and aggregated on the surface of the working electrode 6A. Thereby, a raw milk inspection device is known in which the active oxygen released by somatic cells (especially neutrophils) reacts with the SOD enzyme, and the oxidation-reduction current is sent to the apparatus main body to calculate the somatic cell count.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in such a conventional raw milk inspection device, it is necessary to activate somatic cells in raw milk and measure the reactive oxygen species thereof. Therefore, not only is it necessary to provide a stimulation chamber and irradiate or apply electromagnetic waves or an electric field as a stimulation means, but also a measurement chamber is provided, and the somatic cells activated in the measurement chamber are adhered and aggregated on the surface of the working electrode 6A, and it is necessary to measure the redox current resulting from the reaction between the reactive oxygen species released by the somatic cells (particularly neutrophils) and the SOD enzyme.

[0006] Thus, in the conventional raw milk inspection device, there has been a problem that the process and the device configuration are large-scale and not suitable for practical use.

[0007] On the other hand, although a simple raw milk inspection device can be considered, it has been difficult to stably evaluate the somatic cell count of raw milk, and it has been desired to stably maintain the measurement accuracy.

[0008] Therefore, an object of the present invention is to provide a method for evaluating somatic cells in raw milk and a system for evaluating somatic cells in raw milk that can simply and reliably evaluate the somatic cells of raw milk to be inspected stably.

Means for Solving the Problems

[0009] The method for evaluating somatic cells in raw milk according to the first invention is a method for evaluating the somatic cell count in raw milk extracted from a mammal, a centrifugation step of centrifuging the raw milk to remove the supernatant, a collagen gelation step of mixing the raw milk obtained by the centrifugation step with a collagen gel to gelate it, an immobilization chip production step of spotting the raw milk to be gelled in the collagen gelation step onto an immobilization well to produce a raw milk immobilization chip, the immobilization chip Production an oxygen reduction current measurement step of scanning the surface of the raw milk immobilization chip produced by the step by approaching and separating the electrode of a scanning electrochemical microscope, and measuring the oxygen reduction current value at a position near the raw milk immobilization chip and the oxygen reduction current value at a separated position, A somatic cell count evaluation step of evaluating the somatic cell count of raw milk from the difference between the oxygen reduction current value at the position near the raw milk-immobilized chip and the oxygen reduction current value at the separated position, which are measured by the oxygen reduction current measurement step is characterized by being executed.

[0010] According to the method for evaluating somatic cells in raw milk of the first invention, the somatic cell count can be easily evaluated only from the difference between the oxygen reduction current value depending on the oxygen concentration of the raw milk on the chip and the oxygen reduction current value at the separated position.

[0011] Here, when measuring the oxygen concentration of the raw milk on the chip with the electrode of a scanning electrochemical microscope, there is a risk that proteins will adsorb on the electrode surface and the measurement accuracy will decrease. When manufacturing the raw milk-immobilized chip, by combining a centrifugation step and a collagen gelation step, it is possible to prevent proteins from adhering to the electrode surface, maintain the state of the electrode well, and ensure the measurement accuracy.

[0012] Thus, according to the method for evaluating somatic cells in raw milk of the first invention, the somatic cell count can be stably evaluated simply and reliably.

[0013] The method for evaluating somatic cells in raw milk of the second invention is, in the first invention, the somatic cell count evaluation step is based on a conversion formula that defines the somatic cell count of raw milk with respect to the difference between the oxygen reduction current value at the position near the raw milk-immobilized chip and the oxygen reduction current value at the separated position, which are prepared from raw milk samples with different somatic cell counts, and calculates the somatic cell count of the raw milk from the difference between the oxygen reduction current value at the position near the raw milk-immobilized chip to be evaluated and the oxygen reduction current value at the separated position.

[0014] According to the method for evaluating somatic cells in raw milk of the second invention, in advance, the difference between the oxygen reduction current value at the position near the raw milk-immobilized chip and the oxygen reduction current value at the separated position, which are prepared from raw milk samples with different somatic cell counts, is obtained, and by using this as a calibration curve that defines the somatic cell count of raw milk, a conversion formula that defines the relationship between the difference between the oxygen reduction current value at the position near the raw milk-immobilized chip and the oxygen reduction current value at the separated position and the somatic cell count of raw milk can be obtained.

[0015] Based on such a conversion formula, the somatic cell count of raw milk can be simply and reliably calculated from the difference between the oxygen reduction current value at the position near the milk-immobilized chip to be evaluated and the oxygen reduction current value at the separated position.

[0016] Thus, according to the method for evaluating somatic cells in raw milk of the second invention, the somatic cell count can be stably calculated and evaluated simply and reliably.

[0017] The method for evaluating somatic cells in raw milk of the third invention is the same as that of the first or second invention, before measuring the oxygen reduction current value by the oxygen reduction current measurement step, an electrode refresh step of applying a cleaning pulse voltage to the electrode of the scanning electrochemical microscope is performed.

[0018] According to the method for evaluating somatic cells in raw milk of the third invention, by combining the centrifugation step and the collagen gelation step, it is possible to prevent proteins from adhering to the electrode surface, maintain the state of the electrode well, and ensure the measurement accuracy. However, by applying a cleaning pulse voltage to the electrode surface regularly or irregularly, the state of the electrode can be maintained better and the measurement accuracy can be improved.

[0019] Thus, according to the method for evaluating somatic cells in raw milk of the third invention, the somatic cell count can be evaluated more stably simply and reliably.

[0020] The system for evaluating somatic cells in raw milk of the fourth invention is characterized by evaluating the somatic cell count in raw milk extracted from mammals by the method for evaluating somatic cells in raw milk of the first to third inventions.

[0021] According to the raw milk somatic cell evaluation system of the fourth invention, the somatic cell count can be easily evaluated only from the difference between the oxygen reduction current value depending on the oxygen concentration of the raw milk on the chip and the oxygen reduction current value at the separated position. Furthermore, by combining the centrifugation step and the collagen gelation step, it is possible to prevent proteins from adhering to the electrode surface, maintain the state of the electrode well, and ensure the measurement accuracy.

[0022] Thus, according to the raw milk somatic cell evaluation system of the fourth invention, the somatic cell count can be stably evaluated simply and reliably.

Brief Description of the Drawings

[0023]

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Modes for Carrying Out the Invention

[0024] As shown in Fig. 1, the method for evaluating somatic cells in raw milk of the present embodiment is based on the finding by the inventor of the present application that since somatic cells in raw milk respire and consume oxygen, it is possible to measure this oxygen consumption and estimate the number of somatic cells in raw milk by using a scanning electrochemical microscope (SECM) that can perform measurements with high sensitivity on a minute sample.

[0025] Specifically, the method for evaluating somatic cells in raw milk of the present embodiment is a method for evaluating the number of somatic cells in raw milk extracted from mammals, mainly cows, and includes a centrifugation step, a collagen gelation step, a production step of an immobilization chip, an oxygen reduction current measurement step, and a somatic cell number evaluation step. Further, an electrode refresh step may be performed when the oxygen reduction current measurement step is performed.

[0026] The outline of each step will be described.

[0027] First, in the centrifugation step, a process of centrifuging raw milk to remove the supernatant is performed.

[0028] In the collagen gelation step, the raw milk obtained by the centrifugation step is mixed with a collagen gel and gelled.

[0029] In the production step of the immobilization chip, the raw milk to be gelled in the collagen gelation step is spotted on an immobilization well to produce a raw milk immobilization chip.

[0030] In the oxygen reduction current measurement step, with respect to the surface of the raw milk immobilization chip produced by the production step of the immobilization chip, the electrode of the scanning electrochemical microscope is scanned while approaching and separating from the surface, and the oxygen reduction current value at a position near the raw milk immobilization chip and the oxygen reduction current value at a separated position are measured.

[0031] In the somatic cell count evaluation step, the somatic cell count of raw milk is evaluated from the difference between the oxygen reduction current value at the vicinity position and the oxygen reduction current value at the separated position of the raw milk immobilization chip measured by the oxygen reduction current measurement step.

[0032] Furthermore, in the somatic cell count evaluation step, based on the conversion formula that defines the somatic cell count of raw milk with respect to the difference between the oxygen reduction current value at the vicinity position and the oxygen reduction current value at the separated position of the raw milk immobilization chip created from raw milk samples with different somatic cell counts, a process is executed to calculate the somatic cell count of raw milk from the difference between the oxygen reduction current value at the vicinity position and the oxygen reduction current value at the separated position of the milk immobilization chip to be evaluated.

[0033] Note that in the electrode refresh step, before measuring the oxygen reduction current value by the oxygen reduction current measurement step, a cleaning pulse voltage is periodically or irregularly applied to the electrode of the scanning electrochemical microscope to refresh the electrode. Next, details of an example of each step of this embodiment are shown.

[0034] As shown in FIG. 2, first, in the centrifugation step, 5 mL of a blood cell separation solution was placed in a centrifuge tube, and 5 mL of raw milk was added on top of it. This was centrifuged (500×g, r = 15 cm, 30 min), and 9 mL of the supernatant was removed. Then, 3 mL of a 11.4 mM glucose-containing phosphate buffer was added, and it was centrifuged (400×g, 10 min), and 2.9 mL of the supernatant was removed.

[0035] Next, in the collagen gelation step, the cell aggregation solution and the collagen gel were mixed at a ratio of 1:9.

[0036] Then, in the immobilization chip production step, the mixed solution was put into a microsyringe and spotted in a well for raw milk immobilization at 2 μL, incubated at 37 °C for 15 minutes to gelate, and a raw milk immobilization chip was produced. As a result, the number of somatic cells present in the well becomes 1 / (5.0×10 3 ) times that of the original raw milk sample.

[0037] In Fig. 2, (A1) and (A2) show well photos and dimensional drawings, (B) shows a method for producing a raw milk chip for mastitis inspection, and (C) shows a photo of the raw milk chip for mastitis inspection.

[0038] Next, in the oxygen reduction current measurement step, the fabricated raw milk chip for mastitis inspection is measured using a scanning electrochemical microscope (hereinafter referred to as SECM) with a microelectrode as a probe, as shown in Fig. 3.

[0039] In the oxygen reduction current measurement step, as shown in the conceptual diagram of the microelectrode scan in Fig. 4(A), the fabricated raw milk immobilization chip was fixed in a petri dish with the detection outlet facing up, and 15 mL of a measurement solution, a phosphate buffer solution containing 11.4 mM glucose (pH = 7.4), was placed in the petri dish.

[0040] Apply -0.5 V vs. Ag / AgCl to the platinum microelectrode (φ20μm) of SECM, and the reaction O 2 + 4H + + 4e - → 2H 2 O to measure the oxygen reduction current value. The microelectrode for measuring the oxygen concentration was scanned 3 times at 50μm / s between two points: the point (Surface) where the oxygen concentration decreased due to the respiration of somatic cells and the point (Bulk) not affected by the current value respiration.

[0041] Here, the method for evaluating the somatic cell count in the somatic cell count evaluation step will be described. As shown in the schematic diagram of the oxygen concentration measurement results in Fig. 4(B), at times S 0 , S 1 , S 2 , S 3 the microelectrode is at Surface, and at times B 0 , B 1 , B 2 , B 3 the microelectrode is at Bulk. The oxygen reduction current value difference formula ΔI = |(I S ) - (I B from the oxygen reduction current value I B ) - (IS ) It is obtained from |. Calculate the average ΔI(―) of the oxygen reduction current value differences obtained from three operations. Assume the dissolved oxygen concentration at room temperature of 25 °C is 248 μM, and the oxygen reduction current value at this time is -1.7 nA. Calculate ΔC from ΔI(―) to evaluate the somatic cell count.

[0042] Next, with reference to FIGS. 5 and 6, the evaluation results of the somatic cell count based on the actual measurement results will be described. FIG. 5(A) shows the result of scanning the area near the offshore-chip three times. n represents the somatic cell count in raw milk. The value of n is the value before immobilizing the raw milk on the chip. ΔI 1 from ΔI 3 The average of is taken as ΔI, and the oxygen concentration difference is calculated. FIG. 5(B) is a calibration curve created from the measurement results of three samples with different somatic cell counts in raw milk. The calibration curve is linear over 10 4 ~ 10 6 cells / mL, and ΔC (μM) = (1.3×10 -5 ) n + 2.2 (1) It is expressed as. From this, it is shown that it is possible to discriminate the somatic cell count in raw milk of 10 4 ~ 10 6 cells / mL required for bovine mastitis testing.

[0043] Furthermore, FIG. 6 shows a comparison between a raw milk sample (4.2×10 6 cells / mL) from a cow diagnosed with mastitis and a raw milk sample (2.1×10 5 cells / mL) from a cow not diagnosed with mastitis. The somatic cell counts of the samples are the results of flow cytometry in the regular mastitis check-ups conducted by the Miyagi Prefectural Livestock Association. By calculating ΔI(―) from the graph, converting it to ΔC, and estimating the somatic cell count from Equation (1), the somatic cell count of the mastitis cow is 2.6×10 6 cells / mL, and that of the non-mastitis cow is 8.4×10 5 cells / mL. This result is of the same order as the somatic cell count measurement results of flow cytometry, indicating the effectiveness of this system.

[0044] Note that flow cytometry for measuring somatic cells stained by fluorescence staining measures both dead cells and live cells without distinction, while the oxygen concentration measurement method measures only live cells. Since raw milk from cows with advanced mastitis contains many dead cells, it is considered that a difference occurred in the results of both measurement methods.

[0045] Indicating the usefulness of this system, it enables the detection of bovine mastitis by performing oxygen concentration detection that has not been performed as a mastitis test until now. By immobilizing raw milk samples using a collagen gel and micro-wells, diffusion into the measurement solution of milk components is suppressed, and furthermore, by limiting the surface area of the measurement target, the sensitivity required for bovine mastitis testing can be obtained.

[0046] Furthermore, the following three points can be cited as the features of this method. (1) High-sensitivity measurement is possible In the conventional conductivity measurement method, errors due to temperature changes and differences in fat content in raw milk for each individual cow are large. This method estimates the cell count from the respiratory activity by comparing the oxygen reduction current value near somatic cells with the oxygen reduction current value at a position not affected by cells. However, when the raw milk sample is liquid, it is difficult to perform high-sensitivity measurement due to the diffusion of milk components. With the raw milk well for mastitis testing, this system enables the discrimination of somatic cell counts in raw milk of 10 4 ~ 10 6 cells / mL. (2) Reagent costs can be reduced The conventional fluorescence method requires a fluorescent reagent. Also, the reactive oxygen species (ROS) detection method, which has been under research in recent years, requires an enzyme for high-sensitivity, and the enzyme-linked immunosorbent assay (ELISA) method for cytokine measurement requires an antibody for measurement, resulting in very high reagent costs. This method does not require expensive reagents and can suppress the inspection cost per time. (3) It is easy to be made into a device The colorimetric method of the conventional method is a technique of mixing a pH indicator with raw milk and observing it, and it is difficult to quantify. On the other hand, this method uses the amperometry method, and since the measurement result is output as a current value, quantification and digital processing are easy. In the future, it is highly developable to be miniaturized and linked with an IC tag, leading to early detection.

[0047] Thus, according to the method for evaluating somatic cells in raw milk and the system for evaluating somatic cells in raw milk of this embodiment, the somatic cell count can be evaluated simply and reliably.

[0048] Next, with reference to FIGS. 7 to 10, the measurement stability of this method will be described.

[0049] FIGS. 7 and 8 show the results of a comparative experiment of measuring (CV) the oxygen reduction current values for (A) raw milk and (B) collagen-embedded somatic cells before and after oxygen concentration measurement.

[0050] Note that the content of the sample treatment is the same as the above-described centrifugation step, collagen gelation step, and immobilized chip production step, and the conditions for measuring (CV) the oxygen reduction current value are as follows: Measurement solution: 4 mM K 4 Fe(CN) 6 Working electrode: Platinum microelectrode (φ20 μm) Sweeping speed: 20 mV / s When it is as described above, as shown in FIGS. 7(A) and (B), the sample with collagen gelation (collagen embedding) shows almost no change in CV before and after measurement. In contrast, the untreated raw milk has changed significantly. This is considered to be because the surface state of the electrode has changed significantly due to the milk components.

[0051] Furthermore, as shown in FIG. 8, the error rate with respect to the average value of ΔI is ±0.1% for the sample with collagen gelation (collagen embedding), while it changes significantly to ±13% for the untreated raw milk.

[0052] From these experimental results, it can be seen that the collagen gelation step of the present invention contributes to stable measurement.

[0053] Next, FIGS. 9 and 10 show the experimental results regarding the effectiveness of preventing protein adsorption onto the electrode surface by centrifugation of raw milk and collagen gelation (collagen embedding) treatment, and the experimental results regarding the effectiveness of the electrode refresh process for applying a cleaning pulse voltage.

[0054] Note that the content of the sample treatment is the same as the above-mentioned centrifugation process, collagen gelation process, and immobilized chip fabrication process. Cyclic voltammetry (CV) is an evaluation of the electrode state by CV measurement using a potassium ferrocyanide solution, and the conditions are as follows.

[0055] Measurement solution: 4 mM K 4 Fe(CN) 6 (3 mL) Working electrode: Platinum microelectrode (φ20μm) Sweeping rate: 20 mV / s Sampling interval: 100 ms First, FIG. 9 shows the results of the change over time during amperometry.

[0056] From the change over time according to the presence or absence of the treatment of the samples shown in the table, samples A and B treated with collagen gelation (collagen embedding) can maintain the electrode state for a relatively long time. Furthermore, sample A treated with both centrifugation and collagen gelation (collagen embedding) can maintain the electrode state the best.

[0057] Next, FIG. 10 shows the results of the change according to the number of cleaning pulses.

[0058] When the cleaning pulses of ±1.1 V shown in the figure are repeatedly applied to the electrode 1, 3, 5, 10, and 12 times, it can be seen that regardless of the presence or absence of the treatment of the sample, the surface state is generally improved as the number of times increases. Note that the surface state of the sample treated only with centrifugation is greatly improved by the cleaning pulse.

[0059] From the above, according to the raw milk somatic cell evaluation method and the raw milk somatic cell evaluation system of the present embodiment, it is possible to simply and surely evaluate the somatic cell count stably.

Claims

1. A method for evaluating somatic cells in raw milk for evaluating the number of somatic cells in raw milk extracted from a mammal, comprising the steps of: A centrifugation step of centrifuging the raw milk and removing the supernatant; a collagen gelation step of mixing the raw milk obtained by the centrifugation step with a collagen gel to form a gel; An immobilization chip production step of spotting the raw milk to be gelled in the collagen gelation step onto an immobilization well to produce a raw milk immobilization chip; an oxygen reduction current measuring step of scanning an electrode of a scanning electrochemical microscope by moving it close to and away from the surface of the raw milk immobilization chip produced by the immobilization chip production step, and measuring the oxygen reduction current value at a position near the raw milk immobilization chip and at a position away from the raw milk immobilization chip; a somatic cell number evaluation step of evaluating the somatic cell number of raw milk from the difference between the oxygen reduction current value at a position near the raw milk immobilization chip and the oxygen reduction current value at a position away from the raw milk immobilization chip, which are measured in the oxygen reduction current measurement step; A method for evaluating somatic cells in raw milk, comprising carrying out the steps of:

2. The method for evaluating somatic cells in raw milk according to claim 1, The somatic cell count evaluation process is a method for evaluating somatic cells in raw milk, characterized in that the somatic cell count of raw milk is calculated from the difference between the oxygen reduction current value at a position near the milk immobilization chip to be evaluated and the oxygen reduction current value at a distant position, based on a conversion formula that specifies the somatic cell count of raw milk relative to the oxygen reduction current value at a position near the raw milk immobilization chip and the oxygen reduction current value at a distant position, created from raw milk samples with different somatic cell counts.

3. The method for evaluating somatic cells in raw milk according to claim 1 or 2, A method for evaluating somatic cells in raw milk, comprising carrying out an electrode refresh step of applying a cleaning pulse voltage to the electrodes of the scanning electrochemical microscope before measuring an oxygen reduction current value in the oxygen reduction current measuring step.

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