WO2011093039A1

WO2011093039A1 - Raw milk inspection method and raw milk inspection device

Info

Publication number: WO2011093039A1
Application number: PCT/JP2011/000292
Authority: WO
Inventors: 綾野賢; 二階堂祐子; 岡桂子
Original assignee: 株式会社クラレ
Priority date: 2010-01-27
Filing date: 2011-01-20
Publication date: 2011-08-04

Abstract

Provided is a convenient, highly-sensitive inspection method for determining whether or not given raw milk comes from a mammal infected with mastitis. Also provided is a device that can be used in said method. The provided method includes a step (A) in which a voltage is applied to raw milk from a mammal and a step (B) in which a current that flows when the applied voltage oxidizes oxidizable substances is measured. Said method can be implemented by the provided raw milk inspection device, which contains: at least one pair of electrodes comprising a working electrode and a counter electrode, with a hydrophilic compound exposed on the surface of the working electrode; a circuit that applies a fixed voltage between said electrodes; and a means that measures a current that flows when the applied voltage oxidizes oxidizable substances in the raw milk.

Description

Raw milk inspection method and raw milk inspection device

The present invention relates to a method for inspecting raw milk of mammals, particularly dairy cows, and a raw milk inspection apparatus for performing the inspection.

In dairy farming, mastitis of livestock is a major issue. Milk from livestock infected with mastitis has a reduced commercial value due to a decrease in lactose percentage, an increase in salinity, and other changes in milk components. Infection with mastitis also damages livestock animals and breast tissue, leading to reduced lactation capacity and reduced productivity. Therefore, it is very important to detect mastitis and start treatment while the infection level is low.

Currently, the number of somatic cells in milk is generally used as an indicator of infection. The number of somatic cells in milk is the total number of blood-derived cells including white blood cells and mammary epithelial cells. The rapid increase in the number of somatic cells indicates that leukocytes have moved from blood to milk due to the invasion of bacteria into the breast and proliferated, and that white blood cells have increased in milk. It is useful as an indicator of milk in livestock infected with mastitis.

Although the number of somatic cells can be measured with high reliability by microscopic observation or a cell sorter, the devices used for these measurements are usually expensive, large-sized, and complicated to operate. At the stage of milk collection, the number of somatic cells is measured at a specific laboratory. Therefore, when the results of somatic cell counts are revealed, the milk of livestock infected with mastitis is mixed with normal milk, and the milk of livestock infected with mastitis deteriorates the quality of large quantities of milk in the same tank. As a result, the milk of the tank unit is discarded. In addition, since the milk of livestock with low infectivity is diluted with normal milk, early detection of mastitis is difficult. Therefore, a simple method and apparatus that can measure the number of somatic cells during milking is desired.

Therefore, Patent Document 1 discloses a mastitis comprising a milk heating means, a reactive oxygen species sensor, and a mastitis diagnosis means as an apparatus capable of accurately diagnosing mastitis by a simple method. A diagnostic device is disclosed. The apparatus focuses on the amount of active oxygen generated from neutrophils that increase when infected with mastitis, and determines whether or not mastitis is infected based on the current value detected by the active oxygen sensor. However, the apparatus described in Patent Document 1 has a problem in sensitivity because the current value based on the amount of active oxygen is small.

On the other hand, in the past, it has been reported that the amount of ascorbic acid (reducing vitamin C) in raw milk decreases as the suspicion of latent mastitis in dairy cows increases (see Non-Patent Document 1).

JP 2005-106490 A

In view of the above problems, an object of the present invention is to easily and highly sensitively test whether the milk of a mammal infected with mastitis is raw.

The present invention that has solved the above problems includes a step of applying a voltage to raw milk of a mammal (A), and a step of measuring a current generated when an easily oxidizable substance is oxidized by the applied voltage (B).
Is a method for inspecting raw milk.

The present invention also includes at least one set of electrodes including a working electrode including a hydrophilic compound exposed on the surface, and a counter electrode;
A circuit for applying a constant voltage between the electrodes;
And a means for measuring a current generated when an easily oxidizable substance in raw milk is oxidized by an applied voltage.

According to the present invention, it is possible to easily and highly sensitively check whether the milk is a mammal infected with mastitis.

It is a top view which shows an example of the raw milk test | inspection apparatus of this invention. It is a perspective view which shows the sensor which comprises the raw milk test | inspection apparatus shown in FIG. FIG. 3 is a sectional view taken along line III-III in FIG. 2. It is a top view which shows the sensor which abbreviate | omitted the cover plate. It is a block diagram of the raw milk test | inspection apparatus shown in FIG. It is a top view which shows another example of the raw milk test | inspection apparatus of this invention. 2 is a graph showing the relationship between the current value obtained in Example 1 and the ascorbic acid concentration. It is a graph which shows the relationship between the electric current value obtained in Example 1, and the number of somatic cells.

Healthy milk cow's raw milk contains about 1.5 to 2.0 mg / 100 mL of ascorbic acid (reducible vitamin C) biosynthesized in the body. It has been reported that the amount of ascorbic acid in raw milk decreases as the suspicion of latent mastitis in dairy cows increases (see Non-Patent Document 1). The present inventors pay attention to this phenomenon and electrochemically oxidize an easily oxidizable substance typified by ascorbic acid in raw milk. From the oxidation current value, the raw milk of a mammal infected with mastitis It was found that it can be determined whether or not. Further, the present inventors have found that the oxidation current value is correlated with the number of somatic cells, and the number of somatic cells can be estimated from the oxidation current value.

First, the raw milk inspection method of the present invention will be described. The method for inspecting raw milk of the present invention includes the step (A) and the step (B) described above (method A).

Step (A)
In step (A), a voltage is applied to the milk of the mammal. Step (A) can be performed, for example, by immersing an electrode including a working electrode and a counter electrode in raw milk and applying a voltage between the electrodes.

The working electrode preferably contains a hydrophilic compound exposed on the surface. In this case, the hydrophilic compound functions as an electrode catalyst, and the sensitivity of current measurement can be improved.

The kind of the hydrophilic compound is not particularly limited as long as it has an electrode catalytic action, and may be appropriately determined according to the kind of the easily oxidizable substance. The hydrophilic compound preferably has a cationic group and more preferably has an amino group from the viewpoint of electrocatalysis. Specific examples include cysteine, aminoethanethiol, aminobutanethiol, aminononanethiol, and the like. Since a material such as gold is usually used for the working electrode, the hydrophilic compound preferably has a sulfur atom-containing group (particularly a thiol group) from the viewpoint of affinity with the working electrode. In particular, cysteine is preferred.

The applied voltage may be appropriately selected according to the kind of the easily oxidizable substance so that the easily oxidizable substance is oxidized, and is preferably in the range of +0.1 to + 0.6V. With an applied voltage in this range, ascorbic acid can be oxidized among easily oxidizable substances.

As mammals, those raised as livestock are preferred, and examples include dairy cows and goats.

Process (B)
In the step (B), a current generated when the easily oxidizable substance is oxidized by the applied voltage is measured.

By applying voltage in step (A), the easily oxidizable substance is oxidized, and an oxidation current flows between the working electrode and the counter electrode. Step (B) can be performed by measuring the value of the oxidation current flowing between the working electrode and the counter electrode.

In the present invention, the easily oxidizable substance is a substance that exists in raw milk and is easily oxidized electrochemically, and its concentration is correlated with mastitis. As the easily oxidizable substance, ascorbic acid is preferable.

The current value measured in step (B) has a correlation with the concentration of the easily oxidizable substance. Therefore, since there is a correlation between the mastitis and the concentration of the easily oxidizable substance, it can be determined from the magnitude of the current value whether the milk is milk of a mammal infected with mastitis. For example, when the easily oxidizable substance is ascorbic acid, when the current value is small, it can be determined that the milk is raw milk of a mammal infected with mastitis. Specifically, when ascorbic acid is electrochemically oxidized, the current value of raw milk in a healthy dairy cow is about 50 to 70 nA per square millimeter of electrode area, but the current of raw milk in a dairy cow infected with mastitis. The value is about 20 to 50 nA per square millimeter of electrode area.

The current value can be measured with a known ammeter such as a galvanometer, for example.

As described above, the raw milk inspection method of the present invention can be carried out by a simple operation. In the raw milk inspection method of the present invention, the oxidation current value of an easily oxidizable substance is measured. Since this oxidation current value is larger than the current value based on active oxygen, the raw milk can be inspected with high sensitivity. .

In the conventional method, mastitis infection was examined using active oxygen. However, the lifetime of active oxygen is short, the time that can be measured is limited, and it is necessary to perform measurement immediately after milking. However, oxidizable substances represented by ascorbic acid exist in raw milk with a longer life than active oxygen. Therefore, the method of the present invention also has an advantage that the inspection can be performed even after the time after milking has elapsed (usually, the method of the present invention can be performed for about 24 hours from immediately after milking).

Conventionally, it has been determined whether the milk of a mammal infected with mastitis is based on the number of somatic cells in the raw milk. Therefore, it is preferable to know the number of somatic cells in determining whether the milk is a mammal infected with mastitis. Therefore, it is preferable that the raw milk inspection method of the present invention further includes the following step (C) (Method B).

Process (C)
In step (C), the number of somatic cells in the raw milk is calculated from the measured current value. Since the concentration of the easily oxidizable substance is related to mastitis, the oxidation current value indicating the concentration of the easily oxidizable substance is also related to the number of somatic cells. For example, when the easily oxidizable substance is ascorbic acid, there is a negative correlation between the oxidation current value and the number of somatic cells. Therefore, a calibration curve can be created for raw milk by obtaining a somatic cell value by a conventional method and a current value by the method of the present invention. Step (C) can be performed by converting the number of somatic cells from the measured current value using this calibration curve.

For example, when examining raw milk of dairy cows, the number of somatic cells in raw milk of healthy dairy cows is 20,000 to 100,000 per mL, whereas the number of somatic cells in raw milk of dairy cows infected with mastitis is , 300,000 pieces / mL or more. Therefore, when the number of somatic cells converted from the measured current value is 300,000 cells / mL or more, it can be determined that the milk is milk of a dairy cow infected with mastitis.

Also, for example, a dairy cow has four breasts, but mastitis is infected with each breast independently, and all four breasts are infected with mastitis. Moreover, the amount of oxidizable substances in raw milk is slightly different for each cow, and there are individual differences. Therefore, if raw milk obtained from another breast of the same individual is used as a comparison target, the influence of individual differences in the amount of oxidizable substances can be eliminated, and a more accurate examination can be performed.

Therefore, in a preferred embodiment of the raw milk test method of the present invention, the steps (A) and (B) are further performed on raw milk obtained from another breast of the mammal (the same individual), And (D) comparing the current values obtained from the raw milk of each breast (Method C).

In the step (D), when the current values are compared and there is a large difference in the current values, it can be determined that the examined raw milk contains raw milk of the breast infected with mastitis. For example, when the easily oxidizable substance is ascorbic acid, it can be determined that raw milk having a small current value is raw milk of a breast infected with mastitis.

In another preferred embodiment of the method for examining raw milk of the present invention, the steps (A) and (B) are further performed on raw milk obtained from another breast of the mammal (the same individual), The method includes a step (E) of obtaining a difference between current values obtained from raw milk of each breast (Method D).

When there is a large difference in current values obtained in step (E), it can be determined that raw milk infected with mastitis is included in the examined raw milk.

Also, in the step (E), the magnitude of the difference in the current value obtained from the raw milk of each breast is related to the number of somatic cells. For example, when the easily oxidizable substance is ascorbic acid, there is a positive correlation between the difference in oxidation current value and the number of somatic cells. Therefore, a preferred embodiment of the method (D) further includes a step (F) of calculating the number of somatic cells in raw milk obtained from each breast from the difference in current values obtained in the step (E) ( Method E). A calibration curve can be created for raw milk by determining the difference between the somatic cell value by the conventional method and the current value by the method of the present invention. Step (F) can be performed by converting the number of somatic cells from the difference in the measured current values using this calibration curve. Method E may further include comparing the calculated number of somatic cells in the raw milk of each breast.

In another preferred embodiment of the method for examining raw milk of the present invention, the steps (A) to (C) are further performed on raw milk obtained from another breast of the mammal (the same individual), And (G) comparing the number of somatic cells in raw milk obtained from each breast (Method F).

In step (G), if the number of somatic cells is compared, and there is a large difference in the number of somatic cells, it can be determined that the examined raw milk contains raw milk from a breast infected with mastitis. At this time, it can be determined that raw milk having a large number of somatic cells is raw milk of a breast infected with mastitis.

In the present invention, it is useful to know the current value and / or the number of somatic cells of raw milk in a healthy state for each individual to be measured.

The present invention is an examination method of raw milk, but can also be applied as a mastitis diagnosis method.

Next, the raw milk inspection apparatus of the present invention will be described. The raw milk test apparatus of the present invention includes a working electrode including a hydrophilic compound exposed on the surface, at least one set of electrodes including a counter electrode, a circuit for applying a constant voltage between the electrodes, and easy oxidation in raw milk. Means for measuring a current generated when the sex substance is oxidized by the applied voltage (apparatus A). The raw milk test | inspection method of this invention mentioned above can be performed with the raw milk test | inspection apparatus of this invention.

A set of electrodes includes a working electrode and a counter electrode, but may further include a reference electrode. The electrodes may be in two or more sets to allow simultaneous measurement of two or more raw milks, particularly two or more raw milks obtained from different breasts of the same mammal (same individual). A conductive material such as gold, silver, silver chloride, platinum, copper, aluminum, and stainless steel can be used for the electrode.

The hydrophilic compound contained in the working electrode is as described above. From the viewpoint of sensitivity, the working electrode preferably contains the hydrophilic compound as a layer having a thickness of 20 mm or less. In order to form a hydrophilic compound layer on the surface of the working electrode, a coating solution containing the hydrophilic compound may be applied on the working electrode by a known method such as a dipping method, an ink jet method, or an injection method, and dried.

The circuit electrically connects the working electrode and the counter electrode. If an internal power supply or an external power supply is connected directly or indirectly to this circuit, a voltage can be applied. The circuit is preferably configured so that a voltage of about +0.1 to +0.6 V can be applied.

There is no particular limitation on the means for measuring the current generated when the oxidizable substance in raw milk is oxidized by the applied voltage, and it is preferably a means capable of measuring a current of about 0.01 to 10 μA, such as a galvanometer. Any known ammeter can be used.

The raw milk inspection apparatus of the present invention preferably has means for notifying the measurement result. Examples of the means include a display unit such as a monitor and a lamp, and a speaker.

Since it is preferable to know the number of somatic cells in raw milk, the raw milk test apparatus of the present invention calculates the number of somatic cells in the raw milk from the current value measured at the applied voltage at which the oxidizable substance is oxidized. It is preferable to further include an arithmetic unit for calculation (apparatus B). With the apparatus B, the above method B can be carried out. The calculation unit includes, for example, a CPU and a storage unit such as a memory and a hard disk. A calibration curve indicating the correlation between the number of somatic cells and the current value is input to the storage unit, and the number of somatic cells is calculated by the CPU.

The raw milk inspection apparatus of the present invention may have a calculation unit for determining whether the milk is a mammal infected with mastitis from the current value or the number of somatic cells. For example, the calculation unit includes a CPU, a storage unit, and the like, and a correlation between the current value or the number of somatic cells and mastitis infection is input to the storage unit. This calculation unit may be a calculation unit that calculates the number of somatic cells.

As mentioned above, if raw milk obtained from different breasts of the same mammal (same individual) is used as a comparison target, the effects of individual differences in the amount of oxidizable substances can be eliminated, and more accurate testing should be performed. Can do. Therefore, a preferred embodiment of the raw milk test apparatus according to the present invention includes a storage unit for storing a measured current value in addition to the electrode, the circuit, and the current measuring unit, and a current value obtained by newly measuring. And a data processing unit for comparing the stored current values (device C). According to the apparatus of this embodiment, the method C can be performed by sequentially measuring raw milk obtained from another breast of the same individual.

Another preferable embodiment of the raw milk test apparatus of the present invention is the storage unit for storing the measured current value in addition to the electrode, the circuit, and the current measuring unit, and the current value obtained by newly measuring. Is further included (apparatus D). According to the apparatus of this embodiment, the method D can be performed by sequentially measuring raw milk obtained from another breast of the same individual. The calculation unit can be constituted by a CPU or the like, for example.

The device D may further include a calculation unit that calculates the number of somatic cells in raw milk from the difference between the newly obtained current value obtained by measurement and the stored current value (device E). In this case, the method E can be performed by sequentially measuring raw milk obtained from another breast of the same individual. The calculation unit includes, for example, a CPU and a storage unit such as a memory and a hard disk. A calibration curve indicating a correlation between the number of somatic cells and the difference between the current values is input to the storage unit, and the number of somatic cells is calculated by the CPU. As the CPU, a CPU of a calculation unit that obtains the difference between the current values may be used. The apparatus E may further include a data processing unit that compares the calculated number of somatic cells in the raw milk of each breast.

Another preferred embodiment of the raw milk test apparatus of the present invention is a memory for storing the calculated number of somatic cells, in addition to the electrode, the circuit, the current measuring means, and the calculation unit for calculating the number of somatic cells. And a data processing unit for comparing the number of somatic cells newly obtained by measurement and the stored number of somatic cells (apparatus F). According to the apparatus of this embodiment, the method F can be performed by sequentially measuring raw milk obtained from another breast of the same individual.

Another preferable embodiment of the raw milk inspection apparatus of the present invention is an apparatus including the electrode, the circuit, and the current measuring means, and the apparatus includes two or more sets of the electrodes, and each set It further includes a data processing unit that compares the current values measured with respect to the electrodes (device G). According to the apparatus of this embodiment, the method C can be performed in one measurement by using each set of electrodes for each raw milk obtained from another breast of the same individual.

Another preferable embodiment of the raw milk inspection apparatus of the present invention is an apparatus including the electrode, the circuit, and the current measuring means, and the apparatus includes two or more sets of the electrodes, and each set It further includes a calculation unit for obtaining a difference between the current values measured with respect to the electrodes (apparatus H). According to the apparatus of this embodiment, the above method D can be carried out in one measurement by using each set of electrodes for each raw milk obtained from another breast of the same individual. The calculation unit can be constituted by a CPU or the like, for example.

The apparatus H may further include a calculation unit that calculates the number of somatic cells in raw milk from the difference between the obtained current values (apparatus I). At this time, the method E can be carried out in one measurement by using each set of electrodes for each raw milk obtained from another breast of the same individual. The calculation unit includes, for example, a CPU and a storage unit such as a memory and a hard disk. A calibration curve indicating a correlation between the number of somatic cells and the difference between the current values is input to the storage unit, and the number of somatic cells is calculated by the CPU. As the CPU, a CPU of a calculation unit that obtains the difference between the current values may be used.

Another preferable embodiment of the raw milk test apparatus of the present invention is an apparatus including the electrode, the circuit, the current measuring means, and a calculation unit for calculating the number of somatic cells, wherein the apparatus includes the A data processing unit that includes two or more sets of electrodes and compares the number of somatic cells calculated for each set of electrodes is further included (apparatus J). According to the apparatus of this embodiment, the method F can be performed in one measurement by using each set of electrodes for each raw milk obtained from another breast of the same individual.

The storage unit that stores the measured current value and the storage unit that stores the calculated number of somatic cells can be configured by a memory, a hard disk, or the like. When the calculation unit that calculates the number of somatic cells has a storage unit that stores a calibration curve, the storage unit that stores the calibration curve of the calculation unit may be used as these storage units, or separately A storage unit may be provided. The data processing unit can be configured by a CPU or the like, and when the raw milk inspection apparatus has a calculation unit that calculates the number of somatic cells in raw milk, the CPU or the like of the calculation unit may be used as the data processing unit. A separate data processing unit may be provided. Therefore, the calculation unit that calculates the number of somatic cells may function as the storage unit and the data processing unit, or the storage unit and the data processing unit may be provided separately.

The raw milk inspection apparatus of the present invention can be reduced in size and cost, and can easily and rapidly inspect raw milk with high sensitivity.

The present invention is a raw milk inspection apparatus, but can also be applied as a mastitis diagnosis apparatus.

Hereinafter, specific examples of the raw milk inspection apparatus of the present invention will be shown.

Fig. 1 shows a specific example of a raw milk inspection device. The raw milk inspection apparatus 1 </ b> A includes a sensor 2, a connector 5, and a main body 6, and the sensor 2 is detachable from the main body 6 at the connector 5. As will be described later, the sensor 2 includes electrodes, and by making the sensor 2 detachable, the measurement can be easily repeated simply by replacing the sensor 2. This configuration corresponds to the devices A to F described above.

Next, the configuration of the sensor 2 will be described in detail with reference to FIGS. The sensor 2 is of a substantially rectangular plate type having an examination room 20 for holding raw milk, and the plate type sensor can easily optimize the electrode arrangement and the like. Raw milk is introduced into the examination room 20 by capillary action. The volume of the examination room 20 is preferably 0.01 to 5 mL, and more preferably 0.05 to 1 mL. A more preferable volume of the examination chamber 20 is 0.1 to 0.3 mL.

Specifically, the sensor 2 includes a substantially rectangular base plate 21 and three working electrodes 31, a reference electrode 32, and a counter electrode 33 supported by one surface (one surface in the thickness direction) 21 a of the base plate 21. An electrode and a substantially rectangular cover plate 22 fixed to one surface 21a of the base plate 21 with the electrodes 31 to 33 interposed therebetween are provided. In the following description, for convenience of explanation, one of the longitudinal directions of the base plate 21 (upper right direction in FIG. 2) is referred to as the front, and the other (lower left direction in FIG. 2) is referred to as the rear. (Lower right direction in FIG. 2) is called the right side, and the other (upper left direction in FIG. 2) is called the left side.

The width of the cover plate 22 is the same as the width of the base plate 21, but the length of the cover plate 22 is set shorter than the length of the base plate 21 so as to expose the rear end portion of the one surface 21 a of the base plate 21. Yes. The size of the base plate 21 is, for example, 60 mm long, 30 mm wide, and 1 mm thick. The size of the cover plate 22 is, for example, 50 mm long, 30 mm wide, and 3 mm thick.

The material of the base plate 21 and the cover plate 22 is not particularly limited as long as it is insulative. For example, acrylic resin, polylactic acid resin, polyglycolic acid resin, styrene resin, methacryl-styrene Copolymer resin (MS resin), polycarbonate resin, polyester resin such as polyethylene terephthalate, polyvinyl alcohol resin, ethylene-vinyl alcohol copolymer resin, thermoplastic elastomer such as styrene elastomer, vinyl chloride resin, poly Examples thereof include silicone resins such as dimethylsiloxane, vinyl acetate resins, and polyvinyl butyral resins. The material of the base plate 21 is preferably selected as appropriate in consideration of adhesion with an electrode material described later, and the material of the cover plate 22 is preferably selected as appropriate in consideration of adhesion with the base plate 21. .

In the cover plate 22, a groove 23 is formed in the end surface of the cover plate 22 along the base plate 21, and the inspection chamber 20 is configured by the groove 23 and one surface 21 a of the base plate 21. The examination room 20 is automatically filled with raw milk from the opening 23a by capillary action. That is, the opening 23 a of the groove 23 constitutes an introduction port to the examination room 20. In order to utilize capillary action, the width of the groove 23 is preferably 1 to 50 mm, the depth is 0.05 to 5 mm, and the length is preferably 2 to 100 mm, more preferably the width of the groove 23 is 3 to 30 mm. The depth is 0.1 to 3 mm, and the length is 10 to 70 mm. For example, the width of the groove 23 may be 10 mm, the depth may be 0.5 mm, and the length may be 40 mm.

The groove 23 can be formed by machining, injection molding, or the like, and the base plate 21 and the cover plate 22 can be joined by heat fusion, laser fusion, solution adhesion technique, or the like. By applying a method established as such an industrial production technique, the accuracy of the volume of the examination room 20, that is, the accuracy of the amount of raw milk introduced into the sensor 2 can be increased. Thereby, if it is the same raw milk, the amount of easily oxidizable substances in the examination room 20 can be made constant, and the measurement accuracy can be increased.

In this specific example, since the capillary phenomenon is used, it is possible to fill the examination room 20 with the raw milk in a short time, for example, within 5 seconds, simply by immersing the end of the sensor 2 on the opening 23a side in the raw milk. . In addition, the sensor 2 does not necessarily need to utilize a capillary phenomenon, and an examination room may be provided by a through hole that penetrates the cover plate in the thickness direction. At this time, the sensor 2 corresponds to the spotting method.

The working electrode 31, the reference electrode 32, and the counter electrode 33 are arranged in the left-right direction, and each extends linearly in the front-rear direction from the rear end of the base plate 21 to a predetermined position. In this specific example, a circular first electrode part (positive electrode part) 31a is provided at the tip of the working electrode 31, and a circular second electrode part (negative electrode part) larger than the first electrode part 31a is provided at the tip of the counter electrode 33. ) 33a is provided. Also, the tip 32a of the reference electrode 32 has a small circular shape. The first electrode portion 31 a and the second electrode portion 33 a and the tip 32 a of the reference electrode 32 all face the internal space of the examination room 20. Further, portions of the electrodes 31 to 33 that are not covered with the cover plate 22 constitute wide terminal portions 31b to 33b, and these terminal portions 31b to 33b are inserted into the connector 5 with the sensor 2. When attached, it is electrically connected to a terminal (not shown) of the connector 5.

On the surface 31c of the first electrode part 31a, the hydrophilic compound 4 (eg, cysteine) is immobilized. This immobilization can be performed, for example, by immersing the electrode in a solution of the hydrophilic compound 4. The reference electrode 32 is used as a reference electrode. A voltage of, for example, 1 V DC is applied uniformly between the working electrode 31 and the counter electrode 33 and between the reference electrode 32 and the counter electrode 33. In addition, when an additional voltage of, for example, 0.3 V is applied to the working electrode 31 in a superimposed manner, the oxidizable substance is oxidized on the first electrode portion 31 a to cause hydrolysis, and the working electrode 31 and the counter electrode 33 are oxidized. Current flows between them. By measuring the amount of current flowing between the working electrode 31 and the counter electrode 33, it becomes possible to measure the amount of easily oxidizable substance in raw milk and consequently the number of somatic cells in raw milk. In order to efficiently detect the oxidation current, the area of the second electrode portion 33a is preferably larger than the area of the first electrode portion 31a. Here, “the area of the second electrode portion 33a” and “the area of the first electrode portion 31a” are the areas when the second electrode portion 33a and the first electrode portion 31a are viewed from a direction orthogonal to the base plate 21. Say. The area of the first electrode portion 31a is preferably 0.7 to 500 mm ² , and more preferably 4 to 100 mm ² .

The electrodes 31 to 33 can be formed by vapor deposition, sputtering, electrolytic plating, electroless plating, silk screen printing, metal paste injection, or the like. By adopting such an industrially established method, the electrodes 31 to 33 can be formed with high accuracy, and the reproducibility of measured values can be improved in mass production. Examples of the conductive material used for the electrodes include gold, silver, silver chloride, platinum, copper, aluminum, and stainless steel. In this specific example, in order to detect ascorbic acid as an easily oxidizable substance, the working electrode 31 is made of gold, the reference electrode 32 is made of silver / silver chloride, and the counter electrode 33 is made of platinum.

Preferably, the width of each electrode 31 to 33 is 0.5 to 20 mm, the length is 1 to 100 mm, and the thickness is 0.003 to 300 μm. More preferably, the width of each electrode 31 to 33 is 1 to 10 mm, The length is 2 to 20 mm, and the thickness is 0.02 to 200 μm.

In order to increase the surface area of the electrode, for example, an uneven structure or the like may be provided in the electrode portion.

Next, the configuration of the apparatus body 6 will be described in detail with reference to FIG. The apparatus body 6 calculates the number of somatic cells contained in the raw milk based on the current flowing between the working electrode 31 and the counter electrode 33. Specifically, the apparatus main body 6 is configured such that the current flowing between the working electrode 31 and the counter electrode 33 when a voltage is applied between the working electrode 31 and the reference electrode 32 via the power supply 65 and the voltage application circuit 62. And a calculation unit 64 that calculates the number of somatic cells from the current value. The number of somatic cells calculated by the calculation unit 64 is displayed on the display unit 61.

The power source 65 may be an internal power source such as a battery or a battery, or may be an external power source such as a household power source. As the ammeter 63, a galvanometer or the like can be used. The calculation unit 64 includes a CPU, a storage unit (RAM), and the like. The storage unit stores a calibration curve that associates the current value with the number of somatic cells, and can also store the measured current value and the calculated number of somatic cells. The computing unit 64 calculates the number of somatic cells according to the current value sent from the ammeter 63. Further, the calculation unit 64 can compare the current value and the number of somatic cells obtained by the new measurement with the stored current value and the number of somatic cells. Alternatively, the calculation unit 64 may be configured to calculate the difference between the current value newly measured and the stored current value, and the storage unit stores the current value difference and the number of somatic cells. It is also possible to store a calibration curve that relates to the number of somatic cells and calculate the somatic cell count by the calculation unit 64. When a plurality of types of sensors 2 having different sensitivities are used, a calibration curve may be stored in the storage unit for each sensor 2.

Next, a method for measuring the number of somatic cells using the raw milk test apparatus 1A will be described. The sensor 2 is attached to each connector 5 arranged in the sampling container for raw milk. Next, the on button 6a is pressed to turn on the power switch. When milking is started, the measurement button 6c is pushed to make it measurable. When raw milk is filled in the sampling container, raw milk is filled into the examination room 20 from the opening 23a of the sensor 2 by capillary action. If it does so, the easily oxidizable substance in the raw milk hold | maintained at the test | inspection room 20 will react, the oxidation current will be sent to the apparatus main body 6, and the number of somatic cells will be calculated. The calculated number of somatic cells is displayed on the display unit 61. A configuration in which the oxidation current value is displayed instead of the number of somatic cells is also possible. It is also possible to input a reference somatic cell number into the apparatus main body 6 in advance, and when the calculated somatic cell number exceeds the reference somatic cell number, the lamp can be turned on or notified by voice.

When milking and the measurement of the number of somatic cells are completed, the milker and the raw milk sampling container are washed, and the sensor 2 used for the measurement is replaced with a new sensor 2 in preparation for the next measurement.

If the measurement result of the somatic cell count is stored in the apparatus main body 6, the measurement is performed by the new sensor 2, and the measurement result of the newly obtained somatic cell count is compared with the measurement result of the previous somatic cell count. Can do. When it is desired to end the measurement, the off button 6b is pressed, and thereby the power is turned off.

The raw milk inspection apparatus 1A of this specific example is suitable for batch measurement, and the examiner etc. quickly uses the raw milk sampled by the dairy farmers, the raw milk collected and delivered to the certification association, etc., the raw milk sampled in units of tanks, etc. Effective when measuring. The raw milk inspection apparatus 1A can have outer dimensions of, for example, a height of 120 mm, a width of 80 mm, and a weight of about 300 g so as to be excellent in portability.

FIG. 6 shows another specific example of the raw milk inspection apparatus of the present invention. This raw milk inspection apparatus 1B is composed of four sensors 2 corresponding to the number of dairy cow quarters and an apparatus main body 6 connected to these sensors 2 by a cable 71. The apparatus main body 6 is provided with four display sections 61 corresponding to each sensor 2 and various push buttons 6a to 6c.

This configuration corresponds to the above-described apparatuses G to J. According to this configuration, the raw current obtained from four breasts of the same individual is simultaneously measured for the oxidation current value of an easily oxidizable substance, and the number of somatic cells is determined. Can be found and compared. Since raw milk obtained from four breasts of the same individual can be compared simultaneously, in this configuration, it is not necessary to provide a storage unit that stores the measured current value and a storage unit that stores the calculated number of somatic cells.

Hereinafter, the present invention will be described in detail with reference to examples and comparative examples, but the present invention is not limited to these examples.

[Manufacture of sensor plate for raw milk inspection equipment]
[Production Example 1]
A base plate having a length of 60 mm, a width of 30 mm, and a thickness of 1 mm was formed by injection molding using polyethylene terephthalate resin (Kuraray KS710B-8S). Next, the base plate is masked, and the working electrode (gold), the reference electrode (silver / silver chloride), and the counter electrode (platinum) are patterned as shown in FIG. Formed. Specifically, the diameter of the first electrode part is 6 mm, and the diameter of the second electrode part is 8 mm. Next, the base plate on which the electrode part was formed was immersed in a 50 mM cysteine aqueous solution for 3 hours to form a cysteine self-assembled film on the surface of the first electrode part. Next, using polyethylene terephthalate resin (Kuraray, Kurapet KS710B-8S), it has a length of 40mm, width of 10mm, and depth of 0.5mm by injection molding method, length 50mm, width 30mm, thickness 3mm A cover plate was formed. Next, a base plate and a cover plate were welded using a laser resin welding machine (Miyachi Technos, model: ML-5220B) to obtain a sensor plate.

[Production Example 2]
In Production Example 1, a sensor plate was obtained in the same manner as in Production Example 1 except that a sensor plate whose surface was not modified with a compound was used.

[Production Example 3]
A sensor plate was obtained in the same manner as in Production Example 1 except that the surface of the first electrode part in Production Example 1 was modified with 1-propanethiol, which does not correspond to a hydrophilic compound, instead of cysteine.

[Example 1]
[Current measurement of raw milk sample]
The sensor plate obtained in Production Example 1 was connected to a main body having a power source, a voltage application circuit, and a microammeter. Twenty kinds of raw milk samples were dropped on the sensor plate obtained in Production Example 1, and a current flowing when a voltage of +0.3 V (vs. silver / silver chloride reference electrode) was applied to the first electrode part was measured. Further, the concentration of ascorbic acid contained in the same raw milk sample was quantified by high performance liquid chromatography. The relationship between the current value obtained from each raw milk sample and the ascorbic acid concentration is shown in FIG. From the result of FIG. 7, it can be seen that the higher the ascorbic acid concentration contained in the raw milk, the larger the current value obtained. This is because ascorbic acid in raw milk is oxidized near the surface of the first electrode portion by the applied voltage.

[Measurement of somatic cell count of raw milk sample]
In addition, the number of somatic cells contained in the same raw milk sample was measured by a direct microscopic method using a Burker-Turk type hemocytometer. The relationship between the current value obtained from each raw milk sample and the number of somatic cells is shown in FIG. From the results of FIG. 8, it can be seen that the larger the number of somatic cells contained in the raw milk sample, the lower the current value obtained. This is because ascorbic acid contained in raw milk is oxidized by the increase of somatic cells in raw milk, and as a result, the concentration of ascorbic acid in raw milk decreases.

As described above, if the result of FIG. 7 or FIG. 8 is used as a calibration curve, the content of ascorbic acid or the number of somatic cells in the raw milk sample can be indirectly quantified from the amount of current, and based on these results. Thus, mastitis can be diagnosed.

[Example 2]
[Comparison by quarter]
Raw milk samples were collected for each quarter from two dairy cows (dairy cow A and dairy cow B) suspected of having mastitis infection, and the current value of each sample was measured using the apparatus of Example 1. The number of somatic cells contained in the same sample was measured by the same direct microscopic method as in Example 1. The measurement results of the current value and the number of somatic cells are shown in Table 1. In dairy cow A, the rear left quarter milk and in dairy cow B the left front quarter milk show significantly higher somatic cell counts, and mastitis infection is suspected. At this time, it can be seen that the current value of the raw milk in each abnormal quarter shows a low value exceeding the range of variation between the normal quarters. In this way, abnormal quarters that are suspected of infection can be detected more accurately by comparing each quarter.

[Comparative Example 1]
Table 2 shows the results obtained by selecting four types of the 20 types of raw milk used in Example 1 and measuring the current values of these four types of raw milk using the sensor plate obtained in Production Example 2. When an unmodified working electrode was used, only a small current flowed regardless of the ascorbic acid concentration, and an accurate test could not be performed.

[Comparative Example 2]
Table 2 shows the results of measuring current values for four types of raw milk using the sensor plate obtained in Production Example 3. Also in this case, since the current value was low, an accurate inspection could not be performed. This is presumed that when the electrode was modified with hydrophobic 1-propanethiol, the ionic ascorbic acid did not easily approach the electrode in the raw milk, so the amount of current did not increase.

The present invention is used for examining whether or not a mammal infected with mastitis is raw milk, and can also be used for diagnosis of mastitis infection in mammals.

Claims

A step of applying a voltage to the raw milk of the mammal (A), and a step of measuring a current generated when the easily oxidizable substance is oxidized by the applied voltage (B).
Raw milk inspection method.
The method for examining raw milk according to claim 1, further comprising a step (C) of calculating the number of somatic cells in the raw milk from the measured current value.
The method further comprises the step (D) of further performing the steps (A) and (B) on raw milk obtained from another breast of the mammal, and further comparing a current value obtained from the raw milk of each breast. 2. The method for examining raw milk according to 1.
Further comprising the step (E) of further performing the steps (A) and (B) on raw milk obtained from another breast of the mammal, and further obtaining a difference in current values obtained from the raw milk of each breast. Item 2. The method for inspecting raw milk according to Item 1.
The method for inspecting raw milk according to claim 4, further comprising a step (F) of calculating the number of somatic cells in the raw milk obtained from each breast from the difference between the current values obtained in the step (E).
Further comprising the step (G) of further performing the steps (A) to (C) on raw milk obtained from another breast of the mammal and comparing the number of somatic cells in the raw milk obtained from each breast The method for inspecting raw milk according to claim 2.
The method for inspecting raw milk according to claim 1, wherein the oxidizable substance is ascorbic acid.
A working electrode including a hydrophilic compound exposed on the surface, and at least one set of electrodes including a counter electrode;
A circuit for applying a constant voltage between the electrodes;
And a means for measuring a current generated when an easily oxidizable substance in raw milk is oxidized by an applied voltage.
The raw milk test apparatus according to claim 8, further comprising a calculation unit that calculates the number of somatic cells in the raw milk from a current value measured at an applied voltage at which the easily oxidizable substance is oxidized.
The raw milk test apparatus according to claim 8, further comprising: a storage unit that stores the measured current value; and a data processing unit that compares the current value newly obtained by measurement with the stored current value.
The raw milk test apparatus according to claim 8, further comprising: a storage unit that stores the measured current value; and a calculation unit that obtains a difference between the current value newly measured and the stored current value.
The raw milk test apparatus according to claim 11, further comprising a calculation unit that calculates the number of somatic cells in raw milk from the difference between the obtained current value obtained by measurement and the stored current value.
The raw milk test | inspection apparatus of Claim 9 which further contains the memory | storage part which memorize | stores the calculated number of somatic cells, and the data processing part which compares the number of somatic cells newly obtained by measuring with the somatic cell number memorize | stored.
The raw milk test | inspection apparatus of Claim 8 which further contains the data processing part which contains the said electrode 2 or more and compares the electric current value measured with respect to each set of electrodes.
The raw milk test | inspection apparatus of Claim 8 which further contains the calculating part which calculates the difference of the electric current value measured with respect to each set of electrodes including two or more sets of said electrodes.
The raw milk test apparatus according to claim 15, further comprising a calculation unit that calculates the number of somatic cells in raw milk from the difference between the obtained current values.
The raw milk test | inspection apparatus of Claim 9 which further contains the data processing part which contains two or more sets of said electrodes, and compares the somatic cell count calculated with respect to each set of electrodes.
The raw milk inspection apparatus according to claim 8, wherein the hydrophilic compound is a compound having a cationic group.
The raw milk inspection device according to claim 8, wherein the easily oxidizable substance is ascorbic acid.