JP2010256231A - Milk inspection method, milk inspection device, and mastitis diagnosis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a milk inspection method capable of discovering at low cost and simply the mastitis at an earlier stage. <P>SOLUTION: This milk inspection method includes a first process for applying processing for generating an activation factor from a processing portion to the processing portion which is a part of milk; a second process for activating somatic cells existing in mixed liquid of the processing portion, to which the processing is applied and a non-processing portion which is a residual part of the milk by the activation factor included in the processing portion; and a third process for measuring the number of somatic cells in the milk by using the mixed liquid after the second process. Consequently, somatic cells can be activated, without having to use expensive activation drug requiring complex storage. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for inspecting milk of a mammal, particularly a dairy cow, and a milk inspection apparatus for performing this inspection. Furthermore, the present invention relates to a method for diagnosing a mammal's mastitis infection using the milk test method.

  In dairy farming, livestock mastitis is a major issue. Milk of livestock infected with mastitis has a reduced commercial value due to a decrease in lactose rate, 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 index 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 increase in the number of somatic cells is caused by the invasion and proliferation of bacteria into the breast, the transfer of white blood cells from blood to milk, the increase of white blood cells in milk, and the damage caused by toxins produced by the bacteria. This means that mammary epithelial cells have fallen into the milk from the mammary tissue.

  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. Furthermore, the milk is discarded in units of tanks. In addition, since the milk of livestock with low infectivity is diluted with normal milk, early detection of mastitis is difficult. Therefore, a simple, inexpensive and portable small device that can measure the number of somatic cells during milking is desired.

  Thus, a method has been proposed in which active oxygen produced by somatic cells in milk infected with mastitis is detected by luminol chemiluminescence (see Patent Document 1). This is a method in which a luminol reagent is excited by active oxygen, and the amount of chemiluminescence generated is amplified and quantified with a photomultiplier tube.

  There has also been proposed a method of electrochemically detecting active oxygen by reacting an active oxygen and an electrode that electrochemically reacts. For example, Patent Document 2 discloses a mastitis diagnostic apparatus using a working electrode and a counter electrode in which a polymer film of a metal porphyrin complex is formed on the surface of a conductive member.

  These methods for detecting active oxygen can be easily inspected with a smaller apparatus than conventional methods using a microscope or cell sorter.

Japanese Patent Laid-Open No. 2000-041696 JP 2005-106490 A

  However, in the method for detecting active oxygen, sufficient sensitivity cannot be obtained at an early stage of mastitis having a low number of somatic cells, and early detection of mastitis is difficult. Therefore, it is conceivable to increase the sensitivity by activating somatic cells with an activating drug such as opsonized zymosan or calcium ionophore, thereby increasing the amount of active oxygen produced by somatic cells.

  However, since the activated drug as described above is expensive and unstable, not only the cost is increased, but also the storage of the activated drug itself (usually storing in the freezer) and the work itself are increased.

  In view of the above problems, the present invention provides a milk inspection method capable of easily and inexpensively detecting mastitis at an early stage, a milk inspection apparatus for performing the milk inspection method, and a breast using the milk inspection method. An object is to provide a flame diagnosis method.

  In order to achieve the above object, the present invention provides a method for inspecting milk, wherein a first step of performing a process of generating an activator from a processed part of the milk, which is a part of the milk, and the process A second step of activating somatic cells present in the mixed solution of the treated portion subjected to the treatment and the non-treated portion which is the remainder of the milk, with the activator contained in the treated portion; and the second step. And a third step of measuring the number of somatic cells in the milk using the later mixed solution.

  Further, the present invention is a milk inspection apparatus for inspecting milk, and includes a first container into which a processing portion that is a part of the milk is input, and the processing portion that is input into the first container. Activating means for performing a process for generating an activating factor from the processed part, a second container into which the non-processed part that is the remainder of the milk and the processed part subjected to the process are charged, and the second container And a measuring means for measuring the number of somatic cells in the milk using a mixed liquid of the untreated portion and the treated portion.

  Furthermore, the present invention records the number of somatic cells in milk obtained by the above milk test method, and diagnoses infection of mastitis in the individual from which the milk has been extracted from the transition of the number of somatic cells. Provide a method of diagnosing flame

  According to the milk test method of the present invention, an activation factor is generated using a part of milk to be tested, so that somatic cells can be activated without using an expensive and cumbersome activation drug. can do. And by activating somatic cells in this way, the number of somatic cells can be measured with high sensitivity even if the concentration of somatic cells is low. Therefore, the early stage mastitis can be found inexpensively and easily.

  Moreover, the milk inspection apparatus of the present invention that executes the milk inspection method can be miniaturized. Therefore, this milk inspection apparatus can be brought into a milking place and milk can be inspected simultaneously with milking.

It is a perspective view of the milk test | inspection apparatus which concerns on one Embodiment of this invention. It is a figure which shows the bottom face of a 2nd container. It is a block diagram of the milk test | inspection apparatus shown in FIG. It is a graph (scatter diagram) showing the relationship between the number of somatic cells and the current value.

<Milk testing method>
In the milk test method of the present invention, the treatment for generating the activator in the first step may be performed by applying various energy to a part of the milk. Such energy applying means can be provided at a position where it does not come into contact with milk. In this case, since the activator can be generated in a state where the milk is blocked from the outside (for example, in a sealed state), it is possible to greatly suppress the risk of contamination due to contamination of milk.

  Also, if the first step is performed inside the container and the means for giving energy is provided outside the container, the first step can be performed on a plurality of samples by exchanging the container, which is advantageous for simplicity and cost reduction. .

  The energy is not particularly limited, and examples thereof include heat, electromagnetic waves, pressure, vibration, ultrasonic waves, and shear stress. These energies can be generated by various general means, and are more advantageous for miniaturization, energy amount control, manufacturing cost reduction, and the like. Of these energies, heat and electromagnetic waves are particularly desirable because the maintenance of the apparatus is easy.

  The milk inspection method of the present invention is characterized in that a certain amount of milk to be inspected is once divided into a part (processed part) and a remaining part (non-processed part) and then mixed. The partial amount is preferably 0.1% or more and 50% or less, and more preferably 1% or more and 10% or less of the total amount of milk (the constant amount).

  Specifically, the milk inspection method of the present invention includes a first step of performing a process for generating an activation factor from a processed part that is a part of milk, and the processed part for the remaining part of the milk. The second step of mixing somatic cells in the mixed solution and activating the somatic cells in the mixed solution by the activator contained in the treated component, and using the mixed solution after the second step in the milk A third step of measuring the number of somatic cells.

  The number of somatic cells in milk measured in the third step indicates the total number of mammary epithelial cells and white blood cells or the number of white blood cells contained in the milk. At present, the number of somatic cells used for milk quality examination is the total number of mammary epithelial cells and white blood cells. Usually, it is said that the number of somatic cells is about 1 to 200,000 / mL at the time of non-infection. The number of somatic cells increases at the time of infection, and 20 to 300,000 cells / mL or more is regarded as a standard for infection. It is said that most of the somatic cells that increase during mastitis infection are leukocytes, especially neutrophils. Therefore, since it is considered that early detection of a sign of an increase in white blood cell count is important in reducing damage due to mastitis, it is preferable to measure the white blood cell count.

  The milk to be examined in the present invention is raw milk secreted from a mammal's breast. In the first step, an activator is generated from a processed portion that is a part of milk. The source of the activator existing in the processed portion includes fat, protein, vitamins, minerals originally contained in raw milk, In addition to components such as carbohydrates, somatic cells that vary depending on the physical condition of mammals are also included.

  In the first step, the treatment can be concentrated while generating the activator. For example, when the activation factor is generated by heat, the water can be removed as water vapor, and when the activation factor is generated by pressure, the water can be removed as a separated supernatant.

  The activation of the somatic cells present in the mixed solution performed in the second step means increasing the amount of the product from the somatic cells or increasing the adhesion of the somatic cells. Types of somatic cells to be activated include mammary epithelial cells, neutrophils, monocytes, lymphocytes, and macrophages, which are said to be somatic milk cells. As an example of these activations, for example, neutrophils, which are said to occupy most of the somatic cells in the milk at the time of infection, increase the production amount of active oxygen or increase the adhesiveness to the product detection site ( For example, it can be accumulated in a working electrode described later. This leads to improved measurement sensitivity at the stage of mild mastitis.

  Next, the activator generated in the first step will be described. The activator is a substance that induces the expression of the somatic activation effect described above, and includes secretions from somatic cells, cell membrane components, and degradation products of milk components. Examples of such activators include cytokines, antibacterial proteins, fatty acids and the like. In addition, cytokine, antibacterial protein, and fatty acid may be produced | generated simultaneously by the process given to a process part by a 1st process.

  Cytokines are proteins secreted from cells that transmit information to specific cells. For example, there are interleukin, chemokine, cytotoxic factor, tumor necrosis factor, and more specifically, IL-1β, IL-8, TNF-α and the like can be mentioned.

  The antibacterial protein is a protein having a bacteriostatic action for suppressing the growth of bacteria or a bactericidal action for damaging the cell membrane and metabolic system of the bacteria, and examples thereof include lactoferrin and lysozyme.

  As said fatty acid, what shows the leukocyte activation effect is preferable. As such fatty acid, C10 or higher saturated or unsaturated fatty acid is preferable, C18 or higher saturated or unsaturated fatty acid is more preferable, and C18 or higher unsaturated fatty acid is more preferable.

  Next, in the first step, a method for generating a activating factor from a processed portion that is a part of milk will be described in detail.

  In the case of producing at least one of cytokine and antibacterial protein as an activator, it stimulates somatic cells existing in the treated portion to cause cell death, and decomposes somatic cells to produce fragments and intracellular A method of releasing the activator contained in the above may be used.

  In order to give the stimulation as described above to somatic cells, for example, energy selected from heat, electromagnetic waves, pressure, vibration, ultrasonic waves, and shear stress may be given to the treatment. Alternatively, a bead contact impact, a freeze-thaw method, a surfactant, a homogenizer, or a method of applying a pulsed electric field can be used.

  The method of applying energy can provide energy with low cost and high accuracy using an element established in industrial technology, so that the manufacturing cost of the apparatus can be reduced.

  When a fatty acid is produced as an activator, a method may be used in which fat is liberated from fat globules existing in the treated portion and this fat is hydrolyzed.

  The fat in milk is a spherical micelle of 0.1 to 10 μm in fat with a structure in which a C4 to C18 saturated or unsaturated fatty acid and triglyceride are combined, and the periphery is covered with a membrane composed mainly of protein. It exists as a broken fat globule. For this reason, the action of lipase, which is usually a lipolytic enzyme, is hardly affected and exists stably. Fat globules are weakened in structure by stimulation such as heating and stirring, and are easily affected by lipase.

  By applying energy from the outside to the treated portion, the structure of fat globules is weakened, and fat can be decomposed into glycerol and fatty acids by the action of lipase contained in milk. The liberated fatty acid acts on the cell membrane of neutrophils, which are one of the somatic cells in the milk, in the third step, and can increase the amount of active oxygen produced from neutrophils. A sufficient amount of fat globules and lipase are present in milk compared to the number of somatic cells. The composition ratio of fat in milk is about 90 mol% at C10 or higher, and further 48 mol% at C18 or higher, so that it is sufficient to activate neutrophils simply by decomposing fat globules. Fatty acids can be obtained.

  The energy for weakening the structure of the fat globules is preferably strong enough not to deactivate the lipase. As the energy, energy selected from heat, electromagnetic waves, pressure, vibration, ultrasonic waves, and shear stress can be used.

  Next, the suitable range of the energy given to a process part at a 1st process is demonstrated. In addition, the range shown below is the same even when producing | generating at least one of a cytokine and an antimicrobial protein, and when producing | generating a fatty acid.

  When heat is used as the energy, it is preferable to heat the treatment to 40 to 80 ° C., and to 42 to 60 ° C. from the viewpoints of temperature controllability, simplicity of the apparatus, and thermal stability of the activator. More preferably.

  When electromagnetic waves are used as the energy, it is preferable to irradiate the treated portion with ultraviolet rays having a wavelength of 10 to 400 nm that acts on proteins constituting cell nuclei and fat globules. A more preferable wavelength of ultraviolet rays is 200 to 360 nm.

  As the energy, microwaves, infrared rays, and visible rays, which are electromagnetic waves having an effect of enhancing the molecular motion of water and destroying the structure of cell membranes and fat globules, can also be used.

  Usually, there is no particular limitation, but when using a microwave, a microwave with a frequency of 1 to 10 GHz is preferable, and a microwave with a frequency of 2 to 4 GHz is more preferable. When using visible light, visible light having a wavelength of 380 to 750 nm is preferable, and visible light having a wavelength of 430 to 600 nm is more preferable. When infrared rays are used, infrared rays having a wavelength of 0.7 to 1000 μm are preferable, and infrared rays having a wavelength of 2.0 to 500 μm are more preferable.

  When pressure is used as the energy, osmotic pressure, air pressure, water pressure, or vacuum pressure can be used as a method of destroying the structure of cell membranes or fat globules by pressure.

  Usually, although there is no restriction | limiting in particular, it is preferable that the osmotic pressure is the range of 10-1000 mOsm, and it is more preferable that it is the range of 20-500 mOsm. The air pressure, water pressure, and vacuum pressure are preferably in the range of 0.01 Pa to 100 MPa, and more preferably in the range of 0.1 Pa to 10 MPa.

  When vibration is used as the energy, it is preferable to put the treated portion in a container and vibrate the container at 50 to 10000 Hz, and more preferably vibrate at 100 to 1000 Hz.

  When ultrasonic waves are used as the energy, 10 kHz to 1 GHz is preferable and 20 kHz to 100 MHz is more preferable as a preferable range when the structure of the cell membrane or fat globules is destroyed by ultrasonic waves.

When using the shear stress as the energy, it is preferable to apply a shear stress of 10~10000dynes / cm ² to the processing content, it is more preferable to apply a shear stress of 50~5000dynes / cm ^2.

  Next, the second step will be described. The 2nd process mixes the processed part which is a part of milk processed at the 1st process with the non-processed part which is the remainder of milk, and makes the activator contained in the processed part into the processed part and the non-processed part. It is the process of making it contact and act on the somatic cell which exists in the liquid mixture. This second step is preferably performed while maintaining the mixture at an appropriate temperature in order to maintain a state in which somatic cells can produce a product. The appropriate temperature is preferably in the range of 35 ° C to 45 ° C, more preferably in the range of 37 ° C to 42 ° C.

  Further, in order to increase the opportunity for contact between the activator and the somatic cell and reduce the time required for the second step, stirring or shaking may be performed in the second step.

  Next, the third step will be described.

  Production capacity of active oxygen such as superoxide anion radical, hydrogen peroxide, hypochlorous acid, protein, peptide, lactic acid, fatty acid, etc. is increasing from somatic cells in the mixture activated in the second step. . In the third step, the product is detected and quantified, and the amount is converted into the number of somatic cells in the milk to be examined.

  Any known detection method may be used as the product detection method, and examples thereof include an electrochemical method, a colorimetric method, a chemiluminescence method, and an absorptiometry method. In view of the fact that fat globules and casein micelles are present in milk and light is scattered, it is more preferable to use an electrochemical method.

  When the electrochemical method is used, the current that flows when the mixed solution after the second step is brought into contact with the working electrode on which the enzyme is applied and the counter electrode, and a voltage is applied between the working electrode and the counter electrode in that state. Based on the above, it is preferable to calculate the number of somatic cells contained in milk. The working electrode to which the enzyme is applied is also called an enzyme electrode.

  In addition, the activated somatic cells have an increased amount of product and enhanced adhesion. Neutrophils, one of the somatic cells in the milk, form a pseudo-scaffold when touching a hydrophobic surface and are likely to adhere, so that somatic cells are likely to adhere to the vicinity of the product detection site in the third step. By forming a hydrophobic surface, somatic cells are accumulated and products from somatic cells can be detected with higher sensitivity. The water contact angle of the hydrophobic surface is preferably in the range of 30 ° to 110 °, more preferably in the range of 40 ° to 100 °. When the contact angle is less than 30 °, the adhesion of neutrophils decreases. On the other hand, if the contact angle exceeds 110 °, the choice of materials is limited, which adversely affects manufacturing costs.

  The milk inspection method of the present invention is highly reproducible when executed by the apparatus, and enables a quick test. Next, a milk inspection apparatus for executing the milk inspection method of the present invention will be described.

<Milk inspection device>
In the milk inspection apparatus for executing the milk inspection method of the present invention, parts for performing each step of the milk inspection method may be arranged in one component, or at least one of these parts is a separate body. It may be configured as. However, it is desirable for the downsizing of the apparatus that the portions for performing the respective steps are arranged in one component. In addition, when the part which implements a 1st process is made into a different body, since the part can be reduced in size, it may become advantageous when the structure which gives energy is employ | adopted.

  For example, the first step may be performed with the first component, the second step may be performed with the second component, and the third step may be performed with the third component. Further, for example, the first process may be performed with the first structure, and the second and third processes may be performed with the second structure. Further, for example, the first and second steps may be performed with the first structure, and the third process may be performed with the second structure. Further, for example, the first and third steps may be performed with the first structure, and the second process may be performed with the second structure. Alternatively, one of the first to third steps may be performed with a plurality of components.

  The milk inspection apparatus includes a first container into which a processing portion that is a part of milk is charged, and an activating unit that performs processing for generating an activation factor from the processing portion on the processing portion that has been put into the first container; A somatic cell in milk using a non-processed portion that is the remainder of milk and a second container into which the processed component that has been subjected to the above-described processing is charged, and a mixture of the non-processed component and the processed component in the second container It is desirable to have a measuring means for measuring the number.

  The shape of the first container and the second container is usually not particularly limited, and may be a circular or square box shape in plan view, and the size may be wide horizontally or vertically long. The material of the container is usually not particularly limited, and plastic, glass, metal or the like can be used. The container may be washed and used repeatedly, but may be a disposable container from the viewpoint of contamination due to adhesion of milk components such as fat and protein and the effect of reduced sensitivity.

  The activating means, which is a means for performing the first step in the first container, may be an element that generates energy (for example, an LED, a heater, etc.) disposed outside the first container, for example. Good. Alternatively, the activating means may have a rotating blade disposed in the first container for applying a shear stress to the process charged in the first container. Alternatively, the activating means may be a surfactant applied to the inner surface of the first container.

  The activating means is preferably an energy applying means that gives energy selected from heat, electromagnetic waves, pressure, vibration, ultrasonic waves, and shear stress to the process charged in the first container.

  The energy generation source of the energy applying means may be arranged inside or outside the first container. If the energy generation source is arranged outside the first container, it is advantageous for use in which only the first container is replaced and the energy generation source is repeatedly used. A general-purpose element or the like can be used as the energy generation source. Examples of the heat source include a Peltier element, a heating wire heater, a silicon rubber heater, a mica heater, a heating cable, a thermos heater, and an infrared heater. Examples of the electromagnetic wave source include an LED, an ultraviolet lamp, a high-pressure mercury lamp, a halogen lamp, an infrared lamp, and the like, and the LED is particularly desirable from the viewpoints of handleability and apparatus miniaturization. Examples of the pressure source include changes in osmotic pressure due to the addition of a substance, air pressure / water pressure due to a piston, vacuum pressure due to a vacuum pump, centrifugal force, air pressure due to gravity, and the like. Examples of the vibration source include a piezoelectric element and a ceramic oscillator. Examples of the ultrasonic source include piezoelectric ceramics, lead zirconate titanate (PZT) electrostrictive oscillators, and bolted Langevin ultrasonic transducers. Examples of the shear stress source include passage through a narrow strain passage, stirring by a rotary blade, and the like.

  The milk test apparatus preferably includes a temperature control means for keeping the temperature of the second container in which the second step and the third step are performed at a constant temperature because the activity of somatic cells varies greatly depending on the temperature. . Also, by providing a stirring means for stirring in the second container, the activation factor is more likely to come into contact with somatic cells, and the time required for activation is shortened, but the device cost increases and the device becomes larger. Therefore, it may be provided arbitrarily.

  The measuring means, which is a means for carrying out the third step, may comprise means for adding a certain amount of a coloring reagent or a luminescent reagent in the second container, and means for detecting a change in color or luminescence amount. Good. Alternatively, the measuring means may have a working electrode and a counter electrode provided with the enzyme, provided in the second container. Considering the influence of contamination due to the adhesion of milk components, it is preferable that the working electrode and the counter electrode are disposable together with the second container because the operation is simple. A configuration in which a working electrode and a counter electrode are provided is preferable because it does not require reagent addition operation or management of the reagent itself, and can be measured easily and stably.

  When employing the measuring means having the working electrode and the counter electrode, it is preferable that the milk inspection apparatus includes an apparatus main body that applies a voltage between the working electrode and the counter electrode. Further, it is preferable that at least one of the first container and the second container is detachable from the apparatus main body. If the first container is attachable to and detachable from the apparatus main body, the first container can be detached from the apparatus main body, and the processing amount in the first container can be directly put into the second container.

  In addition, it is preferable that a hydrophobic surface made of a hydrophobic material for accumulating somatic cells is formed at least in the vicinity of the working electrode on the inner surface of the second container. If it is this structure, the product from a somatic cell can be detected more sensitively.

  The hydrophobic material constituting the hydrophobic surface is not particularly limited, but the contact angle of the hydrophobic surface with water is preferably in the range of 30 ° to 110 °, more preferably in the range of 40 ° to 100 °. . When the contact angle is less than 30 °, the adhesion of neutrophils decreases. On the other hand, if the contact angle exceeds 110 °, the choice of materials is limited, which adversely affects manufacturing costs. Specific materials include hydrophobic resins (acrylic resins, styrene resins, acrylic-styrene copolymer resins, polyester resins, polyolefin resins, etc.), metals (gold, aluminum, platinum, copper, nickel, tin, etc.) ), Glass (borosilicate glass, soft glass, quartz glass, etc.) and the like, and acrylic resins, styrene resins, acrylic-styrene copolymer resins, and polyester resins are particularly preferable.

  From the viewpoint of simplifying the apparatus, the processing part may be put into the second container from the first container by a human hand, or the processing part in the first container is sucked with a syringe, pipette or the like, and then the second container. It may be automatically performed by discharging the ink. However, it is preferable to provide a flow path for guiding the processed portion from the first container to the second container. This is because impurities in milk and diffusion of milk can be prevented.

  When the flow path is provided, it is preferable that the first container is disposed at a position higher than the second container. With such a configuration, it is possible to move the processed portion in the first container to the second container by gravity through the flow path. In this case, if the on-off valve is provided in the flow path, the second step can be started simply by opening the on-off valve.

  The capacity of the first container is preferably 1/1000 to 1/2 of the capacity of the second container. If it is smaller than 1/1000, since the amount of the activator produced in the first container is small, the activation effect is small and the measurement sensitivity does not increase. On the other hand, if the ratio is larger than 1/2, the ratio of somatic cells that are damaged increases, so the sensitivity does not increase. The capacity of the first container is more preferably 1/100 to 1/10 of the capacity of the second container.

  If it is a milk | loop inspection apparatus demonstrated above, since the 1st container for implementing a 1st process and the 2nd container for implementing a 2nd and 3rd process are divided, size reduction is easy. . In addition, if a configuration in which the temperature of the second container is kept constant is adopted, the numerical value is stabilized because milk is difficult to denature during the third step. Moreover, by putting a part of milk into the first container, it is possible to employ an activator producing means that can damage somatic cells, shortening the time required for the first step, and a highly effective activator Can be produced.

  A plurality of first containers and second containers may be provided. In particular, if four each of the first container and the second container are provided, it is possible to inspect all four quadrants at one time, and it is possible to more rapidly measure the number of somatic cells.

(Concrete example)
Next, a specific example of the above-described milk inspection apparatus will be described with reference to the drawings.

  FIG. 1 shows a milk test apparatus 1 as a specific example. This milk test | inspection apparatus 1 detects a superoxide anion radical especially among the products from a somatic cell, and measures the number of somatic cells.

  Specifically, the milk inspection apparatus 1 includes an apparatus body 10 that houses a first container 2 and a second container 3. A tray 45 for receiving milk is provided on the upper surface of the apparatus body 10. A display panel 11 for displaying the number of somatic cells as a measurement result is provided on the upper surface of the apparatus main body 10. The first container 2 and the second container 3 are preferably detachable from the apparatus main body 10.

  The tray 45, the first container 2, and the second container 3 are arranged in this order from top to bottom so as to overlap each other in the vertical direction. The tray 45 and the first container 2 are connected by a first flow path 41, the first container 2 and the second container 3 are connected by a second flow path 42, and the tray 45 and the second container 3 are connected by a third flow. They are connected by a path 43. The first to third flow paths 41, 42, 43 all extend in the vertical direction. When milk is poured into the tray 45, a part of milk (processed part) is introduced into the first container 2 through the first flow path 41, and the remaining part of milk (unprocessed part) is the third milk. It is introduced into the second container through the flow path 43.

  Outside the first container 2 is a first heater (activation means) 5 for generating an activating factor from the processed part by heating the processed part put into the first container 2 through the first container 2. It is arranged. It is preferable that power is supplied in advance to the first heater 5 from a power supply unit 105 described later, and the processing portion is heated at the same time as the processing portion is charged into the first container 2. The processed portion heated by the first heater 5 is then put into the second container 3 through the second flow path. Thereby, the non-processed part already put into the 2nd container 3 and the heated process part are mixed, and it becomes a liquid mixture.

  The second flow path 42 is preferably provided with an open / close valve that can be operated automatically or manually. In this way, the residence time in the first container 3 for processing, in other words, the heating time can be set freely. In addition, even if it does not provide an on-off valve, the residence time for the process in the 1st container 3 may be ensured by making the cross-sectional area of the 2nd flow path 42 small.

  A second heater 6 for keeping the temperature of the second container 3 constant is disposed outside the second container 3. Electric power is supplied to the second heater 6 from an electric power supply unit 105 to be described later so that the second container 3 is maintained at a predetermined temperature before the non-processed portion is charged into the second container 3. It is preferable that

  As shown in FIG. 2, a working electrode 7A, a reference electrode 7B, and a counter electrode 7C are provided on the bottom surface 31 of the second container 3. These electrodes 7A to 7C are connected to a power detection circuit 102, which will be described later, via wirings 71 to 73 extending from the bottom surface 31 to the outside of the second container 3 (only the portion on the bottom surface 31 is shown in FIG. 2). Yes. An enzyme (superoxide dismutase) is applied to the surface of the working electrode 7A. In addition, it is preferable that the 2nd container 3 is comprised so that the circumference | surroundings of the bottom face 31 or the working electrode 7A of them may become a hydrophobic surface.

  The apparatus main body 10 calculates the number of somatic cells contained in milk based on the current that flows when a voltage is applied between the working electrode 7A and the counter electrode 7C. Specifically, as shown in FIG. 3, the apparatus main body 10 applies a voltage between the power source 101, the working electrode 7A and the counter electrode 7C, and between the reference electrode 7B and the counter electrode 7C. A current detection circuit 102 that detects a flowing current, a current-voltage conversion circuit 103 that converts the detected current into a voltage, and a calculation unit 104 that calculates the number of somatic cells from the voltage value are calculated by the calculation unit 104. The number of somatic cells thus displayed is displayed on the display panel 11. Further, the apparatus main body 10 includes a power supply unit 105 that supplies power to the first heater 5 and the second heater 6 (not shown in FIG. 3).

  The power source 101 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. The current detection circuit 102 includes a switching element or the like. The current-voltage conversion circuit 103 includes a current-voltage conversion element, an operational amplifier that amplifies the voltage, and the like. The calculation unit 104 includes a CPU, a storage unit (ROM and RAM), and the like. The storage unit stores a calibration curve that associates the voltage value with the number of somatic cells, and the calculation unit 104 calculates the number of somatic cells according to the voltage value sent from the current-voltage conversion circuit 103. In this specific example, the working electrode 7A, the reference electrode 7B, the counter electrode 7C, the current detection circuit 102, the current-voltage conversion circuit 103, and the calculation unit 104 constitute the measuring means of the present invention.

  In this specific example, since the reference electrode 7B is provided, the voltage value when calculating the number of somatic cells is a value considering the reference electrode 7B. That is, the device body 10 obtains a working voltage value by converting a current that flows when a voltage is applied between the working electrode 7B and the counter electrode 7C into a voltage, and a voltage is applied between the reference electrode 7B and the counter electrode 7C. A reference voltage value is obtained by converting a current flowing when applied to a voltage. Then, when calculating the number of somatic cells, the apparatus body 10 calculates the number of somatic cells from a voltage value obtained by subtracting the reference voltage value from the working voltage value. In this way, the voltage value based on the reaction between the active oxygen contained in the mixed liquid and the enzyme can be accurately obtained.

  When the milk inspection apparatus 1 described above is used, the milk inspection method is executed as follows. First, a certain amount of milk to be examined is poured into the tray 45. As a result, the processing portion that is a part of milk is put into the first container 2 and the non-processing portion that is the remaining portion of milk is put into the second container 3. In the 1st container 2, a 1st process is implemented simultaneously with injection | throwing-in. Next, the processed part after the first step is introduced from the first container 2 to the second container 3 through the second flow path 42 and mixed with the non-processed part in the second container 3. Two steps are performed. Then, a third step of measuring the number of somatic cells in milk by detecting activated somatic cells in the mixture is performed.

(Modification)
In the specific example, the first heater 5 is used as the activating means. However, the activating means is applied to the means for applying pressure to the process charged in the first container 2 or applied to the inner surface of the first container 2. It may be a surfactant. As a means for applying pressure, for example, in the case of osmotic pressure, a means for adding water or salts may be provided, or a piston for pressurization or decompression may be provided.

  In the specific example, the measuring means including the electrodes 7A to 7C is adopted. However, the measuring means includes a coloring reagent or a luminescent reagent, and a means for detecting a change in color or luminescence amount. Also good.

  Further, for example, the first container 2 and the third container 3 are arranged side by side, and instead of the second flow path 42, the second portion of the processing in the first container 2 is sucked and discharged by an electric syringe. You may move to the container 3. Alternatively, from the viewpoint of device simplification, such movement may be performed by a human hand.

<Mastitis diagnosis method>
In the mastitis diagnosis method of the present invention, it is desirable to record the number of somatic cells for each individual, and more desirably for each quarter. Usually, if the number of somatic cells is 200,000 cells / mL or less, healthy cattle are diagnosed, and if the number of somatic cells is 200,000 cells / mL or more, it is diagnosed that there is a high possibility of mastitis infection. Furthermore, if the somatic cell count is calculated based on the amount of product produced from leukocytes among somatic cells, the increase or decrease in the ratio of leukocytes can be determined from the somatic cell count measured by this test method. For this reason, if the measured number of somatic cells is recorded, it becomes possible to diagnose the presence or absence of mastitis infection at an early stage from the transition of the number of somatic cells in the individual from which the raw milk has been extracted.

  Examples will be described below. The apparatus shown in this embodiment is an example, and the present invention is not limited to these embodiments.

[Test method for somatic cell count in raw milk samples]
A raw milk sample is smeared onto a fixed area on a slide glass, dried, stained, and microscopically examined. The number of cells is counted, and the area of the field of view of the microscope (observation of oil immersion lens) is related to the raw milk sample. The number of somatic cells present in was calculated. In addition, the representative visual field measured 16 visual fields or more, and the number of nucleated somatic cells in the entire visual field was measured.

  When raw milk was sampled from 2 healthy cattle (a, b) and 3 cattle infected with mastitis (c, d, e), the number of somatic cells in the raw milk was a: 40,000 / mL, b: 150,000 / mL, c: 420,000 / mL, d: 900,000 / mL, e: 2.1 million / mL.

[Production of enzyme electrode]
In this example and comparative example, an enzyme electrode capable of specifically detecting superoxide anion radicals produced from somatic cells was used.

  Masking is performed on a base plate made of acrylic resin having a length of 60 mm, a width of 30 mm, and a thickness of 1 mm, and a working electrode (gold, diameter: 4 mm), reference electrode (deposited by ULVAC, Inc., model: UEP) ( Silver / silver chloride, diameter 1 mm) and counter electrode (platinum, diameter 8 mm) were formed.

  Next, the working electrode was immersed in a cysteine solution to form a self-assembled monolayer of cysteine on the surface of the working electrode. Thereafter, an enzyme (superoxide dismutase) was applied to the surface of the working electrode by an immersion method to obtain an enzyme electrode.

[Example 1] -Activation by somatic cell-derived material crushed by ultraviolet rays-
About milk extracted from each cow ae mentioned above, each milk 1mL (processed part) is put into a polystyrene dish with a diameter of 3 cm, and a wavelength of 254 nm (200 mW / cm ² ) is set in an incubator set at 37 ° C. Light was passed through the milk in the dish and irradiated for 15 seconds, and a somatic cell disruption operation was performed as the first step to produce cytokine as an activator. Next, 5 mL of the milk that had not been irradiated with ultraviolet rays (non-treated portion) was added to the dish, and held at 37 ° C. for 60 seconds to perform the second step.

  Thereafter, as the third step, the milk (mixed solution) after the second step was dropped onto the enzyme electrode, reference electrode and counter electrode, and the current value derived from activated somatic cells was measured. For the measurement of the current value, a potentiostat galvanostat (Hokuto Denko Co., Ltd., model: HA-151) was used. In the measurement method, DC 1 V was applied to the working electrode and the reference electrode, and then an additional voltage of 0.3 V was applied to the working electrode. At the same time as oxidative degradation occurred on the working electrode, enzyme reduction occurred on the counter electrode, and current flowed between the working electrode and the counter electrode. The current value was measured. The current value was measured by fixing the base plate supporting each electrode on a plate-type heater set to a predetermined temperature.

[Example 2] -Activation by somatic cell-derived material crushed by heat-
About milk extracted from each cow ae mentioned above, each milk 5mL is poured into the tray 45 of the milk test | inspection apparatus 1 of FIG. 1, 1 mL (processed part) to the 1st container 2, and 4 mL to the 2nd container 3 (Non-treated portion) was charged. Next, the milk in the 1st container 2 (capacity | capacitance 1mL) was heated at 60 degreeC for 30 second with the 1st heater 5, The somatic cell was crushed as a 1st process, and the cytokine which is an activator was produced. Thereafter, the opening / closing valve (not shown) was opened, and the contents of the first container 2 were introduced into the second container 3 (capacity 5 mL) through the second flow path 42. Next, the 2nd container 3 maintained at 37 degreeC with the 2nd heater 6 was maintained as it was for 60 seconds, and the 2nd process was performed. Next, the current value derived from somatic cells was measured with the enzyme electrode, the reference electrode, and the counter electrode provided in the second container 3. Each electrode of Example 2 was configured the same as that of Example 1.

[Comparative example]
About milk extracted from each of the cows a to e, derived from somatic cells in the same manner as in Example 1 except that the milk was directly dropped onto the enzyme electrode, reference electrode and counter electrode without performing the first and second steps. The current value of was measured.

  The measurement results of current values were as shown in Table 1 and FIG.

  From Table 1 and FIG. 4, it can be seen that in Examples 1 and 2, the measured current value is increased due to the action of the activating factor.

DESCRIPTION OF SYMBOLS 1 Milk test | inspection apparatus 2 1st container 3 2nd container 41 1st flow path 42 2nd flow path 43 3rd flow path 5 1st heater (activation means)
6 Second heater 7A Working electrode 7B Reference electrode 7C Counter electrode 10 Device body

Claims (15)

A method for examining milk,
A first step of performing a process of generating an activator from the processed part of the milk;
A second step of activating somatic cells present in a mixed solution of the treated portion subjected to the treatment and a non-treated portion which is the remainder of the milk by an activating factor contained in the treated portion;
A third step of measuring the number of somatic cells in the milk using the mixed solution after the second step;
A method for examining milk.   The milk test method according to claim 1, wherein in the first step, at least one of a cytokine and an antibacterial protein is generated as the activator by decomposing somatic cells existing in the treated portion.   The milk test method according to claim 1, wherein in the first step, fat is liberated from fat globules existing in the treated portion and the fat is hydrolyzed to generate fatty acid as the activator.   The said 1st process WHEREIN: The activation factor is generated from the said process part by giving the energy chosen from a heat | fever, electromagnetic waves, a pressure, a vibration, an ultrasonic wave, and a shear stress to the said process part. The milk test method according to any one of the above.   In the third step, the mixed solution is brought into contact with a working electrode coated with an enzyme and a counter electrode, and based on a current flowing when a voltage is applied between the working electrode and the counter electrode in the state, The milk test method according to any one of claims 1 to 4, wherein the number of somatic cells contained in the milk is calculated. A milk inspection device for inspecting milk,
A first container into which a processing portion that is a part of the milk is charged;
Activating means for performing a treatment for generating an activation factor from the treated portion on the treated portion put into the first container;
A second container into which the untreated portion that is the remainder of the milk and the treated portion that has been subjected to the treatment;
Measuring means for measuring the number of somatic cells in the milk using a mixed liquid of the untreated portion and the treated portion in the second container;
A milk inspection apparatus.   The milk test according to claim 6, wherein the activating means is a means for giving energy selected from heat, electromagnetic waves, pressure, vibration, ultrasonic waves, and shear stress to the processing portion put into the first container. apparatus.   The milk test apparatus according to claim 6 or 7, wherein the measuring means includes a working electrode and a counter electrode provided with an enzyme and provided in the second container.   The milk inspection apparatus according to claim 8, wherein a hydrophobic surface is formed at least in the vicinity of the working electrode on the inner surface of the second container.   The milk test | inspection apparatus of Claim 8 or 9 further provided with the apparatus main body which applies a voltage between the said working electrode and the said counter electrode with which at least one of the said 1st container and the said 2nd container can be attached or detached.   The milk test | inspection apparatus as described in any one of Claims 6-10 further provided with the flow path which guides the said processed part to which the said process was performed from the said 1st container to the said 2nd container.   The milk inspection apparatus according to claim 11, wherein the first container is disposed at a position higher than the second container.   The capacity | capacitance of a said 1st container is a milk test | inspection apparatus as described in any one of Claims 6-12 which is 1 / 1000-1 / 2 of the capacity | capacitance of a said 2nd container.   The milk inspection apparatus according to any one of claims 6 to 13, wherein a plurality of the first container and the second container are provided.   The number of somatic cells in the milk obtained by the milk test method according to any one of claims 1 to 5 is recorded, and from the transition of the number of somatic cells, mastitis infection of the individual from which the milk has been extracted Diagnose the mastitis diagnosis method.

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