JP2012208006A - Method for detecting mastitis, and measuring instrument for use in method for detecting mastitis - Google Patents
Method for detecting mastitis, and measuring instrument for use in method for detecting mastitis Download PDFInfo
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本発明は、例えば牛の乳汁における過酸化水素の分解反応による乳房炎検出方法、及びこの乳房炎検出方法に用いる測定用器具に関する。 The present invention relates to a mastitis detection method based on, for example, a decomposition reaction of hydrogen peroxide in cow's milk, and a measurement instrument used in this mastitis detection method.
乳房炎防除のため、乳房内への細菌感染を早期に発見し、治療するなどの早期対策が求められている。
従来の乳房炎検出法としてのＣＭＴ変法（ＰＬテスター法）は、酪農家が簡便に検査できるが、乳房炎発症後でないと検知できない方法である。また、ＣＬ法（化学発光法）は、乳房炎発症前の感染を捉えられるが、酪農家が簡便に検査できる方法ではない。
乳房炎に感染すると、活性酸素の一種である過酸化水素（Ｈ２Ｏ２）の分解酵素であるグルタチオンペルオキシダーゼやカタラーゼの量が増加する。
本発明者らは、酪農家が現場において、乳汁中の過酸化水素分解酵素の活性を測定することで、乳房炎感染を簡易に判定する方法を既に提案している。
この方法は、サイクリックボルタンメトリー法を代表例とし、乳汁の過酸化水素消去能を、過酸化水素分解により発生する酸素濃度から検出するものであり、日々の搾乳作業の中で、搾乳インラインから乳汁サンプルを採取し、短時間で自動的に測定、診断することにより、通常の乳房炎検出に加え、感染初期の乳房炎および外見上異常の認められない潜在性乳房炎を検出することができる。
In order to control mastitis, early measures such as early detection and treatment of bacterial infection in the breast are required.
A modified CMT method (PL tester method) as a conventional mastitis detection method is a method that can be easily inspected by dairy farmers, but can only be detected after the onset of mastitis. Moreover, although the CL method (chemiluminescence method) can catch infection before the onset of mastitis, it is not a method that can be easily examined by dairymen.
When infected with mastitis, the amount of glutathione peroxidase and catalase, which are degrading enzymes of hydrogen peroxide (H 2 O 2 ), which is a kind of active oxygen, increases.
The present inventors have already proposed a method in which a dairy farmer can easily determine mastitis infection by measuring the activity of a hydrogen peroxide-degrading enzyme in milk on site.
This method uses a cyclic voltammetry method as a representative example, and detects the hydrogen peroxide scavenging ability of milk from the oxygen concentration generated by hydrogen peroxide decomposition. By collecting a sample and automatically measuring and diagnosing it in a short time, in addition to normal mastitis detection, mastitis in the early stage of infection and latent mastitis with no apparent abnormality can be detected.
しかし、乳汁の過酸化水素消去能を、過酸化水素分解により発生する酸素濃度から検出する方法では、乳汁中の溶存酸素濃度に影響を受ける場合があることが分かってきた。
また、サイクリックボルタンメトリー法を用いる場合には、炭素電極と銀塩化銀電極を用いるが、銀塩化銀電極は、ガラス製であるため破損しやすく、また、測定後に食塩水に浸漬して保管する必要があるため、酪農家にとっては扱いにくいという問題を有している。
However, it has been found that the method for detecting the hydrogen peroxide scavenging ability of milk from the oxygen concentration generated by hydrogen peroxide decomposition may be affected by the dissolved oxygen concentration in the milk.
In addition, when using the cyclic voltammetry method, a carbon electrode and a silver-silver chloride electrode are used, but the silver-silver chloride electrode is easily broken because it is made of glass, and is immersed in saline after measurement and stored. Because it is necessary, it has a problem that it is difficult for dairymen to handle.
そこで本発明は、乳汁中の溶存酸素濃度に影響を受けない乳房炎検出方法、及びこの乳房炎検出方法に用いる測定用器具を提供することで乳房炎発症の予防を目的とする。 Then, this invention aims at prevention of mastitis onset by providing the mastitis detection method which is not influenced by the dissolved oxygen concentration in milk, and the measuring instrument used for this mastitis detection method.
請求項１記載の本発明の乳房炎検出方法は、乳汁に所定の量の過酸化水素を供給し、前記乳汁の過酸化水素消去能を電気化学的方法によって検出する乳房炎検出方法であって、過酸化水素分解酵素活性に応じて一定時間に減少する過酸化水素濃度を測定することを特徴とする。
請求項２記載の本発明は、請求項１に記載の乳房炎検出方法において、前記電気化学的方法が、クロノアンペロメトリー法であることを特徴とする。
請求項３記載の本発明は、請求項１又は請求項２に記載の乳房炎検出方法において、前記乳汁に前記過酸化水素を供給する前の前記過酸化水素濃度を測定する第１の測定ステップと、前記第１の測定ステップの後に前記乳汁に前記過酸化水素を供給して混合する供給ステップと、前記供給ステップの所定時間経過後に前記過酸化水素濃度を測定する第２の測定ステップと、を有し、前記第１の測定ステップで測定した前記過酸化水素濃度から前記第２の測定ステップで測定した前記過酸化水素濃度を減じて前記過酸化水素消去能を算出することを特徴とする。
請求項４記載の本発明は、請求項１から請求項３のいずれかに記載の乳房炎検出方法において、体細胞数をあらかじめ測定し、前記体細胞数が所定値以下の場合に、前記過酸化水素消去能を検出することを特徴とする。
請求項５記載の本発明の乳房炎検出方法に用いる測定用器具は、請求項１から請求項４のいずれかに記載の乳房炎検出方法に用いる測定用器具であって、前記乳汁に供給する前の前記過酸化水素を収容する第１の空間と、前記過酸化水素を供給する前の前記乳汁を収容する第２の空間と、前記第１の空間と前記第２の空間とを連通させる連通手段と、前記第１の空間内の前記過酸化水素濃度を測定するとともに、前記連通手段で連通させた後の前記第１の空間内又は前記第２の空間内の前記過酸化水素濃度を測定する測定手段と、を備えたことを特徴とする。
請求項６記載の本発明は、請求項５に記載の乳房炎検出方法に用いる測定用器具において、複数の前記第１の空間と、複数の前記第１の空間にそれぞれ対応する複数の前記第２の空間とを備え、前記測定手段では、それぞれの前記第１の空間内の前記過酸化水素濃度を同時に個別に測定するとともに、前記連通手段で連通させた後のそれぞれの前記第１の空間内又はそれぞれの前記第２の空間内の前記過酸化水素濃度を同時に個別に測定することを特徴とする。
The mastitis detection method of the present invention according to claim 1 is a mastitis detection method for supplying a predetermined amount of hydrogen peroxide to milk and detecting the hydrogen peroxide scavenging ability of the milk by an electrochemical method. The method is characterized in that the concentration of hydrogen peroxide that decreases in a certain time according to the hydrogen peroxide-degrading enzyme activity is measured.
The present invention according to claim 2 is the mastitis detection method according to claim 1, wherein the electrochemical method is a chronoamperometry method.
According to a third aspect of the present invention, in the mastitis detection method according to the first or second aspect, a first measurement step of measuring the hydrogen peroxide concentration before the hydrogen peroxide is supplied to the milk. A supply step of supplying and mixing the hydrogen peroxide to the milk after the first measurement step; a second measurement step of measuring the hydrogen peroxide concentration after a predetermined time elapses of the supply step; The hydrogen peroxide scavenging ability is calculated by subtracting the hydrogen peroxide concentration measured in the second measurement step from the hydrogen peroxide concentration measured in the first measurement step. .
According to a fourth aspect of the present invention, in the mastitis detection method according to any one of the first to third aspects, when the number of somatic cells is measured in advance and the number of somatic cells is equal to or less than a predetermined value, the excess is detected. It is characterized by detecting hydrogen oxide scavenging ability.
A measurement instrument used in the mastitis detection method of the present invention according to claim 5 is the measurement instrument used in the mastitis detection method according to any one of claims 1 to 4, and is supplied to the milk. The first space for storing the previous hydrogen peroxide, the second space for storing the milk before supplying the hydrogen peroxide, and the first space and the second space are communicated with each other. The hydrogen peroxide concentration in the first space or in the second space after the communication means and the hydrogen peroxide concentration in the first space are measured and the communication means communicates with the hydrogen peroxide concentration in the first space. And measuring means for measuring.
According to a sixth aspect of the present invention, in the measurement instrument used in the mastitis detection method according to the fifth aspect, the plurality of first spaces and the plurality of first spaces respectively corresponding to the plurality of first spaces. And the measurement means measures the hydrogen peroxide concentration in each of the first spaces at the same time individually and communicates with each other by the communication means. The hydrogen peroxide concentration in or in each of the second spaces is measured individually at the same time.
本発明によれば、過酸化水素が分解されて減少する過酸化水素の濃度自体を測定することで、乳汁中に溶存する酸素量の影響を受けることなく、乳汁の過酸化水素消去能を正確に検出することができる。 According to the present invention, the hydrogen peroxide scavenging ability of milk is accurately measured without being affected by the amount of oxygen dissolved in milk by measuring the concentration of hydrogen peroxide itself, which decreases as hydrogen peroxide is decomposed. Can be detected.
本発明の第１の実施の形態による乳房炎検出方法は、過酸化水素分解酵素活性に応じて一定時間に減少する過酸化水素濃度を測定するものである。本実施の形態によれば、過酸化水素が分解されて減少する過酸化水素の濃度自体を測定することで、過酸化水素の分解により発生する酸素濃度を測定する場合のように、乳汁中に溶存する酸素量の影響を受けることなく、乳汁の過酸化水素消去能を正確に検出することができる。
本発明の第２の実施の形態は、第１の実施の形態による乳房炎検出方法において、電気化学的方法が、クロノアンペロメトリー法であるものである。本実施の形態によれば、過酸化水素の分解により減少する過酸化水素濃度を電流値として検出するため、データ処理を容易に行うことができ、また、サイクリックボルタンメトリー法と比較すると、カタラーゼとグルタチオンペルオキシダーゼとの双方の酵素活性を測定でき、また、電極の取り扱いが容易となる。
本発明の第３の実施の形態は、第１又は第２の実施の形態による乳房炎検出方法において、乳汁に過酸化水素を供給する前の過酸化水素濃度を測定する第１の測定ステップと、第１の測定ステップの後に乳汁に過酸化水素を供給して混合する供給ステップと、供給ステップの所定時間経過後に過酸化水素濃度を測定する第２の測定ステップとを有し、第１の測定ステップで測定した過酸化水素濃度から第２の測定ステップで測定した過酸化水素濃度を減じて過酸化水素消去能を算出するものである。本実施の形態によれば、毎回の測定時に電極などの測定器による誤差を補正できるため、測定器によるばらつきの影響を受けることなく、より正確な検出を行うことができる。
本発明の第４の実施の形態は、第１から第３の実施の形態による乳房炎検出方法において、体細胞数をあらかじめ測定し、体細胞数が所定値以下の場合に、過酸化水素消去能を検出するものである。本実施の形態によれば、既に乳房炎が発生している検査対象をあらかじめ除外することで、乳房炎の早期発見が行いやすくなる。
本発明の第５の実施の形態による乳房炎検出方法に用いる測定用器具は、第１から第４の実施の形態による乳房炎検出方法に用いる測定用器具であって、乳汁に供給する前の過酸化水素を収容する第１の空間と、過酸化水素を供給する前の乳汁を収容する第２の空間と、第１の空間と第２の空間とを連通させる連通手段と、第１の空間内の過酸化水素濃度を測定するとともに、連通手段で連通させた後の第１の空間内又は第２の空間内の過酸化水素濃度を測定する測定手段とを備えたものである。本実施の形態によれば、乳汁及び過酸化水素の定量分注を容易に行え、また、第１の測定ステップでの過酸化水素濃度の測定と、第２の測定ステップでの過酸化水素濃度の測定を迅速に行うことができる。
本発明の第６の実施の形態は、第５の実施の形態による乳房炎検出方法に用いる測定用器具において、複数の第１の空間と、複数の第１の空間にそれぞれ対応する複数の第２の空間とを備え、測定手段では、それぞれの第１の空間内の過酸化水素濃度を同時に個別に測定するとともに、連通手段で連通させた後のそれぞれの第１の空間内又はそれぞれの第２の空間内の過酸化水素濃度を同時に個別に測定するものである。本実施の形態によれば、例えば検査対象が牛の場合には、４つの乳房を同時に個別に測定することができ、いずれかの乳房が感染している場合であっても、確実に乳房炎を検出することができるため、乳房炎の早期発見が行いやすくなる。
The mastitis detection method according to the first embodiment of the present invention measures the hydrogen peroxide concentration that decreases in a certain time according to the hydrogen peroxide-degrading enzyme activity. According to the present embodiment, by measuring the concentration of hydrogen peroxide itself, which decreases as hydrogen peroxide is decomposed, the oxygen concentration generated by the decomposition of hydrogen peroxide is measured, as in the case of measuring the concentration of oxygen in milk. Without being affected by the amount of dissolved oxygen, it is possible to accurately detect the hydrogen peroxide scavenging ability of milk.
The second embodiment of the present invention is the mastitis detection method according to the first embodiment, wherein the electrochemical method is a chronoamperometry method. According to this embodiment, since the hydrogen peroxide concentration that decreases due to the decomposition of hydrogen peroxide is detected as a current value, data processing can be easily performed, and, compared with the cyclic voltammetry method, catalase and Both enzyme activities with glutathione peroxidase can be measured, and handling of the electrode becomes easy.
According to a third embodiment of the present invention, in the mastitis detection method according to the first or second embodiment, a first measurement step of measuring a hydrogen peroxide concentration before supplying hydrogen peroxide to milk A supply step of supplying and mixing hydrogen peroxide to the milk after the first measurement step, and a second measurement step of measuring the hydrogen peroxide concentration after a predetermined time elapses of the supply step, The hydrogen peroxide scavenging ability is calculated by subtracting the hydrogen peroxide concentration measured in the second measurement step from the hydrogen peroxide concentration measured in the measurement step. According to the present embodiment, an error caused by a measuring instrument such as an electrode can be corrected at each measurement, so that more accurate detection can be performed without being affected by variations caused by the measuring instrument.
According to a fourth embodiment of the present invention, in the mastitis detection method according to the first to third embodiments, the number of somatic cells is measured in advance, and when the number of somatic cells is equal to or less than a predetermined value, hydrogen peroxide is erased. Is to detect the ability. According to the present embodiment, early detection of mastitis is facilitated by excluding an examination target in which mastitis has already occurred.
The measuring instrument used in the mastitis detection method according to the fifth embodiment of the present invention is a measuring instrument used in the mastitis detection method according to the first to fourth embodiments, and is used before being supplied to milk. A first space for storing hydrogen peroxide, a second space for storing milk before supplying hydrogen peroxide, a communication means for communicating the first space and the second space, And measuring means for measuring the concentration of hydrogen peroxide in the space and measuring the concentration of hydrogen peroxide in the first space or the second space after being communicated by the communicating means. According to the present embodiment, the quantitative dispensing of milk and hydrogen peroxide can be easily performed, the hydrogen peroxide concentration in the first measurement step, and the hydrogen peroxide concentration in the second measurement step. Can be measured quickly.
According to a sixth embodiment of the present invention, in the measurement instrument used in the mastitis detection method according to the fifth embodiment, a plurality of first spaces and a plurality of first spaces respectively corresponding to the plurality of first spaces. The measurement means measures the hydrogen peroxide concentration in each of the first spaces individually and simultaneously in each first space or each of the first spaces after being communicated by the communication means. The hydrogen peroxide concentration in the space 2 is measured individually at the same time. According to the present embodiment, for example, when the inspection target is a cow, four breasts can be measured individually at the same time, and even if any of the breasts is infected, mastitis is ensured. This makes it easier to detect early mastitis.
以下本発明の一実施例による乳房炎検出方法について説明する。
乳房炎原因菌（以下、細菌）に感染すると、白血球が活性酸素を放出して殺菌するが、同時に自身を傷めないよう活性酸素分解酵素が増加する。
本実施例による乳房炎検出方法は、活性酸素の一種である過酸化水素を乳汁に定量添加し、過酸化水素分解酵素の活性に応じて一定時間に減少する過酸化水素濃度を活性酸素消去能として電気化学センサで測定することで、乳房炎発症前の細菌感染を検出する方法である。
A mastitis detection method according to an embodiment of the present invention will be described below.
When infected with mastitis-causing bacteria (hereinafter referred to as bacteria), white blood cells release active oxygen to kill them, but at the same time, active oxygen-degrading enzymes increase so as not to damage themselves.
In the mastitis detection method according to the present embodiment, hydrogen peroxide, which is a kind of active oxygen, is quantitatively added to milk, and the concentration of hydrogen peroxide that decreases in a certain time according to the activity of hydrogen peroxide-degrading enzyme is reduced. This is a method for detecting bacterial infection before the onset of mastitis by measuring with an electrochemical sensor.
図１は、クロノアンペロメトリー測定（以下ＣＡ測定）による時間経過にともなう電流特性を示す図である。図１では、横軸に測定開始からの時間を、縦軸にはＣＡ測定による電流値（μＡ）を示している。
作用極、対極ともに白金電極を用い、電位を０．７Ｖとし、４サンプル（ＣＨ１、ＣＨ２、ＣＨ３、ＣＨ４）について測定を行った。測定温度は２８〜３０℃とした。
図１に示すように、４つのサンプルはいずれも同じ特性を示し、測定開始後６０秒までは電流値が大きく変動するが、その後は一定値に収束し、１２０秒経過後の変化量は少ない。２７０秒ではほぼ一定値に収束しており、その後の変化量は極めて少ないことから、以降の測定には、２７０秒経過後の電流値を用いた。
FIG. 1 is a diagram showing current characteristics over time according to chronoamperometry measurement (hereinafter CA measurement). In FIG. 1, the horizontal axis represents the time from the start of measurement, and the vertical axis represents the current value (μA) by CA measurement.
A platinum electrode was used for both the working electrode and the counter electrode, the potential was 0.7 V, and measurements were performed on four samples (CH1, CH2, CH3, and CH4). The measurement temperature was 28-30 ° C.
As shown in FIG. 1, the four samples all show the same characteristics, and the current value fluctuates greatly until 60 seconds after the start of measurement, but after that it converges to a constant value, and the amount of change after 120 seconds is small. . Since it converged to a substantially constant value at 270 seconds and the amount of change thereafter was very small, the current value after 270 seconds elapsed was used for the subsequent measurements.
図２は、過酸化水素濃度とＣＡ測定による電流値との関係を示す特性図である。図２では、横軸に添加した過酸化水素の濃度を、縦軸には２７０秒後電流値（μＡ）を示している。
電位を０．７Ｖとし、２７０秒後の電流値を測定した。測定には、過酸化水素を混合した生理食塩水を供し、測定温度は２８〜３０℃とした。
図２に示すように、過酸化水素濃度とＣＡ測定における２７０秒後電流値は正の比例関係にあることがわかる。
FIG. 2 is a characteristic diagram showing the relationship between the hydrogen peroxide concentration and the current value obtained by CA measurement. In FIG. 2, the horizontal axis represents the concentration of hydrogen peroxide added, and the vertical axis represents the current value (μA) after 270 seconds.
The electric potential was 0.7 V, and the current value after 270 seconds was measured. For the measurement, physiological saline mixed with hydrogen peroxide was used, and the measurement temperature was 28 to 30 ° C.
As shown in FIG. 2, it can be seen that the hydrogen peroxide concentration and the current value after 270 seconds in the CA measurement are in a positive proportional relationship.
図３は、カタラーゼ混合量とＣＡ測定による電流値の関係を示す特性図である。図３では、横軸に０．１％カタラーゼ混合量（μｌ）を、縦軸には２７０秒後電流値（μＡ）を示している。ここで、縦軸の２７０秒後電流値（μＡ）は過酸化水素濃度を表している。
分解酵素であるカタラーゼ（以下ＣＡT）試薬と生理食塩水を混合した後、過酸化水素を添加し測定した。生理食塩水は１２ｍｌ、過酸化水素は２ｍｌ、１００ｍＭ（ｍｍｏｌ／ｌ）、測定温度は２８〜３０℃とした。
図３に示すように、過酸化水素分解酵素であるカタラーゼの混合量と２７０秒後電流値は負の比例関係にあった。従って、過酸化水素分解酵素の活性に応じて一定時間に減少する過酸化水素濃度、即ち過酸化水素消去能は、ＣＡ測定による電流値で推定できることが分かる。
FIG. 3 is a characteristic diagram showing the relationship between the amount of catalase mixture and the current value obtained by CA measurement. In FIG. 3, the horizontal axis represents the 0.1% catalase mixture amount (μl), and the vertical axis represents the current value (μA) after 270 seconds. Here, the current value (μA) after 270 seconds on the vertical axis represents the hydrogen peroxide concentration.
A catalase (hereinafter referred to as CAT) reagent, which is a degrading enzyme, and physiological saline were mixed, and hydrogen peroxide was added for measurement. The physiological saline was 12 ml, the hydrogen peroxide was 2 ml, 100 mM (mmol / l), and the measurement temperature was 28-30 ° C.
As shown in FIG. 3, the mixing amount of catalase, which is a hydrogen peroxide decomposing enzyme, and the current value after 270 seconds were in a negative proportional relationship. Therefore, it can be seen that the hydrogen peroxide concentration that decreases in a certain time according to the activity of the hydrogen peroxide decomposing enzyme, that is, the hydrogen peroxide scavenging ability, can be estimated by the current value by CA measurement.
図４は、乳汁中の活性酸素消去能と体細胞数の測定結果を示す図である。図４では、横軸に体細胞数（万個／ｍｌ）を、縦軸には活性酸素消去能（μＡ）を示している。ここで、縦軸では数値が大きい場合には活性酸素消去能が高く、数値が小さい場合には活性酸素消去能が低いことを表している。
乳汁中の活性酸素消去能と体細胞数（ＳＣＣ）や細菌数等の関係を調査するため、過酸化水素と乳汁（細胞膜を破壊し反応速度を上げるため界面活性剤を添加）を混合してＣＡ測定を行った。乳汁は１２ｍｌ、過酸化水素は２ｍｌ、１００ｍＭ、測定温度は２８〜３０℃とし、併せて乳汁中の体細胞数と細菌数を測定した。なお、活性酸素消去能は、測定器差補正と温度補正を行っている。測定器差補正については後述する。
図４に示すように、体細胞数が高くなると乳汁中の活性酸素消去能が高くなることが分かる。また、体細胞数が低くても消去能が高い場合があり、この場合は、細菌に感染し、殺菌のために活性酸素が放出されたことにより、消去能が高くなったと推察される。
従って、図４において、領域Ａは乳房炎に感染し、自然免疫では防御できず体細胞（白血球）が増加した群であり、領域Ｂは乳房炎に感染し、自然免疫により活性酸素を放出して殺菌している群であると推察される。
FIG. 4 is a diagram showing the results of measuring the active oxygen scavenging ability and the number of somatic cells in milk. In FIG. 4, the horizontal axis represents the number of somatic cells (10,000 cells / ml), and the vertical axis represents the active oxygen scavenging ability (μA). Here, on the vertical axis, when the numerical value is large, the active oxygen scavenging ability is high, and when the numerical value is small, the active oxygen scavenging ability is low.
In order to investigate the relationship between the ability to scavenge reactive oxygen in milk and the number of somatic cells (SCC) and bacteria, mix hydrogen peroxide and milk (add surfactant to break down cell membranes and increase reaction rate) CA measurement was performed. The milk was 12 ml, the hydrogen peroxide was 2 ml, 100 mM, the measurement temperature was 28-30 ° C., and the number of somatic cells and bacteria in the milk were also measured. The active oxygen scavenging ability is corrected by measuring instrument difference and temperature. The instrument difference correction will be described later.
As shown in FIG. 4, it can be seen that the active oxygen scavenging ability in milk increases as the number of somatic cells increases. Moreover, even if the number of somatic cells is low, the scavenging ability may be high. In this case, it is presumed that the scavenging ability is enhanced by infecting bacteria and releasing active oxygen for sterilization.
Therefore, in FIG. 4, region A is a group infecting mastitis and cannot be protected by innate immunity, but has increased somatic cells (leukocytes), and region B infects mastitis and releases active oxygen by innate immunity. It is inferred that the group is sterilized.
次に、本実施例による乳房炎検出方法に用いる測定用器具について説明する。
図５は本実施例による乳房炎検出方法を機能実現手段で示すブロック図、図６は同測定用器具の構成と使用方法を示す図、図７は、図６（ｅ）の状態を模式的に示した構成図、図８は同測定用器具を用いた測定フロー図である。
Next, a measuring instrument used in the mastitis detection method according to the present embodiment will be described.
FIG. 5 is a block diagram showing the mastitis detection method according to the present embodiment in terms of function realization means, FIG. 6 is a diagram showing the configuration and use method of the measurement instrument, and FIG. 7 is a schematic view of the state of FIG. FIG. 8 is a measurement flowchart using the same measuring instrument.
図５に示すように、本実施例による乳房炎検出方法は、測定用器具１０と、測定用コントローラ２０とを用いて行う。
測定用器具１０は、乳汁に供給する前の過酸化水素を収容する第１の空間（過酸化水素容器）１１と、過酸化水素を供給する前の乳汁を収容する第２の空間（乳汁容器）１２と、過酸化水素容器１１と乳汁容器１２とを連通させる連通手段（押棒）１３と、過酸化水素容器１１内又は乳汁容器１２内の過酸化水素濃度を測定する測定手段であるセンサプレート１４と測定ユニット１４’、乳汁容器１２内に乳汁を定量する乳汁定量手段１５とを備えている。
ここで、過酸化水素容器１１は、独立した複数の空間を備え、また乳汁容器１２も、複数の過酸化水素容器１１にそれぞれ対応する複数の乳汁容器１２を備えていることが好ましい。
また、センサプレート１４は、温度センサ１４ａとＣＡ測定用電極１４ｂを備えている。ＣＡ測定用電極１４ｂは、作用極と対極ともに白金電極を用いることが好ましい。ただし、白金電極以外に、イリジウムや、イリジウムとジルコニウムを含有する電極、又はカーボンを電極とするものであってもよい。
As shown in FIG. 5, the mastitis detection method according to this embodiment is performed using the measurement instrument 10 and the measurement controller 20.
The measuring instrument 10 includes a first space (hydrogen peroxide container) 11 for storing hydrogen peroxide before being supplied to milk, and a second space (milk container for storing milk before supplying hydrogen peroxide). ) 12, a communication means (push bar) 13 for communicating the hydrogen peroxide container 11 and the milk container 12, and a sensor plate which is a measuring means for measuring the hydrogen peroxide concentration in the hydrogen peroxide container 11 or the milk container 12. 14, a measurement unit 14 ′, and a milk quantitative means 15 for quantitatively determining the milk in the milk container 12.
Here, it is preferable that the hydrogen peroxide container 11 includes a plurality of independent spaces, and the milk container 12 also includes a plurality of milk containers 12 respectively corresponding to the plurality of hydrogen peroxide containers 11.
The sensor plate 14 includes a temperature sensor 14a and a CA measurement electrode 14b. The CA measurement electrode 14b is preferably a platinum electrode for both the working electrode and the counter electrode. However, in addition to the platinum electrode, iridium, an electrode containing iridium and zirconium, or carbon may be used.
測定用コントローラ２０は、センサプレート１４と測定ユニット１４’で測定するデータを読み取るデータ取込手段２１と、センサプレート１４と測定ユニット１４’での測定開始からの時間を計測する計時手段２２と、データ取込手段２１で取り込んだデータを基に活性酸素消去能を演算する演算手段２３と、この演算手段２３での演算結果によって細菌感染を判定する判定手段２４と、この判定手段２４での判定に用いる閾値を記憶する閾値記憶手段２５と、判定結果を出力する出力手段２６と、データ取込手段２１で取り込んだデータ及び判定手段２４での判定結果を記憶するデータ記憶手段２７とを備えている。 The measurement controller 20 includes a data capturing unit 21 that reads data measured by the sensor plate 14 and the measurement unit 14 ′, a time measuring unit 22 that measures time from the start of measurement by the sensor plate 14 and the measurement unit 14 ′, Calculation means 23 for calculating the active oxygen scavenging ability based on the data acquired by the data acquisition means 21, determination means 24 for determining bacterial infection based on the calculation result of the calculation means 23, and determination by the determination means 24 Threshold storage means 25 for storing the threshold value used for the output, output means 26 for outputting the determination result, and data storage means 27 for storing the data acquired by the data acquisition means 21 and the determination result of the determination means 24. Yes.
次に、図６及び図７を用いて、同測定用器具の構成と使用方法を説明する。
図６（ａ）は、搬送具内空間に、４つの乳汁容器１２と１つの捨て乳容器１２’を備えていることを示している。４つの乳汁容器１２には、乳汁定量手段１５によって、乳牛１頭の４分房の乳汁がそれぞれ定量収容されている。
図６（ｂ）は、図６（ａ）の状態において、それぞれの乳汁容器１２内に、あらかじめ過酸化水素を定量分注している過酸化水素容器１１を投入した状態を示している。
Next, the configuration and method of use of the measurement instrument will be described with reference to FIGS. 6 and 7.
Fig.6 (a) has shown that the milk container 12 and one discarded milk container 12 'are provided in the space in a conveyance tool. Each of the four milk containers 12 contains a fixed quantity of four-quarter milk from one dairy cow by the milk quantification means 15.
FIG. 6B shows a state in which the hydrogen peroxide container 11 in which hydrogen peroxide is quantitatively dispensed in advance is put in each milk container 12 in the state of FIG. 6A.
図６（ｃ）は、図６（ｂ）の状態にある搬送具とともに、センサプレート１４と、測定ユニット１４’とを示している。センサプレート１４には、温度センサ１４ａとＣＡ測定用電極１４ｂを備えている。また、測定ユニット１４’には、温度センサ用コンタクトピン１４’ａとＣＡ測定用コンタクトピン１４’ｂが設けられている。
それぞれの乳汁容器１２内に、過酸化水素容器１１を浮かべた状態で、センサプレート１４を乳汁容器１２にかぶせ、乳汁容器１２を、センサプレート１４が当接するように測定ユニット１４’に設置する。後述するように、乳汁容器１２底面に押棒１３が設けられているために、センサプレート１４を乳汁容器１２にかぶせた段階で過酸化水素容器１１内には、温度センサ１４ａ及びＣＡ測定用電極１４ｂが位置する。
FIG.6 (c) has shown the sensor plate 14 and measurement unit 14 'with the conveyance tool in the state of FIG.6 (b). The sensor plate 14 includes a temperature sensor 14a and a CA measurement electrode 14b. Further, the measurement unit 14 ′ is provided with a temperature sensor contact pin 14′a and a CA measurement contact pin 14′b.
With the hydrogen peroxide container 11 floating in each milk container 12, the sensor plate 14 is placed on the milk container 12, and the milk container 12 is installed in the measurement unit 14 'so that the sensor plate 14 abuts. As will be described later, since the push bar 13 is provided on the bottom surface of the milk container 12, the temperature sensor 14 a and the CA measurement electrode 14 b are placed in the hydrogen peroxide container 11 when the sensor plate 14 is placed on the milk container 12. Is located.
図６（ｄ）は、測定ユニット１４’に乳汁容器１２を設置した状態を示している。
図６（ｄ）に示すように、乳汁容器１２底面には、押棒１３が設けられている。
図６（ｅ）は、図６（ｄ）の状態にある乳汁容器１２に、保温カバー１６をかぶせた状態を示している。
FIG. 6D shows a state where the milk container 12 is installed in the measurement unit 14 ′.
As shown in FIG. 6 (d), a push bar 13 is provided on the bottom surface of the milk container 12.
FIG.6 (e) has shown the state which covered the heat retention cover 16 in the milk container 12 in the state of FIG.6 (d).
図７（ａ）は、図６（ｅ）の状態を、模式的に示した構成図である。
また、図７（ｂ）は、図７（ａ）の状態から、押棒１３を押し下げて過酸化水素容器１１の底面を割り、過酸化水素容器１１内の過酸化水素と乳汁容器１２内の乳汁を混合する状態を、模式的に示した構成図である。
FIG. 7A is a configuration diagram schematically showing the state of FIG.
7B, from the state of FIG. 7A, the push rod 13 is pushed down to split the bottom surface of the hydrogen peroxide container 11, and the hydrogen peroxide in the hydrogen peroxide container 11 and the milk in the milk container 12 are used. It is the block diagram which showed the state which mixes typically.
次に図８を用いて同測定用器具を用いた測定フローについて説明する。
まず、乳汁を乳汁容器１２に採取する（ステップ１）。ステップ１における乳汁は、乳牛１頭の４分房の乳汁を混合することなく採取する。ステップ２では、ステップ１で採取した４分房の乳汁の内、余計な乳汁が測定用器具を斜めに傾けることで乳汁容器１２の外側にある捨て乳容器１２’に排出され、各乳汁容器１２内の乳汁は一定量にされる。
そして、所定濃度の過酸化水素を過酸化水素容器１１に定量分注する（ステップ３）。
ステップ３において所定濃度の過酸化水素が定量分注された過酸化水素容器１１は、所定位置に設置される（ステップ４）。図６に示す実施例では、過酸化水素容器１１を乳汁容器１２内に浮かべる。
そして、センサプレート１４を過酸化水素容器１１を浮かべた乳汁容器１２の開口に設置し、センサプレート１４を設置した乳汁容器１２を測定ユニット１４’に設置する（ステップ５）。
Next, a measurement flow using the measuring instrument will be described with reference to FIG.
First, milk is collected in the milk container 12 (step 1). The milk in step 1 is collected without mixing the four-quarter milk from one cow. In Step 2, extra milk out of the four-quarter milk collected in Step 1 is discharged into the discarded milk container 12 ′ outside the milk container 12 by tilting the measuring instrument at an angle. The milk inside is made a certain amount.
Then, a predetermined concentration of hydrogen peroxide is quantitatively dispensed into the hydrogen peroxide container 11 (step 3).
The hydrogen peroxide container 11 into which a predetermined concentration of hydrogen peroxide has been dispensed in step 3 is placed at a predetermined position (step 4). In the embodiment shown in FIG. 6, the hydrogen peroxide container 11 is floated in the milk container 12.
And the sensor plate 14 is installed in the opening of the milk container 12 which floated the hydrogen peroxide container 11, and the milk container 12 in which the sensor plate 14 was installed is installed in measurement unit 14 '(step 5).
ステップ５が終了した後に、ＣＡ測定による電流値の測定を開始する（ステップ６）。
ステップ６における測定開始からの経過時間を計時手段２２にて計時し、ステップ７において、あらかじめ設定した時間（２７０秒）が経過した場合には、設定時間経過時点での過酸化水素容器１１内の過酸化水素濃度を測定する（ステップ８）。すなわち、ステップ８（第１の測定ステップ）では、乳汁に過酸化水素を供給する前の過酸化水素濃度をセンサプレート１４と測定ユニット１４’で測定し、測定したデータをデータ取込手段２１で取り込む。センサプレート１４と測定ユニット１４’では、それぞれの過酸化水素容器１１内の過酸化水素濃度を同時に個別に測定する。
ステップ８（第１の測定ステップ）における測定終了後に、押棒１３を操作することで、過酸化水素容器１１を破損し、過酸化水素容器１１内の過酸化水素と乳汁容器１２内の乳汁とを混合する（ステップ９）。
After step 5 ends, measurement of the current value by CA measurement is started (step 6).
The elapsed time from the start of measurement in step 6 is measured by the time measuring means 22, and in step 7, when a preset time (270 seconds) has elapsed, the hydrogen peroxide container 11 in the hydrogen peroxide container 11 at the time when the set time has elapsed. The hydrogen peroxide concentration is measured (step 8). That is, in step 8 (first measurement step), the hydrogen peroxide concentration before supplying hydrogen peroxide to the milk is measured by the sensor plate 14 and the measurement unit 14 ′, and the measured data is obtained by the data capturing means 21. take in. In the sensor plate 14 and the measurement unit 14 ′, the hydrogen peroxide concentration in each hydrogen peroxide container 11 is simultaneously measured individually.
After the measurement in step 8 (first measurement step) is completed, the hydrogen peroxide container 11 is broken by operating the push rod 13, and the hydrogen peroxide in the hydrogen peroxide container 11 and the milk in the milk container 12 are removed. Mix (step 9).
ステップ９における押棒１３の操作、すなわち過酸化水素容器１１内の過酸化水素と乳汁容器１２内の乳汁との混合からの経過時間を計時手段２２にて計時し、ステップ１０において、あらかじめ設定した時間（２７０秒）が経過した場合には、設定時間経過時点での過酸化水素濃度を測定する（ステップ１１）。すなわち、ステップ１１では、過酸化水素を供給した乳汁の過酸化水素濃度をセンサプレート１４と測定ユニット１４’で測定し、測定したデータをデータ取込手段２１で取り込む。センサプレート１４と測定ユニット１４’では、押棒１３で連通させた後のそれぞれの過酸化水素容器１１内又はそれぞれの乳汁容器１２内の過酸化水素濃度を同時に個別に測定する。
ステップ１２では、活性酸素消去能を演算手段２３において演算する。活性酸素消去能の演算は、ステップ８（第１の測定ステップ）で測定した過酸化水素濃度からステップ１１（第２の測定ステップ）で測定した過酸化水素濃度を減じて算出する。演算手段２３での活性酸素消去能の演算は、それぞれの乳汁容器１２別に行う。なお、この演算において、温度センサ１４ａで検出した温度を基に補正を行う。
ここで、演算手段２３での活性酸素消去能の演算を、それぞれの乳汁容器１２別に行うことで、検査対象が牛の場合には、４つの乳房を同時に個別に測定することができ、いずれかの乳房が感染している場合であっても、確実に乳房炎を検出することができるため、乳房炎の早期発見が行いやすくなる。
The elapsed time from the operation of the push bar 13 in step 9, that is, the mixing of the hydrogen peroxide in the hydrogen peroxide container 11 and the milk in the milk container 12 is measured by the time measuring means 22, and the time set in advance in step 10 When (270 seconds) has elapsed, the hydrogen peroxide concentration when the set time has elapsed is measured (step 11). That is, in step 11, the hydrogen peroxide concentration of the milk supplied with hydrogen peroxide is measured by the sensor plate 14 and the measurement unit 14 ′, and the measured data is captured by the data capturing means 21. In the sensor plate 14 and the measurement unit 14 ′, the hydrogen peroxide concentration in each hydrogen peroxide container 11 or each milk container 12 after being communicated by the push rod 13 is simultaneously measured individually.
In step 12, the active oxygen scavenging ability is calculated by the calculation means 23. The active oxygen scavenging ability is calculated by subtracting the hydrogen peroxide concentration measured in step 11 (second measurement step) from the hydrogen peroxide concentration measured in step 8 (first measurement step). The calculation of the active oxygen scavenging ability in the calculation means 23 is performed for each milk container 12. In this calculation, correction is performed based on the temperature detected by the temperature sensor 14a.
Here, the calculation of the active oxygen scavenging ability in the calculation means 23 is performed for each milk container 12, so that when the test object is a cow, four breasts can be measured individually, Even if the breast is infected, mastitis can be reliably detected, so that early detection of mastitis is facilitated.
ステップ１２で演算した結果に基づき、閾値記憶手段２５にあらかじめ記憶している閾値と比較し感染判定を行う（ステップ１３）。
ステップ１３での判定結果は出力される（ステップ１４）。また、ステップ８において測定された過酸化水素濃度、ステップ１１において測定された過酸化水素濃度、及びステップ１３における判定結果はデータ記憶手段２７に記憶される。
Based on the result calculated in step 12, an infection determination is made by comparing with the threshold value stored in advance in the threshold value storage means 25 (step 13).
The determination result in step 13 is output (step 14). Further, the hydrogen peroxide concentration measured in step 8, the hydrogen peroxide concentration measured in step 11, and the determination result in step 13 are stored in the data storage means 27.
図９は、乳房炎感染の簡易判定方法を示す図である。
図４で示した測定結果から、体細胞数が高くなると乳汁中の活性酸素消去能が高くなること、及び体細胞数が低くても活性酸素消去能が高い場合があることが分かった。
この結果から、図９に示すように、体細胞数が所定値以上の場合には、乳房炎であり、体細胞数が所定値以下の場合で活性酸素消去能が所定値より高い場合に初期段階の乳房炎原因菌感染と判断できる。
従って、体細胞数をあらかじめ測定し、体細胞数が所定値以下の場合に、過酸化水素消去能を検出することが好ましい。
このように、体細胞数が所定値以下の検体に限ることで、乳房炎発症の検査負担を減らし、予防検査を確実に行うことができる。
FIG. 9 is a diagram showing a simple determination method for mastitis infection.
From the measurement results shown in FIG. 4, it was found that the active oxygen scavenging ability in milk increases as the number of somatic cells increases, and that the active oxygen scavenging ability may be high even if the number of somatic cells is low.
From this result, as shown in FIG. 9, when the number of somatic cells is greater than or equal to a predetermined value, mastitis, and when the number of somatic cells is less than or equal to the predetermined value and the active oxygen scavenging ability is higher than the predetermined value, It can be determined that the infection is due to mastitis causing bacteria.
Therefore, it is preferable to measure the number of somatic cells in advance and detect hydrogen peroxide scavenging ability when the number of somatic cells is a predetermined value or less.
In this way, by limiting the number of somatic cells to a sample having a predetermined value or less, it is possible to reduce the examination burden for the development of mastitis and reliably perform a preventive examination.
以上のように本発明によれば、乳汁に所定の量の過酸化水素を供給し、乳汁の過酸化水素消去能を電気化学的方法によって検出する乳房炎検出方法において、ＣＡ測定により、過酸化水素分解酵素活性に応じて一定時間に減少する過酸化水素濃度を測定することで、乳汁中の溶存酸素濃度の影響を排除し、かつ過酸化水素分解酵素であるカタラーゼとグルタチオンペルオキシダーゼ両方の消去能が測定できる。
また、本発明では、最初に、既知濃度の過酸化水素をＣＡ測定により行った後、続いて、過酸化水素と乳汁とを混合してＣＡ測定を行うため、測定器差の補正が測定毎に行える。
As described above, according to the present invention, in a mastitis detection method of supplying a predetermined amount of hydrogen peroxide to milk and detecting the hydrogen peroxide scavenging ability of milk by an electrochemical method, peroxidation is performed by CA measurement. By measuring the hydrogen peroxide concentration that decreases in a certain time according to the hydrogenolytic enzyme activity, the effect of dissolved oxygen concentration in milk is eliminated, and the ability to eliminate both catalase and glutathione peroxidase, which are hydrogen peroxide decomposing enzymes, is eliminated. Can be measured.
In the present invention, first, hydrogen peroxide having a known concentration is measured by CA measurement, and then CA measurement is performed by mixing hydrogen peroxide and milk. Can be done.
本発明は、牛や山羊、その他動物の乳房炎検出方法に適用できる。 The present invention can be applied to a method for detecting mastitis in cattle, goats and other animals.
１０ 測定用器具
１１ 第１の空間（過酸化水素容器）
１２ 第２の空間（乳汁容器）
１３ 連通手段（押棒）
１４ 測定手段（センサプレート）
１４’ 測定手段（測定ユニット）
２０ 測定用コントローラ
２１ データ取込手段
10 Measuring instrument 11 First space (hydrogen peroxide container)
12 Second space (milk container)
13 Communication means (push bar)
14 Measuring means (sensor plate)
14 'Measuring means (measuring unit)
20 Measuring controller 21 Data acquisition means
Claims (6)
前記第１の測定ステップの後に前記乳汁に前記過酸化水素を供給して混合する供給ステップと、
前記供給ステップの所定時間経過後に前記過酸化水素濃度を測定する第２の測定ステップと、を有し、
前記第１の測定ステップで測定した前記過酸化水素濃度から前記第２の測定ステップで測定した前記過酸化水素濃度を減じて前記過酸化水素消去能を算出することを特徴とする請求項１又は請求項２に記載の乳房炎検出方法。 A first measurement step of measuring the hydrogen peroxide concentration before supplying the hydrogen peroxide to the milk;
A supply step of supplying and mixing the hydrogen peroxide to the milk after the first measurement step;
A second measurement step of measuring the hydrogen peroxide concentration after a predetermined time of the supply step,
The hydrogen peroxide scavenging ability is calculated by subtracting the hydrogen peroxide concentration measured in the second measurement step from the hydrogen peroxide concentration measured in the first measurement step. The mastitis detection method according to claim 2.
前記乳汁に供給する前の前記過酸化水素を収容する第１の空間と、
前記過酸化水素を供給する前の前記乳汁を収容する第２の空間と、
前記第１の空間と前記第２の空間とを連通させる連通手段と、
前記第１の空間内の前記過酸化水素濃度を測定するとともに、前記連通手段で連通させた後の前記第１の空間内又は前記第２の空間内の前記過酸化水素濃度を測定する測定手段と、
を備えたことを特徴とする乳房炎検出方法に用いる測定用器具。 A measuring instrument for use in the mastitis detection method according to any one of claims 1 to 4,
A first space for storing the hydrogen peroxide before being supplied to the milk;
A second space for containing the milk before supplying the hydrogen peroxide;
Communication means for communicating the first space and the second space;
Measuring means for measuring the hydrogen peroxide concentration in the first space and measuring the hydrogen peroxide concentration in the first space or the second space after being communicated by the communicating means. When,
A measuring instrument for use in a mastitis detection method, comprising:
前記測定手段では、それぞれの前記第１の空間内の前記過酸化水素濃度を同時に個別に測定するとともに、前記連通手段で連通させた後のそれぞれの前記第１の空間内又はそれぞれの前記第２の空間内の前記過酸化水素濃度を同時に個別に測定することを特徴とする請求項５に記載の乳房炎検出方法に用いる測定用器具。 A plurality of the first spaces and a plurality of the second spaces respectively corresponding to the plurality of the first spaces;
In the measurement means, the hydrogen peroxide concentration in each of the first spaces is measured individually and simultaneously, and each of the first spaces after being communicated by the communication means or each of the second spaces. The measurement instrument used for the mastitis detection method according to claim 5, wherein the hydrogen peroxide concentration in the space is individually measured simultaneously.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011073704A JP2012208006A (en) | 2011-03-29 | 2011-03-29 | Method for detecting mastitis, and measuring instrument for use in method for detecting mastitis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011073704A JP2012208006A (en) | 2011-03-29 | 2011-03-29 | Method for detecting mastitis, and measuring instrument for use in method for detecting mastitis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2012208006A true JP2012208006A (en) | 2012-10-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2011073704A Withdrawn JP2012208006A (en) | 2011-03-29 | 2011-03-29 | Method for detecting mastitis, and measuring instrument for use in method for detecting mastitis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2012208006A (en) |
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2011
- 2011-03-29 JP JP2011073704A patent/JP2012208006A/en not_active Withdrawn
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