JP2012181192A - Method for detecting bacterium contamination level of fish meat and sensor used for detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method with which it can be detected easily with naked eyes and for each fish meat without requiring any technical knowledge or skills for bacterium examination whether "the number of bacteria (viable bacteria) per specimen of 1 g is less than one million (according to official laws)" frequently used as an independent reference value regarding the number of bacteria of fresh fish for raw meal supplied in distribution, without damaging article value and regardless of a packaged state for raw fish meat (including non-seasoned fish processed article) being refrigerated, and to provide a simple sensor used for the detection method.SOLUTION: A method for detecting a bacterium contamination level of fish meat includes, for raw fish meal being refrigerated, attaching a sensor which includes azo dye (edible red No. 102 or the like) in contact with drip exudated from the fish meat, and observing the presence/absence of a change in the color of the sensor with naked eyes. For the detection method, a sensor is used in which the azo dye is adsorbed onto a planar medium such as cloth or dissolved and solidified in a solid medium such as agar.

Description

  The present invention relates to a method for detecting the level of bacterial contamination of raw fish meat and a sensor used in the detection method. Specifically, for refrigerated raw fish meat (including unseasoned processed fish products), a method for detecting the level of bacterial contamination with the naked eye without taking a sample and inspecting it and without opening the package And a simple sensor used for the detection method.

  In general, even when raw fish meat is stored in a refrigerated state, bacteria gradually grow and gradually become unfit for consumption. In other words, in raw fish meat that has been refrigerated, if the number of bacteria per gram of the sample counted by the official method (35 ° C culture counting method) exceeds 10 7, rotified nitrogen and volatile nitrogen such as amine and ammonia When the compound starts to be generated and further exceeds 10 8, the volatile nitrogen compound increases, and becomes rotted and unfit for consumption. In addition, when food poisoning bacteria multiply, food poisoning occurs.

  Therefore, the Food Sanitation Law defines bacteriological component standards for many foods, and regulates the distribution of foods that have been contaminated with bacteria beyond the standards. In addition, the Ministry of Health, Labor and Welfare has established a hygiene standard for foods that do not have ingredient standards (for example, bento, side dishes, pickles, raw confectionery, raw noodles, etc.), and is instructed to prevent the distribution of food that exceeds that standard. . For raw fish without ingredient standards or sanitary standards, voluntary standards are set by many retailers and voluntarily restricted so that fish exceeding that standard does not circulate.

  According to the component standards of the Food Sanitation Law and the Ministry of Health, Labor and Welfare's hygiene standards, the allowable level of bacterial contamination is determined by the “number of bacteria per gram of sample counted by the official method”. The bacterial contamination level of foods such as frozen rice cakes and boiled noodles is set to 100,000 or less per gram of sample, and the number of bacteria for raw noodles and utensils is set to 3 million / g or less according to hygiene standards.

  As mentioned above, distributors often set their own standards based on official laws for foods that do not have ingredient standards or hygiene standards. For example, according to the distribution standards of Sakai Living Cooperatives, the number of bacteria in chilled fillets and fresh fish for ingestion after heating should be less than 3 million / g, and the number of bacteria in refrigerated fillets and fresh fish for raw consumption should be less than 100,000 / g. Yes. In another cooperative, the number of bacteria such as octopus, salmon roe, fresh edible fish, and octopus is set at 10 6 or less per gram of specimen. Further, according to the investigations of the present inventors, there are many cases where the management standard for raw fish meat is voluntarily determined as “do not sell fish meat whose bacterial density per gram exceeds 10 6”.

  However, there are doubts as to whether foods sold in the refrigerated state meet these standards. That is, at present, for the extracted food, the bacterial contamination level at the time of manufacture and the increasing tendency of the number of bacteria during the storage period are examined, and based on that, the storage conditions, the expiration date and the expiration date are determined, and stored under the conditions, And it is only said that it does not exceed the standard within that period. However, since the level of bacterial contamination of food varies widely depending on the state of manufacture, even if it is within the expiration date or expiration date, the bacterial contamination level exceeds the component standards, hygiene standards, or voluntary standards. The possibility of being sold is undeniable.

  In fact, in Tokyo, the number of general bacteria per gram of sushi seeds and sashimi exceeded 1 million in the “unsuitable standard for bacterial test results of food and containers”, which was enforced until 2008. Has been judged to be unsuitable, but hygiene guidance has been carried out, but among 893 sushi seeds and sashimi removed in the Tama area between 1993 and 2002, the number of bacteria exceeds 1 million per gram Therefore, it has been reported that the number of specimens deemed unfit for sales reached 6.3% (published in 2004, Tokyo Health and Safety Research Center Annual Report 55).

  From this situation, it is desirable to confirm bacterial safety for all fish before eating fish. Conventionally, in order to examine the number of bacteria in fish meat, a bacteria test is performed by collecting a sample from fish meat. Bacteria testing includes colony counting using an agar plate for bacterial culture, MPN using liquid media, ATP measurement, etc., but once the test is made, the fish meat is sampled. If the packaging is removed, the product value will be damaged, making it unsuitable for sale. Moreover, in the test | inspection method using a bacterial culture medium, two days or more are required by determination. In other words, currently, no method has been developed for inspecting the total number of fish for distribution as they are and knowing the results of the inspection instantly. Therefore, it is impossible to confirm the bacterial contamination level of the fish.

  On the other hand, several methods have been publicly disclosed in which consumers themselves check freshness with the naked eye without attaching a sensor to a food or beverage package and collecting and inspecting a sample. For example, in Patent Document 1, an indicator that changes color with an acid gas is attached to an opening of a packaging container filled with a neutral beverage, and the color of the indicator is checked to confirm the presence or absence of alteration of the contents. A package made in this manner is disclosed. However, this package is applicable only to beverages or semi-fluid foods because the opening is a screw cap. It is unclear whether it can be applied to refrigerated foods. In addition, when this neutral beverage is fermented or spoiled to generate acid gas, the number of bacteria has reached the 10th power level of 10 per mL, exceeding the level for confirming the commercial value. Therefore, this package only shows an application example to a liquid food in a sealed state, and cannot be applied to beverages and foods with ingredient standards, hygiene standards, and even voluntary standards. In other words, this indicator cannot be used for the management of raw fish meat or fish meat foods that need to detect the number of bacteria per gram of fish meat at a level of at least 10 6.

  Patent document 2 targets foods that are easily spoiled and diffuses carbon dioxide generated when bacteria grow into the container to react with a sensor containing a pH sensing solution, so that the sensor changes from green to orange. It discloses a food freshness sensor that can be visually observed. However, this sensor detects carbon dioxide generated from food that has been spoiled in a sealed package, and the time until the concentration of carbon dioxide reaches 0.5% is affected by the volume of the package. Therefore, it is expected that the volume of the package and the ratio of the food to be encapsulated need to be kept constant, and the range of the target food is extremely limited. Further, in the data of FIG. 4 showing the performance, the number of bacteria per 1 g of the specimen is shown at the seventh power level of 10. Therefore, as in the technique of Patent Document 1, the component standard, the hygiene standard, and the self-standard Not applicable to beverages and foods with In other words, this sensor cannot be used for management of raw fish meat and fish foods that need to detect the number of bacteria per gram of fish meat at a level of at least 10 6.

  Further, page 75 of Non-Patent Document 1 discloses a nondestructive freshness sensor for fish meat or livestock meat. This sensor is intended to measure the amount of low molecular weight compounds that increase as the freshness of fish and livestock meat declines from the respiratory activity of spoilage bacteria. The spoilage bacteria are cultured and adsorbed and fixed on a membrane filter. The cell with the filter attached to the tip of the oxygen electrode is pressed against the surface of the fish meat or livestock meat, and the low molecular weight compounds in the fish meat or livestock meat are infiltrated into the cell by diffusion to be assimilated by spoilage bacteria. Is used to evaluate the quality of fish or livestock meat. However, although this sensor is a non-destructive type, it requires specialized knowledge and calculation to determine freshness, and consumers themselves cannot easily determine freshness visually. Thus, no method has yet been developed for confirming the bacterial contamination level of raw fish meat in a refrigerated state with the naked eye, whether sealed or unsealed, without requiring specialized knowledge or skills.

Next, fish meat drip, which occupies an important role in the present invention, will be described.
In general, when a fish dies and oxygen supply by respiration stops, fermentation and decomposition of glycogen in fish meat begins. When the pH of the fish meat begins to drop due to this action, the fish meat composed of proteins with an isoelectric point on the acidic side has a reduced ability to retain moisture, and the water hydrated to the fish protein exudes. start. This exudate is called “drip”. The dipping liquid that covers the surface of the fish is also drip. Because the drip contains free amino acids produced by the degradation of fish protein, bacteria are more prone to grow in the drip than in the fish meat. In other words, since the initial drip is for when the decomposition of fish meat is not progressing much, the amount of free amino acids is small and the number of bacteria is small from the number of bacteria in fish meat, but as time passes, The number of bacteria is larger than that of fish meat.

  In the process of studying the bacteria present in fish meat and its drip, the present inventors have obtained the knowledge that when the fish dies and the decomposition of fish protein proceeds, the number of bacteria in the drip becomes higher than the number of bacteria in the fish meat. It was. In addition, the bacteria in the refrigerated fish meat drip were counted by a 20 ° C. culture counting method, and it was found that when the number of bacteria exceeded 10 8 per mL, the pigment began to degrade rapidly. . Furthermore, the bacteria that preferentially grow in chilled fish meat are psychrophilic bacteria, but the number of bacteria is higher when counted using the 20 ° C. culture counting method than with the official method (35 ° C. culture counting method). I learned that It was also noticed that this psychrophilic bacterium had sufficient growth activity even at 4 ° C, comparable to that at 20 ° C. Based on these findings, the present inventors have continued research on the fish and drip bacteria and the number of bacteria, and have completed the present invention.

JP 2004-359319 A Special table 2008-523391

Edited by Naryo Watanabe, “Freshness and Processing / Storage of Seafood” February 8, 1998

  In view of the above situation, the present invention can be applied to raw fish meat (including unseasoned processed fish meat) that has been refrigerated without damaging the commercial value of the fish meat and regardless of its packaging state. Whether the number of bacteria (live bacteria) per gram of the sample is less than 1 million (according to the official method), which is frequently used as a voluntary standard value for the number of bacteria in fresh fish for serving, that is, the number of bacteria is 999, To provide a method that can be easily detected with the naked eye and for each fish meat, and a simple sensor that is used for the detection method, without requiring specialized knowledge and skills relating to bacterial testing, whether or not it is 999 / g or less. Is an issue.

  Among the present inventions for solving the above-mentioned problems, the invention described in claims 1 and 2 includes an azo dye so that raw fish meat that has been refrigerated comes into contact with a drip that exudes from the fish meat. This is a method of detecting the level of bacterial contamination of the fish meat by attaching a sensor and observing the color change of the sensor with the naked eye. In the present invention, “refrigeration” means storage at a temperature of approximately 10 ° C. or less without freezing.

  The invention described in claim 2 is the method for detecting the bacterial contamination level of fish meat according to claim 1, wherein the fish meat to be detected is in a hermetically sealed state.

  The invention described in claim 3 is a method for detecting the bacterial contamination level of fish meat according to claim 1 or 2, wherein an azo dye adsorbed on a flat medium such as paper or cloth is used as a sensor. is there.

  The invention described in claim 4 is a method for detecting the level of bacterial contamination in fish meat according to claim 1 or 2, wherein the sensor is obtained by dissolving and solidifying an azo dye in a solid medium such as agar or gelatin. It is.

  The invention described in claim 5 uses edible red No. 2, edible red No. 40, edible red No. 102, edible yellow No. 4, or edible yellow No. 5 as the azo dye. It is a method to detect the bacterial contamination level of fish meat.

  In the invention described in claim 6, the azo dye is adsorbed on a flat medium such as paper or cloth, or is dissolved and solidified in a solid medium such as agar or gelatin, and the bacterial contamination level of fish meat It is a sensor used for detecting.

  According to the detection method described in any one of claims 1 to 5, the raw fish meat being refrigerated has a level of bacterial contamination without damaging its commercial value and irrespective of the packaging state of the fish meat. Whether or not “the number of bacteria (viable bacteria) per gram of the sample is less than 1 million (according to the official method)”, which is frequently used as a voluntary standard value for distribution, that is, the number of bacteria is 999,999 / g or less Whether or not the sensor attached to the fish meat is discolored can be easily confirmed for each fish meat simply by observing with the naked eye. Therefore, according to these detection methods, even if it is not a person who has specialized knowledge and skills regarding bacterial inspection and fish freshness determination, even if it is dripping, the bacterial contamination level of the fish It is possible to determine whether or not the number of bacteria exceeds the voluntary standard of “does not sell those exceeding 10 6 per g of fish”.

  That is, according to the detection method according to any one of claims 1 to 5, when the bacterial contamination level of raw fish meat reaches a certain value or more, the sensor attached to the fish meat changes color. Can be confirmed by their own eyes at the time of purchasing the fish or eating the fish without being caught by the expiration date or the expiration date.

  In particular, the detection method according to claim 2 is intended for raw fish meat in a sealed packaging state in which the contents cannot be easily inspected, and bacterial contamination of the raw fish meat in the sealed packaging state. It is extremely useful in that the level can be detected.

  The detection method according to claim 3 or 4 uses a sensor including an azo dye. However, since the azo dye has a property of being stably decomposed by bacteria in the drip, the bacterial contamination level of fish meat Can be confirmed accurately.

  In addition, when the sensor according to claim 6 is attached to the raw fish meat so as to come into contact with the drip, the number of bacteria per 1 g of the fish meat reaches “10 6 (the official method)”. Since it changes color at the time, simply attaching this sensor makes it easy to confirm the bacterial safety of fish meat with the naked eye. In addition, since this sensor is safe and can be manufactured inexpensively and easily, even if it is attached to the total number of fish or fish food to be inspected, the cost does not increase.

It is the graph which showed the growth of the fish drip bacteria in an azo dye addition culture medium and a non-addition culture medium. It is the graph which showed the change of the bacteria count of the fish meat of the yaz refrigerated at 4 degreeC, and its drip. It is the graph which showed the change by investigating the change in the number of bacteria of the fish meat of the yaz refrigerated at 4 degreeC by a different culture | cultivation counting method. It is the graph which showed the growth at the time of "4 degreeC 48 hours" culture | cultivation and the "20 degreeC 48 hours" culture | cultivation of the fish-derived bacteria isolate | separated by the 4 degreeC culture | cultivation counting method. It is the graph which showed the growth at the time of "48 degreeC at 48 degreeC" culture | cultivation at the time of "48 degreeC at 20 degreeC" of the bacteria derived from the fish meat isolate | separated by the 20 degreeC culture | cultivation counting method. It is the graph which showed the decomposition | disassembly of edible red No. 102, and the relationship of bacterial density. It is explanatory drawing of the method of attaching the solid medium sensor provided with the drip introduction part to fish meat.

  In the present invention, “fish meat” is a general term for meat chunks and pieces of meat taken out from fish bodies. In other words, in addition to fillets, “processed fish meat products that are not seasoned” such as fillets for grilled fish and boiled fish, fillets for steak, raw sashimi, and minced processed foods are included. Moreover, “raw fish meat” means unheated and undried fish meat, and includes a thawed product. As a result of trial production and testing, the detection method according to the present invention is sufficiently applicable to smoked salmon made using the cold-cooking method, so the term “unseasoned / unheated / undried” in the present invention is used. “Raw fish meat” includes processed smoked salmon fish.

  The present invention is mainly intended for non-sterilized fish meat and fish meat foods in view of its gist, but even if it has been sterilized, the sterilization may be insufficient. It is not limited to raw fish that have not been applied. The raw fish meat targeted by the present invention may be unwrapped or packaged (vacuum packaged or aerated packaged product).

  The present invention utilizes the property that bacteria derived from fish meat rapidly decompose pigment when the density exceeds a certain level, and an increase in the number of drip bacteria exuded from fish meat is detected by a sensor containing the pigment. There is a big feature in judging the number of bacteria of fish meat. Therefore, first, the sensor used in the present invention will be described.

  The sensor used in the present invention basically adsorbs an azo dye approved as a food additive to a flat medium with good water absorption, such as kraft paper, silk cloth, nylon cloth, polyester cloth, non-woven cloth, and microfiber cloth. Or dissolved and solidified in a solid medium such as agar or gelatin. In addition, since the sensor using a solid medium is easy to collapse, it is preferable to use it by covering one side with a strong and transparent reinforcing material such as a polyethylene film or a stomat bag film.

  As described above, an azo dye is used in the sensor of the present invention. The reason why the azo dye is used is that the azo dye has no influence on the growth of bacteria in the fish drip and the present inventors have used many dyes (flavonoid dyes, monascus dyes, iridoid dyes, quinoid dyes). This is because, as a result of testing with respect to dyes and azo dyes, it was found that only azo dyes were stably decomposed by bacteria in the drip. Therefore, if an azo dye is used, the change in the number of bacteria in the drip can be grasped more accurately. As the azo dye, it is preferable to use Food Red No. 2, Food Red No. 40, Food Red No. 102, Food Yellow No. 4, and Food Yellow No. 5.

  In the present invention, the azo dye is preferably used by diluting a commercially available bulk powder to about 50 to 100 μM (1 / 30,000 to 1 / 5,000). The reason is that if the concentration of the dye is decreased, the color may be changed even when the number of bacteria in the sample is small. On the other hand, if the concentration is too high, it is uneconomical and the sensitivity of the sensor tends to decrease. In the Food Sanitation Law, when an azo dye is used as a colorant for food, the bulk is diluted to 10,000 to 1 / 50,000 or less.

The sensor used in the present invention needs to be attached to the fish meat so as to come into contact with the drip that exudes from the fish meat. For example, the following method can be used to attach the sensor.
(1) In the case of a sensor in which an azo dye is adsorbed on a planar medium (hereinafter referred to as “planar medium sensor”), the simplest method is to attach the sensor to the surface of fish meat. When a flat medium sensor is attached to the surface of the fish meat, the drip that has exuded slightly on the surface of the fish meat and the sensor easily come into contact. In addition, when packaging the fish meat to which the sensor is attached, it is preferable to use at least a portion covering the sensor in the packaging material so that the state of the sensor can be confirmed without peeling off the package.

(2) In the case of a sensor in which an azo dye is dissolved and solidified in a solid medium such as agar (hereinafter referred to as “solid medium sensor”), one surface thereof is covered with a transparent reinforcing material, and the other surface is covered with a fish meat surface. What is necessary is just to take the method of sticking. Alternatively, the surface to be affixed to the fish meat can be stacked on a cloth having good water absorption, such as a nylon cloth, and the surface of the cloth can be affixed to the fish meat. If this method is adopted, the sensor does not have to be in direct contact with the fish meat. The drip that oozes on the surface of the fish meat comes into contact with the sensor through the cloth.
(3) In addition, an azo dye is adsorbed on one end of the cloth having good water absorption, or a cross formed by adsorbing an azo dye or agar dissolved and solidified is overlapped to form a sensor on the part, You may make it affix on fish meat the edge which is not adsorb | sucking a pigment | dye as an introduction part of a drip (FIG. 7). When this method is adopted, the drip is transferred from the portion (introduction portion) attached to the fish meat through the cloth to contact the sensor forming portion where the dye is adsorbed.

(4) A method for attaching the sensor will be described with reference to FIG. FIG. 7 is an explanatory view of a method for attaching a sensor having a drip introduction part to fish meat. In FIG. 7, a line formed by connecting black squares in a dotted line represents a microfiber cross, and the left half of the cross is the back side of the fish meat as a drip introduction part (a part that absorbs the drip and introduces it into the sensor). It is pasted on. In addition, a solid medium sensor made of 0.9% agarose obtained by dissolving and solidifying edible red No. 102 having a concentration of 50 μM is placed on the right half of the cloth, and the entire periphery of the sensor is a transparent packaging film. It is covered so that the sensor does not touch the fish meat. Fish meat is covered with a packaging film. When the sensor is attached to the fish meat as shown in FIG. 7, the drip travels through the microfiber cloth from the introduction portion attached to the fish meat and contacts the sensor forming portion.

(5) In the case where the fish meat is a tray packaged product or a vacuum packaged product, in addition to the above methods, a method of providing a recess in the bottom of a container such as a tray and attaching a sensor to the recess can be employed. The drip drops from the fish into the recess and contacts the sensor. It is preferable to make the recess or the entire bottom surface transparent so that the reaction of the sensor can be confirmed from the bottom surface of the container. In this method, either a flat medium sensor or a solid medium sensor can be used.
(6) Another method of attaching the sensor to fish meat is to attach the sensor in advance to the inside of a transparent wrapping material (eg food wrap film) that comes into contact with the fish meat, and cover the fish meat with the wrapping material. You may make it a water absorption surface contact the surface of fish meat. In this method, either a flat medium sensor or a solid medium sensor can be used.

  Exemplifying how to make the sensor used in the present invention, in the case of making a flat medium sensor, a flat medium (corresponding to 0.5 g) cut and formed into an appropriate size according to the size of fish meat is used as a raw material for a commercially available azo dye. After immersing the powder in a solution diluted to about 1 / 10,000 of the density to sufficiently absorb and dye the dye, it is washed and dried by an appropriate method, and the dye is fixed by a method such as heating. . In the case of making a solid medium sensor, for example, a commercially available azo dye bulk powder is added to an agar melt or gelatin melt prepared to an appropriate concentration so that the final concentration is about 50 μM (about 1 / 30,000). What is necessary is just to cool and solidify after melt | dissolving the thing diluted to (2) and mixing well. When increasing the concentration of the azo dye, it is better to confirm that the dye does not transfer from the sensor to the fish meat, or to confirm that the level is negligible even if transferred.

Another method for producing a sensor by dissolving an azo dye in a solid medium is as follows. First, azo dye powder is dissolved in distilled water so as to have a concentration of 5 mM, and this is filtered and sterilized with a Nuclepore filter having a pore size of 0.2 μM, and this solution is designated as solution A. On the other hand, 0.9 to 1.2 g of agar powder is put in 99 mL of distilled water, dissolved and sterilized by autoclaving to obtain solution B. Next, if 1 mL of solution A is aseptically added before solution B solidifies, a solid medium sensor containing a dye at a concentration of 50 μM can be produced. The amount of the agar powder is preferably adjusted so that the final gel strength is 700 g / cm ² in consideration of the characteristics of the agar used.

Next, a method for detecting the level of bacterial contamination of fish meat according to the present invention will be described.
As a result of research on bacteria existing in fish meat and its drip, the present inventors have obtained some knowledge as described above, and based on this knowledge, developed a detection method according to the present invention.

  When the sensor containing the azo dye was attached so as to be in contact with the drip of the fish meat, even if the fish meat was refrigerated, the number of bacteria in the drip exceeded 10 8 per mL (according to the 20 ° C. culture counting method). At that point, the dye in the sensor is rapidly degraded and its color changes. In the case of refrigerated fish meat with a product temperature of 10 ° C. or lower, psychrotrophic bacteria isolated from the colonies on the plate used in the 4 ° C. culture count method or the 20 ° C. culture count method (hereinafter “20 ° C. culture count method” The number of bacteria is larger than that of mesophilic bacteria isolated by the official method (35 ° C. counting culture method).

  When the number of drip bacteria counted by 20 ° C. culture counting method is 10 8 per mL, the number of fish meat bacteria counted by official method is 10 6 per g. In other words, the sensor changes color when the number of bacteria in the fish exceeds “1 million / g (according to the official method)”, so the number of bacteria in the fish is “per g It can be estimated that it is less than 1 million (by official method). Therefore, when the voluntary standard value of bacterial contamination level of raw fish meat is set to “less than 1 million / g”, it is only necessary to check whether the sensor attached to touch the fish drip is discolored or not. Whether or not the bacterial contamination level is within the voluntary standard can be easily determined by sagging.

That is, if the number of bacteria required for the sensor to react is 200,000,000 by 20 ° C. culture counting method, that is, 2 × 10 ⁸ , the number of fish bacteria counted by the official method is the number of bacteria. If it is 1/40 to 1/200, the number of bacteria in the fish meat will be between 5 × 10 ⁶ and 1 × 10 ⁶ . That is, the sensor reacts when the number of bacteria in the fish meat counted by the official method reaches the sixth power of 10 per gram. Thus, the accuracy of the sensor is determined by the number of bacteria required for the reaction and the magnitude of the “expansion of the ratio between the number of drip bacteria and the number of fish bacteria”.

  In the present invention, as a result of various tests, as shown in the following test examples, the “number of drip bacteria counted by the 20 ° C. culture counting method” divided by “the number of fish bacteria counted by the official method” is always about 50. As long as the number of drip bacteria counted by the 20 ° C. culture counting method is 10 8 per mL, the number of fish meat bacteria counted by the official method is 1 g. It can be judged that it is 10 6 per hit.

It is confirmed by the following test examples and examples that the method for detecting the bacterial contamination level of fish meat according to the present invention is accurate. That is,
(1) From Test Example 1 and FIG. 1, it is confirmed that the azo dye has no influence on the growth of the fish drip bacteria.
(2) It is confirmed from Test Example 2 and FIG. 2 that in the second half of refrigeration, the number of drip bacteria counted by the 20 ° C. culture counting method is higher than the number of fish meat bacteria.
(3) According to Test Example 3 and FIG. 3, since the psychrophilic bacteria are activated in the refrigerated fish meat, the bacterial density is determined by the 4 ° C. culture counting method or the 20 ° C. culture counting method rather than counting by the official method. It has been confirmed that counting is higher. Further, it has been confirmed that the number of bacteria counted by the 4 ° C. culture counting method is almost the same as the number of bacteria counted by the 20 ° C. culture counting method.
(4) From Test Example 4 and FIGS. 4 and 5, it is confirmed that the bacteria in the drip have sufficient activity to react the sensor even at 4 ° C.
(5) From Test Example 5 and FIG. 6, it is confirmed that the bacteria in the drip rapidly change the color of the pigment when the number of bacteria reaches 10 8 per mL.
(6) According to Test Example 6 and Table 1, when the number of drip bacteria counted by 20 ° C. culture counting method is 10 8 per mL, the number of fish bacteria counted by official method is 10 6 per g. It has been confirmed that it is a power.
(7) From the results of trial manufacture and trial use shown in Examples 1 to 7, it has been confirmed that both the “planar medium sensor” and the “solid medium sensor” can be sufficiently put into practical use.
Hereinafter, the present invention will be described in more detail with test examples.

<< Test Example 1 >>
(1) Test purpose :
Confirmation of the effect of azo dye on bacterial growth of fish drip (2) Test method :
When an azo dye is used for the sensor, the number of bacteria in the fish drip cannot be accurately measured if the azo dye has any influence on the growth of the fish drip bacteria. Therefore, Yazu drip was inoculated into a liquid medium (100 ppm: pH 7.0) of fish meat powder (Wako Pure Chemical Industries), which was prepared by imitating the fish meat drip, and the liquid medium was designated as “Commercial Edible Red No. 102 at a concentration of 50 μM. Dispense equal amounts into test tube A filled with 0.3% agar gel containing sensor to 1/3 height and empty test tube B, and incubate at 10 ° C did. During the culture period, the culture solution was collected aseptically and timely and used as a stock solution. This stock solution was repeatedly diluted 10 times with sterilized physiological saline, and 0.1 mL each of the stock solution and the diluted solution was smeared and inoculated on a normal agar plate medium, and this plate medium was cultured at 20 ° C. for 5 days, The colonies that appeared on the plate were counted, and changes in the bacterial density in the fish meat powder liquid medium were examined over time.

(3) Test results :
The test results are as shown in FIG. That is, there was no difference in bacterial growth between the test tube A medium and the test tube B medium. In this test example, edible red No. 102 was used as the azo dye, but the test using edible red No. 2, edible red No. 40, edible yellow No. 4 and edible yellow No. 5 was also the same as this test example. The same result is obtained.
(4) Consideration :
From this test result, it was confirmed that the azo dye had no effect on the growth of bacteria in the fish drip and could accurately measure the bacterial density.

<< Test Example 2 >>
(1) Test purpose :
Confirmation of change in bacterial count of refrigerated fish meat and its drip (2) Test method :
The raw meat for horse mackerel was divided into 6 to 7 pieces, each packaged separately with air and refrigerated at 4 ° C., and one package was opened at a time during the refrigeration period, and the fish meat and drip were taken out. About 1 g of fish meat was weighed and immersed in 9 times the amount of sterile physiological saline, and homogenized with a stomacher. Using the supernatant of this homogenized solution as a stock solution, dilution was repeated 10 times using sterile physiological saline, and 0.1 mL each of the stock solution and the diluted solution was smeared and inoculated on a normal agar plate medium. The drip is aseptically removed 1 mL at a time and used as a stock solution. This stock solution is diluted 10 times with sterile physiological saline, and 0.1 mL each of the stock solution and the diluted solution is smeared on a normal agar plate medium. Vaccinated. Next, these plate culture media were cultured at 20 ° C. for 5 days, colonies that appeared on the plate were counted, and changes in the number of bacteria in fish meat and drip were examined over time.

(3) Test results :
The result of the test is as shown in FIG. That is, the bacterial density of the drip is higher than that of the fish meat, and in the second half of the refrigeration, the difference was about 10 times.
(4) Consideration :
From this test result, in order to detect the number of bacteria in fish meat, it is confirmed that it is more sensitive and efficient to detect the number of bacteria in drip and indirectly know the number of bacteria in fish meat. It was done.

<< Test Example 3 >>
(1) Test purpose :
Confirmation of bacterial growth in chilled fish meat (2) Test method :
Raw edible fish meat was divided into 6 to 7 pieces, each was aerated and packaged separately, refrigerated at 4 ° C., and the number of bacteria in the fish meat was examined by the same method used in Test Example 2. The plate medium was cultured separately at 4 ° C. for 10 days, at 20 ° C. for 5 days, and at 35 ° C. for 2 days, and the colonies that appeared on the plate were counted. I investigated.

(3) Test results :
The result of the test is as shown in FIG. In other words, in the case of a yam refrigerated at 4 ° C., the number of fish bacteria (thermophilic bacteria) counted by the 4 ° C. culture count method and the 20 ° C. culture count method is almost the same, but whichever method is used. It was confirmed that the number of bacteria counted was larger than the number of bacteria (medium-temperature bacteria) counted by the 35 ° C. culture counting method (official method) defined by the Food Sanitation Law.
(4) Consideration :
From this test result, it was found that when fish were refrigerated, there were more psychrophilic bacteria than mesophilic bacteria. In other words, it was confirmed that when the fish meat was refrigerated, it was psychrotrophic bacteria that proliferated well at 20 ° C. or lower that caused the fish meat to deteriorate or decay. Therefore, it was confirmed that the method for detecting the bacterial contamination level of fish meat according to the present invention is useful for fish meat that has been refrigerated because the sensor mainly reacts with cold bacteria at low temperatures.

<< Test Example 4 >>
(1) Test purpose :
Confirmation of activity of psychrophilic bacteria under refrigerated temperature (2) Test method :
Bacteria separated by 4 ° C. culture counting method and bacteria separated by 20 ° C. culture counting method were inoculated into ordinary liquid medium from yaz fish meat refrigerated at 4 ° C., respectively, and 48 ° C. at 4 ° C. and 20 ° C. respectively. After culturing for a time, the turbidity of the culture solution was measured using a spectrophotometer at a wavelength of 700 nm.

(3) Test results :
The results of the test are as shown in FIGS. That is, looking at the graph showing the growth of the bacterial strain isolated by the 4 ° C. culture counting method in FIG. 4, the culture solution whose turbidity was below the measurement limit immediately after inoculation was found in 48 hours at 4 ° C. , The turbidity reaches a value between 0.1 and 0.4, while 48 hours growth at 20 ° C. of the same strain results in a turbidity value between 0.1 and 0.5. Has reached. That is, the growth level at 4 ° C. of the bacterial strain isolated by the 4 ° C. culture counting method is quite close to the degree of growth at 20 ° C. Furthermore, in the graph showing the growth of the bacterial strain isolated by the 20 ° C. culture counting method in FIG. 5, the culture solution whose turbidity was below the measurement limit immediately after inoculation was found in 48 hours at 4 ° C. , The turbidity reached a value between 0.2 and 0.3, while growth of the same strain at 20 ° C. for 48 hours caused the turbidity to be between 0.2 and 0.5. The value has been reached. That is, the growth level at 4 ° C. of the bacterial strain isolated by the 20 ° C. culture counting method is quite close to the growth level at 20 ° C. Therefore, it was confirmed that the bacteria isolated by the 20 ° C. culture counting method have a growth activity comparable to 20 ° C. even at 4 ° C.
(4) Consideration:
From the results of this test, it was clarified that the psychrotrophic bacteria that deteriorate and rot fish meat have sufficient activity to react the sensor even at 4 ° C.

<< Test Example 5 >>
(1) Test purpose:
Confirmation of bacteria density range of fish drip when decomposing azo dye (2) Test method :
Three normal liquid media (meat extract 10 g, peptone 10 g, NaCl 3 g, pH 7.2) containing commercially available edible red No. 102 at concentrations of 50 μM, 10 μM, and 5 μM were inoculated with drip of raw edible yam, respectively. Cultured at 0 ° C. The color of this culture was examined by measuring the absorbance at a wavelength of 507 nm with a spectrophotometer. Further, at the same time as examining the change in color tone, the number of bacteria in the culture solution was examined by the same method as described in Test Example 2. In addition, 507 nm is the light absorption maximum value of edible red No. 102, and the residual ratio of edible red No. 102 remaining in the medium was calculated from this absorbance.

(3) Test results :
The results of the test are as shown in FIG. That is, when the concentration of edible red No. 102 is 50 μM, red begins to decrease when the number of bacteria in the liquid medium exceeds 10 7, and decreases rapidly when it exceeds 10 8. Almost disappeared. The tendency is similar when the dye concentration is 10 μM and 5 μM. Therefore, the sensor used in the present invention accurately changes the color when the number of bacteria in the fish drip during refrigeration is counted by 20 ° C. culture counting method and reaches 10 8 per mL. For example, there is no risk of misjudging the fish meat that is 9th power or higher, such as 10 6th power or 10th 8th power. In this test example, food red No. 102 is used as the azo dye, but the test using food red No. 2, food red No. 40, food yellow No. 4, and food yellow No. 5 is the same as FIG. The result is obtained. In such a test, it is desirable to use an agar gel imitating a sensor, but since it is difficult to examine the change in absorbance of the agar gel, a liquid medium was used in this test example.
(4) Consideration :
From the results of this test, it was confirmed that the bacterial density exhibited by the sensor was reliable at the order level.

<< Test Example 6 >>
(1) Test purpose:
Confirming the proportion of bacteria in chilled fish and its drip
(2) Test method:
In order to determine the bacteria count of fish drip, 1 mL or more of drip is required, but it is impossible to collect such a large amount of drip with a normal fish packaging method. Therefore, 200 g of fish meat was put in a transparent plastic bag formed into a funnel shape, and the tube portion of the funnel was filled with solid agar (concentration 0.9%) dissolved in edible red No. 102 at a concentration of 50 μM. The number of bacteria in fish meat and drip when the food red No. 102 in the test tube changed color was examined by the same method used in Test Example 2. The plate medium was cultured at 20 ° C. for 5 days and at 35 ° C. for 2 days.

(3) Test results:
The test results are shown in Table 1. That is, for fish meat stored at 4 ° C. or 10 ° C., the 50 μM concentration of edible red No. 102 in the test tube is discolored by counting by the 20 ° C. culture counting method at 10 8 power per mL of drip. However, the number of bacteria when the same fish meat was counted by the official method was 10 6 per gram of fish meat. The ratio of the number of bacteria in the drip counted by the 20 ° C. culture counting method and the number of bacteria in the fish meat counted by the official method at this time was between 47 and 130. Further, even when the number of bacteria in the drip was divided by a value between 47 and 130, the number of bacteria was always 10 6.
(4) Consideration:
Even if the ratio of the number of bacteria in the drip by the 20 ° C culture counting method to the number of bacteria in the fish meat by the official method varies between about 50 and 130, the number of bacteria in the fish meat when examined by the official method is 10 6 or more. It is presumed that the sensor reacts because the number of bacteria in the drip when the sensor reacts converges within a narrow range. In addition, even if the number of bacteria in the drip is divided by a value between about 50 and 130, the number of bacteria is always 10 to the sixth power. It was confirmed that the color changed when it reached 10 6. That is, it was confirmed that if the method of detecting the growth of drip bacteria by changing the pigment was used, it was possible to accurately detect whether the number of bacteria in fish meat was less than 10 6.
The present invention will be further described below with reference to examples.

<Example of “Flat media sensor” and its usage>
(1) Production of sensor :
A. Distilled water (40 mL) was placed in a shot bottle, and a small amount of acetic acid was added to adjust the pH to 4.
B. Add 0.02 g of sodium sulfate to the shot bottle, and add 20 white microfiber cloths (20% nylon, 80% polyester: 2 cm × 1 cm) (corresponding to 0.2 g) for 60 minutes at 30 ° C. Heated.
C. Thereafter, 10 mL of an azo dye solution (0.002 g / 10 mL: pH 4) was added and heated at 100 ° C. for 60 minutes. As shown in Table 2, the azo dyes were used separately for food red No. 2, food red No. 40, food red No. 102, food yellow No. 4, and food yellow No. 5 depending on the fish species.
D. After being allowed to cool overnight in a shot bottle, the cloth was taken out, washed with water, and air-dried to produce a “red sensor”.
E. When the amount of dye remaining in the staining solution after staining of the cloth and the amount of dye eluted in the washing solution after washing with water were measured by the method shown in Test Example 5, the concentration of the dye on the cloth was about 15,000 times. , Equivalent to 100 μM.

(2) Sensor mounting :
Each of the above flat medium sensors was attached to the surface of 8 types and 13 slices of commercially available fish meat, and each was placed in a nylon and polyethylene laminate bag and stored at 4 ° C. or 10 ° C. Observation was continued during the storage period, and the fish meat was removed from the bag when the sensor color changed from red to white.
(3) Confirmation test of sensor detection effect :
A sample of about 10 g was aseptically collected from each fish meat taken out from the bag, placed in a stomacher bag, added with 9 times the amount of sterile physiological saline, and homogenized for 2 minutes to prepare a fish meat slurry.
B. Each slurry was transferred to a Corning tube and centrifuged at 500 rpm for 10 seconds.
C. The resulting supernatant was used as a stock solution, and after 10-fold dilution with sterilized physiological saline, 1 mL of the diluted solution was inoculated into each sterilized petri dish for each dilution step, and cooled to 55 ° C after sterilization. A standard agar medium or a normal agar medium was added to perform pour culture.
D. In the case of a standard agar medium, culture was performed at 35 ° C. for 48 hours in accordance with the provisions of the Food Sanitation Law, and the number of each bacteria was examined.

(4) Test results :
Table 2 shows the number of bacteria for each fish species when the flat medium sensor changes color. According to Table 2, the number of bacteria at the time when the sensor reacted was determined by the official method for both fish preserved at 10 ° C and fish preserved at 4 ° C. "Met.
(5) Consideration :
That is, when the raw fish meat attached with the sensor made in Example 1 in contact with the drip is sold side by side in a showcase such as a retail store managed at 4 to 10 ° C., the bacterial count of the raw fish meat Confirms that consumers can inspect the total number of fish sold with their own eyes whether or not it exceeds the voluntary standard of “do not sell if it reaches 10 6 (per official law)” It was.

<Example of how to use the "Flat media sensor">
(1) Sensor mounting and storage test :
Each of the nine types of commercially available fish fillets was divided into three, and the number of bacteria in each group of fillets was examined by the same method as in Example 1. The remaining two were divided into Group A and Group B, both of which were placed on a fillet with a flat medium sensor produced by the same method as in Example 1, and each was placed in a transparent polyethylene bag. Group A fillets were stored at 0 ° C. for 12 hours from the start of the test and then transferred to a room at 4 ° C. or 10 ° C. for continued storage. Group B fillets were stored in a 4 ° C. or 10 ° C. room from the beginning of the test. The presence or absence of a change in sensor color was continuously observed for each fillet, and when the group B fillet sensor turned white, the number of bacteria in the fillet was examined in the same manner as in Example 1.

(2) Test results :
Table 3 shows the number of bacteria for each fish species when the B group fillet sensor changes color. According to Table 3, the number of bacteria at the time when the sensor reacted was in the order of “10 6 / g (according to the official method)”, and the difference from the unreacted fillet was about 10 times. The minimum difference was twice. In addition, the fillet of group A did not reach “sensor positive” before the fillet of group B.
(3) Consideration :
According to Example 2, it was confirmed that the flat medium sensor made in Example 1 can detect that the number of bacteria in fish meat has reached a certain value or more within an error range of about 10 times.

<Example of detection of bacterial contamination level of "vacuum packaged fish meat">
(1) Sensor mounting and storage test :
A sensor was prepared using a microfiber cloth in the same manner as in Example 1 and attached to four types of fish fillets (red pine, kissgo, amadai, oyster) in the same manner as in Example 1, and then these fillets. Each was packaged in vacuum and stored in a room at 10 ° C., and observation was continued until the color of the sensor changed, and the number of bacteria at the time of color change was counted using an official method.
(2) Test results :
Table 4 shows the number of bacteria for each fish species when the vacuum-packed fillet sensor changes color. According to Table 4, even when raw fish meat that had been vacuum-packed was stored at 10 ° C., it was confirmed that all the sensors were discolored at the level of “10 6” as a result of examination by the official method. It was.
(3) Consideration :
According to Example 3, it was confirmed that the flat medium sensor made in Example 1 is effective even when fish meat is vacuum-packaged.

<Example 1 of production and use method of “solid medium sensor”>
(1) Production of sensor :
A. 0.9 g of agar (Agarose S) was added to 99 mL of distilled water and heated at 121 ° C. for 20 minutes. To this agar melt, 1 mL of 5 mM edible red No. 102 filtered and sterilized with a 0.2 μm pore membrane filter was added, and 15 mL thereof was dispensed into a petri dish having an inner diameter of 9 cm.
B. A section of 2.4 × 1.2 cm 2 was cut out from the agar having a thickness of 2.4 mm which was cooled and solidified while being sheared.
C. This section was sandwiched between microfiber cloth and nylon film, and heat sealed on all sides with a sealer to complete the “red sensor”. This sensor attaches the surface of the microfiber cloth to the fish meat and sucks out the drip.
D. The concentration of Edible Red No. 102 in the sensor is 50 μM, which corresponds to a weight ratio of 0.003%. In addition, the “use standard of pigment” defined in the Food Sanitation Law is 0.01 to 0.002%.

(2) Sensor mounting :
A. I purchased Thai and flatfish from Yamaguchi Prefecture at a retail store in Shimonoseki City and dropped them to 3 in the laboratory. The above-mentioned solid medium sensor was placed on the surface of this fillet, and the fillet was put in a polyethylene bag and stored at 10 ° C.
B. During the storage period, when the red color of the sensor disappeared and became white, the fillet was removed from the bag, and the bacterial count was examined using the same method as in Example 1.
(3) Sensor detection results :
The time when the sensor changed color and the number of bacteria were 1.5 × 10 6 per gram of fish for 96 hours after the start of storage in Thailand, and 1.3 × 10 6 after 94 hours for flounder. there were.
(4) Consideration :
According to Example 4, it is confirmed that even if a solid medium sensor is used, whether or not the number of bacteria in fish meat has exceeded “the sixth power of 10 per g of fish (according to the official method)” can be detected by the presence or absence of discoloration of the sensor. It was.

<Example of detection of bacterial contamination level of "unpackaged fish meat">
(1) Sensor installation and display :
We asked a nearby 24-hour supermarket fresh fish department to make a slice of about 300g each from Kintokitai, Hontai (Madai), Japanese flounder, and Itoyori that arrived in the early morning. This fillet was arranged on the end of a showcase adjusted to 10 ° C., and a sensor prepared by the same method as in Example 1 was attached to the surface of each fillet at 12:00 am.
(2) Number of bacteria at sensor discoloration:
When I asked the fresh fish department to observe the progress, I was informed that the sensor of Kintokitai was discolored at 9:00 am on the third day. All the fillets were immediately collected and tested for bacteria by the same method as used in Example 1. As a result, the number of bacteria per gram of Kintokitai fish whose sensor had changed color was 10 6, that is, “1.3 × 10 ⁶ / g ”, and the remaining number of bacteria in the hontai, Japanese flounder, and cynomolgus were between“ 3.0 to 7.4 × 10 ⁴ / g ”.
(3) Consideration:
According to Example 5, it was confirmed that the method for detecting the bacterial contamination level of fish meat according to the present invention is useful in an actual store.

<Example 2 of production and usage method of “solid medium sensor”>
(1) Production of sensor :
A. 0.9 g of agar (Agarose S) was added to 99 mL of pH buffer (10 mL: HEPES, pH 7.0) prepared with distilled water, and heated at 121 ° C. for 20 minutes. To this agar melt, 1 mL of 5 mM red No. 102 filtered and sterilized with a membrane filter having a 0.2 μm pore was added, and 15 mL thereof was dispensed into a petri dish having an inner diameter of 9 cm.
B. A section of 2.4 × 1.2 cm 2 was cut out from the agar having a thickness of 2.4 mm which was cooled and solidified while being sheared.
C. This agar slice was sandwiched between a “long microfiber cloth” and a “nylon film that is the same length as the slice” and heat sealed on all sides with a sealer to complete the “red sensor”. In this sensor, only the surface of the microfiber cloth that protrudes in the long length is attached to the bottom surface of the fish meat as a drip introduction part so as to suck out the drip.
(2) Sensor mounting :

The surface of the cross that protruded in a long length was attached to the lower surface of a commercially available red sea bream fillet as an underlay (FIG. 7), and stored in a 10 ° C. room. Observation was continued throughout the storage period, and when the color of the sensor changed from red to white, fish meat was taken out and the number of bacteria in the fillets was counted in the same manner as used in Example 1.
(3) Sensor detection results :
The sensor was discolored when 98 hours had passed since the start of storage, and the bacterial count was 5.6 × 10 ⁶ .
(4) Consideration :
From the results of Example 6, even if a solid medium sensor equipped with a drip introduction part was used, it was determined whether or not the number of bacteria in fish meat reached “the sixth power of 10 per 1 g of fish (according to the official method)”. It was found that this can be confirmed by the presence or absence of discoloration.

<Example of detecting the level of bacterial contamination in "smoked salmon">
(1) Production of sensor :
Four planar medium sensors having the same size were produced by the same method as used in Example 1.
(2) Sensor mounting :
The above sensors were placed in close contact with four pieces of commercially available smoked salmon (prepared by a cold-cooking method) and packaged separately with air.
(3) Measurement of the number of bacteria :
These four packages were arranged in a showcase adjusted to 10 ° C., and the presence or absence of a change in the color of the sensor was observed. After 198 hours, the sensor of one package changed to white. At that time, all the packages were opened, the sensor and the fillet were taken out, and the same test method used in Example 1 was used for each fillet. The bacterial count was examined.
(4) Test results :

As a result of the test, the number of bacteria in the fillet of the package whose color changed by the sensor reached “9.6 g × 10 6 (per official method) per 1 g of fish meat”.
(5) Consideration :
The effectiveness of the sensor was also confirmed in smoked salmon, but the time to reaction was 198 hours, more than twice as long as the other cases. This is thought to be because the growth of bacteria in smoked salmon is suppressed by chilling or salt treatment.

The method for detecting the bacterial level of fish according to the present invention and the sensor used in the detection method are applicable to all raw fish being refrigerated, and are not limited to fish, but also beef, pork, etc., and chicken, chicken, etc. It can also be applied to bacterial control of these processed foods. That is, the present invention is extremely useful because it can be applied to bacterial management of all raw meat.

Claims (6)

  For raw fish meat that has been refrigerated, a sensor containing an azo dye is attached so that it touches the drip that exudes from the fish meat, and the level of bacterial contamination in the fish meat is detected by visually observing whether the sensor color has changed. Method.   The method for detecting the bacterial contamination level of fish meat according to claim 1, wherein the target fish meat for detecting the bacterial contamination level is hermetically packaged.   The method for detecting the bacterial contamination level of fish meat according to claim 1 or 2, wherein the sensor is one in which an azo dye is adsorbed on a flat medium such as paper or cloth.   The method for detecting the bacterial contamination level of fish meat according to claim 1 or 2, wherein the sensor is prepared by dissolving and solidifying an azo dye in a solid medium such as agar or gelatin.   The method for detecting the bacterial contamination level of fish meat according to claim 3 or 4, wherein any one of Food Red No. 2, Food Red No. 40, Food Red No. 102, Food Yellow No. 4, and Food Yellow No. 5 is used as the azo dye. .   A sensor used to detect the level of bacterial contamination of fish meat, which is obtained by adsorbing an azo dye on a flat medium such as paper or cloth, or by dissolving and solidifying in a solid medium such as agar or gelatin.