WO2005078120A1

WO2005078120A1 - Device for salmonella detection and method of use thereof

Info

Publication number: WO2005078120A1
Application number: PCT/JP2005/002512
Authority: WO
Inventors: Yutaka Midorikawa; Satoshi Nakamura; Kazue Kishimoto; Akiko Makino; Yumiko Takenaka
Original assignee: Mie Tlo Co., Ltd.
Priority date: 2004-02-12
Filing date: 2005-02-10
Publication date: 2005-08-25
Also published as: JPWO2005078120A1; JP4427634B2

Abstract

[PROBLEMS] To provide a device for rapidly screening pathogenic enterobacteria, such as salmonella, in a convenient manner; provide a method of detecting salmonella with the use of the device; and provide a method of qualitative analysis of the concentration of ascorbic acid or citric acid. [MEANS FOR SOLVING PROBLEMS] Salmonella, etc. can be easily detected by means of a device for salmonella detection comprising an agar medium containing a sulfur source, such as DHL agar medium, and a carrier containing an aqueous solution of ascorbic acid or citric acid. Further, qualitative analysis of the concentration of ascorbic acid or citric acid can be carried out by stationing a carrier containing ascorbic acid or citric acid on an agar medium containing salmonella and by conducting an image analysis of iron sulfide formed on the periphery of the carrier.

Description

[Document Name] Statement

TECHNICAL FIELD The present invention relates to a device for detecting Salmonella and a method of using the same.

[0 0 0 1]

The present invention relates to a simple and efficient analytical device for detecting Salmonella, a method for detecting Salmonella, and a method for analyzing the concentration of ascorbic acid or citrate.

[Background Art]

[0 0 0 2]

Among the Enterobacteriaceae, pathogenic bacteria, such as Salmonella, cause serious damage to human life, such as inducing food poisoning. Therefore, several food hygiene inspection guidelines and methods have been proposed (for example, See Non-Patent Documents 1 and 2 below).

In accordance with the above guidelines, etc., the method of detecting bacteria is usually that after culturing Salmonella bacteria in EEM medium / Selenite medium, DHL (Deoxycholate Hydrogen sulfide Lactose) agar medium or Puma SS—SB (Salmonella Shigella Sucrose Bromcresol purple) Bacteria are spread on an agar medium and separated and cultured. Among the intestinal bacteria, Salmonella, Ptoteus, and Citrobacter produce hydrogen sulfide on these media and react with the iron components contained in the media to produce black ferrous sulfide. Generate Therefore, in order to identify Salmonella, a method of screening bacterial species by further inoculating a TSI agar medium / LIM agar medium Z simmons citrate medium or the like has been adopted.

However, the above-mentioned detection method has problems that the process is complicated and the analysis requires a long time, so that it is not possible to respond quickly.

[0 0 0 3]

On the other hand, although an improved medium in which sucrose is added to SS agar medium is commercially available, the separation efficiency of Salmonella was not sufficient, and further improvement was needed. 2 2005/002512

In addition, as a qualitative test method, an inspection method applying the ELISA method has been developed. For example, Patent Literature 1 discloses an alternative method for detecting a Salmonella strain from a nucleic acid sequence specific to a pathogenic bacterium, and Patent Literature 2 provides an external factor such as light or electromagnetic waves to bacteria present in a sample, Patent Document 3 discloses a method for detecting a product produced from a bacteriophage by making use of bacteriopacteriophage parasitic on the inside of a bacterium, and Patent Document 3 discloses a method for detecting a bacterium introduced from a sample introduction section. There is provided a method of binding to an immunochromatographic device provided with an antibody that specifically binds to the bacterium, and then growing the bound bacterium in a medium to detect the bacterium.

[0 0 0 4]

All of the above-mentioned improved test methods aim to detect bacteria quickly and with high accuracy. However, in these known methods, the method for detecting bacteria is still complicated, and there has been a demand for a device and method for detecting intestinal bacteria which is simpler, faster and cheaper.

[0 0 0 5]

[Patent Document 1] Japanese Patent Application Publication No. 2000-01- 5 2 1 7 2 1

[Patent Document 2] Japanese Patent Application Laid-Open No. 2000-27799

[Patent Document 3] Japanese Patent Application Laid-Open No. 2000-018

[Non-Patent Document 1] Supervised by the Ministry of Health and Welfare, Ministry of Health and Welfare: Food Sanitation Inspection Guidelines, Japan Food Sanitation Association, Tokyo, 199

[Non-patent document 2] Ministry of Health and Welfare: Guidelines for sanitary management in poultry slaughterhouses using the HACCP method, No. 71, March 30, 1992

DISCLOSURE OF THE INVENTION

[Problems to be solved by the invention]

[0 0 0 6]

The present invention has been made in view of the above problems, and an object of the present invention is to provide a device for easily and rapidly screening Salmonella bacteria by improving existing bacterial detection methods. 3 TJP2005 / 002512

It is to provide a chair, and to provide a method for detecting Salmonella using the device. Still another object is to provide a method for qualitatively analyzing the concentration of ascorbic acid or citric acid using the technical gist that led to the invention of the depises.

[Means for Solving the Problems]

[000 7]

In order to solve the above problems, the present inventors isolated and cultured pathogenic enterobacteria including Salmonella on a DHL agar medium, and placed various fruits and vegetables cut into slices on the DHL agar medium. Then, the phenomenon appearing on the DHL agar medium was visually observed, and it was found that a certain tendency was obtained, and the present invention was reached.

[0008]

That is, according to the findings of the present inventors, it was placed on a DHL agar medium containing Salmonella / Proteus / Citropactor for several hours, and a few mm in width was placed on the outer periphery of a certain kind of roasted fruit or vegetable. A black reaction zone of iron sulfide (approximately 4 to 5 mm) appeared, and a phenomenon in which the inside of the black zone changed color tone in a blue color was found. On the other hand, clear phenomena as described above were not observed in Proteus and Citrobacter.

[0009]

Next, the present inventors mounted a filter paper impregnated with an aqueous solution of ascorbic acid or cuenic acid on a DHL agar medium in order to confirm that ascorbic acid or cunic acid plays a major role in the above phenomenon. And observed the production of iron sulfide (Π). As a result, as the ascorbic acid or cunic acid concentration was higher, a clear black band of iron sulfide (化) was confirmed on the outer periphery of the filter paper, and the present invention was basically completed.

[00 1 0]

Specifically, it can be placed on a DHL agar medium containing pathogenic enteric bacteria such as Salmonella or on an agar medium containing a sulfur source such as SS agar medium or TSI agar medium. When a carrier containing an aqueous solution of ascorbic acid or cunic acid with a scorbic acid or cunic acid concentration of 0.1 g / L or more (concentration of ascorbic acid per liter of aqueous solution of 0.1 g or more) is placed and left for more than half a day, When Salmonella is present, a black band of iron sulfide (Π) with a width of about 4 mm is visually observed around the carrier on the agar medium. However, in the case of Proteus bacterium and Citrobacter, etc., the formation of a black band is unclear and can be clearly distinguished from Salmonella. Therefore, in order to observe the above phenomenon, it is necessary that the outer dimensions of the carrier be several mm smaller than the outer dimensions of the DHL agar medium at any position.

[001 1]

Here, the aqueous solution of ascorbic acid or cuenic acid refers to fruit juice, vegetable juice, tap water, groundwater, or other naturally derived water or bodily fluid such as urine, sweat, milk, or artificially synthesized artificial water or physiological buffer. It means an aqueous solution containing ascorbic acid or citric acid as a solvent or a dispersion medium. When the concentration of ascorbic acid or cunic acid in the aqueous solution is less than 0.1 gZL, a black band of iron sulfide is not clearly observed.

[001 2]

The carrier is an organic or inorganic porous material such as filter paper, impregnated paper, cloth, nonwoven paper, inorganic porous material, etc., and has an external dimension several mm or more smaller than the external dimension of the agar medium. And a thickness of 0.1 mm or more. Also, it is possible to impregnate an aqueous solution of ascorbic acid or citrate with an ascorbic acid or taenoic acid concentration of 0.1 g / L or more almost uniformly over the entire surface, or citrus fruits such as lemon and tangerine, kiwi roots, acerola, strawberry It may be any of natural products containing ascorbic acid or citric acid in an amount of 0.1 g / L or more. By the way, ascorbic acid or citric acid is generally easily decomposed, and among the fruits or vegetables, citrus fruits such as lemon having an action of stabilizing ascorbic acid or cunic acid are particularly preferably used.

[001 3]

Therefore, when using carriers other than fruits or vegetables, filter paper, impregnated paper, cloth, etc. 5 02512

The carrier containing an aqueous solution of ascorbic acid or citric acid contains carbohydrates, proteins, lipids, ash, lime, calcium, iron, which are components normally contained in fruits and vegetables in addition to asconolevic acid or citric acid. , Magnesium or the like, niacin, vitamin B1, vitamin B2, vitamin E or the like such as vitamin E, fatty acid or vegetable fiber, and the like, and may contain one or more thereof in a complex manner. Here, it is generally desirable that the water content be 850 gZL or more, but it is not necessarily limited.

[00 14]

Further, the next invention is directed to placing and holding a carrier containing an aqueous solution containing ascorbic acid or taenoic acid as an essential component on an agar medium containing a sulfur source such as a DHL agar medium containing Salmonella, A method for qualitatively analyzing the concentration of ascorbic acid or cuenic acid by image-analyzing the color tone change of the iron sulfide (Π) band formed around the carrier and the agar medium expressed therein. I do.

[00 1 5]

That is, a carrier whose concentration of ascorbic acid or cuenic acid is known in advance is placed on an agar medium containing a sulfur source, such as a DHL agar medium containing Salmonella, and observed on the outer periphery of the carrier on the agar medium. A database was constructed with images of the black band and the color change formed in the black band and the color tone formed inside the black band, and used as a standard sample. Then, the image obtained from a carrier whose ascorbic acid or taenoic acid concentration was unknown was obtained. Is compared with the above database, and image analysis is performed to qualitatively analyze ascorbic acid or citrate concentration. Here, the image analysis may be performed using computer software, or more simply by visual observation, and is not particularly limited.

[00 1 6]

By the way, Salmonella, Proteus, and Citrobacter produce hydrogen sulfide (H ₂ S) on DHL agar medium, SS agar medium, SS-SB agar medium, or TSI agar medium, which is rich in sulfur, and produce hydrogen sulfide and hydrogen sulfide. The iron ions contained in the agar medium react to form iron sulfide (硫化). Then, on the agar medium, iron sulfide (Π) was clearly black on the outer periphery of the carrier containing ascorbic acid or cunic acid. Regarding the mechanism by which the color zone is formed and the color of the agar medium inside the color zone changes, it is considered that the reducing action of ascorbic acid or citrate exerts some influence and thus is reduced. 【The invention's effect】

[00 1 7]

ADVANTAGE OF THE INVENTION By this invention, it becomes possible to provide the Nakome device which screens Salmonella easily and quickly, and the detection method of Salmonella. As a result, the presence of Salmonella in food can be easily analyzed in the food industry, hospitals, catering services, grocery stores, and ordinary households, and food hygiene management is significantly improved. In addition, since it is possible to provide a method for qualitatively and quickly analyzing the concentration of ascorbic acid or citric acid, the concentration of ascorbic acid or citric acid in food can be confirmed at low cost, which is useful for quality control. [Brief description of the drawings]

[0018]

FIG. 1 is a diagram showing the change in color tone of iron sulfide (II) and agar medium expressed by the reaction of a DHL agar medium containing Salmonella, Proteus, and Citropactor with a sliced lemon. .

FIG. 2 is a diagram showing the change in color tone of iron sulfide (Π) and agar medium expressed by the reaction of DHL agar medium and SS agar medium containing Salmonella with sliced lemon.

FIG. 3 is a diagram showing the change in color tone of iron sulfide (Π) and agar medium expressed by the reaction between DHL agar medium containing Salmonella and various fruits and vegetables.

FIG. 4 is a diagram showing the change in color tone of iron sulfide (Π) and agar medium expressed by the reaction of DHL agar medium containing Salmonella with filter paper containing various concentrations of ascorbic acid. BEST MODE FOR CARRYING OUT THE INVENTION

[001 9]

Hereinafter, embodiments of the present invention will be described in detail with reference to Examples, but the technical scope of the present invention is not limited by the following embodiments, and the gist of the present invention is not changed. Various modifications can be made. The technical scope of the present invention extends to an equivalent range.

[0020]

DHL agar medium (manufactured by Eiken Chemical Co., Ltd.) was used as the agar medium containing a sulfate source. Among the bacteria cultured on the DHL agar medium, the bacteria that produced iron sulfide (Π) (Salmonella, Proteus Bacteria, citropactor) was smeared directly on a DHL agar medium with a cotton swab or suspended in physiological saline and then smeared. Then, the transferred lemon (ascorbic acid concentration: 0.4 gZL) was placed on the DHL agar medium, and kept at 35 ° C for 18 hours. Thereafter, the state of iron sulfide (Π) expressed on the outer periphery of the carrier on the agar medium was visually observed.

[0021]

The results are shown in FIG. In the presence of Salmonella, a black band of iron sulfide (Π) with a width of 4 to 5 mm was clearly observed around the carrier. On the other hand, in the case of Proteus bacteria and Citrobacter bacteria, the black band was unclear and undefined. Also, the inside of the black belt was almost uniformly changed to pink.

[0022]

In order to examine whether iron sulfide (Π) expression and the change of the color of the agar medium into pink occur on other agar mediums containing sulfur sources, DHL agar medium and SS agar medium (Eiken Chemical Co., Ltd.) 2005/002512

Made by the company). These agar media were inoculated with Salmonella in the same manner as described above, and as ascorbic acid-containing carriers, sliced lemons (ascorbic acid concentration of 0.4 g / L) were used.

The results are shown in FIG. In all the agar media, the expression of iron sulfide (Π) and the color change of the agar media in pink were clearly observed.

[0023]

Example 3: Comparison of Carrier Containing Ascorbic Acid Aqueous Solution>

Since the effect of lemon was clarified as described above, the following commercially available raw fruits and vegetables having various concentrations of ascorbic acid other than lemon were sliced, and those with a clear ascorbic acid concentration were used. We compared whether it was possible to identify Salmonella. Kiwifruit (0.3 gZL), Acerola (2.0 gZL), Guava (1.0 g / L), Strawberry (0.3 g / L), Paprika (0.5 gZL), Sencha leaf (0.6 g / L), Broccoli (0.4 g / L) / L), curry flour (0.2 g / L) and green apple with low ascorbic acid concentration (0.02 gZL), whose cross section is at least 5 mm smaller than the external dimensions of the agar medium did.

[0024]

The results are shown in FIG. The phenomenon of iron sulfide (Π) expression and the change of the color of the agar medium to pink was the clearest pattern when lemon was used, followed by kiwi roots, strawberries, acerola, and paprika, followed by guava, Broccoli, cauliflower and apples did not show very clear patterns, and the tea leaves were the least sharp. Thus, the clarity of the black band pattern depends on the distribution of ascorbic acid and the stability of ascorbic acid, as well as the concentration of ascorbic acid.

Lemon and kiwifruits with stable ascorbic acid on L agar medium tended to produce clearer patterns.

[0025] 9 02512

As carrier containing Asukorubin acid, filter paper having a diameter of 2 Omm, Omo 1 / L ( control), 0.2 5 mo 1 /, 0.5 mo \ / L N 1 mo 1 / L 1. Asukorubin acid aqueous solution 5MO 1 / L After impregnating with 50 pieces of water, it was dried for 24 hours.

Next, the bacteria identified as Salmonella BG1 are suspended in physiological saline using a kit for identifying Salmonella bacteria, then smeared on a DHL agar medium with a sterile cotton swab, and the above-described carrier containing ascorbic acid is further placed thereon. It was placed and left at 38 ° C for 24 hours.

[0026]

The results are shown in FIG. When the ascorbic acid concentration power S was 0.5 mo1 / L or more, a black band of iron sulfide (Π) was observed, and a clear pattern was observed as the concentration increased. As a result, it was proved that the amount of ascorbic acid contained in foods, health foods, pharmaceuticals, feeds and the like can be easily and qualitatively analyzed using the device of the present invention. Here, there is a difference of more than 100 times in the ascorbic acid concentration between the case where Salmonella is identified and the case where the ascorbic acid concentration is analyzed qualitatively. It is considered that the dry condition of the acid has an effect.

[00 2 7]

Using the same method as above, the formation of black bands was tested using Salmonella typhi and sliced lemon. No black band was formed. This clearly shows that Salmonella and Salmonella typhi can be distinguished using the device of the present invention.

Claims

[Document Name] Claims

1. A agar medium containing the sulfur source, is also Asukorubin acid (C _₆ H ₈ 0 _6), characterized in that it is composed of a carrier containing Kuen acid aqueous solution, Salmonella bacteria detection device.

2. The device for detecting Salmonella according to claim 1, wherein the concentration of ascorbic acid or citric acid in the aqueous solution is 0.1 g Zl (lit ^^) or more.

3. The method according to claim 1, wherein the agar medium containing the sulfur source is any one of a DHL agar medium, an SS agar medium, an SS-SB agar medium, and a TSI agar medium. Item 3. The device for detecting Salmonella according to Item 2.

4. The carrier contains an aqueous solution containing at least one of carbohydrates, proteins, lipids, ash, minerals, vitamins, and fatty acids, in addition to ascorbic acid or citrate. The device for detecting Salmonella according to any one of claims 1 to 3.

5. The device for detecting Salmonella according to claim 1, wherein the carrier is filter paper, impregnated paper, cloth, nonwoven fabric, or a porous inorganic substance.

6. The Salmonella bacterium according to claim 1, wherein the carrier is a slice of any of citrus, acerola, kiwifruit, strawberry, pepper, and paprika. Device for detection.

7. The expression state of iron sulfide (ネ) on an agar medium and the change in color tone of Z or agar medium using the device for detecting Salmonella according to any one of claims 1 to 6. A method for detecting Salmonella by analysis.

8. A carrier containing an aqueous solution of ascorbic acid or citrate is placed and held on an agar medium containing Salmonella and containing a sulfur source, and iron sulfide (される) formed on the agar medium is contained. And) a method of analyzing the color tone change of the agar medium expressed inside the iron sulfide (II) by image analysis to determine the concentration of ascorbic acid or taenoic acid.