Medium for culturing microorganisms using succinate as carbon source

Abstract

The present invention relates to a medium for culturing microorganisms used in a dry film method, which is a microbial test method for testing bacteria and, more specifically, to a medium capable of rapidly testing microorganisms by using succinate as a carbon source. The dry film medium for detecting microorganisms of the present invention contains succinate as a carbon source to promote the growth of microorganisms, preferably gram-negative bacteria, in the dried film medium, thereby enabling colony formation within 24 hours, and thus microorganisms to be tested can be rapidly detected. In particular, the dry film medium of the present invention can rapidly form colonies within 12 hours by further promoting the growth of microorganisms, preferably gram-negative bacteria, if succinate and pyruvate are contained at a specific ratio as carbon sources, and thus can be effectively used as a medium for simple detection for inspecting harmful bacteria in raw materials or finished products such as food or cosmetics. In addition, using the dry film medium of the present invention has the advantage that even Pseudomonas aeruginosa (P. aeruginosa), which grows relatively slowly compared to other bacteria, can be detected through colony formation within 24 hours.

Description

Medium for microbial cultivation using succinate as a carbon source

[0001] The present invention relates to a medium for culturing microorganisms used in the dry film method, which is a microorganism test method for examining bacteria, and more specifically, to a medium that enables rapid microorganism testing by using succinate as a carbon source.

[0002] All food companies must conduct mandatory microbial testing in accordance with the Food Code's microbial testing regulations before product shipment or during the process as needed to ensure quality control against microbial contamination. Violations may result in disciplinary actions such as suspension or cancellation of business. The dry film method is an official test method specified in the Food Code for the detection of Gram-negative bacteria and is used worldwide. It is integrated and applied to inspections of each food manufacturing process, including raw materials, manufacturing process, manufacturing environment, and finished products, and is being expanded to industrial fields such as environment, cosmetics, and pharmaceuticals. The film-type bacterial detection strip market is monopolized by the multinational corporation 3M, which holds a 93% share of the global market, including Korea (Source: https: / www.nextround.kr / startup / detail / 2559). 3M's film-type bacterial detection strip (3M Petrifilm) from the U.S. company 3M TM ) is the only standardized product widely used across industries, from bacterial testing during international import and export customs clearance to environmental impact assessments and food safety testing.

[0003] In the case of 3M's Petrifilm sold on the market, it is a domestically certified dry film medium and is listed in the Food Code of the Ministry of Food and Drug Safety (Chapter 7. General Test Methods > 4. Microbiological Test Methods > 4.4 Medium and Reagents > 4.4.1 Medium, 53) Bacterial Count Dry Film Medium I), and the composition of the medium listed in the Food Code discloses sodium pyruvate as the main component as a carbon source.

[0004] Meanwhile, when using Petrifilm, microorganisms are detected by culturing them at a temperature of approximately 35°C for 24 to 48 hours, although this varies slightly depending on the type of microorganism being cultured. Therefore, there is a need to develop a rapid drying film medium that promotes microbial growth and allows for observation of results within 12 to 24 hours.

[0005] Against this backdrop, the inventors of the present invention sought to develop a medium capable of culturing and identifying microorganisms more quickly than conventional commercially available Petrifilm, and confirmed that in the case of a dry film medium containing succinate as a carbon source, microorganisms can quickly form colonies within 12 to 24 hours, thereby enabling rapid detection (inspection) of microorganisms.

[0006] The purpose of the present invention is to provide a dry film medium capable of rapid microbial detection.

[0007] In order to achieve the above-mentioned purpose, the present invention provides a dry film medium for detecting microorganisms, in which a coating layer including a medium composition, a gelling agent, and an indicator is formed on a substrate, wherein the medium composition includes animal protein hydrolysate and yeast extract as a nitrogen source, and succinate as a carbon source.

[0008] According to one embodiment of the present invention, the succinate may be the sole carbon source in the medium composition.

[0009] According to one embodiment of the present invention, the medium composition may further include at least one selected from pyruvate, malate, fumarate, and oxaloacetate as a carbon source.

[0010] According to one embodiment of the present invention, the medium composition may contain succinate and pyruvate in a weight ratio of 1:9 to 9:1.

[0011] According to one embodiment of the present invention, the medium composition may contain succinate and pyruvate in a weight ratio of 4:6 to 7:3.

[0012] According to one embodiment of the present invention, the medium composition may further include glucose as a carbon source.

[0013] According to one embodiment of the present invention, the medium composition may contain succinate and glucose in a weight ratio of 100:5 to 10.

[0014] According to one embodiment of the present invention, the animal protein hydrolysate may be a pancreatic digest of casein.

[0015] According to one embodiment of the present invention, the medium composition may further include dipotassium phosphate, potassium phosphate, or a mixture thereof as a phosphorus.

[0016] According to one embodiment of the present invention, the gelling agent may be at least one selected from agar, sodium alginate, guar gum, xanthan gum, gellan gum, locust bean gum, hyaluronic acid, and sodium hyaluronate.

[0017] According to one embodiment of the present invention, the indicator may be at least one selected from triphenyltetrazolium chloride, phenol red, neutral red, eosin, methylene blue, bromothymol blue, and bromoxylenol blue.

[0018] According to one embodiment of the present invention, the microorganism may be a gram-negative bacterium.

[0019] According to one embodiment of the present invention, the Gram-negative bacteria may be at least one selected from bacteria of the genus Escherichia, bacteria of the genus Pseudomonas, bacteria of the genus Salmonella, bacteria of the genus Vibrio, bacteria of the genus Yersinia, bacteria of the genus Campylobacter, and bacteria of the genus Cronobacter.

[0020] According to one embodiment of the present invention, the Gram-negative bacteria may be Escherichia coli or Pseudomonas aeruginosa.

[0021] The dry film medium for detecting microorganisms of the present invention contains succinate as a carbon source, thereby promoting the growth of microorganisms, preferably gram-negative bacteria, within the dry film medium, enabling the formation of colonies within 24 hours, thereby enabling the rapid detection of microorganisms to be tested. In particular, when the dry film medium of the present invention contains succinate and pyruvate as carbon sources at a specific ratio, it further promotes the growth of microorganisms, preferably gram-negative bacteria, enabling the rapid formation of colonies within 12 hours, thereby enabling the medium to be usefully used as a simple detection medium for testing harmful bacteria in finished products or raw materials such as food or cosmetics. In addition, when the dry film medium of the present invention is used, even P. aeruginosa, which grows relatively slowly compared to other bacteria, has the advantage of enabling the detection of colonies within 24 hours.

[0022] Figure 1 is a photograph of a dry film medium for detecting microorganisms according to one embodiment of the present invention.

[0023] Figure 2 is a photograph showing whether colonies were formed when E. coli was inoculated onto each of the dry film media for detecting microorganisms according to Examples 4 to 6 of the present invention and Comparative Example 1 and cultured for 12 hours.

[0024] Figure 3 is a photograph showing whether colonies were formed when Pseudomonas aeruginosa was inoculated onto a dry film medium for microbial detection according to Examples 4 to 6 of the present invention and Comparative Example 1, respectively, and cultured for 12 hours and 24 hours.

[0025] Hereinafter, the present invention will be described in detail.

[0026] The present invention relates to a dry film medium for detecting microorganisms, wherein a coating layer including a medium composition, a gelling agent, and an indicator is formed on a substrate, wherein the medium composition includes animal protein hydrolysate and yeast extract as a nitrogen source, and succinate as a carbon source.

[0027] In this specification, “dry film medium” may also be referred to as a thin film culture plate, and refers to a film-type bacterial detection medium developed to detect microorganisms, preferably Gram-negative bacteria, contained in finished products such as food and cosmetics or their raw materials by coating the film with nutrients and specific components necessary for the growth of microorganisms.

[0028] The above dry film medium is a biological film coated with nutrients to enable bacteria and fungi to proliferate. When a sample is inoculated onto the dry film medium, it absorbs moisture and forms a gel like an agar medium, allowing microorganisms to form colonies. It is a dry rehydratable film medium. 3M is the only authorized product in Korea. TM Petrifilm is a dry film medium that contains indicators that take into account the growth characteristics of microorganisms, making it easier to read bacteria than agar media. 3M TM Petrifilm uses the same nutrients as agar medium, so the experimental principle is the same as the traditional agar medium method, but it is a new microbial testing method developed to minimize the preparation of the medium and the culture time, allowing for many experiments to be performed in a short period of time.

[0029] In one specific example, the substrate may be a thin plate, but is preferably a transparent film made of a waterproof polymer material that does not absorb water, such as polyester, polypropylene, polystyrene, or polyethylene.

[0030] In one specific example, the substrate preferably has an adhesive layer formed thereon so that a coating layer can be formed by applying a powder of the medium composition, a gelling agent, and an indicator, etc., to the upper surface and then fixing the powder, and the adhesive is preferably a transparent material that does not inhibit the growth of microorganisms and does not interfere with the observation of microbial colonies. Specifically, the adhesive is not particularly limited, but is preferably at least one selected from isooctyl acrylate / acrylamide, N-vinylpyrrolidone, and lauroyl peroxide in terms of promoting microbial growth.

[0031] In one specific example, the dry film medium for detecting microorganisms of the present invention may be manufactured by applying a mixture of medium composition powder, gelling agent powder, and indicator powder onto a substrate having the adhesive layer formed thereon and then pressing it.

[0032] In one specific example, it is preferred that the medium composition is included in the coating layer including the medium composition, gelling agent and indicator at 10 to 20 wt%.

[0033] In one specific example, the thickness of the dry film medium for detecting microorganisms of the present invention may be 20 to 400 μm, preferably 50 to 200 μm.

[0034] In one specific example, the dry film medium may be a dry film medium for detecting Gram-negative bacteria.

[0035] In one specific embodiment, the medium composition of the present invention comprises succinate as a carbon source.

[0036] In one embodiment, the succinate may be the sole carbon source in the medium composition.

[0037] In one specific embodiment, the carbon source may further include at least one selected from pyruvate, malate, fumarate, and oxaloacetate, and preferably further includes pyruvate.

[0038] When the medium composition of the present invention further comprises pyruvate, the succinate and pyruvate may be in a weight ratio of 1:9 to 9:1, preferably in a weight ratio of 2:8 to 8:2, and more preferably in a weight ratio of 4:6 to 7:3. When the weight ratio of the succinate and pyruvate is within the above range, the growth promotion effect of microorganisms on the dried film medium of the present invention can be maximized. In particular, when succinate and pyruvate are included in a weight ratio of 4:6 to 7:3, colonies can be formed within 12 hours of culturing microorganisms, preferably Escherichia coli or Pseudomonas aeruginosa, enabling detection.

[0039] In one embodiment, the carbon source may further comprise glucose.

[0040] When the medium composition of the present invention contains glucose, the weight ratio of succinate and glucose may be 100:5 to 10. When the weight ratio of succinate and glucose is within the above range, the microbial growth promotion effect on the dry film medium of the present invention can be maximized, and when it is outside the above range, the microbial growth promotion effect of the present invention may be minimal.

[0041] In the medium composition of the present invention, the animal protein hydrolysate may be a pancreatic digest of casein.

[0042] In one specific example, it is preferable to contain both the animal protein hydrolysate and yeast extract as the nitrogen source in order to promote microbial growth on the dried film medium of the present invention. If either the animal protein hydrolysate or yeast extract is omitted, the microbial growth promotion effect on the dried film medium of the present invention may be minimal.

[0043] In one specific example, the medium composition may further include dipotassium phosphate, potassium phosphate or a mixture thereof as the agent, preferably a mixture of dipotassium phosphate and potassium phosphate, and more preferably a mixture of dipotassium phosphate and potassium phosphate in a weight ratio of 1:0.2 to 0.5.

[0044] In one specific example, the gelling agent can absorb moisture contained in the culture medium when the culture medium is inoculated onto a dry film medium to form a gel like an agar medium, thereby allowing the microorganism to grow. Specifically, the gelling agent may be at least one selected from agar, sodium alginate, guar gum, xanthan gum, gellan gum, locust bean gum, hyaluronic acid, and sodium hyaluronate, and preferably guar gum.

[0045] In one specific example, the indicator is for visualizing a microbial colony, and specifically, may be a compound capable of detecting a metabolite produced by metabolism by a microorganism. Specifically, the indicator may be a redox indicator, an organic acid detection indicator, an ammonia detection indicator, an amino acid detection indicator, a hydrogen ion concentration detection indicator, etc., and more specifically, it may be at least one selected from triphenyltetrazolium chloride, phenol red, neutral red, eosin, methylene blue, bromothymol blue, and bromoxylenol blue, and 2,3,5-triphenyltetrazolium chloride is preferable.

[0046] The dry film medium according to the present invention can be easily used for the culture and detection of various microorganisms, preferably Gram-negative bacteria.

[0047] In one specific example, the microorganism may be a gram-negative bacterium, and the gram-negative bacterium may be at least one selected from the group consisting of Escherichia bacterium, Pseudomonas bacterium, Salmonella bacterium, Vibrio bacterium, Yersinia bacterium, Campylobacter bacterium, and Cronobacter bacterium, and preferably Escherichia coli or Pseudomonas aeruginosa.

[0048]

[0049] Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited to the following examples.

[0050]

[0051] <Example>

[0052] ingredient

[0053] Escherichia coli (E. coli ATCC 25922) and Pseudomonas aeruginosa (P. aeruginosa ATCC9027) were purchased from Global bioresource center.

[0054]

[0055] Manufacturing Example 1: Manufacturing of a medium composition

[0056] (1) A carbon source solution was prepared by adding 6.8 g of succinate to 500 mL of distilled water.

[0057] (2) 3.4 g of pancreatic digest of casein, 2.4 g of yeast extract, 1.3 g of dipotassium phosphate, 0.4 g of potassium phosphate, and 0.6 g of dextrose were added to 500 mL of distilled water, and then mixed to prepare a basic medium (same ingredients as the bacterial count dried film medium I in the Food Codex).

[0058] (3) A medium composition was prepared by mixing the above basic medium and carbon source solution in a volume ratio of 1:1.

[0059]

[0060] Manufacturing Example 2: Succinate: Pyruvate = 9:1

[0061] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate at a weight ratio of 9:1 was added in equal weight to prepare a medium composition.

[0062]

[0063] Manufacturing Example 3: Succinate: Pyruvate = 8:2

[0064] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 8:2 was added in equal weight to prepare a medium composition.

[0065]

[0066] Manufacturing Example 4: Succinate: Pyruvate = 7:3

[0067] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 7:3 was added in equal weight to prepare a medium composition.

[0068]

[0069] Manufacturing Example 5: Succinate: Pyruvate = 5:5

[0070] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 5:5 was added in equal weight to prepare a medium composition.

[0071]

[0072] Manufacturing Example 6: Succinate: Pyruvate = 4:6

[0073] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 4:6 was added in equal weight to prepare a medium composition.

[0074]

[0075] Manufacturing Example 7: Succinate: Pyruvate = 3:7

[0076] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 3:7 was added in equal weight to prepare a medium composition.

[0077]

[0078] Manufacturing Example 8: Succinate: Pyruvate = 2:8

[0079] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate in a weight ratio of 2:8 was added in the same weight to prepare a medium composition.

[0080]

[0081] Manufacturing Example 9: Succinate: Pyruvate = 1:9

[0082] The same procedure as in Manufacturing Example 1 was followed, but instead of using succinate alone in step (1), a mixture of succinate and pyruvate at a weight ratio of 1:9 was added in equal weight to prepare a medium composition.

[0083]

[0084] Comparative manufacturing example: Pyruvate only

[0085] The same procedure as in Manufacturing Example 1 was followed, but in step (1), a medium composition was prepared using pyruvate instead of succinate.

[0086]

[0087] Preliminary Test Example 1: Microbial Growth Promotion Effect - Liquid Culture - Absorbance Measurement

[0088] Escherichia coli (E. coli ATCC 25922) was inoculated into 10 mL of TSB Broth medium and cultured at 37°C for 19-24 hours to activate. Thereafter, the strain was inoculated into 200 mL of Broth medium and cultured at 37°C for 5 hours to reach the early stationary phase. Next, 5 mL of the strain was mixed with 95 mL of the basic medium of Preparation Example 1 to prepare a 5% strain culture solution.

[0089] The medium composition according to the above manufacturing example or comparative manufacturing example and the strain culture solution were dispensed at 1 mL / well each into a 24-well plate. At this time, the viable count of E. coli contained in each well was approximately 7.80 log CFU / mL. As a control, a mixture of sterile distilled water and the strain suspension was used instead of the medium composition.

[0090] Afterwards, each well-plate was cultured at 37°C for 24 hours, and the absorbance at 600 nm was measured at regular intervals using a Varioskan™ LUX multimode microplate reader. The results are shown in Table 1 below.

[0091] Succinate: Pyruvate Absorbance (OD) over incubation time 600)0 hours8 hours12 hours18 hours24 hoursControl-0.030.030.030.030.03 Manufacturing Example 110: 00.070.580.931.291.17 Manufacturing Example 29: 10.070.550.891.001.11 Manufacturing Example 38: 20.070.490.840.991.01 Manufacturing Example 47: 30.070.540.831.071.23 Manufacturing Example 55: 50.070.460.500.791.03 Manufacturing Example 64: 60.070.440.500.540.50 Manufacturing Example 73: 70.070.360.440.480.49 Manufacturing Example 82: 80.070.370.420.430.43 Manufacturing Example 91: 90.070.340.360.340.35 Comparative Manufacturing Example 0: 100.070.350.350.330.33

[0092] Looking at Table 1 above, in the case of Manufacturing Examples 1 to 4, the absorbance was found to increase rapidly after 8 hours of culture, and in Manufacturing Example 5, the absorbance was found to increase rapidly from 18 hours of culture, whereas in the case of Manufacturing Examples 6 to 9 and the comparative manufacturing example, no rapid increase was found even after 24 hours of culture. From the above results, it can be inferred that the medium composition according to Manufacturing Examples 1 to 5 shortens the lag phase of the growth curve of the strain.

[0093]

[0094] Preliminary Test Example 2: Microbial Growth Promotion Effect - Liquid Culture - Measurement of Viable Cell Count

[0095] After measuring the absorbance in the above preliminary test example 1, the number of viable cells in each well-plate was measured using the standard plate method, and is shown in Table 2 below.

[0096] Succinate: Pyruvate Viable cell count according to culture time (Log CFU / mL) 0 hours 8 hours 12 hours 24 hours 48 hours Control -7.807.807.807.807.80 Manufacturing example 110: 07.808.618.209.268.34 Manufacturing example 29: 17.808.558.209.218.36 Manufacturing example 47: 37.808.559.009.258.97 Manufacturing example 55: 57.808.489.059.119.07 Manufacturing example 64: 67.808.518.878.879.25 Manufacturing example 73: 77.808.398.409.008.51 Manufacturing example 91 : 97.808.318.328.408.40Comparative Manufacturing Example 0 : 107.808.328.308.318.28

[0097] Based on the results in Table 2 above, the increase rate of viable cell count based on the initial cell count was calculated and shown in Table 3 below.

[0098] Succinate: Pyruvate Increase in viable cell count over time (%) 0 hours 8 hours 12 hours 24 hours 48 hours Control group----- Manufacturing example 110: 0-10.38 5.13 18.7 26.92 Manufacturing example 29: 1-9.6 25.13 18.0 87.18 Manufacturing example 47: 3-9.6 215.38 18.59 15.00 Manufacturing example 55: 5-8.7 216.03 16.80 16.28 Manufacturing example 64: 6-9.10 13.7 213.7 218.59 Manufacturing example 73: 7-7.5 67.69 15.38 9.10 Manufacturing example 91: 9-6.5 46.67 7.69 7.69 Comparative Manufacturing example 0: 10-6.676.416.546.15

[0099] Looking at Table 3 above, it can be seen that when the medium compositions of Manufacturing Examples 4 to 6 were treated, the increase rate in the number of viable cells significantly increased from 12 hours after incubation, and the increased number of cells was maintained or further increased until 48 hours after incubation. In addition, when the medium compositions of Manufacturing Examples 1, 2, and 7 were treated, it can be seen that the increase rate in the number of viable cells significantly increased from 24 hours after incubation. On the other hand, when the medium compositions of Manufacturing Example 9 and Comparative Manufacturing Example were treated, it can be seen that the increase rate in the number of cells did not increase rapidly even until 48 hours after incubation.

[0100] Example 1: Preparation of dry film medium

[0101] (1) 3.4 g of pancreatic digest of casein, 2.4 g of yeast extract, 1.3 g of dipotassium phosphate, 0.4 g of potassium phosphate, and 0.6 g of dextrose were added to 1 L of distilled water, and then mixed to prepare a basic medium (same ingredients as the bacterial count dried film medium I in the Food Codex).

[0102] (2) A medium composition was prepared by adding 6.8 g of sodium succinate to the above basic medium.

[0103] (3) 91.4 g of guar gum (gelling agent) and 0.0205 g of 2,3,5-triphenyltetrazolium chloride (indicator) were added to the above medium composition and then mixed.

[0104] (4) The above mixture was homogenized, dried under reduced pressure, and then freeze-dried to powder.

[0105] (5) The mixture powder of step (4) was uniformly applied and pressed onto a transparent polystyrene film on which an adhesive layer was formed by applying an adhesive (N-vinylpyrrolidone). Afterwards, excess powder was removed and dried to produce a dry film medium with a thickness of approximately 150 μm.

[0106]

[0107] Example 2: Succinate: Pyruvate = 9:1

[0108] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 9:1 was added in the same weight to prepare a dry film medium.

[0109]

[0110] Example 3: Succinate: Pyruvate = 8:2

[0111] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 8:2 was added in the same weight to prepare a dry film medium.

[0112]

[0113] Example 4: Succinate: Pyruvate = 7:3

[0114] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 7:3 was added in equal weight to prepare a dry film medium.

[0115]

[0116] Example 5: Succinate: Pyruvate = 5:5

[0117] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 5:5 was added in the same weight to prepare a dry film medium.

[0118]

[0119] Example 6: Succinate: Pyruvate = 4:6

[0120] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 4:6 was added in the same weight to prepare a dry film medium.

[0121]

[0122] Example 7: Succinate: Pyruvate = 3:7

[0123] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 3:7 was added in the same weight to prepare a dry film medium.

[0124]

[0125] Example 8: Succinate: Pyruvate = 2:8

[0126] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 2:8 was added in the same weight to prepare a dry film medium.

[0127]

[0128] Example 9: Succinate: Pyruvate = 1:9

[0129] The same procedure as in Example 1 was followed, but in step (2), instead of using sodium succinate alone, a mixture of sodium succinate and sodium pyruvate in a weight ratio of 1:9 was added in the same weight to prepare a dry film medium.

[0130]

[0131] Comparative Example 1: 3M's dry film badge

[0132] Dry film medium (3M Petrifilm) from 3M Korea Co., Ltd. TM ) was purchased, and the composition is identical to that of the food code bacterial count dry film medium 1.

[0133]

[0134] Comparative Example 2: Pyruvate only

[0135] The same procedure as Example 1 was followed, but in step (2), sodium pyruvate was used instead of sodium succinate to prepare a dry film medium.

[0136]

[0137] Comparative Example 3: Fumarate

[0138] The same procedure as Example 1 was followed, but in step (2), sodium fumarate was used instead of sodium succinate to prepare a dry film medium.

[0139]

[0140] Comparative Example 4: Malate

[0141] The same procedure as Example 1 was followed, but in step (2), sodium malate was used instead of sodium succinate to prepare a dry film medium.

[0142]

[0143] Comparative Example 5: Omission of animal pancreatic digest

[0144] The same procedure as Example 1 was followed, but in step (1), animal pancreatic digest was omitted as a nitrogen source and only 5.8 g of yeast extract was used to prepare a dried film medium.

[0145]

[0146] Test Example 1: Confirmation of detection of Gram-negative bacteria

[0147] The purpose of this study was to determine whether it is possible to detect Gram-negative bacteria within 24 hours using the dry film medium according to the present invention.

[0148] Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC9027) were each inoculated into 10 mL of TSB Broth medium and cultured at 37°C for 19-24 hours to activate them. Afterwards, the strains were inoculated into 200 mL of Broth medium and cultured at 37°C for 5 hours to reach the early stationary phase. Next, 5 mL of the strains were mixed with 95 mL of the basic medium of Example 1 to prepare a 5% strain culture solution.

[0149] After inoculating 1 mL of the E. coli culture solution onto the dried film medium according to the examples, control group, and comparative examples, the culture was incubated at 35°C for 24 hours, and the number of colonies was counted according to the incubation time, and detection was confirmed based on this (see Table 4 and Fig. 2). If 15 or more red colonies were found with the naked eye as the incubation time elapsed after the inoculation of the microorganism, the microorganism was considered to be detected.

[0150] Detection of E. coli after 12 and 24 hours of culture Succinate: Pyruvate After 12 hours of culture After 24 hours of culture Number of colonies detected Number of colonies detected Example 110: 0--197O Example 29: 1--180O Example 38: 213-163O Example 47: 3105O184O Example 55: 5117O192O Example 64: 6102O175O Example 73: 76-144O Example 82: 8--116O Example 91: 9--103O Comparative Example 10: 10--97O Comparative Example 20: 10--95O Comparative Example 3 Fumarate----Comparative Example 4 Malate----Comparative Example 5 10: 0--42-

[0151] Looking at Table 4 and Figure 2 above, when culturing E. coli on the dried film medium according to Comparative Examples 1 to 5, microbial colonies are not formed or are only minimally formed for up to 24 hours of culturing, whereas when culturing E. coli on the dried film medium according to Examples 1 to 9, it can be confirmed that a sufficient number of colonies are formed within 24 hours of culturing, enabling strain detection. In particular, in the case of Examples 4 to 6, it can be confirmed that colonies are formed within 12 hours of culturing, enabling more rapid detection.

[0152] In addition, 1 mL of the Pseudomonas aeruginosa culture solution was inoculated onto the dried film medium according to the examples, control group, and comparative examples, and then cultured at 35°C for 24 hours. The number of colonies was counted over the culture time, and detection was confirmed based on this (see Table 5 and Fig. 3).

[0153] Detection of Pseudomonas aeruginosa after 12 and 24 hours of culture Succinate: Pyruvate After 12 hours of culture After 24 hours of culture Number of colonies detected Number of colonies detected Example 110: 0--152O Example 29: 1--150O Example 38: 278-145O Example 47: 3210O216O Example 55: 5208O210O Example 64: 6226O230O Example 73: 7105O151O Example 82: 882-114O Example 91: 9--103O Comparative Example 10: 10--100O Comparative Example 20: 10--98O Comparative Example 3 Fumarate----Comparative Example 4 Malate----Comparative Example 510: 0--21-

[0154] Looking at Table 5 and Figure 3 above, it can be confirmed that when Pseudomonas aeruginosa is cultured on the dried film medium according to Examples 1 to 9, a sufficient number of colonies are formed within 24 hours of culture, enabling strain detection. In particular, in the case of Examples 4 to 7, it can be confirmed that colonies are formed within 12 hours of culture, enabling faster detection. On the other hand, when Pseudomonas aeruginosa is inoculated and cultured on the dried film medium according to Comparative Examples 1 to 5, no microbial colonies are formed even after 24 hours of culture. Through the above results, it can be seen that the dried film medium according to the present invention promotes colony formation by shortening the lag phase of Gram-negative bacteria compared to the conventional dried film medium.

[0155]

[0156] Although the present invention has been described with reference to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims encompass such modifications and variations as fall within the spirit of the present invention.

Claims

1. In a dry film medium for detecting microorganisms, a coating layer including a medium composition, a gelling agent and an indicator is formed on the medium, The above badge composition is, Contains animal protein hydrolysate and yeast extract as nitrogen sources, A dried film medium for detecting microorganisms containing succinate as a carbon source.

2. In paragraph 1, A dry film medium characterized in that the above succinate is the only carbon source in the medium composition.

3. A dried film medium according to claim 1, characterized in that the medium composition further comprises at least one selected from pyruvate, malate, fumarate, and oxaloacetate as a carbon source.

4. In paragraph 3, The above-mentioned badge composition is a dry film badge characterized in that succinate and pyruvate are contained in a weight ratio of 1:9 to 9:

1.

5. In paragraph 3, The above-mentioned badge composition is a dry film badge characterized in that it contains succinate and pyruvate in a weight ratio of 4:6 to 7:

3.

6. In paragraph 1, A dried film medium characterized in that the above medium composition further contains glucose as a carbon source.

7. In paragraph 6, The above-mentioned badge composition is a dry film badge characterized in that it contains succinate and glucose in a weight ratio of 100:5 to 10.

8. In paragraph 1, A dried film medium characterized in that the animal protein hydrolysate is a pancreatic digest of casein.

9. In paragraph 1, A dry film medium characterized in that the above medium composition further comprises dipotassium phosphate, potassium phosphate or a mixture thereof as a personnel.

10. In paragraph 1, A dried film medium characterized in that the gelling agent is at least one selected from agar, sodium alginate, guar gum, xanthan gum, gellan gum, locust bean gum, hyaluronic acid, and sodium hyaluronate.

11. In paragraph 1, A dry film medium characterized in that the indicator is at least one selected from triphenyltetrazolium chloride, phenol red, neutral red, eosin, methylene blue, bromothymol blue, and bromoxylenol blue.

12. In paragraph 1, The above microorganism is a gram-negative bacteria. A dried film medium characterized in that the above-mentioned Gram-negative bacteria are at least one selected from the group consisting of Escherichia genus bacteria, Pseudomonas genus bacteria, Salmonella genus bacteria, Vibrio genus bacteria, Yersinia genus bacteria, Campylobacter genus bacteria, and Cronobacter genus bacteria.

13. In paragraph 12, A dried film medium characterized in that the above gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa.

14. In a film-type bacterial detection paper having a coating layer formed on a substrate including a badge composition, a gelling agent, and an indicator, The above badge composition is, Contains animal protein hydrolysate and yeast extract as nitrogen sources, A film-type bacterial detection paper containing succinate as a carbon source.

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