KR102144710B1 - Medium composition reduced degradation of ingredient by gamma-ray irradiation - Google Patents

Abstract

The present invention relates to a medium composition that reduces or prevents component denaturation by gamma-ray irradiation and a method for preparing the same, and in particular, it includes cysteine, glutathione, and sodium thioglycolate as active ingredients. By doing so, the composition is reduced or prevented from denaturation of the composition by irradiation with gamma rays, and sterilization is possible through irradiation with gamma rays, and to a method for producing the same.

Description

Medium composition with reduced denaturation by gamma irradiation {MEDIUM COMPOSITION REDUCED DEGRADATION OF INGREDIENT BY GAMMA-RAY IRRADIATION}

The present invention relates to a medium composition for reducing or preventing component denaturation due to gamma-ray irradiation and a method for producing the same.

In Vitro Diagnostics (IVD) is used for testing using samples such as tissues, blood, urine, etc. collected from the human body for the purpose of diagnosing diseases and determining prognosis, assessing health conditions, determining treatment effects of diseases, and preventing. As a medical device, it is used in the sense of including reagents. In such medical devices, the sterilization process to prevent contamination is very important.

Radiation irradiation technology using gamma rays, electron beams and X-rays is a cold and hot sterilization and insecticide technology that can remove harmful components without increasing the temperature in the complete packaging state of the product. It is being used as an effective technology for Among the radiations used in the food and public health industries, gamma rays account for more than 80% and electron rays account for less than 20%, and X-rays are mostly used only for medical diagnosis.

Gamma rays are very short, very short wavelengths, and have very strong penetrating power, so they can be processed continuously in a completely packaged state, thereby increasing energy efficiency and reducing secondary pollution of products as they do not require repackaging operations. There is an advantage. In addition, it is a cold-temperature sterilization and insecticide method that minimizes component destruction due to the increase in product temperature and prevents changes in appearance. Unlike methods using chemical fumigants or chemical additives, it does not generate harmful ingredients and does not leave residual ingredients. The advantage is that it is hardly affected by conditions. In addition, it is known to be advantageous in terms of economics and product quality maintenance as it requires far less energy.

The sterilization business for medical supplies has conventionally been sterilization with ethylene oxide gas, but a sterilization method using gamma irradiation, which has been recognized for safety since 1990, and has been increasing as the safety and health issues for the human body have arisen. Is increasing. However, in many compounds, radical formation occurs by irradiation with gamma rays, and representative examples thereof include OH, H, and O radicals. When irradiating medical devices, the most important thing to pay attention to is the deterioration of the product material, and plastics often lose their strength and become brownish by irradiation. In order to prevent this action, a material of a radiation-resistant material to which a stabilizer layer is added has been developed.

Medium contains nutrients necessary for cells, and is used for cultivation, selection, and transport of cells or microorganisms. The medium is classified into a solid medium and a liquid medium according to its shape, and the solid medium is prepared by adding gelatin or agar to the liquid medium to solidify it.

Transport Medium for molecular diagnosis is a medium designed to maintain its original state for a long time without killing or excessive proliferation of bacteria among clinically derived test materials, and is used for transport and temporary storage of specimens. . In molecular diagnostics widely practiced in the field of medicine and biology, it is absolutely required to stably store clinical samples for accuracy of diagnosis. Currently, there are many products of multinational companies for the transport medium products sold in Korea, and multinational companies that produce transport medium include Copan, Beton&Dickinson, and Puritan.

A typical transport medium is UTM (Universal Transport Medium), which can transport samples such as virus, chlamydia, mycoplasma, and ureaplasma in a living state. In the case of gamma-ray sterilization, the UTM medium has a problem in that the shape and components of the medium change and the specimen storage capacity is poor, and thus, it is manufactured through filtering sterilization. However, filtering sterilization has a problem in that the contamination frequency during processing is higher than that of gamma ray sterilization. In addition, filtering sterilization has a problem in that it is difficult to use an automated production device, so that only manual production is possible, so that productivity in the production of a medium is significantly lowered and the cost of the final product is increased.

MacConkey Medium is a selective discrimination medium that selectively grows Gram-negative bacteria and intestinal bacillus, and discriminates lactose fermenting bacteria, and Salmonella Shigella medium is used to detect Salmonella and Shigella bacteria in clinical samples and foods. It is a selective discrimination medium for separation. Macconkey's medium and Salmonella Shigella's medium also have a problem of changing their properties and components upon gamma irradiation, and are thus manufactured through filtering sterilization.

KR 1020090085202 A

JK Kim et al. Sterilization Characteristics of Ionizing Irradiation and Its Industrial Application. J Kor Musculoskelet Transplant Soc 13(2):49~57. Dec, 2013

An object of the present invention is to provide a medium composition and a method of manufacturing the same, which maintains properties and performance even after sterilization with gamma rays.

According to an aspect of the present invention, a medium composition comprising cysteine, glutathione, and sodium thioglycolate as active ingredients, and component denaturation by gamma ray irradiation is reduced or prevented, to be provided. I can.

In addition, the composition may contain 0.05 to 2wt% of the active ingredient with respect to the total 100wt% of the composition.

In addition, the composition may be for preparing at least one selected from the group consisting of a sample transport medium, a Macconkey medium, and a Salmonella Shigella medium.

In addition, the composition may reduce or prevent a change in pH due to gamma ray irradiation.

According to another aspect of the present invention, a method for preparing a medium composition in which component denaturation by gamma-ray irradiation is reduced or prevented, comprising the step of adding cysteine, glutathione, and sodium thioglycolate Can be provided.

According to another aspect of the invention, the steps of preparing a composition comprising cysteine, glutathione, and sodium thioglycolate; Encapsulating the composition in a container; And sterilizing a container containing the composition by irradiation with gamma rays. A method of preparing a sample transport medium may be provided.

The medium composition according to an aspect of the present invention may reduce or prevent denaturation of the composition by irradiation with gamma rays. Through this, even after sterilization through gamma irradiation, the excellent effect of the medium can be maintained.

In addition, since a sterilization method using gamma rays can be applied during sterilization, a separate repackaging process for sterilization treatment is not required, so that the occurrence of contamination during product manufacturing is reduced, and a product with excellent reliability can be manufactured. have.

In addition, since the product can be manufactured using an automated production system using gamma ray irradiation, continuous production is possible, and since a separate repackaging process is not required, it can be manufactured with excellent productivity.

According to the manufacturing method according to another aspect of the present invention, pollution is reduced while the number of process steps is reduced, so that a medium can be manufactured with high productivity and reliability.

1 is a result of confirming components before and after gamma-ray irradiation in a general UTM medium,
Figure 2 is a result of confirming that the pH change did not occur after gamma-ray irradiation of the UTM medium according to the present invention,
3 is a result of confirming that the UTM medium according to the present invention maintains excellent performance even after gamma-ray irradiation,
4 is a result of confirming that the UTM medium according to the present invention maintains an excellent sterilization state after gamma irradiation.

The present invention relates to a medium composition comprising cysteine, glutathione, and sodium thioglycolate as active ingredients, and reducing or preventing component denaturation by irradiation with gamma rays, and a method for producing the same.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, a medium composition may be provided.

The medium composition is for preparing a medium for transporting, selecting, or culturing cells or viruses, and may contain useful ingredients adjusted according to the type and purpose of the application object. The application object may be an animal cell, a plant cell, a microorganism or a virus.

The medium may be at least one selected from the group consisting of a sample transport medium, a McConkie medium, and a Salmonella Shigella medium, but is not limited thereto. In the sample transport medium, the sample may be a microorganism or a virus. The microorganisms may be pathogenic bacteria. For example, the microorganism may be at least one selected from the group consisting of chlamydia, mycoplasma, and ureaplasma, but is not limited thereto.

The medium composition may include cysteine and sodium thioglycolate as active ingredients. The medium composition according to an aspect of the present invention may reduce or prevent the destruction or denaturation of useful components in the medium composition according to gamma-ray irradiation by including the active ingredient. Through this, even after the medium made of the medium composition undergoes gamma ray irradiation, the original purpose can be achieved without significantly deteriorating its effect. Preferably, the medium composition may further include glutathione.

In the medium composition, the active ingredient may be included to be 0.05 to 2wt% based on the total 100wt% of the composition. Specifically, a mixture in which the cysteine and the sodium thioglycolate are mixed at a ratio of 1:0.8 to 1.2 by weight may be included to be 0.05 to 2 wt% based on the total 100 wt% of the medium composition. Preferably, it may be included to be 0.05 to 1.5wt%, more preferably 0.1 to 1.5wt%. When the medium further includes glutathione as the active ingredient, the glutathione may be included in a 2 to 4 weight ratio based on the cysteine. Specifically, the active ingredient may be a mixture of the cysteine, the sodium thioglycolate, and the glutathione in a weight ratio of 1: 0.8 to 1.2: 2 to 4. When the active ingredient is contained in an amount within the above-described range, stability against gamma-ray irradiation can be increased without reducing the effect on the original cell or virus of the medium composition to be prepared.

According to another aspect of the present invention, a manufacturing method for preparing the medium composition may be provided.

The manufacturing method may include adding cysteine and sodium thioglycolate. At this time, preferably, glutathione may be added together. Specifically, it may be performed by adding cysteine, glutathione, and sodium thioglycolate in the step before, after, or in the same step of the step of adding useful ingredients for preparing a medium, Through this, it is possible to prepare a medium composition in which changes in properties and components are reduced or prevented by irradiation with gamma rays.

The useful ingredients include, for example, in the case of a medium composition for preparing a sample transport medium, sucrose, glutamic acid, carbonate, and BSA (1% Bovine Serum Albumin). ), and may further include an antifungal agent or antibiotic, but is not limited thereto. The carbonate-based compound may be sodium bicarbonate, but is not limited thereto. As the antifungal agent or antibiotic, for example, one selected from the group consisting of vancomycin, colistin, and gentamicin may be used, but is not limited thereto. Or, as another example, in the case of a medium composition for preparing a Macconkey medium, peptone, lactose, bile salt, NaCl, neutral red as a pH indicator, crystal violet, and It may include agar, but is not limited thereto.

Before using the prepared medium composition, the sterilization process is essentially performed.In the case of the conventional sample transport medium, McConkie medium and Salmonella Shigella medium, when irradiated with gamma rays, the components contained in the medium are destroyed or There is a problem in that the properties and performance are changed due to degeneration, and it was manufactured through filtering sterilization. In the case of using this sterilization method, it is difficult to produce by an automated process because it is performed by hand, and there is a problem that frequent contamination occurs during the process. However, when the above-described manufacturing method is applied, since the medium composition to be produced is denatured by gamma ray irradiation, gamma ray irradiation can be applied by a sterilization method. Through this, preparing a medium composition containing cysteine, glutathione, and sodium thioglycolate, sealing the prepared medium composition in a container, and sterilizing it by irradiating with gamma rays. Since it is possible to manufacture a medium through a simplified process including, productivity is increased, and since sealing is performed at a relatively early stage, contamination problems are reduced, and reliability of the manufactured product may be increased.

The container may be made of a material that does not cause denaturation by irradiation with gamma rays. For example, it may be made of at least one material selected from the group consisting of acetal polymer, silicone, and nylon, but is not limited thereto.

Hereinafter, preferred embodiments are presented to aid in understanding the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the examples.

Example

<Performance check of UTM (Universal transport medium) medium composition>

Copan's UTM medium and UTM medium developed by the company (Noble Bio) were prepared, and their performances were compared and shown in Tables 1 and 2 below. Sample Samples Five specimens were prepared and compared. For performance comparison, influenza virus samples were treated at a concentration of 1000 to 10000 copies/ml and stored separately for 24 hours, 3 days or 7 days under 4℃, room temp (RT 25±2℃) temperature conditions, and then real time cDNA It was confirmed by performing PCR analysis under synthetic conditions.

Ct of Epsterin-Barr Virus
(Copan Corporation) Ct of Epsterin-Barr Virus
(Noble Bio) Within 24 hours 3 days 7 days Within 24 hours 3 days 7 days One 21.67 21.99 22.74 22.2 21.76 22.38 2 23.1 23.55 24.89 21.67 22.2 23.6 3 27.6 24.66 24.68 27.63 25.45 25.22 4 19.39 19.85 19.96 19.44 20.2 20.22 5 32.36 32.38 32.74 32.22 31.86 32.89

Delta Rn of Epsterin-Barr Virus
(Copan Corporation) Delta Rn of Epsterin-Barr Virus
(Noble Bio) Within 24 hours 3 days 7 days Within 24 hours 3 days 7 days One 14,336 14,204 18,788 13,620 13,410 13,998 2 9,837 10,899 13,851 12,693 12,030 13,269 3 8,927 9,626 15,809 9,733 9,731 13,703 4 16,587 15,614 18,731 14,365 13,621 14,940 5 6,705 4,491 6,750 4,515 6,023 5,589

Looking at Tables 1 and 2, it can be seen that Copan's UTM medium and its UTM medium are similar in terms of their performance.

Thereafter, Copan's UTM medium and its UTM medium were requested to Soya Green Tec to sterilize through irradiation with gamma rays, and the following properties are compared and shown in Table 3 below.

Copan's badge Noble Bio Company Badge Color before gamma ray sterilization Red color Red color Color after gamma ray sterilization yellow yellow

Looking at Table 3, it can be seen that the color of all UTM medium compositions, regardless of manufacturer, changed from red to yellow after gamma irradiation. The UTM medium used as a sample sample contains phenol red, a pH indicator, through which the pH of the medium can be checked.Through the change in color, the pH of the medium composition changes to acidity after gamma irradiation. I can confirm.

Thereafter, an experiment was performed to confirm the components and the function of the medium together with the sample sample before gamma irradiation. Components were confirmed by sending each sample sample before and after gamma ray irradiation on the company's UTM medium to the analysis center of the Korea Research Institute of Chemical Technology to request component analysis (LC-MS analysis), and the results are shown in FIG. In FIG. 1, (a) means before gamma ray sterilization, and (b) means after gamma ray sterilization.

Referring to FIG. 1, it can be seen that the composition of the UTM medium is changed by irradiation with gamma rays.

<Performance check of MacConkey and SS (Salmonella shigella) plate medium with active ingredient added>

Changes in properties and performance according to gamma irradiation were confirmed using MacConkey and SS (Salmonella shigella) medium. MacConkey medium is peptone 17g, proteose peptone 3g, lactose 10g, bile salts 1.5g, neutral red 0.03g, crystal violet 0.001g, agar 13.5g to 1 liter of water and adjusted to 25℃ pH 7.1. Was prepared. SS medium is peptone 5g, lactose 10g, bile salts 8.5g, sodium citrate 10.0g, sodium thiosulphate 8.5g, ferric citrate 1.0g, brilliant green 0.00033g, Lab-lemco powder 5.0g, neutral red 0.025g, agar 15g with water Was added until 1 liter and adjusted to 25° C. pH 7.0 to prepare. Using these as comparative examples, gamma-ray irradiation was performed under the same conditions as described above, and the shape and medium performance were checked by distinguishing the group before and after the gamma irradiation, and the results are shown in Tables 4 and 5 below. The change in the shape of the medium was performed by observing the change in color with the naked eye, and the change in the medium performance was performed by treating Salmonella as a sample for each sample sample medium, and incubating for 24 hours under 35°C temperature condition, and then counting the number of colonies formed. And compared.

In addition, for use as an example, a 1:3:1 weight ratio mixture of cysteine, glutathione and sodium thioglycolate as additives to each medium composition before hardening by putting agar is 1.5wt% based on 100wt% of the total medium composition After the content was added and stirred to prepare and prepare a medium composition according to an aspect of the present invention, gamma irradiation was performed under the same conditions as in the comparative examples, and the results are shown in Tables 4 and 5 below.

MacConkey Badge Comparative example
(Before gamma ray sterilization) Comparative example
(After gamma ray sterilization) Example
(After gamma ray sterilization) shape Red color yellow Red color Number of colony formed 292 pcs 55 pcs 351 pcs

SS badge Comparative example
(Before gamma ray sterilization) Comparative example
(After gamma ray sterilization) Example
(After gamma ray sterilization) shape Orange Orange Orange Number of colony formed 35 pieces 32 pcs 59 pieces

Referring to Tables 4 and 5, the medium composition according to an aspect of the present invention did not show any change in properties after gamma-ray irradiation, and it was found to maintain excellent functions. Therefore, as a medium composition capable of gamma ray sterilization, it can be confirmed that it is suitable to be manufactured by applying it to an automated production line.

<Confirmation of properties and performance of UTM medium containing active ingredients>

An experiment was conducted to confirm the properties and performance of UTM medium prepared by adding a 1:3:1 weight ratio mixture of cysteine, glutathione and sodium thioglycolate as active ingredients in an amount of 2.0 wt% with respect to 100 wt% of the total medium composition. Performed.

The prepared UTM medium was divided into groups and sterilized by filtering and gamma ray sterilization at an intensity of 10 kGy, respectively, and the pH meter was measured and the color difference was visually observed, and the results are shown in FIG. 2. In FIG. 2, (a) is a visual color observation and pH measurement result of UTM medium not subjected to gamma-ray sterilization, and (b) is a visual color observation and pH measurement result of UTM medium subjected to gamma-ray sterilization.

2, the UTM medium contains phenol red, which is a pH indicator, so that the color varies greatly depending on the difference in pH. However, the UTM medium according to the present invention has a pH change and color according to gamma ray irradiation. It can be seen that there is no change.

For the performance experiment of the prepared UTM medium, Influenza A virus stock solution was prepared so that the cycle threshold (Ct) value was 22 to 25 (10,000 ~ 1,000 copies/ml), and each inoculated into the sterilized medium by a different method. Then, it was stored for 7 days under the conditions of 4°C, RT, and 37°C. After separating total nucleic acid, cDNA was synthesized and real-time PCR was performed. PCR was performed 45 times at 95 ℃ for 2 minutes, followed by 2 seconds at 95 ℃ and 30 seconds at 60 ℃ using the ABI 7500 real time system. The performance test results are shown in FIG. 3. In Figure 3, (a) is 4 ℃, (b) at RT, (c) is the performance results of the medium stored at 37 ℃.

3, it can be seen that the UTM medium according to the present invention exhibits excellent sample storage performance at a level similar to that before gamma radiation even after gamma radiation irradiation. The lower the storage temperature, the less damage the influenza A virus sample was, and the higher the storage temperature, the more damage the influenza A virus sample was.

In order to confirm the degree of sterilization, 0.5 ml of UTM gamma-ray sterilized with an intensity of 10 kGy was added to Tryptic Soy Broth (TSB), followed by incubation at 37° C. for 7 days, and the results are shown in FIG. 4. In FIG. 4, NTC means a negative control group, samples 1 to 5 mean a mixture of TSB and gamma-ray sterilized UTM medium, and PTC is a positive control inoculated with TSB, gamma-ray sterilized TUM medium, and E.coli . it means.

4, as a result of sterilization, it can be seen that the UTM medium prepared according to the present invention and irradiated with gamma rays exhibits excellent sterilization.

<Application of production line>

The UTM medium composition according to the present invention was produced by applying it to an automated production line, and the incidence and production amount of product contamination were compared with those before the application. Specifically, in the case of the previous UTM medium composition, since sterilization through gamma rays was not possible, a medium composition was first prepared, filtered and sterilized by hand, and then put in a container to be prepared as a sealed product. The composition was immediately put into a container and sealed, and then gamma ray sterilization was performed by requesting a company to produce a product. The results of comparing the incidence of product contamination before and after application and the production amount are shown in Table 6 below.

Before application After application Product contamination rate (%) 5% 0.1% or less Production (day) 4,000ea 20,000ea

Referring to Table 6, it can be seen that product quality and productivity were remarkably increased by introducing a gamma-ray sterilization process.

Claims (6)

delete delete delete A medium containing 0.05 to 2 wt% of active ingredients in which cysteine, glutathione, and sodium thioglycolate are mixed in a weight ratio of 1: 2 to 4: 0.8 to 1.2 with respect to the total 100 wt% of the composition As a composition,
The medium composition is for preparing at least one or more selected from the group consisting of a sample transport medium, a Macconkey medium, and a Salmonella Shigella medium,
When the medium prepared by the medium composition is a sample transport medium,
Constituent denaturation by gamma-ray irradiation containing 2wt% of active ingredients in which cysteine, glutathione and sodium thioglycolate are mixed in a weight ratio of 1:3:1 with respect to the total 100wt% of the composition Medium composition to be reduced.
A medium containing 0.05 to 2 wt% of active ingredients in which cysteine, glutathione, and sodium thioglycolate are mixed in a weight ratio of 1: 2 to 4: 0.8 to 1.2 with respect to the total 100 wt% of the composition As a composition,
The medium composition is for preparing at least one or more selected from the group consisting of a sample transport medium, a Macconkey medium, and a Salmonella Shigella medium,
When the medium prepared by the medium composition is McConkie medium or Salmonella Shigella medium,
Component modification by gamma-ray irradiation containing 1.5 wt% of active ingredients in which cysteine, glutathione, and sodium thioglycolate are mixed in a weight ratio of 1:3:1 with respect to the total 100wt% of the composition The medium composition in which this is reduced.
Preparing a medium composition according to claim 4 or 5;
Sealing the medium composition in a container; And
A method for producing a medium in which component denaturation by gamma ray irradiation is reduced, comprising the step of sterilizing a container containing the medium composition by irradiation with gamma radiation.

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