KR20030004864A - Selective culture medium composition for pure acido-halophilic microorganisms lived in semi-fluidal conditions and a manufacturing method of selective culture medium - Google Patents

Abstract

PURPOSE: Provided are a selective culture medium composition for pure acido-halophilic microorganisms viable in semi-fluid conditions and a manufacturing method the selection medium using the same composition, thereby culturing the microorganisms with higher growth rate than in an MRS medium or a YM medium. CONSTITUTION: The selective culture medium composition for pure acido-halophilic microorganisms viable in semi-fluid conditions comprises 3 to 7 g/l of yeast extract, 1.5 to 4 g/l of malt extract, 1.5 to 4 g/l of meat extract, 8 to 12 g/l of tryptone or peptone, 15 to 25 g/l of glucose or lactose, 0.5 to 1.5 g/l of polysorbate 80, 1 to 3 g/l of citric acid or malic acid, 25 to 35 g/l of sodium chloride, and 7 to 9 g/l of agar, wherein 25 to 35 ml/l of glycerol is optionally added to the composition. The method for producing the selective culture medium comprises the steps of: mixing yeast extract, malt extract, meat extract, tryptone or peptone, glucose or lactose, polysorbate 80, citric acid or malic acid, sodium chloride, and agar and inserting the mixture into a flask; adding water into the mixture; slightly heating the mixture; adding glycerol into the heated mixture; and sealing the flask and sterilizing the mixture.

Description

Selective culture medium composition for pure acido-halophilic microorganisms lived in semi-fluidal conditions and a manufacturing method of selective culture medium}

The present invention relates to a selective medium composition for pure culture of microorganisms in semi-fluid, high salt, acidic conditions, and a selective medium manufacturing method using the same. More specifically, the present invention includes citric acid or maleic acid, sodium chloride, agar, glycerol, and the like. The present invention relates to a selective medium composition capable of culturing a high concentration of microorganisms grown in a special condition of a high salt content and a low hydrogen ion concentration in a semi-flow state, and to a method for producing a selective medium using the same.

In the 21st century biotechnology era, the development of high-tech biotechnology, which introduces "Biomimetic Technology", which can industrially imitate, manipulate, and apply various functions of microorganisms, makes its materials visible. Competition among countries to secure their own microbial resources is becoming fierce.

These useful microbial resources mainly have colony-forming characteristics under special environmental conditions, for example, many kinds in special environmental conditions such as fermented kimchi and leftovers (heavy, eosinophil, mid-temperature and semi-fluid). Useful microbial resources are known to exist.

However, in the case of pure culture or preservation of living microorganisms exposed to the above-mentioned special environment using commercially available commercial medium, the titer is not only sharply lowered but also the microbial inherent physiological activity There are problems that are lost or easily denatured.

Therefore, there is an urgent need for the development of selective modified medium to prevent the degradation or denaturation of the microorganism's titer when passing or preserving microorganisms under special environmental conditions.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a selective medium composition capable of purely cultivating useful microbial resources that grow under special environmental conditions such as leftover food or kimchi.

Another object of the present invention is to provide a method for producing a selective medium capable of purely cultivating microorganisms grown under environmental conditions that are physically semi-flowing and chemically have high concentrations of hydrogen ions and high salt contents. will be.

1 is a comparison table of Candida growth rate in the medium of Examples 7, 8, 9 and Comparative Examples 1, 2,

2 is a comparison table of Lactobacillus growth rate in the medium of Examples 7, 8, 9 and Comparative Examples 1, 2,

3 is a comparative table of growth rate of bifidus in the medium of Examples 7, 8, 9 and Comparative Examples 1, 2,

4 is a mixed microorganism growth rate comparison table in the medium of Examples 7, 8, 9 and Comparative Examples 1, 2,

5 is a growth rate comparison table after 9 hours of culture of the microorganisms in the medium of Examples 7, 8, 9 and Comparative Examples 1 and 2.

The present invention is a yeast extract 3-7 g / L, malt extract 1.5-4 g / L, meat extract 1.5-4 g / L, tryptone or peptone 8-12 g / L, glucose or lactose 15- 25 g / L, polysorbate 80 0.5-1.5 g / L, citric acid or maleic acid 1-3 g / L, sodium chloride 20-30 g / L, agar 7-9 g / L The present invention relates to a selective medium composition for pure culture of microorganism under semi-flow, high salt and acidic conditions.

The present invention also provides a mixture of yeast extract, malt extract, meat extract, tryptone or peptone, glucose or lactose, polysorbate 80, citric acid or malic acid, sodium chloride, and agar into a flask. Steps; Adding water to the flask; Heating the flask and heating it gently until a first bubble is produced; Adding glycerol before cooking after the heating; The present invention relates to a method for preparing a selective medium for pure culture of microorganisms in a semi-flow, high salt, and acidic condition, including completely closing and sterilizing a lid of a flask.

In the present invention, glycerol, which is used as an auxiliary additive in order to secure semi-fluidity of pure culture medium, is a liquid material having a high viscosity and is designed to be added after the medium is prepared with a medium composition and then sterilized.

The ecosystem that best represents the special environmental conditions of the present invention, such as semi-flow, high salt and acid conditions, is leftover food. The remaining foods are high in water content and rich in nutrients, which is suitable for microorganisms to grow. Therefore, when the leftover foods are left, the pH is drastically reduced due to the secretion of fermentation products by the growth of microorganisms.

Table 1 below shows the general pH of the remaining foods measured by leaving time, and it can be seen that the pH is in the range of about 4-5.

Changes in pH over Types of Food Leftovers Kinds Lapse of neglect time 14:00 17:00 21:00 09:00 11:00 14:00 17:00 21:00 fried rice 5.99 4.21 4.14 3.97 3.92 3.86 3.85 3.84 rice 5.98 5.07 4.88 4.12 3.84 3.82 3.84 3.84 mix 4.39 4.21 4.17 4.07 3.96 3.93 3.83 3.83 Japchae 4.61 4.61 4.32 4.24 4.12 4.09 4.08 4.06 fish 5.40 6.28 6.33 6.71 6.81 6.86 7.06 7.04 Fish Soup Rice 4.56 4.32 4.24 4.17 4.13 4.08 4.09 3.83 Kimchi 4.42 4.24 4.15 4.07 4.03 4.02 4.01 3.93 Whole greens 4.82 4.62 4.58 4.61 4.78 4.86 4.97 5.04 Fruits 4.26 4.05 4.05 3.95 3.84 3.86 3.85 3.84

The present inventors add citric acid or malic acid to the medium to create an acidic environment and sodium chloride to produce a desired salt content, and then the amount of agar added in consideration of the hydrogen ion concentration (pH 4-5 range). And glycerol was added as an adjuvant to ensure semi-fluidity of the medium.

Hereinafter, the selective medium composition for pure culture of microorganism under semi-fluid, high salt, and acid conditions of the present invention and a selective medium manufacturing method using the same will be described in detail by way of examples, but the present invention is not limited thereto.

Examples 1 to 3

Yeast extract, malt extract, meat extract, tryptone, glucose, polysorbate 80, citric acid and distilled water were each quantified in the composition of Table 2 to prepare a medium.

Examples 4-6

Yeast extract, malt extract, meat extract, tryptone, glucose, polysorbate 80, malic acid and distilled water were each quantified in the composition of Table 2 to prepare a medium.

The selective medium of the present invention used citric acid (matric acid) and malic acid (malic acid) to adjust the pH to the remaining food level.

Hydrochloric acid and sulfuric acid are excluded because they are toxic and have a very small effect on pH as well as form salts with other components of the medium. Citric acid and malic acid promote the growth of microorganisms. It was chosen because of the material properties involved.

Composition of Medium Composition of Development Medium (Example) and Commercial Medium (Comparative Example) for pH Control Medium composition Example Comparative example One 2 3 4 5 6 One 2 Yeast extract 5 g 5 g 5 g 5 g 5 g 5 g 5 g 3 g Malt Extract 2.5g - 5 g 2.5g - 5 g - 3 g Meat Extract 2.5g 5 g - 2.5g 5 g - 10 g - Trypton 10.0 g 10.0 g - 10.0 g 10.0 g - 10 g 5 g glucose 20.0 g 20.0 g - 20.0 g 20.0 g - 20 g 10 g Polysorbate 80 1.0 g - - 1.0 g - - 1.0 g - Citric acid 1.0 g 2.0 g 2.0 g - - - - - Malic acid - - - 1.0 g 2.0 g 2.0 g - - Ammonium citrate - - - - - - 2 g - Sodium acetate - - - - - - 5 g - Magnesium sulfate - - - - - - 0.1g - Manganese sulfate - - - - - - 0.05g - Potassium phosphate - - - - - - 2 g - Distilled water 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml

Test Example 1

Hydrogen ion concentration control in medium

The medium of Examples 1 to 6 and Comparative Example 1 {commercially available commercial medium, MRS liquid medium} and Comparative Example 2 {commercially available commercial medium, YM liquid medium} having the composition of Table 2 After the preparation, the pH of the medium was measured at each preparation step.

As a result of the test, citric acid was more effective than maleic acid to reduce the pH of the medium as shown in Table 3 below. When citric acid was added at a level of 1 to 2 g / L and maleic acid was at least 2 g / L, the pH was maintained at a level similar to that of the remaining food.

On the other hand, commercial mediums of Comparative Examples 1 and 2 exhibited a pH range of 6.5 to 7.0.

Changes in pH for each medium production step of Examples 1 to 6 and Comparative Examples 1 and 2 Medium manufacturing step Example Comparative example One 2 3 4 5 6 One 2 After mixing liquid medium 5.073 4.487 4.523 5.965 4.679 4.743 6.437 6.954 After liquid medium sterilization 5.245 4.352 4.432 5.478 4.589 4.643 6.409 6.831 Solid media after melting 5.254 4.433 4.507 5.877 4.509 4.774 6.425 6.907

Test Example 2

Control of salinity

The remaining foods were collected by the source and the nutritional content, including the salt content of the remaining foods, was investigated.

As shown in Table 4, the salinity of the remaining foods varied from 0.45%, which is a sample of a Japanese restaurant, to 4.93%, which was a sample of salted vegetables, but averaged about 2.57%.

Nutrient Contents of Each Food Discharge Company (Unit:%) division moisture protein Crude fat Crude fiber View minutes Salinity University Restaurant 9.45 27.41 27.45 1.82 10.15 1.09 General restaurant 1 9.36 28.07 26.37 1.91 8.81 1.01 General restaurant 2 8.31 27.84 13.37 1.69 32.54 1.68 General restaurant 3 9.01 11.84 14.04 4.33 18.26 4.53 Meat-oriented 7.26 28.01 14.90 1.89 31.32 1.93 Haejangguk 7.26 27.74 14.93 2.07 31.22 1.86 Vegetable-oriented 8.16 18.67 7.34 8.81 20.63 4.93 One expression house 13.62 21.27 9.68 4.89 13.59 4.53 Buffet Restaurant 14.37 28.10 29.68 3.77 8.38 3.73 Japanese Restaurant 7.16 43.21 17.99 3.35 6.70 0.45 Mean ± SD 9.39 ± 2.57 26.22 ± 8.12 17.57 ± 7.69 3.45 ± 2.21 18.16 ± 10.30 2.57 ± 1.68

The salt content in the culture medium was easily adjusted using sodium chloride, so that the salt content in the culture medium was determined to be similar to the salinity of the remaining foods.

Test Example 3

Grant of semi-liquidity conditions-agar

Many microorganisms are motility and the growth rate of microorganisms varies depending on the concentration of water, so it is necessary to create a semi-fluid condition in order to prepare a medium to be used in the desired environment.

The material used to impart semi-fluidity to the desired medium is agar. Agar hardens depending on the presence of acid, and coagulation may not occur at all if the pH is too low. Therefore, when agar was prepared by adding agar to the basic medium composition of Examples 1 to 6 at 5 g / L, 8 g / L, 12 g / L, and 15 g / L, and then the pH levels were different. The impact on the liquidity of agar was investigated.

As shown in Table 5, there was a clear correlation between the amount of agar added and the hydrogen ion concentration (pH).

That is, when pH falls below 3, coagulation, which is an inherent function of agar, did not occur.

In addition, when the desired concentration of hydrogen ions was fixed in the range of 4 to 5, the amount of agar added was about 8 g / L, but coagulation sometimes occurred when 8 g / L was added.

Control degree of fluidity by adding agar by concentration of hydrogen ion Agar content Hydrogen ion concentration (pH) 3 4 5 6 5 g / L liquidity Liquidity / semi-fluidity Semi-liquidity Semi-liquidity 8 g / L liquidity Semi-liquidity Semi-Liquidity / Coagulation coagulation 12 g / L liquidity coagulation coagulation coagulation 15 g / L liquidity coagulation coagulation coagulation

Test Example 4

Grant of Semi-fluid Conditions-Glycerol

In order to examine the auxiliary effect of glycerol on the flowability of agar, 30 ml / L of glycerol was further added to each medium composition of Test Example 3 to prepare a medium, and then the fluidity of each medium according to the hydrogen ion concentration was observed.

In Test Example 3, when the amount of agar added was 8 g / L, coagulation sometimes occurred depending on the conditions of the medium preparation, but when glycerol was used as an auxiliary additive together with agar, as shown in Table 6, coagulation by agar The phenomenon did not occur.

Therefore, in order to secure semi-fluidity of the medium, it was evaluated that it is most preferable to use 30 ml of glycerol per 1000 ml of medium as an auxiliary additive and add agar at 8 g / L level of the medium.

Control of Fluidity by Addition of Glycerol and Agar by Concentration of Hydrogen Ion division Hydrogen ion concentration 3 4 5 6 5g / L agar + 30ml / L glycerol added liquidity liquidity Semi-liquidity Semi-liquidity 8g / L agar + 30ml / L glycerol added liquidity Semi-liquidity Semi-liquidity coagulation 12g / L agar + 30ml / L glycerol added liquidity Semi-Liquidity / Coagulation Semi-Liquidity / Coagulation coagulation Agar 15g / L + Glycerol 30ml / L addition liquidity coagulation coagulation coagulation

The present inventors have conducted various tests as described above to prepare selective medium for pure culture of useful microbial resources that grow under special environmental conditions such as fermented kimchi or leftover food (heavy, eosinophil, mid-temperature and semi-fluid environment). As a result, in the present invention, the yeast extract 5 g / L, malt extract 2.5 g / L, meat extract 2.5 g / L, tryptone or peptone 10 g / L, glucose or lactose 20 g / L , Polysorbate 80 consists of 1 g / L, citric acid or maleic acid 1 g / L or 2 g / L, sodium chloride 30 g / L, agar 8 g / L, glycerol 30 ml / L It can be seen that the most preferred embodiment.

Examples 7-9

1. Yeast extract, malt extract, meat extract, tryptone, glucose, polysorbate 80, citric acid, sodium chloride, agar was quantified in the composition ratio of Table 7 and put in a Erlenmeyer flask.

2. 1000 ml of distilled water was slowly added along the inner wall of the flask.

3. The heat was cut once only until the first bubble was formed.

4. Quickly add 30 ml of glycerol before the medium cools down.

5. The cap of the Erlenmeyer flask was completely closed and sterilized to prepare a selective medium.

Examples 7, 8, and 9 and Comparative Examples 1 and 2 Composition of medium Example 7 Example 8 Example 9 Comparative Example 1 Comparative Example 2 Yeast extract 5 g 5 g 5 g 5 g 3 g Malt Extract 2.5g 5 g - 3 g Meat Extract 2.5g 5 g 10 g Trypton 10.0 g 10.0 g - 10 g 5 g glucose 20.0 g 20.0 g - 20 g 10 g Polysorbate 80 1.0 g - - 1.0 g - Citric acid 1.0 g 2.0 g 2.0 g - - Ammonium citrate - - - 2 g - Sodium acetate - - - 5 g - Magnesium sulfate - - - 0.1g - Manganese sulfate - - - 0.05g - Potassium phosphate - - - 2 g - Sodium chloride 30.0 g 20.0 g 15.0 g - - Glycerol 30 ml 30 ml 30 ml - - Agar 8.5 g 8.5 g 8.5 g - - Distilled water 1000 ml 1000 ml 1000 ml 1000 ml 1000 ml

Test Example 5

Yeast (Candy) Growth Rate Comparison

Candida was inoculated in the medium of Examples 7, 8 and 9 having the composition of Table 7 and the commercially available MRS medium of Comparative Example 1 and the YM medium of Comparative Example 2 Next, the growth rate of microorganisms was measured as the incubation time progressed.

The growth rate was calculated by collecting a predetermined amount of microbial samples, washing three times with a buffer, and measuring absorbance at 650 nanometers.

As shown in FIG. 1, the growth rate was the highest in Example 7 and the lowest in Comparative Example 2.

In Examples 8 and 9, the growth rate was similar to that of Comparative Example 1, and the possibility of development medium was sufficiently demonstrated.

Test Example 6

Comparison of Growth Rates of Bacteria (Lactobacillus)

After inoculating Lactobacillus classified as bacteria into the medium of Examples 7, 8 and 9 and Comparative Examples 1 and 2, growth rate was measured in the same manner as in Test Example 5.

Figure 2 shows the test results, the growth rate was significantly higher in Comparative Example 2, the following was Example 7 and the rest were similar.

This result is because Comparative Example 2 is a medium for exclusively culturing Lactobacillus as a commercially available YM medium.

However, the growth rate of Lactobacillus in Example 7 was higher than in the commercial MRS medium of Comparative Example 1 to demonstrate the superiority of the present invention.

Test Example 7

Comparison of Growth Rates of Acid-Soluble Microorganisms (Bifiders)

Example 7, 8, 9 and Comparative Examples 1, 2 After inoculating the bifidus separated from the remaining food in the medium was measured the growth rate in the same manner as in Test Example 5.

As shown in FIG. 3, Examples 7, 8, and 9 show higher growth rates than Comparative Examples 1 and 2, indicating that the present invention is a much more effective culture medium than commercial media.

Test Example 8

Comparison of Growth Rates of Candida, Lactobacillus and Bifidus Mixed Microorganisms

After inoculating Candida, Lactobacillus, bifidus mixed microorganisms into the medium of Examples 7, 8, and 9 and Comparative Examples 1 and 2, growth rates were measured in the same manner as in Test Example 5.

As shown in FIG. 4, the growth rate of the mixed microorganisms grown in the medium of Example 7 was the highest, and the growth rate of the mixed microorganisms grown in the commercial medium of Comparative Example 1 was the lowest.

FIG. 5 shows lactobacillus as a growth table after 9 hours of incubation of Candida, Lactobacillus, Bifidus and mixed microorganisms in the media of Examples 7, 8, 9 and Comparative Examples 1 and 2, and the YM-only medium of Comparative Example 2 Except when cultured in the growth rate of all the microorganisms appeared to be the highest when grown in the medium of Example 7.

These results demonstrate that the medium composition of Example 7 is superior to the composition of the commercial medium for culturing microorganisms that grow at least in specific environments (semi-flow, high salt, acidic conditions).

Test Example 9

Growth Rate of Microorganisms Growing Under Semi-flow, High Salt, and Acid Conditions

The selective medium of Example 7 was inoculated with various microorganisms grown under semi-fluid, high salinity, acidic conditions, and the growth rate and multiplication time were measured.

As a result of the test, the overall growth rate and the doubling time were shown as shown in Table 8.

Growth Rate and Doubling Time of Microorganisms in the Medium of Example 7 microbe Growth rate of microorganisms OD at 650 nm cfu or rfu / ml medium D-time 1h 6h 12h 1h 6h 12h (h) Bacillus circulanse 0.158 1.125 2.147 3.13 × 10 ⁸ 2.78 × 10 ⁹ 16.78 × 10 ¹⁰ 〈0.5 Bacillus megaterium 0.097 0.662 1.987 0.24 × 10 ⁸ 1.51 × 10 ⁹ 8.67 × 10 ¹⁰ 〈0.5 Lactobacillus amylophilus 0.138 0.551 2.016 3.01 × 10 ⁸ 1.28 × 10 ⁹ 13.46 × 10 ¹⁰ 〈0.5 Lactobacillus paracasei 0.191 1.883 2.461 3.78 × 10 ⁸ 6.79 × 10 ⁹ 26.21 × 10 ¹⁰ 〈0.5 Lactobacillus plantarum 0.147 0.678 1.569 3.54 × 10 ⁸ 1.76 × 10 ⁹ 4.42 × 10 ¹⁰ 〈0.5 Weissella confusa 0.026 1.347 2.345 0.09 × 10 ⁸ 6.78 × 10 ⁹ 23.12 × 10 ¹¹ <One Candida utilis 0.148 0.774 2.026 4.21 × 10 ⁸ 2.05 × 10 ⁹ 73.17 × 10 ¹⁰ <One Candida sp. CF-5 0.097 0.506 0.987 1.07 × 10 ⁸ 1.16 × 10 ¹⁰ 2.19 × 10 ¹⁰ 〈0.5 Bifidus sp. RF-5 0.109 1.088 1.876 4.65 × 10 ⁸ 3.88 × 10 ⁹ 12.97 × 10 ¹⁰ 〈0.5

As described above, the microorganisms that grow under special environmental conditions such as semi-fluid, high salt, and acidic conditions may be cultured at a higher growth rate than commercially available MRS medium or YM medium.

In another aspect, the present invention has the effect of minimizing the impact on the hydrogen ion concentration, salinity concentration, fluidity, etc. of the culture microorganisms to reduce the biological activity of the microorganisms or degeneration.

Claims (6)

Yeast extract 3-7 g / L, malt extract 1.5-4 g / L, meat extract 1.5-4 g / L, tryptone or peptone 8-12 g / L, glucose or lactose 15-25 g / L L, polysorbate 80 0.5-1.5 g / L, citric acid or maleic acid 1-3 g / L, sodium chloride 25-35 g / L, and agar 7-9 g / L , Selective medium composition for pure culture of microorganism under semi-fluid, high salt, acidic conditions. The selective medium composition for pure culture of microorganisms according to claim 1, which further comprises 25 to 35 ml / L of glycerol. According to claim 2, wherein the yeast extract 5 g / L, malt extract 2.5 g / L, meat extract 2.5 g / L, tryptone or peptone 10 g / L, glucose or lactose 20 g / L, Polysorbate 80 is 1 g / L, citric acid is 1 g / L, sodium chloride is 30 g / L, agar is 8 g / L, and glycerol is 30 ml / L. Selective medium composition for pure culture of microorganism in acidic condition. According to claim 2, wherein the yeast extract 5 g / L, malt extract 2.5 g / L, meat extract 2.5 g / L, tryptone or peptone 10 g / L, glucose or lactose 20 g / L, Polysorbate 80 is 1 g / L, malic acid is 2 g / L, sodium chloride is 30 g / L, agar is 8 g / L, and glycerol is 30 ml / L. Selective medium composition for pure culture of conditions microorganisms. Mixing yeast extract, malt extract, meat extract, tryptone or peptone, glucose or lactose, polysorbate 80, citric acid or malic acid, sodium chloride, and agar into a flask; Adding water to the flask; Heating the flask and heating it gently until a first bubble is produced; Adding glycerol before cooking after the heating; A method for producing a selective medium for pure culture of microorganisms in a semi-flow, high salt and acidic condition, comprising the step of completely closing and sterilizing the cap of the flask. According to claim 5, wherein the yeast extract is 3-7 g / L, malt extract is 1.5 to 4 g / L, meat extract is 1.5 to 4 g / L, tryptone or peptone is 8 to 12 g / L, glucose Or 15 to 25 g / L lactose, 0.5 to 1.5 g / L for polysorbate 80, 1 to 3 g / L for citric or malic acid, 25 to 35 g / L for sodium chloride, 7 to 10 g for agar / L, Glycerol is 25 to 35 ml / L, a method for producing a selective medium for pure culture of microorganism under semi-fluid, high salt, acidic conditions.