KR20140050832A - Culture media containing garlic extract and plant growth regulator, and the method for cultivating chlorella using the same - Google Patents

Abstract

The present invention relates to a method for cultivating a chlorella which is green microalgae, and more specifically, to a chlorella culture medium containing a garlic extract and a plant growth regulator, and the method for cultivating a green microalgae chlorella using the medium. According to the present invention, a general microbial pollution is prevented during a chlorella cultivation, to ease a pure separation, and growth and population of a chlorella is increased, therefore, a high density of a chlorella cultured product can be effectively.

Description

(Culture Media Containing Garlic Extract and Plant Growth Regulator, and Method for Cultivating Chlorella Using the Same)

     The present invention relates to a method for culturing chlorella, a microalgae, more precisely, a chlorella culture medium characterized by containing a garlic extract and a plant growth regulator, and a culture method of microalgae chlorella using the medium .

    Chlorella is a single-celled organism with a diameter of 2-10 ㎛, which is a kind of natural fresh green algae. It is known as 'mysterious plant' and 'future food' in foreign countries because of its photosynthetic activity and excellent cell division ability. It has a large amount of chlorophyll And eight essential amino acids necessary for the human body, vitamins, minerals and various nutrients as well as a large amount of CGF, a physiologically active substance that is beneficial to health, beauty food, and animal feed.

In order to purely separate chlorella, which is a microalgae in natural seawater and fresh water, pure strains are obtained from an agar medium containing antibiotics. Also, in order to cultivate pure cells, they are cultured in a liquid medium containing a small amount of antibiotics. In conventional chlorella culture, chlorella seeds are firstly cultured and inoculated into a culture medium at a concentration of 4 x 10 < ⁴ > cfu / ml in a culture medium in indoor or outdoor breeding stocks, After about 7-10 days, it grows to a concentration of 1.2 × 10 ⁷ cfu / ml, and it is usually commercialized through processing.

   Unlike bacteria, which are generally prokaryotic, chlorella has a long growth period and low cell density, making it economical to commercialize chlorella. To solve this problem, various methods for culturing chlorella at a high concentration by controlling the composition of the medium and the physical culture conditions have been tried. The method of culturing chlorella with natural light by adding glucose and nutrients as a carbon source to 50% of ground water, storing the culture at 20 to 30 ° C for a certain period of time and stirring at 15 to 25 ° C for a certain period of time (Korean Patent Publication No. 0590296) Population was low. It is known that freshwater green algae chlorella high density culture medium (Korean Patent Publication No. 051835) in which silicon or iron is excluded is a method in which chlorella is cultured in a culture medium by excluding silicon or iron from allele medium. The culture medium containing lactoferrin and the culture method of chlorella using the same (Korean Patent Publication No. 0514919) will be difficult to use in large-scale culture because lactoferrin is an expensive natural substance.

   Currently, around 3,000 tons of chlorella are cultivated and produced annually around the world. Chlorella is widely used as a source of health foods, feeds, and medicines because it contains a lot of plant nutrients. In recent years, chlorella has been used to produce bio-energy such as biodiesel. In order to meet this increase in demand, several methods for culturing high concentrations of chlorella have been attempted.

KR 0590296 B1 KR 0514919 B1

 However, Type and Optimum Concentration of Antibiotics for Aseptic Culture of Marine Microalgae. Alage. 2007. Vol.22 (3). 229-234

   Microalgae, such as chlorella, are clustered together to form colonies or surrounded by mucous materials. Many extracellular mucous materials are attached with many bacteria. For this reason, it is difficult to cultivate chlorella at a high concentration in a pure manner. It is also well known in the art that the rate of growth of chlorella is slower than that of microorganisms such as E. coli and yeast. A chlorella culture medium containing a garlic extract having excellent antimicrobial activity and a plant growth regulator for promoting growth of chlorella for pure culture of high concentration of chlorella.

     In order to accomplish the above object, the present invention provides a culture medium for pure, high concentration of micro green algae. In the present invention, the microalgae culture medium is characterized in that the concentration of garlic extract for pure culture is 0.1 to 0.4%, and the concentration of gibberellin as a plant growth regulator for high-concentration culture is 10 to 150 ppm. In the present invention, the microalgae may be chlorella.

      The present invention provides a culture medium for chlorella, which comprises garlic extract and a plant growth regulator, and a method for separating and culturing the same. The present invention enables a fresh chlorella pure separation and can shorten the culture time by promoting the growth of chlorella. As a result of mass culture with the culture medium according to the present invention, 0.1% garlic extract and 10 ppm plant growth regulator gibberellin-containing medium were found to have an effect of about 9 times increase in the number of the population. Therefore, it is expected that when chlorella is cultured with the medium proposed in the present invention, chlorella of high concentration is secured within a short time.

Growth Effects of Chlorella on Garlic Extract and Gibberellin-Containing Medium

    In order to achieve the above object, the present invention provides a microalgae culture medium comprising garlic extract and plant growth regulator.

    The present invention also provides a pure culture method of chlorella, which is a microalgae, using the above culture medium.

    Garlic has been used for the treatment of infectious diseases before antibiotics and antibiotics. The antimicrobial activity of raw garlic has been known for a very long time and it has been shown that it has growth inhibitory effect on bacteria, yeast, mold and protozoa. The antimicrobial activity of garlic is made up of allicin, which is produced by the decomposition of alliin by alliinase. The antimicrobial mechanism of alicin has been reported to inhibit the important protein synthesis of bacteria, and it has been reported that it reacts with enzymes involved in bacterial respiration or inhibits enzymes involved in fatty acid synthesis.

    Plant growth regulators are substances that promote the growth of photosynthetic plants. Gibberellin is a typical substance. Gibberellin is one of the plant growth regulators that promotes the growth and germination of higher plants and promotes flowering. It is named after Gibberella fujikuroi, a fungus that makes this material.

   In the present invention, the concentration of the garlic extract added to the medium is preferably 0.1 to 0.4%. If the concentration of added garlic extract is more than 0.5%, growth of chlorella is inhibited, and if it is less than 0.1%, the antibacterial activity is weak. The concentration of gibberellin as a plant growth regulator is preferably 10 to 150 ppm. When the concentration of gibberellin is 150 ppm or more or 10 ppm or less, the growth rate of chlorella is lowered.

   Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments. Particularly, in the following examples, only the cultivation of chlorella is described, but it is obvious to those skilled in the art that cultivation of micro green algae other than chlorella can be applied. In addition, although the culture method is described in the following examples, it will be apparent to those skilled in the art that the present invention can also be applied to pure separation of chlorella or other fine green algae.

(1) Chlorella liquid medium

The liquid medium used for the culture of seawater and freshwater Chlorella vulgaris is BG11 medium, and the composition is shown in Table 1.

Table 1. BG11 medium composition
Solution number
Ingredients
Storage solution (g / L)
Usage (ml / 1L)
One
NaNO3
15.0
100
2
K2HPO4
2.0
10
3
MgSO4. 7H2O
3.75
10
4
CaCl2.2H2O
.80
10
5
Ammonium ferric citrate green
0.3
10
6
Citric acid
0.3
10
7
EDTA Na2
0.05
10
8
Na2CO3
1.0
10
9
H3BO3
2.86
One

MnCl 2 .4H 2 O
1.81
ZnSO4 .7H2O
0.22
Na2MoO.42H2O
0.39
CuSO4. 5H2O
0.08
Co (NO3) 2 .6H2O
0.05

After preparing according to the above-mentioned compositional table, titrating to pH 7.1, the prepared medium was autoclaved and used at room temperature for cooling.

(2) Chlorella solid medium

BG11 solid medium was prepared by adding 15 g / L of agar to the liquid medium and sterilized by autoclaving, and then cooled in a petri dish and solidified.

(1) Chlorella liquid culture

In the case of chlorella liquid culture, chlorella seedlings were inoculated at a concentration of about 1.0 × 10 ⁵ cfu / ml to 1.0 × 10 ⁶ cfu / ml, cultured at 25 ° C and shaken at 200 rpm. During the incubation, the light intensity was maintained at about 1,000 lux and the light period was maintained at day: night = 16: 8 hours.

(2) Chlorella solid culture

      In the case of chlorella solid culture, 0.1 ml of the chlorella culture was dispensed into the BG11 solid medium and plated. The smoked petri dishes were placed in a clear culture container containing a carbon dioxide gas pack and cultured. The incubation temperature was maintained at 25 ° C, and the light intensity was maintained at about 1000 lux during incubation, and maintained the light period of day: night = 16: 8 hours.

(1) Production of garlic extract

      10 g of raw garlic was ground in a sterilized mortar and the garlic residue was removed by sterile gauze to obtain garlic juice. The garlic residue was removed by centrifugation, and the supernatant was removed by filtration with a 0.45 μm filter. The sterilized garlic extract was stored in a -20 ° C freezer.

       (2) Manufacture of plant growth regulators

      As a plant growth regulator, gibberellin (GA3), which is frequently used in agriculture, was used. The concentration of gibberellin storage solution was set at 10,000 ppm and stored in a refrigerator at 4 ° C.

(1) Effect of Garlic Extract on Growth of Chlorella

      The chlorella liquid medium was prepared in the same manner as in Example 1, and the stock solution of garlic extract was added at concentrations of 0.1%, 0.5%, 1%, 5% and 10%, respectively, and cultured for shaking as described in Example 2 for 7 days. Culture experiments were repeated three times. The number of chlorella populations was counted by cultivating the solid medium described in Example 1 using the flat culture method and the solid culture method described in Example 2. The results of cultivating chlorella by adding garlic extract are shown in Table 2.

       Table 2. Average number of chlorella extracts per garlic extract concentration (unit: cfu / ml)
Pause

Control group

Garlic extract
0.1%
0.5%
1.0%
5.0%
10%
0
5.0 × 10 ⁵
4.9 × 10 ⁵
5.2 × 10 ⁵
4.9 × 10 ⁵
5.5 × 10 ⁵
4.7 × 10 ⁵
One
7.1 x 10 ⁵
6.3 × 10 ⁵
1.8 x 10 ⁴
6.4 × 10 ³
3.1 x 10 ³
1.0 x 10 ³
2
8.6 × 10 ⁵
8.4 × 10 ⁵
7.3 x 10 ³
5.2 × 10 ²
1.0 × 10 ²
9.1 × 10 ¹
3
9.3 × 10 ⁵
9.9 × 10 ⁵
2.9 × 10 ²
7.9 × 10 ¹
2.2 × 10 ¹
-
4
1.4 × 10 ⁶
2.8 × 10 ⁶
3.5 × 10 ¹
-
-
-
5
3.5 × 10 ⁶
6.6 × 10 ⁶
-
-
-
-
6
5.1 × 10 ⁶
8.2 × 10 ⁶
-
-
-
-
7
7.6 × 10 ⁶
1.6 × 10 ⁷
-
-
-
-

      As a result of adding garlic extract to the culture medium of chlorella, it was confirmed that growth of chlorella was inhibited at a concentration of 0.5% or more of garlic extract. However, chlorella growth did not inhibit chlorella growth at 0.1% of garlic extract, and it was about twice as high as that of the control.

      Microalgae such as chlorella have a mucous layer around the cells. Since symbiotic microorganisms coexist here, it is difficult to cultivate pure water. Until now, antibiotics have been used for pure cultivation, but it is possible to purely cultivate micro green algae by replacing them with garlic extract having antibacterial activity.

(1) Effect of Garlic Extract and Plant Growth Regulating Substrate on Growth of Chlorella

      10%, 50 ppm, 100 ppm, 150 ppm, and 200 ppm of 0.1% garlic extract and gibberellin (GA3) as a plant growth regulator were added to 100 ml of the BG11 liquid medium prepared as in Example 1 to prepare a liquid medium. The prepared liquid medium was inoculated with chlorella and incubated for 14 days in liquid medium shaking culture as described in Example 2. Culture experiments were repeated three times.

     For the determination of the number of chlorella populations, a flat plate method was used for the solid medium as described in Example 1. And cultured in the solid culture method described in Example 2 to recover the number of chlorella. The results of cultivating chlorella in a medium containing garlic extract and plant growth regulator gibberellin are shown in Table 3 and Fig.

     Table 3. Average number of chlorella cells per garlic extract and gibberellin-containing medium (unit: cfu / ml)
Pause

Control group

0.1% garlic extract + GA3
10 ppm
50 ppm
100 ppm
150 ppm
200 ppm
0
4.4 × 10 ⁶
4.5 × 10 ⁶
4.0 × 10 ⁶
4.1 × 10 ⁶
4.0 × 10 ⁶
4.7 × 10 ⁶
2
7.3 × 10 ⁶
9.6 × 10 ⁶
8.3 × 10 ⁶
7.6 × 10 ⁶
6.6 × 10 ⁶
6.2 × 10 ⁶
4
1.6 × 10 ⁷
4.2 × 10 ⁷
2.5 x 10 ⁷
1.9 × 10 ⁷
1.8 x 10 ⁷
9.4 × 10 ⁶
6
5.4 × 10 ⁷
1.3 x 10 ⁸
6.8 × 10 ⁷
6.5 × 10 ⁷
5.8 x 10 ⁷
2.0 × 10 ⁷
8
8.4 × 10 ⁷
2.4 × 10 ⁸
1.3 x 10 ⁸
1.4 x 10 ⁸
1.4 x 10 ⁸
8.6 × 10 ⁷
10
9.3 × 10 ⁷
7.3 × 10 ⁸
3.0 × 10 ⁸
3.6 × 10 ⁸
1.7 x 10 ⁸
1.2 × 10 ⁸
12
7.3 × 10 ⁷
7.0 × 10 ⁸
4.0 x 10 ⁸
4.6 × 10 ⁸
1.3 x 10 ⁸
9.5 × 10 ⁷
14
8.1 × 10 ⁷
7.6 × 10 ⁸
4.5 × 10 ⁸
4.1 × 10 ⁸
1.7 x 10 ⁸
9.1 × 10 ⁷

Chlorella was cultivated in garlic extract and plant growth medium containing gibberellin for 14 days. The highest number of cultivars was 0.1% garlic extract + 10ppm gibberellin treated at 7.6x10 ⁸ cfu / ml, which was about 9 times higher than the control Respectively. In addition, 0.1% garlic extract + 50ppm gibberellin treatment increased the population by 5.5 times compared to the control, 0.1% garlic extract + 100ppm gibberellin treatment increased by 5 times, and 0.1% garlic extract + 150ppm gibberellin It was confirmed that the treatment increased about twice the population.

     It will be apparent to those skilled in the art that these specific details have been set forth in order to provide a more thorough understanding of the invention and are therefore not to be considered limiting of its scope will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

cfu / ml: number of microorganism colony forming units per ml;

Claims (6)

A green algae culture medium characterized by containing garlic extract and gibberellin as a plant growth regulator The microalgae culture medium according to claim 1, wherein the garlic extract concentration is 0.1 to 0.4% The microalgae culture medium according to claim 1, wherein the concentration of gibberellin as a plant growth regulator is 10 to 150 ppm. A culture method of micro green algae characterized by using the culture medium of claim 1 The culture method according to claim 4, wherein the microalgae are chlorella A method for purely isolating micro green algae using the culture medium of any one of claims 1 to 3

Images