KR100514919B1 - Culture Media Containing Lactoferrin and the Method for Cultivating Chlorella Using the Same - Google Patents

Abstract

The present invention relates to a method for culturing chlorella, and more particularly, to a chlorella culture medium, characterized in that it contains lactoferrin, and to a chlorella culture method using the medium.

According to the present invention, the separation of a new chlorella species prevents the contamination of other microorganisms, thereby facilitating the pure separation, resulting in the rapid growth of chlorella, the increase in the population and the viability, and the greater the amount of chlorella without the expansion of existing culture facilities. Can be obtained.

Description

Culture Media Containing Lactoferrin and the Method for Cultivating Chlorella Using the Same}

Technical Field

The present invention relates to a method for culturing chlorella, and more particularly, to a chlorella culture medium, characterized in that it contains lactoferrin, and to a chlorella culture method using the medium.

Background

Chlorella is a spherical or oval green algae plant with a diameter of less than 10 micrometers in diameter of single cells, naturally growing in fresh and sea water. These chlorella contain functional materials such as chlorophyl, CGF (Chlorella Growth Factor) and beta glucan, as well as high-quality proteins, carbohydrates, various vitamins and minerals, and have recently been spotlighted as health foods. have. In addition, it is being used as a feed organism of Rotifer, which is used as a feed food for fry, and there is a great demand.

In order to separate new chlorella species from natural seawater and freshwater, the waters grown in the chlorella were collected, diluted to a certain concentration, incubated, and then incubated again in agar medium to separate single colony. A method of obtaining strains is used. And if you look at the existing chlorella mass cultivation method, first cultivated chlorella seedlings and inoculated the seedlings in the culture medium at indoor or outdoor breeding ground at a concentration of 4 × 10 ⁴ cells / ml, maintaining the water temperature 20 ℃, oxygen supply In order to incubate slowly in order to grow to a concentration of 12 million cells / ml after about 7-10 days, it is common to use this product through the processing process.

Unlike general bacteria, chlorella has a long incubation period and low cell concentration, which is costly to produce. In order to solve this problem, various methods of culturing chlorella at high concentration have been attempted by adjusting the culture conditions. In other words, incubation at a high growth rate at the initial high temperature, gradually cooling the temperature and adding pure oxygen at low temperature to cultivate relatively high yield of chlorella (Korean Patent 2001-0019217) and culturing high concentration chlorella under cancer culture conditions. There is a method (Korean Patent Publication No. 1997-0043017), but they still have the disadvantage of slow growth.

On the other hand, as a method of culturing by changing the composition of chlorella culture medium, using pure organic materials such as perilla leaf, seaweed extract and aquatic product extract to commercialize chlorella without separation process when using a chemical medium (Korea Patent Publication 1998-025344) and It is known to add natural minerals such as ginseng extract and elvan to the culture to increase the functionality (Korean Patent Publication 1984-0000010).

Currently, 3,000 tonnes of chlorella are cultured annually worldwide. Chlorella nutritionally contains antioxidants such as phytoproteins, essential amino acids, essential fatty acids, glycoproteins, vitamins, minerals, dietary fiber, chlorophyll, nucleic acids (DNA, RNA), beta carotenoids, flavonoids, and leucine. (Phytochemical, phytosterol), sterols, gamma linolenic acid and other nutrients, including a wide range of uses for health foods, food additives, feed, pharmaceutical ingredients, etc., the demand is increasing every year.

In recent years, there has been global competition to secure new biological resources. Therefore, there is a need for technology development to isolate and increase yield of new chlorella species. With the conventional techniques mentioned above, many microorganisms contained in the sample collected at the time of separation of the new species cannot be removed, making pure separation and cultivation of the new species very difficult. In addition, in order to meet the increasing demand for phytoplankton such as chlorella, various methods such as expansion of production facilities have been proposed, but there are limitations.

Therefore, the present inventors have made intensive efforts to increase the cultivation efficiency of chlorella, and as a result, using lactoferrin having physiological activity in the culturing of chlorella, it is possible to secure a large number of chlorella in a shorter culture period than conventional culture methods, and survivability It was confirmed that this enhanced chlorella could be obtained and completed the present invention.

After all, the main object of the present invention is to provide a culture medium of chlorella characterized by containing lactoferrin and a method for separating and culturing chlorella using the same.

In order to achieve the above object, the present invention provides a phytoplankton culture medium comprising lactoferrin.

The present invention also provides a method for culturing phytoplankton, characterized in that using the culture medium.

The present invention also provides a pure separation method of phytoplankton, characterized in that using the above culture medium.

In the present invention, the phytoplankton may be characterized in that chlorella.

Lactoferrin, which is used as a main component of the culture medium in the present invention, is a powerful antiviral and antimicrobial substance contained in external secretions of mammals, that is, breast milk, tears, and sweat. Lactoferrin is a protein that binds iron and plays an important role in binding and releasing iron. In other words, by binding to the iron absorbed in the body by blocking the iron supply required for bacterial reproduction to inhibit bacterial growth, the combined iron serves to catalyze the supply of oxygen to all cells in the body. Lactoferrin is also known to play a protective role against disease infections, malignant tumors, AIDS and the like. These lactoferrins play an antimicrobial role against microbes, which are common prokaryotes, but have not been identified for unicellular eukaryotes.

In the present invention, it is preferable that the concentration of lactoferrin added to the culture medium is 0.1 to 1,000 mg / L. If the concentration of the added lactoferrin is less than 0.1mg / L, it is difficult to expect the growth effect of chlorella, and if more than 1,000mg / L, the growth rate and concentration of chlorella relative to the raw material value is low, resulting in low economic efficiency.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, the following examples describe only the culture of chlorella, but it will be apparent to those skilled in the art that the present invention can be applied to the culture of phytoplankton other than chlorella.

In addition, although the following examples describe the culture method, 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 phytoplankton.

Example 1 Culture of Chlorella

1) Chlorella culture medium

F / 2 liquid medium commonly used for culturing seawater chlorella chlorella ellipsoidea (Table 1).

F / 2 liquid medium composition division Furtherance density Main nutrients NaNO ₃ 75mg / ml NaH2PO4H2O 5mg / ml Na ₂ SiO ₃ · 9H ₂ O 15-30mg / ml Trace metal mixture Na ₂ · EDTA 4.36mg / ml FeCl ₃ · 6H ₂ O 3.15mg / ml CuSO4, 5H2O 0.01mg / ml CoCl ₂ · 6H ₂ O 0.01mg / ml MnCl ₂ 4H ₂ O 0.18mg / ml Na ₂ MoO ₄ 2H ₂ O 0.006mg / ml ZnSO ₄ · 7H ₂ O 0.022mg / ml Vitamin Blend Thiamin HCl 0.1mg / ml Biotin 0.5 µg / ml B12 0.5 µg / ml

The culture medium was prepared by adding 1 ml each of the main nutrients having the composition and concentration shown in Table 1 to 1 liter of filtered seawater, and adding 1 ml of the trace metal mixture having the composition and concentration shown in Table 1. The prepared medium was autoclaved (121 ° C., 15 minutes), cooled to room temperature, and 0.5 ml of the vitamin mixture having the composition and concentration shown in Table 1 was used as a chlorella culture medium. The filtered seawater used for the production of the culture medium was used after sterilization using a filter having a pore size of 0.2 μm.

2) Culture of Chlorella

Chlorella culture was inoculated to the culture medium prepared above to the concentration of about 4 × 10 ⁴ ~ 1 × 10 ⁵ individuals / ml of chlorella seedlings, the culture temperature was maintained at 20 ~ 24 ℃, and cultured. The amount of light in the culture was maintained at about 1000 lux, day: night = 18 hours: 6 hours photoperiod was maintained.

Example 2. Effect of Lactoferrin on Chlorella Growth

Chlorella culture medium F / 2 medium was prepared in the same manner as in Example 1, and then lactoferrin was added at concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml, respectively. The lactoferrin used was purchased from a lactoferrin prototype isolated from bovine colostrum, and dissolved in PBS (phosphate buffered saline, pH7.4) buffer at a concentration of 10 mg / ml to make a concentrated solution. / ml, 10 µg / ml, 100 µg / ml was added and used.

The prepared lactoferrin-added culture medium was inoculated with the same amount of Chlorella species, and the number of populations was performed. The inoculum was incubated according to the culture conditions of Example 1 above, and the population was measured using a hemocytometer on a microscope every 24 hours. The results of culturing chlorella by adding lactoferrin at different concentrations are shown in FIGS. 1 and 2.

Chlorella population by lactoferrin concentration Category Day Control Lactoferrin Lactoferrin Lactoferrin (1 μg / ml) (10 µg / ml) (100 µg / ml) Inoculation day 1.0 × 10 ⁶ 1.0 × 10 ⁶ 1.0 × 10 ⁶ 1.0 × 10 ⁶ 1 day 2.5 × 10 ⁶ 2.8 × 10 ⁶ 2.8 × 10 ⁶ 2.8 × 10 ⁶ 2 days 4.8 × 10 ⁶ 4.5 × 10 ⁶ 3.8 × 10 ⁶ 4.3 × 10 ⁶ 3 days 6.2 × 10 ⁶ 6.5 × 10 ⁶ 7.5 × 10 ⁶ 4.5 × 10 ⁶ 4 days 7.4 × 10 ⁶ 1.1 × 10 ⁷ 1.2 × 10 ⁷ 7.7 × 10 ⁶ 5 days 1.2 × 10 ⁷ 2.1 × 10 ⁷ 2.2 × 10 ⁷ 1.6 × 10 ⁷ 6 days 9.3 × 10 ⁶ 3.0 × 10 ⁷ 2.6 × 10 ⁷ 2.4 × 10 ⁷ 7 days 1.2 × 10 ⁷ 3.5 × 10 ⁷ 3.3 × 10 ⁷ 3.0 × 10 ⁷ 8 days 1.4 × 10 ⁷ 4.0 × 10 ⁷ 4.3 × 10 ⁷ 3.7 × 10 ⁷ 9 days 1.5 × 10 ⁷ 5.5 × 10 ⁷ 5.0 × 10 ⁷ 4.5 × 10 ⁷ 10 days 1.7 × 10 ⁷ 6.3 × 10 ⁷ 5.2 × 10 ⁷ 5.7 × 10 ⁷ 11 days 2.6 × 10 ⁷ 6.1 × 10 ⁷ 8.3 × 10 ⁷ 6.0 × 10 ⁷ 12 days 3.0 × 10 ⁷ 5.7 × 10 ⁷ 6.2 × 10 ⁷ 5.7 × 10 ⁷ 13 days 2.5 × 10 ⁷ 6.9 × 10 ⁷ 6.2 × 10 ⁷ 7.5 × 10 ⁷ 14 days 2.5 × 10 ⁷ 6.8 × 10 ⁷ 5.7 × 10 ⁷ 9.2 × 10 ⁷   15th 2.7 × 10 ⁷ 6.4 × 10 ⁷ 6.6 × 10 ⁷ 1.0 × 10 ⁸ 16 days 2.8 × 10 ⁷ 8.6 × 10 ⁷ 7.9 × 10 ⁷ 1.2 × 10 ⁸ 17 days 3.0 × 10 ⁷ 7.9 × 10 ⁷ 7.8 × 10 ⁷ 1.1 × 10 ⁸ 18 days 2.8 × 10 ⁷ 8.2 × 10 ⁷ 7.7 × 10 ⁷ 1.1 × 10 ⁸ 19 days 2.4 × 10 ⁷ 8.4 × 10 ⁷ 7.6 × 10 ⁷ 1.2 × 10 ⁸ 20 days 2.4 × 10 ⁷ 8.3 × 10 ⁷ 7.5 × 10 ⁷ 1.2 × 10 ⁸ 21 days 2.5 × 10 ⁷ 8.1 × 10 ⁷ 7.3 × 10 ⁷ 1.2 × 10 ⁸ 22 days 2.5 × 10 ⁷ 9.3 × 10 ⁷ 8.2 × 10 ⁷ 1.1 × 10 ⁸

As a result of the addition of lactoferrin to the chlorella culture medium, the maximum population of the experimental group (100 µg / ml) was 1.1 × 10 ⁸ population / ml in terms of the population, up to about four times the maximum population of the control group (2.5 × 10 ⁷ ). Population increase was shown. And the time to reach the maximum population of 2.5 × 10 ⁷ population / ml of the control group was reduced to half the control group to 11 days, the experimental group to 6 days.

Among the experimental groups, the growth rate of the logarithmic growth stage of chlorella was higher in the low concentration group than in the high concentration group, but the population at the stationary phase was about 10% higher in the high concentration group. As a result, the addition of lactoferrin 1 ㎍ / ml concentration in chlorella culture was able to secure more than three times the population in half the time compared to the conventional culture method.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing a culture medium of chlorella characterized by the addition of lactoferrin and a method for separating and culturing chlorella using the same.

When the present invention is used in the separation of new chlorella species, it is possible to obtain pure chlorella strains by inhibiting the growth of bacteria, viruses, and the like contained in the sample while promoting the growth of chlorella.

As a result of using the culture medium according to the present invention for the mass culture of chlorella, not only a pure inoculum not contaminated with other microorganisms can be obtained at a short time even in the incubation step of the inoculum for mass culture, and the lactoferrin of 1 µg / ml The addition of low concentrations was able to secure more than three times the population while reducing the incubation time by more than half compared to conventional culture methods.

Therefore, when the present invention is applied to large scale production of a plant unit, it will be possible to obtain a large amount of chlorella in a short time by increasing productivity without expanding the facility.

1 is a graph of the growth curve of chlorella according to the concentration of lactoferrin.

Claims (6)

Phytoplankton culture medium containing lactoferrin. The culture medium according to claim 1, wherein the phytoplankton is chlorellaline. The culture medium according to claim 1, wherein the concentration of lactoferrin is 0.1 to 1,000 mg / L. Method for culturing phytoplankton, characterized in that using the culture medium of claim 1. The method of claim 4, wherein the phytoplankton is chlorellaline. The pure water separation method of phytoplankton, characterized by using the culture medium of any one of claims 1 to 3.