KR20150075775A - Process for the cryostorage of chlorella - Google Patents

Abstract

The present invention relates to a chlorella cryopreservation method. The method utilizes the mixture of skim milk and dimethylsulfoxide as a freezing preservative.

Description

Process for the cryostorage of chlorella [

The present invention relates to a method of cryopreservation of chlorella, and more particularly to a method of cryopreservation of chlorella using a combination of dimethyl sulfoxide and skim milk as a cryopreservative.

 Chlorella, one of the fine freshwater green algae, contains nutrients such as chlorophyll, vitamins and minerals as well as beneficial carbohydrates, proteins and fats, and is known as a nutritious food source. Chlorella, which is widely used as a raw material for health functional foods, is known to be beneficial to skin cosmetics and antioxidant activity, and to promote immunity and defecation. It is also known as a natural food that can be eaten safely by everyone because it helps improve the constitution and nutrition and is effective for growth and development.

Chlorella is cultured by an autotrophic culture, a heterotrophic culture, or a combination thereof, and can be cultured in a medium containing a carbon source, a nitrogen source, a trace metal, a trace element, and the like. For example, Korean Patent Registration No. 10-0193748 discloses a method for producing high-concentration chlorella by heterotrophic culture. For long-term storage of chlorella strains, periodic subculture is usually performed. However, there is a problem in that it is exposed to external pollution due to repetitive change of the medium during repeated passaging, and there is a problem that deformation of the strain such as microevolution of the strain itself may occur.

In order to solve the problem of long-term storage of chlorella through subculture, a method of cryostorage or cryopreservation by freezing the chlorella strain has been proposed. The cryopreservation method is a method of freezing and storing the chlorella culture at about -70 ° C to -80 ° C and thawing the chlorella culture as needed to maintain the physiological activity of chlorella for a long period of time. It has been disclosed that dimethylsulfoxide as a cryoprotectant is added to a chlorella culture to perform lyophilization in order to avoid problems such as destruction of the strain and physiological activity caused by the freezing process (Ben-Amotz and A. Gilboa, Mar. Ecol. Prog., 2: 157-161, 1980).

However, when the dimethylsulfoxide is added as a cryopreservative to the culture solution of chlorella according to the conventional method and lyophilized and stored for a certain period of time and defrosted, the physiological activities of the obtained chlorella still show great differences. That is, there is no significant difference in the dry cell weight (DCW), crude protein content, and chlorophyll content of the chlorella obtained at the time of cryopreservation for a relatively short period of time (for example, about 8 weeks) There is a problem in that there is a large difference in the dry cell weight (DCW), crude protein content, and chlorophyll content of the obtained chlorella when stored frozen for a long period of time (for example, 16 weeks or more).

The present inventors have conducted various studies to develop a method of keeping chlorella frozen for a long time while maintaining the physiological activity of chlorella. The present inventors have variously analyzed the cryopreservation agent and the freezing conditions. As a result, when freezing is carried out using a combination of dimethylsulfoxide and skim milk as cryopreservatives, The physiological activity of chlorella is maintained.

Accordingly, it is an object of the present invention to provide a method of freezing chlorella by using dimethylsulfoxide and skim milk as a cryopreservative.

According to one aspect of the present invention, there is provided a method for producing chlorella culture, comprising the steps of: (a) adding dimethylsulfoxide and skim milk as cryopreservatives to a chlorella culture; And (b) freezing the mixed liquor obtained in step (a).

The chlorella culture solution may be a culture solution having an absorbance at 610 nm in a range of 0.5 to 1.0.

In the method for cryopreserving chlorella of the present invention, 1 to 10 parts of dimethylsulfoxide and 5 to 15 parts of skim milk may be added to 100 parts of the chlorella culture solution; Preferably, 3 to 5 parts of dimethylsulfoxide and 5 to 10 parts of skim milk may be added to 100 parts of the chlorella culture solution; More preferably, 3 parts of dimethylsulfoxide and 10 parts of skim milk may be added to 100 parts of the chlorella culture solution.

In addition, step (b) is performed by placing the mixed solution obtained in step (a) in a freezing vial or a freezing tube, immersing the freezing vial or freezing tube containing the mixed solution in isopropyl alcohol and freezing .

The cryopreservation method according to the present invention uses chlorella as a cryopreservative agent in combination with dimethyl sulphoxide and skim milk to produce chlorella for a long period of time (for example, about 16 weeks or more) while maintaining the physiological activity of chlorella Can be stored frozen. In particular, the frozen storage method according to the present invention can minimize the change in the dry cell weight (DCW), crude protein content, and chlorophyll content of chlorella to within 3% even when stored for a long period of time longer than 16 weeks.

Fig. 1 shows the results of evaluating the culture efficiency after freezing for 4 weeks by applying freezing method using isopropyl alcohol and general freezing method.

(A) adding dimethylsulfoxide and skim milk as a cryoprotectant to a chlorella culture; And (b) freezing the mixture obtained in step (a).

The chlorella may be, for example, Chlorella vulgaris , Chlorella pyrenoidosa), Chlorella regyulra less (Chlorella regularis), pillars (Chlorella as Chlorella Sako saccharophila), Chlorella Thoreau Kearney Ana (Chlorella sorokiniana ), and the like, including all that are available to those skilled in the art.

As the chlorella culture solution used in the freeze storage method of the present invention, a culture solution obtained by culturing chlorella with an ordinary culture method (for example, Korean Patent Registration No. 10-0193748) can be used. For example, the cultivation can be performed in an aseptic tank with an aseptic, organic (e.g., using anhydrous glucose as a sugar source), with air at 0.3-1.0 vvm, stirring at 200-500 rpm, pH 6.5 To 7.5 and can be cultured in a feed-like manner. The Chlorella Chlorella content in the culture medium may be but are not limited particularly, preferably used when using a spectral measurement system measured the absorbance at 610 nm absorbance (OD _610nm) of the culture solution of the 0.5 to 1.0 range.

The method for cryopreserving chlorella according to the present invention is characterized by using dimethyl sulphoxide and skim milk as a cryopreservative. Step (a) of the cryopreservation method according to the present invention comprises adding 1 to 10 parts of dimethylsulfoxide and 5 to 15 parts of skim milk to 100 parts of chlorella culture solution; Preferably 100 parts of chlorella culture solution, 3 to 5 parts of dimethylsulfoxide and 5 to 10 parts of skim milk to skin; More preferably, it can be carried out by adding about 3 parts of dimethyl sulfoxide and about 10 parts of skim milk to 100 parts of the chlorella culture solution.

 Step (b) of the cryopreservation method according to the present invention can be carried out according to a conventional freezing method. For example, the mixed solution obtained in step (a) may be placed in a freezing vial or a freezing tube and the temperature may be lowered to -70 ° C to -80 ° C at a rate of about -1 ° C per minute in the freezer. Surprisingly, the present inventors have found that when the freezing process is carried out in the presence of a specific solvent, that is, isopropyl alcohol, the culture efficiency of chlorella obtained after thawing can be further enhanced. Accordingly, in the step (b) of the cryopreservation method according to the present invention, the mixed solution obtained in the step (a) is placed in a freezing vial or a freezing tube, and the freezing vial containing the mixed solution or the freezing tube is immersed in isopropyl alcohol Followed by immersion, followed by freezing.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

In the following examples, after the Chlorella vulgaris (Chlorella vulgaris) were cultured according to the Republic of Korea Patent No. 10-0193748 arc, the resultant culture solution was used as a chlorella culture medium. In order to adjust a constant content in the chlorella chlorella culture medium, using the medium it was adjusted to an absorbance (OD _610nm) at 610 nm of about 0.75 ± 0.25.

Example 1. Evaluation of cryopreservation agent

As cryopreservation agents, skim milk (SM), glycerol (Gly), bethane, dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) After freezing for 4 weeks, the culture efficiency was evaluated. Specifically, a cryopreservative was added to the chlorella culture solution, and the mixture was dispensed into a freezing tube and dropped in the freezer at a rate of -1 캜 / minute to be frozen to about -75 캜. The resulting frozen strains were kept frozen for 4 weeks. The strain obtained by thawing was inoculated into a medium supplemented with 1% (w / v) of glucose (0.7 g / L KH ₂ PO ₄ , 0.3 g / LK ₂ HPO ₄ , 0.34 g / L MgSO ₄ .7H ₂ (W / v) residual sugar content in the treated group was confirmed by incubation for 48 hours in the presence of 0.5% (w / v, 0.06 g / L EDTA-2Na, 0.4 ml / ] and ΔOD - the (OD _610nm after inoculation when the culture OD _610nm) was measured. The results are shown in Table 1 below.

Skim milk Glycerol Betten DMSO BSA Skim milk NC 12% (v / v) SM +
10% (v / v) Gly
(0.119%, [Delta] OD = 22) NC 10% (v / v) SM + 3% (v / v) DMSO
(0%, [Delta] OD = 25) NC Glycerol NC NC 8% (v / v) Gly + 3% (v / v) DMSO
(0.407%, [Delta] OD = 14) NC Betten NC NC NC DMSO 3% (v / v) DMSO
(0%, [Delta] OD = 27) 5% (v / v) DMSO + 3% (v / v) BSA
(0.293%, [Delta] OD = 17) BSA NC

* NC: not cultured

As can be seen from the results of Table 1, most of the cryopreservation alone did not succeed in cryopreservation, and 3% DMSO treatment as a cryoprotectant showed optimal cryopreservation function. Also, the method of adding skim milk was evaluated as good sugar consumption and cell growth.

Example 2: Evaluation of efficiency different from ratio of cryopreservative agent

Dimethyl sulfoxide (DMSO) and skim milk (SM) were used as cryopreservation agents in the ratio shown in Table 2 below for storage for 8 weeks, and then the culture efficiency was evaluated. Specifically, dimethylsulfoxide (DMSO) and skim milk (SM) as cryopreservatives were added to the chlorella culture according to the ratios shown in the following Table 2, and the resulting mixture was dispensed into a freezing tube and dropped at a rate of -1 ° C per minute -75 < 0 > C. The resulting frozen strains were stored frozen for 8 weeks. The strain obtained by thawing was inoculated into a medium supplemented with 1% (w / v) of glucose (0.7 g / L KH ₂ PO ₄ , 0.3 g / LK ₂ HPO ₄ , 0.34 g / L MgSO ₄ .7H ₂ The chlorella content [% (w / w)] of chlorella obtained from the cultured group was confirmed by incubation for 48 hours in 0.06 g / L EDTA-2Na and 0.4 ml / L trace metal solution. Were measured. The results are shown in Table 2 below.

DMSO [% (v / v)] 0 One 3 5 10 12 Skim milk
[% (v / v)] 0 NC 12.11 13.85 12.54 12.11 10.95 5 NC 12.72 13.97 13.27 12.91 11.20 10 NC 13.22 14.12 14.02 13.67 13.00 15 NC 13.11 13.92 13.62 13.24 10.52 20 NC 11.94 12.07 12.42 11.86 10.22

* NC: not cultured

As can be seen from the results in Table 2, DMSO showed a difference in culture efficiency depending on the addition rate even when it was used alone, but the sole use of skim milk was not cultured even when the addition rate was changed. From the results shown in Table 2, it can be seen that the combination of 3% DMSO and 10% skim milk shows the best results.

Example 3. Evaluation of Storage by Freezing with Isopropyl Alcohol

A combination of skim milk 10% (v / v), dimethyl sulfoxide 3% (v / v), skim milk 10% (v / v) and dimethyl sulfoxide 3% (v / v) After freezing for 4 weeks by using freezing method using isopropyl alcohol and general freezing method, the culture efficiency was evaluated. The evaluation of the culture efficiency was carried out for three lots each. In the general freezing method, each of the cryopreservatives was added to the chlorella culture, and the resulting mixture was dispensed into a freezing tube, which was then dropped in the freezer at a rate of -1 ° C per minute and frozen to about -75 ° C. In the freezing method using isopropyl alcohol, each of the cryopreservatives was added to the chlorella culture, and the resulting solution was dispensed into a freezing tube, and the solution was dropped in a freezer at a rate of -1 ° C per minute in a state immersed in isopropyl alcohol and frozen to about -75 ° C . Each of the obtained frozen strains was kept frozen for 4 weeks. The strain obtained by thawing was inoculated into a medium supplemented with 1% (w / v) of glucose (0.7 g / L KH ₂ PO ₄ , 0.3 g / LK ₂ HPO ₄ , 0.34 g / L MgSO ₄ .7H ₂ O, cultured for 48 hours in a 0.06 g / L EDTA-2Na, 0.4 ml / L trace metal solution) were measured by OD _610nm. The results are shown in Fig.

From the results shown in Fig. 1, there was a difference in the culture efficiency depending on the type of the cryopreservative, and it was found that the cultivation efficiency was relatively high in the group treated with isopropyl alcohol. In particular, in the test group treated with skim milk alone, chlorella growth did not occur in the group obtained from the freezing method not using isopropyl alcohol. Thus, it can be seen that more stable cryopreservation can be achieved when freezing is carried out using isopropyl alcohol.

Example 4. Analysis of the strain obtained after freezing storage

The combination of 3% (v / v) dimethyl sulphoxide and 10% (v / v) skim milk and 3% (v / v) dimethyl sulphoxide was used as a cryopreservation agent and freezing using isopropyl alcohol was applied (Dry cell weight, DCW, crude protein content, and chlorophyll content) of chlorella after freezing for 8 weeks and 16 weeks were measured. Each measurement was performed on three lots each. Specifically, each cryoprotectant (i.e., dimethylsulfoxide / skim milk + dimethylsulfoxide) was added to the chlorella culture, and the cells were divided into frozen tubes and immersed in isopropyl alcohol. Deg.] C and frozen to about -75 [deg.] C. Each of the obtained frozen strains was stored frozen for 8 and 16 weeks. The strain obtained by thawing was inoculated into a medium supplemented with 1% (w / v) of glucose (0.7 g / L KH ₂ PO ₄ , 0.3 g / LK ₂ HPO ₄ , 0.34 g / L MgSO ₄ .7H ₂ (RS), dry cell weight (DCW), crude protein content, chlorophyll content, and chlorophyll content were measured after 48 hours of incubation for 48 hours in a buffer solution (0.06 g / L EDTA-2Na, And the content thereof was measured. The results are shown in Tables 3 and 4 below.

Frozen for 8 weeks DCW
[g / L] Crude protein
[% (w / w)] chlorophyll
[% (w / w)] Initial strain of storage start 43.12 ± 0.30 53.58 ± 1.23 14.17 ± 1.11 Frozen preservation agent-free frozen storage strain NC NC NC 3% DMSO-added freeze-storage strain 42.80 ± 0.19 54.17 ± 3.25 13.46 ± 3.61 3% DMSO + 10% skim milk added
Frozen storage 42.10 ± 0.20 52.83 + - 3.15 14.12 ± 2.72

* NC: not cultured

Frozen for 16 weeks RS
[% (w / v)] DCW
[g / L] Crude protein
[% (w / w)] chlorophyll
[% (w / w)] Initial strain of storage start 0% 43.12 ± 0.30 53.58 ± 1.23 14.17 ± 1.11 Frozen preservation agent-free frozen storage strain NC NC NC NC 3% DMSO-added freeze-storage strain 0.4% 29.61 ± 3.81 48.81 + - 5.26 11.43 + - 2.64 3% DMSO + 10% skim milk added
Frozen storage 0% 42.38 ± 3.19 53.17 + - 2.40 13.80 ± 1.19

* NC: not cultured

From the above results, it can be seen that DCW, crude protein, and chlorophyll contents were kept within 3% when stored frozen for 8 weeks (Table 3). However, when stored frozen for 16 weeks, DMSO alone was significantly lower in dry weight (DCW) and started to grow at an initial 1% (w / v) % Remained, indicating that the growth of the cells was correspondingly slow (Table 4). In addition, not only crude protein content (-9%) but also chlorophyll content fell to 80% or less. On the contrary, in the group using the combination of skim milk and DMSO, all of the sugar was consumed and both the crude protein and the chlorophyll were stored within 3% (Table 4).

Claims (6)

(a) adding dimethylsulfoxide and skim milk as cryopreservatives to the chlorella culture; And (b) freezing the mixture obtained in step (a). The method for cryopreservation of chlorella according to claim 1, wherein the chlorella culture solution is a culture solution having an absorbance at a range of 0.5 to 1.0 at 610 nm. The method for cryopreservation of chlorella according to claim 1, wherein 1 to 10 parts of dimethylsulfoxide and 5 to 15 parts of skim milk are added to 100 parts of the chlorella culture solution. The method for cryopreservation of chlorella according to claim 1, wherein 3 to 5 parts of dimethylsulfoxide and 5 to 10 parts of skim milk are added to 100 parts of the chlorella culture solution. The method for cryopreservation of chlorella according to claim 1, wherein 3 parts of dimethylsulfoxide and 10 parts of skim milk are added to 100 parts of the chlorella culture solution. The method according to any one of claims 1 to 5, wherein the step (b) comprises the step of placing the mixed solution obtained in step (a) in a freezing vial or a freezing tube, and adding a freezing vial or freezing tube Followed by immersion in isopropyl alcohol, followed by freezing.

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