KR102123805B1 - Process for the cryostorage of chlorella - Google Patents

Abstract

The present invention provides a cryopreservation method of chlorella, characterized by using a combination of dimethyl sulfoxide and skim milk as a cryopreservative.

Description

How to store chlorella frozen {Process for the cryostorage of chlorella}

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

Chlorella, one of the finest fresh green algae, contains nutrients such as chlorophyll, vitamins, and minerals as well as carbohydrates, proteins, and fats, which are beneficial to the human body, and is known as a source of nutritionally valuable food. In addition to this, Chlorella, which is widely used as a raw material for health functional food, is known to help in skin beauty and antioxidant, and to promote immunity and defecation. In addition, it is known as a natural food that can help everyone improve their constitution and nutrition, and is effective in growth and development, so everyone can take it with confidence.

Chlorella is cultured by independent nutrient culture, heterotrophic culture, or a combination thereof, and may be cultured in a medium containing a carbon source, a nitrogen source, a trace metal, and a trace element. For example, Korean Patent Registration No. 10-0193748 discloses a method for producing high concentration chlorella by heterotrophic culture. For long-term storage of the Chlorella strain, periodic passage culture is usually performed. However, not only is there a problem of being exposed to external contamination due to repeated medium replacement in the repeated passage culture process, there is also 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 passage culture, a method of freezing and storing chlorella strain (cryostorage or cryopreservation) has been proposed. The cryopreservation method is a method in which chlorella culture is stored by freezing at about -70°C to -80°C, thawing it when necessary, and maintaining the physiological activity of chlorella for a long period of time. In order to avoid problems such as destruction of strains occurring in the freezing process and degradation of physiological activity, it has been disclosed to perform lyophilization by adding dimethyl sulfoxide as a cryoprotectant to chlorella culture (Ben-Amotz and A. Gilboa, Mar. Ecol. Prog. Ser. 2: 157-161, 1980).

However, when dimethyl sulfoxide is added as a cryopreservative to a chlorella culture medium according to a conventional method and lyophilized and stored for a certain period of time and then thawed, it still shows a large difference in the physiological activity of chlorella obtained. That is, when frozen for a relatively short period of time in a frozen state (for example, about 8 weeks), there is no significant 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 (eg, 16 weeks or more), there is a problem in that a large difference in dry cell weight (DCW), crude protein content, and chlorophyll content of the obtained chlorella is obtained.

The present inventors 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 analyzed a variety of cryopreservatives and freezing conditions, and as a result, when cryopreservation was performed using a combination of dimethyl sulfoxide and skim milk as a cryopreservation agent, even when stored for a longer period of 16 weeks It was found that the physiological activity of chlorella was maintained.

Accordingly, an object of the present invention is to provide a cryopreservation method for chlorella, characterized in that a combination of dimethyl sulfoxide and skim milk is used as a cryopreservative.

According to an aspect of the present invention, (a) adding dimethyl sulfoxide and skim milk as a cryopreservative to a chlorella culture; And (b) freezing the mixed solution obtained in step (a).

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

In the frozen storage method of chlorella of the present invention, 1 to 10 parts by volume of dimethyl sulfoxide and 5 to 15 parts by volume of milk of milk can be added to 100 parts by volume of the chlorella culture medium; Preferably, it is possible to add 3 to 5 parts by volume of dimethyl sulfoxide and 5 to 10 parts by volume of milk of milk with respect to 100 parts by volume of the chlorella culture medium; More preferably, 3 parts by volume of dimethyl sulfoxide and 10 parts by volume of scheme milk may be added to 100 parts by volume of the chlorella culture.

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

In the cryopreservation method according to the present invention, by using dimethyl sulfoxide and skim milk as a cryopreservative, chlorella is maintained for a long period of time (eg, about 16 weeks or more) while maintaining the physiological activity of chlorella. Can be stored frozen. In particular, the cryopreservation method according to the present invention can minimize changes in the dry cell weight (DCW), crude protein content, and chlorophyll content of chlorella within 3% even when stored for a long period of time for 16 weeks or more.

Figure 1 shows the evaluation of the cultivation efficiency after freezing for 4 weeks by applying the freezing method using isopropyl alcohol and the general freezing method.

The present invention comprises the steps of (a) adding dimethyl sulfoxide and skim milk as cryopreservatives to the chlorella culture; And (b) freezing the mixed liquid obtained in step (a).

The chlorella may be, for example, Chlorella vulgaris (Chlorella vulgaris ), Chlorella pyrenoidosa ) , Chlorella regularis , Chlorella saccharophila ), Chlorella sorokiniana ) and the like, and includes everything available to those skilled in the art.

As the culture medium for chlorella used in the cryopreservation method of the present invention, a culture medium obtained by culturing chlorella by a conventional culture method (for example, Korean Patent Registration No. 10-0193748) can be used. For example, the culture may be performed in a sterile tank aseptically and organically (for example, using anhydrous glucose as a sugar source), air is 0.3 to 1.0 vvm, agitation is 200 to 500 rpm, pH is 6.5 It can be cultured in fed-batch by adjusting to ~7.5. 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 cold storage method of chlorella of the present invention is characterized by using a combination of dimethyl sulfoxide and skim milk as a cryopreservative. Step (a) of the cryopreservation method according to the present invention is to add 1 to 10 parts by volume of dimethyl sulfoxide and 5 to 15 parts by volume of milk of milk with respect to 100 parts by volume of chlorella culture; Preferably, 3 to 5 parts by volume of dimethyl sulfoxide and 5 to 10 parts by volume of milk of milk are added to 100 parts by volume of chlorella culture; More preferably, it can be carried out by adding about 3 parts by volume of dimethyl sulfoxide and about 10 parts by volume of scheme milk relative to 100 parts by volume of chlorella culture.

Step (b) of the freezing storage method according to the present invention may be performed according to a conventional freezing method. For example, the mixture obtained in step (a) may be performed by placing the frozen vial or freezing tube in a freezer and lowering the temperature from -70°C to -80°C at a rate of -1°C per minute in the freezer. Surprisingly, the inventors have found that when the freezing process is carried out in the presence of a specific solvent, i.e. isopropyl alcohol, the cultivation efficiency of chlorella obtained after thawing can be further enhanced. Therefore, in step (b) of the cryopreservation method according to the present invention, the mixed solution obtained in step (a) is placed in a freezing vial or a freezing tube, and a freezing vial or freezing tube containing the mixed solution is added to isopropyl alcohol. After immersion, it can be preferably carried out by freezing.

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

In the following examples, after culturing Chlorella vulgaris according to Korean Patent Registration No. 10-0193748, the obtained culture was used as a chlorella culture. In order to adjust a constant content in the chlorella chlorella culture medium, using the medium was adjusted to an absorbance (OD _610nm) at 610 nm of about 0.75 ± 0.25.

Example 1. Evaluation of cryopreservatives

As a cryopreservative, skim milk (SM), glycerol (Gly), betaine, dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) can be used alone or in combination. After freezing for 4 weeks, the culture efficiency was evaluated. Specifically, a cryopreservative was added to the chlorella culture medium, dispensed into a freezing tube, and lowered at a rate of -1°C per minute in a freezer to freeze to about -75°C. The obtained frozen strain was stored frozen for 4 weeks. 1% (w/v) sugar added to the strain obtained by thawing (0.7 g/L KH ₂ PO ₄ , 0.3 g/LK ₂ HPO ₄ , 0.34 g/L MgSO ₄ ·7H _{2 in} purified water) O, 0.06 g/L EDTA-2Na, 0.4 ml/L trace metal solution) was incubated for 48 hours to confirm cultivation, and the cultured group was treated with residual sugar content [%(w/v) ] and ΔOD - the (OD _610nm after inoculation when the culture OD _610nm) was measured. The results are shown in Table 1 below.

Scheme milk Glycerol Betaine DMSO BSA Scheme milk NC 12% (v/v) SM +
10%(v/v) Gly
(0.119%, ΔOD=22) NC 10% (v/v) SM + 3% (v/v) DMSO
(0%, ΔOD=25) NC Glycerol NC NC 8% (v/v) Gly + 3% (v/v) DMSO
(0.407%, ΔOD=14) NC Betaine NC NC NC DMSO 3% (v/v) DMSO
(0%, ΔOD=27) 5% (v/v) DMSO + 3% (v/v) BSA
(0.293%, ΔOD=17) BSA NC

* NC: not cultured

As can be seen from the results of Table 1, most of the cryopreservatives alone did not succeed in culturing after cryopreservation, and the 3% DMSO treated group as the cryopreservative showed the optimal cryopreservative function. In addition, the method of adding the scheme milk was also evaluated to have good sugar consumption and cell growth.

Example 2. Evaluating the efficiency of different freezing preservatives

As a cryopreservative, dimethyl sulfoxide (DMSO) and skim milk (SM) were used in combination in the ratio shown in Table 2 below and stored frozen for 8 weeks to evaluate the culture efficiency. Specifically, dimethyl sulfoxide (DMSO) and skim milk (SM) as cryopreservatives were added to the Chlorella culture according to the ratio in Table 2 below, and dispensed into a freezing tube to drop at a rate of -1°C per minute in the freezer. Frozen to -75 °C. The obtained frozen strain was stored frozen for 8 weeks. 1% (w/v) sugar added to the strain obtained by thawing (0.7 g/L KH ₂ PO ₄ , 0.3 g/LK ₂ HPO ₄ , 0.34 g/L MgSO ₄ ·7H _{2 in} purified water) O, 0.06 g/L EDTA-2Na, 0.4 ml/L trace metal solution) was incubated for 48 hours to confirm cultivation, and chlorophyll content [% (w/w)] of chlorella obtained from the treatment group where the culture was confirmed. Was measured. The results are shown in Table 2 below.

DMSO [%(v/v)] 0 One 3 5 10 12 Scheme 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 of Table 2, DMSO showed a difference in cultivation efficiency depending on the addition rate even when used alone, but the use of Scheme Milk alone was not cultivated even if the addition rate was changed. In addition, from the results of Table 2, it can be seen that the combination of DMSO 3% and skim milk 10% showed the best performance.

Example 3. Evaluation of storage according to freezing using isopropyl alcohol

As a cryopreservative, skim milk 10% (v/v), dimethyl sulfoxide 3% (v/v), and a combination of scheme milk 10% (v/v) and dimethyl sulfoxide 3% (v/v) After freezing by using the freeze method using isopropyl alcohol and the normal freeze method for 4 weeks, the culture efficiency was evaluated. Evaluation of the culture efficiency was performed for each of three lots. In the general freezing method, each cryopreservative was added to the chlorella culture medium, dispensed into a freezing tube, and lowered at a rate of -1°C per minute in a freezer to freeze to about -75°C. In the freezing method using isopropyl alcohol, each cryopreservative was added to the chlorella culture medium, dispensed into a freezing tube, and immersed in isopropyl alcohol to drop at a rate of -1°C per minute in the freezer to freeze to about -75°C. . Each obtained frozen strain was stored frozen for 4 weeks. 1% (w/v) sugar added to the strain obtained by thawing (0.7 g/L KH ₂ PO ₄ , 0.3 g/LK ₂ HPO ₄ , 0.34 g/L MgSO ₄ ·7H _{2 in} purified water) O, 0.06 g/L EDTA-2Na, 0.4 ml/L trace metal solution) was incubated for 48 hours to measure OD _{610 nm} . The results are shown in FIG. 1.

From the results of Figure 1, it can be seen that the culture efficiency was different according to the type of the cryopreservative, and showed relatively high culture efficiency in the group treated with isopropyl alcohol. In particular, in the case of the test group treated with skim milk alone, the growth of chlorella did not occur in the group obtained from the freezing method without isopropyl alcohol. Therefore, it can be seen that when freezing is performed using isopropyl alcohol, more stable cryopreservation can be achieved.

Example 4. Analysis of strains obtained after cryopreservation

As a cryopreservative, a combination of dimethyl sulfoxide 3% (v/v) and scheme milk 10% (v/v) and dimethyl sulfoxide 3% (v/v) was used, and a freezing method using isopropyl alcohol was applied. Then, after freeze storage for 8 and 16 weeks, the dry cell weight (DCW), crude protein content, and chlorophyll content of chlorella obtained by thawing were measured. Each measurement was performed for 3 lots each. Specifically, each cryopreservative (i.e., dimethyl sulfoxide / scheme milk + dimethyl sulfoxide) was added to the chlorella culture medium, dispensed into a freezing tube, and immersed in isopropyl alcohol at -1°C per minute in the freezer. It was lowered at a rate and frozen to about -75°C. Each obtained frozen strain was stored frozen for 8 and 16 weeks. 1% (w/v) sugar added to the strain obtained by thawing (0.7 g/L KH ₂ PO ₄ , 0.3 g/LK ₂ HPO ₄ , 0.34 g/L MgSO ₄ ·7H _{2 in} purified water) After incubation for 48 hours in O, 0.06 g/L EDTA-2Na, 0.4 ml/L trace metal solution, residual sugar (RS), dry cell weight (DCW), crude protein content, and chlorophyll Content was measured. The results are shown in Tables 3 and 4 below.

Frozen storage for 8 weeks DCW
[g/L] Crude protein
[%(w/w)] chlorophyll
[%(w/w)] Storage initial strain 43.12 ± 0.30 53.58 ± 1.23 14.17 ± 1.11 Freeze preservative-free frozen storage strain NC NC NC Frozen storage strain with 3% DMSO 42.80 ± 0.19 54.17 ± 3.25 13.46 ± 3.61 Add 3% DMSO + 10% scheme milk
Frozen storage strain 42.10 ± 0.20 52.83 ± 3.15 14.12 ± 2.72

* NC: not cultured

Frozen storage for 16 weeks RS
[%(w/v)] DCW
[g/L] Crude protein
[%(w/w)] chlorophyll
[%(w/w)] Storage initial strain 0% 43.12 ± 0.30 53.58 ± 1.23 14.17 ± 1.11 Freeze preservative-free frozen storage strain NC NC NC NC Frozen storage strain with 3% DMSO 0.4% 29.61 ± 3.81 48.81 ± 5.26 11.43 ± 2.64 Add 3% DMSO + 10% scheme milk
Frozen storage strain 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 when stored for 8 weeks, the contents of DCW, crude protein, and chlorophyll were preserved within 3%, respectively, compared to the passage culture strain (Table 3). However, when stored frozen for 16 weeks, the dry storage strain (DMW) of DMSO alone was significantly lowered, and incubation was started at the initial 1% (w/v) level, but after 48 hours, only 60% was consumed and 40 % Remained, indicating that the cells were slow to grow as much (Table 4). In addition, not only the content of crude protein (-9%), but also chlorophyll was lowered to 80% or less. On the other hand, in the group using the combination of skim milk and DMSO, all sugars were consumed, and both crude protein and chlorophyll were preserved within 3% (Table 4).

Claims (6)

(a) adding dimethyl sulfoxide and skim milk as cryopreservatives to the chlorella culture; And (b) freezing the mixture obtained in step (a) by lowering the temperature to -70°C to -80°C at a rate of -1°C per minute to freeze the chlorella. The method of claim 1, wherein the chlorella culture solution is a culture medium with absorbance at 610 nm in the range of 0.5 to 1.0. The method of claim 1, wherein 1 to 10 parts by volume of dimethyl sulfoxide and 5 to 15 parts by volume of skim milk are added to 100 parts by volume of the chlorella culture solution. The method of claim 1, wherein 3 to 5 parts by volume of dimethyl sulfoxide and 5 to 10 parts by volume of skim milk are added to 100 parts by volume of the chlorella culture solution. The method of claim 1, wherein 3 parts by volume of dimethyl sulfoxide and 10 parts by volume of skim milk are added to 100 parts by volume of the chlorella culture solution. The freezing solution vial or freezing tube containing the mixture solution according to any one of claims 1 to 5, wherein step (b) puts the mixture obtained in step (a) into a freezing vial or a freezing tube. After immersion in isopropyl alcohol, the freezing storage method of chlorella, characterized in that it is carried out by freezing by lowering the temperature to -70 ℃ to -80 ℃ at a rate of -1 ℃ per minute.

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