Method for frozen preservation of brown algae
Patent Information
- Application Number
- JP2023130133
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-08-09
- Publication Date
- 2025-02-21
Smart Images
Figure 2025025400000001_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to a method for freezing and preserving brown algae. [Background technology]
[0002] When conducting ongoing research using various cells and tissues, the technology to freeze and preserve them without damaging their inherent properties is extremely important. The same is true in research on algae, and various cryopreservation methods, such as a two-stage cooling method, are known (for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]
[0003] [Non-Patent Document 1] Kuwano, W., 2002, "Cryopreservation of algae", in "Current status of phycology at the beginning of the 21st century" edited by Hori, K., Ohno, M. and Horiguchi, T., The Phycological Society of Japan, Yamagata, pp. 108-111. Summary of the Invention [Problem to be solved by the invention]
[0004] However, the technology for freezing and preserving algae has not yet been fully established, and there are issues with the survival rate of algae when thawed after preservation. The present inventors therefore discovered that appropriate freezing and preservation conditions may vary depending on the type of algae, and focused in particular on establishing a freezing and preservation technique for brown algae belonging to the Laminariales order.
[0005] The present invention has been made in consideration of the above circumstances, and aims to provide a novel technique for freezing and preserving brown algae belonging to the Laminariales order. [Means for solving the problem]
[0006] The present inventors have discovered a new finding that the post-thaw survival rate of brown algae belonging to the Laminariales order can be improved by combining a two-step cooling method with a specific preservation solution, and have completed the present invention. Specifically, the present invention provides the following.
[0007] (1) A method for freezing and preserving brown algae, comprising the steps of: The freezing and preserving method comprises: A soaking step of placing brown algae and a preservation solution in a container and soaking the brown algae in the preservation solution; After the immersion step, the container is cooled at a rate of -0.2°C / min or more and -2°C / min or less; and A rapid cooling step of immersing the container in liquid nitrogen after the slow cooling step, The brown algae are brown algae belonging to the order Laminariales, The preservation solution comprises two or more sugars, The saccharides consist of one or more selected from the group consisting of ethyleneglycerol, sucrose, and trehalose, and glycerol; Freezing preservation method.
[0008] (2) The freezing and preservation method described in (1), wherein the total amount of the sugars is 10 v / v% or more and 40 v / v% or less of the preservation solution. Effect of the Invention
[0009] According to the present invention, a novel technique for cryopreservation of brown algae belonging to the Laminariales order is provided. [Brief description of the drawings]
[0010] [Figure 1] FIG. 1 is a diagram showing a flow of a cryopreservation method according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0011] Hereinafter, embodiments of the present invention will be described in detail, however, the present invention is not limited to the following embodiments.
[0012] <How to freeze and preserve brown algae> The method for freezing and preserving brown algae of the present invention (hereinafter also referred to as the "method of the present invention") corresponds to the so-called two-stage cooling method.
[0013] The two-stage cooling method is a method in which the object to be preserved is slowly cooled at a slow cooling rate in the first stage, and then rapidly cooled with liquid nitrogen in the second stage. It is known that such a method makes it possible to freeze the preserved subject while suppressing cell damage, etc., and furthermore, can improve the survival rate of the preserved subject after thawing.
[0014] On the other hand, the inventors noticed that there is insufficient knowledge regarding appropriate cooling conditions for the two-stage cooling method depending on the type of preservation object, and in particular, no established knowledge has been established for brown algae belonging to the Laminariales order. After extensive research, the inventors discovered that by adopting specific cooling conditions in a two-stage cooling method and using a preservation solution containing specific sugars, it is possible to stably improve the post-thaw survival rate of brown algae belonging to the Laminariales order.
[0015] In the present invention, the term "post-thaw survival rate" refers to the ratio of the total number of cells (living cells) at the time of thawing after freezing to the total number of cells (living cells) before freezing.
[0016] In the present invention, "freezing" includes a state in which ice is formed in cells.
[0017] In the present invention, "thawing" includes returning ice in a frozen cell to a water state.
[0018] The method of the present invention will now be described in detail. In the following, unless otherwise specified, the temperature refers to the ambient temperature and does not necessarily correspond to the product temperature of the brown algae or the like itself.
[0019] (1) Brown algae The subject to be preserved (subject to be frozen and preserved) in the method of the present invention is brown algae belonging to the order Laminariales (LAMINARIALES Migula).
[0020] Brown algae belonging to the Laminariales order include, but are not limited to, Eisenia bicyclis (Kjellman) Setchell, Ecklonia stolomifera Okamura, and the like.
[0021] The form of brown algae when frozen and preserved is not particularly limited, but forms in which the effects of the present invention can be easily obtained include gametophytes (male gametophytes, female gametophytes), gametophyte fragments, isolated cells, sporophytes, zoospores, etc. The disrupted gametophytes can be obtained, for example, by the method using a homogenizer, mixer, or the like as shown in “(3) Immersion of brown algae in a preservation solution” in the Examples.
[0022] (2) Soaking process The brown algae to be preserved are placed in a container together with a preservation solution and immersed in the solution. The preservation solution in the present invention has a function of preventing freeze damage to cells, and is technically characterized in that it contains sugars, which will be described later.
[0023] In the soaking step, any means can be used that allows the entire brown algae to be soaked in the preservation solution. For example, the container containing the brown algae and the preservation solution may be centrifuged or rotated. However, it is preferable to adjust the rotation speed of these means from the viewpoint of not giving excessive impact to the brown algae. For example, in the case of centrifugation, the rotation speed may be 5000 rpm or more and 10000 rpm or less, and the time may be 1 minute or more and 10 minutes or less. In the case of rotary stirring, the rotation speed may be 10 rpm or more and 15 rpm or less, and the time may be 5 minutes or more and 30 minutes or less.
[0024] Preferable conditions for the soaking step include the conditions shown in “(3) Soaking brown algae in a preservation solution” in the Examples.
[0025] The immersion step is usually carried out at room temperature (for example, from 0° C. to 40° C.).
[0026] (2-1) Container The size, shape, etc. of the container are not particularly limited as long as it has sufficient cold resistance and physical strength as a storage container and can store and seal the desired brown algae and preservation solution, and can be set appropriately depending on the total amount of brown algae and preservation solution, etc.
[0027] The container to be used is preferably made of resin (such as polypropylene).
[0028] As the container, for example, a commercially available freezer tube can be used.
[0029] (2-2) Storage solution The preservation solution of the present invention contains two or more types of saccharides. The sugars are ethyleneglycerol (C2H6O2), sucrose (C 12 H 22 O 11 ), and trehalose (C 12 H 22 O 11 ) and glycerol (C3H8O3).
[0030] The sugars are not particularly limited as long as they are two or more types, but from the viewpoint of easily achieving the effects of the present invention, the sugars preferably consist of two types of sugars (i.e., one selected from the group consisting of ethyleneglycerol, sucrose, and trehalose, and glycerol).
[0031] In a preferred embodiment of the present invention, the sugars contained in the preservation solution include the following combinations: -Sugars consisting of glycerol and ethyleneglycerol -Sugars consisting of glycerol and sucrose -Sugars consisting of glycerol and trehalose
[0032] The content (total amount) of sugars contained in the preservation solution is not particularly limited, but from the viewpoint of easily achieving the effects of the present invention, it is preferably 10 v / v% or more and 40 v / v% or less, and more preferably 15 v / v% or more and 30 v / v% or less, relative to the preservation solution. However, although the above values are expressed as volume percent, in the present invention, the preferred concentrations overlap with the above values regardless of whether the sugar is in liquid or solid form, and therefore, the units of sugar concentration in the present invention may be used interchangeably as "v / v%" and "w / v%". Examples of sugars that are liquid at room temperature include glycerol and ethylene glycerol, and examples of sugars that are solid at room temperature include sucrose and trehalose.
[0033] The ratio of the saccharides contained in the preservation solution is not particularly limited, but may satisfy the following mass ratio (or volume ratio), for example. Glycerol:Ethylene glycerol = 1:0.5~1:1.5 Glycerol:sucrose = 1:0.5~1:1.5 Glycerol:Trehalose = 1:0.5~1:1.5
[0034] The present invention does not exclude embodiments in which the preservation solution contains sugars other than the above four types, but from the viewpoint of easily achieving the effects of the present invention, it is preferable that the preservation solution does not contain sugars other than the above four types.
[0035] The solvent of the preservation solution is not particularly limited as long as it does not impede the survival of the brown algae. Preferred solvents include, for example, seawater and artificial seawater.
[0036] The amount of preservation solution is not particularly limited, but usually, a sufficient amount is used to evenly immerse the entire brown algae to be frozen and preserved.
[0037] (3) Slow cooling process The slow cooling step is a step in which the sealed container containing the brown algae and the preservation solution is cooled at a rate of -0.2°C / min or more and -2°C / min or less after the soaking step. The present inventors have found that performing slow cooling at such a cooling rate while immersing in the above-mentioned preservation solution is particularly suitable for suppressing cell damage and the like of brown algae belonging to the Laminariales order.
[0038] In the present invention, "cooling at a rate of -n° C. / min" means cooling at a rate of n° C. decrease per minute.
[0039] The cooling rate in the slow cooling step is −0.2° C. / min or more and −2° C. / min or less, preferably −0.2° C. / min or more and −1° C. / min or less.
[0040] As a means for carrying out the slow cooling step, any means capable of controlling the temperature can be adopted. Such means include, for example, commercially available program freezers.
[0041] The temperature at the start of the slow cooling step is usually room temperature (for example, 0° C. or higher and 40° C. or lower).
[0042] The end point of the slow cooling step may be any point at which adequate freezing and dehydration occurs, from the viewpoint of reducing cell damage. In a preferred embodiment of the present invention, the slow cooling step is terminated when the temperature reaches preferably −35° C. to −45° C., more preferably −39° C. to −41° C., and most preferably −40° C. Usually, when such a temperature is reached in the slow cooling step, the rapid cooling step is immediately (i.e., without any other operations or steps).
[0043] (4) Rapid cooling process The rapid cooling step is a step in which the sealed container containing the brown algae and the preservation solution after the slow cooling step is immersed in liquid nitrogen.
[0044] The equipment and conditions employed in the rapid cooling step may be the same as those employed in the rapid cooling method using liquid nitrogen in conventional freezing techniques.
[0045] After the rapid cooling process, the brown algae can be stored frozen in a container immersed in liquid nitrogen for a desired period of time. The frozen storage period is not particularly limited and can be, for example, from a few days to a few years.
[0046] (5) Thawing process After the rapid cooling process, the brown algae can be removed from the liquid nitrogen while still in the container and thawed after the desired frozen storage period has elapsed.
[0047] From the viewpoint of suppressing cell damage, the thawing in the present invention is preferably rapid thawing. Rapid thawing includes, for example, quickly immersing the entire container containing frozen brown algae in warm water or the like (for example, warm water of 35° C. or higher and 45° C. or lower). EXAMPLES
[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
[0049] Brown algae belonging to the Laminariales order were frozen and preserved using the following method. The survival rate of the brown algae after thawing after freezing was also examined. The flow of the freezing and preservation method used in this study is shown in Figure 1.
[0050] (1) Preparation of brown algae The following brown algae were prepared. All of the brown algae listed below belong to the Laminariales order. Eisenia bicyclis (Kjellman) Setchell, collected from the sea off Saga Prefecture Ecklonia stolomifera Okamura, collected from the sea off Akita Prefecture
[0051] Each brown alga was stored in a thermostatic chamber (4 to 20°C) until it was subjected to the freezing and preservation process.
[0052] (2) Preparation of storage solution As the preservation solutions, three types of solutions having the following compositions were prepared.
[0053] [Table 1]
[0054] (3) Immersion of brown algae in a preservation solution Each brown alga (male or female gametophyte) was disrupted in sterilized seawater using a homogenizer (dial 11 strength). The resulting crushed material was placed in a container (1 mL tube) containing each preservation solution, and rotary agitation (10 to 15 rpm) was performed for 20 minutes. Through this process, the brown algae and the preservation solutions were dispersed within the container. Next, centrifugation (9000 rpm) was performed for 7 minutes, the supernatant was discarded, and the preservation solution was newly added, followed by rotary stirring (10 to 15 rpm) for 20 minutes. This operation was repeated twice to immerse the brown algae in the preservation solution. Hereinafter, the brown algae immersed in the preservation solution were subjected to a two-stage cooling treatment while still in a sealed container.
[0055] (4) Freezing preservation Each brown alga was subjected to a two-stage cooling process by cooling at two different rates using the following method.
[0056] (4-1) Slow cooling process The container containing the brown algae was cooled from room temperature to -40°C at a cooling rate of 1°C per minute (-1°C / min) using a small programmable freezer. This process corresponds to the "slow cooling process."
[0057] (4-2) Rapid cooling process When the temperature reached -40°C in the slow cooling step, the container was immediately immersed in liquid nitrogen and cooled to -196°C in one go. This step corresponds to the "rapid cooling step." The container was placed in liquid nitrogen and left undisturbed for several days.
[0058] (5) Thawing brown algae The brown algae that had been cooled in two stages were thawed in the following manner, and it was confirmed whether the brown algae were viable. First, the container was removed from the liquid nitrogen and the entire container was immediately immersed in warm water (40°C). This process corresponds to the "rapid thawing process." Next, centrifugation (9000 rpm) was performed for 7 minutes, the supernatant was discarded, and sterilized seawater was added, followed by rotary mixing (10 to 15 rpm) for 20 minutes. This operation was repeated twice to immerse the brown algae in the sterilized seawater.
[0059] (6) Observation Each thawed brown algae was placed in a 12-well multiplate, and 4 mL of PESI medium-added 50% sterilized seawater was poured in. The water temperature was 24°C, and the photon flux density was 40 μmol / s / m 2 The brown algae were cultured under conditions of 12 hours light and 12 hours dark. Seven, 14, and 28 days after the start of culture, the survival and death of each brown algae and cell elongation were observed under a microscope.
[0060] As a result, regardless of which preservation solution was used, the brown algae (cells) survived and normal cell elongation was observed. These results demonstrate that brown algae belonging to the Laminariales order can be successfully frozen and preserved by combining the preservation solution of the present invention with two-stage cooling.
[0061] In this test, the cooling rate in the slow cooling step was set to -1°C / min, but it was confirmed that similarly favorable effects could be obtained if the cooling rate was in the range of -0.2°C / min to -2°C / min.
Claims
1. A method for freezing and preserving brown algae, comprising the steps of: The freezing and preserving method comprises: A soaking step of placing brown algae and a preservation solution in a container and soaking the brown algae in the preservation solution; After the immersion step, the container is cooled at a rate of −0.2° C. / min or more and −2° C. / min or less. A rapid cooling step of immersing the container in liquid nitrogen after the slow cooling step, The brown algae are brown algae belonging to the order Laminariales, The preservation solution comprises two or more sugars, The sugars consist of one or more selected from the group consisting of ethyleneglycerol, sucrose, and trehalose, and glycerol; Freezing preservation method.
2. 2. The method of claim 1, wherein the total amount of the sugars is 10% v / v or more and 40% v / v or less relative to the preservation solution.