JP2006025651A - Method for improving storage stability of algaecidal virus against red tide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving storage stability of an algaecidal virus against red tide, in order to prevent reduction in algaecidal ability to algae to be infection object, since a large-sized DNA virus having algaecidal activity against red tide-causing algae is readily deactivated by light and a water temperature, although activity is kept to some extent by preservation under light shielding and low-temperature conditions and in order to further improve preservability, wherein antibiotics and glycerol as a protecting agent are added to the algaecidal virus against red tide. <P>SOLUTION: The method for improving storage stability of the virus comprises adding streptomycin sulfate (50-5,000 ppm) and chloramphenicol (5-500 ppm) and glycerol (1-20%) to a suspension of HaV (Heterosigma akashiwo virus) being an algaecidal virus against Heterosigma akashiwo of harmful red tide-causing alga and HcV (Heterocapsa circularisquama virus) being an algaecidal virus against Heterocapsa circularisquama. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a virus HaV ( Heterosigma akashiwo virus ) having a strong algicidal activity against the harmful red tide causing alga Heterosigma akashio and a virus HcV ( Heterocapsa circularisquama viru s) having an algicidal activity against the heterocapsa circulariskama. When formulated and stored for practical use as a biological red tide control agent, storage stability is improved by adding antibiotics and protective agents in a method for long-term storage while maintaining activity. How to make.

Useful red tide algae viruses HaV and HcV are icosahedral double-stranded DNA viruses having a diameter of about 200 nm. These red tide algae viruses are strong algicides isolated from the natural seas where the red tide-causing algae heterosigma, acacio, or heterocapsae, which cause enormous fishery damage to bivalves, cause fishery damage to cultured fish. It is a red tide algae virus that is resident in active natural sea areas and is expected to be put to practical use as a useful red tide control factor (Patent Documents 1 and 2).

However, these large DNA viruses easily inactivate algicidal activity under conditions where temperature or light is present in the absence of host cells (Non-patent Document 1). Therefore, in order to preserve these red tide algae viruses for a long period of time, a method for semipermanently storing them in liquid nitrogen using a cryoprotectant such as 10-20% dimethyl sulfoxide has been developed (non-patented). In order to use these red tide-killing algae viruses commercially, it is unsuitable for providing them to users, that is, aquaculture fishers, and for storage in order to use these red tide algae viruses.

Therefore, in order to supply these useful red tide algae viruses as red tide control agents, users who need red tide control, such as aquaculture fishermen, can easily use and disseminate as red tide control (control) preparations. In addition, inexpensive long-term storage technology is required.

These useful red tide algae viruses HaV and HcV are sealed in a light-shielding container against the effects of light and temperature, which are the main causes of loss of algaecidal activity, and stored at a temperature that does not freeze, that is, refrigerated conditions. Thus, it is possible to maintain algaecidal activity to some extent.

However, in storage under refrigerated conditions, if other bacteria such as bacteria and filamentous fungi are present, the environment deteriorates or the red tide algae virus is degraded by enzymes produced by the contaminated bacteria, and the algaecidal activity is reduced. There is a need for a method that can reduce these contaminations, which can lead to a decrease. Conventionally, when red tide algae virus is preserved as a material for testing and research, a method of adding about 0.1% of sodium azide has been used. There is also an effect on the red tide algae virus, such as a decrease in activity, and it is considered that it is difficult to use from the viewpoint of safety because of its high human toxicity.

On the other hand, the reason why these useful red tide algae viruses lose their algicidal activity due to the effects of light has not been elucidated at present, but red tide algae viruses infect host cells, inject genomes, In view of a series of infectious mechanisms of proliferation in the red tide algae virus particle surface layer, it is considered to have an infection structure specific to the host cell. As a factor of inactivation, the surface structure of these red tide algae viruses may be destroyed by factors such as light and temperature.

Therefore, it is considered that the storage period can be further extended by finding an effective additive having an effect of protecting the surface layer structure related to infection with such red tide algae virus from external factors.

JP-A-11-99879 JP 2001-231550 A Yuji Totomaru et al. Abstracts of the 2003 Annual Meeting of the Fisheries Science Society of Japan NAGASAKI et al. , Cryopreservation of Virus (HaV) Infectinga Halmful Bloom Causing Microalga, Heterosigma akashiwo (Raphidophyceae). Vol.65 No.2319-320

An object of the present invention is to provide a method for stably storing these red tide algae viruses for a long period of time in order to practically use useful red tide algae viruses such as HaV and HcV as a red tide control agent. .

In addition to storing these red tide killing algae viruses under light-shielding and low temperature conditions, the present inventors have conducted extensive studies on a method for suppressing spoilage caused by various bacteria and protecting the surface structure of the red tide killing algae virus particles. went. As a result, it was found that it is effective to add a plurality of antibiotics at a concentration that does not affect the algicidal activity of the red tide algae virus, and to add glycerin to protect the surface layer structure, and the present invention was completed. It came to do.
That is, the present invention
[1] A method for improving storage stability by adding an antibiotic and a protective agent to a red tide algae virus useful as a biological red tide control agent. [2] A useful red tide algae virus is a heterosigma acacio algicidal virus. The method according to 1 above, wherein the antibiotics to be added are Hav ( Heterosigma akashiwo virus ) and HcV ( Heterocapsa circularisquama viru s), which is an algicidal virus of heterocapsa circulariskama [3] The antibiotics added are streptomycin sulfate and chloramphenicol [4] The method according to 1 above, wherein the concentration of streptomycin sulfate to be added is 50 to 5000 ppm and the concentration of chloramphenicol is 5 to 500 ppm [5] Preferably, the streptomycin sulfate to be added Concentration of 100-1000 ppm and chloramphenicol The method according to 3 above, wherein the concentration of the glycerol is 10 to 100 ppm [6] The method according to 1 above, wherein the protective agent to be added is glycerin [7] The amount of glycerin to be added is 1 to 20%. [8] The method according to 6 above, wherein the amount of glycerin added is preferably 5 to 10%.

In the present invention, the red tide algae virus HaV, HcV that can easily inactivate its useful algaecidal activity can be stably maintained for a long period of time. Leads to improved versatility for commercial use as a biological red tide control agent.

Hereinafter, each step will be described in detail as a preferred example for carrying out the present invention.
As for HaV and HcV, red tide-causing algae serving as the respective hosts are cultured using a general seawater medium for algae (SWM3 medium, ESM medium, F / 2 medium, ASP medium, etc.), and at the initial stage of logarithmic growth, respectively. Inoculated with the red tide killing algae virus, the culture solution in which the host dissolved by viral infection is made as a useful red tide killing algae virus suspension. The obtained red tide killing algae virus suspension is concentrated by removing unnecessary culture solution by a method such as hollow fiber membrane filtration to prepare a high density red tide killing algae virus suspension. It is desirable to culture the host cell and the red tide algae virus only in a two-part culture, and to carry out the filtration process under aseptic conditions so that various bacteria are not mixed in the red tide algae virus suspension during these processes. . The thus obtained high-density red tide algae virus suspension free from various germs is mixed with 50 antibiotic streptomycin sulfate as a secondary germ-inhibiting growth inhibitor (at a concentration below this level, the effect of inhibiting germs is completely eliminated. To 5000 ppm (at higher concentrations, it adversely affects the algicidal activity of the red tide algae virus) Preferably, the most stable effect of inhibiting the growth of miscellaneous bacteria is seen, and there is no adverse effect on the red tide algae virus 100 1000 ppm, 5 to 500 ppm of chloramphenicol (as long as the effect of suppressing various bacteria can be expected and does not adversely affect the red tide algae virus) Preferably, the most stable growth inhibitory effect of bacteria can be obtained, Add and dissolve to a concentration of 10 to 100 ppm, and as a protective agent, For the purpose of protecting the surface layer structure of the particles, 1 to 20% of glycerin, which can be expected to have a protective effect, compared with the case where glycerin is not added, preferably 5 to 10%, which is effective in improving storage stability due to the protective effect, is added. After mixing well, enclose in a light-shielding material container and store under refrigerated conditions that do not freeze.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[Example 1]
1) Efficacy of streptomycin sulfate and chloramphenicol In order to clarify the effective components for suppressing the contamination of bacteria during storage of HcV, the effects of some drugs and their effects on the algicidal activity of HcV investigated.
Heterocapsa circulariskerma that is an HcV-infected host is a modified SWM3 medium containing ₂ nM Na ₂ SeO ₃ (Chen et al., 1969, J. Physol 5: 211-220; Itoh & Imai, 1987, Shuwa, Tokyo, p. 122- 130) 10 L inoculated to an initial density of 6000 cells / ml, light / dark cycle conditions of 16 hours light and 8 hours dark, temperature 25 ± 2 ° C., light intensity 100 μmol photons m ⁻² s ⁻¹ (illumination by fluorescent lamp) ) For 3 days, and a culture solution was prepared in which it was confirmed that the number of host cells in the early logarithmic growth was 10 ⁵ cells / ml or more. 1000 ml of HcV suspension cultured in a Falcon 750 ml volume tissue culture square flask with a vent hole was inoculated into the virus production tank filled with the culture solution of the resulting heterocapsa circulariskerma. Thereafter, the HcV solution obtained by static culture at 23 ± 2 ° C. for 4 days was concentrated and recovered by filtering through a hollow fiber membrane (Asahi Kasei microza (registered trademark) MF module PSP-113), and HcV high-density concentrated suspension was obtained. A turbid liquid (9.71 × 10 ² infectious units / ml) was obtained. To this HcV high-density concentrated suspension, a solution in which contamination with germs capable of growing in this solution was confirmed in advance was added so that the germicidal effect of antibiotics could be easily discriminated. About 50 ml of the prepared HcV suspension is put in a light-shielding aluminum laminate bag, and after refrigerated overnight, (a) streptomycin sulfate 100 ppm, (b) chloramphenicol 10 ppm, (c) streptomycin sulfate 1000 ppm + chloramphenicol 100 ppm, (d) 100 ppm streptomycin sulfate + 10 ppm chloramphenicol, (e) sodium azide 1000 ppm, (f) sodium azide 100 ppm, (g) penicillin G potassium 1000 ppm, (g) non-additive stock solution HcV high-density concentrated suspension) was added to each reagent, and immediately, the virus titer of each suspension immediately after adjustment was examined by a limiting dilution method. The remaining sample was stored refrigerated in the dark, and after one month, the virus titer was investigated by the same method to investigate the degree of deterioration of the active red tide algae virus.
As a result, in the (ku) non-addition stock solution and (e) sodium azide 1000 ppm addition group, the titer decreased significantly after one month (6.81 × 10 ⁵ infectious units / ml at the time of adjustment, 3.01 after one month) × 10 ² infection units / ml). That is, it was found that the addition of sodium azide at a high concentration causes direct damage to the algicidal activity of the red tide algae virus. In addition, the virus titer was detected relatively high in the group (f) added with 100 ppm sodium azide and the group added with (ki) penicillin G potassium, but ((f) 5.15 × 10 ⁵ infection). Unit / ml, (ki) 5.82 × 10 ⁴ infectious units / ml), many bacteria were detected, and it was found that there was no effect of improving the storage stability of HcV by long-term bacteria suppression. In contrast, suppression of various bacteria and suppression of viral titer deterioration were observed in the treatment group of streptomycin sulfate and chloramphenicol (single month (A) 1.38 × 10 ⁵ infectious units / ml). (A) 7.1 × 10 ⁵ infectious units / ml, (U) 9.38 × 10 ⁵ infectious units / ml and 1.61 × 10 ⁶ infectious units / ml). In particular, the effect was higher when both bacteria suppression and virus titer were mixed. In addition, when mixed, mixing at a low concentration (streptomycin sulfate 100 ppm + chloramphenicol 10 ppm) is less damaging to the virus titer of the drug and at the same time has a sufficient anti-proliferative effect on contaminating bacteria. It was confirmed to be effective for stable preservation of the algicidal virus. (Figure 1)

[Example 2]
2) Evaluation of several protective agents against HcV With respect to HcV that is difficult to store for a long period of time, the effect of addition of several protective agents that can be expected to improve storage stability was investigated. The protective agents tested were 10% polyethylene glycol (# 6000), 50% and 5% glycerin, 12% sucrose, and 0.2% magnesium sulfate, so that the final concentration was as described above. It was adjusted.
Immediately after the preparation, an appropriate amount was taken from each solution, and the viral infection unit amount per 1 ml was examined by the limiting dilution method for Heterocapsa circulariskerma HU9433P strain (a strain isolated from Uchi Bay in Kochi Prefecture). Thereafter, the aluminum laminate pack was filled with each solution, kept in a light-shielded state, and stored at 5 ° C. One month and two months after the start of storage, the algicidal activity of the red tide algavirus was measured in the same manner, and the storage stability under each condition was investigated.
As a result, in both glycerin 50% and sucrose 12%, the HcV density was 100 infectious units / ml or less immediately after adjustment, and the algicidal activity was significantly reduced (immediately after adjustment, glycerin 50% was 49.1 infectious units / ml, Only 40.6 infectious units / ml were detected with 12% sucrose). Furthermore, after one month, all were below the detection limit (3.01 infectious units / ml). This revealed that these treatments do not function as protective agents. On the other hand, the addition of polyethylene glycol and magnesium sulfate is equivalent to the non-treated group (2500 infectious units / ml) after 1 month (magnesium sulfate 0.2% added group, 2180 infectious units), although deterioration immediately after adjustment is not recognized. / Ml) or less (10% polyethylene glycol added group, 5.15 infectious units / ml), and two months later, 103.2 infectious units / ml polyethylene in 0.2% magnesium sulfate group In the glycol addition section, the deterioration progressed to below the detection limit.
Only in the group with 5% glycerin, the storage stability up to 2 months later was observed compared to the untreated group, and 1.19 × 10 ⁵ infectious units / ml immediately after adjustment was 7. The result was 08 × 10 ⁵ infectious units / ml, and 3.73 × 10 ⁴ infectious units / ml after 2 months. That is, it was revealed that the addition of 5% glycerin is effective for preserving the red tide algae virus (FIG. 2).

[Example 3]
3) Optimization of glycerin concentration The optimum concentration of glycerin to be added as a protective agent was evaluated together with the effect of combined use with antibiotics (streptomycin sulfate, chloramphenicol).
In the test, as in Example 1), the heterocapsa circulariskerma used as the infected host was modified with 2 nM Na ₂ SeO ₃ -containing modified SWM3 medium (Chen et al., 1969, J. Physol 5: 211-220; Itoh & Imai, 1987). , Shuwa, Tokyo, p. 122-130) 10 L was inoculated so as to have an initial density of 6000 cells / ml, light / dark cycle conditions of 16 hours light and 8 hours dark, temperature 25 ± 2 ° C., light intensity 100 μmol photons m ^−. A culture solution was prepared by culturing for 3 days under the condition of ² s ⁻¹ (illumination with a fluorescent lamp) and confirming that the number of host cells in the early logarithmic growth was 10 ⁵ cells / ml or more. 1000 ml of HcV suspension cultured in a Falcon 750 ml volume tissue culture square flask with a vent hole was inoculated into the virus production tank filled with the culture solution of the resulting heterocapsa circulariskerma. Thereafter, the red tide algae virus HcV suspension obtained by stationary culture at 23 ± 2 ° C. for 4 days was concentrated and recovered by filtering through a hollow fiber membrane (Asahi Kasei microza (registered trademark) MF module PSP-113). HcV high density concentrated suspension was obtained. In this test, an HcV suspension that was confirmed to be free from contamination was used, and each component was prepared according to the following procedure. That is, glycerin was added to the prepared HcV suspension so as to have addition concentrations of 2.5%, 5.0%, and 10.0%, and as a control, modified SWM3 medium containing ₂ nM Na ₂ SeO ₃ The HcV suspension (untreated section) adjusted to have the same dilution concentration was used in the experiment. In addition, a section was added to which 100 ppm of streptomycin sulfate and 10 ppm of chloramphenicol, which were thought to be effective in improving storage stability by controlling miscellaneous bacteria, and the protective effect when used in combination was also evaluated. . Since the ratio of dilution of the HcV solution used in the experiment varies depending on the amount of glycerin added, in order to increase the accuracy of the experiment, instead of glycerin and antibiotic solution for each concentration of glycerin added section An untreated section by adding SWM3 medium was set.

As a result, in the glycerin 2.5% comparison group, when no antibiotic was added, 9.4 × 10 ⁴ infectious units / ml and 1. 99 × 10 ⁵ infectious units / ml, 1.03 × 10 ⁵ infectious units / ml and 3.35 × 10 ⁵ infectious units / ml with and without glycerin added and with 2.5% added, respectively There is almost no difference from ml, and in the glycerin 5.0% comparative group, when no antibiotic is added, glycerin is not added, and 5.0% glycerin is added, 4.45 × 10 ⁴ infectious units / ml and 6.98, respectively. × 10 ⁵ infectious units / ml, 1.11 × 10 ⁵ infectious units / ml and 8.6 when glycerin is not added and 5.0% is added, respectively, when antibiotics are added It became 7 × 10 ⁵ infectious units / ml, and a tendency was found that the density of viral infectious units increased in the antibiotic and 5% glycerol-added sections. On the other hand, glycerol in 10.0% compared ku, appears this tendency more remarkably, without the addition of antibiotics, glycerol was not added and 10.0% glycerol, respectively, below the detection limit and 3.01 × 10 ³ in the case of addition Infectious units / ml, when antibiotics were added, the glycerin-free and 10.0% additions were 7.18 × 10 ³ infectious units / ml and 9.82 × 10 ³ infectious units / ml, respectively. . In the glycerin 10% comparison group, the virus infection unit density after 1 day of adjustment is lower than in the other test groups. This is due to the difference in the HcV suspension used in the test and the storage suspension for each study. This is because the ratio of dilution during the adjustment of the liquid was higher than in other sections.

In the investigation after 3 months, in the glycerin 2.5% comparison group, the antibiotic-free system, the glycerin-free system and the 2.5% -added system, 3.01 infectious unit / ml and 18 infectious unit / ml, Even in the antibiotic addition system, the glycerin-free and 2.5% additions were 30.1 infectious units / ml and 70.2 infectious units / ml, respectively, and when the antibiotic and glycerin were added simultaneously, the red tide algavirus Infectious unit density was highest. This tendency is also observed in the glycerin 5.0% comparison group, in the case of no antibiotics added, 3.85 infectious units / ml and 9.89 × 10 ^{2 in} the case of no glycerin and 5.0% glycerin, respectively. Infectious units / ml, antibiotic addition system, 30.1 infectious units / ml and 2.32 × 10 ⁴ infectious units / ml, respectively, with no glycerin added and 5.0% glycerin added. In the glycerin 10.0% comparative group, this tendency becomes more prominent. In the non-antibiotic-added system, 9.82 infectious units / ml and 44.5 infectious cases with no glycerin and 10.0% glycerin, respectively. In the case of the unit / ml, antibiotic addition system, in the case of no glycerin addition and 10.0% addition, respectively, it was 30.1 infectious units / ml and 1.39 × 10 ² infectious units / ml. Although the active red tide algae virus density immediately after the start of the test was as low as 1/10 to 1/100 of the glycerin 5% addition group, the active red tide algae virus density after 3 months was almost equal. From this, it was revealed that the protective effect with 10% glycerin is high. (Figure 3).

[Example 4]
4) Storage stability at optimal concentrations of streptomycin sulfate, chloramphenicol, and glycerin The long-term storage stability at optimal concentrations of streptomycin sulfate, chloramphenicol, and glycerin was investigated.
The red tide algae virus HcV suspension produced using a 20 L virus production culture vessel was concentrated and recovered by filtration through a hollow fiber membrane, and the HcV high-density concentrated suspension (the number of infected virus particles was about 1.8 × 10 ¹⁰ infectious units / ml) was obtained.
To the resulting HcV high-density concentrated suspension, 100 ppm of streptomycin sulfate, 10 ppm of chloramphenicol (hereinafter abbreviated as antibiotic mixture) and 10% glycerin are added, and 100 ml is filled in a light-shielding aluminum laminate bag and refrigerated (5 ° C.). saved. In addition, as a comparative control, a test group in which only an antibiotic mixture was added to a HcV high-density concentrated suspension, a test group in which only 10% glycerin was added, and an untreated group without addition were prepared, and 100 ml each was prepared for the experiment. I tried it. Immediately after the start of storage, after 1 day of adjustment, 2 weeks, 1 month, 3 months, and 6 months, the virus infection unit density was measured by the limiting dilution method to confirm long-term virus storage stability.

When only the HcV concentrated suspension stock solution was refrigerated and stored in the dark, a significant decrease in the density of viral infectious units was observed after 1 month (6.45 × 10 ⁶ infectious units / ml), and below the detection limit after 6 months ( 0 infection units / ml). In contrast, in the group to which 10% of glycerin mixed with antibiotics was added, almost no deterioration was observed until the first month after the start of storage (1.82 × 10 ¹⁰ infectious units / ml at the time of adjustment, one month later) 1.77 × 10 ¹⁰ infectious units / ml), and thereafter, after 3 months and 6 months, they were 1.09 × 10 ⁹ infectious units / ml and 1.82 × 10 ³ infectious units / ml, respectively. On the other hand, in the test group to which the antibiotic mixture was added alone, it was 3.05 × 10 ⁷ infectious units / ml and 16.3 infectious units / ml after 3 months and 6 months, respectively, and the test in which glycerin was added alone In the ward, it decreased to 1.07 × 10 ⁷ infectious units / ml and 8.8 infectious units / ml after 3 months and 6 months, respectively (FIG. 4).
From the above results, the storage stability of HcV is improved by controlling the contamination of bacteria after the start of storage by adding an antibiotic mixture and adding 10% glycerin to the protective effect against HcV particles. Became clear.

It is explanatory drawing which showed the effect of streptomycin sulfate and crumbphenicol. (Example 1) It is explanatory drawing which showed the effectiveness of the protective agent. (Example 2) It is explanatory drawing which showed the protective effect of glycerol at the time of using together with antibiotics. Example 3 It is explanatory drawing which showed the long-term storage stability in the optimal addition density | concentration of antibiotics and glycerol. Example 4

Claims (8)

  Method of improving storage stability by adding antibiotics and protective agents to red tide algae virus useful as a biological red tide control agent The useful red tide algae virus is Heterosigma akashiwo virus ( Heterosigma akashiwo virus ) and HcV ( Heterocapsa circularisquama viru s) which is a Heteroscapsa circulariska algavirus . Method   The method according to claim 1, wherein the antibiotics added are streptomycin sulfate and chloramphenicol.   The method according to claim 3, wherein the concentration of streptomycin sulfate to be added is 50 to 5000 ppm and the concentration of chloramphenicol is 5 to 500 ppm.   The method according to claim 3, wherein the concentration of streptomycin sulfate to be added is 100 to 1000 ppm and the concentration of chloramphenicol is 10 to 100 ppm.   The method according to claim 1, wherein the protective agent to be added is glycerin.   The method according to claim 6, wherein the amount of glycerin added is 1 to 20%.   The method according to claim 6, wherein the amount of glycerin added is 5 to 10%.

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