JP2021078476A - Microalgal proliferation-promoting agents and methods of making - Google Patents

Abstract

To provide microalgal proliferation-promoting agents of which production is easy and low cost, as well as methods for producing the microalgal proliferation-promoting agents.SOLUTION: Disclosed is an agent for promoting the proliferation of microalgae, which comprises pulverized cells of a photosynthetic bacterium as an effective ingredient. Also disclosed is a method for producing the microalgal proliferation-promoting agent, which comprises: pulverizing cells of a photosynthetic bacterium such as a purple non-sulfur bacterium Rhodobacter spheroids; and collecting a fraction of insoluble matter from the pulverized cells by centrifugation. Addition of the obtained microalgal proliferation-promoting agent to a culture fluid containing microalgae promotes the microalgal proliferation.SELECTED DRAWING: Figure 1

Description

The present invention relates to a microalgae growth promoter and a method for producing the same.

In recent years, there has been increasing interest in using microalgae as food or feed, and using substances produced by microalgae as pharmaceuticals, cosmetics, and fuels. In order to commercialize the use of such microalgae, it is indispensable to cultivate the microalgae in large quantities, and for that purpose, a technique for efficiently growing the microalgae is required.

For example, in Patent Document 1, by adding a fulvic acid-containing composition having specific physical characteristics contained in brackish water (groundwater having a higher salinity than fresh water) to a medium of microalgae, the growth of the microalgae is effective. It is stated that it can be promoted. Further, Patent Document 2 describes a method for promoting the growth of microalgae by adding a pyrrole compound containing a tetrapyrrole compound to a medium for microalgae.

JP-A-2015-78155 JP-A-2019-50731

However, in order to produce the fluboic acid-containing composition used in the invention described in Patent Document 1, first, the rot plantous substance (after the death of the organism, the bioorganic substance is decomposed by microbiological or thermochemical action. After preparing irrigation water containing abundantly generated organic substances whose chemical structure is not specified) and having specific characteristics, the irrigation water is made acidic, and the obtained acidic water is filled with a porous adsorbent. After purification by the column, it is necessary to fractionate with at least two types of MF membranes and / or UF membranes, which causes a problem that a large amount of labor and cost are required. Further, in order to realize the invention described in Patent Document 2 on a commercial basis, special Escherichia coli described in the same document (Escherichia coli in which the gene ypjD (b2611) cannot be expressed due to mutation) is obtained and described in the Escherichia coli. It was necessary to bioproduce the tetrapyrrole compound, which was not easily realized.

The present invention has been made in view of the above points, and an object of the present invention is to provide a microalgae growth promoter that can be easily produced at low cost and a method for producing the microalgae growth promoter. is there.

The microalgae growth-promoting agent according to the present invention, which has been made to solve the above problems, contains a crushed cell of a photosynthetic bacterium as an active ingredient.

Further, the microalgae growth promoter according to the present invention made to solve the above problems may contain an insoluble fraction of a photosynthetic bacterium as an active ingredient.

In addition, the microalgae growth promoter according to the present invention made to solve the above problems may contain a soluble fraction of a photosynthetic bacterium as an active ingredient.

Further, the microalgae growth promoter according to the present invention made to solve the above problems may contain lipopolysaccharide as an active ingredient.

The method for producing a microalgae growth promoter according to the present invention, which has been made to solve the above problems, is
It has a step of producing a crushed bacterial cell by crushing the bacterial cell of a photosynthetic bacterium.

Further, the method for producing a microalgae growth promoter according to the present invention further
A step of extracting an insoluble fraction from the disrupted cell product by centrifugation,
It may have.

Further, the method for producing a microalgae growth promoter according to the present invention further
Step of extracting the soluble fraction from the crushed cells by centrifugation,
It may have.

Further, the method for producing the microalgae growth promoter according to the present invention further describes.
The step of extracting lipopolysaccharide from the insoluble fraction,
It may have.

In the method for producing the microalgae growth-promoting agent and the microalgae growth-promoting agent according to the present invention, it is desirable that the photosynthetic bacterium is a purple non-sulfur bacterium, and among them, it is particularly a photosynthetic bacterium of the genus Rhodobacter. desirable. Further, it is desirable that the photosynthetic bacterium of the genus Rhodobacter is Rhodobacter sphaeroides.

According to the present invention, it is possible to provide a microalgae growth promoter that can be easily produced at low cost and a method for producing the same.

The schematic diagram which shows the outline of the preparation method of the supernatant liquid and the pellet liquid in Example 1. FIG. The graph which shows the relationship between the supernatant concentration and the growth rate of algae in Euglena which was cultured by administering the supernatant solution at various concentrations. The graph which shows the relationship between the pellet concentration and the growth rate of algae in Euglena which was cultured by administering the pellet solution at various concentrations. The graph which shows the relationship between the supernatant concentration and the growth rate of algae in Chlamydomonas cultured by administering the supernatant solution at various concentrations. The graph which shows the relationship between the pellet concentration and the growth rate of algae in Chlamydomonas cultured by administering the pellet solution at various concentrations. The graph which shows the relationship between the pellet concentration and the growth rate of algae in Euglena which was cultured by administering the pellet solution prepared using the pellet treated with hydrochloric acid at various concentrations. The graph which shows the relationship between the lipopolysaccharide concentration and the growth rate of algae in Euglena cultured by administering lipopolysaccharide (LPS) derived from a photosynthetic bacterium at various concentrations.

As a result of diligent studies to solve the above problems, the present inventor has finely divided both the supernatant (soluble fraction) and pellets (insoluble fraction) obtained by centrifuging the disrupted product of photosynthetic bacteria. It was found that it has the effect of promoting the growth of algae. Furthermore, the present inventor has different concentrations in which the effect of promoting the growth of microalgae is observed between the soluble fraction and the insoluble fraction, and the insoluble fraction is solubilized and then administered to the microalgae to make fine particles. It was found that the effect of promoting the growth of algae can be enhanced, and that lipopolysaccharide contained in the insoluble fraction functions as a factor for promoting the growth of microalgae. The microalgae growth promoter and the method for producing the same according to the present invention were conceived based on the above findings.

That is, the first aspect of the microalgae growth promoter according to the present invention contains a crushed cell of a photosynthetic bacterium as an active ingredient.

Since photosynthetic bacteria are commercially available at low cost and easy to culture, the microalgae growth promoter and the method for producing the same according to the present invention can be easily carried out at low cost.

Further, as described above, both the supernatant (soluble fraction) and the pellet (insoluble fraction) obtained by centrifuging the crushed cells have the effect of promoting the growth of microalgae, and both are effective. Since there is a difference in the concentration at which the above-mentioned substances are exhibited, the microalgae growth-promoting agent according to the present invention has a concentration suitable for administration of either one or both of the soluble fraction and the insoluble fraction. It may be contained in.

That is, the second aspect of the microalgae growth promoter according to the present invention contains an insoluble fraction of a photosynthetic bacterium as an active ingredient.

In addition, the third aspect of the microalgae growth promoter according to the present invention contains a soluble fraction of a photosynthetic bacterium as an active ingredient.

Here, the cell crushed product can be obtained by crushing the cells of a photosynthetic bacterium using, for example, an ultrasonic crusher, a homogenizer (French press), a bead-type cell crusher, an enzyme, or the like. The insoluble fraction and the soluble fraction can be obtained by centrifuging the obtained pulverized product, respectively.

That is, the first aspect of the method for producing a microalga growth promoter according to the present invention includes a step of producing a crushed cell product by crushing the cell cells of a photosynthetic bacterium.

In addition, the second aspect of the method for producing a microalga growth promoter according to the present invention includes a step of producing a cell crushed product by crushing a bacterial cell of a photosynthetic bacterium, and the cell crushing by centrifugation. It includes a step of extracting an insoluble fraction from a substance.

It is desirable that the second aspect of the method for producing a microalgae growth promoter according to the present invention further has a step of solubilizing the insoluble fraction.

The solubilization method includes, for example, treatment with a strong acid such as hydrochloric acid, sulfuric acid or nitric acid, treatment with a weak acid such as lactic acid, phosphoric acid or acetic acid, and treatment with a strong base such as sodium hydroxide or potassium hydroxide. , Treatment with a weak base such as calcium hydroxide or ammonia, or treatment with an organic solvent such as ethanol or acetone, but is not limited thereto. By performing such a solubilization treatment, the active ingredient contained in the insoluble fraction can be eluted and activated, and the effect (effect of promoting the growth of microalgae) can be enhanced.

In addition, the third aspect of the method for producing a microalgae growth promoter according to the present invention includes a step of producing a cell crushed product by crushing a bacterial cell of a photosynthetic bacterium, and the cell crushing by centrifugation. It includes a step of extracting a soluble fraction from a product.

In addition, the fourth aspect of the microalga growth promoter according to the present invention contains lipopolysaccharide as an active ingredient.

Any lipopolysaccharide may be used, but it is desirable to use one extracted from an insoluble fraction of a photosynthetic bacterium.

That is, the fourth aspect of the method for producing a microalga growth promoter according to the present invention is a step of producing a cell crushed product by crushing a bacterial cell of a photosynthetic bacterium, and a step of producing a bacterial cell crushed product by centrifugation, and from the bacterial cell crushed product by centrifugation. It includes a step of extracting an insoluble fraction and a step of extracting lipopolysaccharide from the insoluble fraction.

The type of the photosynthetic bacterium is not particularly limited in the method for producing the microalgae growth-promoting agent or the microalgae growth-promoting agent according to each of the above aspects, but it is desirable that the photosynthetic bacterium is a purple non-sulfur bacterium. Above all, it is particularly desirable that it is a photosynthetic bacterium of the genus Rhodobacter. As the photosynthetic bacterium of the genus Rhodobacter, for example, Rhodobacter sphaeroides can be preferably used. In addition, as Rhodobacter spharoides, the bacterial cells marketed under the names of "PSB frozen bacterial cells", "Aures PSB", "Hikari Aures", "NEW Panaores", or "Ores Midori" (Distributor: Co., Ltd.) Matsumoto Microbial Research Institute) can be preferably used.

The microalgae growth promoter according to each of the above aspects may be in any dosage form such as a liquid agent, a powder agent, a granule agent, or a tablet. For example, when the ease of administration to microalgae is important, it is desirable to make this drug a liquid (that is, a liquid), and when the ease of transportation and storage is important, this drug is in a solid state. It is desirable to use a product (that is, a powder, a granule, or a tablet). When the microalgae growth-promoting agent according to the present invention is used as a liquid preparation, the liquid preparation is prepared by using a disrupted substance, an insoluble fraction, a soluble fraction, or a lipopolysaccharide of the photosynthetic bacterium in water or an aqueous dispersion medium or an aqueous dispersion medium. It can be produced by adjusting the concentration to a predetermined value by suspending, dissolving, or dispersing in a solvent (hereinafter referred to as water or the like). When the microalgae growth promoter according to the present invention is used as a powder, the powder is produced by drying the cell disrupted product, insoluble fraction, soluble fraction, or lipopolysaccharide of the photosynthetic bacteria. Will be done. When the microalgae growth-promoting agent according to the present invention is a granule or tablet, the granule or tablet is designated as the dried cell crushed product, insoluble fraction, soluble fraction, or lipopolysaccharide. It is manufactured by adding additives such as excipients, binders and disintegrants of the above.

The powder, granules, or tablets are administered to microalgae (specifically, after being dissolved in water or the like by a user (worker who administers to microalgae) to an appropriate concentration at the time of use. , Administration into a culture solution containing microalgae) is desirable. Further, the liquid preparation may be administered to microalgae after being diluted to an appropriate concentration by the user with water or the like at the time of use, and is manufactured so as to have an appropriate concentration in advance and is not diluted (as is, as the undiluted solution). It may be administered to microalgae.

The microalgae growth promoter according to each of the above aspects can be used to promote the growth of various microalgae. The "microalgae" are typically unicellular eukaryotes or eubacteria that perform oxygen-generating photosynthesis, but in the present invention, in addition to this, two of the unicellular eukaryotes It also includes sub-unicellular algae that do not photosynthesize. Examples of the microalgae to which the microalgae growth promoter according to the present invention is applied include Euglenidae algae (Euglenidae, etc.), Cramidomonas algae, Chlorella algae, Boturiococcus algae, and Suizenjinori. Examples include, but are not limited to, orchid algae (such as Suizenjinori) and orchid algae of the genus Euglenida (such as spirulina).

Pellet solution and supernatant solution containing pellets (insoluble fraction) and supernatant (soluble fraction) obtained from crushed cells of photosynthetic bacteria are administered to a culture solution containing Euglena, which is a microalgae. , The effects of them on the proliferation of Euglena were investigated. As the photosynthetic bacterium, commercially available Rhodobacter sphaeroides (sold by Matsumoto Microbial Co., Ltd., trade name: PSB frozen bacterium) was used, and the Euglena was a microbial line preservation facility of the National Institute for Environmental Studies. Euglena gracilis NIES-48 strain stored in (NIES collection) was used.

The outline of the method for preparing the pellet solution and the supernatant solution is shown in FIG. First, the culture solution of photosynthetic bacteria (turbidity OD ₆₆₀ = 20, about 60 mg fresh weight / mL) was crushed with an ultrasonic crusher at an output of 60 W, ON / OFF repeatedly for 30 seconds four times, and obtained. The crushed solution was separated into pellets and a supernatant by centrifugation (15560 × g, 10 min, 25 ° C.). The obtained pellets and supernatant were added to 1 liter of tap water so as to have the following concentrations, respectively, to prepare a pellet solution or a supernatant solution. The concentrations were all calculated from the number of colony forming units (cfu) before crushing.
Supernatant concentration: 0 (without addition of supernatant), 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ (all equivalent to cfu / mL)
Pellet concentration: 0 (without pellet addition), 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ (all equivalent to cfu / mL)

Culture solution (turbidity OD) obtained by culturing the Euglena in CM medium (Cramer-Myers medium, pH 3.5, PADILLA, GM and JAMES, TW (1960) Exp. Cell Res. 20, 401-415.). ₆₈₀ = 0.05) was placed in each well of a 12-well plate in an amount of 1800 uL, and 200 uL of the supernatant or pellet solution having any of the above concentrations was added thereto and cultured for 12 days (25 ° C., 24-hour light condition). went. Since the supernatant or the pellet was diluted 10-fold by adding the Euglena to the culture, the final concentration of the supernatant (concentration in the culture) was ⁰ , 100, 10 ¹ , 10 ^2, 10 ^3, 10 ^4, 10 ^5, 10 ^6, 10 ⁷ (both cfu / mL equivalent), and the final concentration of the pellets (concentration in culture medium) is 0,10 ^4, 10 ^5, 10 ^6, They were 10 ⁷ and 10 ⁸ (both equivalent to cfu / mL).

The results of the above tests are shown in FIGS. 2 and 3. FIG. 2 is a graph showing the growth rate of Euglena cultured by administering the supernatant, and FIG. 3 is a graph showing the growth rate of Euglena cultured by administering the pellet solution. _{The growth rate is determined by measuring the turbidity (OD 680} ) of the culture sample (that is, the liquid in each well) at the start and end of the culture with a spectrophotometer, and the value of the turbidity at the end of the culture. Was calculated by dividing by the value of turbidity at the start of culturing (hereinafter, the same applies to all examples).

As apparent from FIG. 2, is obtained by administering the supernatant, growth promoting effect of Euglena was seen in the supernatant concentration ^{10 3 ~10 7 (cfu / mL} ). On the other hand, as is clear from FIG. 3, when the pellet solution was administered, a significant Euglena growth promoting effect was observed at a ^{pellet concentration of 10 8 (cfu / mL).} From the above, it was found that the supernatant of the photosynthetic bacterium crushed solution and the pellet had different concentrations showing the growth promoting effect of microalgae (the supernatant had a low concentration and the pellet had a high concentration).

The supernatant and the pellet were administered to a culture medium containing the microalga Chlamydomonas, and their effects on the growth of Chlamydomonas were investigated. As the Chlamydomonas, the Chlamydomonas reinhardtii CC-124 strain stored in the Chlamydomonas Resource Center (Duke University, USA) was used.

Culture medium (turbidity OD) obtained by culturing the Chlamydomonas in Tris-Minimal medium (pH 7.0, Gorman, DS, and RP Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669). ₆₈₀ = 0.05) was placed in each well of a 12-well plate in an amount of 1800 uL, and 200 uL of the supernatant or pellet solution prepared in the same manner as in Example 1 was added thereto and cultured for 5 days (25 ° C, 24 hours). Condition) was performed. The final concentration of the supernatant (concentration in the culture solution) was 0, 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ (all equivalent to cfu / mL) of the pellets. the final concentration (concentration in culture medium) was set to 0,10 ^1, 10 ^2, 10 ^3, 10 ^4, 10 ^5, 10 ^6, 10 ^7, 10 ⁸ (both cfu / mL equivalent).

The results of the above tests are shown in FIGS. 4 and 5. FIG. 4 is a graph showing the growth rate of Chlamydomonas cultured by administering the supernatant, and FIG. 5 is a graph showing the growth rate of Chlamydomonas cultured by administering the pellet solution. As apparent from FIG. 4, is obtained by administering the supernatant, significant growth-promoting effect on Chlamydomonas were seen in the supernatant concentration ^{10 1 ~10 7 (cfu / mL} ). On the other hand, as is clear from FIG. 5, when the pellet solution was administered, a ^{significant growth promoting effect on Chlamydomonas was observed at pellet concentrations of 10 1} to 10 ⁸ (cfu / mL), and the most effective concentration was 10 ⁵ It was (cfu / mL). From this, the concentration range in which the supernatant and the pellet solution are effective differs depending on the type of microalgae, and Chlamydomonas is more sensitive (that is, lower concentration) to the supernatant and the pellet solution than Euglena. It was found that the growth promoting effect can be obtained.

In Example 1 above, the reason why the effect of promoting Euglena growth by the pellets was smaller than that of the supernatant was considered to be that the active ingredient in the pellets was not dissolved in the pellet solution. Therefore, it was examined whether or not the growth promoting effect can be enhanced by solubilizing the cell wall component of the pellet by hydrochloric acid treatment.

First, the pellets obtained in the same manner as in Example 1 are treated with 0.1 M HCl at 100 ° C. for 30 minutes, and the treated pellets are added to 1 liter of tap water to have the following concentrations. The liquid was prepared.
Pellet concentration: 0 (without pellet addition), 10 ⁵ , 10 ⁹ (all equivalent to cfu / mL)

The above pellet solution was added to the culture solution of Euglena (Euglena gracilis, NIES-48 strain) in the same manner as in Example 1 and cultured for 12 days (final concentration of pellets: 10 ⁴ , 10 ⁸ (cfu / mL). Equivalent)). As a result, as shown in FIG. 6, not only a remarkable growth promoting effect was observed at ^{10 8 cfu / mL, but also a growth promoting effect of Euglena was observed at 10 4} cfu / mL.

Among the components contained in the insoluble fraction of photosynthetic bacteria (centrifugated pellets of crushed cells), lipopolysaccharide (LPS), which is a component of the outer membrane of Gram-negative bacteria, produces reactive oxygen species in plants. Is known to promote. Furthermore, it is known that reactive oxygen species are toxic to plants at high concentrations, but conversely promote plant growth at low concentrations. Therefore, the present inventor predicts that the microalga growth-promoting substance contained in the insoluble fraction is LPS, and administers LPS derived from photosynthetic bacteria to Euglena (Euglena gracilis, NIES-48 strain) at various concentrations. The effect on proliferation was investigated.

As the LPS, a commercially available standard product (derived from Rhodobacter sphaeroides) was used, and an LPS solution was prepared by adding the LPS to 10 mL of tap water so as to have the following concentration.
LPS concentration: 0 (without LPS added), 10 ^-17 , 10 ^-16 , 10 ^-15 , 10 ^-14 , 10 ^-13 , 10 ^-12 , 10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 , 10 ^-4 (all g / mL)

The Euglena culture solution (turbidity OD ₆₈₀ = 0.05) was placed in each well of a 12-well plate in an amount of 1800 uL, and 200 uL of an LPS solution having one of the above concentrations was added thereto. (LPS final concentration (concentration in culture solution) [g / mL]: 10 ^-18 , 10 ^-17 , 10 ^-16 , 10 ^-15 , 10 ^-14 , 10 ^-13 , 10 ^-12 , 10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 ).

The results of the above test are shown in FIG. As is clear from the figure, LPS derived from photosynthetic bacteria showed a growth promoting effect of Euglena at a concentration of ^{10 -7} g / mL (0.1 ug / mL) to 10 ^{-5 g / mL (10 ug / mL).} ..

Claims (15)

A microalgae growth promoter containing crushed cells of photosynthetic bacteria as an active ingredient. A microalgae growth promoter containing an insoluble fraction of photosynthetic bacteria as an active ingredient. A microalgae growth promoter containing a soluble fraction of photosynthetic bacteria as an active ingredient. The microalgae growth promoter according to any one of claims 1 to 3, wherein the photosynthetic bacterium is a purple non-sulfur bacterium. The microalgae growth promoter according to claim 4, wherein the purple non-sulfur bacterium is a bacterium belonging to the genus Rhodobacter. The microalgae growth promoter according to claim 5, wherein the bacterium of the genus Rhodobacter is Rhodobacter spharoides. A microalgae growth promoter containing lipopolysaccharide as an active ingredient. A method for producing a microalgae growth promoter, which comprises a step of producing a crushed cell product by crushing the cell of a photosynthetic bacterium. In addition
A step of extracting an insoluble fraction from the disrupted cell product by centrifugation,
The method for producing a microalgae growth promoter according to claim 8, wherein the agent is characterized by having. In addition
Step of solubilizing the insoluble fraction,
9. The method for producing a microalgae growth promoter according to claim 9. In addition
Step of extracting the soluble fraction from the crushed cells by centrifugation,
The method for producing a microalgae growth promoter according to claim 8, wherein the agent is characterized by having. In addition
The step of extracting lipopolysaccharide from the insoluble fraction,
9. The method for producing a microalgae growth promoter according to claim 9. The method for producing a microalga growth promoter according to any one of claims 8 to 12, wherein the photosynthetic bacterium is a purple non-sulfur bacterium. The method for producing a microalga growth promoter according to claim 13, wherein the purple non-sulfur bacterium is a bacterium belonging to the genus Rhodobacter. The method for producing a microalga growth promoter according to claim 14, wherein the bacterium of the genus Rhodobacter is Rhodobacter spharoides.

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