TW202239964A - Method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from plankton wherein the plankton are collected from seawater - Google Patents

Abstract

An object of the present invention is to provide a method of collecting seawater containing plankton and producing antioxidant substance DHMBA by the plankton contained in the seawater. The present invention is characterized by filtering the collected seawater containing the plankton by using a filter, extracting cell contents of the plankton remained in the filter, and then heating and pressurizing the extracted cell contents, and producing 3,5-dihydroxy-4-methoxybenzyl alcohol from the heated/pressurized product after being heated/pressurized, and the plankton are diatoms.

Description

Method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol from plankton

The present invention relates to 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) which produces antioxidant substances from plankton The production method of 3,5-dihydroxy-4-methoxybenzyl alcohol.

The inventor of this article has discovered a novel antioxidant substance 3,5-dihydroxy-4-methoxybenzyl alcohol from heated oyster meat: 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA (Diba) ), and successfully synthesized and identified it. Furthermore, DHMBA (Diba) was not detected from raw oyster meat. Here, in terms of the ecological characteristics of oysters, it is generally known that they inhale a large amount of seawater and ingest plankton as bait from the inhaled large amount of seawater. [Prior Art Literature] [Patent Document]

[Patent Document 1] Japanese Patent Laid-Open No. 2016-42825

[Problem to be Solved by the Invention]

Thus, the inventors of the present invention established a hypothesis that DHMBA (Diba) is contained in plankton in the present invention. Therefore, the plankton was collected and filtered, and after the DHMBA (Diba) in the plankton was measured, DHMBA (Diba) was not detected. Then, the plankton was collected, filtered, and heated to detect DHMBA (Diba). Also, after the plankton was collected, filtered, and pressurized, DHMBA (Diba) was detected.

Therefore, the present inventors considered the above-mentioned DHMBA (diba) to be a useful substance derived from plankton, and invented a method for producing DHMBA (diba) from the plankton. [Means to solve the problem]

The present invention is characterized by For the seawater containing the collected plankton, use a filter to filter, and for the plankton remaining on the filter, remove the cell contents, and then heat the removed cell contents at a temperature of 60°C for more than 1 hour, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heated material, The aforementioned plankton are diatoms, diatom subphylum (Subphylum Bacillariophytina), diatom class (Class Bacillariophyceae), diatom subclass (Subclass Bacillariophycidae), diatomaceous (Surirellales (order), Diatomaceous Diatoms of the family Nipponaceae (Entomoneidaceae), diatoms of the genus Amygdalus (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, add the extract to the plankton remaining on the filter to break it up, extract the cell contents from the plankton above, and heat at 60°C for 1 hour The above is formed by generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from the heated material, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, and for the plankton remaining on the filter, take out the cell content, and then heat the taken out cell content at a temperature of 80°C for more than 30 minutes, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the heated material, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, add the extract to the plankton remaining on the filter to break it up, extract the cell contents from the plankton above, and heat at 80°C for 30 minutes The above is formed by generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from the heated material, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, and for the plankton remaining on the above-mentioned filter, take out the cell content, and then pressurize the taken out cell content at 2 atmospheres for 30 minutes, by The pressurized product is produced from 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by The seawater containing the collected plankton is filtered with a filter, and the plankton remaining on the filter is added with an extract to break it up. After extracting the cell contents from the plankton above, pressurize at 2 atmospheres for 30 minutes. , from the pressurized product to generate 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, and for the plankton remaining on the above-mentioned filter, take out the cell content, and then pressurize the taken out cell content at 2.5 atmospheric pressure for more than 30 minutes, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the pressurized product, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, add an extract to the plankton remaining on the filter to break it up, extract the cell contents from the plankton above, and pressurize at 2.5 atmospheres for 30 minutes The above is formed by generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from the pressurized product, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by For the seawater containing the collected plankton, use a filter to filter, and for the plankton remaining on the filter, take out the cell content, and then pressurize the taken out cell content at 3 atmospheres for more than 30 minutes, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is produced from the pressurized product, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus), or, characterized by Filter the seawater containing the collected plankton with a filter, add the extract to the plankton remaining on the filter to break it up, extract the cell contents from the plankton above, and pressurize at 3 atmospheres for 30 minutes The above is formed by generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from the pressurized product, The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). [Effect of Invention]

According to the present invention, the following excellent effect can be exerted: it is possible to provide a method of taking seawater containing plankton and producing the antioxidant substance DHMBA (Diba) from the plankton contained in the seawater.

First, collect seawater for a specific period of time in a sea area where oyster farming is mainly carried out (such as the Hiroshima sea area), filter the collected seawater, take out the plankton 1 remaining on the filter used for filtration, and heat or pressurize it , Carry out exploration/analysis of whether DHMBA (Diba) is detected.

First, detection of DHMBA (Diba) by heating will be described. Seawater collected from the sea area where the oyster is cultured (such as the sea area of Hiroshima).

(collection of seawater containing plankton) Seawater containing plankton in September in a sea area where oyster culture is carried out (for example, the sea area of Hiroshima) is sucked with a pump or the like. Furthermore, in this embodiment, the seawater in the sea area of Hiroshima is used, but it is not limited to the seawater in Hiroshima. Also, the time is not limited to seawater in September.

(Filtration of seawater containing plankton) After the seawater containing the plankton 1 is collected, the seawater is filtered by, for example, a GF/C filter 4 made of nonwoven fabric or the like (see FIG. 1 ). Here, there is no limit to the amount of seawater to be filtered, but this time, approximately 3,100 liters of seawater were collected from the sea area of Hiroshima and filtered. Furthermore, a large amount of plankton 1 is attached to the filter after filtering by the above-mentioned method, and the filter with the plankton 1 attached thereto can be frozen and preserved until the extraction operation is performed.

(ultrasonic treatment) Then, for example, the filter with the plankton 1 attached is cryopreserved, and the cryopreserved filter is placed in a container 5 such as a centrifuge tube, and ultrapure water is added in the container 5, and the plankton 1 is further for example ultrasonically Perform ultrasonic vibration for about 1 hour or use a ball mill to destroy the cell wall, so that DHMBA (Diba) can be easily extracted.

(extraction) After destroying the cells of the plankton 1 by the aforementioned method such as ultrasonic vibration, etc., it is heated to remove the contents of the cells. That is to say, the aforementioned plankton and the like are subjected to hot water bath extraction at about 60° C. or about 80° C. (refer to FIG. 2 ). Here, the plankton 1 in the aforementioned container 5 is not directly heated due to the heating carried out by extraction in a hot water bath. That is, heated hot water is stored in a beaker or the like, and the container 5 is placed in the hot water to perform so-called indirect heating. Thereby, there is an advantage that stable heating extraction can be performed, such as heating at a constant temperature.

(sampling) Then, for the aforementioned hot water bath extraction, samples were taken every 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours.

(Useful substance analysis) The concentration of DHMBA (Diba) in the plankton 1 sampled above was measured by LC-MS/MS.

(Results of extraction experiments conducted in a hot water bath at about 60°C) As a result, DHMBA (Diba) was not detected in the plankton before heating, after 30 minutes and after 5 hours of hot water bath extraction. However, after heating, that is, 1 hour after extraction in a hot water bath, it was confirmed that the extraction amount of DHMBA (Diba) was detected to be 0.0053 (ng/L). In addition, 2 hours after the extraction in the hot water bath, it can be confirmed that the extraction amount of DHMBA (Diba) is detected to be 0.0064 (ng/L), and 3 hours after the extraction in the hot water bath, it can be confirmed that the extraction amount of DHMBA (Diba) is detected. Out 0.0059 (ng/L). Furthermore, 4 hours after extraction in a hot water bath, it was confirmed that the amount of DHMBA (Diba) extracted was 0.0063 (ng/L) (see Figure 4).

(Results of extraction experiments conducted in a hot water bath at about 80°C) As a result, DHMBA (Diba) was not detected in the plankton before heating and after 2 hours of hot water bath extraction. However, after heating, that is, after 30 minutes of hot water bath extraction, it can be confirmed that the extraction amount of DHMBA (Diba) is 0.0035 (ng/L), and after 1 hour after hot water bath extraction, it can be confirmed that DHMBA (Diba) ) extraction amount was detected to be 0.0053 (ng/L). Also, after 3 hours of hot water bath extraction, it can be confirmed that the extraction amount of DHMBA (Diba) is detected to be 0.0035 (ng/L), and after 4 hours of hot water bath extraction, it can be confirmed that the amount of DHMBA (Diba) is detected by detection Out 0.0045 (ng/L). Furthermore, 5 hours after extraction in a hot water bath, it was confirmed that the amount of DHMBA (Diba) extracted was 0.0036 (ng/L) (see Figure 4).

In addition, heating time is not limited to the time after elapse of 1 hour. It is also possible to detect DHMBA (Diba) with a heating time shorter than 1 hour. In any case, DHMBA (Diba) was not detected from the plankton when the plankton was not heated, but it was evident that DHMBA (Diba) was detected from the rapidly heated plankton when the plankton was heated.

Next, detection of DHMBA (Diba) by pressurization will be described. First, seawater is collected from the sea area where the oyster is cultured (for example, the sea area of Hiroshima).

(collection of seawater containing plankton) Use pumps, etc. to pump seawater of a specific period in the sea area where oyster farming is carried out (such as the sea area of Hiroshima), that is, sea water containing plankton in September 2019 in the sea area of Hiroshima Prefecture. Furthermore, in this embodiment, the seawater in the sea area of Hiroshima is used, but it is not limited to the seawater in Hiroshima. Also, the time is not limited to seawater in September.

(Filtration of seawater containing plankton) After the seawater containing the plankton 1 is collected, the seawater is filtered by, for example, a GF/C filter 4 made of nonwoven fabric or the like (see FIG. 1 ). Here, there is no limit to the amount of seawater to be filtered, but this time, about 3,100 liters of seawater was collected in the sea area of Hiroshima and filtered. Furthermore, a large amount of plankton 1 is attached to the filter after filtering by the above-mentioned method, and the filter with the plankton 1 attached thereto can be frozen and preserved until the extraction operation is performed.

(ultrasonic treatment) Then, for example, the filter with the plankton 1 attached is cryopreserved, and the cryopreserved filter is placed in a container 5 such as a centrifuge tube, and ultrapure water is added in the container 5, and the plankton 1 is further for example ultrasonically Perform ultrasonic vibration for about 1 hour or use a ball mill to destroy the cell wall, so that DHMBA (Diba) can be easily extracted.

(extraction) Afterwards, the cells of the plankton 1 are destroyed by the aforementioned methods such as ultrasonic vibration, etc., and then pressurized to remove the contents of the cells. That is to say, the aforementioned plankton etc. are pressurized to about 2 atmospheres, about 2.5 atmospheres or about 3 atmospheres for extraction (refer to Figure 3). Here, the pressurization method is not limited in any way. It can also be carried out by using an autoclave in a general method to pressurize. In addition, it is also possible to pressurize by another method.

(sampling) Then, for the aforementioned pressurized extraction, samples were taken every 30 minutes, 1 hour, 3 hours, and 5 hours.

(Useful substance analysis) The concentration of DHMBA (Diba) in the plankton 1 sampled above was measured by LC-MS/MS.

(The result of the extraction experiment with pressurization to about 2 atmospheres) The detection results of DHMBA (Diba) sampled in Hiroshima waters are shown in Figure 5. In Figure 5, it shows the change of the extraction amount of DHMBA (Diba) over time. DHMBA (Diba) was not detected in plankton before pressurization. After pressurizing at 2 atmospheres, that is, after pressurizing at 2 atmospheres for 30 minutes, it can be confirmed that the extraction amount of DHMBA (Diba) is 0.0125 (ng/L).

(Results of extraction experiments with pressurization to about 2.5 atmospheres) DHMBA (Diba) was not detected in plankton before pressurization (see FIG. 5 ). After pressurizing at 2.5 atmospheres, that is, after pressurizing at 2.5 atmospheres for 30 minutes, it can be confirmed that the extraction amount of DHMBA (Diba) is 0.197 (ng/L), and after 1 hour of pressurization, it can be confirmed that DHMBA (Diba ) extraction amount was detected to be 0.355 (ng/L). Also, after pressurizing at 2.5 atmospheres for 3 hours, it can be confirmed that the extraction amount of DHMBA (Diba) is detected to be 0.483 (ng/L), and after pressing at 2.5 atmospheres for 5 hours, it can be confirmed that the extraction amount of DHMBA (Diba) is detected 0.683 (ng/L) was obtained (refer to Figure 5).

(The result of the extraction experiment with pressurization to about 3 atmospheres) DHMBA (Diba) was not detected in plankton before pressurization (see FIG. 5 ). After pressurization at 3 atmospheres, that is, after 30 minutes of pressurization at 3 atmospheres, it can be confirmed that the extraction amount of DHMBA (Diba) is 0.282 (ng/L), and after 1 hour of pressurization, it can be confirmed that DHMBA (Diba) ) extraction amount was detected to be 0.938 (ng/L). Also, after pressurizing at 3 atmospheres for 3 hours, it can be confirmed that the extraction amount of DHMBA (Diba) is detected to be 1.925 (ng/L), and after pressing at 3 atmospheres for 5 hours, it can be confirmed that the extraction amount of DHMBA (Diba) is detected The yield was 1.341 (ng/L) (refer to FIG. 5 ).

In any case, DHMBA(Diba) was not detected from the plankton when the plankton was not pressurized, but DHMBA(Diba) was clearly detected from the rapidly pressurized plankton when the plankton was pressurized .

(Identification of plankton 1 that can detect DHMBA (Diba)) There are many types of plankton 1 in seawater, and it is not clear what kind of plankton 1 will detect DHMBA (Diba). Therefore, the present inventors identified and detected the plankton 1 of DHMBA (Diba).

The inventor of the present invention took seawater as mentioned above, and obtained the plankton 1 therein. About 200 kinds of plankton 1 are selected from the large number of kinds of plankton 1 obtained, and the plankton 1 of about 200 kinds are cultured respectively. Then, about 200 types of plankton that were cultured separately were subjected to the aforementioned DHMBA (Diba) extraction treatment. That is to say, the above-mentioned ultrasonic treatment, the above-mentioned extraction treatment by heating or pressurization, etc.

As a result, plankton 1 of DHMBA (Diba) was detected as 4 strains of microalgae. Then, after detailed observation of the morphology of the 4 strains of microalgae, these can be identified as diatoms. Furthermore, diatoms are the most abundant plant plankton, and there are many types. In this way, the present inventor detected the morphology of the 4 strains of DHMBA (Diba) microalgae through microscopic observation in detail, and all 4 strains were diatoms. Here, the magnification of the microscope used is 1,000 times (eyepiece: 10 times, objective lens: 100 times). The aforementioned four strains of microalgae, diatoms, are the same type of marine diatoms widely distributed in the seawater of the bay, and are known to be diatoms that produce abundant unsaturated fatty acids. In addition, it is also known as a hyperplanktonic diatom.

Next, the present inventors conducted DNA analysis of the aforementioned 4 strains of microalgae, that is, diatoms, in an attempt to more specifically identify diatoms. DNA analysis is carried out according to which classification class the diatoms belong to. The aforementioned classification classes are classified into "phylum", "subphylum", "class", "subclass", "order", "family", " genus" and "species". And as the "gate" approaches the "species", it becomes more specific. The process of this DNA analysis will be described below.

Identification of the diatom (strain H-7-09) that detected DHMBA (Diba) It is considered to be a diatom of the subclass Diatoms. However, no grade below "mesh" has been identified.

(Method) Execute megablast search in Genbank (NCBI, NHI) Retrieval conditions (1) Search for all sequences other than those derived from the environment (Fig. 6, Fig. 7) → Consider based on the results here. Retrieval condition (2) Limit the search to sequences derived from standard substances (Figure 8, Figure 9) →Since the information content of the sequence data to be compared is insufficient, the results here are not considered. parts sequence name The length of the base sequence used for analysis combination of sequences Blast analysis results 18S H7-09-18S 1223 bp implement Image 6 28S H7-09-28S-D2R2 474 bp not implemented Figure 7

(megablast search results) •18S It shows about 96.2% homology (homology) with the sequence of Humidophila schmassmannii isolate HYU-D030. → The similarity is low at around 96%. However, considering the second and subsequent sequences detected (hit), it is presumed that they belong to diatoms of the diatom subclass of the diatom class. • 28S D2R2 sequence It shows about 96.1% similarity with the sequence of Pseudo-nitzschia multistriata strain HAB-132. → The similarity is low at around 96%. In addition, the sequence is short, and the score and coverage are low. Compared with the result of 18S, the reliability of the identification result is low. However, there is no contradiction with the possibility of diatoms of the subclass Diatoms deduced from the results of 18S.

(identification result) It is considered to be diatoms of diatoms, diatoms, diatoms, and diatoms. However, grades below the "item" cannot be identified.

Identification of diatom (strain H-9-05) from which DHMBA (Diba) was detected It is considered to be diatoms, diatoms, diatoms, diatoms, diatoms (Surirellales), diatomaceae (Entomoneidaceae), diatoms Diatoms of the genus Rhizoma (Entomoneis genus). However, the level of "species" has not yet been identified.

(Method) Execute megablast search in Genbank (NCBI, NHI) Retrieval conditions (1) Search for all sequences other than those derived from the environment (Fig. 10, Fig. 11, Fig. 12) → Consider based on the results here. Search condition (2) Limit the search to sequences derived from standard substances (Figure 11, Figure 12, Figure 13) → Since the information content of the sequence data to be compared is insufficient, the results here are not considered. parts sequence name The length of the base sequence used for analysis combination of sequences Blast analysis result 18S H9-05-18S 1204 bp implement Figure 10 28S H9-05-28S-D2C2 480 bp not implemented Figure 11 28S H9-05-28S-D2R2 458 bp not implemented Figure 12

(megablast search results) •18S It shows 98% similarity with the sequence of Entomoneis paludosa L431 strain. →Similarity is 98%, which is low, but considering that the second and subsequent Amphipitcs (Entomoneis genus) are also detected, it is presumed that they belong to the diatoms, diatom subphylum, and diatoms , Diatom subclass, Diatomaceae (Surirellales), diatomaceae (Entomoneidaceae family), diatoms of the genus Dibophyta (Entomoneis genus) . • The sequence of 28S D2C2 It shows 92% similarity with the sequence of Entomoneis ornata 27D strain. → The similarity is 92%, which is quite low and cannot be used as a reference. In addition, the sequence is short, and the score and coverage are low. Compared with the result of 18S, the reliability of the identification result is quite low. However, it does not contradict the possibility of being a diatom of the genus Amphipipes (Entomoneis) deduced from the results of 18S. • 28S D2R2 sequence It showed 96% similarity with the sequence of Pseudo-nitzschia multistriata HAB-132 strain. → The similarity is 96%, which is low and cannot be used as a reference. In addition, the sequence is short, and the score and coverage are lower than those of D2C2, and the reliability of the identification result is quite low. However, there is no contradiction with the possibility of diatoms of the subclass Diatoms deduced from the results of 18S.

(identification result) It is considered to be diatoms, diatoms, diatoms, diatoms, diatoms (Surirellales), diatomaceae (Entomoneidaceae), diatoms Diatoms of the genus Rhizoma (Entomoneis genus). However, the level of "species" has not yet been identified.

Identification of diatom (H-9-06 strain) from which DHMBA (Diba) was detected It is considered to be diatoms, diatoms, diatoms, diatoms, diatoms (Surirellales), diatomaceae (Entomoneidaceae), diatoms Diatoms of the genus Rhizoma (Entomoneis genus). However, the level of "species" has not yet been identified.

(Method) Execute megablast search in Genbank (NCBI, NHI) Retrieval conditions (1) Search for all sequences other than those derived from the environment (Fig. 16, Fig. 17, Fig. 18) → Consider based on the results here. Retrieval condition (2) Limit the search to sequences derived from standard substances (Figure 19, Figure 20, Figure 21) →Since the information content of the sequence data to be compared is insufficient, the results here are not considered. parts sequence name The length of the base sequence used for analysis combination of sequences Blast analysis result 18S H9-06-18S 1204bp implement Figure 16 28S H9-06-28S-D2C2 440 bp not implemented Figure 17 28S H9-06-28S-D2R2 609 bp not implemented Figure 18

(megablast search results) •18S It shows 99% similarity with the sequence of Entomoneis paludosa L431 strain. →Considering that the similarity is 99%, and when the second and subsequent species of Amphipena (Entomoneis genus) are also detected, it is presumed that they belong to the diatom phylum, diatom subphylum, diatom class, diatom Subclass, diatoms of the diatoms of the diatoms of the diatoms of the diatoms of the diatomaceae (Surirellales) of the diatomaceae (Entomoneidaceae family) • The sequence of 28S D2C2 It shows 91% similarity with the sequence of Entomoneis ornata 27D strain. → The similarity is 91%, which is quite low and cannot be used as a reference. Also, the sequence is short and the score is low, compared with the result of 18S, the reliability of the identification result is quite low. However, it does not contradict the possibility of being a diatom of the genus Amphipipes (Entomoneis) deduced from the results of 18S. • 28S D2R2 sequence It shows 88% similarity with the amoeba (Vannella septentrionalis (amoeba)). Also, it shows a similarity of 79% or less with fungi that form arbuscular mycorrhizae. → The similarity is less than 90%. In addition, the sequence is short, and the score and coverage are lower than those of D2C2, and the reliability of the identification result is quite low. Therefore, it is excluded from this appraisal inspection.

(identification result) It is considered to be diatoms, diatoms, diatoms, diatoms, diatoms (Surirellales), diatomaceae (Entomoneidaceae), diatoms Diatoms of the genus Rhizoma (Entomoneis genus).

Identification of strain H-9-09 unknown

(Method) Execute megablast search in Genbank (NCBI, NHI) Retrieval conditions (1) Search for all sequences other than those derived from the environment (Fig. 22, Fig. 23, Fig. 24) → Consider based on the results here. Search condition (2) Limit the search to sequences derived from standard substances (Fig. 25, Fig. 26, Fig. 27) → Since the information content of the sequence data to be compared is insufficient, the results here are not considered. parts sequence name The length of the base sequence used for analysis combination of sequences Blast analysis results 18S H9-09-18S 1228 bp implement Figure 22 28S H9-09-28S-D2C2 511 bp not implemented Figure 23 28S H9-09-28S-D2R2 441 bp not implemented Figure 24

(megablast search results) •18S It shows 97% similarity with the sequence of Caecitellus paraparvulus HFCC320 strain (bicosoecida). After the second one, they also showed 97% similarity with diatoms of the same genus. → The similarity is 97%, which is relatively low, but the possibility that the sequence of the dimorpha trichuris is amplified (amplified) cannot be denied. However, in the prior microscopic examination, it was confirmed that the specimen showed a diatom-like shape. • The sequence of 28S D2C2 It shows 88% similarity with the sequence of Caecitellus paraparvulus HFCC71 strain (Diflaflagellate). → The similarity is less than 90%. In addition, the sequence is short, and the score and coverage are low. Compared with the result of 18S, the reliability of the identification result is quite low. Therefore, it is excluded from the investigation of this identification. However, it did not contradict the result of 18S (for dimorphous whipworm). • 28S D2R2 sequence It shows 92% similarity with the sequence of Pseudo-nitzschia multistriata HAB-132 strain (Diatom subclass). → The similarity is 92%, which is low. In addition, the sequence is short, and the score and coverage are low. Compared with the result of 18S, the reliability of the identification result is low. However, it did not contradict the prior observation of the morphology (for diatoms).

(identification result) unknown

1: Plankton 4: GF/C filter 5: container

[Fig. 1] An explanatory diagram of seawater collected by filtration. [Figure 2] An explanatory diagram of ultrasonically vibrating the plankton remaining on the filtered filter and then heating and extracting DHMBA (Diba). [Figure 3] An explanatory diagram of extracting DHMBA (Diba) under pressure after ultrasonic vibration of plankton remaining on the filtered filter. [ Fig. 4 ] An explanatory diagram illustrating whether DHMBA (Diba) is generated when plankton present in seawater collected at a specific time in a specific sea area of Hiroshima is heated. [ Fig. 5 ] An explanatory diagram illustrating whether DHMBA (Diba) is generated when plankton present in seawater collected at a specific time in a specific sea area of Hiroshima is pressurized. [FIG. 6] Explanatory drawing (1) explaining the result of searching all other than the environment-originated sequence by a search condition. [FIG. 7] Explanatory diagram (2) explaining the result of searching all other than the environment-originated sequence by the retrieval condition. [ Fig. 8 ] An explanatory diagram (1) illustrating the results of a search performed by limiting search conditions to sequences derived from reference substances. [ Fig. 9 ] An explanatory diagram (2) illustrating the results of a search performed by limiting the search conditions to sequences derived from reference substances. [FIG. 10] Explanatory drawing (3) explaining the result of searching all other than the sequence originating in the environment using a search condition. [FIG. 11] Explanatory diagram (4) explaining the result of searching all other than the environment-originated sequence by the retrieval condition. [FIG. 12] Explanatory diagram (5) explaining the result of searching all other than the environment-originated sequence by the retrieval condition. [ Fig. 13 ] An explanatory diagram (3) illustrating the results of a search performed by limiting search conditions to sequences derived from standard substances. [FIG. 14] An explanatory diagram (4) illustrating the results of a search performed by limiting the search conditions to sequences derived from standard substances. [ Fig. 15 ] An explanatory diagram (5) illustrating the results of a search performed by limiting search conditions to sequences derived from standard substances. [FIG. 16] Explanatory drawing (6) explaining the result of searching all other than the environment-originated sequence by a search condition. [FIG. 17] An explanatory diagram (7) for explaining the result of searching the whole except the sequence derived from the environment by the search condition. [FIG. 18] An explanatory diagram (8) for explaining the result of searching the whole except the environment-derived sequence by the retrieval condition. [ Fig. 19 ] An explanatory diagram (6) illustrating the results of a search performed by limiting the search conditions to sequences derived from standard substances. [ Fig. 20 ] An explanatory diagram (7) illustrating the results of a search performed by limiting search conditions to sequences derived from standard substances. [ Fig. 21 ] An explanatory diagram (8) illustrating the results of a search performed by limiting search conditions to sequences derived from standard substances. [FIG. 22] An explanatory diagram (9) for explaining the result of searching the whole except the environment-derived sequence by the retrieval condition. [ Fig. 23 ] An explanatory diagram (10) for explaining the result of searching the whole except the environment-derived sequence by the search condition. [ Fig. 24 ] An explanatory diagram (11) for explaining the result of searching the whole except the environment-derived sequence by the search condition. [FIG. 25] An explanatory diagram (9) illustrating the results of a search performed by limiting the search conditions to sequences derived from standard substances. [ Fig. 26 ] An explanatory diagram (10) explaining the results of a search performed by limiting the search conditions to sequences derived from standard substances. [ Fig. 27 ] An explanatory diagram (11) explaining the results of a search performed by limiting search conditions to sequences derived from reference substances.

1: Plankton

4: GF/C filter

Claims (10)

A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and for the plankton remaining on the aforementioned filter, the cell content is taken out, and then the taken out cell content is heated at a temperature of 60° C. for 1 hour to 4 hours, and 3, 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtrate, add extract to the plankton remaining on the filter to break it up, extract the cell contents from the above-mentioned plankton, heat at 60°C for 1 hour to 4 hours, and generate from the heated material 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and for the plankton remaining on the aforementioned filter, the cell content is taken out, and then the taken out cell content is heated at a temperature of 80° C. for 30 minutes to 1 hour or 3 hours to 5 hours, and the heated The heated product generates 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and the plankton remaining on the filter is added with an extract to break it up. After the above-mentioned plankton extracts the cell content, it is heated at a temperature of 80° C. for 30 minutes to 1 hour or 3 hours to 5 hours. The heated material generates 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is performed, and the cell contents of plankton remaining on the filter are removed, and the removed cell contents are pressurized at 2 atmospheres for 30 minutes, and 3,5 -Dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and the plankton remaining on the filter is added with an extract to break it up. After extracting the cell contents from the above-mentioned plankton, pressurize at 2 atmospheres for 30 minutes, and produce 3 ,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and the cell content is taken out of the plankton remaining on the aforementioned filter, and then the taken out cell content is pressurized at 2.5 atmospheres for more than 30 minutes, and 3 is produced from the pressurized pressurized product. 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is performed, and the plankton remaining on the filter is added with an extract to break it up. After extracting the cell contents from the above-mentioned plankton, pressurize at 2.5 atmospheric pressure for more than 30 minutes, and produce from the pressurized pressurized product. 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and the cell content is taken out of the plankton remaining on the aforementioned filter, and then the taken out cell content is pressurized at 3 atmospheres for more than 30 minutes, and 3, 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus). A method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) from plankton, characterized in that, for seawater containing the plankton taken, using a filter Filtration is carried out, and the plankton remaining on the filter is added with an extract to break it up. After extracting the cell contents from the above-mentioned plankton, pressurize at 3 atmospheres for more than 30 minutes, and produce from the pressurized product. 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), The above-mentioned plankton are diatoms, diatoms, diatoms, diatoms, diatomes (Surirellales), and diatomaceae (Entomoneidaceae) , diatoms of the genus Dinomones (Entomoneis genus).

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