Production method for fucoxanthin-containing composition, fucoxanthin-containing composition, use for fucoxanthin-containing composition, diatom

Abstract

Provided is a technology that can improve the antibacterial properties of a composition that includes fucoxanthin. According to the present invention, a production method for a fucoxanthin-containing composition includes a step for culturing a diatom in a medium that has a pH of 1.0–4.0. The diatom has 18S rRNA that comprises (a) or (b). (a) A polynucleotide that comprises the base sequence indicated by SEQ ID NO:1. (b) A polynucleotide that comprises a base sequence that has at least 85% homology with the base sequence indicated by SEQ ID NO:1.

Description

Method for producing fucoxanthin-containing composition, fucoxanthin-containing composition and its use, diatom

[0001] The present disclosure relates to the production of a fucoxanthin-containing composition. This application is based on Japanese Patent Application No. 2023-026634, filed on February 22, 2023, the contents of which are incorporated herein by reference.

[0002] Fucoxanthin is a type of carotenoid found in large brown algae such as kelp and wakame, and in microalgae such as diatoms, haptophytes, raphidophytes, and dinoflagellates. Fucoxanthin is known to have excellent antioxidant and anti-obesity effects (e.g., Non-Patent Document 1). Diatoms are sometimes used to produce fucoxanthin. For example, Patent Document 1 discloses a method for culturing diatoms by irradiating them with green light.

[0003] Japanese Patent Application Laid-Open No. 2020-074733

[0004] Sho Nishikawa and two others, "Anti-obesity and anti-diabetic effects and mechanisms of fucoxanthin derived from brown algae," Chemistry and Biology, 2016, Vol. 54, No. 8, pp. 580-585

[0005] The fucoxanthin-containing algae obtained by the method described in Patent Document 1 have room for improvement in terms of antibacterial properties. Therefore, there has been a demand for a technology that can improve the antibacterial properties of compositions containing fucoxanthin.

[0006] The present invention can be realized as the following aspects.

[0007] (1) One aspect of the present invention provides a method for producing a fucoxanthin-containing composition. This method includes culturing a diatom in a medium having a pH of 1.0 or higher and a pH of 4.0 or lower, wherein the diatom has 18S rRNA consisting of either (a) or (b) below: (a) a polynucleotide having the base sequence represented by SEQ ID NO: 1, or (b) a polynucleotide having a base sequence that is 85% or higher identical to the base sequence represented by SEQ ID NO: 1. This method for producing a fucoxanthin-containing composition involves culturing a diatom having 18S rRNA consisting of either (a) or (b) above in a medium having a pH of 1.0 or higher and a pH of 4.0 or lower to produce a fucoxanthin-containing composition, thereby enabling the production of a fucoxanthin-containing composition with improved antibacterial properties.

[0008] (2) In the method for producing a fucoxanthin-containing composition described in (1) above, the medium may contain silicon equivalent to 100 mg / L to 10 g / L of sodium metasilicate nonahydrate. This method for producing a fucoxanthin-containing composition can prevent a shortage of silicic acid, which is necessary for the synthesis of diatom silica frustules. As a result, diatom growth can be promoted, thereby improving the production efficiency of the fucoxanthin-containing composition.

[0009] (3) In the method for producing a fucoxanthin-containing composition according to (1) or (2) above, the pH of the medium may be between pH 1.5 and pH 2.5. According to this method for producing a fucoxanthin-containing composition, the fucoxanthin-containing composition is produced by culturing the 18S rRNA-containing diatom of (a) or (b) above in a medium having a pH of 1.5 to 2.5, thereby preventing a decrease in productivity in the production of a fucoxanthin-containing composition with improved antibacterial properties.

[0010] (4) In the method for producing a fucoxanthin-containing composition according to any one of (1) to (3) above, the medium may contain seawater. According to this form of the method for producing a fucoxanthin-containing composition, the fucoxanthin-containing composition is produced using a medium containing seawater, which makes it possible to suppress the growth of organisms that have difficulty growing in seawater. As a result, contamination during production can be suppressed, and a decrease in productivity of the fucoxanthin-containing composition can be suppressed.

[0011] (5) In the method for producing a fucoxanthin-containing composition according to any one of (1) to (4) above, the sodium ion concentration of the medium may be from 0.1 M to 0.6 M. According to this form of the method for producing a fucoxanthin-containing composition, the fucoxanthin-containing composition is produced using a medium with a sodium ion concentration of from 0.1 M to 0.6 M, which prevents contamination during production and, as a result, prevents a decrease in productivity of the fucoxanthin-containing composition.

[0012] (6) In the method for producing a fucoxanthin-containing composition according to any one of (1) to (5) above, the medium may be a liquid medium, and the method may further include, after the culturing step, a sedimentation step of allowing the liquid medium to stand to allow the diatoms to settle, and a step of removing the liquid medium after the sedimentation step. According to this form of the method for producing a fucoxanthin-containing composition, the liquid medium is allowed to stand to allow the diatoms to settle, and then the liquid medium is removed, thereby preventing the process for producing a fucoxanthin-containing composition from becoming complicated.

[0013] (7) Another aspect of the present disclosure provides a fucoxanthin-containing composition comprising a diatom and fucoxanthin, wherein the diatom has 18S rRNA consisting of either (a) or (b) below and is viable at a pH of 1.0 or higher and 4.0 or lower: (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1, or (b) a polynucleotide consisting of a base sequence that is 85% or higher identical to the base sequence represented by SEQ ID NO: 1. The fucoxanthin-containing composition of this aspect has excellent antibacterial properties.

[0014] (8) The fucoxanthin-containing composition described in (7) above may further contain silicon. This form of the fucoxanthin-containing composition makes it possible to omit or simplify the step of removing silicon from the fucoxanthin-containing composition.

[0015] (9) In the fucoxanthin-containing composition according to (7) or (8), the ratio of the fucoxanthin content to the diatom content may be 0.1% by mass or more and 5% by mass or less, calculated on a dry mass basis. This fucoxanthin-containing composition can prevent a decrease in the stability of fucoxanthin.

[0016] (10) In the fucoxanthin-containing composition according to (7) or (8), the ratio of the fucoxanthin content to the diatom content may be 50% by mass or more and 99.9% by mass or less, calculated on a dry mass basis. This fucoxanthin-containing composition can provide a high concentration of fucoxanthin.

[0017] (11) In the fucoxanthin-containing composition according to any one of (7) to (10) above, the diatom may be viable at a pH of 1.5 or higher and 2.5 or lower, providing the fucoxanthin-containing composition with even more excellent antibacterial properties.

[0018] (12) According to another aspect of the present disclosure, there is provided a food product containing the fucoxanthin-containing composition according to any one of (7) to (11) above.

[0019] (13) According to another aspect of the present disclosure, there is provided a cosmetic product containing the fucoxanthin-containing composition according to any one of (7) to (11) above.

[0020] (14) According to another aspect of the present disclosure, there is provided a diatom. The diatom has 18S rRNA consisting of the following (a) or (b), and is viable at a pH of 1.0 or higher and 4.0 or lower: (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1, or (b) a polynucleotide consisting of a base sequence that is 85% or higher identical to the base sequence represented by SEQ ID NO: 1. This form of diatom can be used to produce a fucoxanthin-containing composition with improved antibacterial properties.

[0021] The present invention can be realized in various forms, such as the use of diatoms to produce a fucoxanthin-containing composition, a pharmaceutical product containing a fucoxanthin-containing composition, a quasi-drug product containing a fucoxanthin-containing composition, a topical skin preparation containing a fucoxanthin-containing composition, a supplement containing a fucoxanthin-containing composition, a method for producing a food product containing a fucoxanthin-containing composition, or a method for producing cosmetics containing a fucoxanthin-containing composition.

[0022] Microscopic image showing diatoms used in Experiment 1. Explanatory diagram showing the results for freshwater medium in Experiment 1. Explanatory diagram showing the results for seawater medium in Experiment 1. Explanatory diagram showing the results of cell density measurement. Explanatory diagram showing the results of chlorophyll concentration measurement. Explanatory diagram showing cell production amount. Chromatogram showing the results of HPLC analysis. Explanatory diagram showing the production rate of fucoxanthin. Explanatory diagram showing the results of cell density measurement in Experiment 4. Explanatory diagram showing the results of chlorophyll fluorescence measurement in Experiment 4. Explanatory diagram showing the results of chlorophyll fluorescence / cell density in Experiment 4. Explanatory diagram showing the results of measurement of the pH of the culture medium in Experiment 4.

[0023] According to one embodiment of the present disclosure, there is provided a method for producing a fucoxanthin-containing composition. This method for producing a fucoxanthin-containing composition includes a step of culturing a diatom in a medium having a pH of 1.0 or higher and a pH of 4.0 or lower. The diatom has 18S rRNA consisting of either (a) or (b) below: (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; or (b) a polynucleotide consisting of a base sequence that is 85% or more identical to the base sequence represented by SEQ ID NO: 1.

[0024] The medium used in the culturing step (hereinafter also referred to as the "culturing step") is not particularly limited, and may be, for example, CSi medium, f / 2 medium, IMK medium, etc., and may be prepared from freshwater or contain seawater. By including seawater in the medium, the growth of organisms that have difficulty growing in seawater can be suppressed. More specifically, the growth of organisms that are not acid-tolerant, as well as organisms such as protozoa and microorganisms that are not salt-tolerant, can be suppressed. As a result, contamination during culture can be suppressed, and a decrease in productivity of the fucoxanthin-containing composition can be prevented. Furthermore, since the medium can be prepared using seawater even in environments where freshwater is not available, the fucoxanthin-containing composition can be produced in a wide range of environments. Examples of seawater include, but are not limited to, harvested seawater and artificial seawater. The sodium ion concentration of the medium is not particularly limited, but is preferably 0.05 M or more from the viewpoint of suppressing contamination during culture, and preferably 1.0 M or less from the viewpoint of improving the production efficiency of the fucoxanthin-containing composition. The sodium ion concentration of the medium is more preferably 0.1 M or more and 0.6 M or less, and even more preferably 0.2 M or more and 0.5 M or less.

[0025] The medium preferably contains silicon from the viewpoint of promoting diatom growth and improving the production efficiency of the fucoxanthin-containing composition. The silicon concentration contained in the medium is not particularly limited, but is preferably 10 mg / L or more when converted into sodium metasilicate nonahydrate from the viewpoint of preventing a shortage of silicic acid necessary for the synthesis of diatom silica frustules. The silicon concentration contained in the medium is preferably 10 g / L or less when converted into sodium metasilicate nonahydrate from the viewpoint of preventing the addition of an excessively high concentration of silicic acid and the aggregation and precipitation of medium components. The silicon concentration contained in the medium is preferably 10 mg / L or more and 8 g / L or less, more preferably 50 mg / L or more and 6 g / L or less, and even more preferably 100 mg / L or more and 4 g / L or less, when converted into sodium metasilicate nonahydrate. The silicon concentration contained in the medium may be 100 mg / L or more and 10 g / L or less when converted into sodium metasilicate nonahydrate. The medium may also contain nutrients such as nitrogen and phosphoric acid, vitamins, and trace elements.

[0026] By setting the pH of the medium to 1.0 or higher and 4.0 or lower, the growth of microorganisms that have difficulty growing in acidic environments can be inhibited, thereby suppressing contamination during culture. From the viewpoint of efficiently inhibiting the growth of microorganisms that have difficulty growing in acidic environments, the pH of the medium is preferably 3.5 or lower, more preferably 3.0 or lower, and even more preferably 2.5 or lower. Furthermore, from the viewpoint of improving the production efficiency of the fucoxanthin-containing composition, the pH of the medium is preferably 1.5 or higher. Therefore, the pH of the medium is preferably 1.5 or higher and 3.5 or lower, more preferably 1.5 or higher and 3.0 or lower, and even more preferably 1.5 or higher and 2.5 or lower. In particular, when the sodium ion concentration of the medium is 0.1 M or higher and 0.6 M or lower, for example, when the medium contains seawater, the pH of the medium is preferably 1.0 to 3.5, more preferably 1.2 to 3.5, even more preferably 1.5 to 3.5, still more preferably 1.5 to 3.0, and particularly preferably 1.5 to 2.5. Furthermore, when the sodium ion concentration contained in the medium is less than 0.1 M, the pH of the medium is preferably 1.5 to 3.5, more preferably 1.5 to 3.0, even more preferably 1.5 to 2.5, and still more preferably 2.0 to 2.5.

[0027] The form of the culture medium is not particularly limited, and may be a liquid medium or a solid medium, but a liquid medium is preferable from the viewpoint of facilitating large-scale culture. Furthermore, the culture method in the culture step is not particularly limited. For example, a nutrient source may be continuously or intermittently added to the medium during culture. Furthermore, the culture may be performed using a solid support device, or may be performed two-dimensionally or three-dimensionally using a substrate for cell fixation, etc. The culture medium may be changed or replaced during the culture step.

[0028] The culture step is preferably carried out by a method with enhanced aeration, and may be, for example, continuous or intermittent aeration culture, shaking culture, agitation culture, aeration and agitation culture, etc. Furthermore, the culture step may be carried out under light irradiation conditions, for example, continuous or periodic irradiation with white or green light. The culture temperature in the culture step is not particularly limited, but may be, for example, 10°C to 30°C. Furthermore, the culture period in the culture step is not particularly limited, but may be, for example, 1 week to 8 weeks. Furthermore, the culture location in the culture step is not particularly limited, but may be, for example, a greenhouse, outdoors, or indoors.

[0029] In the culturing step, diatoms having 18S rRNA consisting of (a) or (b) above are cultured. The base sequence identity described in (b) above refers to the maximum identity (%) of the base sequences obtained by aligning the two base sequences to be compared, optionally with gaps introduced. The base sequence identity can be calculated, for example, using blastn in NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ), which implements the BLAST algorithm, or Homolog search in Genetyx Win.

[0030] From the viewpoint of increasing the production efficiency of the fucoxanthin-containing composition, the diatom preferably has, as 18S rRNA, a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence represented by SEQ ID NO: 1, more preferably a polynucleotide consisting of a base sequence that is 95% or more identical to the base sequence represented by SEQ ID NO: 1, and even more preferably a polynucleotide consisting of a base sequence that is 97% or more identical to the base sequence represented by SEQ ID NO: 1.

[0031] The diatom having the 18S rRNA consisting of (a) or (b) above is not particularly limited, but is preferably a pennate diatom. The pennate diatom is not particularly limited, but is preferably a diatom belonging to the genus Pinnularia. The diatom having the 18S rRNA consisting of (a) or (b) above is not particularly limited, but may be, for example, a diatom with the accession number FERM P-22467.

[0032] The amount of cell increase during the culture step, i.e., the growth rate, is not particularly limited, but is preferably 0.5 mg / L / day or more, more preferably 5 mg / L / day or more, and even more preferably 50 mg / L / day or more, in terms of dry weight. The amount of fucoxanthin increase during the culture step, i.e., the fucoxanthin production rate, is not particularly limited, but is preferably 0.002 mg / L / day or more, more preferably 0.02 mg / L / day or more, and even more preferably 0.2 mg / L / day or more, in terms of dry weight. The amount of fucoxanthin increase during the culture step can be determined by comparison with the growth rate of the diatom.

[0033] The method for producing a fucoxanthin-containing composition may further include a step of removing the medium after the culturing step. The step of removing the medium is not particularly limited, but, when the medium is a liquid medium, it may include, for example, a step of allowing the liquid medium to stand to settle the diatom cells, or a step of centrifuging the liquid medium to settle the diatom cells. From the perspective of simplifying the diatom cell recovery step, the step of removing the medium preferably includes a sedimentation step of allowing the liquid medium to stand to settle the diatoms, and a step of removing the liquid medium after the sedimentation step. This method can prevent the production steps of the fucoxanthin-containing composition from becoming too complicated, thereby preventing an increase in the production cost of the fucoxanthin-containing composition.

[0034] The method for producing a fucoxanthin-containing composition may further include, after the culturing step, an extraction step for extracting fucoxanthin or a purification step for purifying fucoxanthin. The extraction and purification steps can be carried out according to known methods. The extraction step is not particularly limited, but examples include a method in which the culture medium is allowed to stand to allow diatom cells to settle and be collected, followed by extraction with a solvent such as an organic solvent; a method in which the diatom cells are collected on a filter such as a glass fiber filter, dried, and then extracted with a solvent such as an organic solvent; and a method in which the cells are disrupted using a French press or homogenizer, followed by extraction with a solvent. The solvent used to extract fucoxanthin from diatoms is not particularly limited as long as it can extract fucoxanthin. Examples of the solvent include alcohols such as methanol, ethanol, propanol, isopropanol, and n-butanol; ketones such as methyl ethyl ketone and acetone; esters such as methyl acetate and ethyl acetate; organic chlorinated hydrocarbons such as chloroform; aliphatic hydrocarbons such as hexane; aromatic hydrocarbons such as benzene and toluene; and acetone, which can be used alone or in combination. The purification step is not particularly limited, but for example, the solvent extract obtained in the extraction step may be subjected to reverse phase chromatography using an open column, a flash column, or a high performance liquid chromatograph, either directly or after removing the residue.

[0035] According to the method for producing a fucoxanthin-containing composition of this embodiment described above, a fucoxanthin-containing composition is produced by culturing a diatom having 18S rRNA comprising either (a) or (b) in a medium having a pH of 1.0 or higher and a pH of 4.0 or lower, thereby producing a fucoxanthin-containing composition with improved antibacterial properties. Furthermore, since the fucoxanthin-containing composition is produced by culturing a diatom in a medium having a pH of 1.0 or higher and a pH of 4.0 or lower, the growth of acid-intolerant organisms is suppressed, allowing fucoxanthin to be produced under conditions with reduced contamination. This suppresses contamination by, for example, protozoa that prey on diatoms, or bacteria and fungi that compete with diatoms for nutrients, thereby preventing a decrease in productivity of the fucoxanthin-containing composition. Furthermore, as a result of the suppression of contamination, the culture can be performed using a simple culture device, such as an open culture, thereby preventing an increase in the production cost of the fucoxanthin-containing composition. Furthermore, since the fucoxanthin-containing composition is produced by causing diatoms to produce fucoxanthin, a decrease in productivity of the fucoxanthin-containing composition can be suppressed.

[0036] According to a second aspect of the present disclosure, there is provided a fucoxanthin-containing composition comprising a diatom and fucoxanthin. In this fucoxanthin-containing composition, the diatom has 18S rRNA consisting of either (a) or (b) below, and is viable at a pH of 1.0 or higher and 4.0 or lower: (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; or (b) a polynucleotide consisting of a base sequence that is 85% or higher identical to the base sequence represented by SEQ ID NO: 1.

[0037] From the viewpoint of enhancing the antibacterial properties of the fucoxanthin-containing composition, the diatom preferably survives at a pH of 1.0 or higher and 3.0 or lower, and more preferably at a pH of 1.5 or higher and 2.5 or lower. The diatom preferably has, as its 18S rRNA, a polynucleotide consisting of a base sequence that is 90% or higher identical to the base sequence represented by SEQ ID NO: 1, more preferably a polynucleotide consisting of a base sequence that is 95% or higher identical to the base sequence represented by SEQ ID NO: 1, and even more preferably a polynucleotide consisting of a base sequence that is 97% or higher identical to the base sequence represented by SEQ ID NO: 1. Diatoms having 18S rRNA of (a) or (b) above are not particularly limited, but are preferably pennate diatoms. Pennate diatoms are not particularly limited, but are preferably diatoms belonging to the genus Pinnularia. Diatoms having 18S rRNA of (a) or (b) above are not particularly limited, but may be, for example, diatoms with accession number FERM P-22467.

[0038] A fucoxanthin-containing composition containing diatoms and fucoxanthin may be obtained, for example, by recovering cells from the medium after the culture step in the above-mentioned method for producing a fucoxanthin-containing composition. The cell recovery method is not particularly limited, and examples include a method in which the liquid medium is left to stand to allow the diatom cells to settle and then the cells are recovered, a method in which the culture solution is filtered through a mesh filter to recover the cells, and a method in which the cells are recovered using a continuous centrifuge equipped with a nozzle-type separator. By this method, cells in a slurry can be obtained.

[0039] The fucoxanthin-containing composition may be in a liquid form, a solid form, or a dry form such as a powder, etc. The method for obtaining a dry fucoxanthin-containing composition is not particularly limited, but examples include methods in which the cells collected by the above-mentioned method are dried using a spray dryer, freeze dryer, vacuum dryer, etc.

[0040] In a fucoxanthin-containing composition, the ratio of the fucoxanthin content to the diatom content is not particularly limited, but from the viewpoint of preventing a decrease in fucoxanthin stability, it is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, even more preferably 0.5% by mass or more, and even more preferably 1.0% by mass or more, calculated on a dry mass basis. Furthermore, from the viewpoint of preventing a decrease in fucoxanthin stability, it is preferably 10% by mass or less, more preferably 7% by mass or less, and even more preferably 5% by mass or less, calculated on a dry mass basis. Therefore, the ratio of the fucoxanthin content to the diatom content may be, for example, 0.1% by mass or more and 5% by mass or less, calculated on a dry mass basis.

[0041] From the viewpoint of inhibiting the decomposition of fucoxanthin, a preferred embodiment of the fucoxanthin-containing composition is one in which diatom cells that have produced fucoxanthin are directly dried and powdered. This embodiment stabilizes fucoxanthin compared to an embodiment in which fucoxanthin is extracted, thereby inhibiting the decomposition of fucoxanthin. For example, compared to an embodiment in which fucoxanthin is extracted with a solvent such as ethanol and then powdered, the decomposition of fucoxanthin can be inhibited for a longer period of time. Furthermore, compared to an embodiment in which fucoxanthin is extracted with a solvent such as ethanol, powdered, and then dissolved in a solvent such as ethanol, the decomposition of fucoxanthin can be inhibited for an even longer period of time.

[0042] The fucoxanthin concentration of the fucoxanthin-containing composition may be increased by undergoing the extraction process described above, or may be further increased by undergoing the purification process described above. Although the amount of diatoms contained in the fucoxanthin-containing composition that has undergone the extraction and purification processes is expected to be very small, they can be detected using highly sensitive analytical techniques such as PCR using primers designed based on the 18S rRNA, i.e., the base sequence represented by SEQ ID NO: 1, or time-of-flight mass spectrometry.

[0043] In a fucoxanthin-containing composition in which the fucoxanthin concentration has been increased by an extraction process or the like, the ratio of the fucoxanthin content to the diatom content is not particularly limited, but from the viewpoint of providing a high concentration of fucoxanthin, it is preferably 50% by mass or more, more preferably 60% by mass or more, even more preferably 70% by mass or more, even more preferably 80% by mass or more, even more preferably 90% by mass or more, particularly preferably 95% by mass or more, and especially preferably 97% by mass or more, calculated on a dry mass basis. From the viewpoint of productivity, it is preferably 99.9% by mass or less, more preferably 99.8% by mass or less, and even more preferably 99.5% by mass or less. Therefore, the ratio of the fucoxanthin content to the diatom content may be, for example, 50% by mass or more but 99.9% by mass or less, calculated on a dry mass basis.

[0044] The fucoxanthin-containing composition may further contain silicon. In other words, the fucoxanthin-containing composition may contain a component derived from diatom frustules. In an embodiment in which the fucoxanthin-containing composition contains silicon, the step of removing silicon (i.e., the component derived from diatom frustules) from the fucoxanthin-containing composition can be omitted or simplified. As a result, the production process for the fucoxanthin-containing composition can be prevented from becoming complicated, thereby preventing an increase in production costs. The step of removing the component derived from diatom frustules is not particularly limited, but examples thereof include a step of extracting fucoxanthin using a solvent. The concentration of silicon in the fucoxanthin-containing composition is not particularly limited, but is preferably 20% by mass or more and 80% by mass or less, and more preferably 50% by mass or more and 70% by mass or less. The silicon concentration in a fucoxanthin-containing composition can be determined using the molybdenum blue method in accordance with the method described in J. Grad. Sch. Biosp. Sci., Hiroshima Univ. (2006), 45: 21-29. The fucoxanthin-containing composition may contain other optional components, such as additives such as dextran, or stabilizers such as antioxidants.

[0045] The fucoxanthin-containing composition of this embodiment may be used as an ingredient in various foods and beverages. The form of the ingredient for foods and beverages is not particularly limited, but examples include powder, granules, tablets, soft capsules, hard capsules, tablets, and liquids.

[0046] A third aspect of the present disclosure provides a food product containing a fucoxanthin-containing composition. Examples of such foods include, but are not limited to, breads such as white bread and butter rolls, confectioneries such as baked goods and cakes, noodles such as udon and buckwheat, cooked rice dishes such as rice balls and rice porridge, mochi rice cakes such as kusamochi and kinako mochi, pastas such as spaghetti, fettuccine, penne, erike, ravioli, and lasagna, dressings such as mayonnaise, creamy salad dressing, semi-solid dressing, emulsified liquid dressing, and separated liquid dressing, and foods for the sick or elderly such as mousse, jelly, and soup. Furthermore, foods containing the fucoxanthin-containing composition may be so-called health foods, or may be supplements, foods for specified health uses, nutritional supplements, or nutrients.

[0047] In foods containing a fucoxanthin-containing composition, the fucoxanthin-containing composition is preferably in the form of a dried powder of cells recovered by the method described above. The amount of the fucoxanthin-containing composition contained in the food is not particularly limited and may be appropriately determined depending on the food. The amount of the fucoxanthin-containing composition contained in the food is preferably 0.1% by mass to 98% by mass, more preferably 0.5% by mass to 95% by mass, and even more preferably 1% by mass to 90% by mass. For example, when the food is pasta, the amount is preferably 0.5% by mass to 20% by mass, and more preferably 1% by mass to 10% by mass. Furthermore, when the food is a supplement, the amount is preferably 50% by mass to 98% by mass, and more preferably 70% by mass to 95% by mass. For applications where a high fucoxanthin concentration is desirable, such as supplements and health foods, the fucoxanthin-containing composition may be in a form in which the fucoxanthin concentration has been increased by an extraction process such as those described above. For example, when the food is a health food, the fucoxanthin concentration in the health food is preferably 10% by mass or more and 98% by mass or less, and more preferably 20% by mass or more and 95% by mass or less.

[0048] The fucoxanthin-containing composition of the present disclosure may also be used as a cosmetic ingredient.

[0049] According to a fourth aspect of the present disclosure, there is provided a cosmetic product containing a fucoxanthin-containing composition. The cosmetic product is not particularly limited, but may be intended for purposes such as preventing skin dullness, rough skin, blemishes, wrinkle improvement, skin rejuvenation, and acne prevention. The cosmetic product may take any form, but is not particularly limited, and may be in the form of a makeup cosmetic product such as an emulsion, cream, lotion, serum, pack, cleanser, foundation, blush, or lipstick; a scalp cosmetic product such as a hair care product, hair tonic, shampoo, or rinse; a dispersion, ointment, liquid, aerosol, or patch.

[0050] In cosmetics containing a fucoxanthin-containing composition, the fucoxanthin-containing composition may be in the form of a dried powder of cells recovered by the method described above, but is preferably in a form in which the fucoxanthin concentration has been increased by an extraction process or the like as described above. The amount of the fucoxanthin-containing composition contained in the food product is not particularly limited and may be appropriately determined depending on the cosmetic. The amount of the fucoxanthin-containing composition contained in the cosmetic is preferably 0.001% by mass or more and 10% by mass or less, more preferably 0.005% by mass or more and 5% by mass or less, and even more preferably 0.01% by mass or more and 2% by mass or less. For example, when the cosmetic is a beauty serum, the fucoxanthin concentration in the beauty serum is preferably 0.001% by mass or more and 1% by mass or less, and more preferably 0.005% by mass or more and 0.5% by mass or less.

[0051] The fucoxanthin-containing composition of the present disclosure may also be used as a raw material for pharmaceuticals and quasi-drugs. Its uses are not particularly limited, but include, for example, anti-inflammatory agents, angiogenesis inhibitors, therapeutic agents for virus-related malignant tumors, antioxidants and cell activity activators, neutral fat absorption regulators, anti-obesity agents, therapeutic and preventative agents for lipid metabolism disorders, antitumor agents, cancer cell proliferation inhibitors, blood glucose level increase inhibitors, scalp topical medications, antidiabetic drugs, therapeutic and preventative agents for hyperlipidemia, topical medications for skin, neuroprotective drugs, and veterinary drugs. The composition may be in a variety of forms, such as tablets, powders, and capsules, as needed. The fucoxanthin-containing composition of the present disclosure can also be used for various purposes, such as research reagents. In pharmaceuticals and research reagents containing the fucoxanthin-containing composition, the fucoxanthin-containing composition is preferably in a form in which the fucoxanthin concentration has been increased by the extraction and purification processes described above. For example, when used in a research reagent, the fucoxanthin concentration in the research reagent is preferably 90% by mass or more and 99.9% by mass or less, and more preferably 95% by mass or more and 99.5% by mass or less.

[0052] A solution containing the fucoxanthin-containing composition of the present disclosure is expected to be acidic. Therefore, the fucoxanthin-containing composition of the present disclosure has excellent antibacterial properties. As a result, the antibacterial properties can be improved in the various applications described above, such as foods and cosmetics containing the fucoxanthin-containing composition.

[0053] According to a fifth aspect of the present disclosure, there is provided a diatom. The diatom has 18S rRNA consisting of the following (a) or (b), and is viable at a pH of 1.0 or higher and 4.0 or lower: (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; or (b) a polynucleotide consisting of a base sequence having 85% or more identity to the base sequence represented by SEQ ID NO: 1.

[0054] Using this type of diatom, a fucoxanthin-containing composition with improved antibacterial properties can be produced. From the perspective of enhancing the antibacterial properties of the fucoxanthin-containing composition, the diatom preferably survives at a pH of 1.0 or higher and 3.0 or lower, and more preferably at a pH of 1.5 or higher and 2.5 or lower. The diatom preferably has, as its 18S rRNA, a polynucleotide consisting of a base sequence that is 90% or higher identical to the base sequence represented by SEQ ID NO: 1, more preferably a polynucleotide consisting of a base sequence that is 95% or higher identical to the base sequence represented by SEQ ID NO: 1, and even more preferably a polynucleotide consisting of a base sequence that is 97% or higher identical to the base sequence represented by SEQ ID NO: 1. Diatoms having 18S rRNA consisting of (a) or (b) above are not particularly limited, but are preferably pennate diatoms. Pennate diatoms are not particularly limited, but are preferably diatoms belonging to the genus Pinnularia. The diatom having the 18S rRNA consisting of (a) or (b) above is not particularly limited, but may be, for example, the diatom having the accession number FERM P-22467.

[0055] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0056] 1. Experiment 1 (Experiment on Diatom Growth as a Function of pH) (1) Method The pH at which diatoms possessing 18S rRNA as described above can grow was investigated. The diatom strain designated by accession number FERM P-22467 was used. Freshwater media with the compositions shown in Table 1 below and seawater media with the compositions shown in Table 2 below were used as culture media. The pH of the media was adjusted to 1.5, 2.0, 3.0, 4.0, 5.0, and 6.0, respectively. Seawater media with pH 1.0, pH 1.1, pH 1.2, pH 1.3, and pH 1.4 were also used. 3 mL of the medium was dispensed into a 24-well plate. Static culture was performed for 10 days at a culture temperature of 26°C under a light intensity of 10 μE using white fluorescent lamps. Using a Qubit3 Fluorometer (ThermoFisher Scientific) as a fluorometer, chlorophyll fluorescence of the culture medium was measured at 665-720 nm using blue light excitation at 430-495 nm and a red fluorescence filter, to quantify the cell proliferation rate.

[0057]

[0058]

[0059] (2) Results and Discussion Figure 1 is a microscopic image of the diatom used in Experiment 1. Figure 2 is an explanatory diagram showing the results of Experiment 1 in freshwater medium. Figure 3 is an explanatory diagram showing the results of Experiment 1 in seawater medium. In Figures 2 and 3, the horizontal axis represents pH, and the vertical axis represents chlorophyll fluorescence intensity (B excitation). The results shown in Figure 2 indicate that the diatom used in Experiment 1 can grow in freshwater medium at least in the pH range of 1.5 to 6.0 where the experiment was conducted. This suggests that it can survive at least in the pH range of 1.5 to 6.0. The results shown in Figure 3 indicate that the diatom used in Experiment 1 can grow in seawater medium at least in the pH range of 1.0 to 6.0 where the experiment was conducted. This suggests that it can survive at least in the pH range of 1.0 to 6.0.

[0060] 2. Experiment 2 (Experiment Concerning the Production of Fucoxanthin-Containing Compositions) (1) Method <Confirmation of Cultivation and Cell Density / Chlorophyll Concentration> A fucoxanthin-containing composition was produced using a freshwater medium and a seawater medium for the diatoms having 18S rRNA as described above. As in Experiment 1, the diatom strain designated by Accession Number FERM P-22467 was used as the diatom. As in Experiment 1, a strongly acidic freshwater medium (pH 2.5) having the composition shown in Table 1 above and a strongly acidic seawater medium (pH 2.5) having the composition shown in Table 2 above were used as the media. Both media contained 2.273 g / L of sodium metasilicate (Na ２ SiO ３ ・9H ２ The diatom strain was subcultured in 60 mL of strongly acidic seawater medium in a 100 mL baffled flask and cultured at 150 rpm for 12 days under a 40 μE light intensity using a white fluorescent lamp at 26°C. During the culture period, the cell density and chlorophyll concentration in the culture medium were monitored every two days. Cell density was determined using a V-650 spectrophotometer (JASCO Corporation) based on scattering at a wavelength of 750 nm in the culture medium. Chlorophyll concentration was determined using a Qubit3 Fluorometer (ThermoFisher Scientific) by measuring fluorescence (665-720 nm) primarily derived from chlorophyll excited by blue light (430-495 nm).

[0061] <Cell Recovery> The cultured cells were centrifuged at room temperature at 5,000 g for 5 minutes to recover the cells. The volume (mL) of the culture medium was also confirmed at the time of cell recovery. The recovered cells were freeze-dried using a freeze dryer VD-800R (Tomy Seiko Co., Ltd.), and the dry weight of the cells was measured.

[0062] <Extraction of fucoxanthin> 1.0 mL of acetone was added to 20 mg of dried and powdered cells, and the mixture was vigorously shaken at 30 Hz for 2 minutes using a TissueLyser II osmotic homogenizer (Qiagen). The homogenate was centrifuged at 21,600 g for 10 minutes at room temperature, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter to obtain an extract.

[0063] <Analysis of Fucoxanthin> The extract was analyzed using a high-performance liquid chromatograph mass spectrometer, Alliance HPLC / 2998PDA / qDa System (Waters) under the following analytical conditions. As a fucoxanthin standard, 95%-grade fucoxanthin (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted to 100 μg / mL, 50 μg / mL, and 10 μg / mL and used. The peak at 445 nm, which is the absorption maximum of fucoxanthin, was confirmed on the chromatogram. Injection sample volume: 10 μL Column used: XBridge C18 3.5 μm, 4.6 mm × 150 mm Column temperature: 35 ° C Flow rate: 1 mL / min Solvent composition Solvent A: 80% methanol, 19.9% ​​water, 0.1% formic acid Solvent B: 99.9% methanol, 0.1% formic acid Solvent C: 100% acetonitrile Gradient 0 → 10 min: Solvent A 100% → Solvent B 100% 10 min → 20 min: Solvent B 100% 20 min → 30 min: Solvent C 100%

[0064] (2) Results and Discussion Figure 4 is an explanatory diagram showing the results of measuring cell density. In Figure 4, the horizontal axis represents the culture period (days), and the vertical axis represents the cell density (OD ７５０ ) are shown. Figure 5 is an explanatory diagram showing the measurement results of chlorophyll concentration. In Figure 5, the horizontal axis represents the culture period (days), and the vertical axis represents chlorophyll fluorescence intensity. As shown in Figure 4, regardless of whether the culture was performed in a strongly acidic freshwater medium or a strongly acidic seawater medium, the cell density increased linearly over 12 days of culture. Furthermore, as shown in Figure 5, the chlorophyll concentration also increased linearly over 12 days of culture. These results indicate that the FERM AP-22467 strain can grow well in both a strongly acidic freshwater medium and a strongly acidic seawater medium.

[0065] Figure 6 is an explanatory diagram showing cell production. In Figure 6, the vertical axis shows cell production (g / L / day). Cell production was calculated from the volume (mL) of the culture medium at the time of cell recovery, the weight (mg) of dried and powdered cells, and the number of days of culture. Note that cell production corresponds to the cell proliferation rate. The cell production was found to be 0.074 g / L / day in the strongly acidic freshwater medium and 0.077 g / L / day in the strongly acidic seawater medium.

[0066] FIG. 7 shows chromatograms showing the results of HPLC analysis. A shows the results of HPLC analysis of 50 μg / mL of fucoxanthin standard, B shows the results of an extract of cells cultured in a strongly acidic freshwater medium, and C shows the results of HPLC analysis of an extract of cells cultured in a strongly acidic seawater medium. A clear fucoxanthin peak was observed at the same retention time as the fucoxanthin standard in both the strongly acidic freshwater medium and the strongly acidic seawater medium. Comparison with the peak obtained from the calibration curve revealed that the fucoxanthin concentration in the cells was 4.7 mg / g (0.47% (w / w)) per dry weight under strongly acidic freshwater medium culture conditions and 2.5 mg / g (0.25% (w / w)) under strongly acidic seawater medium culture conditions.

[0067] Fig. 8 is an explanatory diagram showing the fucoxanthin production rate. In Fig. 8, the vertical axis represents the fucoxanthin production rate (mg / L / day). The fucoxanthin production rate is calculated from the cell growth rate and the fucoxanthin concentration contained in the cells. The fucoxanthin production rate was found to be 0.35 mg / L / day in the strongly acidic freshwater medium and 0.19 mg / L / day in the strongly acidic seawater medium.

[0068] 3. Experiment 3 (Antibacterial Experiment) (1) Method 0.75 mL of water was added to 0.5 g of the freeze-dried powder of cells obtained in Experiment 2 under the culture conditions of a strongly acidic seawater medium, and the mixture was mixed thoroughly to form a paste. The pH of the surface of the paste was measured using a flat pH composite electrode 6261-10C (manufactured by Horiba, Ltd.). 0.75 mL of water was then added to the paste and mixed thoroughly to form a slurry, and the pH was measured using the same electrode.

[0069] (2) Results and Discussion The pH of the cells in paste form was 4.59, and the pH of the cells in slurry form was 4.67. This indicates that even when a certain amount of water is added to the freeze-dried powder of the diatom strain designated by Accession Number FERM P-22467, which was cultivated under strongly acidic conditions, the pH remains on the acidic side. Since acidic conditions are generally known to inhibit the growth of various bacteria, this freeze-dried powder was also thought to have the effect of inhibiting contamination. This suggests that this freeze-dried powder, i.e., the fucoxanthin-containing composition of this example, has excellent antibacterial properties.

[0070] 4. Experiment 4 (Experiment on scale-up) (1) Method A 100 L aquarium (Sunlight Tank SLP-100) was placed in a greenhouse, and diatoms were cultured in approximately 90 L of freshwater medium while bubbling with an air pump. A 30 cm x 50 cm board was placed on the upper opening of the aquarium, but it did not completely cover the opening, creating an environment where insects and other insects could easily enter. No filter or the like for preventing contamination was installed in the air pump, and unsterilized air was ventilated. The diatom strain designated by accession number FERM P-22467 was used as the diatom. As the medium, a freshwater medium having the composition shown in Table 1 above was added with MgSO ４ ・7H ２The medium used contained 0 (0.5 g / L). Culture was initiated on November 17 and continued for the period shown in Table 3 below. On November 29, the pH of the culture was adjusted to 2.7 by adding dilute sulfuric acid. During the culture period, the chlorophyll fluorescence, cell density, pH, and temperature of the culture were measured. The chlorophyll fluorescence of the culture was determined by measuring the fluorescence (665-720 nm) excited by blue light (430-495 nm) using a Qubit3 Fluorometer (ThermoFisher Scientific) for the culture diluted 10-fold with medium. The cell density of the culture was determined based on the scattering of the culture at a wavelength of 750 nm using a V-650 spectrophotometer (JASCO Corporation). The pH of the culture solution was measured using a pen-type pH meter (pH-222, manufactured by Mother Tools Co., Ltd.) Table 3 also shows the time when the temperature of the culture solution was measured.

[0071]

[0072] (2) Results and Discussion Figure 9 is an explanatory diagram showing the cell density measurement results in Experiment 4. Figure 10 is an explanatory diagram showing the chlorophyll fluorescence measurement results in Experiment 4. Figure 11 is an explanatory diagram showing the chlorophyll fluorescence / cell density results in Experiment 4. Figure 12 is an explanatory diagram showing the culture solution pH measurement results in Experiment 4. Figures 9 to 12 show the results after adjusting the pH of the culture solution by adding dilute sulfuric acid. As shown in Figures 9 and 10, as the culture period increased, the cell density and chlorophyll fluorescence intensity increased. Note that because the culture period was in winter, the culture solution temperature did not increase, and the cell growth rate tended to be somewhat slow. As shown in Figure 11, the amount of chlorophyll per cell density remained almost constant. This indicates that the growth of other organisms in the culture solution was suppressed, and only chlorophyll-containing diatoms grew. Furthermore, as shown in Figure 12, the culture solution pH was maintained at 2.5 to 3.0 throughout the culture period. Therefore, it has been demonstrated that the diatoms disclosed herein are useful for mass cultivation in environments such as outdoors where other microorganisms, insects, etc. may be present, by using a strongly acidic medium.

[0073] The present invention is not limited to the above-described embodiments and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A method for producing a fucoxanthin-containing composition, comprising: The method comprises culturing diatoms in a medium having a pH of 1.0 or more and a pH of 4.0 or less, The diatom has 18S rRNA consisting of the following (a) or (b): Method for producing a fucoxanthin-containing composition; (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; (b) A polynucleotide consisting of a base sequence having an identity of 85% or more with the base sequence represented by SEQ ID NO:

1.

2. The method for producing a fucoxanthin-containing composition according to claim 1, The medium contains silicon equivalent to 100 mg / L or more and 10 g / L or less of sodium metasilicate nonahydrate, A method for producing a fucoxanthin-containing composition.

3. The method for producing a fucoxanthin-containing composition according to claim 1 or 2, The pH of the medium is pH 1.5 or higher and pH 2.5 or lower. A method for producing a fucoxanthin-containing composition.

4. The method for producing a fucoxanthin-containing composition according to claim 1 or 2, The medium comprises seawater. A method for producing a fucoxanthin-containing composition.

5. The method for producing a fucoxanthin-containing composition according to claim 1 or 2, The sodium ion concentration of the medium is 0.1 M or more and 0.6 M or less. A method for producing a fucoxanthin-containing composition.

6. The method for producing a fucoxanthin-containing composition according to claim 1 or 2, The medium is a liquid medium, After the culturing step, a sedimentation step in which the liquid culture medium is allowed to stand to allow the diatoms to settle; removing the liquid medium after the settling step; Including, A method for producing a fucoxanthin-containing composition.

7. A fucoxanthin-containing composition comprising a diatom and fucoxanthin, The diatom is It has 18S rRNA consisting of the following (a) or (b): It can survive at pH 1.0 or higher and pH 4.0 or lower. fucoxanthin-containing composition; (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; (b) A polynucleotide consisting of a base sequence having an identity of 85% or more with the base sequence represented by SEQ ID NO:

1.

8. The fucoxanthin-containing composition according to claim 7, further comprising: Contains silicon, A fucoxanthin-containing composition.

9. The fucoxanthin-containing composition according to claim 7 or 8, the ratio of the fucoxanthin content to the diatom content is 0.1% by mass or more and 5% by mass or less in terms of dry mass; A fucoxanthin-containing composition.

10. The fucoxanthin-containing composition according to claim 7 or 8, the ratio of the content of the fucoxanthin to the content of the diatom is 50% by mass or more and 99.9% by mass or less in terms of dry mass; A fucoxanthin-containing composition.

11. The fucoxanthin-containing composition according to claim 7 or 8, The diatom is viable at a pH of 1.5 or higher and 2.5 or lower. A fucoxanthin-containing composition.

12. A food product comprising the fucoxanthin-containing composition according to claim 7 or 8.

13. A cosmetic comprising the fucoxanthin-containing composition according to claim 7 or 8.

14. A diatom, The bacterium has 18S rRNA consisting of the following (a) or (b) and is viable at a pH of 1.0 or higher and 4.0 or lower: diatom; (a) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1; (b) A polynucleotide consisting of a base sequence having an identity of 85% or more with the base sequence represented by SEQ ID NO: 1.