WO2017195870A1

WO2017195870A1 - L-hydroxyproline-containing yeast cell or cell culture product or extract of same, use thereof, and method for producing l-hydroxyproline

Info

Publication number: WO2017195870A1
Application number: PCT/JP2017/017932
Authority: WO
Inventors: 幸彦成田; 祐子福井; 龍雄松岡
Original assignee: サントリーホールディングス株式会社
Priority date: 2016-05-12
Filing date: 2017-05-11
Publication date: 2017-11-16
Also published as: CN109196090A; KR102265246B1; JP6966431B2; TW201809262A; KR20190006954A; JPWO2017195870A1

Abstract

The yeast cell, cell culture product and extract of the same according to the present invention are a cell of at least one kind of yeast selected from the group consisting of Kodamaea ohmeri, Metschnikowia reukaufii, Meyerozyma caribbica, Meyerozyma guilliermondii and Clavispora lusitaniae, a cell culture product thereof and an extract of the same, characterized in that: the yeast cell, cell culture product or extract of the same as described above contains L-hydroxyproline; and the ratio [100 × Hyp/(Pro + Hyp)] of the content (μg/mL) of L-hydroxyproline (Hyp) to the total content (μg/mL) of L-proline (Pro) and L-hydroxyproline is 35-100.

Description

Yeast cells or cell cultures containing L-hydroxyproline or extracts thereof, uses thereof, and method for producing L-hydroxyproline

The present invention relates to a yeast cell or a cell culture or an extract thereof containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline. The invention also relates to the use of yeast for producing L-hydroxyproline. The present invention also relates to a food / beverage product, a cosmetic, a cosmetic raw material, a composition for reinforcing L-hydroxyproline, and the like containing a yeast cell or cell culture or an extract thereof.

L-hydroxyproline (hydroxy-L-proline) is an amino acid having a structure in which a hydroxyl group is bonded to the 4-position carbon atom of L-proline. L-hydroxyproline has the following effects: promotion of collagen production in fibroblasts, promotion of epidermal cell proliferation, moisturizing effect equivalent to or better than collagen, prevention of skin aging, transdermal absorbability higher than tripeptide, wrinkle improvement effect, The improvement effect of atopic dermatitis etc. are mentioned. Since L-hydroxyproline is safe for the human body, it can be used by being contained in foods and drinks, cosmetics, pharmaceuticals, etc., and its usefulness is very high.

L-hydroxyproline can be produced by an organic synthesis method, but a production method using a microorganism is also being studied. For example, Patent Document 1 discloses that a transformant obtained by introducing a polynucleotide encoding L-proline cis-4-hydroxylase derived from Rhizobium rhizobia into a host cell is cultured in a medium, and the cis- A method for producing cis-4-hydroxy-L-proline is described in which 4-hydroxy-L-proline is produced and accumulated, and cis-4-hydroxy-L-proline is collected from the culture.

Japanese Patent No. 5506668

By the way, yeast is a microorganism which has been tried for various industrial uses from the old days in the food and drink field, etc., and yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are L -It is useful as a raw material for cosmetics, foods and drinks where the effect of hydroxyproline is expected. However, a yeast that accumulates L-hydroxyproline in bacterial cells or bacterial cell cultures has not yet been reported.

The main object of the present invention is to provide a yeast cell or cell culture or yeast extract containing L-hydroxyproline, its use, and a method for producing L-hydroxyproline.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that when a certain type of yeast such as Kodamaea ohmeri is aerobically cultured, L or L -It was found to accumulate hydroxyproline. In addition, the obtained yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are L-hydroxyproline based on the total weight content of L-proline (Pro) and L-hydroxyproline (Hyp). It was found that the ratio of the weight content of (100 × Hyp / (Pro + Hyp)) could be in a specific range. The present inventors have further studied based on these findings and have completed the present invention.

Cells or cell cultures or extracts thereof yeast of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and a Kurabisupora-Rushitanie (Clavispora lusitaniae) at least one cell or cell culture of the yeast is selected from the group consisting of or extracts thereof,
The yeast cells or cell cultures or extracts thereof contain L-hydroxyproline, and L-hydroxy to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio (100 × Hyp / (Pro + Hyp)) of hydroxyproline content (μg / mL) is 35 to 100.

The yeast cells or cell cultures or extracts thereof of the present invention preferably have an L-hydroxyproline content of 10 μg / mL or more. The yeast cell or cell culture of the present invention or an extract thereof has a value (μg / mL / OD660) obtained by dividing the L-hydroxyproline content (μg / mL) by OD660 (20 or more). It is preferable.

Another aspect of the bacteria or bacterial cultures or extracts thereof yeast of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Giri Erumondi a (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie least one cell or cell culture of the yeast is selected from the group consisting of (Clavispora lusitaniae) or extracts thereof,
The content of L-hydroxyproline is 10 μg / mL or more.

The method of producing L- hydroxyproline invention Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie ( At least one yeast selected from the group consisting of Clavispora lusitaniae ) in an aerobic culture in a liquid medium containing a carbon source and a nitrogen source, so that L-hydroxy is contained in the yeast cell or cell culture. A step of accumulating proline, wherein the nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.

In the production method of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000.
In the production method of the present invention, the aerobic culture is preferably performed for 10 to 100 hours.

The present invention is selected from the group consisting of Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) Also included is the use of at least one selected yeast for producing L-hydroxyproline.

The use of the present invention includes accumulating L-hydroxyproline in the yeast cell or cell culture by aerobically culturing the yeast in a liquid medium containing a carbon source and a nitrogen source. The nitrogen source is preferably a nitrogen source containing an L-hydroxyproline-containing peptide.
In the use of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000. In the use of the present invention, the aerobic culture is preferably performed for 10 to 100 hours.

The composition of the present invention is characterized in that it comprises the yeast or cell culture of the yeast of the present invention or an extract thereof.
The food / beverage products of this invention are characterized by including the microbial cell or microbial cell culture of this invention, or these extracts.

The cosmetic or cosmetic raw material of the present invention is characterized by containing the yeast or bacterial culture of the yeast of the present invention or an extract thereof.
The cosmetic or cosmetic raw material of the present invention comprises collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle prevention or improvement and It is preferably used for applications selected from the improvement of atopic dermatitis.
In one embodiment, the cosmetic or cosmetic raw material of the present invention is a cosmetic raw material, and preferably has an L-hydroxyproline content of 5 to 300 ppm.
The cosmetic or cosmetic raw material of the present invention is a cosmetic and preferably has an L-hydroxyproline content of 0.01 to 20 ppm. In this specification, ppm means weight ppm.

L- hydroxyproline reinforcement composition of the present invention contain L- hydroxyproline, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi ( Meyerozyma guilliermondii ) and Clavispora lusitaniae ( At least one yeast selected from the group consisting of Clavispora lusitaniae ) or a cell culture, or an extract thereof.

In the L-hydroxyproline reinforcing composition of the present invention, the yeast cell or cell culture or extract thereof contains the total content (μg of L-proline (Pro) and L-hydroxyproline (Hyp)). The ratio (100 × Hyp / (Pro + Hyp)) of L-hydroxyproline content (μg / mL) to (mL / mL) is preferably 35 to 100. Further, the yeast cells or cell cultures or extracts thereof preferably have an L-hydroxyproline content of 10 μg / mL or more.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a yeast cell or bacterial cell culture or an extract thereof and its use containing L-hydroxyproline, a method for producing L-hydroxyproline, and the like. The yeast cell or cell culture of the present invention or an extract thereof is suitably used as a raw material for foods and beverages, cosmetics and the like.

FIG. 1 is a diagram showing the measurement results of L-hydroxyproline content of culture samples by HPLC. FIG. 2 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 μmol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection).

Hereinafter, embodiments of the present invention will be specifically described. However, the present invention is not limited to the following embodiments, and can be applied with appropriate modifications without departing from the scope of the present invention.
In this specification, the genus species of yeast were described by the genus name described in The Yeasts, a Taxonomic Study Fifth Edition (issued by Elsevier, 2011).

The first cell or cell culture of yeast aspects or these extracts of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima - Girierumondi a (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie least one cell or cell culture of the yeast is selected from the group consisting of (Clavispora lusitaniae) or extracts thereof, cells of the yeast or The cell culture or these extracts contain L-hydroxyproline, and the content of L-hydroxyproline (μg / mL) relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). / ML) ratio (100 × Hyp / (Pro + Hyp)) is 35 to 00 is.

Second cells or cell cultures or extracts thereof yeast aspect of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima - Girierumondi a (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie least one cell or cell culture of the yeast is selected from the group consisting of (Clavispora lusitaniae) or extracts thereof, the content of L- hydroxyproline Is 10 μg / mL or more.
Hereinafter, the yeast cells or cell cultures of the yeast according to the first aspect and the second aspect of the present invention, or extracts thereof are collectively referred to as the yeast cells or cell cultures of the yeast of the present invention or extracts thereof. Also called.

Cells or cell cultures or extracts thereof yeast of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and a Kurabisupora-Rushitanie (at least one of the cells or cell culture of the yeast is selected from the group consisting of Clavispora lusitaniae) or extracts thereof.
The yeast in the present invention may be any yeast of any of the above genus species. Yeast may use only 1 type and may use 2 or more types.
The yeast is available from various depository institutions. Examples of depository organizations include the National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). It can also be separated from nature.
Among them, as the yeast in the present invention, Kodamaea ohmeri is preferable because of its high L-hydroxyproline content. Cells or cell cultures or extracts thereof yeast of the present invention is preferably a bacterial cell or cell culture Kodamaea-Oumeri (Kodamaea ohmeri) or extracts thereof.

The yeast or cell culture of the yeast of the present invention or an extract thereof contains L-hydroxyproline (Hyp). L-hydroxyproline in the present invention is 4-hydroxy-L-proline. In the present specification, L-hydroxyproline contained in yeast cells or cell cultures or extracts thereof refers to free L-hydroxyproline. The L-hydroxyproline content or accumulated amount in yeast cells or cell cultures or extracts thereof refers to the amount of free L-hydroxyproline. No yeast has been reported that accumulates free L-hydroxyproline in its cells or cell cultures.

The yeast or cell culture of yeast according to the first aspect of the present invention, or an extract thereof, contains L-hydroxy with respect to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio of proline content (μg / mL) (100 × Hyp / (Pro + Hyp)) is 35 to 100. Such yeast cells or cell cultures or extracts thereof are suitably used for foods and beverages and cosmetic raw materials for which L-hydroxyproline is expected to be effective. “Ratio of L-hydroxyproline content (μg / mL) to total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp)” (100 × Hyp / (Pro + Hyp)) Then, it is also referred to as “(Hyp / (Pro + Hyp)) ratio”. In the present specification, the L-proline content in the above ratio (Hyp / (Pro + Hyp)) refers to the free L-proline content contained in yeast cells or cell cultures or extracts thereof.
In a preferred embodiment, the yeast cell or cell culture of the second embodiment of the present invention or an extract thereof has a (Hyp / (Pro + Hyp)) ratio of 35 to 100.
The (Hyp / (Pro + Hyp)) ratio is preferably 40 to 100, more preferably 50 to 100, more preferably 60 to 100, still more preferably 70 to 100, and still more preferably. 80 to 100, particularly preferably 90 to 100. The above yeast (Hyp / (Pro + Hyp)) ratio of such yeast cells or cell cultures or extracts thereof have a high Hyp content ratio and are expected to be effective for L-hydroxyproline. It is particularly suitable as a raw material for the material.

The yeast cell or cell culture or the extract thereof according to the second aspect of the present invention has an L-hydroxyproline content of 10 μg / mL or more. The yeast cell or cell culture or extract thereof according to the first aspect of the present invention preferably has an L-hydroxyproline content of 10 μg / mL or more. A yeast cell or cell culture or an extract thereof having a content of L-hydroxyproline within the above range is suitable as a raw material for foods and beverages and cosmetics for which the effect of L-hydroxyproline is expected. The content of L-hydroxyproline in the yeast or cell culture of the yeast of the present invention or these extracts is more preferably 15 μg / mL or more, more preferably 17 μg / mL or more, and more preferably 20 μg / mL or more. More preferably, 30 μg / mL or more is further preferable, 40 μg / mL or more is further preferable, 50 μg / mL or more is further preferable, 100 μg / mL or more is further preferable, 150 μg / mL or more is further preferable, and 200 μg / mL or more is particularly preferable. 250 μg / mL or more is particularly preferable, and 300 μg / mL or more is particularly preferable.
The upper limit of the content of L-hydroxyproline in yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably large, but is usually 6000 μg / mL or less, 3000 μg / mL or less, or 2000 μg. / ML or less may be sufficient.
According to the present invention, it is possible to provide a yeast cell or cell culture, or an extract thereof, wherein the content of L-hydroxyproline derived from the yeast cell or cell culture is in the above range. .

The L-proline content (μg / mL) and the L-hydroxyproline content (μg / mL) in yeast cells or cell cultures or extracts thereof are measured by high performance liquid chromatography (HPLC). be able to. When yeast cells or cell cultures or extracts of these contain cells, use the one that crushes cells by heating, etc. by self-digestion or enzymatic degradation, and elutes the cell contents. Then, the contents of L-proline and L-hydroxyproline are measured. For example, a method in which a primary amino group is derivatized with mercaptopropionic acid or o-phthalaldehyde (OPA) and then a secondary amino acid is derivatized with chloroformic acid-9-fluorenylmethyl (FMOC) and analyzed by HPLC (excitation L-proline and L-hydroxyproline can be quantified by detection at a wavelength of 266 nm and a fluorescence wavelength of 305 nm. As a method for measuring L-proline and L-hydroxyproline content, HPLC measurement conditions, and the like, the methods and conditions described in the Examples may be employed.

The yeast cells or cell cultures or extracts thereof of the present invention are obtained by aerobically culturing the above yeast in a liquid medium and crushing the cells as necessary.
The yeast cell culture preferably contains yeast cells and / or culture supernatant, and may contain yeast cell contents. The yeast cells may be live or dead. As a bacterial cell culture containing yeast cells and / or culture supernatant, yeast cells (cultured cells) obtained by aerobic culture of the yeast and cell culture liquid containing the culture supernatant, the bacteria Examples include yeast cells collected from a body culture solution (bacteria) or culture supernatants obtained by removing the cells from the cell culture solution. The culture supernatant of the cell culture medium is simply referred to as the culture supernatant. The cell culture is preferably a cell culture solution or culture supernatant containing yeast cells and culture supernatant.
Moreover, the extract of a microbial cell or a microbial cell culture contains a microbial cell content normally, and it is preferable that a microbial cell content and a culture supernatant are included. As an extract of the microbial cell or the microbial cell culture, the microbial cell culture or a microbial cell culture containing the microbial cell (preferably microbial cell culture solution) is subjected to microbial cell disruption treatment such as self-digestion treatment or enzymatic degradation treatment, What eluted yeast cell contents in the culture solution (broken cell), removed the cell residue from the broken cell or cell culture (broken cell) Preferably, the microbial cell culture solution (bacterial cell crushed material) that has been subjected to the cell disruption treatment or an extract containing the cell contents and the culture supernatant obtained by removing the cell residue from the cell crushed material It is a thing.

As described above, the yeast cells or cell cultures or extracts thereof of the present invention usually contain yeast cells and culture supernatant obtained by aerobic culture of the yeast in a liquid medium. It is prepared by subjecting the bacterial cell culture solution to treatment such as collection and disruption of the bacterial cells as necessary.
The L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is preferably derived from the yeast cell or cell culture obtained by the above aerobic culture. It is preferable that the L-hydroxyproline contained in the yeast cell or cell culture of the present invention or the extract thereof is substantially absent before the aerobic culture.
The yeast cells or cell cultures or extracts thereof of the present invention can be suitably used as raw materials for foods and drinks including cosmetics and liquors, for example.

The yeast cell or cell culture of the present invention or an extract thereof has a value (μg / mL / OD660) obtained by dividing the L-hydroxyproline content (μg / mL) by OD660 (20 or more). It is preferable. A value (μg / mL / OD660) obtained by dividing the content of L-hydroxyproline (μg / mL) by OD660 (μg / mL / OD660) is hereinafter also referred to as a Hyp / OD660 value. A higher Hyp / OD660 value is preferable because the content of L-hydroxyproline per cell is larger. The upper limit of the Hyp / OD660 value of yeast cells or cell cultures or extracts thereof is not particularly limited and is preferably as large as possible, but is usually 300 or less. The Hyp / OD660 value is more preferably 25 or more, further preferably 30 or more, still more preferably 40 or more, particularly preferably 50 or more, particularly preferably 60 or more, and most preferably 80 or more.

OD is an abbreviation for optical density and refers to optical density. OD represents a cell concentration or the like. In general, absorbance OD600 or OD660 with respect to visible light having a wavelength of 600 nm or 660 nm is measured (Bio Experiment Illustrated (7) Yeast that can be used Two Hybrid, Shujunsha, published in 2003).
The OD660 used for the calculation of the Hyp / OD660 value is the cell culture solution (cells and culture supernatant) containing the cells and culture supernatant used for the preparation of the cells or cell cultures or extracts thereof. Is an absorbance at 660 nm.
More specifically, when the yeast cell culture solution obtained by aerobic culture of the above yeast in a liquid medium is used as the cell culture as it is, OD660 is the cell culture solution (cells). Absorbance OD660 of the culture). In the case of yeast cells or cell cultures or extracts thereof, the microbial cells or cell culture extract obtained by crushing yeast cells and eluting the cell contents, OD660 is: It is the light absorbency OD660 of the microbial cell culture solution (the microbial cell culture solution containing the microbial cell of the yeast before microbial cell disruption, and a culture supernatant) used for the preparation. OD660 can be measured with a spectrophotometer, for example.

The yeast cells or cell cultures or extracts thereof of the present invention preferably have an ethanol content of 1 v / v% or less. When the ethanol content is 1 v / v% or less, it can be particularly preferably used as a raw material for various foods and cosmetics. If ethanol exceeds 1 v / v%, there may be adverse effects on yeast growth and the like. The ethanol content of the yeast or the cell culture of the yeast of the present invention or the extract thereof is more preferably 0.8 v / v% or less, and further preferably 0.5 v / v% or less. The ethanol content can be measured by a known method.

The form of the yeast cells or cell cultures or extracts thereof of the present invention is not particularly limited, and examples thereof include pastes, suspensions, extracts, and liquids.
The yeast cells or cell cultures of the present invention or extracts thereof can be suitably used as a raw material for cosmetics, foods and drinks, etc. as described later. The yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be used after being powdered by drying or the like.

Cells or cell cultures or extracts thereof yeast of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and at least one yeast selected from the group consisting of Kurabisupora-Rushitanie (Clavispora lusitaniae), cultured aerobically in a liquid medium comprising carbon and nitrogen sources, be disrupted cell or the like, if necessary Can be obtained. More specifically, the nitrogen source includes an L-hydroxyproline-containing peptide.
By aerobically culturing the yeast in a liquid medium containing a nitrogen source containing a carbon source and an L-hydroxyproline-containing peptide, L-hydroxyproline accumulates in the yeast or the cell culture of the yeast, A yeast cell or cell culture containing L-hydroxyproline is obtained. The yeast is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, thereby accumulating L-hydroxyproline in the yeast or the culture of the yeast. The method including the steps is preferable as a method for producing the above-described yeast cell or cell culture of the present invention or an extract thereof, or a method for producing L-hydroxyproline.

The method of producing L- hydroxyproline invention Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie ( At least one yeast selected from the group consisting of Clavispora lusitaniae ) in an aerobic culture in a liquid medium containing a carbon source and a nitrogen source, so that L-hydroxy is contained in the yeast cell or cell culture. A step of accumulating proline (hereinafter also referred to as a Hyp accumulation step). The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide.
The production method of the present invention may have steps other than the Hyp accumulation step as desired. For example, you may have 1 or 2 or more processes, such as the preculture process mentioned later, a microbe collection process, and a microbial cell crushing process.

In the method for producing L-hydroxyproline of the present invention, the above-described yeast cell or cell culture of the present invention or an extract thereof can be obtained.
By performing the Hyp accumulation step, a yeast cell or cell culture containing L-hydroxyproline is obtained. The obtained yeast cells or cell cultures usually have the above (Hyp / (Pro + Hyp)) ratio of 35 to 100. In addition, the yeast cell or cell culture obtained by the Hyp accumulation step usually has an L-hydroxyproline content of 10 μg / mL or more. Such yeast cells or cell cultures can be used as the yeast cells or cell cultures of the present invention described above. Further, the obtained bacterial cells or bacterial cell culture is further subjected to treatment such as cell disruption as required to prepare a yeast bacterial cell or bacterial cell culture extract containing L-hydroxyproline. You can also
The method for producing L-hydroxyproline including the Hyp accumulation step is also preferable as a method for producing the above-described yeast cells or cell cultures of the present invention or extracts thereof. When obtaining the yeast cells or cell cultures or extracts thereof of the present invention, the Hyp accumulation step and preferred embodiments thereof are the same as the Hyp accumulation step and preferred embodiments thereof in the method for producing L-hydroxyproline.

In the Hyp accumulation step, yeast can be added to the liquid medium by inoculating a small amount of cells directly in the liquid medium containing the carbon source and nitrogen source. In order to raise the amount, it is preferable to inoculate a pre-cultured bacterial solution. The medium used for the pre-culture is not particularly limited, and may be the same medium as the liquid medium used in the Hyp accumulation step (usually main culture), or a known medium that can be used for yeast. The preculture time is usually 10 to 72 hours, preferably 12 to 48 hours. The preculture temperature is preferably 15 to 40 ° C. The amount inoculated with the pre-cultured bacterial solution is usually 1/10000 to 1/2 of the amount of medium used in the Hyp accumulation step, preferably 1/1000 to 1/10, and preferably 1/200 to 1/10. Is more preferable, and 1/200 to 1/20 is even more preferable. When the inoculation amount is in the above range, the yeast grows rapidly in the Hyp accumulation step, and L-hydroxyproline can be accumulated efficiently.

The nitrogen source of the liquid medium used in the Hyp accumulation step is a nitrogen source containing an L-hydroxyproline-containing peptide. When the yeast is aerobically cultured using such a nitrogen source, L-hydroxyproline accumulates in the cells or the cell culture. Such nitrogen sources may be used alone or in combination of two or more. The number of L-hydroxyproline-containing peptides may be one, or two or more.
The L-hydroxyproline-containing peptide may be any peptide that contains L-hydroxyproline as a constituent amino acid, but is preferably a peptide in which 10% by weight or more of the constituent amino acid is L-hydroxyproline. In one embodiment, the nitrogen source containing the L-hydroxyproline-containing peptide is also preferably an L-hydroxyproline-containing peptide.

A nitrogen source containing an L-hydroxyproline-containing peptide can be obtained, for example, by hydrolyzing an L-hydroxyproline-containing protein. The L-hydroxyproline-containing protein may be a protein containing L-hydroxyproline as a constituent amino acid, but is preferably a protein in which 10% by weight or more of the constituent amino acid is L-hydroxyproline. As the L-hydroxyproline-containing protein, collagenous protein and the like are preferable. Examples of the collagenous protein include proteins prepared from tissues containing collagen such as viscera, skin, fish scales, and bones; collagen and gelatin. The origin of the collagen protein is not particularly limited. For example, collagen-derived proteins derived from animals such as cow-derived, pig-derived and fish-derived can be preferably used. As the collagenous protein, a commercially available product can be used. Hydrolysis of the L-hydroxyproline-containing protein can be performed by a known method using an enzyme or the like.

As a nitrogen source containing an L-hydroxyproline-containing peptide, for example, animal-derived peptone can be preferably used. Peptone derived from cow, pig or fish is preferred, and peptone derived from cow or fish is more preferred. In one embodiment of the present invention, animal peptone, myocardial peptone, and gelatin peptone are also preferable as peptone.
An example of a commercial product of a nitrogen source containing an L-hydroxyproline-containing peptide that can be used in the present invention is the product name Pepton (# 211677) (Bacto).

In one embodiment, the L-hydroxyproline-containing peptide is preferably a collagen peptide. Collagen peptide means hydrolyzed collagen, which can be either gelatin modified by heat treatment of natural collagen, collagen peptide hydrolyzed from natural collagen, or those chemically or enzymatically modified. Good. Hydrolysis can be performed with an enzyme, acid, alkali, or the like, and preferably with an enzyme. Preferably, gelatin modified by heat treatment of natural collagen or collagen peptide hydrolyzed from natural collagen is used. The origin of the collagen peptide is not particularly limited. For example, animal-derived collagen peptides such as cow-derived, pig-derived and fish-derived can be preferably used. Preferably, it is a collagen peptide derived from fish. A commercially available collagen peptide can be used.

Examples of commercially available collagen peptides that can be used in the present invention include, for example, “Collagen Peptide Iquos HDL-50SP” (product name) (average molecular weight 5000), “Collagen Peptide Type S” (product) manufactured by Nitta Gelatin Co., Ltd. Name) (average molecular weight 1200), “super collagen peptide SCP-2000” (product name) (average molecular weight 2000), “collagen peptide P-5000” (product name) (average molecular weight 5000) manufactured by Yasu Chemical Co., Ltd. "Collagen Peptide F-5000" (product name) (average molecular weight 5000), "Marine Collagen Oligo CF" (product name) (average molecular weight 900-1100) manufactured by Nissho Co., Ltd., "Marine Collagen Oligo MF" ( Product name) (average molecular weight 900-1500) and the like. Among these, “collagen peptide Type S” (average molecular weight 1200), “collagen peptide Iquos HDL-50SP” (average molecular weight 5000) and the like are preferable.
Among these, for example, “collagen peptide Iquos HDL-50SP”, “collagen peptide Type S”, “collagen peptide F-5000”, “marine collagen CF”, “marine collagen oligo MF” are derived from fish. “Collagen Peptide P-5000” and “Super Collagen Peptide SCP-2000” are derived from pigs.

In a preferred embodiment of the invention, the nitrogen source comprising the L-hydroxyproline-containing peptide is preferably a collagen peptide (more preferably a collagen peptide derived from fish) and / or a peptone (preferably an animal, more preferably a cow, Pigs or fish-derived, more preferably cattle or fish-derived peptone), particularly preferably collagen peptides. When a nitrogen source containing such an L-hydroxyproline-containing peptide is used, the amount of L-hydroxyproline accumulated in yeast cells or cell cultures increases. The peptone may be animal meat peptone, heart muscle peptone, gelatin peptone.
An example of a preferred embodiment of the method for producing L-hydroxyproline of the present invention is that the yeast is aerobically cultured in a liquid medium containing a carbon source and a collagen peptide and / or peptone. A step of accumulating L-hydroxyproline in the bacterial cell or the bacterial cell culture.

In one embodiment, the nitrogen source containing the L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example. The L-hydroxyproline-containing peptide preferably has an average molecular weight of 10,000 or less, and preferably has an average molecular weight of 100 to 10,000, for example. In addition, the L-hydroxyproline-containing peptide preferably has a molecular weight of 10,000 or less.
The collagen peptide preferably has an average molecular weight of 1000 to 10,000. When a collagen peptide having an average molecular weight in the above range is used as a nitrogen source, the amount of L-hydroxyproline accumulated in the bacterial cells or bacterial cell culture increases.
The average molecular weight of the L-hydroxyproline-containing peptide is calculated by gel filtration or the like. The average molecular weight of the collagen peptide is usually a value calculated by the method described in “20-2 Average Molecular Weight” of the 10th edition of Photographic Gelatin Test Method (PAGI Method). The average molecular weight of a peptide refers to a weight average molecular weight.

The average molecular weight of the collagen peptide is more preferably 1000 to 6000, still more preferably 1000 to 5500, and particularly preferably 1000 to 5000. When such a collagen peptide is used as a nitrogen source, the amount of L-hydroxyproline accumulated in the microbial cells or microbial cell culture is increased. In one embodiment, the average molecular weight of the collagen peptide is more preferably 1000 to 3000, and even more preferably 1000 to 1500. In another preferred embodiment of the present invention, the average molecular weight of the collagen peptide is more preferably 2000 to 5500, further preferably 3000 to 5000.

The concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is usually preferably 0.1 to 10% by weight, more preferably 0.25 to 5% by weight, and 1 to 5% by weight. Further preferred. When the concentration of the nitrogen source is within the above range, L-hydroxyproline accumulates in the bacterial cells or bacterial cell culture. Therefore, for example, a yeast cell or cell culture or an extract thereof having an L-hydroxyproline content of 10 μg / mL or more can be obtained. In addition, cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 can be obtained. The concentration of the nitrogen source in the liquid medium is still more preferably 1.5 to 4.5% by weight, particularly preferably 2 to 4% by weight. The concentration of the nitrogen source may be the above concentration at the start of culture. In one embodiment, the concentration of the collagen peptide or peptone in the liquid medium is preferably in the above range.

The carbon source is not particularly limited. Sugars or sugar alcohols; organic acids such as acetic acid, citric acid, or gluconic acid. A carbon source may be used individually by 1 type, and may mix and use 2 or more types. Among them, the carbon source is preferably a sugar such as glucose, fructose, or sucrose, and glucose is particularly preferable.

The concentration of the carbon source in the liquid medium is preferably 0.1 to 20% by weight, preferably 0.5 to 15% by weight, more preferably 1 to 10% by weight, more preferably 1 to 5% by weight, more preferably 2-5% by weight. It is preferable that the concentration of the carbon source in the liquid medium is 1% by weight or more because the growth rate of the bacterial cells is high. In addition, the density | concentration of a carbon source should just be the said density | concentration at the time of a culture | cultivation start.

In the production method of the present invention, the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20. A weight ratio of C / N within the above range is preferable because the amount of L-hydroxyproline accumulated in the bacterial cells or bacterial cell culture increases. The C / N weight ratio is more preferably 0.25 to 5, more preferably 0.3 to 3, and further preferably 0.4 to 1.5. When the weight ratio of C / N is within the above range, the amount of L-hydroxyproline accumulated in the microbial cells or microbial cell culture is increased. In one embodiment, for example, when a collagen peptide (preferably having an average molecular weight of 1000 to 5000) or peptone is used as the nitrogen source (N) containing the L-hydroxyproline-containing peptide, the C / N weight ratio is 0. 25 to 5 is more preferable, 0.3 to 3 is more preferable, 0.4 to 1.5 is still more preferable, and 0.5 to 1.3 is particularly preferable. In another preferred embodiment, the C / N weight ratio is preferably 0.5 to 20. The weight ratio of C / N may be in the above range at the start of culture.

The liquid medium may contain components other than the above-described carbon source and a nitrogen source containing an L-hydroxyproline-containing peptide, and preferably contains, for example, a yeast extract. Yeast extracts usually do not contain L-hydroxyproline-containing peptides and are not included in nitrogen sources that contain L-hydroxyproline-containing peptides. The yeast extract is not particularly limited as long as it can be used for yeast culture, and a commercially available product can be used. For example, the product name Bacto yeast Extract (# 212750) (Bacto) can be preferably used.
When using a yeast extract, the concentration of the yeast extract is preferably 0.1 to 3% by weight, more preferably 0.5 to 3% by weight, based on the liquid medium. The concentration of the yeast extract may be the above concentration at the start of culture.

In one embodiment, the pH of the liquid medium is preferably 3 to 9, preferably 4 to 9, more preferably more than 4 and 9 or less, further preferably 4.5 to 8.8, and particularly preferably 5 to 8.7. preferable. The pH of the liquid medium can be adjusted as appropriate. A known acid or alkali agent can be used for pH adjustment, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, glutamic acid, acetic acid, butyric acid, lactic acid, formic acid, succinic acid, maleic acid, malic acid, oxalic acid, citric acid, Examples thereof include sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, and sodium glutamate.

The culture temperature is preferably 15 to 45 ° C, more preferably 20 to 40 ° C, and further preferably 25 to 35 ° C. When the culture temperature is within this temperature range, the yeast grows rapidly and the amount of L-hydroxyproline accumulated in the cells or cell cultures increases.

The method for aerobic culture is not particularly limited, and the liquid medium inoculated with the bacteria may be, for example, shake culture or stirring culture. The speed of shaking or stirring is not particularly limited, but is preferably 30 to 600 rpm, and in one aspect, more preferably 30 to 500 rpm, still more preferably 50 to 500 rpm, and particularly preferably 50 to 300 rpm. Particularly preferred is 50 to 100 rpm. In another preferred embodiment, the shaking or stirring speed is preferably 50 to 600 rpm, more preferably 100 to 600 rpm. Shaking culture or stirring culture at such a rate is preferable because the amount of accumulated L-hydroxyproline increases. More preferably, the shaking culture is performed at the above speed. In addition, bubbling may be performed with sterilized air or oxygen if desired. The culture format may be batch culture, fed-batch culture, or continuous culture, but batch culture is preferred. In the production method of the present invention, static culture may be performed.

The culture time is not particularly limited and may be set as appropriate. For example, it is preferable to perform aerobic culture for 10 to 100 hours. When the culture time is within the above range, L-hydroxyproline accumulates in the yeast cells or cell culture. In addition, usually, yeast cells or cell cultures or extracts thereof having a (Hyp / (Pro + Hyp)) ratio of 35 to 100, yeast cells having an L-hydroxyproline content of 10 μg / mL or more. Alternatively, a bacterial cell culture or an extract thereof can be obtained. In addition, yeast cells or cell cultures or extracts thereof having a low ethanol content (for example, 1 v / v% or less) can be obtained. When the culture time is less than 10 hours, the amount of accumulated L-hydroxyproline is small, or the yeast cell or cell culture obtained or the extract (Hyp / (Pro + Hyp)) ratio is less than 35. It may become. When the culture time exceeds 100 hours, the ethanol concentration of the obtained yeast or bacterial cell culture or the extract thereof may exceed 1 v / v%. In addition, contamination may easily occur, and coloring due to self-digestion may occur after the death of the yeast. The culture time is more preferably 10 to 80 hours, more preferably 12 to 72 hours, still more preferably 20 to 60 hours, still more preferably 24 to 55 hours, and particularly preferably 24 to 60 hours. 50 hours.
In the production method of the present invention, it is preferable to perform aerobic culture until the L-hydroxyproline content in the microbial cells or the microbial cell culture becomes 10 μg / mL or more.
In the production method of the present invention, aerobic culture is usually performed as main culture, but it may be preculture or aerobic culture may be performed in preculture and main culture.

By carrying out the above aerobic culture, L-hydroxyproline accumulates in the yeast cells or cell cultures. The cell culture may be a cell culture solution containing yeast cells and culture supernatant, may be yeast cells, or may be a culture supernatant of a cell culture solution. Good. By performing the Hyp accumulation step, yeast cells or cell cultures containing L-hydroxyproline and having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 can be obtained. In addition, yeast cells or cell cultures having an L-hydroxyproline content of 10 μg / mL or more can be obtained. An extract of yeast cells or cell cultures can be obtained by subjecting yeast cells or cell cultures to a cell disruption treatment, for example.

In the present invention, when preparing a yeast cell or cell culture or an extract thereof containing L-hydroxyproline in the present invention, for example, a cell culture comprising a yeast cell obtained in the Hyp accumulation step and a culture supernatant. The solution can be used as it is as a yeast cell culture containing L-hydroxyproline. Alternatively, yeast cells may be collected from the cell culture medium, and the obtained cell bodies may be used as yeast cells or cell cultures. It can also be a body culture. Further, the cells or the cell culture medium is subjected to a treatment for crushing the cells as necessary, and the cell contents are eluted in the culture solution to prepare a cell or cell culture extract. You can also In the preparation of an extract of bacterial cells or bacterial cell cultures, a step of removing bacterial cell residues may be performed after disrupting the bacterial cells. Moreover, you may perform processes, such as disinfection and a heating, to a microbial cell or a microbial cell culture, or these extracts as needed. The production method of the present invention may include one or more steps such as such a collection process, a microbial cell disruption process, a microbial cell residue removal process, and a sterilization process.

A method for collecting the cells from the cell culture solution is not particularly limited, and a commonly used method can be employed, and examples thereof include centrifugation.
The method for disrupting the cells is not particularly limited, and a commonly used method can be employed, and examples thereof include an autolysis method, an enzymatic decomposition method, and an alkali extraction method. Of these, the autolysis method is preferred. In the self-digestion method, for example, the cells or the cell culture may be heated at 40 to 60 ° C. for 60 to 180 minutes. In the autolysis method, for example, heating may be performed at 95 to 100 ° C. for 5 to 15 minutes.
The method for removing the cell residue is not particularly limited. For example, what is necessary is just to remove a microbial cell residue by well-known methods, such as filtration and centrifugation.
In the case of sterilization, it is preferable to heat yeast cells or cell cultures or extracts thereof at 75 to 90 ° C. (more preferably 80 ° C.) and 45 to 90 minutes (more preferably 60 minutes). . When removing the microbial cell residue and sterilizing, either may be performed first.

The yeast cells or cell cultures or extracts thereof obtained in the present invention, and preferred embodiments thereof are the yeast cells or cell cultures or extracts thereof of the present invention described above, and the extracts thereof. This is the same as the preferred embodiment. According to the production method of the present invention, for example, a yeast or a cell culture of yeast having a (Hyp / (Pro + Hyp)) ratio of 35 to 100 and an L-hydroxyproline content of 10 μg / mL or These extracts can be produced.
L-hydroxyproline derived or synthesized from natural products may be further added to yeast cells or cell cultures or extracts thereof obtained by the above method. In a preferred embodiment, yeast cells are used. Alternatively, the L-hydroxyproline contained in the cell culture or the extract thereof is composed of L-hydroxyproline derived from the yeast cell or cell culture obtained by the above Hyp accumulation step.

The yeast cells or cell cultures or extracts thereof containing L-hydroxyproline obtained in the present invention can be used as raw materials for foods and beverages, cosmetics and the like described later. Further, in the method for producing L-hydroxyproline of the present invention, a step of purifying L-hydroxyproline from the obtained yeast cell or cell culture or an extract thereof may be performed. Purification of L-hydroxyproline may be performed by a known method such as column chromatography.

The yeast is also suitably used for producing a yeast cell or cell culture or an extract thereof containing L-hydroxyproline. The yeast cells or cell cultures or extracts thereof containing L-hydroxyproline preferably have a (Hyp / (Pro + Hyp)) ratio of 35 to 100. It is also preferable that the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline have an L-hydroxyproline content of 10 μg / mL or more. In addition, it is preferable that the above-mentioned Hyp / OD660 value is 20 or more for the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline. Preferred embodiments of the yeast cells or cell cultures or extracts thereof containing L-hydroxyproline are the same as the preferred embodiments of the yeast cells or cell cultures or extracts thereof of the present invention described above. It is.

The use of the present invention includes accumulating L-hydroxyproline in the yeast cells or cell cultures by aerobic culture of the yeast in a liquid medium containing a carbon source and a nitrogen source. It is preferable. The nitrogen source is preferably a nitrogen source containing an L-hydroxyproline-containing peptide.

In the use of the present invention, the L-hydroxyproline-containing peptide is preferably a collagen peptide. The average molecular weight of the collagen peptide is preferably 1000 to 10,000.

In the use of the present invention, the concentration of the nitrogen source containing the L-hydroxyproline-containing peptide in the liquid medium is preferably 1 to 5% by weight. The weight ratio (C / N) of the carbon source (C) and the nitrogen source (N) containing the L-hydroxyproline-containing peptide is preferably 0.25 to 20. In one embodiment, the C / N ratio is preferably 0.5 to 20. In the use of the present invention, the aerobic culture is preferably performed for 10 to 100 hours, more preferably for 10 to 80 hours.

In the use of the present invention, the liquid medium, the carbon source and the nitrogen source containing the L-hydroxyproline-containing peptide and preferred embodiments thereof are the same as those in the above-described method for producing L-hydroxyproline. The aerobic culture conditions and preferred embodiments thereof are also the same as those in the above-described method for producing L-hydroxyproline. The use of the present invention may include one or more processes such as the above-described collection process, microbial cell disruption process, microbial cell removal process, and sterilization process.

The yeast cell or cell culture of the present invention described above or an extract thereof can be blended in various compositions such as cosmetics, foods and drinks, and pharmaceuticals. The yeast cell or cell culture of the present invention or a composition containing an extract thereof is also encompassed in the present invention.
The composition of the present invention may include any one of the yeast cell or cell culture of the yeast of the first aspect and the second aspect of the present invention described above, or an extract thereof, and may include both. The yeast cell or cell culture of the present invention or a composition containing these extracts contains L-hydroxyproline derived from the yeast cell or cell culture or these extracts. Examples of the composition of the present invention include cosmetics (cosmetic compositions), foods and drinks (food and beverage compositions), pharmaceuticals (pharmaceutical compositions), quasi drugs (quasi drugs) and the like. . The composition may be these raw materials. In one embodiment, the composition is preferably a cosmetic or a food or drink, or a raw material thereof.

The content of the yeast cell culture or cell culture or the extract thereof in the composition of the present invention is not particularly limited, and can be appropriately set according to the type and use of the composition. For example, the solid content of the yeast cell or cell culture or extract thereof is preferably 0.00001 to 50% by weight, preferably 0.00005 to 20% by weight, based on the composition. % Is more preferable, and 0.0001 to 10% by weight is more preferable.

When the composition of the present invention is a cosmetic or a pharmaceutical, the dosage form is not particularly limited, and any dosage form such as a solution, paste, gel, solid, or powder can be used.
Cosmetics are not particularly limited. , Perfume, powder, eau de cologne, body soap, soap, bath salt, sunscreen cream and the like.

The cosmetics or cosmetic raw materials containing the above-described yeast cells or cell cultures of the present invention or extracts thereof are one of the preferred embodiments of the present invention. The cosmetics and cosmetic raw materials may contain components other than the yeast cells or cell cultures or extracts thereof. Various ingredients that are usually blended in cosmetics can be blended in cosmetics and cosmetic raw materials. For example, oils, fragrances, surfactants, humectants, antioxidants, ultraviolet absorbers, preservatives, pigments, dyes and the like can be appropriately blended. What is necessary is just to select these compounding ratios suitably. The cosmetic raw material of the present invention is suitably used for producing the cosmetic of the present invention.
The usage and dosage of the cosmetic can be appropriately determined according to the type of cosmetic.
Since the cosmetic or cosmetic raw material of the present invention contains L-hydroxyproline, for example, promotion of collagen production, promotion of epidermal cell proliferation, skin moisturization, prevention of skin aging, prevention or improvement of skin sagging, It is preferably used for applications selected from the improvement of skin firmness, prevention or improvement of wrinkles and the improvement of atopic dermatitis, and is preferably used for applications selected from improvement of skin firmness and prevention or improvement of wrinkles. .

The content of the yeast cells or cell culture or the extract thereof in the cosmetic is preferably 0.00001 to 10% by weight, preferably 0.0001 to 10% by weight in terms of solid content with respect to the cosmetic. It is preferably 0.0001 to 5% by weight, more preferably 0.001 to 5% by weight, still more preferably 0.01 to 3% by weight, and particularly preferably 0.05 to 2% by weight. In another preferred embodiment, the content of the yeast cell culture or cell culture or extract thereof in the cosmetic is 0.00005 to 1% by weight in terms of solid content relative to the cosmetic. Preferably, 0.0001 to 0.5% by weight is more preferable.
The content of the yeast cell or cell culture or extract thereof in the cosmetic raw material is preferably 0.001 to 20% by weight, for example, in terms of solid content, based on the cosmetic raw material, Is more preferably from 10 to 10% by weight, further preferably from 0.05 to 5% by weight, particularly preferably from 0.1 to 2% by weight. The L-hydroxyproline content in the cosmetic raw material is, for example, preferably from 5 to 300 ppm, more preferably from 10 to 200 ppm, still more preferably from 50 to 100 ppm. In one embodiment, the L-hydroxyproline content in the cosmetic can be, for example, 0.01 to 20 ppm, preferably 0.03 to 15 ppm, and more preferably 0.05 to 10 ppm. It is preferable to blend the yeast cells or cell cultures or extracts thereof so that the L-hydroxyproline content falls within the above range.

When the composition of the present invention is a food or drink (food or drink composition), the food or drink is not particularly limited. The form of the food or drink may be any of liquid, semi-liquid or solid, and paste, and may be any of general food and drink, health food, functional food, and the like.
General food and drink is not particularly limited, and includes alcoholic beverages. Healthy food means food that is considered healthy or healthy, and includes nutritional supplements, natural foods, and the like. Nutritional supplements refer to foods that are enriched with specific nutritional components. Functional foods refer to foods for supplementing nutritional components that fulfill the body's regulatory functions, and include foods for specified health use and functional nutritional foods.

Examples of dietary supplements include beauty drinks and supplements. The food and drink of the present invention may be in the form of a pharmaceutical preparation such as a capsule, a drink or the like.
Various components that are permitted to be blended in food and drink can be blended in the food and drink. Examples of such components include binders, thickeners, colorants, stabilizers, emulsifiers, dispersants, disintegrants, suspending agents, surfactants, preservatives, sweeteners, and sour agents. .
The above-described yeast cell or cell culture of the present invention or a food or drink containing these extracts is one of the preferred embodiments of the present invention.

The content of the yeast cells or cell culture or the extract thereof in the food or drink is preferably 0.0001 to 10% by weight in terms of solid content with respect to the food or drink. 001 to 5% by weight is more preferable, and 0.01 to 1% by weight is more preferable. Further, the L-hydroxyproline content in the food and drink is preferably 0.0001 to 0.01% by weight, and the yeast cell or cell culture or the culture of the yeast so that the L-hydroxyproline content falls within the above range. It is preferable to blend these extracts.
The L-hydroxyproline content in the cosmetics, cosmetic raw materials, foods and beverages, etc. is a free L-hydroxyproline content. L-hydroxyproline is preferably derived from the above-mentioned yeast cells or cell cultures or extracts thereof.

Compositions such as cosmetics, foods and drinks, and these raw materials containing yeast cells or bacterial cell cultures or extracts thereof according to the present invention are the types of raw materials, additives, etc. that are usually used in these. The yeast cells or cell cultures of the yeast of the present invention or extracts thereof can be blended and produced by a known technique.

The present invention is selected from the group consisting of Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) Also included are cosmetics or cosmetic ingredients containing at least one yeast cell or cell culture or extract thereof.
The yeast cell or cell culture or the extract thereof is preferably the yeast cell or cell culture of the present invention described above or an extract thereof.

The present invention is selected from the group consisting of Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) The aerobic culture of at least one yeast produced in a liquid medium containing a carbon source and a nitrogen source, thereby accumulating L-hydroxyproline in the yeast cells or cell culture (Hyp accumulation) And a process for producing an L-hydroxyproline-containing cosmetic raw material. The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide. The preferred embodiment of the method for producing an L-hydroxyproline-containing cosmetic raw material of the present invention is the same as the preferred embodiment of the method for producing L-hydroxyproline described above. The method for producing the L-hydroxyproline-containing cosmetic raw material is preferable as the method for producing the cosmetic raw material of the present invention.
By performing the Hyp accumulation step, a yeast cell or cell culture containing L-hydroxyproline is obtained. In addition, by performing the above-described cell disruption treatment on yeast cells or cell cultures, an extract of yeast cells or cell cultures containing L-hydroxyproline can be obtained. Thus obtained yeast cells or cell cultures or extracts thereof, and preferred embodiments thereof are the yeast cells or cell cultures of the present invention described above or extracts thereof, and The preferred embodiment is the same. L-hydroxyproline-containing cosmetic raw material containing L-hydroxyproline-containing yeast cells or cell cultures or extracts thereof, if desired, containing additives or the like commonly used in cosmetics Can be used as In addition, L-hydroxyproline purified from yeast cells or cell cultures or extracts thereof containing L-hydroxyproline may be blended with additives ordinarily used in cosmetics, if desired. A hydroxyproline-containing cosmetic raw material can also be produced. The cosmetic raw material containing L-hydroxyproline obtained by the present invention has the following effects: collagen production promotion, epidermal cell growth promotion, skin moisturization, skin aging prevention, skin sagging prevention or improvement, skin firmness improvement, wrinkle It is suitably used for cosmetics for uses selected from prevention or improvement and improvement of atopic dermatitis.

The present invention, L- hydroxyproline containing, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie ( A composition for reinforcing L-hydroxyproline comprising at least one yeast cell culture or cell culture selected from the group consisting of Clavispora lusitaniae ) or an extract thereof is also included.
The yeast cell or cell culture or extract thereof contains L-hydroxyproline, but the L relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The ratio (100 × Hyp / (Pro + Hyp)) of the hydroxyproline content (μg / mL) is preferably 35-100. The yeast cells or cell cultures or extracts thereof described above preferably have an L-hydroxyproline content of 10 μg / mL or more. A composition for reinforcing L-hydroxyproline containing such yeast cells or cell cultures or extracts thereof is an additive for reinforcing, supplementing or strengthening L-hydroxyproline in cosmetics, foods and drinks, etc. Can be used particularly preferably. Preferred embodiments of yeast cells or cell cultures or extracts thereof are the same as the yeast cells or cell cultures or extracts thereof of the present invention described above and preferred embodiments thereof. The L-hydroxyproline reinforcing composition can be suitably used as an additive composition for reinforcing L-hydroxyproline in foods and drinks, cosmetics and the like. The composition for reinforcing L-hydroxyproline can also be referred to as a composition for supplementing L-hydroxyproline or a composition for reinforcing L-hydroxyproline.

The L-hydroxyproline reinforcing composition of the present invention may contain the above-mentioned yeast cell or cell culture or an extract thereof containing L-hydroxyproline, and the cell or cell culture Alternatively, the content of these extracts may be 100% by weight, but may contain other components as desired. For example, when the L-hydroxyproline reinforcing composition is used as a food additive, it may contain one or more known additives used in foods.

The L-hydroxyproline reinforcing composition of the present invention is also suitably used as a cosmetic additive, for example. When the L-hydroxyproline reinforcing composition is used as a cosmetic additive, the composition may contain the yeast cells or cell cultures or extracts thereof as described above. The content of the bacterial cell culture or the extract thereof may be 100% by weight, but may contain one or more known additives used in cosmetics as desired.

Method for producing L- hydroxyproline reinforcement composition of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and By aerobically culturing at least one yeast selected from the group consisting of Clavispora lusitaniae in a liquid medium containing a carbon source and a nitrogen source, It is preferable to include a step of accumulating L-hydroxyproline. A method for producing an L-hydroxyproline reinforcing composition including such steps is also encompassed by the present invention. The nitrogen source is a nitrogen source containing an L-hydroxyproline-containing peptide. The production method of L-hydroxyproline reinforcing composition of the present invention and preferred embodiments thereof are the same as the above-described production method of L-hydroxyproline and preferred embodiments thereof. The method for producing an L-hydroxyproline reinforcing composition of the present invention comprises a step of adding a known food additive, cosmetic additive, etc. to yeast cells or cell cultures or extracts thereof, if desired. May be included.

Another manufacturing method of embodiments of the L- hydroxyproline invention Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and By aerobically culturing at least one yeast selected from the group consisting of Clavispora lusitaniae in a liquid medium containing a carbon source and a nitrogen source, The step of accumulating L-hydroxyproline.
In the production method of another aspect of the present invention, the nitrogen source preferably contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide. In the production method of another aspect of the present invention, the L-hydroxyproline-containing protein is preferably a collagenous protein, and the L-hydroxyproline-containing peptide is preferably a collagen peptide. In the production method of another aspect of the present invention, the average molecular weight of the collagenous protein and collagen peptide is preferably 1,000 to 100,000. In the production method of another aspect of the present invention, the concentration of the nitrogen source in the liquid medium is 1 to 5% by weight, and the weight ratio (C / N) of the carbon source (C) and the nitrogen source (N). Is preferably 0.5 to 20. The aerobic culture is preferably performed for 10 to 100 hours, more preferably 10 to 80 hours.

Use of another aspect of the present invention, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae The use of at least one yeast selected from the group consisting of) for producing L-hydroxyproline, wherein the yeast is aerobically cultured in a liquid medium containing a carbon source and a nitrogen source. It is preferable that L-hydroxyproline is accumulated in the yeast cells or cell culture, and the nitrogen source contains an L-hydroxyproline-containing protein or an L-hydroxyproline-containing peptide.
In the use of another aspect of the present invention, the L-hydroxyproline-containing protein is preferably a collagenous protein, and the L-hydroxyproline-containing peptide is preferably a collagen peptide. In the use of another aspect of the present invention, the collagen protein and collagen peptide preferably have an average molecular weight of 1,000 to 100,000. In another embodiment of the present invention, the concentration of the nitrogen source in the liquid medium is 1 to 5% by weight, and the weight ratio (C / N) of the carbon source (C) to the nitrogen source (N) is It is preferably 0.5 to 20. The aerobic culture is preferably performed for 10 to 100 hours, more preferably 10 to 80 hours.

Examples of the L-hydroxyproline-containing protein include the aforementioned collagenous protein. The average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is preferably more than 10,000 and not more than 100,000. The average molecular weight of protein refers to the weight average molecular weight. The average molecular weight of the L-hydroxyproline-containing protein such as collagenous protein is calculated by gel filtration or the like.

Hereinafter, test examples for more specifically explaining the present invention will be shown. The present invention is not limited only to these test examples.

In the test examples, L-4-hydroxyproline manufactured by Nacalai Tesque Co., Ltd. was used to prepare an L-hydroxyproline (Hyp) standard solution used for preparing a calibration curve. For the preparation of L-proline (Pro) standard solution, L-proline manufactured by Nacalai Tesque Co., Ltd. was used. L-hydroxyproline and L-proline measured in the test examples are both free L-hydroxyproline and L-proline.

<Test Example 1>
Screening for Bacteria Accumulating L-Hydroxyproline (hereinafter Hyp) In order to screen for genus species that accumulate Hyp in the cell culture medium, the 1st to 3rd selection tests were conducted under the following selection conditions.
Primary selection test: Selection condition: Ethanol production, growth rate Secondary selection test: Selection condition: Hyp accumulation amount (colorimetric method)
Third selection test: Selection conditions: Hyp accumulation (HPLC)
Reproducibility: Hyp accumulation (HPLC)

Strains with different genus species shown in Tables 1 to 3 were used for the test.

In the first to third selection tests, preculture and main culture were performed under the following conditions. The YPD liquid medium used in the preculture and the main culture has a Y: P: D ratio of 1: 2: 2 (weight ratio) (Y: yeast extract (product name Yeast Extract (# 212750) manufactured by Bacto)). 0% by weight, P: peptone (product name Pepton (# 211677) manufactured by Bacto, which is obtained by digesting bovine cells with an enzyme derived from porcine pancreas) 2.0% by weight, D: 2.0% by weight glucose) It is.

(Pre-culture)
One platinum loop of each strain was inoculated into 3 mL of YPD liquid medium, and statically cultured at 30 ° C. for 1 day to obtain a preculture solution.

(Main culture)
100 μL of the preculture solution was inoculated into 5 mL of YPD liquid medium, and shake culture (60 rpm) was performed at 30 ° C. for 1 day to obtain a yeast cell culture solution (cell culture).

The culture sample used for the measurement of the Hyp content in the secondary selection test and the tertiary selection test was prepared by performing the following self-digestion process and sterilization process on the yeast cell culture solution obtained in the main culture.
(Self digestion process)
The yeast cell culture solution was incubated at 50 ° C. for 2 hours, and the cell contents were eluted into the culture solution by autolysis.
(Sterilization process)
The culture solution self-digested above was incubated at 80 ° C. for 1 hour.
The cell residue was removed from the obtained extract by centrifugation (3000 rpm, 5 min, 1 ° C.). This prepared the culture sample for measuring the amount of Hyp accumulation.

1.1 Primary selection test (1) Amount of ethanol produced For the yeast strains 116 shown in Tables 1 to 3, the ethanol concentration in the supernatant of the cell culture broth obtained by main culture (30 ° C, 1 day, shaking culture) Was measured. Biosensor BF5 (Oji Scientific Instruments) was used for ethanol measurement.

Tables 1 to 3 show the ethanol concentration of the culture supernatant of the yeast used in the test.
In all yeasts (116 strains) used in the test, the ethanol concentration of the cell culture supernatant was 1 v / v% or less.

(2) Growth rate Next, the growth curve of the yeast was measured. For the measurement, the absorbance (660 nm) of the cell culture broth was measured using TVS 062CA (ADVANTEC).
Among these, 100 strains (strains other than 16 strains described below) having a high growth rate were used for the secondary selection test and the tertiary selection test.
(Strains that were not used in the second and third rounds)
Zygosaccharomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces siamensis , Candida etchellsii, Starmerella bombicola, Starmerella sp, Candida holmii, Rahnella aquatilis, Candida railenensis, Candida ernobii, Debaryomyces nepalensis, Candida sake, Peterozyma toletana, Trigonopsis cantarellii, Brettanomyces bruxellensis, Candida oleophila

2.2 Secondary selection test Using 100 strains selected in the primary selection test, the amount of Hyp accumulated was compared by the colorimetric method described below.

Add 0.2 mL of 0.5 M borate buffer and 0.4 mL of culture sample (or Hyp standard solution) to a 2 mL Eppendorf tube, stir well, let stand at room temperature for 5 minutes, then place in an ice bath (0 ° C). Let stand for 5 minutes. Next, 0.2 mL of 0.2 M chloramine T solution was added and placed in an ice bath (0 ° C.) for 120 minutes (oxidation reaction) with occasional stirring. To the solution after the oxidation reaction, 0.4 mL of a 3.6 M aqueous sodium thiosulfate solution was added, sealed, and heated in a boiling water bath for 30 minutes (decarboxylation reaction). The solution after the deoxidation reaction was cooled to room temperature with tap water, 0.6 mL of ethyl acetate was added, and the mixture was shaken vigorously with a shaker for 20 minutes. At the time of decarboxylation and ethyl acetate extraction, a cap was put on to prevent opening.
Centrifugation (1500 rpm, 5 minutes) of the resulting solution, 0.4 mL of ethyl acetate layer (upper layer: colorless) was taken into a 1.5 mL Eppendorf tube, and 0.16 mL of Erich reagent (yellow) was added thereto, The sample was allowed to stand at room temperature for 30 minutes to give a sample (color reaction: red when Hyp is contained). The blank was prepared by adding 0.16 mL of Erich reagent to 0.4 mL of ethyl acetate and allowing to stand at room temperature for 30 minutes.
200 μL of the sample was dispensed into a glass plate (96-well, transparent, flat bottom), and after adjusting the baseline at 650 nm to 0 with a plate reader (Tecan; infinite M200 Pro), the absorbance at 560 nm was measured (volatilization and A film was put on the plate to prevent acid corrosion of the equipment).
A calibration curve was prepared with a Hyp standard solution (Hyp concentration 2.5 to 0.25 μg / mL), and the Hyp concentration of the culture sample was determined. Among the strains subjected to the test, 22 strains having a high Hyp content in the culture sample obtained by the colorimetric method were selected.

3.3 Third Selection Test Using a culture sample of the same strain as the second selection test, the amount of accumulated Hyp was measured by a HPLC method with higher accuracy than the colorimetric method.

In HPLC, a system in which primary amino acids (primary amino groups) and secondary amino acids (secondary amino groups) were fluorescently labeled with an autosampler and analyzed by reversed-phase HPLC was used. O-phthalaldehyde (OPA) was used for the fluorescent labeling of the primary amino group, and chloroformate-9-fluorenylmethyl (FMOC) was used for the fluorescent labeling of the secondary amino group.

The culture sample was diluted with 0.1N HCl to obtain a sample. The sample was filtered through a 0.45 μm filter into a sample vial and set in an autosampler. An empty vial was charged with 30 μL of MPA (mercaptopropionic acid) reagent, 15 μL of OPA reagent, and 5 μL of the above sample and allowed to stand for 1 minute, and then 5 μL of FMOC reagent was added and 1 μL of the reacted solution was injected into HPLC. OPA reacts with primary amino acid, and Hyp and Pro having the remaining secondary amino group react with FMOC. By detecting each fluorescence wavelength with two channels, it is possible to detect all amino acids simultaneously.

The apparatus and conditions used for the HPLC analysis are shown below.
(apparatus)
High-performance liquid chromatograph Nexera X2 system (product name) manufactured by Shimadzu Corporation
System controller: CBM-20A, liquid feeding unit: LC-30AD (2 units), deaeration unit: DGU-20A5R, mixer: MR180 μL II, autosampler: SIL-30AC, column oven: CTO-20AC, fluorescence detector: RF-20AXS, workstation: LabSolutions LC / GC

(Measurement condition)
Column: Inertsil ODS-4 100 mm × 3.0 mmφ S-2 μm (GL Sciences Inc.)
Guard column: UHPLC Fitting (product name, GL Sciences Inc.) (Max. Pressure: 130 MPa)
Mobile phase A solution: 15 mmol / L KH ₂ PO ₄ and 5 mmol / L K ₂ HPO ₄ (pH 6.5)
Liquid B: 15/45/40 (v / v / v) = water / acetonitrile / methanol R0 (rinse liquid): water / methanol = 20/80 (v / v)
R3 (rinse solution): water / acetonitrile = 80/20 (v / v)
Initial solution B concentration: 10% (v / v)
Flow rate: 0.8mL / min
Column oven temperature: 35 ° C
Injection volume: 1 μL
Detection: Ch1: excitation wavelength 350 nm, fluorescence wavelength 450 nm
Ch2: excitation wavelength 266 nm, fluorescence wavelength 305 nm
Cell temperature: 25 ° C, Gain: × 4, Sensitivity: Midium

Gradient program (B solution concentration (%) is v / v%)
0-1.5 min. Conc 10%
1.5-6 minutes: B.I. Conc 10% → 30% gradient 6-11 min. Conc 30% → 40% gradient 11-15 minutes: Conc 100%
20-21.5 min. Conc 100% → 10%
25 min: Controller stop calibration curve: 6.25 μmol / L, 25 μmol / L, 50 μmol / L, and 100 μmol / L 0.1N HCl solutions of each amino acid were prepared. Each of these solutions was reacted with each reagent of MPA, OPA and FMOC by the above method, and analyzed by HPLC to draw a calibration curve.

An amino acid mixed standard solution H type solution and a 0.1N hydrochloric acid solution containing L-hydroxyproline were also reacted with each reagent in the same manner as described above, and analyzed by HPLC using the above apparatus and measurement conditions.
FIG. 2 is an HPLC chart obtained by analyzing a 0.1N hydrochloric acid solution containing amino acid mixed standard solution H and L-hydroxyproline (each amino acid concentration 20 μmol / L) ((a): Ch1 excitation wavelength 350 nm, fluorescence wavelength. Detection at 450 nm, (b): Ch2 excitation wavelength 266 nm, fluorescence wavelength 305 nm detection). The amino acid mixed standard solution H type used is Wako Pure Chemical Industries, Ltd.

Also by measurement by HPLC, it was confirmed that the cell cultures of 22 strains confirmed to have a large amount of Hyp accumulation in the secondary selection test had a high Hyp content. The measurement results of the Hyp content by HPLC of the culture samples prepared from these 22 cell cultures are shown in FIG. In addition, free Hyp was not detected in each liquid medium before the start of culture.

FIG. 1 is a diagram showing the measurement results of the Hyp content of a culture sample by HPLC.
Among these, reproducibility was confirmed for the following 11 strains having a Hyp content of 90 μg / mL or more as measured by HPLC.
Meyerozyma caribbica (2 species), Candida chrysomelidarum , Candida saopaulonensis , Candida blattae , Clavispora lusitaniae , Metschnikowia reukaufii (2 species), Meyerozyma guilliermondii , Candida morakotiae , Kodamaea eri

4). Confirmation of reproducibility Reproducibility was confirmed for the above 11 strains.
Pre-culture and main culture of each strain were carried out in the same manner as in the third selection test, and a culture sample was prepared by self-digestion of yeast using the cell culture solution obtained in the main culture. About the culture sample, the Hyp content was measured by the same method as the HPLC method of the tertiary selection test. As a result, the same results as in the third selection test were obtained for all 11 strains, and reproducibility could be confirmed.

<Test Example 2>
From the results of Test Example 1, from the viewpoint of the amount of Hyp accumulation, safety when used in foods, cosmetics, etc., in the preparation of yeast cells or cell cultures or extracts thereof containing Hyp Further experiments were carried out on six promising genera, Meyerozyma caribbica (2 species), Clavispor a lusitaniae , Metschnikowia reukaufii , Meyerozyma guilliermondii and Kodamaea ohmeri .

For the above 6 types of bacteria, the main culture was performed with the composition of YPD medium (ratio of Y: P: D and the type of P) and the culture time changed, and the Hyp content and Pro content in the cell culture were measured. did.

(Pre-culture)
YPD liquid medium in which Y: P: D is 1: 2: 2 (weight ratio) (Y: yeast extract 1.0% by weight, P: peptone 2.0% by weight, D: glucose 2.0% by weight) used. The yeast extract and peptone are the same as those used in Test Example 1.
The pre-culture was performed under the same conditions as the primary selection test in Test Example 1.

(Main culture)
100 μL of the preculture solution obtained above was inoculated into 5 mL of the following YPD liquid medium, followed by shaking culture (60 rpm) at 30 ° C. The culture time was 1 day or 2 days to obtain a cell culture solution (cell culture).
In the main culture, peptone or collagen peptide (CP) was used as P in the YPD liquid medium. Collagen peptides include collagen peptide Iquos HDL-50SP (product name, Nitta Gelatin Co., Ltd., average molecular weight 5000) (hereinafter referred to as collagen peptide (CP1)) or collagen peptide Type S (product name, Nitta). Gelatin Co., Ltd., average molecular weight 1200) (hereinafter referred to as collagen peptide (CP2)) was used. In the YPD medium, a medium using a collagen peptide instead of peptone can be said to be a YPD modified medium.
Moreover, the ratio of Y: P: D of the YPD liquid medium in the main culture is (1) Y: P: D = 1: 2: 2 (weight ratio) (Y: 1.0% by weight of yeast extract, P: Peptone or collagen peptide 2.0 wt%, D: glucose 2.0 wt%) or (2) Y: P: D = 1: 4: 5 (weight ratio) (Y: yeast extract 1.0 wt%) %, P: peptone or collagen peptide 4.0 wt%, D: glucose 5.0 wt%). Table 4 shows the culture conditions 1 to 12 used in the main culture.

A culture sample for measuring the Hyp content was prepared by self-digesting and sterilizing the yeast cell culture solution obtained in the main culture in the same manner as in Test Example 1, and removing the cell residue. The obtained culture sample was diluted with 0.1N hydrochloric acid solution to prepare a sample for HPLC. The sample prepared from the cell culture solution with a culture time of 1 day was diluted 10-fold, and the sample prepared from the cell culture solution with a culture time of 2 days was diluted 40-fold. These samples were reacted with MPA, OPA and FMOC reagents in the same manner as in Test Example 1 and analyzed by HPLC.

The apparatus and measurement conditions used in HPLC are the same as in the third selection test.
For the calibration curves, 5 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 250 μmol / L of 0.1 N HCl solutions were prepared for Hyp and Pro, respectively. It was made to react with each reagent of MPA, OPA, and FMOC by the same method, and analyzed by HPLC.

Table 5 shows the Hyp quantification results of the culture samples. In the table, CP1 is the above-described collagen peptide (CP1) (product name collagen peptide Iquos HDL-50SP), and CP2 is collagen peptide (CP2) (product name collagen peptide Type S). Table 6 shows the quantitative results of Pro of the culture samples. The culture conditions in the table are the conditions for main culture. Each liquid medium before the start of culture contained almost no free Hyp.

From the results shown in Table 5 and Table 6, the ratio of the content of Hyp (μg / mL) to the total content of Pro and Hyp (μg / mL) in each culture sample (μg / mL) (100 × Hyp / (Pro + Hyp)) was calculated. . The results are shown in Table 7.

All of the six types of yeast subjected to the test accumulated L-hydroxyproline in the culture by aerobic culture. All of the obtained bacterial cell cultures had a (Hyp / (Pro + Hyp)) ratio of 35 or more.

<Test Example 3>
Meyerozyma caribbica , Clavispora lusitaniae , Metschnikowia reukaufii , Meyerozyma guilliermondii and Kodamaea ohmeri shown in Table 8 were used. Yeasts identified by the NBRC numbers shown in Table 8 were obtained from the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashika, Kisarazu City, Chiba Prefecture, Japan).

(Pre-culture)
The test was performed under the same conditions as in Test Example 2 except that the yeast shown in Table 8 was used.
(Main culture)
100 μL of the preculture solution obtained above was inoculated into 5 mL of the following YPD liquid medium, followed by shaking culture (60 rpm) at 30 ° C. The culture time was 1 or 2 days to obtain a cell culture solution.
The conditions of the main culture were the culture conditions 11 or 12 of Test Example 2. Specifically, collagen peptide Type S (product name, Nitta Gelatin Co., Ltd., average molecular weight 1200) (collagen peptide (CP2) of Test Example 2) was used as P in the YPD liquid medium. The ratio of Y: P: D in the YPD liquid medium is as follows: Y: P: D = 1: 4: 5 (weight ratio) (Y: 1.0% by weight of yeast extract, P: collagen peptide Type S 4.0% by weight) %, D: glucose 5.0% by weight).

A culture sample was prepared from the cell culture solution in the same manner as in Test Example 2. The obtained culture sample was diluted with 0.1N hydrochloric acid solution to prepare a sample for HPLC. The sample prepared from the cell culture solution having a culture time of 1 day was diluted 20 times with the culture sample, and the sample prepared from the cell culture solution having a culture time of 2 days was diluted 50 times. These samples were reacted with MPA, OPA and FMOC reagents in the same manner as in Test Example 2.
Under the same conditions as in Test Example 2, the amino acid content in the culture sample was measured by HPLC. The results are shown in Table 9. Table 9 shows the total amino acid amount (TotalAA) in addition to the Hyp content and the Pro content. All the yeasts used for the test accumulated L-hydroxyproline in the culture by aerobic culture. In addition, Table 6 shows the absorbance (OD660) at 660 nm of the bacterial cell culture solution (culture time 1 day). Absorbance was measured by TVS 062CA (ADVANTEC).

From the results shown in Table 9, the ratio (100 × Hyp / (Pro + Hyp)) of the content (μg / mL) of Hyp to the total content (μg / mL) of Pro and Hyp in each culture sample was calculated. The results are shown in Table 10. Table 10 shows values obtained by dividing the Hyp content (μg / mL) of the daily culture by the absorbance OD660 of the bacterial cell culture solution (μg / mL / OD660) (Hyp / OD660 value).

<Test Example 4>
Using the yeasts used in Test Examples 2 and 3, the cells were cultured in the same manner as in Test Examples 2 and 3, and a cell culture solution was obtained. This was incubated at 50 ° C. for 2 hours to elute the cell contents into the culture solution by autolysis, and then incubated at 80 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 1 ° C. (3000 rpm, 5 min) to obtain an L-hydroxyproline-containing cell culture extract (Hyp-containing cell culture extract) from which the cell residue was removed. The extract of the Hyp-containing cell culture can be used after appropriately diluted.

Hereinafter, an example of the manufacture example of the skin external preparation composition which mix | blended the extract of each Hyp containing microbial cell culture obtained in Test Example 4 is shown.
<Production Example 1> Table 11 shows blending amounts of soap raw materials.
The soap base was mixed and stirred, and then an extract of each Hyp-containing cell culture was added and uniformly mixed, followed by molding.

<Production Example 2> Table 12 shows the blending amounts of the shampoo raw materials.
A preservative dissolved in 1,3-butylene glycol was added to purified water. After uniformly stirring, sodium laureth sulfate and coconut oil fatty acid monoethanolamide were added, and then a pigment, a fragrance and the remaining 1,3-butylene glycol were added, and each Hyp-containing cell culture extract was added, The mixture was uniformly mixed and stirred.

<Production Example 3> Table 13 shows the blending amounts of the conditioner raw materials.
(1) Stearyldimethylbenzylammonium chloride and sodium chloride were added to purified water and heated to 80 ° C. to dissolve.
(2) Cetostearyl alcohol, hydrogenated polyisobutene and glycerin monostearate were heated to 80 ° C. and dissolved.
(3) (2) was added while stirring (1) with a homomixer, and preliminary stirring was performed for 5 minutes after the addition.
(4) After completion of the pre-stirring, the mixture was cooled to 50 ° C. with stirring, each Hyp-containing cell culture extract was added, and the mixture was further cooled to 35 ° C. with stirring.

<Production Example 4> Table 14 shows the blending amounts of the hair tonic raw materials.
Vitamin E and L-menthol dissolved in salicylic acid, glycerin, and ethanol are added to purified water, and dipotassium glycyrrhizinate dissolved in a portion of purified water is added, and then each Hyp-containing cell culture extract is added. And mixed uniformly to prepare.

<Production Example 5> Table 15 shows blending amounts of mist raw materials.
Citric acid and sodium citrate were added to purified water and dissolved. Thereafter, an antiseptic and polysorbate 80 dissolved in ethanol were added. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.

<Production Example 6> Table 16 shows the amounts of the lotion ingredients.
Citric acid and sodium citrate were added to purified water and dissolved. Next, glycerin, 1,3-butylene glycol and trisodium ethylenediaminetetraacetate were sequentially added, and polyoxyethylene (18) oleyl alcohol ether dissolved in ethanol, vitamin E and methylparaben were added and stirred until uniform. Thereafter, each Hyp-containing cell culture extract was added and stirred uniformly.

<Production Example 7> Table 17 shows the amounts of the emulsion raw materials.
(1) Stearic acid, cetyl alcohol, octyldodecyl myristate and liquid paraffin were heated to 80 ° C. and dissolved.
(2) Triethanolamine, sodium hyaluronate, glycerin, 1,3-butylene glycol, polyoxyethylene (10) monooleate and sodium ethylenediaminehydroxytriacetate were added to purified water and heated to 80 ° C.
(3) While stirring (1) with a homomixer, (2) was added and pre-stirred for 5 minutes after the addition.
(4) After completion of preliminary stirring, the mixture was cooled to 50 ° C., an extract of each Hyp-containing cell culture was added, and further cooled to 35 ° C. for preparation.

<Production Example 8> Table 18 shows the blending amounts of the cream raw materials.
(1) Stearic acid, glyceryl monostearate, sorbitan sesquistearate, polyoxyethylene sorbitan monostearate, cetostearyl alcohol, squalane, hexa (hydroxystearic acid / stearic acid / rosinic acid) dipentaerythlit, olive oil, myristic Octyldodecyl acid and methylpolysiloxane were dissolved by heating to 80 ° C.
(2) Glycerin, 1,3-butylene glycol, sodium hydroxide and methylparaben were added to purified water and heated to 80 ° C. to dissolve.
(3) While stirring (1) with a homomixer, (2) was added and pre-stirred for 5 minutes after the addition.
(4) After completion of preliminary stirring, the mixture was cooled to 50 ° C., an extract of each Hyp-containing cell culture was added, and further cooled to 35 ° C. for preparation.

The yeast cells or cell cultures or extracts thereof containing L-hydroxyproline of the present invention are useful as raw materials for cosmetics, foods and drinks and the like.

Claims

Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), at least one selected from the group consisting of Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) A yeast or a bacterial culture of a seed yeast or an extract thereof,
The yeast cells or cell cultures or extracts thereof contain L-hydroxyproline, and L-hydroxy to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). A yeast cell or a cell culture or an extract thereof, wherein the content of hydroxyproline (μg / mL) (100 × Hyp / (Pro + Hyp)) is 35 to 100.
The yeast cell or cell culture or extract thereof according to claim 1, wherein the content of L-hydroxyproline is 10 µg / mL or more.
3. The yeast cell or cell culture of yeast according to claim 1 or 2, wherein the value (μg / mL / OD660) obtained by dividing the L-hydroxyproline content (μg / mL) by OD660 is 20 or more. Extract.
Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), at least one selected from the group consisting of Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) A yeast or a bacterial culture of a seed yeast or an extract thereof,
A yeast cell or cell culture, or an extract thereof, wherein the content of L-hydroxyproline is 10 μg / mL or more.
Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), at least one selected from the group consisting of Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) A step of aerobically culturing a seed yeast in a liquid medium containing a carbon source and a nitrogen source, thereby accumulating L-hydroxyproline in the yeast cells or cell culture, wherein the nitrogen source comprises A method for producing L-hydroxyproline, which is a nitrogen source containing an L-hydroxyproline-containing peptide.
The production method according to claim 5, wherein the L-hydroxyproline-containing peptide is a collagen peptide.
The production method according to claim 6, wherein the collagen peptide has an average molecular weight of 1,000 to 10,000.
The production method according to any one of claims 5 to 7, wherein the aerobic culture is performed for 10 to 100 hours.
Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), at least one selected from the group consisting of Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) Use of a seed yeast to produce L-hydroxyproline.
Aerobically culturing the yeast in a liquid medium containing a carbon source and a nitrogen source, thereby accumulating L-hydroxyproline in the yeast cells or cell culture, wherein the nitrogen source is L Use according to claim 9, which is a nitrogen source comprising a hydroxyproline containing peptide.
The use according to claim 10, wherein the L-hydroxyproline-containing peptide is a collagen peptide.
The use according to claim 11, wherein the collagen peptide has an average molecular weight of 1000 to 10,000.
The use according to any one of claims 10 to 12, wherein the aerobic culture is performed for 10 to 100 hours.
A composition comprising the yeast cell or cell culture or extract thereof according to any one of claims 1 to 4.
A food or drink comprising the yeast cell or cell culture or extract thereof according to any one of claims 1 to 4.
A cosmetic or a cosmetic raw material comprising the yeast cell culture or culture of yeast according to any one of claims 1 to 4 or an extract thereof.
For applications selected from promotion of collagen production, promotion of epidermal cell growth, skin moisturization, prevention of skin aging, prevention or improvement of skin sagging, improvement of skin firmness, prevention or improvement of wrinkles and improvement of atopic dermatitis The cosmetic or cosmetic raw material according to claim 16, which is used.
The cosmetic or cosmetic raw material according to claim 16 or 17, wherein the cosmetic or cosmetic raw material is a cosmetic raw material and has an L-hydroxyproline content of 5 to 300 ppm.
The cosmetic or cosmetic raw material according to claim 16 or 17, wherein the cosmetic or cosmetic raw material is a cosmetic and has an L-hydroxyproline content of 0.01 to 20 ppm.
Containing L- hydroxyproline, Kodamaea-Oumeri (Kodamaea ohmeri), Metschnikowia-Roikaufi (Metschnikowia reukaufii), Meierozaima-Karibika (Meyerozyma caribbica), from Meierozaima-Girierumondi (Meyerozyma guilliermondii) and Kurabisupora-Rushitanie (Clavispora lusitaniae) A composition for reinforcing L-hydroxyproline, comprising at least one yeast cell culture or cell culture or an extract thereof selected from the group consisting of
The yeast cells or cell cultures or extracts thereof have an L-hydroxyproline content (μg / mL) relative to the total content (μg / mL) of L-proline (Pro) and L-hydroxyproline (Hyp). The composition for reinforcing L-hydroxyproline according to claim 20, wherein the ratio (100 × Hyp / (Pro + Hyp)) is 35 to 100.
The composition for reinforcing L-hydroxyproline according to claim 20 or 21, wherein the yeast cell or cell culture or an extract thereof has an L-hydroxyproline content of 10 µg / mL or more.