JP6142058B1 - New strain of Sporidioboras pararoseus, food and drink, method for producing food and drink, skin cosmetic, and method for producing skin cosmetic - Google Patents

Abstract

[Problem] To provide Sporidioborus pararoseus having excellent production efficiency of carotenoids and high safety. The content of the total carotenoid per cell in the fermented product when fermenting tomatoes is Sporidiobolus pararoseus (0.5 mg / g or more). The aspect in which the Sporidioborus pararoseus is Sporidioborus pararoseus accession number NITE P-02236 is preferable. [Selection figure] None

Description

The present invention relates to a new strain excellent in the production efficiency of carotenoids, Sporidioborus pararoseus , and the above-mentioned Sporidioborus pararoseus ( Spodiobiorus pararoseus ), food and drink, method for producing food and drink, skin cosmetics, The present invention relates to a method for producing cosmetics.

Carotenoid is a general term for naturally occurring pigment components, which captures active oxygen and exhibits an antioxidant effect. In particular, plant-derived lutein and algae-derived astaxanthin have been clarified to improve eye diseases and to protect the skin against UV rays, and the demand in Japan is increasing. However, most of the raw materials depend on imports from China, etc., and the use of alternatives and the development of efficient production methods are cited as technical issues in order to achieve self-sufficiency at low cost.
It is known that microorganisms, particularly some yeasts, synthesize carotenoids and accumulate them in cells. It is known that a mixture of carotenoids containing β-carotene or the like reduces erythema on the skin after UV irradiation (see, for example, Patent Document 1).

As a method for fermentative production of carotenoids, a method of culturing a kind of yeast, Rhodotorula glutinis , and collecting carotenoids containing β-carotene, tolralodine and the like from cultured cells has been proposed (for example, Patent Document 2).
However, in the fermentation production method of carotenoids, the yield of total carotenoids can be increased to 0.04 mg / L medium by performing aeration culture by bubbling, but the yield is low and the method is not efficient.

  Therefore, development of a method for producing carotenoids that is excellent in carotenoid production efficiency and high in safety is desired.

JP 2000-511923 A JP-A-6-277093

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide Sporidioborus pararoseus which is excellent in the production efficiency of carotenoids and has high safety.
Another object of the present invention is to provide a food / beverage product containing a high concentration of carotenoids and containing Sporidioborus pararoseus , and a method for producing the food / beverage product.
Another object of the present invention is to provide a skin cosmetic containing a high level of carotenoids and having high safety, including Sporidiobolus pararoseus , and a method for producing the skin cosmetic.

Means for solving the problems are as follows. That is,
<1> content of total carotenoids per cell in the fermentation product when fermented tomato is a Sporidiobolus Pararozeusu, characterized in that at 0.5 mg / g cells or (Sporidiobolus pararoseus).
A <2> Sporidiobolus Pararozeusu (Sporidiobolus pararoseus) Accession No. NITE P-02,236 at which the Sporidiobolus Pararozeusu according to <1> (Sporidiobolus pararoseus).
<3> A food or drink comprising the Sporidioborus pararoseus according to any one of <1> to <2>.
<4> The food and drink according to <3>, which is a fermented product containing lycopene and has a total carotenoid content of 0.001 mg / g or more.
<5> Food and drink having a total carotenoid content of 0.001 mg / g or more by fermenting a lycopene-containing material using Sporidioborus pararoseus according to any one of <1> to <2> It is the manufacturing method of the food-drinks characterized by obtaining.
<6> A skin cosmetic characterized by containing the Sporidioborus pararoseus according to any one of <1> to <2>.
<7> Skin makeup in which lycopene-containing material is fermented using Sporidiobolus pararoseus according to any one of <1> to <2>, and the total carotenoid content is 0.001 mg / g or more A method for producing a skin cosmetic, characterized in that a preparation is obtained.

According to the present invention, the above-mentioned problems in the prior art are solved, a new strain of Sporidioborus pararoseus having excellent carotenoid production efficiency and high safety, and a high concentration of carotenoids, and Foods and drinks including the highly safe Sporidioborus pararoseus , and skin cosmetics, and the use of the Sporidiobolus pararoseus at a high concentration and high safety. The manufacturing method of food-drinks and the manufacturing method of skin cosmetics can be provided.

(New strain of Sporidioboras pararose)
Sporidiobolus Pararozeusu of the present invention (Sporidiobolus pararoseus), the content of total carotenoids per cell in the fermentation product when fermented tomato is 0.5 mg / g cells or more.
Here, the “fermented product when tomato was fermented” was shaken at 25 ° C. and 200 rpm using a tomato paste diluted to 5-fold with sterile water (100 mL) as a tomato and pre-cultured Sporidioborus pararoseus. Under conditions, it can be obtained by culturing for 48 hours and fermenting.
The total carotenoid content means the total content of carotenoids, and can be measured by a known method such as high performance liquid chromatography (HPLC). For example, measuring device name: Prominence (manufactured by Shimadzu Corporation) ).

The Sporidioborus pararoseus of the present invention is a new strain TYC-2190 obtained as a result of intensive studies by the present inventors. It has been deposited on April 13, 2016 at Kazusa Kamashitsu 2-10-8, Kisarazu City, Chiba Prefecture, and its deposit number is NITE P-02236 (reception number: NITE AP-02236). It is also owned by the National Research Institute for Agricultural and Food Industry Technology, and can be sold through a licensing procedure.

The Sporidioborus pararoseus of the present invention is excellent in the production efficiency of carotenoids, and can produce carotenoids at a high concentration.
The carotenoids are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include γ-carotene, tolylene, tolralodine, lycopene, Δ-carotene, lutein, β-carotene, astaxanthin, and fucoxanthin. Can be mentioned.
It is known that the γ-carotene, tolylene, tolralozin, Δ-carotene, lutein, β-carotene, and astaxanthin are biosynthesized from the lycopene. By fermenting, a fermented product containing the carotenoids at a high concentration can be obtained.
In addition, Sporidioboras pararoseus of the present invention, the inventors focused on yeast as an alternative raw material for carotenoids, selected yeast that produces carotenoids at a high concentration and developed efficient fermentation production technology, earnest study As a result, it is based on the obtained knowledge, and the present inventors themselves are yeasts isolated from grapes.
The Sporidioborus pararoseus of the present invention is a yeast isolated from food, is highly safe, and can produce carotenoids at a high concentration. Therefore, foods and drinks and skin cosmetics containing carotenoids at a high concentration It is suitable for.
In addition, the carotenoids were separated from the new strain of Sporidioboras pararoseus by extraction, purification, etc. from the fermented product obtained by fermenting the lycopene-containing material using the new strain of Sporidioboras pararose. The carotenoids that can be obtained in a state may be used for food and drink, skin cosmetics, and the like.

The Sporidioboras pararoseus of the present invention is a kind of yeast belonging to the genus Sporidioboras, and is known to produce carotenoids and accumulate them in the cells.
As described above, the Sporidioboras pararose of the present invention has a total carotenoid content of 0.5 mg / g cells or more in the fermented product when tomato is fermented. It can be selected as an indicator.
When culturing the Sporidioboras pararoseus of the present invention, the medium that can be used is not particularly limited and can be appropriately selected from known yeast culture media according to the purpose, and the culture conditions are particularly limited. However, it can be appropriately selected from known yeast culture conditions for the purpose.

(Food)
The food or drink of the present invention contains at least the Sporidioboras pararose of the present invention, the total carotenoid content is preferably 0.001 mg / g or more, and further contains other components as necessary.

The content of the Sporidioboras pararoseus of the present invention in the food or drink is not particularly limited and may be appropriately selected depending on the application and may be sufficient to produce carotenoids. In addition, the above-mentioned sporidioborus pararose may remain after the carotenoids are produced.
As the Sporidioboras pararoseus of the present invention contained in the food or drink, live bacteria may be used or a sterilized state may be used.
There is no restriction | limiting in particular as a method to confirm that the said food-drinks contain the Sporidioborus pararose of this invention, According to the objective, it can select suitably, For example, between 18S rRNA and 5.8S rRNA Examples include a method of performing sequence analysis after PCR amplification of the spacer region (ITS1) and the spacer region (IGS1) between 26S rRNA and 5S rRNA.

In the foods and beverages, the carotenoids produced by the Sporidioboras pararoseus of the present invention are stable to heat and stable even after autoclaving, etc. Specific examples of the food and drink include various dairy products, various breads, noodles such as udon, various side dishes, confectionery, soft drinks such as confectionery, juices, alcoholic beverages, lunches, and the like. The food and drink may contain other ingredients such as known food additives, seasonings, colorants, preservatives, etc., selected appropriately according to the purpose, in addition to the raw materials and materials of the food and drink. Good. The said food / beverage products contain carotenoids in high concentration, and are suitable for the use as food / beverage products, such as health food and functional display foods containing a carotenoid, by the point of high safety | security.
The food and drink may contain other known edible bacteria within the range that does not impair the effects of the present invention, in addition to the Sporidioboras pararose of the present invention. It is preferable.
There is no restriction | limiting in particular as a method of making the said food / beverage products contain the Sporidioborus pararose of this invention, According to the kind of said food / beverage products, the objective, etc., it can select suitably.

The content of the carotenoids per microbial cell in the food or drink is not particularly limited and can be appropriately selected according to the use etc., but is preferably a high content, with the total carotenoid content. 0.05 mg / g cells or more is preferable, 0.1 mg / g cells or more is more preferable, and 0.5 mg / g cells or more is particularly preferable.
There is no restriction | limiting in particular as content in the said food-drinks of the said total carotenoid, Although it can select suitably according to a use etc., it is preferable that it is high content, 0.001 mg / g or more is preferable, 0 0.004 mg / g or more is more preferable, and 0.01 mg / g or more is particularly preferable.
The food and drink is a fermented product containing lycopene, and the total carotenoid content is preferably 0.001 mg / g or more.

(Method for producing food and drink)
The method for producing a food or drink of the present invention includes fermenting a lycopene-containing material using the Sporidioboras pararose of the present invention, and obtaining a food or drink having a total carotenoid content of 0.001 mg / g or more. Other processes are included depending on the situation.

The raw material for the food and drink is not particularly limited as long as it is a material suitable for food and drink, and can be appropriately selected according to the purpose. By biosynthesis in the cell body of Sporidiobolus pararoseus of the present invention, a high concentration of carotenoids is obtained. Can be manufactured. Among these, a lycopene-containing material containing lycopene, which is a raw material (intermediate) for biosynthesis of carotenoids, is preferable in terms of improving the production efficiency of carotenoids.
The lycopene-containing material is not particularly limited as long as it contains lycopene, and can be appropriately selected according to the purpose. Examples thereof include tomato, watermelon, strawberries, and pink grapefruit.
The lycopene content in the lycopene-containing material is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.005 mg / g or more, more preferably 0.01 mg / g or more, 0.05 mg / G or more is particularly preferable.

The number of viable bacteria of Sporidioboras pararoseus of the present invention used in the method for producing food and drink is not particularly limited as long as carotenoids can be biosynthesized, and can be appropriately selected according to the purpose.
There is no restriction | limiting in particular as Sporidioborus pararoseus of this invention used for the manufacturing method of the said food-drinks, According to the objective, it can select suitably, For example, what was prepared by preculture is mentioned. There is no restriction | limiting in particular as the method of the said preculture, According to the objective, the well-known yeast culture | cultivation method can be selected suitably, For example, the method etc. which culture | cultivate using a yeast complete culture medium (YPD culture medium) etc. are mentioned. .

  The method for fermenting the lycopene-containing material is not particularly limited and may be appropriately selected depending on the purpose.For example, the Sporidioboras pararose of the present invention is added to the lycopene-containing material, and a temperature of 25 ° C. to 35 ° C. It can be carried out by culturing for 24 hours to 96 hours under conditions of 200 rpm to 300 rpm.

<Other processes>
There is no restriction | limiting in particular as said other process, The various processes in manufacture of food-drinks can be selected suitably according to the objective, For example, a sterilization process, a concentration process, a drying process, etc. are mentioned.

There is no restriction | limiting in particular as said sterilization process, According to the objective, the well-known sterilization process in manufacture of food-drinks can be selected suitably, For example, heat sterilization, filtration sterilization, etc. are mentioned.
There is no restriction | limiting in particular as said concentration process, According to the objective, the well-known concentration process in manufacture of food-drinks can be selected suitably, For example, centrifugal concentration, membrane concentration, etc. are mentioned.
There is no restriction | limiting in particular as said drying process, According to the objective, the well-known drying process in manufacture of food-drinks can be selected suitably, For example, freeze-drying, reduced pressure drying, etc. are mentioned.

(Skin cosmetic)
The skin cosmetic of the present invention contains at least the Sporidioboras pararose of the present invention, the total carotenoid content is 0.001 mg / g or more, and contains other components as necessary.
About the said skin cosmetics, the matter described about the said food-drinks can be suitably selected according to the objective.

(Method for manufacturing skin cosmetics)
The method for producing a skin cosmetic of the present invention includes fermenting a lycopene-containing material using the Sporidioboras pararose of the present invention, and obtaining a skin cosmetic having a total carotenoid content of 0.001 mg / g or more, Furthermore, other steps are included as necessary.
About the manufacturing method of the said skin cosmetics, the matter described about the manufacturing method of the said food-drinks can be suitably selected according to the objective.

<Application>
Sporidioboras pararoseus of the present invention is excellent in the production efficiency of carotenoids and high in safety, and therefore contains foods and beverages containing high levels of carotenoids and high safety, including sporidioboras pararoseus, and skin cosmetics. It can be suitably used.
The Sporidioborus pararoseus of the present invention is excellent in safety and has excellent SOD-like action, nitric oxide (NO) production inhibitory action, melanin production inhibitory action, type VII collagen production promoting action, laminin-5 production promoting action, and elastin production. Since it has a promoting action, a skin keratinocyte proliferation promoting action, and a glutathione production promoting action, it is a superoxide scavenger, nitric oxide (NO) production inhibitor, melanin production inhibitor, type VII collagen production promoter, laminin-5 It can be used as a production promoter, elastin production promoter, skin keratinocyte proliferation promoter, and glutathione production promoter.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Sporidiobolus Pararozeusu (Sporidiobolus pararoseus) of Sporidiobolus Pararozeusu of separation the invention of new strains (Sporidiobolus pararoseus) TYC-2190 strain (already deposited with the Patent Microorganisms Depositary Center, accession number: NITE P-02236, receipt number: NITE AP-02236 ) Were separated as follows.
That is, the collected yama grapes were cultured in a YPD liquid medium for 48 hours, and a part of the culture solution was applied to a YPD agar plate containing chloramphenicol and cultured for 5 days to select yeast-like colonies. A single colony was picked, applied to a YPD agar plate, and cultured repeatedly until a single strain was isolated to obtain a TYC-2190 strain.
The colony color tone of the obtained TYC-2190 strain was red. The spacer region (ITS1) between 18S rRNA and 5.8S rRNA of TYC-2190 strain was subjected to PCR amplification and then subjected to sequence analysis. As a result, it was 100% identical with the standard strain of Sporidioboras pararoseus (NBRC 0376).・ Identified as Pararose. Further, IGS1 (spacer region between 26S rRNA and 5S rRNA), which is an index for strain level identification, was subjected to PCR amplification and sequence analysis. As a result, the homology with the standard strain was 94.57%. There were differences such as substitution. In addition, in the nucleotide sequence database managed by NCBI, a sequence that completely matches IGS1 of TYC-2190 strain was not registered.

Example 1
Yeast complete medium (YPD medium) 1,000 mL of the following composition as the medium, and Sporidioboras pararoseus TYC-2190 strain, using TYC-2190 strain obtained by pre-culture using a small amount of YPD medium, 25 ° C., The culture of Example 1 was obtained by culturing for 2 days under the condition of shaking at 200 rpm.
The cells were collected from the culture obtained by centrifugation (6,300 rpm, 15 minutes), and the total carotenoid content in the cells was measured by the measurement method shown below. The results are shown in Table 1.

<Composition of YPD medium>
The YPD medium was prepared using 10 g of yeast extract, 20 g of peptone, 20 g of glucose and 1 L of distilled water, and used after sterilization in an autoclave.

<Measurement method of total carotenoid content>
Dimethyl sulfoxide (DMSO) was added to the collected bacterial cells, and the bacterial cells were crushed with an ultrasonic crusher. This was filtered using a membrane filter, and then the total amount of carotenoids (torralodin, lycopene, tolylene, γ-carotene, β-carotene) was measured using HPLC to obtain the total carotenoid amount.
Column: Thermo Scientific, Synchronis C18, 150 × 4.6 mm I.D. D. , 5μm
Column temperature: 30 ° C
Mobile phase: 90% by volume acetonitrile: ethyl acetate = 50: 50 (volume ratio)
Flow rate: 1 mL / min
Measurement wavelength: 450nm
Sample solution: dimethyl sulfoxide Standard solution: t-butyl methyl ether-methanol mixture containing butylated hydroxytoluene

(Example 2)
In Example 1, it culture | cultivated like Example 1 except having used the tomato as a lycopene containing material instead of the YPD culture medium, and obtained the fermented material of Example 2.
As the tomato, a tomato paste (trade name: Kagome Tomato Paste CB, manufactured by Kagome Co., Ltd., lycopene content 0.56 mg / g) diluted 5 times with sterilized water was used.
The results of measuring the total carotenoid content in the cells in the same manner as in Example 1 are shown in Table 1.

(Comparative Example 1)
The results of Example 2 of Patent Document 2 (Japanese Patent Laid-Open No. 6-277093) are also shown in Table 1 as Comparative Example 1.

  As shown in Test Examples 1 to 8 below, an SOD-like action test (Test Example 1) is performed as an evaluation of antioxidation (reactive oxygen scavenging) action, and nitric oxide (NO) production suppression is performed as an anti-inflammatory action evaluation. An action test (Test Example 2) is conducted, a melanin production inhibitory action test (Test Example 3) is conducted as an evaluation of the whitening action, and an anti-aging action evaluation is conducted as a type VII collagen production promoting action test (Test Example 4), laminin. -5 Production promoting action test (Test Example 5), Elastin production promoting action test (Test Example 6), Skin keratinocyte proliferation promoting action test (Test Example 7), and Glutathione production promoting action test (Test Example 8) It was.

Test Example 1: Super oxide dismutase (SOD) -like action test In a test tube, 0.05 mol / L sodium carbonate buffer solution (pH 10.2) 2.4 mL, 3 mmol / L xanthine 0.1 mL, 3 mmol / L EDTA 0.1 mL Then, 0.1 mL of 1.5 mg / mL bovine serum albumin and 0.1 mL of 0.75 mmol / L nitroblue tetrazolium were added, and 0.1 mL of the test sample solution was added thereto and left at 25 ° C. for 10 minutes. Xanthine oxidase solution (0.1 mL) was added, stirred rapidly, and allowed to react at 25 ° C. for 20 minutes. Thereafter, 0.1 mL of 6 mmol / L copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured. A blank test was performed and corrected in the same manner.

The calculation method of the superoxide erasure rate is as follows.
Erase rate (%) = {1− (A−B) / (C−D)} × 100
A: Absorbance at a wavelength of 560 nm of the test sample solution B: Absorbance at a wavelength of 560 nm of the test sample solution blank C: Absorbance at a wavelength of 560 nm of the control solution D: Absorbance at a wavelength of 560 nm of the control solution blank

Test Example 2: Nitric Oxide (NO) Production Inhibitory Action Test Mouse macrophage cells (RAW264.7) were cultured using 10% by volume FBS-containing Dulbecco MEM, and then cells were collected with a cell scraper. The collected cells were diluted with 10% FBS-containing phenol red-free Dulbecco MEM to a concentration of 3.0 × 10 ⁶ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured for 4 hours. . After completion of the culture, the medium was removed, 100 μL of a test sample dissolved in 10% by volume FBS-containing phenol red-free Dulbecco MEM containing 0.5% by volume DMSO was added to each well, and 10 volumes at a final concentration of 1 μg / mL. 100 μL of lipopolysaccharide (LPS, E. coli 0111; B4, DIFCO) dissolved in% FBS-containing phenol red-free Dulbecco MEM was added and cultured for 48 hours. The amount of NO production was measured using the amount of nitrite ion (NO ²⁻ ) as an index. After completion of the culture, the same amount of grease reagent (1% by mass sulfanilamide, 0.1% by mass N-1-naphthyl ethylenedihydride in 5% by mass phosphoric acid solution) was added to the culture solution of each well, and the room temperature was kept for 10 minutes. It was made to react with. After the reaction, absorbance at a wavelength of 540 nm was measured. A calibration curve was prepared using NO ²⁻ as an index, and the production amount of NO in the culture supernatant was determined.

The calculation method of the NO production suppression rate is as follows.
NO production inhibition rate (%) = {(B−A) / B} × 100
A: NO amount when test sample is added B: NO amount when test sample is not added

Test Example 3: Test for inhibiting melanin production on B16 melanoma cells B16 melanoma cells were cultured using 10% by volume FBS-containing Dulbecco MEM, and then cells were collected by trypsin treatment. The collected cells were diluted with Dulbecco's MEM containing 10% by volume FBS and 1 mmol / L theophylline to a concentration of 24.0 × 10 ⁴ cells / mL, then seeded at 300 μL per well in a 48-well plate, and cultured for 6 hours. . After completion of the culture, 300 μL of a test sample dissolved in Dulbecco's MEM containing 10% by volume FBS and 1 mmol / L theophylline was added to each well and cultured for 4 days. After completion of the culture, the medium was removed from each well, 200 μL of 2 mol / L NaOH solution was added, the cells were disrupted with an ultrasonic crusher, and the absorbance at a wavelength of 475 nm was measured. From the measured absorbance value, the amount of melanin was calculated based on a calibration curve prepared using synthetic melanin.

  In addition, in order to measure the cell viability, 200 μL of neutral red dissolved in Dulbecco MEM containing 10 vol% FBS at a final concentration of 0.05 mg / mL and washed with 400 μL of PBS (−) was similarly cultured. Added. After culturing for 2.5 hours, the neutral red solution was discarded, and 200 μL of ethanol / acetic acid solution (ethanol: acetic acid: water = 50: 1: 49, volume ratio) was added to each well to extract the dye. After extraction, the absorbance at a wavelength of 540 nm was measured. As a blank test, cells cultured only with Dulbecco MEM containing 10% by volume FBS and 1 mmol / L theophylline were tested in the same manner.

The calculation method of melanin production suppression rate and cell viability is as follows.
Melanin production inhibition rate (%) = {1− (B / D) / (A / C)} × 100
Cell viability (%) = (D / C) × 100
A: Melanin amount without addition of test sample B: Melanin amount with addition of test sample C: Absorbance at 540 nm without addition of test sample D: Absorbance at 540 nm with addition of test sample

Test Example 4: Type VII Collagen Production Promotion Action Test Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then cells were collected by trypsin treatment. The recovered cells were diluted with BPE-free KGM (KGM-BPE) to a concentration of 1 × 10 ⁵ cells / mL, seeded in a 24-well plate at 500 μL per well, and cultured for 1 day. After completion of the culture, the medium was removed, and 500 μL of a test sample dissolved in KGM-BPE was added to each well and cultured for 48 hours. After completion of the culture, the amount of type VII collagen in the medium of each well was measured by ELISA.

The calculation method of the type VII collagen production promotion rate is as follows.
VII type collagen production promotion rate (%) = A / B × 100
A: Absorbance at a wavelength of 405 nm when a test sample is added B: Absorbance at a wavelength of 405 nm when no test sample is added

Test Example 5: Laminin-5 Production Promotion Action Test Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then cells were collected by trypsin treatment. The recovered cells were diluted with BPE-free KGM (KGM-BPE) to a concentration of 1 × 10 ⁵ cells / mL, seeded in a 24-well plate at 500 μL per well, and cultured for 1 day. After completion of the culture, the medium was removed, and 500 μL of a test sample dissolved in KGM-BPE was added to each well and cultured for 48 hours. After completion of the culture, the amount of laminin-5 in the medium of each well was measured by ELISA.

The calculation method of the laminin-5 production promotion rate is as follows.
Laminin-5 production promotion rate (%) = A / B × 100
A: Absorbance at a wavelength of 405 nm when a test sample is added B: Absorbance at a wavelength of 405 nm when no test sample is added

Test Example 6: Elastin production promoting action test Human normal fibroblasts (NB1RGB) were cultured using 10% by volume FBS-containing Dulbecco MEM, and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a concentration of 2.2 × 10 ⁵ cells / mL, then seeded at 100 μL per well in a 96-well microplate, and cultured overnight. After completion of the culture, the medium was removed, and 150 μL of a test sample dissolved in Dulbecco's MEM containing 0.25% by volume FBS was added to each well and cultured for 5 days. After completion of the culture, the amount of elastin in the culture supernatant of each well was measured by ELISA.

The calculation method of elastin production promotion rate is as follows.
Elastin production promotion rate (%) = A / B × 100
A: Elastin amount when test sample is added B: Elastin amount when test sample is not added

Test Example 7: Skin Fibroblast Proliferation Promoting Action Test Human normal skin fibroblasts (NB1RGB) were cultured using 10 vol% FBS-containing α-MEM, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 5% by volume FBS to a concentration of 7.0 × 10 ⁴ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured overnight. After completion of the culture, 100 μL of a test sample dissolved in α-MEM containing 5% by volume FBS was added to each well and cultured for 3 days. The skin fibroblast proliferation effect was measured using the MTT assay. After completion of the culture, the medium was removed, and 100 μL of MTT dissolved in PBS (−) at a final concentration of 0.4 mg / mL was added to each well. After culturing for 2 hours, blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced.

The calculation method of the skin fibroblast proliferation promotion rate is as follows.
Skin fibroblast proliferation promotion rate (%) = A / B × 100
A: Blue formazan production amount in cells when test sample is added B: Blue formazan production amount in cells when no test sample is added

Test Example 8: Glutathione production promoting action test (fibroblast)
Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% by volume FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM containing 10% by volume FBS to a concentration of 2.0 × 10 ⁵ cells / mL, then seeded at 200 μL per well in a 48-well plate, and cultured overnight. After culturing, 200 μL of a test sample dissolved in D-MEM containing 1% by volume FBS was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (registered trademark, PIERCE). The total glutathione was quantified using 100 μL of this. Specifically, 100 μL of cell extract dissolved in 96-well plate, 50 μL of 0.1 mol / L phosphate buffer, 2 μL of NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units / mL) were added and added at 37 ° C. for 10 minutes. After warming, 25 μL of 10 mmol / L 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance at a wavelength of 412 nm until 5 minutes later was measured to obtain ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione. The obtained value was corrected to the glutathione amount per total protein amount, and the glutathione production promotion rate was calculated according to the following formula.

Glutathione production promotion rate (%) = A / B × 100
A: Glutathione amount per total protein in the cell when no test sample is added B: Glutathione amount per total protein in the cell when the test sample is added

  Sporidioboras pararoseus of the present invention is excellent in the production efficiency of carotenoids and high in safety. It can be suitably used.

  NITE P-02236

Claims (6)

The content of total carotenoids per cell in the fermentation product when fermented tomatoes, 0.5 mg / g cells or der is, a Sporidiobolus Pararozeusu (Sporidiobolus pararoseus) Accession No. NITE P-02,236 The characteristic Sporidioborus pararoseus ( Sporidiobolus pararoseus ). A food / beverage product comprising the Sporidioborus pararoseus according to claim 1. The food / beverage product according to claim 2, which is a fermented product of lycopene-containing material and has a total carotenoid content of 0.001 mg / g or more. 2. Production of a food or drink characterized by fermenting a lycopene-containing material using the Sporidioborus pararoseus according to claim 1 to obtain a food or drink having a total carotenoid content of 0.001 mg / g or more. Method. A skin cosmetic comprising the Sporidioborus pararoseus according to claim 1. A skin cosmetic comprising: fermenting a lycopene-containing material using the Sporidioborus pararoseus according to claim 1 to obtain a skin cosmetic having a total carotenoid content of 0.001 mg / g or more. Manufacturing method.