JP2019136027A - Fermentation product - Google Patents

Abstract

To provide a collagen raw material having a novel function in addition to the conventional function of reduction of a molecular weight.SOLUTION: This invention relates to a fermentation product of collagen with lactic acid bacteria. This invention also relates to a method for producing ornithine or citrulline including the step of fermenting collagen with lactic acid bacteria. This invention also relates to a skin whitening composition containing the fermentation product.SELECTED DRAWING: None

Description

The present invention relates to a fermented product, and particularly to a fermented product of collagen by lactic acid bacteria.

Collagen is attracting attention as a useful material for cosmetic applications, among which collagen peptides, which have been made low molecularly by enzymes, can be expected to be more absorbable into the body than high molecular weight collagen. It is known as a functional collagen raw material (Patent Documents 1 and 2). However, in order to meet the diversifying needs of consumers, it is not sufficient to reduce the molecular weight alone, and it is required to provide a collagen raw material having a function different from that of reducing the molecular weight.

On the other hand, lactic acid bacteria are used in the production of fermented foods such as yogurt and pickles. It is known that the flavor of food can be improved by lactic acid bacteria fermentation, and various useful components are produced as metabolites. Milk, vegetables and the like are mainly used as fermentation raw materials for lactic acid bacteria, but it has not been known that lactic acid bacteria can be fermented in collagen.

JP 2004-238365 A JP 2006-079122 A

An object of this invention is to provide the collagen raw material which has a function different from molecular weight reduction.

The inventors of the present invention have studied various methods for treating collagen, and have found that lactic acid bacteria can be fermented using a medium mainly composed of collagen. Furthermore, when collagen was fermented with lactic acid bacteria, a metabolite containing ornithine and citrulline was produced in the fermented product, and the fermented product had a whitening action, and the present invention was completed.

That is, the present invention relates to a fermented product of collagen by lactic acid bacteria.

The lactic acid bacteria are preferably Lactococcus microorganisms.

The microorganism of the genus Lactococcus is preferably Lactococcus lactis.

The collagen is preferably fish-derived collagen.

Moreover, this invention relates to the manufacturing method of ornithine or citrulline including the process of fermenting collagen with lactic acid bacteria.

Moreover, this invention relates to the composition for whitening containing the said fermented material.

The whitening is preferably whitening caused by suppression of melanin synthesis.

Suppression of melanin synthesis is preferably caused by suppression of tyrosinase gene expression.

According to the present invention, a metabolite containing ornithine and citrulline is produced in a fermented product by fermenting collagen with lactic acid bacteria. In addition, the fermented product of the present invention includes a collagen component and lactic acid bacteria used in the fermentation process, and the effects of these components can also be enjoyed. Furthermore, the fermented product has a whitening effect.

The cell number of the pigment cell cultured by the fermented material of this invention is shown. The coloring degree of the pigment cell cultured with the fermented material of this invention is shown. The expression level of tyrosinase gene is shown. The expression level of TRP-1 gene is shown. The expression level of TRP-2 gene is shown.

(1) Fermented product The fermented product of the present invention is characterized by being a fermented product of collagen by lactic acid bacteria.

Collagen is not particularly limited, and for example, collagen extracted from animals can be used. There are no particular limitations on the species of animal derived from the animal, and examples include freshwater fish such as tilapia, marine fish such as cod and salmon, mammals such as cows and pigs, and birds such as chickens. Among these, those derived from fish are preferable, and those derived from tilapia are more preferable. Moreover, it does not specifically limit about the site | part which originates, When using fish, for example, a scale, skin, bone, cartilage, a fin, an organ etc. are mentioned. Among these, scale is preferable.

As the collagen, a collagen peptide obtained by reducing the molecular weight of the aforementioned collagen with an enzyme may be used. The enzyme for obtaining the collagen peptide is not particularly limited, and examples thereof include protease, peptidase, collagenase and the like. These enzymes may be used alone or in combination of two or more. The conditions for the enzyme reaction are not particularly limited, but the temperature is preferably 40 ° C. or less, more preferably 35 to 38 ° C. The enzyme reaction product can be further subjected to treatments such as filtration, concentration, sterilization, and spray drying. Examples of the form of the collagen peptide include powder and liquid.

The lactic acid bacterium is not particularly limited as long as it grows in a medium containing collagen. For example, the genus Lactococcus, Lactobacillus, Pediococcus, Enterococcus, Examples include microorganisms belonging to the genus Leuconostoc, but the genus Lactococcus and Lactobacillus are preferred. Examples of lactic acid bacteria belonging to the genus Lactococcus include Lactococcus lactis, Lactococcus lactis subsp. Lactis, and Lactococcus lactis subspice lactis is preferable. As long as it is a lactic acid bacterium corresponding to the above-mentioned genus and species, not only a commercially available product but also a strain isolated independently can be used.

The fermentation method is not particularly limited as long as a medium containing collagen is used. For example, it may be a condition in which the cells grow well, such as stationary culture, neutralized culture with a constant pH, batch culture, continuous culture, etc. There is no particular limitation.

If necessary, fermentation promoting substances, for example, carbon sources such as sugars, starches and dextrins, nitrogen sources such as yeast extract and peptone, vitamins and minerals can be added.

Examples of the saccharide include glucose, arabinose, sucrose, lactose, sorbitol, fructose, and trehalose. Of these, glucose, arabinose, and trehalose are preferably contained.

The concentration of collagen in the medium is preferably 5 to 40% by weight, and more preferably 15 to 30% by weight. When saccharides are used, the amount used is preferably 0.1 to 5% by weight, more preferably 0.5 to 2% by weight in the medium.

Although fermentation temperature is not specifically limited, 20-42 degreeC is preferable and 25-37 degreeC is more preferable. If it exceeds 42 ° C, the culture tends to be impossible, and if it is less than 20 ° C, the culture time tends to be long.

Although fermentation time is not specifically limited, 4 to 120 hours are preferable, 12 to 48 hours are more preferable, and 18 to 36 hours are more preferable.

The atmosphere during fermentation is not limited, but is preferably aerobic.

In order to activate the growth ability and fermentation ability of lactic acid bacteria, it is preferable to pre-culture the lactic acid bacteria before the fermentation step. Pre-culture can be performed under the fermentation conditions described above.

Although the obtained fermented product can be used as it is after completion of the fermentation process, sterilization treatment and concentration treatment may be performed as necessary. You may use what removed the lactic acid bacteria from the fermented material. Examples of methods for removing lactic acid bacteria include filter filtration and centrifugation. Moreover, it does not specifically limit about the form of fermented material, It can use with forms, such as liquid form and powder form.

As a result of the aforementioned fermentation process, the molecular weight of the collagen is reduced and the content of ornithine or citrulline in the fermented product is increased from the content in the raw collagen. Here, with respect to amino acids such as ornithine and citrulline, the content in the fermented product refers to the amount of free amino acid contained in the liquid component of the fermented product.

The weight average molecular weight of the collagen contained in the fermented product of the present invention is preferably 3200 or less, more preferably 3000 or less, and even more preferably 2800 or less.

In the fermented product of the present invention, the number of lactic acid bacteria is preferably 1.0 × 10 ¹⁰ cfu / g or more, more preferably 1.5 × 10 ¹⁰ cfu / g or more, and 2.5 × 10 ¹⁰ cfu / g on a solid basis. The above is more preferable.

In the fermented product of the present invention, the ornithine content is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, further preferably 1.0% by weight or more, and even more preferably 1.5% by weight or more, 1.8% by weight or more is particularly preferable. Ornithine is known to be effective in metabolizing ammonia in the liver and protecting the degraded liver. Ornithine is difficult to take in a necessary amount in a normal meal, but can be efficiently taken in using the fermented product of the present invention.

In the fermented product of the present invention, the citrulline content is preferably 0.01% by weight or more, more preferably 0.05% by weight or more, further preferably 0.1% by weight or more, and still more preferably 0.4% by weight or more. Citrulline is known for its effects such as vasodilation. Citrulline is difficult to ingest in a normal diet, but can be efficiently ingested using the fermented product of the present invention.

The fermented product of the present invention is manufactured from raw materials with dietary experience of collagen and lactic acid bacteria, so it is highly safe, foods, beverages, pharmaceuticals, quasi-drugs, cosmetics, foods for specified health use, functional nutritional foods, functionality It can be applied to labeled foods, dietary supplements (supplements), feeds, etc. Among these, cosmetics, foods and supplements are preferable. As a form of cosmetics, the form mentioned later as a dosage form of the composition for whitening is mentioned. It does not specifically limit as a form of foodstuff, For example, a jelly, a drink, confectionery, nursing care food etc. are mentioned. The supplement form can be applied to solid agents and liquid agents, but solid agents such as tablets, capsules and granules are preferred. By taking such cosmetics, foods, and supplements, intestinal environment improving action, immunostimulating action, beauty / skin-beautifying action and the like can be expected.

The collagen molecular weight reduction method of the present invention is characterized by having a step of fermenting collagen with lactic acid bacteria. In the fermentation process, the aforementioned collagen and lactic acid bacteria can be used, and the fermentation can be performed under the conditions described above.

(2) Method for producing ornithine or citrulline The method for producing ornithine according to the present invention comprises a step of fermenting collagen with lactic acid bacteria. In addition, the method for producing citrulline of the present invention includes a step of fermenting collagen with lactic acid bacteria. In any of the production methods, the aforementioned collagen and lactic acid bacteria can be used, and fermentation can be performed under the aforementioned conditions. Following the step of fermenting collagen with lactic acid bacteria, a step of hydrolyzing the fermented product may be included. In addition, after the step of fermenting collagen with lactic acid bacteria, a step of removing lactic acid bacteria from the fermented product or a step of hydrolyzing the fermented product may be performed. Lactic acid bacteria can be removed from the fermented product by known methods such as filter filtration and centrifugation.

The fermented product obtained through the step of fermenting collagen with lactic acid bacteria contains ornithine and / or citrulline in addition to the degradation product of the raw collagen. Furthermore, the raw material collagen undegraded product may also be included. Since these are all useful ingredients, they can be taken as they are. Ornithine and / or citrulline can also be used after purification. Ornithine and / or citrulline can be purified by a known method for purifying these amino acids, such as separation with an ion exchange resin or fractional crystallization using a difference in solubility.

(3) Whitening composition The whitening composition of the present invention comprises the fermented product. The fermented product has a skin whitening effect by suppressing the synthesis of melanin by pigment cells. The skin whitening effect is preferably due to suppression of tyrosinase gene expression. It is generally known that skin tanning and spots are caused by the deposition of melanin in pigment cells. Melanin is synthesized from tyrosine, a kind of amino acid, by the action of tyrosinase. In the present invention, melanin deposition can be prevented by suppressing the expression of the tyrosinase gene, and a skin whitening effect can be achieved.

In the whitening composition of the present invention, the amount of the fermented product is not particularly limited, but is preferably 0.01 to 30% by weight, and more preferably 0.1 to 10% by weight.

The whitening composition may contain an optional component usually used in cosmetics in addition to the fermented product. Examples of such optional components include surfactants, alcohols, humectants, thickeners, preservatives, antioxidants, chelating agents, pH adjusters, fragrances, dyes, pigments, ultraviolet absorbers, vitamins , Amino acids, medicinal ingredients, plant extracts and the like.

The dosage form of the whitening composition is not particularly limited and can be prepared into a dosage form commonly used in cosmetics. For example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, foundations And spray.

The whitening composition exhibits a skin whitening effect when applied to the skin as a cosmetic. Examples of the application location include skin such as the face, body, and limbs.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited only to these.

(1) Production of fermented material (Example 1)
(1-1) Preparation of Collagen Peptide Decalcification was performed by immersing tilapia scales in dilute hydrochloric acid for 1 hour, followed by neutralization and washing with water using caustic soda. The scales after washing were poured into hot water, and gelatin was extracted at 95 ° C. over 4 hours. 0.4 to 0.8% by weight of protease was added to the solid content of the resulting gelatin solution, and an enzyme reaction was performed at 38 ° C. After completion of the reaction, after adding 1 to 2% by weight of activated carbon to the solid content of the enzyme reaction solution, filter filtration using diatomaceous earth as a filter aid, vacuum concentration and heat sterilization (85 ° C, 15 minutes) The powdered collagen peptide was obtained by spray drying.

(1-2) As a culture before production of a fermented product, the present inventors separated into 100 ml of a medium containing 25% by weight of the collagen peptide obtained in (1-1), 1% by weight of glucose, and the balance water. Lactococcus lactis subspecies lactis strain RT221 (Lactococcus lactis subsp. Lactis RT221) was inoculated and cultured at 30 ° C. for 24 hours. Subsequently, as the main culture, inoculation was performed so that the preculture solution was 2% by weight with respect to a medium containing 25% by weight of the collagen peptide obtained in (1), 1% by weight of glucose, and the balance being water. The cells were cultured at 24 ° C. for 24 hours. After culturing, the mixture was sterilized by heating and spray-dried to obtain a powdered fermented product.

(1-3) Measurement of amino acid composition A sample obtained by filtering lactic acid bacteria from the fermented product obtained in (1-2) above is used as a sample, the sample is hydrolyzed by a conventional method, and the amino acid composition is determined by an amino acid automatic analysis method. It was measured. By the same hydrolysis method and amino acid automatic analysis method, the amino acid composition of the collagen peptide of (1-1) before the fermentation treatment was also measured. The results are shown in Table 1.

In the collagen peptide before lactic acid bacteria fermentation, ornithine and citrulline were both below the detection limit (10 mg / 100 g), whereas 100 g of lactic acid bacteria fermented product contained 1.99 g of ornithine and 439 mg of citrulline. It was confirmed that the content of ornithine and citrulline increased by fermentation.

(2) Measurement of inhibition of pigmentation of fermented product (Examples 2 to 4, Reference Example 1, Comparative Examples 1 and 2)
(2-1) Cultivation of pigment cells Lactic acid bacteria were removed from the fermented product obtained in (1-2) above by filtration to obtain lactic acid fermented collagen. This lactic acid fermented collagen was added to the cell culture medium (derma-life) to a final concentration of 10 μg / mL, 100 μg / mL, or 500 μg / mL (Examples 2 to 4). Normal human cultured pigment cells were cultured in this medium for 7 days. Photographs were periodically taken from the beginning of the culture, and the proliferation and coloring of pigment cells were visually observed.

The same operation as in Examples 2 to 4 was performed except that endothelin 1 was added to the cell culture medium to a final concentration of 50 nM instead of lactic acid fermented collagen (Reference Example 1). Endothelin 1 was used as a positive control having tyrosinase induction ability (melanin production promoting action).

Except not adding lactic acid fermentation collagen, the same operation as Example 2-4 was performed (comparative example 1). Moreover, it replaced with lactic acid fermentation collagen and performed the same operation as Examples 2-4 except having added hydroquinone to the cell culture medium so that it might become a final concentration of 25 micromol (comparative example 2). Hydroquinone was used as a negative control that inhibits tyrosinase activity.

(2-2) Measurement of pigmentation-inhibiting ability On the fifth day after the start of culture, the number of pigment cells was measured by measuring the actual number of melanocytes found in any one visual field under a phase-contrast microscope. The average value of the measured values was calculated. The result is shown in FIG.

On the seventh day after the start of the culture, the cells were fixed with formalin, dye cells having a tyrosinase function were stained by dopa staining, and coloring of the cells was measured by an absorption method. The measurement result was corrected by the number of cells, and the degree of coloring per cell was calculated. The calculation results are shown in FIG.

As shown in FIG. 1, dose dependency was observed in the pigment cell growth inhibitory ability of the lactic acid fermentation collagen addition group (Examples 2 to 4). Further, as shown in FIG. 2, in Example 3, the degree of coloring due to dopa staining was lower than in Comparative Example 1 and Reference Example 1. On the other hand, in Example 3, the degree of coloring by dopa staining was higher than that in Comparative Example 2. Dopa is an intermediate in the biosynthetic pathway from tyrosine to melanin due to the activity of tyrosinase, and the degree of coloration due to dopa staining correlates with the intensity of tyrosinase activity.

(2-3) Measurement of gene expression level Total RNA was extracted from the pigment cells obtained in Examples 2 to 4, Reference Example 1 and Comparative Examples 1 to 2, and tyrosinase gene, TRP- was analyzed by quantitative RT-PCR. The expression levels of 1 gene and TRP-2 gene were measured. The TRP-1 gene and the TRP-2 gene are genes that function downstream of the tyrosinase gene. The results are shown in FIGS. 3 to 5, the vertical axis represents the ratio of the expression level of each gene to the expression level of β-actin.

As shown in FIGS. 3 to 5, the expression of each gene was significantly enhanced in the endothelin 1 added group (Reference Example 1) compared to the non-added group (Comparative Example 1). In contrast, the lactic acid fermented collagen addition group (Examples 2 to 4) decreased gene expression in a dose-dependent manner for the tyrosinase gene (FIG. 3). The hydroquinone-added group (Comparative Example 2) was not significantly different from the non-added group (Comparative Example 1) (FIG. 3). Hydroquinone is known to have a mechanism of action that inhibits only the enzyme activity of tyrosinase, and does not affect tyrosinase gene expression and protein synthesis.

The above results show that lactic acid-fermented collagen suppresses the expression of tyrosinase from pigment cells and controls the enzyme function of tyrosinase, thereby producing a whitening effect.

Claims (8)

Fermented product of collagen by lactic acid bacteria. The fermented product according to claim 1, wherein the lactic acid bacterium is a microorganism of the genus Lactococcus. The fermented product according to claim 2, wherein the Lactococcus microorganism is Lactococcus lactis. The fermented product according to any one of claims 1 to 3, wherein the collagen is fish-derived collagen. A method for producing ornithine or citrulline, comprising a step of fermenting collagen with lactic acid bacteria. The composition for whitening containing the fermented material of any one of Claims 1-4. The whitening composition according to claim 6, wherein the whitening is caused by suppression of melanin synthesis. The whitening composition according to claim 7, wherein the suppression of melanin synthesis is caused by suppression of tyrosinase gene expression.

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