A composition for improving drying containing the culture supernatant of lactic acid bacteria.

Abstract

The objective is to provide a composition that provides excellent dryness-improving effects to mucous membranes or skin. More specifically, the objective is to provide a dryness-improving composition that exhibits a high saliva secretion-promoting effect and an oral dryness-improving effect, regardless of taste components, and a dryness-improving composition that provides a high moisturizing effect to the skin. [Solution] A composition for improving drying, containing the culture supernatant of lactic acid bacteria.

Description

Technical Field

[0001] The present invention relates to a composition for improving dryness, which contains a culture supernatant of lactic acid bacteria.

Background Art

[0002] For humans, dryness is known to have various adverse effects. Examples of dryness that causes adverse effects include dryness of mucous membranes and dryness of the skin.

[0003] Among the dryness of mucous membranes, the state where the inside of the mouth is dry is called xerostomia (dry mouth). Xerostomia is a disease in which saliva is not secreted or the amount of secreted saliva is small, and mainly complains of dryness in the mouth and thirst in the throat. The number of patients in Japan is said to be 8 million. Specific symptoms include dysphagia, increased stickiness in the mouth, pain in the tongue, bad breath, easy biting in the oral cavity, frequent occurrence of dental caries, periodontal disease, cracks on the surface of the tongue, etc. (Non-Patent Document 1).

[0004] As one of the methods for improving xerostomia, activation of functions originally possessed by humans, that is, a method of promoting saliva secretion, has been studied. From this perspective, saliva secretion promoters using umeboshi, organic acids, vitamins, sansho pepper, etc. have been developed (Patent Documents 1 to 4). However, all of these promote saliva secretion by components (taste components) that exhibit tastes such as sourness and spiciness, and there is a problem that they are difficult to use for those with unsuitable palatability.

[0005] In addition, as a saliva secretion promoter that does not contain taste components, a composition containing a mixed culture solution of lactic acid bacteria, yeast, and Bacillus natto has also been developed (Patent Document 5). However, such a composition does not have a sufficient saliva secretion promoting effect.

[0006] On the other hand, dryness of the skin can cause a decrease in the barrier function and itching. In order to maintain the original function of the skin, it is important to appropriately maintain the water content of the skin and maintain the water retention function of the skin (Non-Patent Document 2).

[0007] Compositions containing moisturizers such as petrolatum or glycerin are commonly used as skincare cosmetics or topical skin preparations for moisturizing purposes (Non-Patent Document 3). In addition, topical skin preparations utilizing microorganisms have been developed, including those containing a fermented liquid obtained by fermenting germinated brown rice with Saccharomyces verona (Patent Document 6). However, none of these provide sufficient moisturizing effects. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 56-022719 [Patent Document 2] Japanese Patent Application Publication No. 7-101856 [Patent Document 3] Japanese Patent Publication No. 2006-199670 [Patent Document 4] Japanese Patent Publication No. 2011-068642 [Patent Document 5] Patent No. 6218280 [Patent Document 6] Japanese Patent Publication No. 2018-002607 [Non-patent literature]

[0009] [Non-Patent Document 1] Yoko Ando, ​​"Dry Mouth (Xerostomia) - Lessons Learned from the Discovery of Sjögren's Syndrome," Journal of the Japanese Society of Gnathology, The Science of Occlusion 31.1-2 (2011): pp. 114-118. [Non-Patent Document 2] Kenji Maeda, "Skin and Chemistry - The Boundary Between Inside and Outside," Chemistry and Education 65.2 (2017): pp. 84-85. [Non-Patent Document 3] Tetsuji Hirao, "Moisturizing Mechanism of the Skin," Journal of the Japanese Society of Cosmetic Scientists 37.2(2013):p.95-100. [Non-Patent Document 4] Kenichi Ishibashi et al., "Regulation of Aquaporin 3 Expression in Skin," Cosmetology Research Report 11 (2003): pp. 67-70. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The objective is to provide a composition that provides excellent dryness-improving effects to mucous membranes or skin. More specifically, the objective is to provide a composition that does not contain taste components, exhibits a high saliva secretion-promoting effect and an oral dryness-improving effect, and a composition that provides a high moisturizing effect to the skin.

[0011] In this specification, "improvement of dryness" refers to the effect of promoting saliva secretion and improving dryness in the oral cavity when the dryness-improving composition of the present invention is ingested or administered orally. It also refers to the effect of maintaining or increasing the moisture content of the skin when the dryness-improving composition of the present invention is applied to the skin. [Means for solving the problem]

[0012] The applicant has found that a composition containing the culture supernatant of lactic acid bacteria has an excellent drying improvement effect.

[0013] A first aspect of the present invention is a drying-improving composition comprising the culture supernatant of lactic acid bacteria.

[0014] A second aspect of the present invention is a composition for promoting saliva secretion, comprising the dryness-improving composition of the present invention. Preferably, the composition for promoting saliva secretion of the present invention is a food product.

[0015] A third aspect of the present invention is a composition for moisturizing the skin, comprising the dryness-improving composition of the present invention. Preferably, the composition for moisturizing the skin of the present invention is a cosmetic. [Effects of the Invention]

[0016] The composition for improving drying of the present invention provides a high drying improvement effect. More specifically, when orally ingested or administered, it brings about the effects of promoting saliva secretion and improving dryness in the oral cavity. Furthermore, since the composition of the present invention does not contain flavor components, it has the advantage that it can be used regardless of the taste preference of the food to which it is added and the taste preference of the ingestion or administration subject without affecting the taste of the added food. On the other hand, when the composition of the present invention is applied to the skin, it can also provide a high moisturizing effect on the skin.

Brief Description of the Drawings

[0017] [Figure 1] Shows the measurement results of the relative expression level of AQP3 mRNA in epidermal keratinocytes described in Example 8.

Modes for Carrying Out the Invention

[0018] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

[0019] 1. Composition for improving drying containing culture supernatant of lactic acid bacteria One aspect of the present invention is a composition for improving drying containing the culture supernatant of lactic acid bacteria. The composition for improving drying of the present invention may contain the culture supernatant of lactic acid bacteria in any amount as long as it can exhibit a drying improvement effect. For example, but not limited to this, it can be 0.1% by weight or more. Also, the composition of the present invention may consist only of the culture supernatant of lactic acid bacteria.

[0020] (Culture supernatant of lactic acid bacteria) In the present invention, the "culture supernatant of lactic acid bacteria" refers to the supernatant obtained by removing lactic acid bacteria from the medium in which lactic acid bacteria have been cultured. In the present invention, the culture supernatant of lactic acid bacteria may be prepared by any method. For example, the culture supernatant of lactic acid bacteria of the present invention can be obtained by inoculating lactic acid bacteria into a medium, culturing them, and then recovering the supernatant.

[0021] Lactic acid bacteria are a general term for obligate anaerobic microorganisms that produce lactic acid from sugars through metabolism. In this invention, the lactic acid bacteria used to prepare the culture supernatant of lactic acid bacteria are not limited to these, but include the genera Lactobacillus, Lactiplantibacillus, Lacticaseibacillus, Levilactobacillus, Lactococcus, Streptococcus, and Enterococcus. Examples of bacteria belonging to the genera Enterococcus, Pediococcus, Leuconostoc, Limosilactobacillus, Latilactobacillus, Ligilactobacillus, and Bifidobacterium can be cited, with bacteria belonging to the Lactobacillus genus being preferred. While not limited to these, examples of bacteria belonging to the genus Lactobacillus include Lactobacillus crispatus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus paragasseri, and Lactobacillus johnsonii, with Lactobacillus crispatus being preferred. More preferably, the lactic acid bacteria in this invention is Lactobacillus crispatus strain KT-11. The KT-11 strain is deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) under accession number FERM BP-11332.

[0022] Lactic acid bacteria can be cultured under any conditions as long as they can be cultured. Any culture medium suitable for culturing lactic acid bacteria can be used. The components of the culture medium can be any components commonly used in culture media for lactic acid bacteria, and are not limited to these, but include carbon sources, nitrogen sources, inorganic salts, and organic components. As carbon sources, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and molasses can be used depending on their assimilation properties. As nitrogen sources, for example, ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, as well as organic nitrogen-containing substances such as casein hydrolysates, soy protein hydrolysates, potato hydrolysates, and shochu lees can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium sulfate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Examples of organic components that can be used include peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract. Fatty acids may also be added from the viewpoint of promoting the growth of lactic acid bacteria, and examples of nonionic surfactants that can be used include glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester, propylene glycol fatty acid ester, and sucrose fatty acid ester.

[0023] The amount of lactic acid bacteria inoculated into the culture medium is not particularly limited, but can be, for example, 0.001% by weight or more. The culture temperature can be 20 to 55°C, preferably 25 to 45°C, and more preferably 30 to 40°C. The culture time can be 1 to 72 hours, preferably 24 to 72 hours. Culture can be carried out under either aerobic or anaerobic conditions. When culturing under aerobic conditions, the oxygen concentration can be, for example, 0.5% to 30%. When culturing under anaerobic conditions, the oxygen concentration can be, for example, 0% to less than 0.5%.

[0024] To collect the supernatant from the culture medium in which lactic acid bacteria are cultured, for example, centrifugation can be used. For example, the supernatant can be collected by centrifugation at 4000 rpm for 5 to 20 minutes. The supernatant collected after centrifugation may be used as is as the culture supernatant for the lactic acid bacteria of the present invention. Alternatively, the supernatant after centrifugation can be subjected to additional processing, and the supernatant after additional processing can also be used as the culture supernatant for the lactic acid bacteria of the present invention.

[0025] Additional processing methods include heating, filtration, and concentration. Heating, filtration, and concentration can all be carried out by known methods. Heating can be performed, for example, at a temperature of 80°C to 130°C for 1 to 30 minutes. An autoclave may be used for heating. Filtration can be performed, for example, using filter paper or a filter. Concentration can be performed by boiling or by removing water from the supernatant using a rotary evaporator or centrifugal evaporator. Additional processing can be performed in any order.

[0026] (Additional ingredients) The drying-improving composition of the present invention may contain additional components. These additional components may include, but are not limited to, pH adjusters, preservatives, emulsifiers, thickeners, diluents, flavorings, colorants, sweeteners, antioxidants, and fluidizing agents. The amount of additional components can be any amount as long as the composition of the present invention exhibits a drying-improving effect, for example, 0.01 to 99.9% by weight. The drying-improving composition of the present invention may also consist of the culture supernatant of lactic acid bacteria.

[0027] (Form of composition for improving drying) The drying-improving composition of the present invention may take any form, but is not limited thereto, including solids (powder, granules, tablets, etc.), liquids (solutions, suspensions, emulsions, etc.), and semi-solids (gels, etc.). The drying-improving composition of the present invention can be prepared by any method. For example, but is not limited thereto, treatments such as freeze-drying, spray-drying, granulation, concentration, emulsification, and mixing can be used to prepare the drying-improving composition.

[0028] (Effects of the composition for improving drying) The present invention's composition for improving dryness, by containing the culture supernatant of lactic acid bacteria, can provide an excellent dryness-improving effect. Specifically, when taken orally or administered, it can promote saliva secretion and improve dryness in the oral cavity. Furthermore, when applied to the skin, it can provide an excellent moisturizing effect.

[0029] (Active ingredients in the culture supernatant) As described in the Examples section, the drying improvement effect of the drying improvement composition of the present invention is not theoretically constrained, but it is thought to be brought about by the production of an active ingredient through the cultivation of lactic acid bacteria, and by the action of this active ingredient on mucous membranes or skin. Furthermore, as will be described in detail in the Examples section, when we searched for the active ingredient of the composition of the present invention, the results of the analysis suggested that the active ingredient is a compound that has high water solubility and can be fractionated by column chromatography using TOYOPEAL HW-40c as a packing material.

[0030] 2. Composition for promoting saliva secretion Another aspect of the present invention is a composition for promoting saliva secretion. A composition for promoting saliva secretion includes the dryness-improving composition of the present invention. Compositions for promoting saliva secretion include, but are not limited to, foods and personal care products (mouthwash, oral moisturizing gel, oral spray, toothpaste, etc.). In addition to the foods described later, compositions for promoting saliva secretion can take the form of, but are not limited to, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolvable tablets), granules, powders, pills, capsules, oral liquids (elixirs, suspensions, emulsions, aromatics, limonases), syrups, oral jellies, and other orally administered preparations, oral tablets (gums, sublingual tablets, lozenges, drops, buccal tablets, adhesive tablets), semi-solid oral preparations, ointments, gargles, etc.

[0031] The composition for promoting saliva secretion is preferably a food. In this specification, "food" includes beverages. Foods containing the drying-improving composition of the present invention are not particularly limited as long as they can provide a saliva secretion-promoting effect, but examples include cooked rice, bakery foods (bread, cakes, etc.), confectionery (cookies, gummies, candy, gum, jelly, chocolate, snack foods, rice crackers, etc.), nutritional supplements, health functional foods (foods with functional claims, nutritional functional foods, foods for specified health uses), supplements, and beverages (juices, soft drinks, energy drinks, alcoholic beverages, non-alcoholic beverages, etc.). In the food of the present invention, the content of the drying-improving composition of the present invention is not particularly limited as long as it can exert the drying-improving effect of the drying-improving composition of the present invention. The content of the drying-improving composition can be, for example, 0.1% by weight or more, 0.5% by weight or more, or 1% by weight or more, based on the total weight of the food, but are not limited to these. Furthermore, in the food of the present invention, the content of the drying-improving composition of the present invention as a daily intake amount is, for example, 0.1 to 100 g, preferably 0.5 g to 80 g, and more preferably 1 to 50 g, although this is not limited to these examples.

[0032] The drying-improving composition of the present invention can be added to food in any way. For example, the drying-improving composition of the present invention may be used as a food ingredient, or it may be added to existing food products. By containing the drying-improving composition of the present invention, the food product of the present invention can provide an excellent saliva secretion-promoting effect when ingested orally.

[0033] 3. Compositions for moisturizing the skin Another aspect of the present invention is a composition for moisturizing the skin. The composition for moisturizing the skin includes the dryness-improving composition of the present invention. Examples of compositions for moisturizing the skin include, but are not limited to, cosmetics, moisturizers, and bath additives.

[0034] The composition for moisturizing the skin in the present invention is preferably a cosmetic. The cosmetic is not particularly limited as long as it can provide a moisturizing effect to the skin, but examples include skincare products (lotions, emulsions, serums, sunscreens, hand creams, lip balms, eye creams, etc.) and makeup products (makeup bases, foundations, etc.). In the cosmetic of the present invention, the content of the dryness-improving composition of the present invention is not particularly limited as long as it can exert the dryness-improving effect of the dryness-improving composition of the present invention. The content of the dryness-improving composition is, for example, not limited to these, but can be 0.1% by weight or more, 0.5% by weight or more, or 1% by weight or more, based on the total weight of the cosmetic.

[0035] The dryness-improving composition of the present invention can be added to cosmetics in any way. For example, the dryness-improving composition of the present invention may be used as a cosmetic ingredient, or it may be added to existing cosmetics. Cosmetics containing the dryness-improving composition of the present invention can provide excellent moisturizing effects to the skin.

[0036] 4. Examples The embodiments of the present invention will be described in detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention.

[0037] Example 1. Preparation of a composition for improving drying. Compositions 1 to 3 for improving drying were prepared according to the following procedure.

[0038] (Composition for improving dryness 1) Lactobacillus crispapus KT-11 strain was inoculated into a culture medium for lactic acid bacteria at a concentration of 0.001% by weight and cultured at 37°C for 72 hours. After culturing, the culture was centrifuged (4,000 rpm, 10 minutes) and the supernatant was collected. The supernatant was heat-treated (121°C, 15 minutes), filtered to remove precipitate, and obtained the drying improvement composition 1 of the present invention.

[0039] (Composition for improving dryness 2) The supernatant liquid obtained after heat treatment using the same procedure as for composition 1 for improving drying was boiled and concentrated to 2.5 times the concentration before concentration. The supernatant liquid after concentration was designated as composition 2 for improving drying.

[0040] (Composition for improving dryness 3) The supernatant liquid obtained after heat treatment using the same procedure as for drying-improving composition 1 was boiled and concentrated to five times its original concentration. The supernatant liquid after concentration was designated as drying-improving composition 3.

[0041] Example 2. Evaluation of the saliva secretion promoting effect of the dryness improving composition of the present invention 1 The saliva secretion-promoting effect of the present invention's drying-improving composition was evaluated by sensory testing using cucumbers immersed in various treatment solutions.

[0042] (1) Test method Cucumbers cut to a thickness of 0.5 cm were immersed in the following treatment solutions. After standing in the refrigerator for 3 hours, the cucumbers were removed, placed between sheets of paper, and left to stand for 1 minute to drain the water. The degree of saliva secretion when eating the resulting cucumbers was evaluated by three panelists. The degree of saliva secretion was evaluated on a 7-point scale (7: very good, 6: good, 5: slightly good, 4: average, 3: slightly bad, 2: bad, 1: very bad). The Brix value of the sugar solution refers to the mass percentage of sugar in the solution.

[0043] [Processing solution] Drying Improvement Composition 1: Drying Improvement Composition 1 prepared in Example 1 Drying Improvement Composition 2: Drying improvement composition 2 prepared in Example 1 Drying Improvement Composition 3: Drying Improvement Composition 3 prepared in Example 1 Saltwater: 3 w / w% saline solution (equivalent to the amount of salt in composition 2 for improving drying) Sugar water: 45.5 w / w% sugar water (equivalent to the Brix value of composition 3 for improving drying) Salt and sugar water: Water containing 3 w / w% salt and 45.5 w / w% sugar. Salted rice malt: Commercially available (granular) Liquid salt koji: Commercially available product (liquid) Pickling mix: Commercially available (liquid)

[0044] (2) Results The results are shown in Table 1 below. Each score is the average of the three panelists.

[0045] [Table 1]

[0046] These results demonstrate that the drying-improving composition of the present invention has a saliva-secreting effect equivalent to or greater than that of existing seasonings. In particular, the drying-improving composition of the present invention showed a higher saliva-secreting effect than saline solution with a salt concentration adjusted to the same level as the composition of the present invention, and sugar solution with a Brix value adjusted to the same level as the composition of the present invention. These results suggest that the drying-improving composition of the present invention can promote saliva secretion regardless of the taste components, more specifically, salt and sugar content.

[0047] Example 3. Evaluation of the saliva secretion promoting effect of the present invention's dryness improving composition 2 Gummies containing the drying-improving composition of the present invention were prepared, and the saliva secretion-promoting effect of the drying-improving composition of the present invention was evaluated by sensory testing. The evaluation was performed on a 5-fold concentrate of a culture medium without cultured lactic acid bacteria (uncultured medium) and on drying-improving composition 3 (prepared in Example 1).

[0048] (1) Making gummies The gummies were prepared using the following procedure. The control was prepared similarly, except that the drying-improving composition and a 5-fold concentrate of the uncultured medium were not added. The detailed composition of the gummies is shown in Table 2.

[0049] [procedure] 1. Water and sugar were placed in a beaker and mixed. 2. The uncultured medium or drying-improving composition 3 was further added and mixed to obtain a gummy solution. The amount of uncultured medium and drying-improving composition 3 added was 3% by weight relative to the gummy solution. 3. Divide the gummy solution into 13.5g portions and heat them in a microwave (500W, 30 seconds). 4. Add 2.5g of gelatin powder and stir for 1 minute. 5. Heat further in the microwave (500W, 10 seconds) and stir for 30 seconds. 6. Chilled in the refrigerator for 3 hours. 7. The gummy candies were removed from the beaker, divided into 5 equal parts, and used for evaluation.

[0050] [Table 2]

[0051] (2) Evaluation method The degree of saliva secretion from the prepared gummies was evaluated by five panelists. The degree of saliva secretion was evaluated on a 7-point scale (7: very good, 6: good, 5: slightly good, 4: average, 3: slightly bad, 2: bad, 1: very bad).

[0052] (3) Results The results are shown in Table 3 below. Each score represents the average of the five panelists.

[0053] [Table 3]

[0054] The saliva-stimulating effect of gummies containing the drying-improving composition of the present invention surpassed that of controls without the drying-improving composition, as well as gummies containing uncultured culture medium. This result suggests that the saliva-stimulating effect of the drying-improving composition of the present invention is not due to components derived from the uncultured culture medium, but rather to components produced by the cultivation of lactic acid bacteria.

[0055] Example 4. Evaluation of the concentration dependence of the saliva secretion promoting effect 1 Gummies containing the drying-improving composition of the present invention at various concentrations were prepared, and the effect of the drying-improving composition content in the food on the saliva secretion-promoting effect was evaluated. Orange juice was selected as the base material for the gummies because it has a sour taste. As the drying-improving composition, drying-improving composition 3 prepared in Example 1 was used.

[0056] (1) Making gummies The gummies were prepared using the following procedure. The control was prepared in the same manner, except that the drying-improving composition was not added. The detailed composition of the gummies is shown in Table 4.

[0057] [procedure] 1. 100% orange juice and sugar were placed in a beaker. Drying-improving composition 3 was added and mixed to make a gummy solution. The amounts of drying-improving composition 3 added were 1% by weight, 3% by weight, 5% by weight, and 10% by weight relative to the gummy solution. 2. Divide the gummy solution into 13.5g portions and heat them in a microwave (500W, 30 seconds). 3. Add 1.5g of powdered gelatin and stir for 1 minute. 4. Heat further in the microwave (500W, 10 seconds) and stir for 30 seconds. Repeat this process twice. 5. Chilled in the refrigerator for 3 hours. 6. The gummies were removed from the beaker, divided into 5 equal parts, and used for evaluation.

[0058] [Table 4]

[0059] (2) Evaluation method The degree of saliva secretion from the prepared gummies was evaluated by five panelists. The degree of saliva secretion was evaluated on a 7-point scale (7: very good, 6: good, 5: slightly good, 4: average, 3: slightly bad, 2: bad, 1: very bad).

[0060] (3) Results The results are shown in Table 5 below. Each score represents the average of the five panelists.

[0061] [Table 5]

[0062] All gummies containing the drying-improving composition showed increased saliva secretion compared to the control group. This result suggests that combining the composition with acidity, which is generally known to stimulate saliva secretion, further enhances saliva production. Furthermore, it was suggested that the saliva-promoting effect increased with increasing amounts of the drying-improving composition added to the food.

[0063] Example 5. Evaluation of the concentration dependence of the saliva secretion promoting effect 2 We prepared gummies with a different base flavor than those in Examples 2-4, and evaluated the saliva secretion-promoting effect of the dryness-improving composition of the present invention.

[0064] (1) Method for making gummy candies The gummies were prepared using the following procedure. A 3-fold diluted apple juice concentrate (diluted apple juice) was used as the base for the gummies. For the drying-improving composition added to the gummies, the drying-improving composition 3 prepared in Example 1 was used. The control was prepared in the same manner except that the drying-improving composition was not added. Details of the gummy composition are shown in Table 6.

[0065] [procedure] 1. Diluted apple juice, sugar, and drying-improving composition 3 were placed in a beaker and mixed. The concentrations of the drying-improving composition were 3% by weight, 5% by weight, and 10% by weight relative to the finished gummy. 2. The beaker was heated in a microwave oven (500W, 30 seconds). 3. Add the gelatin powder and stir for 1 minute. 4. Heat in a microwave (500W, 20 seconds) and stir for 1 minute. 5. Heat in a microwave (500W, 10 seconds) and stir for 1 minute. 6. Chilled in the refrigerator for 3 hours. 7. Remove the gummy candy from the beaker, divide it into 5 equal parts, and use them for evaluation.

[0066] [Table 6]

[0067] (2) Evaluation method The degree of saliva secretion from the prepared gummies was evaluated by five panelists. The degree of saliva secretion was evaluated on a 7-point scale (7: very good, 6: good, 5: slightly good, 4: average, 3: slightly bad, 2: bad, 1: very bad).

[0068] (3) Results The results are shown in the table below. Each score is the average of the five panelists.

[0069] [Table 7]

[0070] The results above demonstrate that, even in apple juice-based gummies, the drying-improving composition of the present invention has the effect of promoting saliva secretion compared to the control. Therefore, it is suggested that the drying-improving composition of the present invention can promote saliva secretion regardless of the taste of the food to which the drying-improving composition is added.

[0071] Example 6. Evaluation of the effect of food type on saliva secretion promoting effect. Candy containing the drying-improving composition of the present invention at different concentrations was prepared, and the effect of the type of food on the saliva secretion-promoting effect was evaluated.

[0072] (1) Making candy Candy was prepared using the following procedure. For the drying-improving composition added to the candy, the drying-improving composition 3 prepared in Example 1 was used. The control was prepared in the same manner except that the drying-improving composition was not added. Details of the candy composition are shown in Table 8.

[0073] [procedure] 1. Sugar, water, and drying-improving composition 3 were placed in a pot and mixed to make a candy solution. The concentrations of drying-improving composition 3 were 1% by weight and 3% by weight relative to the candy. 2. The pot was placed over a fire and heated. The heat was turned off when the candy solution began to change color (about 2 minutes). 3. Using a measuring spoon, take some candy and place one spoonful at a time onto a piece of aluminum foil. 4. Chilled in the refrigerator for 1 hour. 5. The candy was peeled off the aluminum foil and used for evaluation.

[0074] [Table 8]

[0075] (2) Evaluation method The degree of saliva secretion from the prepared candy was evaluated by five panelists. The degree of saliva secretion was evaluated on a 7-point scale (7: very good, 6: good, 5: slightly good, 4: average, 3: slightly bad, 2: bad, 1: very bad).

[0076] (3) Results The results are shown in Table 9 below. Each score represents the average of the five panelists.

[0077] [Table 9]

[0078] As shown in Table 9, the drying-improving composition of the present invention was demonstrated to have a saliva-stimulating effect even in candy. Therefore, it is suggested that the drying-improving composition of the present invention has a saliva-stimulating effect without chewing, and that it has a saliva-stimulating effect regardless of the food to which it is added.

[0079] Example 7. Evaluation of the moisturizing effect of the present invention's composition for improving dryness in epidermal keratinocytes. The moisturizing effect of the dryness-improving composition of the present invention on the skin was evaluated by measuring the expression level of the aquaporin 3 (AQP3) gene in epidermal keratinocytes. AQP3 is a membrane protein expressed in epidermal cells and is thought to contribute to the maintenance of skin moisture, elasticity, and barrier function by transporting substances such as water, glycerin, and urea (Non-Patent Literature 4). In this example, the moisturizing effect of the dryness-improving composition of the present invention on the skin was evaluated by measuring the expression level of AQP3 mRNA in epidermal keratinocytes.

[0080] (1) Measurement of the relative expression level of AQP3 mRNA in skin keratinocytes First, PHK16-0b cells were suspended in HuMedia-KG2 medium and seeded into 12-well plates (3 × 10⁶). 5The culture medium was divided into cells / mL / well and incubated at 37°C and 5% CO2 for 24 hours. The medium was removed, and fresh antimicrobial medium containing the antimicrobial agent was added at a volume of 1 mL / well and incubated at 37°C and 5% CO2 for another 24 hours. The antimicrobial medium containing the antimicrobial agent was prepared by adding 0.5 mL of antimicrobial agents (gentamicin 50 mg / mL, amphotericin B 50 μg / mL) to 500 mL of HuMedia-KB2 medium and mixing.

[0081] After incubation, the culture medium was removed from the 12-well plate, and either the drying-improving composition medium or the antibacterial agent medium (control) shown in Table 10 was added at a volume of 1 mL / well. The cultures were then incubated at 37°C and 5% CO2 for 24 hours. As the drying-improving composition, drying-improving composition 1 prepared in Example 1 was used.

[0082] [Table 10]

[0083] After culturing, cells were harvested from each well, and RNA was extracted using the PureLink RNA mini kit (Invitrogen by Thermo Fisher Scientific). Reverse transcriptase was added to the extracted RNA, and cDNA was synthesized by reverse transcription.

[0084] The AQP3 gene was quantified using real-time PCR with the QuantStudio3 real-time PCR system (Thermo Fisher Scientific) based on the synthesized cDNA. The quantification was performed using a relative quantification method, with the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene selected as the internal standard. For the analysis of the results, the amount of the AQP3 gene was corrected for the amount of the GAPDH gene, and based on these results, the relative expression level of AQP3 mRNA in PHK16-0b cells relative to the control was determined. The results are shown in Figure 1.

[0085] (2) Results As is clear from Figure 1, compared to the control, the relative expression level of AQP3 mRNA increased in all cells treated with the culture medium containing the dryness-improving composition. It is thought that an increase in AQP3 expression increases the transport of water and moisturizing components such as glycerin and urea into the cells, thereby increasing the skin's moisture content and improving its moisturizing ability. Therefore, it is suggested that the dryness-improving composition of the present invention provides a moisturizing effect on the skin by increasing the expression of AQP3 mRNA in epidermal keratinocytes. The moisturizing effect of existing topical moisturizing agents is brought about by actions such as replenishing moisture to the skin and preventing evaporation of moisture from the skin. Therefore, the moisturizing effect based on the increased expression of AQP3 mRNA by the dryness-improving composition of the present invention is fundamentally different from the moisturizing effect brought about by existing topical agents in that it enhances the skin's own moisture retention capacity. Furthermore, since an increase in AQP3 mRNA expression leads to an improvement in the skin's own moisture retention capacity, it is expected to provide a long-lasting moisturizing effect, unlike the temporary moisturizing effect of applying topical agents.

[0086] Example 8. Comparison of skin moisturizing effects in humans The skin moisturizing effect of the present invention's dryness-improving composition was compared with existing moisturizing ingredients. The test was conducted on five healthy adult men and women.

[0087] (1) Preparation of test composition The test composition was prepared according to the following procedure. (Contrast) A 20% glycerin solution was prepared by mixing 20 μL of glycerin with 80 μL of water. (1% solution) The drying-improving composition 1 prepared in Example 1 was diluted 10-fold with water. 10 μL of this diluted solution was mixed with 20 μL of glycerin and 70 μL of water to prepare a 1% solution. (5% solution) 5 μL of the drying-improving composition 1 prepared in Example 1 was mixed with 20 μL of glycerin and 75 μL of water to make a 5% solution. (10% solution) 10 μL of the drying-improving composition 1 prepared in Example 1 was mixed with 20 μL of glycerin and 70 μL of water to make a 10% solution.

[0088] (2) Test method The moisturizing effect on the skin was evaluated by measuring the stratum corneum moisture content using the procedure described below. The stratum corneum moisture content was measured inside the forearm using a Moisture Checker MY-808S (Scala Co., Ltd.). Each test composition was tested at two locations per person.

[0089] [procedure] 1. The moisture content of the test area was measured using a moisture checker and used as the data before application. 2. 10 μL of each test composition was applied and held for 5 minutes. 3. The test composition was wiped off, and the moisture content was measured using a moisture checker to obtain the data after application.

[0090] (3) Results The results are shown in Table 11 below. The stratum corneum moisture content values ​​are the average of the two measurement locations.

[0091] [Table 11]

[0092] As is clear from Table 11, the test compositions containing the dryness-improving composition of the present invention showed higher stratum corneum moisture content in almost all subjects compared to the control. This result indicates that the dryness-improving composition of the present invention can provide a moisturizing effect on the skin when applied to the skin.

[0093] Example 9. Search for active ingredients To search for the active ingredient in the drying-improving composition of the present invention, we attempted to isolate the component.

[0094] (1) Solvent extraction fractionation 200 mL of the drying improvement composition 1 prepared in Example 1 was fractionated with 200 mL of hexane, and the lower aqueous phase was separated using a separatory funnel. The aqueous phase was returned to the separatory funnel, and fractionation was performed again with the same amount of hexane to separate the aqueous phase. Subsequently, fractionation was performed with 200 mL of ethyl acetate to separate the aqueous phase. The aqueous phase was returned to the separatory funnel, and fractionation was performed again with the same amount of ethyl acetate to separate the aqueous phase. Subsequently, fractionation was performed with 200 mL of water-saturated butanol to separate the aqueous phase. The aqueous phase was returned to the separatory funnel, and fractionation was performed again with the same amount of water-saturated butanol to separate the aqueous phase. Water-saturated butanol was obtained by mixing 1-butanol with distilled water, allowing it to stand, and collecting the upper layer after it separated into two layers. Since activity was confirmed in the aqueous phase after fractionation with water-saturated butanol, this aqueous phase was used in the next separation operation. The presence or absence of activity was confirmed by measuring the expression level of AQP3 mRNA using epidermal keratinocytes, according to the method described in Example 7.

[0095] (2) Activated carbon gel filtration flask Next, the components were separated by gel filtration chromatography using activated carbon. First, the activated carbon (Fujifilm Wako Pure Chemical Industries, product code 031-02135) was replaced three times with 100% by volume acetone. Subsequently, it was replaced sequentially with 60%, 40%, 20%, 10%, and 5% by volume acetone aqueous solutions. Finally, it was replaced three times with deionized water. The activated carbon was packed into the column, and the aqueous phase obtained by solvent extraction was passed through the column. 100 mL of the aqueous phase recovered after solvent extraction was taken and concentrated to 50 mL using a rotary evaporator before use.

[0096] Next, acetone and deionized water were mixed to prepare acetone aqueous solutions of 0, 5, 10, 20, 40, 60, 80, and 100% by volume. 45 mL of each acetone aqueous solution was passed through the column in order from the lowest concentration, and the fraction was collected each time.

[0097] After removing the solvent from each of the obtained fractions using a rotary evaporator, 3 mL of deionized water was added to collect the separated components. Of the obtained fractions, those whose activity was confirmed were used in the next separation procedure.

[0098] (3) Column chromatography (separation by molecular weight) TOYOPEAL HW-40c (particle size 75 μm, pore size 5 nm, Tosoh Corporation) was used as the packing material. Approximately 40 mL of the packing material was first equilibrated by substituting it three times with 100 vol% methanol. Subsequently, it was sequentially substituted with 40 mL each of 80%, 60%, 40%, 20%, 10%, and 5 vol% methanol aqueous solutions. Finally, it was substituted three times with deionized water.

[0099] A packing material replaced with deionized water was packed into a column (volume 65 mL). 1 mL of fraction 1 or 2 obtained by activated carbon gel filtration chromatography was added. Deionized water was used as the developing solvent, and 30 fractions were collected, with each fraction set to 6.5 mL. The solvent was removed from each of the obtained fractions using a centrifugal evaporator. 1 mL of deionized water was added to each fraction, and the fractions were collected and their activity checked. Active fractions were observed.

[0100] The above results suggest that the active ingredient of the drying-improving composition of the present invention is a compound that has high water solubility and can be fractionated by column chromatography using TOYOPEAL HW-40c as a packing agent.

[0101] summary As described above, the drying-improving composition of the present invention, by containing the culture supernatant of lactic acid bacteria, was shown to promote saliva secretion when ingested orally, regardless of taste components, particularly salt and sugar. Furthermore, it was shown that when the drying-improving composition of the present invention is combined with acidity, which is known to promote saliva secretion, saliva secretion can be further promoted. Moreover, it was shown that the drying-improving composition of the present invention can promote saliva secretion even without chewing. Such a saliva secretion-promoting effect was not observed in compositions containing a culture medium without lactic acid bacteria culture, and therefore is not theoretically constrained, but it was suggested that the drying-improving effect of the composition of the present invention is brought about by components produced by the cultivation of lactic acid bacteria.

[0102] Furthermore, it has been shown that the dryness-improving composition of the present invention provides a moisturizing effect on the skin when applied to it. It has also been suggested that this moisturizing effect is brought about by an increase in AQP3 mRNA in the skin. Since an increase in AQP3 mRNA leads to an improvement in the skin's own moisture retention capacity, the dryness-improving composition of the present invention is expected to provide a long-lasting moisturizing effect on the skin, unlike existing topical preparations. [Industrial applicability]

[0103] The dryness-improving composition of the present invention can be added to food products as an ingredient that provides saliva secretion-promoting effects and oral cavity dryness-improving effects. Furthermore, the dryness-improving composition of the present invention can be added to cosmetics as an ingredient that provides skin moisturizing effects. [Accession Number]

[0104] FERM BP-11332

Claims

1. A composition for improving drying, containing the culture supernatant of lactic acid bacteria.

2. The drying-improving composition according to claim 1, wherein the lactic acid bacteria are bacteria belonging to the genus Lactobacillus.

3. The drying-improving composition according to claim 2, wherein the bacteria belonging to the genus Lactobacillus is a bacterium belonging to Lactobacillus crispapus.

4. The drying-improving composition according to claim 3, wherein the bacterium belonging to Lactobacillus crispapus is strain KT-11.

5. A composition for promoting saliva secretion, comprising the dryness-improving composition described in claim 1.

6. A composition for moisturizing the skin, comprising the dryness-improving composition described in claim 1.

7. The composition according to claim 5, which is a food product.

8. The composition according to claim 6, which is a cosmetic.

Images