Agent for improving balance of normal vaginal microbiota and vaginal composition

Abstract

Provided is an agent for improving the balance of normal vaginal microbiota that is effective in keeping a favorable vaginal state. The present invention provides an agent for improving the balance of normal vaginal microbiota using sophorose and / or laminaribiose. Sophorose and / or laminaribiose have functionality for promoting the growth of beneficial bacteria that have a beneficial vaginal effect while suppressing the growth of bacteria that have the possibility of causing an adverse vaginal effect among normal vaginal microbiota. This agent for improving the balance of normal vaginal microbiota can be suitably used for the purpose of keeping a favorable vaginal state while maintaining the balance of normal vaginal microbiota.

Description

Vaginal microbiome balance improving agent and vaginal composition

[0001] The present invention relates to a vaginal microbiome balance improving agent and vaginal composition using sophorose and / or laminaribiose.

[0002] In healthy women, the vaginal flora is largely dominated by bacteria of the Lactobacillus genus, and the lactic acid produced by these bacteria keeps the vagina acidic. When the balance of the uterine flora is disrupted, bacterial vaginosis (BV) develops, causing itching and inflammation. Bacterial vaginosis (BV) can cause miscarriage and premature birth, and can also spread to other organs, leading to serious complications. Bacterial vaginosis (BV) is estimated to affect one in three women, and it is a disease with a high recurrence rate even after treatment with antibiotics.

[0003] Lactobacillus species that form the bacterial flora of the human vagina include Lactobacillus gasseri, Lactobacillus paragasseri, Lactobacillus crispatus, and Lactobacillus jensenii. These Lactobacillus bacteria produce D-lactic acid and bacteriocins, which help maintain vaginal health.

[0004] On the other hand, among the vaginal resident flora, there are bacteria known to be harmful by excessive growth. For example, Lactobacillus iners produces L-lactic acid, but it is insufficient for inhibiting bacterial vaginitis (BV) and is known to be likely to progress to bacterial vaginitis (BV). Gardnerella vaginalis has been confirmed to grow in patients with bacterial vaginitis (BV), form biofilms, and is known to inhibit the treatment of bacterial vaginitis (BV). Mobiluncus curtisii has been confirmed to grow in patients with bacterial vaginitis (BV) and is known to be resistant to metronidazole, a treatment method for bacterial vaginitis (BV). In addition, Atopobium vaginae is also known to have been confirmed to grow in patients with bacterial vaginitis (BV).

[0005] From the above, the vaginal resident flora is considered extremely important for maintaining good vaginal conditions.

[0006] Regarding the vaginal resident flora, for example, Patent Document 1 discloses an invention of a method of using a combination of a saccharide and at least one of benzoic acid and its sodium salt to maintain the pH value of vaginal secretions within the range of 3.5 to 4.5. Patent Document 2 discloses an invention of a preparation for preventing and / or treating infectious diseases of the vagina, containing a bacteriostatic agent containing phenethyl alcohol as an active ingredient. Patent Document 3 discloses an invention of a lactic acid bacterial strain for preventing and / or treating vaginal infections.

[0007] Japanese Patent Publication No. 2008-539176, Japanese Patent Publication No. 2019-534896, Japanese Patent Publication No. 2024-503366

[0008] Conventionally, attempts have been made to improve the flora of vaginal resident bacteria with bacteriostatic agents and lactic acid bacterial strains, but the development of new materials is desired for the convenience of consumers.

[0009] An object of the present invention is to provide an agent for improving the balance of vaginal resident flora, which is useful for maintaining good vaginal conditions.

[0010] To achieve the above objective, the inventors conducted various studies and, as a result, discovered sophorose and / or laminaribiose as materials that selectively promote the growth of bacteria that have a beneficial effect on the vagina, thus completing the present invention.

[0011] In other words, in its first aspect, the present invention provides a vaginal microbiome balance improving agent comprising sophorose and / or laminaribiose as active ingredients.

[0012] In the present invention, the vaginal microbiota balance improving agent inhibits the activity of one or more species selected from the group consisting of Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, and Atopobium vaginae, while simultaneously inhibiting the activity of Lactobacillus gasseri, Lactobacillus paragasseri, and Lactobacillus crispatus. Preferably, it is for promoting the propagation of one or more species selected from the group consisting of *Lactobacillus crispatus* and *Lactobacillus jensenii*.

[0013] Furthermore, it is preferable that the sophorose and / or laminaribiose used as the active ingredient is in the form of a composition containing sophorose and / or laminaribiose produced using a glycosyltransferase / condensation enzyme.

[0014] On the other hand, in a second aspect thereof, the present invention provides a vaginal composition containing the above-mentioned vaginal microbiome balance improving agent.

[0015] According to the present invention, by using sophorose and / or laminaribiose, it is possible to suppress the growth of bacteria that may have a negative impact on the vagina among the normal vaginal flora, while promoting the growth of beneficial bacteria that have a positive impact on the vagina.

[0016] This figure shows the results of the study in Test Example 1, where various bacteria were cultured as vaginal commensal bacteria under appropriate culture conditions, and the change in bacterial count was investigated when α-gluco-oligosaccharide or β-gluco-oligosaccharide was added to the culture medium. This figure shows the results of the study in Test Example 2, where various bacteria (L. crispatus, L. paragasseri, L. jensenii, L. gasseri) were cultured under appropriate culture conditions, and the change in bacterial count was investigated when high-purity sophorose or laminaribiose was added to the culture medium. This figure shows the results of the study in Test Example 3, where various bacteria (L. crispatus, L. paragasseri, L. jensenii, L. gasseri) were cultured under appropriate culture conditions, and the change in bacterial count was investigated when high-purity sophorose or laminaribiose was added to the culture medium at varying final concentrations. This figure shows the results of an investigation into the changes in bacterial counts when various bacteria (Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, Atopobium vaginae), which are considered normal vaginal flora, were cultured under appropriate culture conditions, and high-purity sophorose or laminaribiose was added to the culture medium at varying final concentrations.

[0017] The present invention will be described in more detail below. In this specification, when a numerical range is indicated by "~", unless otherwise specified, it means from the above to the below, and both of the numerical values ​​at both ends are included. Furthermore, the upper and lower limits can be combined as appropriate, and the numerical range obtained thereby is also included in the disclosure of this specification.

[0018] The vaginal microbiota balance improving agent according to the present invention uses sophorose and / or laminaribiose as its active ingredient.

[0019] Sophorose (also known as 2-O-β-D-glucopyranosyl-D-glucose) is a disaccharide composed of two glucose molecules linked by a β-1,2 bond. It was first discovered as a sugar component of the pigment glycoside in the pods of the legume plant Sophora japonica, but it rarely exists in nature as a free disaccharide and is present in very small amounts in nature. It is only known to be present in small amounts in acid hydrolysates of starch and royal jelly.

[0020] The method of obtaining sophorose is not particularly limited, but for example, it can be prepared enzymatically using glycosyltransferase and condensation reactions. Specifically, as described in detail in Japanese Patent Publication No. 2750374 and Japanese Patent Publication No. 3020583, sophorose can be obtained by reacting microbial β-glucosidase with glucose and / or β-gluco-oligosaccharides and utilizing the condensation and glycosyltransferase reactions of β-glucosidase. Furthermore, as described in detail in Japanese Patent Publication No. 7025941, sophorose can be obtained in higher yields by reacting 1,2-β-oligoglucan phosphorylase with α-glucose-1-phosphate and glucose.

[0021] Laminaribiose (also known as 3-O-β-D-glucopyranosyl-D-glucose) is a disaccharide in which two glucose molecules are linked by a β-1,3 bond, and is known as the minimum structural unit of β-1,3 glucans such as laminaran, curdlan, and pachyman.

[0022] Laminaribiose can be obtained in any way, however there are no particular limitations, and it can be prepared enzymatically, for example, by utilizing a glycosyltransferase / condensation reaction. Specifically, as described in detail in Japanese Patent Publication No. 2750374 and Japanese Patent Publication No. 3020583, laminaribiose can be obtained by reacting microbial β-glucosidase with glucose and / or β-gluco-oligosaccharides and utilizing the condensation / glycosyltransfer reaction possessed by β-glucosidase. Furthermore, as described in detail in Japanese Patent Publication No. 2955590, laminaribiose can be easily obtained in high yield by using sucrose as a raw material and reacting it in the presence of phosphate with a combination of three or four enzymes: sucrose phosphorylase, glucose isomerase, laminaribiose phosphorylase, and / or β-1,3-oligoglucan phosphorylase.

[0023] Compositions containing sophorose and / or laminaribiose can be obtained by these methods. The obtained compositions containing sophorose and / or laminaribiose can be subjected to treatments such as filtration, decolorization, deodorization, and desalting as needed. They may also be subjected to treatments such as extraction, centrifugation, crystallization, microbial assimilation, and further treatments such as chromatographic fractionation using activated carbon, porous carriers, hydrophobic resins, hydrophilic resins, ion exchange resins, adsorption resins, etc., dialysis, and membrane fractionation using extreme filtration. These treatments can increase the purity of sophorose and / or laminaribiose.

[0024] In the present invention, sophorose and / or laminaribiose can be used in a highly purified form, but any form containing sophorose and / or laminaribiose is also usable, that is, it can be used in the form of a sugar composition containing sophorose and / or laminaribiose. In this case, there are no particular restrictions on the content of sophorose and / or laminaribiose in the sugar composition used in the present invention, but for example, it is preferably 1% by mass or more per dry solids, more preferably 5% by mass or more, even more preferably 10% by mass or more, and most preferably 30% by mass or more. On the other hand, if the content of sophorose and / or laminaribiose per dry solids is less than 0.5% by mass, the functionality of sophorose and / or laminaribiose may not be fully exhibited. In this specification, "sophorose and / or laminaribiose content" means the total content of sophorose and laminaribiose, and if each is included individually, it means the content of sophorose or laminaribiose when included individually.

[0025] The sugar composition containing sophorose and / or laminaribiose described above may also contain other components such as sugars and oligosaccharides with different bonding modes and degrees of polymerization. Typically, for example, one or more oligosaccharides with a degree of polymerization of 2 to 10, such as sophorooligosaccharides (excluding sophorose), gentiooligosaccharides, cellooligosaccharides, and laminarioligosaccharides (excluding laminaribiose), can be exemplified by a composition containing each in the dry solid content equivalent shown below. Furthermore, as shown in the examples described later, the sugar composition may also contain β-glucooligosaccharides obtained by adding β-glucosidase to a glucose solution.

[0026] Sophorooligosaccharides: 5% to 50% by mass Genthiooligosaccharides: 5% to 80% by mass Cellooligosaccharides: 5% to 50% by mass Laminarioligosaccharides: 5% to 50% by mass

[0027] As shown in the examples described later, sophorose and / or laminaribiose have the functionality to suppress the growth of bacteria in the vaginal flora that may have a negative impact on the vagina (also known as "bacteriostatic"), while promoting the growth of beneficial bacteria that have a positive impact on the vagina. Specifically, beneficial bacteria that have a positive impact on the vagina include, but are not limited to, Lactobacillus gasseri, Lactobacillus paragasseri, Lactobacillus crispatus, and Lactobacillus jensenii. On the other hand, bacteria that may have adverse effects on the vagina include Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, and Atopobium vaginae.

[0028] Since the present invention uses sophorose and / or laminaribiose, which possess the above-described functionalities, as an active ingredient, it can be suitably used for the purpose of maintaining the balance of the vaginal flora and keeping the vagina in good condition.

[0029] Specifically, in one embodiment, the present invention can be suitably used for the purpose of improving the balance of the vaginal microbiota. When the balance of the vaginal microbiota is disrupted, excessive proliferation of commensal bacteria and invasion and proliferation of other harmful bacteria occur, inducing various symptoms. The present invention can be suitably used to prevent such conditions from occurring, or to alleviate or suppress the severity of such symptoms. In this case, improving the balance may involve, for example, increasing the proportion of beneficial bacteria such as Lactobacillus species or decreasing the proportion of harmful bacteria. Lactobacillus bacteria have an antagonistic effect against pathogenic bacteria, such as Gardnerella vaginalis, and can inhibit the proliferation of such harmful bacteria. Therefore, by promoting the proliferation of Lactobacillus bacteria, they can function as balance adjusters for the vaginal microbiota. Furthermore, improving the balance of the vaginal flora may not affect the growth of beneficial bacteria such as Lactobacillus, but may suppress the growth of harmful bacteria.

[0030] In one embodiment, the present invention can be suitably used for the purpose of maintaining a slightly acidic environment in the vagina and preventing infection by other bacteria. For example, by promoting the growth of beneficial bacteria such as those of the Lactobacillus genus, these bacteria produce lactic acid or antimicrobial peptides that suppress the growth of pathogenic bacteria, and thus can be suitably used for the above-mentioned purposes.

[0031] In one embodiment, the present invention can be suitably used for the purpose of preventing bacterial vaginosis (BV), or reducing or suppressing the severity of its symptoms. For example, by inhibiting the growth of Gardnerella vaginalis, the formation of biofilms caused by this bacterium can be prevented, thereby suppressing the inhibition of BV treatment, and thus the present invention can be suitably used for the purposes described above.

[0032] In one embodiment, the present invention can be suitably used for the purpose of suppressing the recurrence of bacterial vaginosis (BV). For example, Lactobacillus iners usually proliferate after a patient with bacterial vaginosis (BV) receives antibiotic treatment, but by suppressing this, it is possible to prevent the vagina from becoming an environment that is prone to the recurrence of bacterial vaginosis (BV), and therefore the present invention can be suitably used for the above-mentioned purpose.

[0033] (Vaginal Composition) The vaginal microbiome balance improving agent provided by the present invention may be used in the form of various vaginal compositions such as cosmetics, topical pharmaceuticals, and quasi-drugs. Examples of vaginal compositions include solid preparations such as suppositories and capsules; semi-solid preparations such as creams, gels, and ointments; and liquid preparations such as liquids and lotions. The present invention is preferably in the form of a cream, gel, ointment, or suppository. The vaginal microbiome balance improving agent is preferably applied to the vagina, and more preferably applied to the area around the vagina or in direct contact with the vagina.

[0034] When the vaginal microbiota balance improving agent provided by the present invention is used in the form of a vaginal composition, the above-mentioned sophorose and / or laminaribiose, or sugar composition containing sophorose and / or laminaribiose, can be prepared in their respective forms using appropriate pharmaceutically acceptable formulation carriers and components according to conventional methods as appropriate. Examples of such formulation carriers and components include water, other aqueous solvents, petrolatum, glycerin, glyceryl monostearate, glyceryl stearate, self-emulsifying glyceryl monostearate, corn starch, sodium carboxymethyl starch, crystalline cellulose, cellulose, lactose, D-sorbitol, stearic acid, magnesium stearate, palmitic acid, cetyl palmitate, tartaric acid, hard fat, octyldodecanol, paraffin, light liquid paraffin, gelling hydrocarbons, stearyl alcohol, cetanol, cetostearyl alcohol, sucrose fatty acid esters, diethanolamine, dimethylpolysiloxane, squalane, sodium bicarbonate, medium-chain triglyceride, propylene glycol, propylene glycol fatty acid esters, 1,3-butylene glycol, polyethylene glycol, polyoxyethylene cetyl ether, isopropyl myristate, octyldodecyl myristate, cetyl myristate, etc.Other ingredients include stabilizers such as dibutylhydroxytoluene, sodium edetate, sodium sulfite, and anhydrous sodium sulfite; thickeners such as xanthan gum, hydroxypropyl methylcellulose, hydroxypropylcellulose, and carboxyvinyl polymer; surfactants such as sorbitan stearate, sorbitan monostearate, and polyoxyl stearate; preservatives such as butylparaben, methylparaben, propylparaben, ethylparaben, sodium benzoate, and benzyl alcohol; and pH adjusters such as hydrochloric acid, sulfuric acid, and phosphorus. Examples of buffering agents include inorganic acids such as acids, polyphosphates, and boric acid; organic acids such as lactic acid, acetic acid, citric acid, tartaric acid, malic acid, succinic acid, oxalic acid, gluconic acid, fumaric acid, propionic acid, acetic acid, aspartic acid, epsilon-aminocaproic acid, glutamic acid, and aminoethylsulfonic acid; and inorganic bases such as sodium bicarbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, calcium hydroxide, and magnesium hydroxide. Examples of buffering agents include boric acid buffers, phosphate buffers, carbonate buffers, citrate buffers, acetate buffers, epsilon-aminocaproic acid, aspartic acid, and aspartates. These pharmaceutical carriers and components can be processed according to the type and form of product to be added, using conventional methods (for example, crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc., by arbitrarily selecting and combining various materials), and supplied as desired.

[0035] Furthermore, by combining the various pharmacological effects of each known raw material, it is possible to create a product that offers not only the functionality of improving the balance of the vaginal microbiota, which is the objective of this invention, but also multifunctional effects.

[0036] In the vaginal microbiota balance improving agent or vaginal composition provided by the present invention, the content of sophorose and / or laminaribiose may be in the range of 0.01% to 100% by mass per dry solids. If the content per dry solids is less than 0.01% by mass, the functionality of sophorose and / or laminaribiose may not be fully exhibited. The content of sophorose and / or laminaribiose is more preferably 0.1% by mass or more, and even more preferably 0.3% by mass or more.

[0037] When the vaginal microbiota balance improving agent or vaginal composition provided by the present invention is in a liquid, gel, cream, or other form containing water or other medium, the content of sophorose and / or laminaribiose in such form may be set as appropriate and is not particularly limited, but is typically in the range of 0.01 mg / g to 400 mg / g (the denominator is the weight of the preparation), 0.1 mg / g to 100 mg / g, or 1 mg / g to 50 mg / g.

[0038] The vaginal microbiota balance improving agent or vaginal composition provided by the present invention, when applied to the vagina, may be administered in an appropriate amount, and is not particularly limited. For example, the amount of sophorose and / or laminaribiose may be in the range of 0.001 g to 1 g, 0.005 g to 0.5 g, or 0.02 g to 0.05 g.

[0039] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0040] [1. Preparation of α-gluco-oligosaccharides] 50 mg of glucoamylase enzyme preparation per 1 g of solids was added to a 75 (w / w) % glucose solution, the pH was adjusted to 5.0, and the mixture was maintained at 66°C. After 63 hours of reaction, the pH was adjusted to 4.0 and the temperature was raised to 80°C to inactivate the enzyme. Monosaccharides were removed from the product by membrane fractionation to obtain α-gluco-oligosaccharides.

[0041] [2. Preparation of β-gluco-oligosaccharides] 9.3 mg of β-glucosidase enzyme preparation per 1 g of solids was added to a 60 (w / w) % glucose solution, and the pH was adjusted to 6.6 and maintained at 60°C. After 72 hours of reaction, the pH was adjusted to 4.0 and the temperature was raised to 80°C to inactivate the enzyme, and monosaccharides were removed from the product by membrane fractionation to obtain β-gluco-oligosaccharides.

[0042] [3. Preparation of High-Purity Sophorose] 1.0 M sucrose and 1.0 M glucose were dissolved in 100 mM potassium phosphate buffer (pH 7.0). To 1 mL of this substrate, 10 μL each of a protein derived from Bifidobacterium skardovii having 19 mg / mL of 1,2-β-oligoglucan phosphorylase activity (the protein disclosed as "BSGP" in Japanese Patent No. 7025941) and 17 mg / mL sucrose phosphorylase (Sigma-Aldrich) were added and the mixture was reacted at 30°C. After 7 days, the enzyme was inactivated by boiling for 10 minutes, and the reaction mixture was concentrated as appropriate and subjected to activated carbon column chromatography to remove monosaccharides and disaccharides. Hydrochloric acid was added to this mixture to a final concentration of 85 mM, and the mixture was held at 80°C for 17 hours to perform acid hydrolysis. The hydrolyzed sample was subjected to HPLC under the following conditions, and the disaccharide fraction was recovered to obtain high-purity sophorose.

[0043] <HPLC Conditions> Column: ULTRON PS-80NL (Shinwa Chemical Co., Ltd.) Column temperature: 50°C Eluent: Ultrapure water Flow rate: 0.9 mL / min Detector: Differential refractive index detector Analysis time: 20 minutes

[0044] [4. Preparation of High-Purity Laminaribiose] 8.0 g of curdlan (Mitsubishi Corporation Life Sciences Ltd.) and 720 mL of ultrapure water were mixed, and 8 mL of 1 M sodium hydroxide was added and stirred for 10 minutes. 40 mL of 1 M Tris-HCl buffer (pH 7.0) and 8 mL of 1 M hydrochloric acid were added and stirred, and then 80 mg of yeast cell wall lytic enzyme ("Zymolyase (registered trademark)", Nacalai Tesque, Inc.) was added. Cysteine was added to a final concentration of 10 mM, and the mixture was reacted overnight at 37 °C with vigorous stirring. It was heated at 80 °C for 10 minutes to inactivate the enzyme, cooled to room temperature, and then centrifuged to collect the supernatant. This was subjected to HPLC under the same conditions as for the preparation of high-purity sophorose, and the disaccharide fraction was collected to obtain high-purity laminaribiose.

[0045] [5. Sugar Composition Analysis] Sugar composition analysis was performed using HPLC. The content of each degree of polymerization component was determined from the peak area of the obtained chromatogram. The analysis of α-gluco-oligosaccharides was carried out according to HPLC condition A, and the analysis of β-gluco-oligosaccharides and high-purity samples was carried out according to HPLC condition A and HPLC condition B.

[0046] <HPLC Condition A> Column: ULTRON PS-80NL (Showa Denko K.K.) Column temperature: 50 °C Eluent: Ultrapure water Flow rate: 0.9 mL / min Detector: Differential refractive index detector Analysis time: 20 minutes

[0047] <HPLC Condition B> Column: HILICpak VG-50 4E (Showa Denko K.K.) Column temperature: 40 °C Eluent: Acetonitrile: Ultrapure water = 8:2 Flow rate: 0.6 mL / min Detector: Charged particle detector Analysis time: 60 minutes

[0048] Tables 1 and 2 show the results of the sugar composition analysis.

[0049]

[0050] As shown in Table 1, all of the gluco-oligosaccharides prepared above had a sugar composition with a disaccharide as the main component. In addition, β-gluco-oligosaccharides contained 12.4% by mass of sophorose and 10.5% by mass of laminaribiose.

[0051]

[0052] As shown in Table 2, the high-purity preparations of sophorose and laminariviose prepared above all had a purity of at least 90% by mass.

[0053] [Test Example 1] For various bacteria shown in Table 3 as vaginal commensal bacteria, the change in bacterial count was investigated when the α-gluco-oligosaccharide or β-gluco-oligosaccharide prepared above was added to the culture medium while culturing each under appropriate culture conditions.

[0054]

[0055] Specifically, glucose was added to a culture medium suitable for each bacterial species to a final concentration of 0.5 (w / v)%, and each bacterial species was cultured overnight to obtain a pre-culture solution. Then, α-gluco-oligosaccharide or β-gluco-oligosaccharide prepared above was added to 3 mL of fresh medium to a final concentration of 0.5 (w / v), and 0.03 mL of the pre-culture solution was mixed with this and cultured for 24 hours. The bacterial turbidity (OD660) after culturing was measured. In addition, glucose was added to the culture medium as a carbohydrate and the same test was performed as a comparative control. The results are shown as relative values ​​(percentage compared to the sugar-free group) with the bacterial turbidity when sterile ultrapure water was added instead of carbohydrates set to 100%.

[0056] As a result, as shown in Figure 1, significant growth was observed in all bacterial species in the glucose-added group, indicating poor selectivity for specific species. Growth of L. iners was also observed in the α-gluco-oligosaccharide-added group. On the other hand, in the β-gluco-oligosaccharide-added group, growth was observed in L. crispatus, as well as L. paragasseri, L. jensenii, and L. gasseri, but almost no growth was observed in the other four species.

[0057] From the above, it has become clear that β-gluco-oligosaccharides, which are mainly composed of disaccharides, have the effect of suppressing the growth of bacteria harmful to the skin while promoting the growth of beneficial bacteria in the vagina (L. crispatus, L. paragasseri, L. jensenii, and L. gasseri).

[0058] [Test Example 2] The effect of sophorose and laminalibiose, which are β-gluco-oligosaccharides composed of disaccharides, on promoting the growth of L. crispatus, L. paragasseri, L. jensenii, and L. gasseri was evaluated. Specifically, the effect on promoting the growth of L. crispatus, L. paragasseri, L. jensenii, and L. gasseri was evaluated in the same manner as in Test Example 1, except that sophorose or laminalibiose, which were prepared to a high purity as described above, were used as the carbohydrate added to the culture medium.

[0059] As a result, as shown in Figure 2, sophorose and laminaribiose had the effect of promoting the growth of L. crispatus, L. paragasseri, L. jensenii, and L. gasseri.

[0060] [Test Example 3] Using sophorose and laminaribiose, which were prepared to high purity as disaccharides, the same procedure as in Test Example 2 was used, except that the amount added to the culture medium was set to a final concentration of 0.1 (w / v)%, 0.5 (w / v)%, or 1.0 (w / v)%, to evaluate the effect of promoting the growth of L. crispatus, L. paragasseri, L. jensenii, and L. gasseri.

[0061] As a result, as shown in Figure 3, when a preparation containing high purity sophorose or laminaribiose was added to the culture medium to a final concentration of 0.1 (w / v)% and cultured, an effect of promoting the growth of L. crispatus, L. paragasseri, L. jensenii, and L. gasseri was observed. Furthermore, at a final concentration of 1.0 (w / v)%, a greater growth effect was observed than that shown in Test Example 2 at a final concentration of 0.5 (w / v)%.

[0062] [Test Example 4] The effect of promoting the growth of Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, and Atopobium vaginae was evaluated in the same manner as in Test Example 1, except that sophorose or laminaribiose, which was prepared to high purity as described above, was used as the β-glucooligosaccharide consisting of disaccharides, and the amount added to the culture medium was set to a final concentration of 0.1 (w / v)%, 0.5 (w / v)%, or 1.0 (w / v)%.

[0063] As a result, as shown in Figure 4, neither the amount of sophorose or laminaribiose added to the preparations containing high purity showed any effect in promoting the growth of Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, and Atopobium vaginae compared to the control group without added sugar (percentage compared to the sugar-free group).

[0064] Based on the above, and considering the results of Test Examples 2 to 4 together, it has become clear that sophorose and laminaribiose, which are β-glucooligosaccharides composed of disaccharides, have the effect of suppressing the growth of bacteria harmful to the vagina while promoting the growth of bacteria beneficial to the vagina.

[0065] (Formulation Example 1) This is a formulation example in which sophorose or laminaribiose is used as an ointment as a vaginal flora balance improving agent according to the present invention.

[0066] (Ingredients) Content (mass%) Sophorose or laminaribiose 0.5% White petrolatum 25.0% Stearyl alcohol 20.0% Propylene glycol 12.0% Polyoxyethylene hydrogenated castor oil 60 4.0% Glyceryl monostearate 1.0% Purified water Residue

[0067] (Formulation Example 2) This is a formulation example in which β-gluco-oligosaccharide is used in an ointment as a vaginal flora balance improving agent according to the present invention.

[0068] (Ingredients) Content (mass%) β-Gluco-oligosaccharide 1.0% White petrolatum 25.0% Stearyl alcohol 20.0% Propylene glycol 12.0% Polyoxyethylene hydrogenated castor oil 60 4.0% Glyceryl monostearate 1.0% Purified water Residue

[0069] (Formulation Example 3) This is a formulation example in which sophorose or laminaribiose is used as a vaginal suppository as an agent for improving the balance of the vaginal flora according to the present invention.

[0070] (Ingredients) Content (mass%) Sophorose or laminaribiose 11.5% Hard fat 88.5%

[0071] (Formulation Example 4) This is a formulation example in which β-gluco-oligosaccharide is used in a vaginal suppository as a vaginal microbiota balance improving agent according to the present invention.

[0072] (Ingredients) Content (mass%) β-Gluco-oligosaccharide 11.5% Hard fat 88.5%

[0073] (Formulation Example 5) This is a formulation example in which sophorose or laminaribiose is used as a cream to improve the balance of the vaginal flora according to the present invention.

[0074] (Ingredients) Content (mass%) Sophorose or laminaribiose 1.0% Stearyl alcohol 8.0% Behenyl alcohol 2.0% Isopropyl palmitate 6.0% Polysorbate 60 2.0% Sorbitan stearate 1.0% Self-emulsifying glyceryl monostearate 0.3% 1,3-Butylene glycol 20.0% Sodium dihydrogen phosphate 0.3% Hydrochloric acid Appropriate amount Sodium hydroxide Appropriate amount Purified water Residue

[0075] (Formulation Example 6) This is a formulation example in which β-gluco-oligosaccharide is used in a cream formulation as a vaginal flora balance improving agent according to the present invention.

[0076] (Ingredients) Content (mass%) β-Gluco-oligosaccharide 2.0% Stearyl alcohol 8.0% Behenyl alcohol 2.0% Isopropyl palmitate 6.0% Polysorbate 60 2.0% Sorbitan stearate 1.0% Self-emulsifying glyceryl monostearate 0.3% 1,3-Butylene glycol 20.0% Sodium dihydrogen phosphate 0.3% Hydrochloric acid Appropriate amount Sodium hydroxide Appropriate amount Purified water Residue

Claims

1. A vaginal microbiome balance improving agent containing sophorose and / or laminaribiose as active ingredients.

2. While oscillating one or more species selected from the group consisting of Lactobacillus iners, Gardnerella vaginalis, Mobiluncus curtisii, and Atopobium vaginae, Lactobacillus gasseri, Lactobacillus paragasseri, and Lactobacillus crispatus A vaginal microbiota balance improving agent according to claim 1, which is for promoting the growth of one or more species selected from the group consisting of *Crispatus* and *Lactobacillus jensenii*.

3. The vaginal microbiota balance improving agent according to claim 1, wherein the sophorose and / or laminaribiose used as the active ingredient is in the form of a sugar composition containing sophorose and / or laminaribiose produced using a glycosyltransferase / condensation enzyme.

4. A vaginal composition containing a vaginal microbiome balance improving agent according to any one of claims 1 to 3.

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