TWI629361B - Hyaluronidase inhibitor from symbiotic fermented product and application thereof - Google Patents

Abstract

The present invention relates to a hyaluronidase inhibitor comprising a symbiotic yeast product, and the symbiotic yeast product is obtained by using a plurality of fermentation strains in a vegetative medium through a symbiotic fermentation step and a solid-liquid separation step. The resulting hyaluronidase inhibitor can be applied to a skin external composition or to prepare an oral composition or a pharmaceutical composition for inhibiting hyaluronidase.

Description

Hyaluronidase inhibition derived from symbiotic fermentation products Preparation and its application

The present invention relates to a hyaluronidase inhibitor, and more particularly to a hyaluronidase inhibitor derived from a symbiotic yeast product and its use in an oral composition, a skin external composition, and a pharmaceutical composition.

Hyaluronidase (HAase), also known as diffusion factor, is a general term for a group of hydrolyzed hyaluronic acid (HA) glycosidases, mainly found in animal tissues (such as skin, joints, ligaments, eyes, etc.), animal venom And in microorganisms. Hyaluronidase mainly acts on β-1,3,β-1,4 glycosidic bonds between hyaluronic acid N-acetamidoglucose and D-glucuronic acid, depolymerization and hydrolysis into Relatively low molecular mass (Mr) of HA or oligosaccharides. Hyaluronic acid is widely present in various tissues of animals and exhibits various important physiological functions in the body, such as lubricating joints, regulating protein function, and promoting wound healing. More importantly, it has a good moisturizing effect and is a good transdermal absorption enhancer at the same time as moisturizing. Cosmetics containing hyaluronic acid are currently recognized internationally as "bionic cosmetics", "The fourth generation of cosmetics."

Hyaluronidase can be divided into three types according to the optimal reaction pH, amino acid sequence homology and source, structure and mechanism of action, respectively: endo-β-N-acetamid glucosidase, endo-cut -β-glucuronidase, hyaluronan lyase.

Hyaluronidase is a participant in type I allergic reactions and tumor cell proliferation, and it is strongly associated with inflammation and allergies. It can decompose the interstitial tissue of connective tissue, organize edema, increase vascular permeability, and intensify inflammation. These effects are associated with the rapid spread of penetration of bacteria and its products and inflammation. Therefore, inhibiting the activity of hyaluronidase can not only decompose hyaluronic acid to maintain its normal physiological function, but also achieve anti-inflammatory, anti-allergic, anti-tumor effects. Hyaluronidase can also temporarily reduce the viscosity of the intercellular substance, so that subcutaneous injection of infusion, local accumulation of exudate or blood to spread and facilitate absorption, but also relieve the symptoms of degenerative arthritis. Therefore, hyaluronidase can also be used as a drug diffusion agent and in the treatment of degenerative arthritis. Further, by adding an additive which inhibits hyaluronidase activity to a preparation containing hyaluronic acid (particularly, such as a cosmetic enhancer and a tissue filler), the durability of the action of the preparation can be enhanced, and the repeated administration can be avoided. Therefore, the development of additives that inhibit hyaluronidase activity is of great significance in the pharmaceutical, health care, and cosmetic industries.

The breakdown of hyaluronic acid causes many diseases such as allergies, inflammation, and degenerative arthritis. Some hyaluronidase inhibitors, such as polysulfonate mucopolysaccharides, are known to inhibit the activity of hyaluronidase enzymes, so that hyaluronic acid is not decomposed to maintain its normal physiological functions, and resistance is achieved. Inflammation, anti-allergy, and slowing down of degenerative arthritis.

Among the products of natural origin, extracts such as the fruit of Cistanche tubulosa, the fruit of the horse chestnut, and rosemary, which have the activity of inhibiting hyaluronidase activity, are listed. However, the above-mentioned products of natural origin inhibiting hyaluronidase have a limited inhibitory effect on hyaluronidase.

In view of this, there is an urgent need to develop a product of natural origin to provide a product having activity for inhibiting hyaluronidase.

Accordingly, one aspect of the present invention provides a hyaluronidase inhibitor derived from a symbiotic yeast product comprising a symbiotic yeast product, and the symbiotic yeast product is in a plant medium The fermented strain is obtained by a symbiotic fermentation step and a solid-liquid separation step.

Another aspect of the present invention provides a skin external composition comprising the above hyaluronidase inhibitor, wherein a hyaluronidase inhibitor is used as an active ingredient.

Still another aspect of the present invention provides a hyaluronidase inhibitor for producing an oral composition having hyaluronidase inhibitor, which comprises the above hyaluronidase inhibitor as an active ingredient.

A further aspect of the present invention provides a hyaluronidase inhibitor for producing a pharmaceutical composition having hyaluronidase inhibitor, which comprises the above-described hyaluronidase inhibitor as an active ingredient.

According to the above aspect of the present invention, a hyaluronidase inhibitor derived from a symbiotic yeast product is proposed, which is obtained by a symbiotic fermentation step and a solid-liquid separation step in a plant medium. among them The symbiotic fermentation step is carried out at 37 ° C for 70 hours to 100 hours, and the aforementioned fermentation strain is composed of a plurality of lactic acid bacteria strains and at least one non-lactobacillus strain.

According to an embodiment of the present invention, the lactic acid bacteria strain comprises a plurality of Bifidobacterium spp. strains and/or Lactobacillus spp. strains.

According to an embodiment of the present invention, the above-described system of the genus Bifidobacterium bifurcated bifidobacteria (B.bifidum; BCRC 14615); and one of the group consisting of any combination thereof, Bifidobacterium longum (BCRC 14602 B.longum) .

According to an embodiment of the present invention, the Lactobacillus strain is a Lactobacillus delbrueckii subsp. bulgaricus (BCRC 10696), a Lactobacillus subfamily subspecies ( L. paracasei subsp. paracasei ; BCRC 17475) , a group consisting of Lactobacillus brevis ( L. brevis ; BCRC 12187) and Lactobacillus sp. (BCRC 17003) and any combination thereof.

According to an embodiment of the present invention, the at least one non-lactobacillus strain is composed of a strain of Streptococcus spp. and a strain of Saccharomyces spp .

According to an embodiment of the present invention, the Streptococcus sp. strain is Streptococcus salivarius subsp. thermophilus (BCRC 13869).

According to an embodiment of the present invention, the above-mentioned yeast strain is Saccharomyces cerevisia e (BCRC 21680).

According to an embodiment of the invention, the vegetable medium comprises soybean, sesame, alfalfa, sucrose, sodium chloride and water.

According to an embodiment of the present invention, the vegetable medium comprises 1.0 to 10.0% by weight of soybean, 1.0 to 5.0% by weight of sesame, 1.0 to 5.0% by weight of cerium, 1.0 to 5.0% by weight of sucrose, and 0.1 to 2.0% by weight. Sodium chloride.

According to another aspect of the present invention, a hyaluronidase inhibitor is proposed for the preparation of an oral composition having hyaluronidase inhibitor, which comprises the above-described hyaluronidase inhibitor as an active ingredient.

The hyaluronidase inhibitor of the present invention comprises a symbiotic yeast-derived product, and the symbiotic yeast-derived product is a symbiotic fermentation step and solid-liquid separation using a plurality of fermentation strains in a vegetable medium. In the step, the hyaluronidase inhibitor obtained has activity for inhibiting hyaluronidase, and can be applied to a composition for external use on skin, and an oral composition or a pharmaceutical composition having hyaluronidase inhibiting activity can be prepared.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Fig. 1 is a HPLC chart showing the symbiotic yeast products obtained in Experimental Example 1, Comparative Example 1, and Comparative Example 2 according to Example 1 of the present invention.

2A to 2C are symbiotic yeast products showing the experimental examples (Fig. 2A), Comparative Example 1 (Fig. 2B), and Comparative Example 2 (Fig. 2C) of Example 1 having a concentration of 1.0 to 10% according to the present invention. After co-culture with 3T3 fibroblasts, hyaluronic Photograph of the hyaluronidase zymogram of the acid enzyme.

As described above, the present invention provides a hyaluronidase inhibitor derived from a symbiotic fermentation product, which comprises a symbiotic fermentation product, and the symbiotic fermentation product is a plurality of fermentation enzymes for use in a vegetable culture medium. The strain is obtained by a symbiotic fermentation step and a solid-liquid separation step.

In the present invention, the term "symbiotic fermentation product" as used herein refers to a product obtained by culturing various strains in vitro in a fermentation substrate for symbiotic fermentation, followed by solid-liquid separation, etc., wherein Contains residual components of cells, metabolites, etc.

In one embodiment, the aforementioned symbiotic fermentation product can be obtained, for example, in a vegetable culture medium, inoculated with a plurality of fermentation bacterium, and subjected to a symbiotic fermentation step and a solid-liquid separation step. It is explained here that it is of course not the symbiotic fermentation product that inhibits the hyaluronidase activity, which needs to be confirmed by experiments.

In one embodiment, the above vegetable medium may include, for example, soybean, sesame, alfalfa, sucrose, sodium chloride, and water. In one example, the above vegetable medium may comprise 1.0 to 10.0 weight percent soybean, 1.0 to 5.0 weight percent sesame, 1.0 to 5.0 weight percent bismuth, 1.0 to 5.0 weight percent sucrose, and 0.1 to 2.0 weight percent chlorine. Sodium, the rest is water. In another illustration, the above vegetable medium may comprise 1.0 to 6.0 weight percent soybean, 1.0 to 3.0 weight percent sesame, 1.0 to 3.0 weight percent bismuth, 2 to 5 weight percent sucrose, and 0.2 to 1.0 weight percent of sodium chloride, the balance being water.

Here, in the case where the amount of each component of the vegetable culture medium is outside the above range, it has not been confirmed by experiments that it is not expected to obtain a symbiotic fermentation product having activity for inhibiting hyaluronidase.

In one embodiment, the fermentation strain is selected from at least 8 of a population consisting of a plurality of lactic acid bacteria strains and at least one non-lactobacillus strain. In an example, the inoculum amount of the above fermented strain in the vegetable medium may be, for example, 3 to 5 weight percent.

According to an embodiment of the present invention, the lactic acid bacteria strain is composed of a plurality of strains of Bifidobacterium spp. and Lactobacillus spp.

In the above embodiment, the Bifidobacterium may be bifurcated by the bifidobacteria (B.bifidum; BCRC 14615) For example, Bifidobacterium longum (B.longum; BCRC 14602) as one of the group consisting of.

In the above embodiments, the Lactobacillus strain may, for example, be selected from the group consisting of L. delbrueckii subsp. bulgaricus (BCRC 10696), L. paracasei subsp. paracasei ( BCRC) . 17475), Lactobacillus brevis ( L. brevis ; BCRC 12187) and Lactobacillus sp. (BCRC 17003).

In the above examples, at least one non-lactobacillus strain is composed of a strain of Streptococcus spp. and a strain of Saccharomyces spp .

In the above examples, the Streptococcus strain is Streptococcus salivarius subsp. thermophilus (BCRC 13869).

In the above examples, the Saccharomyces strain is Saccharomyces cerevisia e (BCRC 21680).

In one embodiment, the aforementioned symbiotic fermentation step can be carried out, for example, at 37 ° C for 70 hours to 100 hours, and preferably at 37 ° C for 72 hours to 96 hours to obtain a fermentation product. In another illustration, the solid-liquid separation step may be, for example, a filtration step or a centrifugation step to remove the solids of the fermented product to obtain a symbiotic fermentation product.

In other embodiments, between the symbiotic fermentation step and the solid-liquid separation step, the termination of the fermentation step is more selectively performed. In one example, the termination of the fermentation step can be carried out, for example, at 90 ° C for 15 minutes.

In one embodiment, the symbiotic fermentation product obtained above can be selectively subjected to conventional post-treatment steps, such as a concentration step, a decolorization step, and/or a microbial detection step, depending on actual needs.

The symbiotic yeast product obtained above was confirmed to have hyaluronidase activity after being evaluated by an in vitro activity test, and thus can be further applied to compositions of various dosage forms. Suitable compositions can include, but are not limited to, oral compositions, topical compositions for skin, and/or pharmaceutical compositions. In one embodiment, the oral composition, the external composition for skin, and/or the pharmaceutical composition of the above-described symbiotic yeast product are commensal fermentation products as an active ingredient, and the composition of the above dosage form has hyaluronic acid inhibition. Enzyme activity. It is explained herein that, in general, it is known that a natural-derived product having a hyaluronidase-inhibiting activity has a dose- and time-dependent degree of inhibitory activity (dose-and time- Dependency correlation. However, the symbiotic yeast product obtained by the present invention has good water solubility as a hyaluronidase inhibitor, does not have dose-dependent cytotoxicity, and can completely inhibit transparency without dose and time dependence. The enzyme enzyme enzyme activity provides a hyaluronic acid enzyme inhibitor with high biosafety, stability, and good bioabsorbability.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Preparation of symbiotic fermentation products

1. Preparation of plant culture medium

First, the plant culture media of Examples and Comparative Examples 1 to 2 were prepared according to Table 1.

Prior to fermentation, the vegetable medium listed in Table 1 can be first sterilized by conventional autoclaving, and the sterilization process conditions can be carried out, for example, at 121 ° C for 40 minutes. Then, 8 different fermentation strains were inoculated separately into the plant medium of Table 1, wherein the total inoculum of the fermentation strain in the vegetable medium may be 3% (w/v) to 5% (w/v). .

In this embodiment, the bifurcated Po yeast strains include Bifidobacterium (B.bifidum; BCRC 14615), Bifidobacterium longum (B.longum; BCRC 14602), Lactobacillus delbrueckii subsp. Bulgaricus (L.delbrueckii subsp . bulgaricus; BCRC 10696), Lactobacillus paracasei subsp (L.paracasei subsp paracasei;. BCRC 17475 ), Lactobacillus brevis (L.brevis; BCRC 12187), Lactobacillus (Lactobacillus sp;. BCRC17003), thermophilic Streptococcus salivarius subsp. thermophilus (BCRC 13869) and Saccharomyces cerevisia e (BCRC 21680).

Next, the inoculated plant medium is subjected to a symbiotic fermentation step, which is carried out at 37 ° C for 72 hours to 96 hours. The resulting symbiotic yeast product obtains the resulting symbiotic fermentation product and performs subsequent evaluation after the termination of the fermentation step and the solid-liquid separation step.

Example 2: Evaluation of symbiotic fermentation products by high performance liquid chromatography

1. Analytical product extraction of symbiotic fermentation products

The symbiotic yeast products obtained in the experimental examples of the first embodiment, the comparative examples 1 and the comparative example 2 were first extracted with 50% ethyl acetate, and then multi-layered (for example). For example, 6 layers of the filtrate filtered by gauze was dried at 45 ° C for 6 hours to obtain the extracted product of the test sample of the symbiotic fermentation product, and 1 mg of the sample was taken and dissolved in 2 mL of DMSO for high performance liquid chromatography ( High performance liquid chromatography; HPLC) analysis.

2. Analytical method for extracting products from symbiotic fermentation products

The HPLC analysis conditions are as follows, and the results are shown in Figure 1:

(i) Analysis column: Cosmosil C18

(ii) UV-VIS detector: Hitachi L-7420

(iii) Detection wavelength: 243 nm

(iv) Mobile phase: Eluent A: methanol/acetic acid (100/0.5)

(v) Eluent B: methanol/water/acetic acid (80/20/0.5)

(vi) Gradient elution conditions: the methanol concentration increases linearly from 80% to 84% in 0 to 15 minutes, then increases to 86% in 15 to 30 minutes, and increases to 88% in 30 to 40 minutes. It is further increased to 94% in 0 to 50 minutes and further increased to 100% in 50 to 70 minutes.

(vii) Flow rate: 0.7 mL/min.

Please refer to FIG. 1 , which is a HPLC chart of the symbiotic fermentation product obtained in the experimental example of the first embodiment, the comparative example 1 and the comparative example 2. It can be seen from the results of Fig. 1 that the peak pattern of the different plant culture media is different after being treated by the steps of symbiotic fermentation. The peak pattern of the symbiotic fermentation product of the experimental group 1 can be used as a reference and as a quality control standard for the production of symbiotic fermentation products in the future.

Example 3: Evaluation of symbiotic fermentation products inhibiting the activity of hyaluronidase (ie, anti-allergic activity analysis)

Cell culture

First, mouse 3T3 fibroblasts (BCRC 60071) were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum (Hyclone) and 2.5% bovine calf serum (Hyclone). DMEM) medium and added 3.7% (w/v) sodium bicarbonate, 0.03% (w/v) glutamate (L-glutamine), 100 units of penicillin per ml and 100 mg of streptavidin Antibiotics such as streptomycin are cultured in a 10% CO ₂ incubator at 37 ° C, usually inoculated in a 75 cm ² culture dish at a cell density of 1 × 10 ⁶ cells, subcultured every three to four days and replaced with fresh Medium. After that, about the 10th generation of 3T3 cells were taken and cell experiments of symbiotic fermentation products at different concentrations were performed.

2. Hyaluronidase activity inhibition assay method (hyaluronan-based zymography)

3T3 mouse fibroblasts (BCRC 60071) were co-cultured with the symbiotic yeast products of the experimental examples of Examples 1 and 1 to 2, or not with the aforementioned symbiotic fermentation products (control group). to cultivate. After 1 or 2 days of culture (24 or 48 hours), the cell culture medium of each group is taken to prepare an electrophoretic film containing hyaluronic acid (including stacking gel and separating gel), and then subjected to protein electrophoresis analysis, wherein the conditions of protein electrophoresis should be The invention is well known to those skilled in the art and will not be further described herein. After the electrophoresis is finished, the glue in the electrophoresis tank is used. The tablets were taken out, immersed in a washing buffer, and incubated at room temperature for 1 hour, and then the washing buffer solution was removed, and then added to a reaction buffer, and shaken in a 37 ° C water bath for 16 hours. After the reaction was completed, the film was taken out, immersed in a 0.5% Alcian blue solution for 2 hours, and then immersed in a destaining buffer for about 1 to 2 hours. The band is clearly visible. After that, it is fixed on the acrylic plate with a cellophane sheet and left to dry naturally at room temperature, that is, the analysis step of the effect of the extract product of the symbiotic fermentation product on the hyaluronidase activity is completed, and the result is shown in FIG. 2A. Figure 2C.

2A to 2C, which show the symbiotic fermentation of the experimental example (Fig. 2A), the comparative example 1 (Fig. 2B), and the comparative example 2 (Fig. 2C) of Example 1 according to the present invention at a concentration of 1.0 to 10%. A photo of the hyaluronidase zymogram of hyaluronidase after co-culture with 3T3 fibroblasts. In Figures 2A to 2C, the Mth lane represents the protein marker, and the C0, _C24 , and _C48 lanes show the hyaluronidase activity of the untreated 3T3 mouse fibroblasts after ₀ , ₂₄ , and ₄₈ hours of culture, respectively. .

2A to 2C, it can be seen that the symbiotic fermentation product of the experimental example (Fig. 2A) of the first embodiment of the present invention has a strong inhibitory effect on the hyaluronan activity at a concentration of 1.0 to 10% (treatment for 24 hours). And no dose dependence. Herein, FIG. 2A shows not only the symbiotic fermentation product of the experimental example of Example 1 in a low dose (1.0%) experimental example ( FIG. 2A ), but also within 24 hours. Complete inhibition of hyaluronidase activity, significantly inhibiting hyaluronic acid activity at concentrations of 1.0 to 10% Sexuality, and its inhibition does not have a dose dependency between 1.0 and 10% concentration. The inhibitory effect of the symbiotic fermentation product of the experimental example of the present invention on hyaluronic acid is apparently more effective than other hyaluronidase inhibitors known to have a dose- and time-dependent natural source product. As for the symbiotic yeast product of Comparative Example 1 (Fig. 2B) at a concentration within 10% (treatment for 48 hours), there was no inhibition of the enzyme activity of hyaluronidase, and there was no effect of protecting hyaluronic acid. The symbiotic yeast product of Comparative Example 2 (Fig. 2C) had a more effective inhibitory effect (or even complete activity) on hyaluronan activity at a concentration of 10.0% (treatment for 24 hours) and at a concentration of 1.0% (treatment for 48 hours). Inhibition), but the inhibition effect is far less than the experimental example (Fig. 2A).

Since the symbiotic fermentation product of the experimental example of Example 1 can significantly inhibit the activity of hyaluronidase, it has a better effect of protecting hyaluronic acid, that is, the ability to delay the destruction of hyaluronic acid by hyaluronidase due to aging factors. As an active ingredient, it is applied to a composition for external use on the skin, such as a cosmetic composition.

Next, the symbiotic fermentation product of the experimental example of Example 1 was administered at a low dose (3.5 g/kg body weight/day) and a medium dose (7 g/ according to the health food safety assessment method announced by the Food and Drug Administration of the Ministry of Health and Welfare of Taiwan. The dose of kg body weight/day and high dose (11.67g/kg body weight/day) was administered orally to the test animals (male and female rats, at least three replicates) using a gastric tube for 28 days of subchronic toxicity. The trial did not cause death and adverse clinical signs. Daily weight gain and water, feed consumption, morphology, behavioral activity, and organ tissue weight did not produce significant dose-related changes. The results of blood analysis and biochemical indicator test showed that the symbiotic fermentation of the experimental example of Example 1 was fed. There was no significant difference between the product and the comparative example or the control group, and it was confirmed that there was no obvious subchronic toxicity, which met the food safety standard (not shown), and the symbiotic fermentation product of the experimental example of Example 1 was estimated. The no-observed-adverse-effect-level (NOAEL) was observed to be 11.67 g/kg/day.

Since the symbiotic fermentation product of the experimental example of Example 1 can significantly inhibit the activity of hyaluronidase and meet the food safety standards, it can be used as an active ingredient and used for preparing an oral composition having activity of inhibiting hyaluronidase. And/or pharmaceutical composition.

In summary, the above-described several examples demonstrate that the present invention is derived from a hyaluronidase inhibitor of a symbiotic fermentation product, which is successfully produced by a plurality of fermentation strains through a symbiotic fermentation step and a solid-liquid separation step, and The obtained symbiotic yeast product does have an activity of inhibiting hyaluronidase, and can further inhibit allergy, inflammation, and degenerative arthritis, can be applied to a composition for external use on skin, or an oral composition having hyaluronidase inhibiting activity or Pharmaceutical composition.

It should be noted that the present invention exemplifies a symbiotic fermentation product having the activity of inhibiting hyaluronidase and the use thereof according to a specific process, a specific analysis method or a specific instrument, but the technical field to which the present invention pertains It is to be understood by those skilled in the art that the present invention is not limited thereto, and other processes, other processes, may be used for the symbiotic fermentation products of the present invention having the activity of inhibiting hyaluronidase activity without departing from the spirit and scope of the present invention. Analytical methods or other instruments are performed.

It can be seen from the above examples that the hyaluronidase inhibitor derived from the symbiotic fermentation product of the present invention and the use thereof have the advantages of utilizing plural The fermented strain is obtained by the symbiotic fermentation step and the solid-liquid separation step, and the obtained symbiotic yeast product has the activity of inhibiting hyaluronidase, and can be applied to the external composition for skin and prepared for oral administration inhibiting hyaluronidase a composition or a pharmaceutical composition.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims (6)

A hyaluronidase inhibitor derived from a symbiotic fermentation product, which is obtained from a phytochemical medium by a plurality of fermentation strains through a symbiotic fermentation step and a solid-liquid separation step, wherein the symbiotic fermentation The step is carried out at 37 ° C for 70 hours to 100 hours, and the fermentation strains are composed of a plurality of strains of lactic acid bacteria and at least one strain of non-lactobacillus strains, which are composed of a plurality of strains of Bifidobacterium (. Bifidobacterium spp) (. Lactobacillus spp) and a plurality of kinds of strains of Lactobacillus strain composition, the plurality of lines by the genus Bifidobacterium bifurcated bifidobacteria (B.bifidum; BCRC 14615), Bifidobacterium longum (B. longum; BCRC 14602) consisting of, the plurality of lines of Lactobacillus delbrueckii subsp. bulgaricus (L.delbrueckii subsp bulgaricus;.. BCRC 10696), Lactobacillus paracasei subsp (L.paracasei subsp paracasei; BCRC 17475 , Lactobacillus brevis ( L. brevis ; BCRC 12187) and Lactobacillus sp. (BCRC 17003), the at least one non-lactobacillus strain is a strain of Streptococcus spp. and a genus of the genus ( Sac A strain consisting of a strain of Streptococcus salivarius subsp. thermophilus (BCRC 13869), and the strain of the genus Saccharomyces cerevisia e (BCRC 21680). The hyaluronidase inhibitor derived from a symbiotic yeast product according to claim 1, wherein the yeast extract is inoculated in an amount of from 3 to 5 wt% in the plant medium. A hyaluronidase inhibitor derived from a symbiotic yeast product according to the first aspect of the invention, wherein the vegetable medium comprises soybean, sesame, alfalfa, sucrose, sodium chloride and water. The hyaluronidase inhibitor derived from a symbiotic yeast product according to claim 2, wherein the vegetable medium comprises 1.0 to 10.0% by weight of the soybean, 1.0 to 5.0% by weight of the sesame, 1.0 Up to 5.0% by weight of the cerium, 1.0 to 5.0% by weight of the sucrose, and 0.1 to 2.0% by weight of the sodium chloride. A hyaluronidase inhibitor derived from a symbiotic yeast product according to claim 2, wherein the vegetable medium comprises 1.0 to 6.0% by weight of the soybean, 1.0 to 3.0% by weight of the sesame, 1.0 Up to 3.0% by weight of the bismuth, 2 to 5% by weight of the sucrose, and 0.2 to 1.0% by weight of the sodium chloride. A skin external composition comprising the hyaluronidase inhibitor according to any one of claims 1 to 5, wherein the hyaluronidase inhibitor is an active ingredient.