WO2013176320A1

WO2013176320A1 - Human-derived lactobacillus rhamnosus hk-9, and fermented product and cosmetic composition using same

Info

Publication number: WO2013176320A1
Application number: PCT/KR2012/004156
Authority: WO
Inventors: 이현용; 최운용; 서용창; 김지선; 이춘근; 송치호; 윤창순; 임혜원
Original assignee: 강원대학교 산학협력단; 주식회사 세바바이오텍; 서원대학교 산학협력단
Priority date: 2012-05-25
Filing date: 2012-05-25
Publication date: 2013-11-28

Abstract

The present invention relates to a human-derived Lactobacillus rhamnosus HK-9 and an application method thereof, and more specifically, to: Lactobacillus rhamnosus HK-9 which increases physiological activities and has antioxidant effects associated with whitening and antiaging effects through the fermentation of a natural substance; and a fermented product and a cosmetic product using the same. According to the present invention, the Lactobacillus is a strain derived not from common food but from the human body, and is thus very effective for activities such as food fermentation, whitening, and the like. A known problem of the deterioration of fermentation efficiency due to no elution or very small elution from a natural substance is overcome by the Lactobacillus, and thus it is possible to effectively elute active materials and obtain cosmetic materials.

Description

[Specification】

[Name of invention]

Lactobacillus Lactobacillus rhamnosus HK-9 derived from human body and fermented products and cosmetic composition using the same

Technical Field

The present invention relates to lactic acid bacteria Lactobacillus rhamnosus (Hactin) HK-9 derived from the human body and a method of using the same, more specifically, it has a physiological activity enhancing activity through natural fermentation, as well as a whitening effect and anti-aging It relates to a lactic acid bacterium Lactobacillus rhamnosus HK-9 and its fermentation and cosmetic products using the same.

Since the lactic acid bacteria according to the present invention is a strain derived from the human body rather than a general food, it is very effective for not only food fermentation but also whitening activity. Through this, it is not eluted from the existing natural water or very small amount is eluted to overcome the problem of lowering the fermentation efficiency can effectively elute the effective material, it can be effective as a raw material of cosmetics. Background technology

^ Acidophilus (Lactobacillus) is an important microorganism widely used worldwide for the production of fermented dairy products, such as cheese, fermented milk and lactic acid bacteria beverages.

Lactic acid bacteria have long been used for the purpose of treating and preventing the fermentation and intestinal diseases of the food industry. There are several reports on the use of lactic acid bacteria. For example, 70% of patients suffering from diarrhea have been treated since reports of Lactobacillus were administered to infant diarrhea symptoms (RO Clock, JAMA Vol. 61, P.164, 1913). Report (LM Gornpertz, et al., JAMA Vol. 80, P.90, 1923), inhibits the growth of harmful bacteria in the intestine and creates favorable conditions for the growth of beneficial bacteria, preventing food poisoning, preventing intestinal rot, preventing constipation, and immunity It has been found that it has various physiological functions such as increase in the amount of cancer and suppression of carcinogenesis.

Fermented products using these lactic acid bacteria are commercialized in the form of yogurt or cheese. It is also affecting the food industry such as Kimchi and Cheonggukjang.

On the other hand, when fermenting a product using a general lactic acid bacteria, it is true that there is a limit in the production of various bioactive substances produced during the flavor and fermentation of fermented products. In addition, the fermentation yield may vary when fermenting natural products, depending on the type of lactic acid bacteria, and some of them are used in foods, and some cosmetics are used as raw materials for cosmetics. It is also true. By the way, the lactic acid bacteria used to produce fermented products possessing such functionalities are imported most of the fermentation strains from abroad, and the source of lactic acid bacteria is a strain isolated from fermented dairy products such as cheese.

Therefore, securing a new lactic acid bacterium with excellent fermentation ability can have a very economical ripple effect. The present inventors focused on saying that food is a human hand and that there are many lactic acid bacteria in a woman's hand. In fact, there are reports of ^' lactic acid bacteria ^' in women's hands (The influence of sex handedness and washing on the diversity of hand surface bacteria, PNAS 105 (46) 17994-17999). A physiological activity test was conducted, and among them, Lactobacillus spp. Was isolated to isolate Lactobacillus rhamnosus HK-9.

The lactobacillus Lactobacillus rhamnosus HK-9 isolated by the present inventors is a lactic acid bacterium separated from the female hand, taking note that the female hand is finer than the male hand. As a result of the test, the lactic acid bacterium was found only in the female hand. In addition to fermentation ability, which is a function of lactic acid bacteria, the strain has been verified to have an active ability such as whitening, which seems to be related to a phenomenon detected only in female hands.

Ordinary lactic acid bacteria are most used in food fermentation because most of the strains obtained from food, but the strains identified by the present invention can be applied to both food and cosmetics.

[Content of invention]

[Problem to solve]

Although sufficiently suggested by the above description, an object of the present invention is to provide a novel microorganism that can be used for both natural product fermentation and cosmetics.

The inventors isolate strains that meet the above objectives and use them to confirm the increase in effective physiological activity through fermentation of natural products and the improvement of fermentation yield. Increased.

[Measures of problem]

The present invention as described above is characterized in that the lactic acid bacteria Lactobacillus rhamnosus HK-9 (Accession Number: KCCM11254P) isolated from the human body.

The present invention is also characterized in that the fermented product fermented with the lactic acid bacteria.

It is also characterized in that it is a cosmetic composition containing a liquid extracted from the lactic acid bacteria (lactic acid bacteria itself). At this time, the extract of the lactic acid bacteria itself, centrifuged Lactobacillus rhamnosus HK-9 strain (Accession Number: KCCM11254P) cultured in MRS medium to secure the strain, and the resulting strain was sonicated and broken, and then extracted with hot water It is characterized by being obtained by.

In addition, the present invention is characterized in that the cosmetic composition or food containing the above-mentioned fermented product. ^'

【Effects of the Invention】

The present invention relates to the Lactobacillus rhamnosus HK-9 isolated from female hands, the production of effective biologically active substance through natural product fermentation is increased than conventional lactic acid bacteria, the fermentation effect is excellent, the whitening effect and antioxidant associated with anti-aging Since it has the effect, it can be supplied as a raw material of food and cosmetics material and can have a great economic effect. '

[Brief Description of Drawings]

1 is a result of analysis of Lactobacillus rhamnosus HK-9 examined by the Korea Microorganism Conservation Center, 16S rDNA nucleotide sequence of the strain.

Figure 2 is a diagram showing the phylogenetic location of Lactobacillus rhamnosus HK-9.

3 is a view showing the morphological appearance of lactic acid bacteria by taking a SEM photograph of Lactobacillus rhamnosus HK-9.

Figure 4 is a diagram showing the cell concentration over time of Lactobacillus rhamnosus HK-9 using MRS medium.

5 is a view showing the ginsenoside content of ginseng according to the fermentation of Lactobacillus rhamnosus HK-9 strain and other common strains. 6 is a view showing the results of the production of new materials from the fermentation using Lactobacillus rhamnosus HK-9.

7 is a view showing the results of DPPH activity before and after fermentation of the fermentation deodeok. 8 and 9 are results showing the whitening-related efficacy test of Lactobacillus rhamnosus HK-9 strain itself, Figure 8 is a melanin synthesis inhibition activity experiment, Figure 9 Tyrosinase inhibition experiment (Tyrosinase inhibition) experiment The figure which showed the result.

[Specific contents to carry out invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. Example 1 Lactic Acid Bacteria Separation Method For the isolation of new lactic acid bacteria, men and women in their 20s and 40s were asked to participate about 7 days in the laboratory, and the participants who participated in the experiment were not washed and treated separately before the experiment. Experiments were conducted on unspecified days to avoid.

Initial microorganisms were inoculated by dipping the palms of the experimental participants into the prepared MRS Agar medium. After the inoculated initial samples were incubated for 24 hours at 30 ^° C., the obtained microbial colonies were again inoculated in MRS Agar medium to obtain strains. As a result of fermentation by colonies obtained at the second inoculation, the most active microorganisms were selected.

The microorganism obtained by the above method was commissioned by the Korea Microorganism Conservation Center, and the lactic acid bacteria were identified by PCR. The results are shown in FIG. 1 (base sequence of 16S rDNA, and the sequence is attached as SEQ ID NO: 1) and FIG. Location).

As a result of the Korea microbial preservation center was confirmed that the strain obtained in the present invention is a strain of the genus Lactobacillus (Lactobacillus). The inventors named the strain Lactobacius ^ no ^ ^ Lactobaci Uus rhamnosus HK-9, which was deposited with the Korean Culture Center of Microorganism as Accession No. KCCM11254P.

The morphology of the lactic acid bacteria confirmed through the SEM photograph of the novel strain Lactobacillus rhamnosus HK-9 is shown in FIG. 3. Example 2. Lactic Acid Bacteria Culture Method The lactic acid bacteria isolated through Example 1 were cultured in MRS medium to determine the optimum incubation time according to the characteristics of the strain. The culture method was prepared by adding 55% MRS medium and 0.5% L-Cysteine hydrochloride as a general lactic acid bacteria culture method, the culture temperature was 36 ^° C, and was tested in anaerobic conditions in sealed form due to the fermentation strain characteristics. Because it should be incubated at a minimum stirring speed (50 ~ 60rpm) and experimented in a volume of 400ml in a flask of 1L for 72 hours.

4 shows the results of cell concentration over time of the Lactobacillus rhamnosus HK-9 strain cultured by the above method. According to the results, when the cells were incubated for up to 12 hours, the concentration of the cells was present in an amount of about 644 X 10 ⁶ , but when incubated for 24 hours after 12 hours, the concentration was increased to 3255 X 10 ⁶ , which is the highest concentration. Cell concentration is maintained until, and after 48 hours it can be confirmed that the cells gradually decrease. Therefore, the optimal incubation time of the Lactobacillus rhamnosus HK-9 strain of the present invention was determined to be 48 hours. Example 3 Validation of Effective Biological Activity by Fermentation of Natural Products

1) Analysis of ginsenoside content of ginseng fermented ginseng The ginseng was fermented using the isolated Lactobacillus rhamnosus HK-9 strain. As a fermentation method, 100 _g of ginseng was ground and added to a 2 L flask, 1 L of culture medium was made, and 5% of Lactobacillus rhamnosus HK-9 strain was inoculated to ferment for 48 hours.

In addition, Lactobacillus rhamnosus GG, Lactobacillus paracasei, Lactobaci 1 lus case /, Lactobaci 1 lus acidophi lus PI ant arum + Bifidobacterium I act is Longum with Lactobacillus rhamnosus HK-9 strain as a control. The culture was carried out in the same manner as the HK-9 strain.

After ginsenoside analysis, the fermented product was powdered, and the total saponin content (Rg3 + Rg2 + Rh2 + CK + Rgl + Rbl + Rb2 + Rc + Re + Rd) was measured using butanol extraction.

As can be seen from the ginsenoside content results of the fermentation of ginseng according to the type of lactic acid bacteria of Figure 5, the highest amount of Lactobacillus rhamnosus HK-9 strain of the present invention It was confirmed that ginsenosides were detected, and showed higher efficiency up to 200% than normal lactic acid bacteria.

2) Antioxidant Activity of Golden Fermentation The fermentation of golden fermentation was carried out in the same way as the fermentation of ginseng. To a 2 L flask, 100 g of gold was added in a powder form, and then 1 L of culture medium was prepared, followed by fermentation for 48 hours by inoculating 5% of Lactobacillus rhamnosus HK-9 strain.

In addition, Lactobacillus rhamnosus GG, Lactobaci ilus paracasei, Lactobaci J Jus casei strains with the Lactobacillus rhamnosus HK-9 strain as a ^' control ^' was used in the same manner as the Lactobacillus rhamnosus HK-9 strain.

The fermentation broth was lyophilized and powdered for the experiment. The DPPH scavenging activity was measured, the reducing power was measured, and the total phenol content was measured. The results are shown in Table 1.

The antioxidative activity of the gold of Table 1. Through the experiments of the antioxidant activity of the lactobacillus rhamnosus HK-9 strain of lactic acid bacteria of the present invention was confirmed that the excellent antioxidant activity.

In particular, the results of the total phenolic content of the antioxidant activity can be seen that the activity is about 2 to 4 times higher than the gold fermented through other lactic acid bacteria. Based on these results, cosmetic activity of Lactobacillus rhamnosus HK—9 strains of the present invention (anti-aging And antioxidant activity associated with

As a result, it can be seen that when the fermentation is performed through Lactobacillus rhamnosus HK-9 strain, the useful physiological activity of fermentation is easy and the cosmetic activity is superior to other lactic acid bacteria.

3) Generation of New Substances by Fermentation of Deodeok The inventors measured the changes in bioactive substances before and after fermentation of Deodeok using the strain according to the present invention.

Before fermentation, deodeok was dried for 24 hours at a temperature of 40 ^° C using a hot air dryer, and then pulverized to a size of 2-3 mm using a ball mill and extracted with 70% ethanol.

In addition, the fermentation deodeok fermented in the same manner as the fermentation of the ginseng, and dried dry deodeok lOOg in powder form, added to a 2L flask, 1L culture medium, and then inoculated with Lactobacillus rhamnosus HK-9 strain> Fermentation was carried out for 48 hours. The HPLC analysis results of Deodeok prepared by the above method are shown in FIG. 6. As a result of FIG ^. 6, only one peak of about 2.7 minutes was detected before the fermentation, but after the fermentation, a new peak was detected at 3.1 minutes together with the peak detected at about 2.7 minutes.

This new peak of the material is a new physiological activity, as shown in Figure 7 showing the DPPH activity of Deodeok before fermentation and after the fermentation, it can be seen that the DPPH activity of Deodeok increased through fermentation, thereby lactobacillus It can be seen that the novel substance produced through the fermentation of Rhamnosus HK-9 strain affects DPPH activity.

4) Determination of perfume activity according to Lactobacillus rhamnosus HK-9 strain itself. Tyrosinase inhibition experiment was performed. First, in order to disrupt Lactobacillus rhamnosus HK-9 strain, Lactobacillus rhamnosus HK-9 strain cultured in the existing MRS medium was centrifuged to secure a total of 100ml strains. Thereafter, the strain was sonicated for 20 to 50 minutes to crush the strain. The crushed strain and its solution were subjected to hot water extraction at 60 ^° C to obtain a strain extract, and the cosmetic activity was tested.

8 shows the results of melanin synthesis inhibition activity (Melanin synthesis inhibition activity). In order to observe the effect on melanin biosynthesis and cell viability when the sample was administered using Clon-M3, a melanocyte (melanocyte), melanin production inhibition was measured after 24 hours. As a result, it was confirmed that the inhibition rate increased from 12.5 mg / ml to 100 mg / ml, and finally, about 40% inhibition rate was observed. These results show that there is an effect of reducing melanogenesis in the components of the strain itself of the Lactobacillus rhamnosus HK-9 strain. In addition, Figure 9 is the result of measuring Tyrosinase inhibition (Tyrosinase inhibition). Tyrosinase is an enzyme that stimulates the production of skin melanin pigment, and the whitening effect can be recognized by measuring the inhibitory activity of tyrosinase. As a result of FIG. 9, it was confirmed that the inhibition rate was finally about 22%.

The results are lower than the melanin inhibition rate using natural products, but the result is a decisive experiment that contains the components having fragrance activity in the strain itself, which is relatively easier to produce than producing perfume products using expensive natural products. Fermentation strains are expected to be used to produce cosmetics-related raw materials and products more competitively.

[Accession number]

Name of Depositary ： Korea Microorganism Conservation Center (overseas)

Accession number ： KCCM11254P

Date of Trust ： 20120217 ¾ or representative PCTA120 ⁴ 0 ⁷ 0 International application number

Reference number

PCT / KR2012 / 004156 Labeling of deposited microorganisms or other biological crop materials

(Article 13-2 of the Rules of Patent Cooperation Treaty)

A. The following indications shall be deposited on page 3 of the specification, _ second _ line:

Regarding microorganisms or other biological substances.

B. Additional deposits other than the confirmation of deposits shall be listed on a separate page.

Address of the deposit institution of the Korea Microbial Conservation Center (including postal code and country name)

Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seolleung-si, South Korea (Postal code: 120-091) Deposit date February 17, 2012 Deposit number KCCM11254P

C. Additional Markings (Submit blank if left blank) Continue to Additional Page

D. Designated station designated by indication (if indication is not for all designated stations)

E. Submission of Marked Items (Submit blank if not specified)

The following indications will be submitted later to the International Bureau (specify the general nature of the indications).

Receiving Office Use Section International Bureau Use Section

^ The paper was received with the international application. □ The paper was accompanied by the international application. Received: Representative

Form PCT / RO / 134 (July 1998; reprint January 2004)

Alternative Site (Article 26)

Claims

[Patent Claims]

[Claim 1]

Lactobacillus Lactobacillus rhamnosus HK-9 isolated from human body Accession No .: CCM11254P).

[Claim 2]

Fermented product fermented with lactic acid bacteria of claim 1. ·

[Claim 3]

Cosmetic composition containing the extract of lactic acid bacteria itself of Claim 1. ^'

[Claim 4]

The method of claim 3,

The extract of the lactic acid bacteria itself, centrifuged Lactobacillus rhamnosus HK-9 strain (Accession Number: KCCM11254P) cultured in MRS medium to secure the strain, and the resulting strain was sonicated and crushed, hot water extraction Cosmetic composition, characterized in that obtained by.

[Claim 5]

Cosmetic composition containing fermented product of claim 12.

[Claim 6]

Food containing the fermented product of Claim 2.