JPS63179829A - Antibacterial agent for skin - Google Patents

Abstract

PURPOSE:To obtain the titled antibacterium agent containing bacterium cell of a microorganism belonging to the genus Streptococcus, etc., and/or water- extracted product thereof as an active ingredient. CONSTITUTION:A bacterium cell of microorganism belonging to the genus Streptococcus, Lactobacillus or Bifidobactrerium (e.g. Streptococcus faecium, Streptococcus durans, Streptococcus avium, Streptococcus faecalis, Lactobacillus brevis, Lactobacillus salivarius, Bifidobacterium adolecsentis, etc.) and/or water- extracted product thereof is included as an active ingredient to provide the aimed antibacterial agent for skin. The agent has strong antibacterial property to Propionibacterium acnes which is an acnegenic-originated bacterium. The agent includes various kinds of cosmetic such as bathing agent or cosmetic for bath, facial-cleansing agent, anti-suntan cream, hair fixing agent, and, composition for skin coating, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an antibacterial agent for the skin containing as an active ingredient the cells and/or water extract of a microorganism belonging to the genus Streptococcus, a microorganism belonging to the genus Lactobacillus, or a microorganism belonging to the genus Bifidobacterium. The present invention relates to agents, especially anti-loin sore agents, skin disease improving agents, and skin protective agents.

Propionibacterium acnes, the main bacterial cause of FFL acne.
terium acnes or Corynebacterium
Antibacterial substances against Rium parvum include hexachl orophene,
Side effects such as effects on the human body and intestinal bacteria are virtually unknown, and it is difficult to conclude that they are necessarily safe for skin absorption through skin application and oral entry into the body.

In view of the above, the present inventors conducted intensive research and found that microorganisms belonging to the Streptococcus genus, Lactobacillus genus, or Bifidobacterium genus, which are lactic acid bacteria derived from healthy human intestinal bacteria, or their aqueous extracts can be used as probiotics. Pionibacterium・
It has strong antibacterial activity against acne, and the results of oral administration or skin application experiments show that these lactic acid bacteria cells or aqueous extracts are virtually non-toxic, including effects on intestinal bacteria and skin. The present invention was developed based on this knowledge.

Hereinafter, the types and mycological properties of microorganisms that can be used in the present invention, screening methods, cell preparation methods, pharmacological actions, etc. will be discussed in detail.

A microorganism belonging to the genus Streptococcus, Lactobacillus or Bifidobacterium, especially Streptofuccus faecium' (S, faecal).
is), Lactobacillus brevi, etc. can be exemplified as suitable examples. Here, particularly useful bacterial strains in the present invention are shown in Table 1 below, together with their Hui Koken accession numbers.

Table 1 5treptococcus faecium
ADiO05FERN P-8697S
treptococcus avium
AD2001 FERN
P-8698Streptococcus dura
ns AD3003
FERN P-8699Streptococcu
s faecalis AD9002 F
ERN P-8696Lactobacillus
brevis ADOO12FER
NP-8695Lactobaeillus 5a
livarius ADOO13FERM P-8
694Bifidobacterium aclol
essentis ADOO53FERM P-8
693Bifidobacterium breve
ADOO54FERM P-86
92Bifidobacterium forum
ADOO57FERN P-8691
=3- Mycological Properties The screening method, identification, and other mycological properties of the microorganism of the present invention are summarized as follows.

1. Screening method Watanabe + T,, et al, + 5tu
dies on 5trept.

cocci, 1. Distribution of
fecal 5treptococci in man
, old crobio1. Immuno1.25.257-
269 (1981). That is,
As described in the above literature, feces of a healthy person was diluted with a diluent having the following composition (Table 2), and Streptococcus was found to be KMN.
agar [vander WeitoKorstan
je, J, A.

A,, and K, C0Winkder: J,
Med, Microbiol.

8491 (1975): Table 3 1, U9)
) I, siiLBsagar [BBL+ Table 4 1
, Bifidobacterium is MPN agar [T
anaka, R, et al, + 8pp1.
Environ.

Microbiol, 40.866-869 (198
0), Table 5], cultured at 37°C for 48-72 hours under aerobic to anaerobic conditions, counted the colonies produced, and randomly picked them. Colony shape, catalase activity (negative), Durham staining. Positive cocci and bacilli were isolated and classified and identified by examining their physiological and biochemical properties.

Table 2 Composition of diluent K112P0. 4.5gN
a2HPO46, Og L-cystine hydrochloride 0.5° Tween 8
0 0.5g agar
1.0g purified water
1.000ml! Table 3 KMNagar (Recl-Kanamycin-+n
1lk agar) n set (tributose 15g meat extract 3g sodium aceide 0.2g pH 6,8 salt
5g Agar at 121℃ for 10 minutes
1.8g Purified water 800 mβ Skim milk 16g B =, - Toral Red 40mg 100°C1
Purified water 200 mN C Kanamycin 24 mH After separately sterilizing A, B, and C, combine them to form a flat plate.

Table 4 Composition of LBSagar (BBL') Tributose
10g Meat extract 5g KII2PO, 6g Triammonium citrate 2g Glucose 20g Sodium acetate anhydride 15g Tween 801g Mg5O<*7 N20 575mgM
n5O< ・2H20120+oH FeSO+ ・7H2034mg Agar 15g Purified water 100mN pH5,51
Heat sterilized at 21°C for 15 minutes Table 5 Composition of MP Nagar Lactose 2g (N114)2SO, 0,51? 2HPO, 0.1g Mg5L ・7H2020mg Fe50.・7H201mg Mn5'04・2820 0.8mg salt IB O, 1% resazurin 0. IB biotin
0. OIB Calcium pantothenate 0.21 Riboflavin 0.1mg Adenine
O, IB Guanine O, 1mg xanthine
O, 1mg Uracil 0. lll1g tween 8
0 0.1g10% pyruvic acid
0.1ml 18% Na2C0 35mN 3% L-cystine hydrochloride 1mN Nalidixic acid 1010ll 1.6% Bromucresol Purple 0.1mN Agar
2g Purified water 100mn pH6,8
Heat sterilization at 100° C. for 30 minutes without pressure 2. Identification of isolated lactic acid bacteria The general bacteriological properties of the microorganism of the present invention are in the same classification and have the same properties as those shown in various known documents.

That is, selective medium KHN for Streptococcus bacteria
agar medium, selective medium for Lactobacillus bacteria LBS
Agar medium, and a selective medium for Bifidobacterium bacteria MPNagar medium, based on colony shape, Durham staining, morphology, and physiological and biochemical properties, the following documents 1) to 4
).

References 1) Tomozoku Mitsuoka; Japanese Journal of Bacteriology, 24(6), 26)
-2) Poupard, J, ^, + Hus
ain, r, and Norris+R,F,
, : Bacteriol, Rev., 371
36-1653) Bergey's Mannua
l of Determinative Bacter
iology, 8th ed, 490-67
6 (1974) 4) Tomozoku Mitsuoka: Clinical and Bacteria, 2 (3),
55(197)-97(239), (1975) The main mycological properties used as the basis for identification are summarized in Tables 6 to 8.

(Margins below) -] 1-3. Cultivation method These microorganisms are cultured using conventional methods as shown in the above-mentioned documents. For example, Streptococcus microorganisms and Lactobacillus microorganisms are cultured using Rogosa.
) in a liquid medium (Note 1), microorganisms of the genus Bifidobacterium in a CAM liquid medium (note 2), genus Streptococcus and genus Lactobacillus in an aerobic or anaerobic manner, and microorganisms of the genus Bifidobacterium in an anaerobic manner (note 2). This culture is carried out, and the resulting culture solution is centrifuged to collect the bacterial cells.

(Note 1) Trypticase of iH of Rogosa liquid medium in 1 p of distilled water], O, Og yeast extract 5. Og trip
- Su 3
．． 0gK211P0. 3.
OgK)l 2P0.
3.0 g sodium acetate (″
)1. Og triammonium citrate 2.0g Tween 8
0 1.0゜Glucose
20. OgL-cystine hydrochloride
0.2g salt solution ("" 5.0
mN (pH 7, o, heat sterilized at 121°C for 15 minutes) (
Wood) Sodium acetate is not necessary for Streptococcus (* Wood) Salt solution Mg5O in 100 mN of distilled water <・
7) 120 11.5gFe5L ・7H
200,68g Mn5O,・2H202,4g (Note 2) CAM liquid medium composition CAM broth “Nissui Pharmaceutical Co., Ltd.” Food 05422
59, Og (IN) Peptone 10.0g Soybean Peptone 3.0g Proteose Peptone
10.0g digested serum powder 13.0g yeast extract 5.0g meat extract
2.2g liver extract powder 1.
2g Gurufus 3.0gKH2P
0. 2.5g sodium chloride 3.0g soluble starch
5. OgL-cystine hydrochloride 0.3g Sodium thioglycolate 0.3g (pH 7.3±0.
1.Heat sterilization at 115°C for 15 minutes) The obtained microbial cells can be used as drugs as they are, either as live microorganisms or as killed microorganisms by heat treatment, but they can be used as destroyed microbial cells by ultrasonication, etc. may be provided. Therefore, the term "killed bacterial cells" in the present invention includes all or part of these destroyed bacterial cells.

Preparation of an antibacterial agent for the skin The antibacterial agent for the skin of the present invention contains various sterilized cells of the above-mentioned microorganisms or their water extracted fractions as active ingredients. An example is as follows.

1. Hot water oil treatment The collected bacterial cells are heated to 80-130'C (more preferably 1
00 to 120'C), subjected to pressurized or non-pressurized hot water extraction treatment that also serves as sterilization for several minutes to several hours, and then subjected to centrifugation treatment, etc.
By removing the water-insoluble solids, a water-soluble fraction containing the desired active ingredient is obtained. The extraction solvent used is normal saline (
0.9% NaCρ aqueous solution, etc.), various buffer solutions prepared to a predetermined pH value, various salt solutions, water:alcohol (methanol and ethanol) mixed solvent with a water/alcohol > 172 (weight ratio), etc. , various aqueous solvents, and water alone may be used as well.

Furthermore, the whole collected bacterial body was subjected to the sterilized hot water extraction treatment,
It should be noted that freeze-dried, vacuum-dried, spray-dried powders, etc., without further solid content removal treatment such as centrifugation, are also useful as the antibacterial agent for skin of the present invention.

2. Various sterilized bacterial cells obtained by subjecting the collected bacterial cells to heat sterilization such as spray drying or ultrasonic destruction treatment (for example, 15 kc, 60 minutes) can also be used as the active ingredient of the antibacterial agent for skin of the present invention. can be done. Furthermore, these sterilized bacterial cells may be subjected to water extraction treatment to obtain water-soluble components to obtain the desired active fraction.

) Antibacterial activity 1, antibacterial activity As shown in the experimental examples below, the antibacterial agent for skin of the present invention very effectively suppresses or inhibits the growth of Propionibacterium bacteria that are the causative agents of chiasmia. On the other hand, the selection does not have a substantial inhibitory effect on the major lactic acid bacteria of intestinal bacteria (Streptococcus faecalis, Lactobacillus faecium, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacterium plebis, Bifidobacterium adolescentes, Lactobacterium breve). Exhibits a specific antibacterial spectrum.

2. Toxicity Orally, it is virtually non-toxic at all, and its LD. The value was about 5 mg/mouse or more as a hot water extract or sterilized body.

3. Usage mode The antibacterial agent for skin of the present invention can be used as a bath agent or bath cosmetic, a facial cleanser,
It can be used in various cosmetics such as sunscreen creams, hair and scalp preparations, lipsticks and blushers, and compositions for skin application, or as a skin disinfectant to improve skin diseases or to provide skin protection in the form of application powders or IJ-mums. The amount used is usually about 0.001 to 10% by weight (in terms of dry weight) as a treated bacterial cell or aqueous extract.

Hereinafter, the present invention will be explained in more detail using experimental examples.

Experimental Example 1 Propionibacterium acnes (Propionibacterium acnes)
bacterium acnes: 1640001
．． Each lactic acid bacterium of the present invention was inoculated into a Rogosa liquid medium or a GAH liquid medium (distributed from Osaka University Huiken Bacteria Stock Storage Facility) to a viable cell count concentration of approximately 1.067 mN, and then aerobically grown. 1 under target or anaerobic conditions
Cultured at 37°C for 5-24 hours. After culturing, the bacteria were collected by centrifugation, the collected bacteria were suspended in about 10 volumes of physiological saline, and the process was repeated twice to collect the washed bacteria.

This was suspended in 1 volume of distilled water and 110 to +20'
Autoclave for 10-15 minutes at 40°C. Then, centrifugation was performed, and the supernatant itself or its freeze-dried material was used as a sample. GAM agar containing the above sample
The culture medium (Note) was brought to an anaerobic state, and Propionibacterium acnes was inoculated at an amount of approximately 0.03 cm and cultured under anaerobic conditions. 4
The number of colonies produced after 8 hours was counted, and the inhibition rate was measured in comparison with a control containing no sample. The results are shown in Table 9.

(Left below) =20- As is clear from the above results, S, faecium, S,
Three species, P. dudurans, B. and Adolescentes, strongly inhibited the proliferation of P. acnes, and the other six species also inhibited the proliferation.

Among these nine species, especially S. faecium 8 D1005, Funtrol (that is, a group in which only water as a solvent in the bacterial cell extract is added) has 441 P per plate,
Although P. acnes colonies were produced, it also showed a bacteriostatic ability that suppressed the growth of P. acnes by almost 100%, at one colony per plate.

Experimental Example 2 Comparison of the inhibitory effect of Streptoconcus on the growth process of P. acnes A CAM liquid medium containing a sample prepared in the same manner as in Experimental Example 1 was brought into an anaerobic state, and P.
ml! It was inoculated and cultured. And 0°6, 12.24
．． The number of viable bacteria (/mρ) was measured after 30 hours. The results are shown in Figure 1. 8.7 Esium, S, duurance,
The growth of P. acnes was inhibited in the following order: S., E. virum, and 8.7 Ecaris.

In S. faecium AD1005, the growth of P. acnes was suppressed for nearly 30 hours. and 12-15
The time was reduced from the number at 0, and it also showed bacteriostatic ability.

Although omitted here, it should be added that B. Adrecentes ADOO53 also shows almost the same results as S. Durans.

Regarding 8.7 Ecium 8 [11005], the cell aqueous extract prepared in the same manner as in Experimental Example 1 was added to the CAM liquid medium at three concentrations based on the dry bacterial weight, and the experiment was carried out in the same manner as in Experimental Example 2. Ta. The results are shown in Figure 2.

As shown in the figure, P is proportional to the concentration (amount of protein).

The growth of C. acnes was inhibited, and the inhibitory ability was remarkable until about 12 hours of culture.

Antibacterial Spectrum Human intestinal bacteria (
The results of testing the effects on various lactic acid bacteria and Escherichia coli are summarized in Table 10 below. From the table, it can be seen that the hot water extract is inactive against major intestinal bacteria.

(Margins below) Acute toxicity■ I'CR mouse (male 6 weeks old, average weight 33.0±
0.5g), and the hot water extract prepared according to the method for producing the hot water-treated extract was added to 9×109.9×1 per mouse.
0'', 9X10' starting bacteria count in 3 stages (10 bacteria in each group)
A suspension of 0.5 ml (0.5 ml) of physiological saline was administered intraperitoneally, and the mice were observed for 14 days to see if they were alive or dead.

Calculated according to the Behreus-Kmrber method
The 5o values (-g/mouse) are shown in Table 11. In addition, in both cases of daily oral administration and skin application, the drug was completely nontoxic.

Table 11■ ICII mouse (male 6 weeks old, average weight 30.5±
0.6g) and the sterilized body obtained according to the above-mentioned heat-sterilized body preparation example was 1) 9 x 109.9 x 108.
The suspension of physiological saline 0.5 + nA' was intraperitoneally administered to 9X]O' bacteria cells at three stages (10 animals in each group), and 14
The survival of the mice was observed for several days.

L calculated according to Behrens-'arber method
The D5o value (number of bacterial cells/mouse) is 6 x 109 cells/mouse or more (intraperitoneal administration) for all bacteria, and virtually no total fever occurs in either oral administration or skin application. It was non-toxic.

Phantom L [1. Facial cleansing cream fatty acid 36 oleyl alcohol 1.5 lanolin derivative book (E, 0. adduct) 1.0 glycerin
18.0 Potassium hydroxide 6.
0 Water extract of the present invention 0.1. -1.0
Fragrance 0.5 Preservative
Appropriate amount of purified water
36-36.9 100% by weight 2. Bath agent lauryl sulfate triethanolamine 40 Sodium lauryl sulfate 5 Sodium lauroyl sarcosinate 1 function 3 Sodium lauryl imitasoline dicarboxylate 10
Lauric acid diethanolamide 5 Ethylenediaminetetraacetic acid disodium 0.
3 Sodium hexametaphosphate 1 Propylene glyfur 10 Water extract of the present invention 1-5 Flavor, pigment,
Preservative Appropriate amount Purified water 31-35 100% by weight = 27- 3, Liquid coating agent Ethyl lactate 10ml Glycerin
5-10ml water extract of the present invention
1 to 5g ethanol, 100ml in total!4, ointment resorcinol 6g zinc oxide 6g bismuth subnitrate 6g company pine moktar 2g beeswax Log yellow vaseline
27g purified lanolin
26g glycerin 13g water extract of the present invention 4g

[Brief explanation of the drawing]

FIG. 1 compares the inhibitory effects of Streptococcus on the growth process of P and P. acnes, where the horizontal axis shows the culture time and the vertical axis shows the number of P and P. acnes viable bacteria (log/mN). Figure 2 shows the investigation of the growth inhibition effect of S. faecium AD1005 on P. acnes, where the horizontal axis is the culture time;
The vertical axis shows the number of viable bacteria of P. acnes (Iog/mβ). Patent applicant Advance Co., Ltd. Figure 1 0 6 12 24 3゜Figure 2

Claims (2)

[Claims] (1) An antibacterial agent for skin characterized by containing as an active ingredient bacterial cells and/or water extracts of microorganisms belonging to the genus Streptococcus, Lactobacillus, or Bifidobacteria. (2) the microorganism is Streptococcus faecium;
Bifidobacterium durans, Bifidobacterium faecalis, Lactobacillus brevis, Lactobacillus salivarius, Bifidobacterium adolescentes, Bifidobacterium breve, and Bifidobacterium longum. Antibacterial agent for skin.