WO1999007332A2

WO1999007332A2 - A natural eubiotic product for maintenance and treatment of teguments

Info

Publication number: WO1999007332A2
Application number: PCT/RO1998/000014
Authority: WO
Inventors: Rodica Teodorescu
Original assignee: Rodica Teodorescu
Priority date: 1997-08-05
Filing date: 1998-08-03
Publication date: 1999-02-18
Also published as: RO113114B1; WO1999007332A3; AU8753998A

Abstract

The eubiotic product, according to the invention, is constituted of a mixture of three Lactobacillus acidophylus LR, LV and LD strains, registered in the Biosan Centre Collection Bucharest under the nos. 13, 17 and 11, the association being in equal parts of each strain before lyophilisation of mixture. The Lactobacillus acidophylus LD-11 strain ferments the raffinose. The Lactobacillus acidophylus LR-13 strain ferments the trehalose. The Lactobacillus acidophylus LV-17 strain ferments the dextrine. The three associated lactic bacilli strains are used as active ingredients in different cosmetic forms for the maintenance and treatment of teguments, the product being included in amount of 1-10 g under lyophilised form at 100 g of cosmetic product.

Description

A NATURAL EUBIOTIC PRODUCT FOR THE MAINTENANCE AND TREATMENT

OF TEGUMENTS

The present invention relates to an natural eubiotic product for the maintenance and treatment of teguments.

It is known that the skin is not a mere body coating, but a living organ having a vital importance for the body.

By age, the sebum secretion is decreasing and the skin begins to dry, being able no more to accomplish its functions. The sebum, together with other skin secretions, forms the so-called "acid casing" of the skin whose acidity is varying from person to person.

The skin acidity is a defense factor of the body against the environment influences, especially of micro-organisms that do not favourably grow in an acid medium.

If the skin is lacking this protecting acid casing, abscesses, furuncules, mycosis etc. can frequently appear.

The pH of teguments and, especially, that of complexion, easily modifies as a result of the non-respecting of hygiene rules, of sudden temperature variations, inadequate nourishment, constipation, stress. At the same time, the pH of complexion modifies as a result of the excess of soap use, of alkaline face lotions, concentrated glycerine, concentrated alcoholic lotions. li is well known the use of lactic acid in cosmetics, in preparations for skin maintenance, especially for modifying the pH of teguments. The used lactic acid is usually a syntesis product and the skin acidification, in physiological limits, is hard to be obtained as is less biologically compatible with the functions of teguments.

For obtaining the natural lactic acid, it is known, in food industry, the use of Lactobacillus acidophylus strains. For using the natural lactic acid, this one should be isolated from the culture medium, as purely as possible, implying complex technologies.

The common Lb. acidophylus strains are also inadequate for a direct use in cosmetics as they are too less resistant to cosmetic compositions, and because of their firm and spinning aspect, they produce the changement of the cosmetic product, being very important for the product user.

It is also known the use of a drug consisting of a mixture of two Lactobacillus acidophylus and Lactobacillus casei bacteria strains, registered in the IISB collection - Bucharest under nos.3 and 7. The eubiotic product, according to the invention, consists of a mixture of three Lactobacillus acidophylus LD, LV and LD strains, registered in the Biosan Centre Collection Bucharest under nos.13, 17 and 11, the association being in equal parts from each strain before the mixture lyophilisation.

The Lactobacillus acidophylus LD-11 strain ferments the raffinose. The Lactobacillus acidophylus LR-13 strain ferments the trehalose. The Lactobacillus acidophylus LV-17 ferments the dextrine. The three associated lactic bacilli strains are used as active ingredients in different cosmetic forms for the maintenance and treatment of teguments, the product being included in amount of 1 - 10 g. under the lyophilised form at 100 g. of cosmetic product.

The eubiotic advantages, according to the invention, are the following:

- it is easily obtainable from natural, easily accesible ingredients, it destroys the pathogenic microflora and maintains the pH at physiological values, not producing allergies and inflammations;

- it tones up and renders the flexibility of teguments by "ergons" as a result of the activity, it develops a non-specific immunity and inhibits the growing of different microbial agents by the pH that it grows - the no passing barrier for other micro-organisms;

- it is the active compound of cosmetic products being the only that approaches the physiological chemistry of teguments.

It is given hereunder a example of realization of the invention. For obtaining the eubiotic product, according to the present invention, the three bacteria strains are associated, before lyophilisation, in equal parts.

The characteristics of Lactobacillus acidophylus common strains are the following: a) Morphological characteristics:

Following the Gram colouring, Lactobacillus acidophylus appears as sticks with rounded ends, Gram positive, organized in filamentous groups of variable dimensions, giving a filamentous and mucilaginous aspect to the growing medium. b) Cultural characteristics

On solid media (solid MRS medium), they can develop on the surface forming long, tangled, gluey colonies, with filiform, fluffy projections, or in depth, where the colonies are small, irregular, with fine prolongations.

On MRS liquid, they form, after 48 hours, a fine, fluffy sediment. „„„.,„ O 99/07332

c) Biochemical characteristics:

- resistance to inhibitory substances 3,5% - NaCI;

- sodium taurocolate 3%;

- phenol 0,4%; - acid power 140°T;

- limit acidity 1,2% lactic acid:

- they do not fluid the gelatine;

- they do not hydrolize the starch;

- they do not reduce the nitrates;

- they reduce the tincture of litmus and the methylene blue;

- they do not produce indole, sulphuric acid, arginine, ammonia;

- they are urease and catalase negative;

- the culture is not haemolytic on blood media;

- they coagulate the milk and are homofermentative;

- they produce acetylmethylcarbinol;

- they do not use the sodium citrate;

- they ferment the glucose, lactose, saccharose, fructose, galactose, manose, maltose;

- they do not ferment the dextrine, trehalose and raffinose;

- they produce lactic acid optically inactive and volatile acids as a result of sugars fermentation;

- a visible growth on media lacking carbon hydrates is not noticed; d) Temperature relations - The optimum growing temperature is 371. They do not grow under 20°C. The maximum temperature at which they can normally grow is 43...45°C. They can resist 30 minutes at 60°C.

The attempts for using these strains in cosmetic products have not lead to optimum results, therefore, by tests, it has been attempted and realized the selection and isolation of some Lactobacillus acidophylus strains to be compatible for the preparation of cosmetic products for the maintenance of teguments.

The aspect that the present invention solves consists in finding an optimum association of Lactobacillus acidophylus anallergical strains for realizing a natural eubiotic product, capable of improving the pH of teguments, approaching it to the physiological one of 5,5 and to be resistant in cosmetic compositions. The product, according to the invention, consists of a mixture of three Lactobacillus acidophylus strains, in equal parts each, the three strains being registered in the Biosan Centre Collection Bucharest with denomination LR, LV and LD, under the nos. 13, 17 and 11.

Modality for obtaining the strains

The lactobacillus pure strains have been obtained by repeated dispersions and isolations on agar media, known per se, that contain growing substances and inhibitory substances for other strains of micro-organisms.

The Lb. acidophylus strains having the following characteristics are identified and isolated from the culture media:

1. LD strain no.11 a) Morphological characteristics - they are long bacilli, intermittently coloured, immobile but forming short chains. At the colouring with methylene blue, they appear as long bacilli, intermittently coloured, immobile, forming short chains. b) Cultural characteristics - they coagulate the milk on litmus milk and they disturb the medium on a MRS liquid medium. On a solid MRS medium, the colonies are small, whitish and gluey, with irregular margins. c) Bio-chemical characteristics:

- the maximum growing temperature is 47°C;

- they resist 30 minutes at 60°;

- resistance at inhibitory substances a) NaCI - 6%; b) Sodium taurocolate - 3%; c) Phenol - 0,6%

- acid power - final acidity of culture 170°T

- limit acidity in lactic acid 1 ,4%

- they do not fluid the gelatine;

- they do not hydrolize the starch;

- they do not reduce the nitrates;

- they reduce the tincture of litmus and the methylene blue;

- they do not produce indole, sulphuric acid and arginine;

- they are urease and catalase negative;

- the culture is not haemolytic on blood media; - they coagulate the milk and are homofermentative;

- they produce acetylmethylcarbinol;

- they do not use the sodium citrate;

- they ferment the glucose, lactose, saccharose, fructose, galactose, manose. The fermentation of raffinose is a characteristic of this strain.

2. LR strain no.13 a) Moφhological characteristics - long and thin ba lli, forming, nevertheless, short chains, intensely coloured. They are immobile and have rounded ends.

At the colouring with methylene blue, there appear as thin, long bacilli, with rounded ends, immobile. They form short chains, intensely coloured. b) Cultural characteristics

- they coagulate and acidulate the milk on litmus milk;

- they disturb the medium on liquid MRS medium and form a sediment at 48 hours;

- on solid MRS medium, the surface colonies appear delicate, long, with fluffy projections and a small and dark centre. The depth colonies appear small, irregular, whitish with fine ramified or radial prolongations. c) Bio-chemical characteristics

- the maximum growing temperature is at 48°C;

- resistance at 60°;

- the acid power is of Iδδ' and the limit acidity is of 1 ,3% in lactic acid;

- resistance at the following inhibitory substances: a) NaCI - they grow no more over 8%; b) Sodium taurocolate - they grow no more over 4%; c) Phenol - the growth is very poor over 0,8%.

- they do not fluid the gelatine;

- they do not hydrolize the starch;

- they do not reduce the nitrates;

- they reduce the tincture of litmus and the methylene blue;

- they do not produce indole, sulphuric acid, ammonia and arginine;

- they are urease and catalase negative;

- the culture is not haemolytic on blood media;

- they coagulate the milk and are homofermentative;

- they produce acetylmethylcarbinol; „,„„,,.,

WO 99/07332

6

- they do not use the sodium citrate;

- they feiment the glucose, lactose, saccharose, fructose, galactose, manose, maltose.

The fermentation of trehalose is a characteristic of this strain.

3. LV strain no.17 a) Moφhological characteristics - short badlli, weakly coloured, immobile, forming short chains.

At the colouring with methylene blue, there appear short, weakly coloured bacilli, short chains, immobile, with rounded ends. b) Cultural characteristics - they coagulate and acidify the milk on litmus milk, in 18 hours after insemination; on liquid MRS medium, they disturb the medium forming a sediment; on solid MRS medium, the colonies are white, with smooth margins, presenting ramifications. c) Bio-chemical characteristics

- the maximum growing temperature is at 48°C;

- they resist 90 minutes at 60°;

- the acid power is of 173°T and the limit acidity is of 1 ,4% in lactic acid;

- resistance at inhibitory substances: a) NaCI - they grow no more over 8%; b) Sodium taurocolate - they grow no more over 4%; c) Phenol - they grow no more over 0,8%.

- they do not fluid the gelatine;

- they do not hydrolize the starch;

- they do not reduce the nitrates;

- they reduce the tincture of litmus and the methylene blue;

- they do not produce indole, sulphuric acid, ammonia and arginine;

- they are urease and catalase negative;

- the culture is not haemolytic on blood media;

- they coagulate the milk and are homofermentative;

- they produce acetylmethylcarbinol;

- they do not use the sodium citrate;

- they ferment the glucose, lactose, saccharose, fructose, galactose, manose, maltose. The fermentation of dextrine is a characteristic of this strain. The identified and isolated strains, according to the invention, can be characterised as being different from the Lb. acidophylus common strains by the following:

- they form short chains, very adequate for the homogeneity and resistance of cosmetic products where the eubiotic is included;

- they coagulate and acidify the milk more rapidly, leading to a faster diminution of pH from the medium where the strains are induded, either this one is a cosmetic product;

- they disturb evidently the liquid culture media and deposit in abundance, proving a great viability of the strain and a performing production;

- in comparison with the common strains, they ferment other sugars (trehalose, raffinose and dextrine) that the common strains do not ferment, proving a superior enzymatic package during the identification process;

- the grown resistance to dassic inhibitory substances proves a bigger viability of strains obtained according to the invention in comparison with the common strains.

The identified and isolated strains will be maintained on synthetic media such as MRS, having the following composition: 10 g of tryptone, 10 g of extract of meat, 5 g of extract of yeast, 20 g of glucose, 2 g of disodium phosphate, 5 g of sodium acetate, 2 g of sodium citrate, 0,05 g of magnesium sulphate, 1000 ml of distilled water.

Periodically, the strains are submitted to the most important known tests of identification in order to find out the resistance in time of the respective strains, their ageing speed.

In order to obtain the vitality, the strains are periodically passed on solid media, by methods known per se.

In order to prepare the strains for their production stage, these ones are passed on sterile degreased milk, in the association ratio of 1/3 of each strain at 10 litres of milk.

The sterile degreased milk is also the used protecting medium, according to the invention, for the last stage of process, namely the lyophilisation of the three multiplied strains. A pure culture of the three strains is obtained.

There have not been noticed phenomena of antagonism, of elimination of one strain by another between the lactobadllus strains, the number of viable germs from the pure culture being maximum.

The lactobadlli culture, obtained according to the invention, is congelated at -45 O - 50°C, the proper lyophilization being started afterwards. The lyophilzation is realized in vacuum of 3 tonrduring 30 hours, having as protection medium the sterile degreased milk corresponding to physical, chemical and micro-biological standards (lack of heavy metals etc).

The lyophilised product is conditioned in special containers, in asepsis conditions (work under hood) and their closing is realized in vacuum.

The lyophilised product, obtained according to the invention, has the following characteristics that identify it:

- aspect: lyophilised powder without mechanical impurities, hydroscopic;

- colour: white yellowish

- smell: specific

- taste: typically sour

- solubility: insoluble in water, but it easily disperses realizing a homogenous dispersion

- pH of suspension 1% in water: 3, 8...5, 5

- each Lb.acidophylus bacilli strain is identified by the morphological, cultural and bio-chemical characteristics presented according to the invention

- content in Cl^"% in weight = max. 1 ,2

- content in Ca²⁺% in weight = min. 1,2

- content in nitrogen % in weight = min. 4,5

- calcination residues = max. 12% in weight

- absent heavy metals

- microbiological purity - according to Romanian Pharmacopeea

- number of viable germs = min. 5 x 10⁷ germs/0,50 g

- validity period: 2 years

- the lyophilised product may be used for manufacturing the creams for all types of complexion, the demaking-up milk and the cosmetic face masks.

For the treatment of acnea complexion, simultaneously with the use of cosmetic face masks, it is administered per os, in the morning on an empty stomach, 30 minutes before meals (0,50 g or 2 tablets of 0,25 g).

Examples of cosmetics containg the eubiotic product:

Cream for dry complexion with lactic bacilli

2 g of lyophilised eubiotic product, 10 g of lanoline, 2 g of cetaceum, 5 g of vaseline, 0,1 g of cholesterol, 21 g of oleum helianti, 0,5 g of wax, 3 g of boraxed water, 50 g of distilled water. O 99/07332 y

Cream for αreasv complexion with lactic bacilli

10 g of lyophilised eubiotic product, 20 g of anhydre lanoline, 10 g of cetaceum, 10 g of stearine, 10 g of helianti oils, 30 g of boraxed water, 30 g of distilled water.

The above-mentioned cosmetic products do not limit the use of the product according to this invention with a view to obtaining other types of cosmetic products such as the demaking-up milk, the hydration cream etc.

Cosmetic face mask with lactobacilli

The content of 1 - 2 bottles, containing the lyophilised eubiotic product, is dissolved in a small quantity of boiled and cooled water in which a clean gauze tissue is impregnated and it is applied on the face, the complexion being, beforehand, washed with lukewarm water (37...40°C).

The face mask is maintained on the face 30 minutes - 1 hour, the person being at total rest.

For the acnea complexion, a bottle per os is taken in the morning, on an empty stomach, before meals, and the face mask is applied in the evening.

The treatment takes 10...15 days, depending on the complexion alteration.

Claims

C L A I M S

1. A natural eubiotic product for the maintenance and treatment of teguments comprising Lactobacillus, characterized in that is constituted of a mixture of three Lactobacillus acidophylus LR, LV and LD strains, registered in the Biosan Centre Collection - Bucharest under the nos. 13, 17 and, respectively, 11 , the association being made in equal parts of each strain, before lyophilisation of mixture.

2. A eubiotic product, according to claim 1, characterized in that the used Lactobacillus acidophylus LD-11 strain is the strain that ferments the raffinose.

3. A eubiotic product, according to claim 1, characterized in that the used Lactobacillus acidophylus LR-13 strain is the strain that ferments the trehalose.

4. A eubiotic product, according to claim 1 , characterized in that the used Lactobacillus acidophylus LV-17 strain is the strain that ferments the dextrine.

5. A eubiotic product, according to claims 1 - 4, characterized in that the three associated lactic bacilli strains are used as active ingredients in different cosmetic forms for the maintenance and treatment of teguments, the product being included in amount of 1...10 g under lyophilised form at 100 g of cosmetic product.