RU2440096C2 - Method for preparing biologically active component for making skin care product, and component prepared by such method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to cosmetology and represents a method for preparing a biologically active component for making a skin care product, involving lactic culture growing on regulated nutrient mediums, stabiliser addition to the prepared suspension and packing, and differing by the fact that there is used a lactic culture containing lactic organisms of different taxonomic groups, containing 2-50% of bacteria and 98-50% of yeast (in amount of living cells); as stabilisers, the suspension is added with substances specified in: orcin, hexylresorcin, starch, gelatin; it is followed by oxygen-free vacuum packing.

EFFECT: invention provides higher range of probiotic active agents found in the skin care products with improved shelf-life and prolonged action.

8 cl, 3 dwg

Description

The invention relates to the production of funds for improving the condition of the external integument of a person and can be used in cosmetic, pharmaceutical, cosmeceutical and parapharmaceutical products.

Lactic acid organisms (hereinafter MCO) are representatives of the normal microflora (normoflora) of a person, i.e. microorganisms, normal inhabitants of its skin, intestines and mucous membranes (including the genitourinary system). MCOs, inhabitants of the skin and external integuments, perform a number of functions important for humans - they prevent the development of pathogenic microorganisms (due to the release of bacteriocins), maintain a low pH (secrete lactic, acetic and other organic acids), excrete components (metabolites of different nature and enzymes), strengthening the human skin (affecting the biosynthetic and proliferative activity of skin fibroblasts), strengthen the immune system, participate in the creation of specific natural body odors.

Currently, with the excessive and unreasonable distribution of hygiene and antiseptic products, as well as the uncontrolled use of antimicrobial agents, the number of MCOs on human skin can be often reduced, which leads to the development of skin diseases (or dysfunctions), such as pustular diseases, acne, irritation, dryness and others.

The use of therapeutic and cosmetic preparations containing components originating from lactic acid organisms significantly improves and restores the functional activity and structural integrity of the skin.

Known skin cream with antibacterial and anti-inflammatory effects. The cream contains whey obtained by fermenting milk and then separating (filtering) the lactic acid bacteria (RU 2001121077, 07/10/2003). The disadvantage of this method is the use of vital products of lactic acid bacteria and lipophilic base for cream, which limits the ease of use.

A means is known which contains, as an active principle, affecting the biosynthetic and proliferative activity of skin fibroblasts, a combination of products of hydrolysis of the cell wall of lactic acid bacteria by lysozyme and extracellular water-soluble peptidoglycans secreted by growing lactic acid bacteria into the culture medium. The tool is effective for caring for normal and dry skin, especially with signs of aging and aging of the skin, however, the method of obtaining funds is complicated, because consists of a combination of methods for growing bacteria Lactobacillus bulgaricus to a titer of 7.5 × 10 ¹⁰ cells / ml, separating the culture fluid, evaporation, freeze drying, freeze-thaw, centrifugation, treatment with acetone and lysozyme (RU 2197953, 02.10.2003).

A known method of preparing the basis for medicinal or cosmetic products, in which the active component is used, which is a product of the cultivation of lacto and bifidobacteria. The method is as follows. A native mixture of lactobacilli or bifidobacteria is obtained by culturing production strains on regulated nutrient media. Inactivation of the suspension of bacteria is carried out by heating, the culture fluid is obtained by separating the cell suspension by separation or ultrafiltration. The obtained biologically active liquid component is mixed with other components of the base until a homogeneous mass is obtained using generally accepted technological methods for preparing ointment bases (RU 2187994, 08.27.2002).

A known method for producing cosmetic raw materials based on whey, which includes fermenting skim sterile animal milk with starter from lactic acid bacteria, cultivating bacteria, separating bacteria from serum by centrifugation or filtration, heating and membrane sterilization. (RU 2236144, 09/20/2004).

Known probiotic composition with high antagonistic activity against opportunistic and pathogenic microflora, used to create therapeutic and prophylactic agents, and a method for its preparation. As the microbial mass of beneficial microflora, live non-lyophilized microbial mass of Enterococcus faecium, Lactobacillus acidophilus or other lactic acid bacteria is used. The basis of the preparation contains a loose component, neutral for beneficial microflora and for human skin and mucous membranes, which is used as an sorbent for the non-lyophilized mass of bacteria in a ratio of 1: 1-1: 10 with a fat component neutral for beneficial microflora and for human skin and mucous membranes (RU 2227039, 04/20/2004).

The disadvantages of the tool is the use of a limited range of lactic acid organisms - only lactic acid bacteria, as well as a loose sorbent, which are inconvenient for use in creams for everyday care. The use of fat as a cream base can significantly increase the rate of death of live lactic acid bacteria, i.e. lead to a decrease in the shelf life of drugs. The use of dried bacterial biomass also leads to a decrease in the number of living cells.

A means is known which is a hygienic tissue impregnated with a suspension of lactic acid producing bacteria in a lipid. The amount of lipid suspension of lactic acid producing bacteria on the tissue is 0.5-95% wt. The bacterial preparation is preferably presented in dried form, the amount of probiotic bacteria on the tissue is preferably 10 ⁴ -10 ¹¹ colony forming units. The bacterial strain is selected from the genera Pediococcus, Lactobacillus or Lactococcus, including combinations thereof. (RU 2306132, 09.20.2007).

The disadvantage of the obtained funds is the use of a suspension of bacteria on tissues, the use of lipid, which significantly limits the scope and convenience of their use.

There is a method of therapeutic and prophylactic skin care of the face and neck of a patient, which involves cleansing the skin and applying funds to it, which use a daily culture of bifidumbacteria, or lactobacilli, or thermophilic streptococci, or a mixture of culture of lactobacilli and thermophilic streptococci in a ratio of 1: 4 , or a mixture of a culture of bifidumbacteria and lactobacilli in a ratio of 1: 1. The following strains of lactic acid bacteria can be used: B. longum B379 m, B. bifidum 1, B. bifidum 791, L. acidophilus CMPM - B3324, L. acidophilus CMPM - B4425, L.plantarum 8 times, Str.thermophilus T-50 . The given bacterial strains are grown in sterile milk environments: hydrolyzate-milk, milk-yeast, whole milk, milk (RU 2026665, 01/10/1995).

The main disadvantage of the known technical solution is the use of freshly grown cultures of bacteria, which limits its use to specialized medical institutions, and in mass production of the product, the shelf life of the product is reduced to several days, which is commercially unprofitable.

Thus, the known methods for producing skin care products containing MCOs or their metabolic products are divided into two groups: those using lactic acid bacteria vital products (RU 2001121077, RU 2227039, RU 2197953, RU 2187994, RU 2236144) and using live lactic acid bacteria (RU 2306132, RU 2026665, RU 2227039). The methods of both groups have disadvantages. A common drawback of the first group is the non-use (due to exclusion from the composition of living organisms) of the entire spectrum of properties of lactic acid bacteria that are useful for humans (production of bacteriocins, exoenzymes and other substances). In the case of the use of live bacteria (second group), their use is limited either by the inconvenience of using the product (use of fat and solid carriers), or by the extremely short duration of the product. In addition, the spectrum of organisms used is limited only to bacteria, while probiotic yeast organisms also have beneficial properties.

For the prototype of the proposed technical solution selected RU 2187994, 08.27.2002.

The objective (and technical result) of the present invention is to expand the range of probiotic active agents that are part of the skin and skin care products that can be used for medical and cosmetic purposes, which are characterized by an increased shelf life and prolonged action.

The problem is solved by the described method for producing a biologically active component of a product intended for skin care, including growing a mixed culture of lactic acid organisms on regulated nutrient media, introducing stabilizing additives into the resulting suspension and packaging, according to which, after growing to a stationary growth phase, the mixed culture of lactic acid bacteria and the relative content of bacteria is 2-50% and the content of yeast is 98-50% (by the number of living cells current CFU), as stabilizing additives in the slurry is a substance selected from the group of orpin, hexylresorcinol, starch, starch, gelatin, packaging is carried out in a sealed package in the absence of free oxygen access to the facility.

Preferably, the suspension contains lactic acid bacteria Lb. plantarum and C. parapsilosis yeast.

Preferably, the total lactic acid content of the organisms is at least 2 × 10 ⁸ living cells (CFU) / ml.

Preferably, stabilizing additives are introduced into the suspension in the following amounts (wt.%): Orcin - 0.0025-0.01, hexylresorcinol - 0.0005-0.0015, starch - 3, gelatin - 5.

The problem is also solved by the described biologically active component of the product intended for skin care, containing as an active component a composition of lactic acid organisms of different taxonomic groups obtained by the method described above.

The following are examples and experimental results.

The proposed method is based on a comparative study of the use of pure museum cultures of lactic organisms, an association of museum cultures of lactic organisms, selected for 6 months based on long-term survival at room temperature, and the strains included in this association (Table 1). Table 1 lists the lactic acid organisms used in the present invention.

Table 1. Organism strain number Source (collection) Lactobacillus acidophilus B-1660 All-Russian Collection of Microorganisms (VKM) LV.casei B-1144 VKM L.plantarum B-578 VKM L. Pentosus B-1941 VKM Lactococcus lactis TB2 Collection of the State Research Institute of Genetics and Breeding of Industrial Microorganism Strains Candida parapsilosis Y-2645 Bkm Torulaspora delbrueckii Y-879 Bkm Lb.plantarum B-578 (after selection for survival) C.parapsilosis Y-2645 (after selection for survival) Association of Lb.plantarum B-578 and C.parapsilosis Y-2645 (after selection for survival)

An example of obtaining the claimed funds

Mixed (consisting of lactic acid bacteria and yeast) or pure cultures of MCO (i.e. consisting of only bacteria or only yeast) were grown in compliance with aseptic standards and basic microbiological techniques in sterile media - skim milk or MRS, on a rocking chair for 3-5 days at a temperature of 25-30 ° C in 250 ml flasks in 50 ml of medium until the stationary growth phase (concentration of living cells was 2-11 × 10 ⁸ CFU / ml). When growing mixed cultures, the inoculum consisted of bacteria and yeast in different proportions. We used the MCOs listed in Table 1. The concentration of living cells was determined as the concentration of colony forming units (CFU) in 1 ml of medium by plating aliquots of decimal dilutions on solid MRS medium. In stationary phase cultures, the relative content of bacterial and yeast cultures varied from 0 to 100% (pure cultures were used, as well as with a content of 2% or 50% of bacteria). After obtaining the crops of the stationary growth phase, they were divided into portions and one of the additives was added: polysaccharide nature - starch or carboxymethyl cellulose (3%), protein nature - gelatin (5%), antioxidant nature - orcin (0.0025-0.05%) or hexylresorcinol (0.0005 -0.0025%), a sorbent based on wheat bran (10%), milk cream fat (5%), after which it was left for long-term storage at room conditions (temperature 17-25 ° С). Microbiological tubes with cotton-gauze plugs or plastic syringes with hermetically sealed needles (to maintain oxygen-free conditions) were used for the packaging and storage of preparations with MCO. At certain time intervals, a portion of the drug was taken from test tubes and syringes to determine the concentration of viable microorganisms, as well as to determine the purity of the preparations and their qualitative composition, determined microscopically and by the method of seeding on a dense medium of MRS. According to the results of studies, the most effective compositions were chosen.

An example of using the tool

To study the effect of a composition of lactic organisms on human skin, a volunteer test was used.

Suspensions of MCO, consisting of bacteria and yeast, namely (Lb.plantarum + C. parapsilosis) and (L.pentosus + T.delbrueckii) in two different proportions (1 - 50% yeast and bacteria; 2 - 2% bacteria + 98% yeast) grown in skim milk or MRS medium to a titer of 2-11 × 10 ⁸ CFU / ml was used to wipe the skin of the hands, face or femoral area of voluntary testers. Such treatment led to softening of the skin of the hands and face (dry before using the product), to a significant (up to 90%) reduction of acne on the face and to the almost complete disappearance of pustular rashes on the femoral region.

This example shows that the use of suspensions of various MCOs has a pronounced positive (healing) effect on human skin.

The results of the study shelf life of funds

It is known that MCOs, when stored at room temperature, die quickly, their number decreases below concentrations when they show their probiotic, positive properties.

Figure 1 presents the results of a study on the storage of certain cultures of MCO at a temperature of 17-25 ° C without the use of any special measures to maintain their viability in test tubes covered with cotton-gauze plugs.

The presented results show that 1) museum strains die off much faster than strains specially selected by us on the basis of long survival for 6 months (curves 1 + 2 and 3 + 4), and 2) the MCO association survives much more successfully (for more time maintains a higher titer of living cells) than pure cultures, its components separately (curves 5 + 6 and 3 + 4), with different initial composition of the population of organisms - 2% and 50% of yeast. The data obtained illustrate the importance of using precisely the compositions of different MCOs - yeast and bacteria, and not their pure cultures, for long-term maintenance of the viability of organisms. For pure cultures Lv.casei, Lv.pentosus and T.delbrueckii, the results are similar to those for Lb.acidophilus (rapid death). In another binary culture (L. pentosus + T. delbrueckii), the death of bacterial and yeast cultures was slower than in pure cultures.

MCOs are sensitive to the oxygen content in the environment and quickly die during storage. The influence of the method of packaging and storage of crops on the survival of MCO during storage for a long time was investigated. The amount of oxygen was regulated by choosing the method of packaging and storing the cultures of MCOs - in test tubes under cotton-snout plugs (free access of oxygen), or in hermetically sealed plastic syringes without access to air. MCO associations initially contained from 2 to 50% of bacteria Lv.plantarum (or Lv.pentosus), as well as the yeast C. parapsilosis (or T.delbrueckii), respectively. The results presented in Fig. 2 (for the association of L.planfarum + C. parapsilosis with a bacterial content of 50%) indicate that storage under conditions of limited air access for several months allows maintaining the concentration of MCO several orders of magnitude higher than under free conditions oxygen access. For the association of L. plantarum + C. parapsilosis with an initial bacterial content of 2%, the results are similar. For the association L.pentosus + T.delbrueckii, the results are similar.

The data obtained show the significance of the sign regarding the limitation of the contact of the MCO association with atmospheric oxygen during packaging and storage in order to maintain their viability for several months.

Results Concerning Administration of Stabilizing Additives

During the study, we checked the effect of stabilizing additives (antioxidants of orcin and hexylresorcinol, polymers of different nature - starch, carboxymethyl cellulose, gelatin, sorbents - finely ground wheat bran) on the duration of storage of MCOs. Figure 3 presents the results of an experiment on storing the association of lactic acid bacteria and yeast (initial content of 50%) under oxygen-free conditions for 12 months at room temperature. The data presented show that the addition of bran and milk fat significantly reduced the titer of viable cells; in the presence of orcin, hexylresorcinol, starch and gelatin, the titer of living cells was 1-1.5 orders of magnitude higher than in the control; the use of carboxymethyl cellulose gave mixed results. For association with an initial bacterial content of 2%, the data are similar.

This example illustrates the importance of the presence in the tool of stabilizers of different nature to increase the number of viable cells during prolonged storage at room temperature. The most effective was the use in the claimed composition of the use of orcin, hexylresorcinol, starch and gelatin in the declared concentrations.

As a result of our studies, we developed a method that allows you to get the active component, which can be stored in sealed packaging for up to 12 months. During the storage period, the resulting product is suitable for mixing with a carrier to obtain a homogeneous mass of the required consistency using generally accepted technological methods for preparing cream, gel, milk, or other means. After receiving a specific product, it is again packaged in the absence or with restriction of free access of air to an airtight package, minimizing contact of the product with air. The result is a skin care product with high biological activity, namely, softening, moisturizing and regenerating the skin, with antibacterial against pathogenic microflora and at the same time probiotic effect with respect to symbiotic microflora and prolonged duration (12 months or more).

Brief Description of the Drawings

Figure 1. Change in the number of viable cells (CFU / ml) of different MCOs for 6 months 1 - Lb.acidophilus, 2 - Lc.lactis, 3 - Lb.plantarum, 4 - C. parapsilosis, 5 - association of Lb.plantarum and C. parapsilosis (initially containing 50% of bacteria and yeast), 6 - association of Lb.plantarum and C. parapsilosis (initially containing 2% of bacteria and 98% yeast).

Figure 2. Survival of the association of lactic acid bacteria Lb.plantarum and C. parapsilosis yeast (initial content - 50% each) for 12 months under different storage conditions. 1st air access, 2nd air restriction.

Figure 3. Storage of the association of lactic acid bacteria Lb.plantarum and C. parapsilosis yeast under oxygen-free conditions for 12 months at room temperature in the presence of various stabilizing additives: 1 - control; 2 - hexylresorcinol (0.0005%, for 0.0015% the data are similar); 3 - orcin (0.0025%, for 0.01% the data are similar); 4 - starch (3%); 5 - carboxymethyl cellulose (3%); 6 - gelatin (5%); 7 - wheat bran (10%); 8 - milk fat (5%).

Claims (8)

1. A method of obtaining a biologically active component for a product intended for skin care, including growing a culture of lactic organisms on regulated nutrient media, introducing stabilizing additives into the resulting suspension and packaging, characterized in that a lactic acid culture is used, consisting of lactic acid organisms of different taxonomic groups containing 2-50% bacteria and 98-50% yeast (according to the number of living cells), as stabilizing additives, things are introduced into the suspension substances selected from the series: orcin, hexylresorcinol, starch, gelatin, packaging are carried out in airtight packaging in the absence of free access of atmospheric oxygen. 2. The method according to claim 1, characterized in that the suspension contains lactic acid bacteria Lb.plantarum and yeast C. parapsilosis. 3. The method according to claim 1, characterized in that the total content of lactic acid organisms in suspension is at least 2 · 10 ⁸ living cells (CFU) / ml 4. The method according to claim 1, characterized in that the orcins are driven in an amount of 0.0025-0.01 wt.%. 5. The method according to claim 1, characterized in that hexylresorcinol is added in an amount of 0.0005-0.0015 wt.%. 6. The method according to claim 1, characterized in that the starch is driven in an amount of 3 wt.%. 7. The method according to claim 1, characterized in that the gelatin is driven in an amount of 5 wt.%. 8. Biologically active component for a product intended for skin care, containing as an active component a suspension of lactic acid organisms of different taxonomic groups obtained by the method described in claims 1-7.

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