DE19619505A1 - Milk and bacterial lysate for use in cosmetics and drugs - Google Patents

Abstract

Milk/bacterial lysate is obtained by: (a) addition of a bacterial culture to a milk/yeast extract medium; (b) addition of an inhibitor of murein biosynthesis after reaching the log phase;(c) addition of a murein hydrolase lyse the bacteria in the culture, and (d) pasteurisation of the product of (c).

Description

The invention relates to new milk / bacteria lysates, methods for their manufacture, as well as cosmetics and medicines contain the bacterial lysates according to the invention. Insbeson The milk / bacteria lysates according to the invention are used in the cosmetics and medicaments of the invention as anti- inflammatory additive.

Inflammation is that of active connective tissue and blood vessel-borne, complex reaction cascade of the organism to an externally or internally triggered inflammatory stimulus (Adam, Med. Clin. Prax. 78 (1983), 12-21; Floh', Giertz, Rev. Infect. Dis. 9 Suppl. 5 (1987), 553-561; Halliwell, Lancet 344 (1994), 721-724; Grisham, Lancet 344 (1994), 859-861; Cerutti, Lancet 344 (1994), 862-863) with the purpose of to eliminate, inactivate and stimulate it Repair tissue damage. Here are playing chemical Ra dikalreaktionen under the influence of catalytic Factors, high-energy radiation (UV light), reactive  joyous molecules (radicals) and / or heavy metals in Come a fundamental role in the destruction of Cell structures, especially the cell membranes that make up the cells delimit against the environment.

For the treatment of such inflammatory phenomena are number rich substances known as simple substances such as vitamins, un saturated fatty acids, short chain fatty acids like Acetic or lactic acid, but also complex substances animal and of plant origin such as hormones (cortisone), collagen, Elastin and hyaluronic acid (Frese and steel, perfumery and Cosmetics 72. Jg. 9 (1991), 569-574). Also substances bacterial len origin are used as anti-inflammatory agents used as autolysates of Lactobacillus gasseri (Weirich-Schwaiger et al., Drug Research 45 (1995), 342-344), the bifidobacteria repair complex (EP-A-0 043 128, U.S. Patent 4,646,362) or cell wall polysaccharides Bifidobacteria (EP-A-0 201 332). The use of Bakte in the form of probiotics (Lactic Acid Bacteria, ed. Salmins, by Wright, New York, 1993) or for reforestation natural mucosal colonization (Hughes et al., Obstet. Gynecol. 75 (1990), 244-248) is partially even if anti-inflammatory, anti-inflammatory or Radical chain inhibiting activity.

The production of such active ingredients (cortisone, hyaluron acid, collagen, elastin, cell wall components, etc.) complex plant, animal or bacterial material or using complicated breeding Techniques (breeding of bacteria in complex composite nutrient media) is practically invariably expensive Combined extraction and fractionation, since the effective ingredient is effective only in purified form. This is a significant disadvantage. Such starting material rial can potentially cause harmful contamination contain the most diverse kind, z. B. endotoxins, polyamines, Foreign protein etc. Such impurities, but also leftovers Extractants can cause irritative side effects  (Pharm. form. (Innsbruck) 10th ed. (4) (1995), 1-4; Maes et al., Cosmetics and Toiletries 105 (1990), 43).

Especially with animal source material exist due special risks (zoonotic risks (BAnz 164 (1991), 6120; Pharm. Ind. 53 (1991), 206) and risks of dissemination of the spongiform encephalopathy (BAnz 40 (1994), 1851; BAnz, 96 (1994), 5500) strict requirements for pharmaceutical and cosmetically used active ingredients. The extraction and Cleaning procedures are therefore subject to approved waste for certain pathogens and additional inputs Restrictions that often do not provide isolation economically make sense.

This also applies to the recovery of bacterial cells complex nutrient media too, as far as these materials are animal Origin (peptone, liver extract, etc).

These risks, but also a lately stronger a growing demand for new, as far as possible "natural" simple and toxicologically harmless active substances or Products for use in the pharmaceutical sector and Kos have to do intensive research in the field led the "natural" products.

The goal is to use as natural as possible and easy-to-use agents using Substances of defined quality. The use of is known Fractions from plant extracts (Tragni et al., Pharm. Res. Comm. 20 Suppl. 5 (1988), 87-90), extract extracts from Plants, e.g. Chamomile, fennel, etc., of facial masks from freshly cut potato peels, cucumbers etc. as well-known home remedy mostly to support the skin functions of red wine, black tea and green tea (Serafini et al., Lancet 344 (1994), 626) and skimmed Milk (Taylor, Richardson, J. Dairy Res. 63 (1980), 1783-1795) as antioxidants.

The use of milk as a raw material for "naturbelas sene "active ingredients is still barely developed, although their Be  parts are well known (Niederauer, perfumery and Kos 75, Vol. 10 (1994), 666-670; Harnett et al., ZFL 45. Jg., 78 (1994), 14-16). Previous uses described be especially draw on stock obtained from the milk parts, such as on processed fractions of casein, the What Serbindungs-, gel and foaming power and emulsifier features on milk glycoproteins as novel Protective agents as an alternative to hyaluronic acid or to the skin regeneration and on lactoperoxidase and lactoferrin as anti-inflammatory substances.

As a "natural" dairy product come by bacteria acidified milk products therapeutically for use. Ge ordinary yogurt and similar sour milk products Therapeutic in gynecological practice to restore Position of a normal acid vaginal milieu through milk rebildner applied (Berger, Karovic, Arch. Gynäk. 195 (1961), 46-49; Gunston, Fairbrother, S. Afr. Med. J. 49 (1975), 675-676; Fredricsson et al., Lancet (1987), 276; Hughes et al., Obstet. Gynecol. 75 (1990), 244-248), such. B. Acidophilus yogurt, even in pregnant women without Side effects and complications were used (Neri et al., Acta Obstet. Gynecol. Scand. 72 (1993), 17-19). anti oxidative effects of skimmed milk versus unsat saturated fatty acids, eg. B. Linoleate (Taylor, Richardson, J. Dairy Sci. 63 (1980), 1783-1795) and anti-mutagenic in In vitro effects of milk and by Bifidobacterium or Lactobacillus acidified milk (Pool Zobel et al., Nutr. Cancer Res. 20 (1993), 261-270; Cassand et al., J. Dairy Res. 61 (1994), 545-552; Morishita, Shiromizu, Bifidobacteria Microflora 9 (1990), 135-138) are described ben. In contrast, anti-inflammatory effects so far we which in milk is still described for dairy products.

The processing of milk in the pharmaceutical and Kosmetikindu However, strie is partly with considerable problems be liable. Such problems are based inter alia on the  Sensitivity of milk to heat (Bulletin of the Intern. Dairy Federation No. 238/1989, Heat-induced changes in milk, Hsg. P.F. Fox). In the milk processing indu Strie provides the very gentle heat treatment no problem. With further processing of the milk in the Pharmaceutical or cosmetics sector, however, lead again Heat treatments to unsightly brown discoloration (Maillard reactions) and unpleasant-smelling degradation products (Ledl et al., Z. Lebensm. Unters. Forsch. 182 (1986), 19-24; Freimuth, Ludwig, Food 26 (1982), 97-102; Ludwig, food 23 (1979), 531-536).

Furthermore ge when using bacteria acidified milk products to bacterial activities too optically unsightly clotted, inhomogeneous and bad-smelling Lead milk, z. B. in too sour yogurt to deposition of Whey and curdled egg whites or destroying the milk white colloidal suspension, giving the milk a glassy gives a flaky appearance ("peptonization"). Further un desirable activities of bacteria are proteolysis, lipo lysis or amino acid dissimilation, which is also a bad odor due to the formation of amines, Ammonia, sulfhydryl compounds and aldehydes with it bring. The quality of such product is also toxicological worried. Such a modified product is suitable not synonymous for further processing into a knit fabric for cosmetic or pharmaceutical use.

The above-described disadvantages can be found in use mildly acidified milk / bacteria products are avoided. However, in all these milk / bacteria products the effective ingredients included in the cells. Lyse the cells to improve the effectiveness of the bacteria containing product, eg. B. by mechanical treatment (EP-A-0 043 128), by proteolytic treatment (EP-A-0 201 332) or by lysozyme action on the cell wall (Chassy, Giuffrida, Appl. Environm. Microbiol. 39 (1980), 153-158; U.S. Patent 3,929,994) is required. To receive a Efficient lysis thereby become the cells of the  Medium separated. In addition, following that Extraction and cleaning method applied so that a natural-looking "natural" product in the real meaning is no longer present.

Particularly advantageous for a "natural" produced Product would therefore be the application of cell autolysis, so the lysis of bacteria's own enzyme systems, so-called. Auto lysines (Shockman, Barrett, Ann. Rev. Microbiol. 37 (1983), 501-527). However, such methods are still too little known. Unfavorable act on the activation of autolysis high carbohydrate concentrations (Dirar and Collins, J.Gen. Microbiol. 73 (1972), 233-238), and lactose inhibits autolysis (Moustafa et Collins, J. Bac. 95 (1968), 592-602). Thus, the high lactose concentration is in milk a natural inhibitor of the application of the cell prevents autolysis. Also, the use of lysozyme he turns out to be problematic, as abundant in the milk existing N-acetyl-glucosamine-containing glycoproteins Akti inhibit the activity of the lysozyme (Chipman et al., J. Biol. Chem. 242 (1967), 4388-4394; Nicolai et al. Hoppe-Seyler's Z. Physiol. Chem. 362 (1981), 153-162).

So z. B. in the lysozyme-sensitive bacterial species Lactococcus lactis both cell autolysis and the lysozyme-dependent lysis in milk strongly inhibited. This is Not surprisingly, because of milk as the preferred location adapted bacterium to resist lysis in milk "ge learns a lysis of the cell wall of gram-positive bak Milk is a major obstacle to milk production Path.

Thus, the invention has the object, a neuarti to provide milk / bacteria lysate that attracts Has outer appearance and qualitatively as Be Part is suitable for cosmetics and pharmaceuticals. Of Another object of the invention is to provide a To provide milk / bacteria lysate that acts as an anti-inflamma  toric agent suitable in cosmetics and medicines is.

The solution to this problem is achieved by the Bereitstel Development of the characterized in the claims Ausfüh insurance forms.

The invention thus relates to a milk / bacteria lysate, the is available through

• (a) preparing a culture of bacteria in a milk yeast extract medium;
• (b) Placing the medium with an inhibitor of Mureinbio synthesis after reaching the log phase;
• (c) adding a murein hydrolase for lysis of the culture contained bacteria; and
• (d) pasteurizing the milk / bacteria lysate thus obtained.

According to the invention can be for the production of this Milk / bacterial lysates gram-positive lactic acid-forming Use bacteria that are among the inventive Culture conditions well in a milk-based medium without the milk in its external appearance to change too much, including lactic acid bacteria, Cocci or rods with a DNA guanosine cytosine content between 30 and 50 mol%, with the genera Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. In all called genera are suitable candidates with low risk potential included.

As a milk-yeast extract medium is preferred according to the invention wise a gently prepared in a high layer and sterilized mixed solution of 10% (w / v) skimmed milk powder and 0.5% (w / v) yeast extract powder in distilled Water used.

Usually, the log phase is dependent on the Bacterial species, their cell division rate and the Tem reached at different times, at he  after a breeding period of 3 hours at 30 ° C the case.

As Mureinbiosyn inhibitors usable according to the invention These are preferably glycine and amino acids of D-Kon figuration, z. D-serine, D-threonine, D-alanine and D-methionine, but also L-configuration amino acids in Ver used with a racemase. These inhibitors hem in high concentrations by acting on the D-Ala-D-Ala ligase cross-links the murein. They will be in Ab dependence on bacterial species and bacterial strain used in a concentration of 0.1 to 10 wt .-%.

Murein hydrolases according to the invention are N-acetylmuramides containing lysozyme and mutanolysin, N-acetylmuramyl-L-alanine amidases and N-acetylglucosaminidases. you depending on specificity in a concentration of 10³ to 10⁹ enzyme units per gram of bacterial cells used.

The pasteurization of the invention produced Milk / Bacteria lysates in the usual way, for example by heat treatment of the culture at least 70 ° C. for 30 minutes.

In a preferred embodiment of the invention Milk / Bacteria lysates are the bacteria used Genus Lactococcus. Particularly preferred is the strain Lactococcus lactis ssp. lactis DSM 10605. This strain was under the Budapest Treaty on 25 March 1996 at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, under the Deposit no. DSM 10605, deposited. The toxicological Safety of this strain is based on the year Tenth experience in nutritive use of Lactococci as Mesophilic Starter Culture in Sour Milkfa brikation and on the organoleptisch pleasant property of the milk / bacteria lysate. Dangerous mining or Decay products could not be detected. in addition comes that the bacteria in non-viable form are applied and toxic follow-up reactions due physiological activities do not occur.  

Furthermore, it is a preferred embodiment of the erfindungsge milk / bacteria lysates, in its production as Inhibitor of murein biosynthesis to use glycine. It will in a concentration of 0.1 to 10% by weight, preferably 1 % By weight used.

Furthermore, it is preferred according to the invention, as Mureinhydro lase the enzyme lysozyme in a concentration of 10⁶ to 10⁸ units per liter of culture to use.

In another preferred embodiment of the invention the completeness of the lysis of the in culture bacteria before pasteurisation according to step (d) checked. This is usually done by Bestim the number of live germs (CFU / ml) by conventional methods before and after the lysis of the bacteria. The quality of Lysis may also be determined by cell sedimentation by Zen trifugation of an aliquot of the culture at a maximum of 5000 × g, are determined before and after lysis, respectively after lysis no sediment forms.

In a further preferred embodiment according to the invention form the pH of the medium throughout the Kul held at a value between 6 and 7. Possibly Neutralization by addition of conventional alkali shear additives, such as 1 N sodium hydroxide solution, potassium hydroxide solution, disodium car- carbonate solution or solutions of organic buffer substances, for example Trishydroxymethylaminomethane hydrochloride buffer. The verwen Quantities must be avoided to avoid the Maillard reaction and precipitation of milk protein and bacteria low being held. Sedimentation of particulate matter and Increasing ion concentration are for the further verar problematic.

In a further embodiment, the invention relates to a Process for the preparation of the above-explained  Milk / bacterial lysates. This manufacturing method has the features explained above.

Furthermore, the invention relates to cosmetics containing a Milk / bacterial lysate according to the invention. Typically han These cosmetics are products containing the Milk / bacterial lysate either as ointment base or lip pen stock or in liquid suspension. The Application is by applying to the skin and serving generally the care of the skin after sunbathing or after daily light exposure, protection against inflammatory re actions of the lips with favoring a viral Herpes development and use on oily scalp Tendency to develop acne or dermatological eczema men. The amount to be used according to the invention Milk / Bacteria lysate in cosmetics depends on the formu lation and the intended use of the cosmetic.

In a further embodiment, the invention relates Medicaments containing a milk / bacterial lysate according to the invention contain. Preferred embodiments of such drugs are topically applied sunscreens for the care of Skin and lips after sun exposure, lasting UV-Ein effect or general exposure to light; Means for Regenera tion of the skin after sunburn and prophylactic The treatment of radiation-induced degeneration of the skin; Means for the prevention of malignant changes, Be acceleration of epidermal growth after wound treatment, especially with burns.

Also included are protective agents for application to Mucous membranes (mouth, throat, vagina) for the treatment of cracked lips, erythematous, itching spots the skin, in the intimate or anal area and for aftercare tissue damage of various shapes. The An externally, depending on the requirements Apply to the skin or by oral administration in Form of oil emulsions in soft gelatin capsules, as supposi  on a fat basis or in freeze-dried Dry milk-like type, z. B. dosed in hard gelatin plug capsules, lozenges or in chewing gum mass. He according to the invention to be used amount of milk / bacteria lysate in The drugs depend on the formulation and the use intended purpose of the medicinal product.

Preferably, the milk / bacteria of the invention lysates in the cosmetics or medicaments according to the invention used as anti-inflammatory additives.

The application of a product according to the invention Milk / Bacteria lysates on the skin causes a protection ge inflammatory irritation and symptoms of the skin or mucous membrane, such as erythema, edema or itching. The knit In addition, substance can also be used prophylactically become.

The cosmetic ingredient causes by the inflammatory Protecting a strengthening of the epidermis cells and a verbes DNA repair activity, an asset to be accelerated skin aging and teratogenic skin changes. The pharmacologically applied active ingredient causes a ver improved healing of the inflammatory weakened or ge damaged skin.

Served for testing anti-inflammatory efficacy consider the endotoxin test as an exogenous stimulus Macrophage cell cultures or directly on the skin below Use of UV light, allergens or irritants chemical, plant or bacterial origin.

In the macrophage test, the distribution pro-inflammatori substances (endogenous mediators such as cytokines, Leuko triene, oxygen radicals, etc.). It will be the Inhibitory effect of the active ingredient milk / bacterial lysate against milk measured as a control.

The efficacy tests on the skin are performed in vitro at the Mouse skin hairless mice performed. It will be the dermis of freshly killed mice and tissue culture incubated at 37 ° C. From the dermis the epidermis is abge  separates and a test for formation of water-insoluble Formazan from soluble methyltetrazolium salt as a measure of Vi tality (Kietzmann et al., Naunyn Schmiedeberger's Arch. Pharmacol. (Suppl.) R108 (1994), 349). By differently long UV irradiation becomes a kinetically directly correlated weakening of activity gemes sen. The effectiveness of the milk / bacteria lysate is in Ver equal to milk as a control.

A particular advantage of the milk / Bak according to the invention terienlysate is that they are unproblematic can be further processed, for example, to the invention appropriate cosmetics and medicines because they are practical particle-free and pH-neutral and are hardly externally from Skim milk differ. The entire manufacturing process can be fully automatic up to the finished dosage form and computer-controlled.

The examples illustrate the invention.

example 1 Preparation of a milk / bacteria lysate a) Preparation of the milk culture medium

Composition (% w / v): skimmed milk powder (health food store), about 10%; Yeast extract powder (Yeast Extract USP), 0.5%; least. Water, ad 100% (w / v), pH: ca. 6.5.

Milk powder and yeast extract (eg fully soluble yeast Extract powder Oxoid L 21) are each as separate, aqueous solutions, 10% and 20% strength, with a slight Er heated and autoclaved (15 min / 121 ° C). From The cooled to about 30 ° C solutions are 1000 ml or 25 ml in an Erlenmeyer flask (2 liters) aseptically combined into a solution.  

b) culture conditions

Colonies of a pure culture of Lactococcus lactis ssp. lactis DSM 10605 are used to prepare a preculture in 10 ml of the milk culture medium at 30 ° C overnight incubated to early stationary growth phase (pH ca. 4.5; Max. 5 g / L L-lactic acid; about 1 × 10⁹ cfu / ml). 1 ml This preculture is then used to produce the Main culture in about 1000 ml of milk culture medium inoculated.

The main culture is kept at 30 ° C for 3 hours a slight pivoting movement is incubated; the pH is then at 6.35, the germ count is about 3 to 4 × 10⁶ cfu / ml. The formed lactic acid is 1 g / l.

c) lysis

100 ml of 10% glycine solution becomes aseptically the main Culture added. After 20 minutes exposure to 30 ° C 50 ml of 1% lysozyme solution (Lysozym Serva 28262 with a specific activity of 146,500 U / mg) mix well and add culture medium for 2 hours occasional pivoting movement. Then the Living germ count and purity and identity colony-morphologically after smear on blood agar reviewed.

The setpoint for the germ count before pasteurization should be <100 CFU / ml.

d) pasteurization

The culture is incubated in a water bath at 70 ° C for 30 min.

e) Final check on the milk / bacteria lysate

The milk / bacteria lysate has the following properties on:

• 1. fermentation product L-lactic acid (enzymatic): 1 to 2 g / l.
• 2. Identity (organoleptic): milk-like whitish- homogeneous liquid without sediment.
• 3. Purity (microbiological): sterile

f) Storage

The pasteurized product is refrigerated in disinfected Container filled and in the refrigerator until further Use stored. If necessary, this can Milk-like, whitish homogeneous-looking product with Preserved a preservative.

Example 2 Production of a cosmetic raw material % (G / G) 1. Cetylstearyl alcohol | 12,500 2. Vaseline, white 19,500 3. Sorbitol solution (70%) 8 to 13 4. Tween 80 (Polysorbate 80) 5,000 5. least. Water and milk / bacteria lysate ad 100 or @ 1. Emulsifier E 2155 6,000 2. Paraffin oil, viscous 10,000 3. Microwax 3,000 4. isopropyl myristate 3,000 5. Glycerin 5,000 6. least. Water and milk / bacteria lysate ad 100

example 3 Preparation of an ointment and cream based medicament

The recipe or techno given above for a cosmetic logie can continue to be used for a drug.

raw material % (G / G) 1. Lanette N (blend of Lanette O and Lanette E of wool wax) | 10,000 2. 2-octyldodecanol 8,000 3. Paraffin oil, viscous 4,000 4. Glycerol 4,500 5. least. Water and milk / bacteria lysate ad 100 or: @ 1. Glycerol monostearate 4,000 2. cetyl alcohol 6,000 3. Spreading oil (medium chain triglycerides) 7,500 4. Vaseline, white 20 to 25 5. POE glycerol monostearate (Tagat S2) 7,000 6. 1,2-propylene glycol 10,000 7. least. Water and milk / bacteria lysate ad 100

Claims (17)

1. Milk / Bacteria Lysate available through:

• (a) preparing a culture of bacteria in a milk-yeast extract medium;
• (b) placing the medium with an inhibitor of murein biosynthesis after reaching the log phase;
• (c) adding a murein hydrolase to lyse the bacteria contained in the culture; and
• (D) pasteurizing the milk / bacteria lysate thus obtained.

2. milk / bacterial lysate according to claim 1, wherein the Bakte belong to the genus Lactococcus. 3. milk / bacteria lysate according to claim 2, wherein the Bakte of the genus Lactococcus Lactococcus lactis ssp. lactis DSM 10605. 4. milk / bacteria lysate according to one of claims 1 to 3, where the inhibitor of murein biosynthesis is glycine. 5. milk / bacteria lysate according to one of claims 1 to 4, where the murein hydrolase is lysozyme. 6. milk / bacteria lysate according to one of claims 1 to 5, wherein before the pasteurisation in step (d) the full lysis is controlled. 7. milk / bacterial lysate according to one of claims 1 to 6, wherein during the cultivation of the bacteria the pH the medium controlled and optionally the medium is neutralized. 8. A method for producing a milk / bacteria lysate, comprising the steps of:

• (a) preparing a culture of bacteria in a milk-yeast extract medium;
• (b) placing the medium with an inhibitor of murein biosynthesis after reaching the log phase;
• (c) adding a murein hydrolase to lyse the bacteria contained in the culture; and
• (D) pasteurizing the milk / bacteria lysate thus obtained.

9. The method of claim 8, wherein the bacteria for Genus Lactococcus belong. 10. The method of claim 9, wherein the bacterium of the Genus Lactococcus Lactococcus lactis ssp. lactis DSM 10605 is. 11. The method according to any one of claims 8 to 10, wherein the Inhibitor of murein biosynthesis is glycine. 12. The method according to any one of claims 8 to 11, wherein the Murein hydrolase is lysozyme. 13. The method according to any one of claims 8 to 12, wherein before the pasteurization in step (d) the completeness the lysis is controlled. 14. The method according to any one of claims 8 to 13, wherein currency During the cultivation of the bacteria, the pH of the Me controlled medium and, where appropriate, the medium neutra is lisiert. 15. Cosmetic containing a milk / bacterial lysate one of claims 1 to 7. 16. Medicament containing a milk / bacterial lysate one of claims 1 to 7.   17. Cosmetic according to claim 15 or medicaments according to An claim 16, wherein the product contained therein after a of claims 1 to 7 as an anti-inflammatory additive serves.