WO2008031131A1 - Method for the production of a mare milk preparation - Google Patents

Abstract

The present invention relates to a method for the production of hydrolyzed mare milk, comprising the steps of: - providing mare milk, and - incubating the mare milk with at least one protease.

Description

 i

Process for preparing a mare's milk preparation

The present invention relates to processes for the preparation of hydrolyzed mare milk.

The history of nutritional significance of mare's milk dates back to the 8th century BC. (Homer described the horse moths as hypomologists in the Iliad). Also, the Scythians (8th to 3rd century BC), the Mongols under Genghis Khan (12th century AD) and various Russian nomadic tribes used mare's milk and their fermented product "Kumyss" as a nutrient and remedy In the 19th and 20th centuries, mare's milk was used in several dozen Eastern European sanatoriums for the treatment of immunological, dermatological and pulmonary diseases (tuberculosis), especially in the areas of immunological (allergic) and dermatological (eczema, psoriasis, acne) diseases scientific renaissance and was the subject of microbiological, clinical-analytical and clinical research.

As mentioned earlier, mare's milk has been used in the Asian region for centuries for inflammatory diseases and as a dietetic. Today it is known that the milk contains antibacterial, anti-inflammatory and immune system activating ingredients. Various ingredients and immunologically active components (including lysozyme, lactoferrin, secretory IgA) exhibit the bactericidal and dietary effects. Furthermore, it has been demonstrated that mare's milk is particularly suitable for the treatment of skin diseases such as atopic dermatitis and psoriasis (US 2005/170007). The high similarity of mare's milk to breast milk compared to other types of milk makes it possible to use mare's milk as a breastmilk substitute. In particular, the casein of the mare's milk coagulates in the stomach analogous to the mother's milk soft and flaky and passes quickly into the small intestine (low burden of the stomach). Cow's milk, on the other hand, forms solid coagulation particles, which linger longer due to the slow dissolving of digestive juices in the stomach.

Mare's milk is distinguished from cow's milk by a lower content of dry matter, fat, protein and phosphate, as well as by a high content of milk sugar (lactose). Egg- The essential characteristic of mare's milk is the mother's milk-like ratio of casein: main whey protein (albumin / globin) of about 55:45 to cow's milk of about 85:15, and the casein coagulates on acidification with finer fuzziness than that of cow's milk.

The fat of the mare's milk is finer distributed than in cow's milk, the fat globules are about one third smaller. This property and the added presence of lecithins and a high-activity lipase indicate easy fat digestibility and absorption. Furthermore, mare's milk is characterized by the high proportion of unsaturated fatty acids.

Mare's milk, like all other albumin milks, has a much lower acidity than cow's milk. This is primarily due to the very low germ content of the mare's milk. The total germ count is usually less than 10,000 germs per ml. For cow's milk, which is processed into drinking milk, currently a maximum of 100,000 germs per ml are allowed.

The low germ count is mainly due to the small capacity of the udder (2-31) and the frequent milk removal (2-hour milking or about 80 Saugakte per day).

Despite the low number of germs in the mare's milk, this is due to the presence of unsaturated fatty acids only limited storage. To avoid this problem, mare's milk is dried and produced especially in the form of powder or capsules. The drying of mare's milk must be gentle due to the sensitivity of the unsaturated fatty acids to oxidation. In particular, freeze-drying, spray-drying and evaporation drying are used here. It has been found to be particularly advantageous for the preparation of storage-stable mare's milk (preferably at room temperature over a period of more than one year) to have added a highly dispersed matrix (see, for example, AT 393,961).

In US 2002/0192333 a method for the treatment of milk with proteases is described. Treating milk with proteases greatly increases the bioavailability of calcium and makes the milk more resistant to acid coagulation. The protease-treated milk has the same odor and taste as natural and untreated milk. The proteases destroy milk proteins during incubation in such a way that "milk allergens" are degraded, making the milk more compatible with the consumer.

The mare milk preparations described in the prior art have some significant disadvantages. On the one hand, the proteins present in the mare's milk can trigger milk and protein intolerances or allergies. On the other hand, mare's milk has an inherent odor that prevents many consumers from consuming or taking mare's milk supplements.

It is an object of the present invention to provide mare's milk preparations or processes for the preparation of such preparations which do not have the disadvantages of the prior art. In particular, mare's milk preparations are to be made available which do not lead to an allergic reaction during administration, but nevertheless have the advantageous properties of natural mare's milk and conventional mare's milk preparations. Further, although mare's milk is compared to e.g. Cow's milk is known to be better digestible, there is still a need to improve the digestibility and wholesomeness in addition.

Therefore, the present invention relates to a process for the preparation of hydrolyzed mare's milk comprising the steps:

- Provide a mare's milk and

- incubate the mare's milk with at least one protease.

It has surprisingly been found that mare's milk can be incubated or treated with the aid of proteases, without adversely affecting the known properties of mare's milk or mare's milk preparations. By incubating mare's milk with proteases, the proteins contained in the milk can be split. Depending on the selected incubation conditions, which depend on the one hand on the proteases used (eg temperature optimum, pH optimum, etc.) and on the other hand on the desired product properties (eg retention of the unsaturated fatty acids, etc.), the mare's milk may have a different degree of hydrolysis. Already a degree of hydrolysis of at least 10%, preferably of at least 20%, even more preferably of at least 30%, most preferably of at least 40%, in particular of at least 50%, is sufficient according to the invention for "hydrolyzation". The degree of hydrolysis of the proteins present in the mare's milk refers to a non-protease-treated, preferably native, mare's milk which can be determined in a simple manner by methods known in the art (eg electrophoresis, especially SDS). PAGE).

Conventional hydrolysis methods, e.g. The acidic hydrolysis is only of limited suitability for splitting up the mare's milk proteins, since on the one hand they greatly alter the taste of the mare's milk, and on the other hand they destroy the nat- ural enzymes of the mare's milk due to the strong pH changes. Proteases which are substantially active in the pH range of native mare's milk exhibit their hydrolytic activities during incubation, but also in the course of further processing steps, e.g. during evaporation processes. Furthermore, proteases, in particular plant proteases, are common as foodstuffs and marketable. The hydrolyzed end product also has organoleptic (odor, flavor) benefits.

In the method according to the present invention, at least one protease is used, but it is quite possible to use at least two, three, four, five, six or seven different proteases.

By the use of proteases in the method according to the invention hydrolyzed mare's milk preparations are prepared, which in addition to the at least partially hydrolyzed mare's milk components contain proteases, unless they are incubated after incubation, e.g. chromatographically.

Proteases of any type can be used in the process according to the invention, but these are preferably selected from the group consisting of serine proteases, threonine prosteases, cysteine proteases, aspartate proteases, metalloproteases and glutamate protease.

Of course, different combinations of proteases can also be used in the present process.

According to a preferred embodiment of the present invention, the protease is of plant, animal or microbial origin.

The proteases used in the process can come from different sources, but it is particularly preferred to use plant proteases, since they are known per se in the Are not subject to any legal restrictions and are themselves considered to be food or dietary supplements.

The proteases of plant origin are preferably selected from the group consisting of papain, bromelain, ficin and combinations thereof.

Papain and / or bromelain are particularly preferably used in the process according to the invention.

The dried latex of immature papayas contains not only papain (3 subunits), but also the acid-stable chymopain (4 subunits). Similar plant enzymes are the bromelain from pineapple and the ficin from figs.

Papain is an endopeptidase (EC 3.4.22.2) consisting of 211 amino acids belonging to the class of thiol proteases. Papain has a broad substrate specificity, but Arg or Lys are preferentially cleaved in Pl position.

Due to its high temperature and pH stability, papain is used in many biotechnological processes (e.g., as meat delicacies in the food industry, for clarification of beverages, as protein stain removers in the detergent industry, and in the leather industry and in the manufacture of denture cleaners).

The proteases of animal origin are preferably selected from the group consisting of trypsin, pepsin, chymotrypsin and combinations thereof.

According to a further preferred embodiment of the present invention, the protease of microbial origin is nattokinase.

Proteases of microbial origin include, according to the invention, proteases derived from microorganisms, e.g. Bacteria, fungi and yeasts can be isolated and occur naturally in these organisms. That Proteases from other organisms (e.g., animals or plants) recombinantly produced in microorganisms are not considered, according to the invention, to be proteases of microbial origin.

The proteases used in the process according to the invention can either be prepared recombinantly or be of natural origin.

Methods for the recombinant production of polypeptides and proteins, in particular of proteases, are sufficient for the person skilled in the art. (See, eg, Sambrook, J. et al., Molecular Cloning 3rd Ed., CoId Spring Harbor Laboratory Press, Colard Spring Harbor, New York, 2001). Preferred host cells in which the proteases according to the invention are expressed are bacterial cells (eg E. coli, B. subtilis), yeast cells (eg P. Pastoris, H. Polymorpha), animal cells in cell cultures and insect cells (eg baculovirus system). Of course, in recombinant production, it is also possible to provide peptides and polypeptide fragments at the C- or N-terminus of the proteases, which make it possible to purify the proteases from a cell culture supernatant or from the cells.

According to a preferred embodiment of the present invention, the at least one protease is in an amount of 0.1 to 10 g, preferably from 0.2 to 5 g, more preferably from 0.3 to 2 g, particularly preferably from 0.4 to Ig, per Liter of mare's milk added.

The amount of at least one protease used in the method of the invention may vary and is preferably governed by the reaction conditions chosen (e.g., incubation temperature near the temperature optimum of the protease). For example, if the incubation time is to be kept short, a higher amount of enzyme is preferably used.

The incubation of the mare's milk with the at least one protease is preferably carried out for at least 30 minutes, preferably for at least 1 hour, even more preferably for at least 2 hours, more preferably for at least 5 hours, in particular for at least 10 hours.

The incubation period of the protease with the mare's milk depends in particular on the type of protease, the amount of protease used, the incubation temperature and the desired hydrolysis rate.

According to a further preferred embodiment of the present invention, the pH is adjusted before the incubation of the mare's milk with the at least one protease in a range from 4 to 9, preferably in a range from 5 to 8.

The pH, which is adjusted in the course or before the incubation, depends in particular on the pH optimum and the pH stability of the proteases or the mare's milk.

In order to preserve the hydrolysed mare's milk, in particular In particular, in order to preserve their oxidation-sensitive ingredients, the mixture is dried after or during the incubation of the mare's milk with the at least one protease. The enzymatic reactions of the proteases used can of course still take place in the course of drying.

According to a preferred embodiment of the present invention, the drying is carried out by evaporation, preferably by evaporation under reduced atmospheric pressure, in particular by evaporation under vacuum.

Mare's milk and hydrolysed mare's milk include oxidation-sensitive substances such as e.g. unsaturated fatty acids. In order to prevent or largely suppress the oxidation of such substances, the drying is preferably carried out under a reduced oxygen atmosphere.

The drying is preferably carried out at a temperature of less than 80 ^° C., preferably less than 70 ^° C., more preferably less than 60 ^° C., even more preferably less than 50 ^° C., in particular less than 40 ^° C.

In order to protect the ingredients of the hydrolysed mare's milk, the drying is preferably carried out at a temperature of less than 80 ⁰ C.

According to a preferred embodiment of the present invention, prior to drying, the hydrolyzed mare's milk is mixed with a highly dispersed matrix, preferably with fumed silica.

For example, if the hydrolyzed mare's milk at a temperature of below 40 ⁰ C and under exclusion of oxygen to remove the water contained in the milk, dried and thus concentrated, arises due to the content of low molecular weight oligosaccharides, oligopeptides and high-quality oils, a mare's milk concentrate with a viscous-amorphous mass, which in this form would be difficult to process galenically. In order to compensate for this technological disadvantage, it has been disclosed, for example, in AT 393 961 that mare's milk is to be mixed with inert, highly disperse silicon dioxide (silica) as a carrier matrix before vacuum distillation, so that after the vacuum distillation a crystalline, powdery dry concentrate is obtained results.

This mare milk dry concentrates based on highly disperse matrices were ^"thus developed to the production method To maintain the high-quality ingredients to simplify, as well as to store the mare's milk over a long period of time, without causing loss of quality. The silica also gives the product an improved flowability.

By dried in this preferred manner mare's milk, it is possible, the temperature and oxygen-sensitive ingredients, especially the fatty acids, gently and without loss to concentrate and dry, so that the high-quality ingredients are dried temperature-friendly. This provides a mare's milk concentrate which not only has a maximum biological value, but is storage-stable at room temperature and, surprisingly, is better suited than the conventional preparations for the treatment of skin diseases. Compared, for example, to a treatment with spray-dried mare's milk, in the use according to the invention an improvement in the course of the disease rapidly occurs and the healing also lasts longer.

The term "highly dispersed matrix" can be understood according to the invention to mean a matrix having a large surface area of at least 50 μg / g. It is important that the matrix is biologically inert, so that the mare's milk is not chemically altered and thus loses its biological value. The fact that the mare's milk is dried on the highly dispersed matrix, ensures that the mare's milk droplets are deposited finely distributed on the matrix particles and it comes to an important for a gentle drying optimal fine surface distribution of the milk. The milk is thus distributed as much as possible to the lowest possible volume. As a result, the milk is dried quickly and under gentle conditions and can be made available in high storage stability. Not only does the matrix achieve a fine distribution of milk on as large a surface as possible, but the matrix also provides some protection against other substances that attack the delicate components of the milk, such as the unsaturated fatty acids. The milk can be applied, for example, by spraying onto the highly dispersed matrix.

According to a further preferred embodiment of the present invention, before and / or after incubation, the mare's milk with the at least one protease is at least one Antioxidant, preferably ascorbic acid, ascorbates, ascorbic acid esters, tocopherols, in particular α-tocopherol added.

In order to increase or enhance the valence and possible applications of the hydrolyzed mare milk produced by the process according to the invention, further substances can be added in the course of the process.

The dried hydrolyzed mare's milk is preferably ground and optionally packaged. By a post-drying milling, the grain size of the mare's milk powder can be changed and adapted to further processing steps (e.g., tabletting, filling into capsules).

Another aspect of the present invention relates to hydrolyzed mare's milk or hydrolyzed mare's milk powder obtainable by the process according to the invention.

The hydrolyzed mare's milk, which can be prepared by the method of the present invention or by incubation of mare's milk with proteases, contains the same high-quality fats (such as unsaturated fatty acids) as native mare's milk. Furthermore, hydrolysis with proteases cleaves the proteins that occur natively in the mare's milk, so that the allergenic potential of native mare's milk can be partially or completely reduced. The mare dry concentrate obtained by the method according to the invention furthermore has a shelf life of more than 24 months, in particular from 24 to 36 months. Surprisingly, after the hydrolysis process, the typical pungent odor and taste (common in conventional mare milk preparations) disappeared.

A further aspect of the present invention relates to the use of the hydrolyzed mare milk produced according to the invention as a food, in particular as a liquid, solid or pasty food, as a dietary supplement, as a pharmaceutical or as a cosmetic preparation.

Hydrolyzed mare's milk can be used for example as food or nutrition for babies (as breastmilk substitute) due to their properties. The food according to the invention is also suitable for consumption by humans who have a protein intolerance to proteins, for example cow's milk.

Mare's milk can also be used as a dietary supplement or drug, eg in the form of capsules or ointments. the. It is known that mare's milk has a calming effect on skin irritations and redness (thus also as a cosmetic preparation), but also has an immunostimulating effect. It is also known to use mare's milk, for example, against psoriasis and atopic dermatitis (see, for example, WO 03/090728).

The present invention will be further explained with reference to the following figures and examples, but without being limited thereto.

Fig. 1 shows the composition of the individual experimental approaches.

Figure 2 shows an SDS-PAGE gel with mare milk powder and papain hydrolyzed mare milk powder (2 and 10 hours drying time).

Figure 3 shows an SDS-PAGE gel with mare milk powder and mare milk powder hydrolyzed with papain or bromelain.

Figure 4 shows a native PAGE gel with mare milk powder and mare milk powder hydrolyzed with papain or bromelain.

Figure 5 shows a native PAGE gel with mare milk powder and mare milk powder hydrolyzed with papain or bromelain.

Fig. 6 shows a BSA standard curve for protein determination.

EXAMPLES:

Example 1 :

1.1. Hydrolysis and drying:

Milk hydrolysis of mare's milk was carried out with vegetable hydrolases. Several approaches with different amounts of vegetable enzyme (papain and bromelain) and hydrolysis times were tested.

The enzymes papain and bromelain investigated in the example are considered foodstuffs and generally do not require any further toxicological testing.

The drying temperature did not exceed 40 ^° C., which ensured that the proteins or the resulting low molecular weight proteins, polypeptides or peptides of the mare's milk would remain in native form and that the natural properties of the mare's milk were changed as little as possible.

The degree of hydrolysis was varied by the duration of the hydrolysis and by subsequent introduction of the enzyme solution and / or by the addition of different amounts of enzyme.

In detail, the hydrolysis was carried out as follows. The mare's milk was thawed slowly and 250 ml in a pear _ _

transferred piston. Then, after adding the respective amount of enzyme (0.8 g / l mare's milk), the mixture of Rotavapor (Rotavapor Water Bath Büchi 461) at max. 4O ⁰ C dried. The reaction time was controlled with the vacuum, that is, the drying started only by applying a vacuum. The enzyme units in the experimental batch were FIP-1/1 for papain and 384 FIP-1/1 for bromelain.

After drying, the obtained mare milk powder or mare milk hydrolyzate was ground (manually by means of a mortar) and filled into dark vials.

For the further investigations, solutions were prepared from the mare milk powder or mare milk hydrolyzate powder.

In the present example, a total of 6 batches were prepared (see FIG. 1). The starting basis was 250 ml of whole milk. Approach A is pure horse milk powder, which was not hydrolyzed and was included as a reference.

The drying time was set to about 2.5 hours. To see how and if the degree of hydrolysis of the milk changes with increasing duration of drying, the sample C was dried for 10 hours or the drying activated after 7.5 hours.

The 6 batches in powder form were used for the gel electrophoreses in dest. Water dissolved.

1.2. PLC Page

Acrylamide / Bis (30% T, 2.67% C, 1: 37.5): 5x Running buffer pH 8.3:

146.0 g acrylamide 15.15 g Tris base 4.0 g To 72.0 g glycine with dist. Water to 500 mL, filtrate 5.00 g SDS ren with dist. Water to 1000 mL; Store in the dark at 4 "C. Do not adjust pH, store at 4 ° C and use 1: 5 before use

Dilute 1.5 Mris-HCl, pH 8.8 (separating gel).

54, 45 g Tris base

120 mL dist. Water Ix Running buffer pH 8.3: adjust to pH 8.8 with 6N HCl; 160 mL 5x Running buffer pH 8.3 with dist. Make up to 300 mL water 640 mL dist. water

0.5 Mris-HCl, pH 6.8 (collection gel) 2X Sample buffer:

6, 00 g Tris-Base 900 μL Stock-2XSB 60 mL dist. Water 100 μL β-mercaptoethanol with 6N HCl to pH 6.8; with dist. Make up to 100 mL water 10% (w / v) SDS:

10% (w / v) ammonium persulfate (APS): ¹ O 9 ^SDS

10 mL dist. Water 50 mg ammonium persulfate - - -

500 μL dist. water

N, N, N ', N'-tetramethyl-ethylenediamine

Stock-2XSB: (TEMED)

2.0 mL of 0.5 M Tris-HCl pH 6.8

1.6 mL glycerol (20%)

3.2 mL 15% SDS (5%)

0.4 mL 0.5% (w / v) bromophenol blue

The SDS-PAGE gels were prepared as follows:

After casting the release gel, it was overcoated with a little 2-butanol to form a release liner. The gel was polymerized for about 30-45 minutes. Thereafter, the butanol was removed, with dist. Rinsed water and vacuumed the zone for collecting gel with filter paper.

In the next step the collection gel was poured with a height of 1 cm and the combs with 10 slots were used. The polymerization time of the collection gel was about 30-45 min.

After removal of the comb, the electrophoresis apparatus was assembled.

The gels were placed in the electrophoresis chamber and filled with 1 x running buffer. The samples were taken up in the same volume 2x sample buffer, heated for about 4 min at 95 ° C and then centrifuged for about 1 min. The samples were applied from left to right using a Hamilton syringe.

Subsequently, voltage source at const. 200V and max. 70 mA per gel applied.

In order to later understand the direction of sample application, the left corner of the gel was cut off or marked.

Subsequently, the gel was stained (preferably a Coommassie stain). In order to be able to assign the resulting bands to the individual mare milk proteins, an SDS-PAGE marker has been used. This was composed of myosin (rabbit muscle, 205.0 kDa), beta-galactosidase (E. coli, 116.0 kDa), phosphorylase b

(Rabbit muscle, 97.0 kDa), fructose-β-phosphate kinase (rabbit muscle, 84.0 kDa), albumin (bovine serum, 66.0 kDa), glutamate dehydrogenase (bovine liver, 55.0 kDa), ovalbumin (Hühnerei , 45.0 kDa), glyceraldehyde-3-phosphate dehydrogenase (rabbit muscle, 36.0 kDa), carbonic anhydrase (bovine erythrocytes, 29.0 kDa), trypsinogen (bovine pancreas, 24.0 kDa), trypsin inhibitor

(Soybean, 20.0 kDa), alpha-lactalbumin (bovine milk, 14.2 kDa) and aprotinin (bovine lung, 6.5 kDa).

Stock solutions were prepared with concentrations as equal as possible to allow semi-qualitative interpretation of gels (see Figure 2).

On the lane 1, the SDS marker has run. This allows a clear allocation of the bands and the respective proteins.

The high molecular weight immunoglobulins, which make up a small percentage, and serum albumin are found at the upper end of the SDS gel.

In the midfield, the caseins can be found. The caseins represent the largest share with about 55 percent, followed by the whey proteins, which can be found at the lower part of the gel, with about 40 percent.

On track 2 was in dest. Water dissolved mare milk powder applied.

On track 3, stutemilk hydrolyzate was applied, whereby it can be seen that the casein bands have decreased markedly. In the case of lane 4, mare's milk was hydrolyzed for 10 h; here, there is a further reduction of casein bands or no clear bands are recognizable.

Another SDS-PAGE was performed with all 6 approaches (see Figure 3):

1.3. Native-PAGE:

Acrylamide / bis (30% T, 2.67% C, crosslinking 5x running buffer pH 8.3: grade 1: 37.5): 15.15 g Tris base

72.0 g glycine

146.0 g Acrylaraid with dist. Water to 1000 mL; 4.0 g Do not adjust to pH; with dist. Store water at 500 mL, filter at 4 ⁰ C and dilute 1: 5 before use. and store in the dark at 4 ⁰ C.

Ix Running buffer pH 8.3:

1.5 M Tris-citrate, pH 8.8 (separating gel buffer.)

160 mL 5x Running buffer pH 8.3

54.45 g Tris base 640 mL dist. water

120 mL dist. Water with citric acid crystals to pH

8, set θ; B rom phenol blue: with dist. Store water at 300 mL at 4 ^° C.

0.5 M Tris-HCl, pH 6.8 (collection gel) N, N, N ', N'-tetramethyl-ethylenediamine (TE)

6.00 g Tris base MED)

60 mL dist. Water with citric acid crystals to pH

Set 6.8; with dist. Make up to 100 mL water. store at 4 ^° C. - -

10% (w / v) Ammonium Sulphate (APS):

50 mg of ammonium persulfate 500 μL dist. water

sucrose

2.0 mL of 0.5 M Tris-HCl pH 6.8

1, 6 mL glycerol (20%)

3.2 mL 15% SDS (6%)

0.4 mL 0.5% (w / v) bromophenol blue

The native PAGE gels were prepared as follows:

The preparation and use of native PAGE gels is analogous to the SDS-PAGE gels.

Native PAGE was performed on all the following batches (see Figures 4 and 5, in which the samples were diluted 1: 5):

1.4. Bradford protein determination The determination is based on the observation that the absorption maximum for an acidic Coomassie Brilliant Blue G-250 solution migrates from 465 nm to 595 nm when bound to a protein. The dye binds primarily to basic (especially arginine) and aromatic amino acid residues. This observation was first used by Bradford for protein determination. For quantitative evaluation, the Lambert-Beersche law can be used.

Staining solution (B) bovine serum albumin BSA (Sigma)

Dissolve 100 mg Coomassie Blue G in 52 mL 92.4% ethanol Decolorizing solution: 100 mL 85% phosphoric acid 40% (v / v) methanol to 1000 mL with dist. Water 10% (v / v) glacial acetic acid 50% (v / v) dist. water

staining solution:

Sample determination:

60 μl of the sample or standard (zero points with 60 μl distilled water)

150 μl 1 M NaOH. mix well with 3 ml of staining solution (B).

Incubate for 5 min.

Measure absorbance at 595 nm.

Create the default curve:

10 mg of BSA were dissolved in 1 ml of dist. Water dissolved (c = 10,000 μg / ml). From this solution, 5 dilution steps (200, 300, 500, - 750, 1000 μg / ml) were prepared. The evaluation was carried out with Help the standard curve (see Fig. 6).

The standard curve was used to determine the amount of protein in the samples before and after hydrolysis and thus the degree of hydrolysis.

1.5. discussion

Six approaches were tested. The degree of hydrolysis after 2 hours at 37 ⁰ C could be determined with about 35% and after 10 hours with about 65%.

Also, the adaptation of the existing mare's drying method with the new method is possible without additional effort. .. _. , The experimental amounts were determined with 250 ml mare's milk and 0.2 g of enzyme. Variations were made in enzyme species, pH and duration.

The microbial load during the procedure did not play a decisive role, although there was a concentration.

This type of milk hydrolysis has already been used in cheese production. The enzymes papain and bromelain were used as laboratory replacements, but this process was again discarded because the protein splits produced had a bitter taste and thus found no consumer acceptance. The mare hydrochloride obtained by this type of milk hydrolysis surprisingly has a neutral, slightly sweetish smell and taste.

Since these enzymes mainly split only the caseins, the basic properties of the milk are retained. Furthermore, such enzymes were added to the dried milk to achieve a better digestion of the milk proteins.

Example 2:

The effectiveness of the preparation according to the invention is shown in the following case studies:

Case Study 1

M.M., male, b. on 01.08.1998; inpatient stay diagnoses:

1. Bacterially superinfected atopic dermatitis in generalized form, from severe course

2. Mixed form of bronchial asthma

3. Allergic Rhinopathy due to pollen

The patient took a preparation based on hydrolyzed mare's milk.

In the course of therapy, no complaints occurred. The general and skin condition has improved significantly under therapy.

Case study 2

F.A., female, b. on 08.04.1972; inpatient stay diagnosis:

Allergic dermatitis of unknown origin

Ms F. took a preparation based on hydrolyzed mare's milk. There were no complaints after taking it. The general and skin condition has improved under the therapy. Case Study 3

M. L. female, b. on 01.08.2000; inpatient stay diagnosis:

Bacterially superinfected, atopic dermatitis from severe course

The intake of a preparation based on hydrolyzed mare's milk took place for 4 weeks. It was well tolerated. Even after taking it came to no complaints.

The general and skin condition has improved significantly during the stay.

Case Study 4

M.V., female, b. on 28.06.1991; inpatient stay diagnosis:

Bacterially superinfected, atopic dermatitis in generalized form, of severe course

M. V. took a preparation based on hydrolyzed mare's milk. There were no complaints after taking it. The general and skin condition has improved significantly under therapy.

Case Example 5

R.N., female, b. on 19.09.1969; inpatient stay diagnoses:

1. Generalized psoriasis vulgaris intertriginosa et capillitii of the chronically stationary type

2. Impetiginization of some psoriasis with Staphylococcus aureus

The intake of a preparation based on hydrolyzed mare's milk was carried out for 3 weeks. There were no allergic reactions, i. it was well tolerated. Even after taking it came to no complaints.

The patient was able to be discharged in an improved general and skin condition and continued therapy with the product based on hydrolyzed mare's milk at home.

Case Study 6

H.B., female, b. on 08.01.1943

The patient was diagnosed with atopic dermatitis, i.a. with dyshidrosiform skin symptoms, hospitalized. R. N. enjoyed the classical therapy of atopic dermatitis first with a rehabilitation phase, later with anti-inflammatory ointments, i.a. also with pasta exicans on the hands.

Claims

Claims: A process for producing hydrolyzed mare milk comprising the steps of: - Provide a mare's milk and - incubate the mare's milk with at least one protease. 2. The method according to claim 1, characterized in that the protease is a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a metalloprotease or a glutamate protease. 3. The method according to claim 1 or 2, characterized in that the protease is of plant, animal or microbial origin. 4. The method according to claim 3, characterized in that the protease of plant origin is selected from the group consisting of papain, bromelain, ficin and combinations thereof. 5. The method according to claim 3, characterized in that the protease of animal origin is selected from the group consisting of trypsin, pepsin, chymotrypsin and combinations thereof. β. A method according to claim 3, characterized in that the protease of microbial origin is nattokinase. 7. The method according to any one of claims 1 to 6, characterized in that the protease is produced recombinantly or of natural origin. 8. The method according to any one of claims 1 to 7, characterized in that the at least one protease in an amount of 0.1 to 10g, preferably from 0.2 to 5g, more preferably from 0.3 to 2g, more preferably from 0.4 to Ig, per liter of mare's milk is added. 9. The method according to any one of claims 1 to 8, characterized in that the incubation of the mare's milk with the at least a protease for at least 30 minutes, preferably for at least 1 hour, even more preferably for at least 2 hours, more preferably for at least 5 hours, in particular for at least 10 hours. 10. The method according to any one of claims 1 to 9, characterized in that the pH is adjusted before the incubation of the mare's milk with the at least one protease in a range of 4 to 9, preferably in a range of 5 to 8. 11. The method according to any one of claims 1 to 10, characterized in that after or during the incubation of the mare's milk with the at least one protease, the mixture is dried. 12. The method according to claim 11, characterized in that the drying by evaporation, preferably by evaporation under reduced atmospheric pressure, in particular by evaporation under vacuum takes place. 13. The method according to claim 11 or 12, characterized in that prior to drying, the hydrolyzed mare's milk with a highly dispersed matrix, preferably with fumed silica, is added. 14. The method according to any one of claims 11 to 13, characterized in that the drying at a temperature of less than 8O ⁰ C, preferably less than 70 ⁰ C, more preferably less than 60 ⁰ C, even more preferably less than 50 ⁰ C, in particular less than 40 ⁰ C takes place. 15. The method according to any one of claims 1 to 14, characterized in that prior to and / or after incubation of the mare's milk with the at least one protease, at least one antioxidant, preferably ascorbic acid, ascorbic acid, ascorbic acid, tocopherol, in particular α-tocopherol added becomes. 16. The method according to any one of claims 11 to 15, characterized in that the dried hydrolyzed mare's milk is ground and optionally packaged. 17. Hydrolyzed mare's milk or hydrolysed mare's milk powder obtainable according to one of claims 1 to 16. 18. Use of a hydrolyzed mare's milk according to claim 17 as a food, in particular as a liquid, solid or pasty food, as a dietary supplement, as a pharmaceutical or as a cosmetic preparation.