FI120267B - Process for producing therapeutically useful peptides - Google Patents

Description

A method for viable therapeutic peptides

Field of the Invention

The invention relates to a process for the preparation of therapeutically useful peptides. In the method, the protein-rich material is fermented by microorganisms which release short chain peptides from the starting material during fermentation. The peptides are inhibitors of ACE, an angiotensin-converting enzyme, and have been shown to have antihypertensive effects, for example, and to improve bone formation. They are thus useful as health active products as such and as an ingredient or additive in substances intended for human consumption.

Background of the Invention

The preparation of therapeutically useful peptides, in particular antihypertensives, by fermentation has been extensively studied and there is a great deal of Kiris related in the art.

Nakamura et al. describe, for example, the use of an acid containing Lactobacillus helveticus and Saccharomyces cerevisiae sb in J Dairy Soi 78 (1995) 777-783 for the preparation of two ACE inhibitors. The raw material used was Ruma reconstituted from skimmed milk powder (9%), and the fermentation was carried out by conventional fermentation at 37 ° C for 24 h. After fermentation, the ACE inhibitors were purified by chromatography and analyzed. The active compounds were both tripeptides, Val-Pro-Pro and lie-Pro-Pro (VPP and IPP). J Dairy Soi 78 (1995) 1253-1257 Nakamura et al. describe the in vivo antihypertensive effect of said tripeptides and acidified sour milk product.

U.S. Patent No. 5,449,661 to Nakamura et al. describe the preparation and use of a Vai-Pro-Pro (VPP) peptide containing tripeptide sequence for lowering hypertension. The peptide was prepared from skimmed milk powder with Lactobacillus helveticus strain JCM-1004. Here again, traditional acidification was carried out at 37 ° C for 24 hours, after which the peptide was purified by chromatography and freeze-dried.

Yamamoto et al. in J Dairy Sci 82 (1999) 1388-1393 describe the preparation of a yoghurt type product containing the antihypertensive Tyr-Pro peptide (TP). The product was prepared with a traditional strain of Lactobacillus helveticus CPN4. Here again the raw material was Kurri, reconstituted from 2 skimmed milk powder (9%). The acidification was carried out at 37 ° C and the acidification was terminated after the pH had dropped to 4.3. The concentration of Tyr-Pro peptides formed in whey separated from the yogurt product is about 8.1 mg / l. A similar method is also described in U.S. Pat. No. 5,585,029 and EP 0 583 074 B1.

EP-A-1 016 709 B1 to Yamamoto et al. Relates to Lactobacillus fielveticus strains which produce large amounts of so-called. lactotripeptides, i.e. VPP and IPP. The concentration of peptides, calculated as VFP peptides, is greater than 60 mg / l.

The best yield was obtained using a CM 4 strain which, by conventional acidification from 10 skimmed milk powder (9% J on a reconstituted courgette medium, produced 74.4 mg / l of VPP. The acidification conditions were 32 ° C and 20 h. EP stripping 1 142 481 A1,

Kitamura and Ueyama, relates to a method modified from the method described above, wherein the medium is mixed either continuously or intermittently at 50 rpm. According to the publication, this method provides a product with a much lower viscosity and more efficient whey recovery. The yield of the antihypertensive peptides remains approximately the same as that of the unmixed acidification, EP 1 266 267 B1, Valio Oy, discloses a process for the preparation of an antihypertensive peptide product comprising two main steps, fermentation and nanofiltration. The antihypertensive peptides were released from the milk protein by fermentation of the production strain as a medium prepared from skimmed milk powder (9-10%) at 37 ° C for 22-24 hours. During culturing, the pH was allowed to drop to 3.3-3.5. At the end of the acidification, the pH was raised to 4.6 with KOH solution to precipitate the slurry. The concentration of blood pressure peptides produced by this method has been about 24 mg / l.

Common to the methods described above is that the acidification is conventionally performed. Conventional acidification refers to acidification where the pH is generally freely lowered during fermentation and is terminated when the pH has dropped to a certain value. Generally, the acidifiers are added to the milk at a pH of about 6.5.

U.S. Patents 5,449,661 to Nakamura et al. Mention that the pH at the beginning of the fermentation can be adjusted to 6-7, and that the pH can also be adjusted during the fermentation, for example with NaOH or KOH.

35 WO 04/060073, Unilever NV, discloses a fermented milk product containing the tripeptide VPP and / or IPP. The product is prepared by fermenting milk with Lactobacillus helveticus strain CNRZ 244, maintaining the pH at 4.3-5. , With 9 base addition, the strain used in the publication produces large amounts of tripeptides and has an optimum pH of about 4.5.

In free acidification, only a portion of the lactose is fermented to lactic acid. Typically, the concentration of lactic acid formed during the fermentation of skimmed milk is 1.5-2% and the residual lactose content is 2.5-3%. The acidity of the product obtained by conventional acidification is unpleasant to some consumers and high lactose levels cause unpleasant and painful stomach and intestinal symptoms in people who are lactose sensitive. To overcome these problems, the above-described process with the nanofiltration steps described in Valio Oy, EP Patent No. 1 266 267 Bt, provides a solution n.

Alternatives, most often related to the discovery of efficient proteolytic production bacteria, have also been proposed to improve the yield of therapeutically useful peptides. WO Patent Application 03/082019 15 A2, Chr. Hansen A / S describes a method for producing hypertensive peptides using a lytic (autolysable) strain in addition to a proteolytic strain, whereby the peptide enzymes produced by the lytic strain are capable of releasing more hypertensive peptides from the raw material than from using protein alone.

Brief Description of the Invention

It is an object of the present invention to provide a novel process for the preparation of therapeutically useful peptides which provides a higher peptide yield.

It is a further object of the present invention to provide a novel process for the preparation of therapeutically useful peptides by which the lactose content of the final product can be significantly reduced.

The invention thus relates to a process for the preparation of a therapeutically useful peptide-containing product, in particular antihypertensives, by fermentation of a milk protein with a lactic acid bacterium, characterized in that the lactic acid bacterium is a therapeutically useful, in particular, hypertensive and lactic acid producing peptide.

In a preferred embodiment, the fermentation of the present invention involves the use of pH control in addition to lactase to produce a product containing therapeutically useful peptides.

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In another preferred embodiment, the lactic acid bacterium of the genus Lactobacillus is used as the lactic acid bacterium in the preparation of a product containing therapeutically useful peptides according to the invention. More preferably, the strain of Lactobacillus heiveticus is used. Particularly preferred is Lactobacillus heiveticus LB1936, DSM 17754.

The invention also relates to Lactobacillus heiveticus LB 1936, DSM 17754.

It is also an object of the present invention to provide a product containing bioactive peptides or purified bioactive peptides prepared by the process of the invention, either as such as a health active ingredient or in the preparation of ingestible substances such as health food or pharmaceuticals.

It is a further object of the present invention to provide orally administered products, such as health food and pharmaceuticals, which contain, as an active ingredient, a product prepared by the method described herein, supplemented with purified bioactive peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the pH curve obtained by performing conventional fermentation without lactase addition or pH adjustment (comparison).

Figure 2 shows the pH curve obtained by fermentation with a lactase inoculum according to the invention without pH adjustment.

Figure 3 shows the pH curve obtained by performing the fermentation with the lactase addition step of the invention and adjusting the pH.

Figure 4 shows the pH curve obtained by fermentation of the lactam with the lactase addition according to the invention and the pH adjustment,

Figure 5 shows the pH curve obtained by fermentation of the fermentation broth using pH adjustment without addition of lactase.

DETAILED DESCRIPTION OF THE INVENTION Thus, according to the present invention, it has surprisingly been found that the above objects can be achieved by a novel process based on the use of the enzyme lactase to enhance fermentation.

Lactase, or β-D-galactosidase, is commercially available as a variety of enzyme preparations. As examples of yeast-derived commercial lactase preparations, e.g. GODO YNL (Godo Shusei Company, Japan), Maxilakt L2000 (DSM, Gist-Brocades, The Netherlands), Validase (Valley Research inc., USA), Ha-Lactase (Chr. Hansen A / S, Denmark), and Laetozym 3000L ( Novo Nordisk A / S, Novozymes, Denmark). In addition, mold-based lak-5 re-formulations are available, including e.g. GLL-Conc. (Biocon Japan Ltd). Optimal hydrolysis conditions for the enzymes may vary depending on the preparation and are selected according to the manufacturer of the enzyme used.

Lactase is used in the dairy industry for the manufacture of low-lactose and lactose-free products. The milk to be used as the starting material may be pre-treated using various chromatographic or membrane techniques, followed by the addition of lactase (residue) to remove lactose. Such a method for producing lactose-free milk is described in Fi patent 115752, Valio Oy. Valio Oy, a process for preparing therapeutically useful peptides described in EP 1 266 267 B1, describes one alternative use of lactase to cleave lactose prior to the nanofiltration step. The addition of lactase during the fermentation step has not been described in the literature for the preparation of antihypertensive peptide products.

In the fermentation of the invention, any product containing the sequences of the desired therapeutically useful peptides as part of its own peptide or protein sequence can be used as starting material. Preferably the milk protein, especially casein, is used as such or in the form of various preparations. Also suitable are various dairy products containing casein, such as skimmed or semi-skimmed milk as such or in the form of powdered milk and acidified dairy products such as milk, buttermilk, yoghurt, filet, cream cheese, etc. Animal milk may also be used as a parent. and products derived therefrom, for example, soy, wheat or oat milk or proteins derived therefrom.

The fermentation can be carried out with any lactic acid bacterium capable of producing the desired therapeutically useful peptides from the starting material. Suitable lactic acid bacteria can be found e.g. Lactobacillus, Laciococcus, Leuconostoc, Streptococcus and Bifidobacterium. The most lactolactic bacterial proteolytic is Lactobacillus helveticus and is therefore considered particularly suitable for this purpose. Lactic acid bacteria can be used as pure inoculum or mixed cultures, alone or with conventionally used and commercially available acids. Lactic acid bacteria can also be used in combination with other microorganisms. For microbial compound formulations, combinations that provide the best taste to the final product and which eliminate the risk of contamination are appropriately selected.

A preferred Lactobacillus helvetl · cus strain according to the present invention is L. helveticus IB 1936, DSM 17754.

The classification and general characteristics of the bacteria belonging to the genus Lactobacillus and the species Lactobacillus helveticus are presented, inter alia, in Bergey's 10 Manual of Systematic Bacteriology, vol. 2, Sneath et al., Williams & Wilkins, Baltimore, London, Los Angeles, Sydney, 1984, Vol. 14, p. 1208 forth. Lactobacillus helveticus strains are generally isolated from dairy products, such as fermented milk products and cheeses, and are traditionally used as inhibitors in the manufacture of cheeses, particularly Emmental and Gruyere cheeses.

Lactobacillus helveticus LB 1936 is deposited with the Deutsche Sammlung von Mikroorganismen und Zeilkulturen GmbH (DSMZ). It has a strain number of DSM 17754 and a deposit date of November 25, 2005, and has the following characteristics: 20 gram-positive strain of Lactobacillus helveticus, morphologically a short rod. The temperature optimum is about 35-45 ° C, preferably about 37-42 ° C, and the pH optimum is about 4.5-7. The strain is proteolytically active, highly tolerant to acidity and forms DL-lactic acid during fermentation. As a carbon source, it can use e.g. galactose, glucose, fructose, man-25 noose, N-acetylglucoseamine, milk, lactose and trehalose.

Lactobacillus helveticus LB 1936 grows well in lactobacilli commonly used media, such as Rogosa and MRS broth, and in milk, such as untreated milk, reconstituted milk powder or ultrasonically treated milk, and can be produced by growth such as

Lactobacillus helveticus LB 1936 may be used as a health active ingredient in the form of selenium, in the manufacture of ingestible substances or as an ingredient or adjuvant in ingestible substances. It is preferably used in the manufacture of specialized products such as fermented milk products containing bioactive peptides, but is also useful as a conventional acid bacterium in the dairy industry, for example in the manufacture of fermented milk products and cheeses.

According to the present invention, the fermentation is carried out in the presence of lactase. Lactase is added to hydrolyze lactose to glucose and galactose. Monosaccharides, and glucose in particular, are faster and more efficient in fermentation as the carbon source of microorganisms than disaccharide, so hydrolysis of lactose significantly accelerates fermentation.

The advantage of the invention is greatest when using a production strain which is able to produce therapeutically active peptides well from a protein source, but which slowly metabolizes lactose, but the yield is improved by other strains as a result of their lactase addition.

Commercial lactase formulations such as those described above can be used as lactase. In addition to the pure enzyme preparation, impure enzyme preparations and even a microorganism having a high lactase activity may be used as such or in the form of an extract or other combination thereof.

The amount of lactase is used in an amount that provides optimum lactose hydrolysis efficiency. For example, the amount may be from about 10 to about 10,000 microns (milk), preferably from about 50 microns to about 300 microns (milk). The amount used depends on the enzyme preparation selected and its activity. There is no harm in dosing excess, but for reasons of cost, the goal is to keep the amount as low as possible.

In a preferred embodiment of the invention, in addition to the lactase addition, pH is used in the fermentation. In normal fermentation, the pH is not adjusted, whereby, due to the lactic acid produced during the fermentation, it is clearly acidified, usually up to about 3-3.5. Within the framework of the present invention, it has been surprisingly found that pH control also has a significant effect on the yield of the desired therapeutically useful peptides. The pH of the Pi-30 at about 4.6-7, preferably at about 4.8-6, more preferably at about 5-5.5, and especially at 5.1-5.3, according to the present invention, is to ensure that casein is accessible to proteolytic enzymes and thus available for the production of peptides. Although flakes are always formed during fermentation, casein is not as precipitated as Alem-35 at pH, and due to its easy availability, the yield of the process is further improved. Adjustment of pH has been found to be particularly advantageous in the initial stages of fermentation and during the growth of the most efficient microorganism (s). Evenly adjusting the pH is considered a preferred embodiment, but the pH may also be intermittent. At the end of the fermentation, the pH adjustment can be stopped and the pH can be freely lowered without decreasing the yield. However, at that 5 step, the pH does not generally fall below 4.6.

Any suitable alkaline agent may be used to adjust the pH. Commonly used substances include e.g. KOH and NaOH, and may be present in a concentration of about 10-90%. Often about 50% KOH solution is used. The amount required to adjust the pH depends on other parameters of the fermentation. Control and adjustment of pH 10 and determination of the appropriate amount of control chemical to be used to maintain the desired pH are within the skill of the art.

The fermentation conditions are selected according to the requirements of the strain used in the fermentation so that an optimal amount of therapeutically useful peptides is formed. The selection of suitable conditions, such as 15 growth temperature and aeration, is within the skill of the art. The temperature may be, for example, 30-45 ° C.

The fermentation is allowed to continue until the desired amount of peptides is formed. This usually takes about 20-30 hours, preferably 22-25 hours.

During fermentation, a mixture of different peptides is formed. The size of the peptides 20 is dependent on the length of the fermentation, the shorter the fermentation time, the longer the peptides formed, and the longer the fermentation, the more small peptides are formed. When fermentation is long enough, predominantly relatively small di- and tripeptides, such as Val-Pro-Pro (VPP) and lie-Pro-Pro (IPP), are found to have, e.g. 25 excellent antihypertensive effect.

After fermentation, the cell suspension is collected. It is contemplated that the cell suspension may be further treated as desired, for example, by removing any residual casein or all milk proteins by a purification method known in the art, such as precipitation, filtration, or well, or by heat treatment. Co-precipitation to obtain a nearly protein-free, peptide-containing whey can be effected, for example, by addition of CaGl 2, heat treatment or acid addition. Centrifuge treatment may be used if necessary. The cell suspension may also be treated or finalized by nanofiltration, ultrafiltration, or other membrane techniques or chromatography. Peptides can be separated and purified therefrom, for example, by chromatography or membrane techniques.

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The moss suspension can also be used directly or concentrated, for example, by evaporation or partial or total drying, such as applying to a plate, drying and finally grinding to obtain a well-preserved dry powder.

Within the scope of the present invention, a process has been found to be advantageous for the final treatment, wherein the casein precipitate after fermentation is separated by a separator and the whey is clarified by a clarifying centrifuge. The whey is then nano-filtered and the retentate is evaporated to a concentrate. The retentate can also be evaporated and dried as a spray powder.

Thus, the product containing the bioactive peptides obtained by the process of the invention and possibly concentrated, can be used as such as a health active ingredient. The dried product may also be used in the form of a powder or lyophilized preparation. However, in accordance with the present invention, the product is preferably used in the manufacture of health-promoting foods or other products.

The term food has a broad meaning in this publication, encompassing all products intended for ingestion, which may be in solid, gelatinized or liquid form and encompassing both finished products and products to which the product of the invention is added upon ingestion, as an additive or as part of a product. Foodstuffs may be, for example, products of the dairy industry, the pulp industry, the food industry, the beverage industry, the bakery industry and the confectionery industry. Typical are dairy products such as yogurt, frankfurters, quark, buttermilk, buttermilk, other sour milk drinks, cream cheese and matured cheeses, snack fillings, etc. Beverages such as whey drinks, fruit drinks, and beers form another important category.

The product containing the bioactive peptides may be added to the food during the manufacturing process or to the food already prepared. The foodstuffs in question thus contain, in addition to the ingredients contained in the bioactive peptide product of the invention or isolated and / or purified bioactive peptides, ingredients which are similar in taste and usage to those conventional products.

When the final product is a dairy product, it can be pre-packaged by heat treatment, for example by pasteurization at {72 °, 35 for 15 s), ESL (130, for 1-2 seconds) or UHT (138, 2 for 2). 4 s).

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The process according to the invention provides many advantages. Compared to the methods described in the prior art, the yield is significantly improved. The method of the invention has yielded peptides in whey up to 136 mg / l, yielding about 46% of the theoretical yield. For comparison 5, it can be mentioned that the yield using the conventional acidification method is about 24 mg / L. By adding lactase to the culture medium of microorganisms, the hydrolysis of lactose is accelerated, which in turn greatly accelerates the fermentation. Unexpectedly, by adding lactase, poor production strains can also be transformed into highly productive strains, and thus the yield of therapeutically useful peptides is further improved.

The process of the invention can also ensure that casein is continuously available to the producing organism (s), whereby the starting material can release the bioactive peptides much more efficiently than before, up to 3 times more efficiently. Again, this significantly improves the efficiency of the manufacturing process.

Furthermore, in the process according to the invention even a completely lactose-free product can be prepared. By adding lactase and adjusting the pH, the advantage is obtained that all lactose in the culture medium is fermented to the potassium salt of lactic acid (potassium lactate).

The process according to the invention is simple and inexpensive and is suitable for very large scale production.

The invention will be described in more detail by the following examples. These examples are provided for purposes of illustration only and are not to be construed as limiting the scope thereof.

25 Comparative Example 1

Traditional fermentation

Freeze-dried LB 1936 Acid Protein Titer using sterile whey permeate-based medium supplemented with tryptone and yeast extract as well as minerals. The fermentation was carried out at 37 ° C and pH 5.0 with stirring at 100-120 rpm. The growth time was 22 h. After fermentation, the cells were concentrated by microfiltration followed by concentration of the concentrate into Syophilisol.

Conventional fermentation was performed on LB 1936 using non-fat milk medium as starting material. 4 liters of milk were pasteurized at 85 ° C for 10 min 35 and cooled to a fermentation temperature of 42 ° C. The medium was inoculated with LB 1936 acid so that the concentration of living cells at the start of acidification was about 11 1.60 * 10 ° CFU / g. The milk was allowed to acidify at 42 ° C for 24 hours, during which the medium was agitated at 200 rpm. Figure 1 shows the pH curve during acidification. Whey was separated from the prepared milk by centrifugation at 4000 rpm for 15 min and analyzed for VPP and IPP-5 peptides, lactose, glucose, gaffactose and lactic acid.

In the butter prepared according to Comparative Example, the pH dropped from 6.5 to 3.7 as in Figure 1. At the end of acidification in milk, the concentration of live lactic acid bacteria was 1.9 * 10 9 CFU / g. Hera has a VPP + IPP content of 29 rng / l. At the end of acidification, the chemical composition of the whey taken in the art was similar to that shown in Table 1.

Example 1

Fermentation lactase supplementation

Freeze-dried LB 1936 acid was prepared using a sterilized whey permeate-based medium supplemented with trypton 15 and yeast extract as well as minerals. The fermentation was carried out at 37 ° C and pH 5.0 with stirring at 100-120 rpm. The growth time was 22 h. After fermentation, the cells were concentrated by microfiltration, followed by lyophilization.

The fermentation according to the invention was carried out on LB 1936 strain starting from skimmed milk medium. 4 liters of milk were pasteurized at 85 ° C for 10 min and cooled to a fermentation temperature of 42 ° C, 400 µL of GODO lactase enzyme was added to the Milk Set to hydrolyze lactose to glucose and galactose. The medium was inoculated with 60 mg of freeze-dried LB 1936 yeast (Valio Oy). Thus, the concentration of living cells at the start of the fermentation was 1.65 * 10 6 CFU / g. The milk was allowed to acidify at 42 ° C for 24 hours. During acidification, the medium was agitated at 200 rpm. Figure 2 shows the pH curve during acidification. Whey was separated from the prepared milk by centrifugation at 4000 rpm for 15 min and analyzed for VPP and IPP peptides, lactose, glucose, galactose and lactic acid.

In the butter prepared according to Example 1, the pH dropped from 6.5 to 3.7 as shown in Figure 2. At the end of acidification in milk, the concentration of live lactic acid bacteria was 1.9 * 10 9 CFU / g. The VPP + IPP concentration of whey was 40 mg / l. The chemical composition of the whey recovered at the end of acidification is shown in Table 1.

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Table 1,. Chemical composition of whey after fermentation iak-re-addition sieve Sample__Lactose [%] Glucose [% j Glucose f% 1 Lactic acid j '%]

Hera 24h 0.22 [0.80 2.04 12.16]

Example 2 Fermentation Using Lactase and pH Adjustment LB 1936 acid was prepared by silencing 1% of the production vial with 10 ml of sterilized 9% skimmed milk powder and growing at 42 ° C for 17 h. The strain was repeated twice, always using 1% inoculum. . The final fermentation inoculum was prepared at 110 ° C for 10 min in heat-treated 9% skim milk by growth for 22 h and at 37 ° C.

Fermentation with LB 1936 was carried out on non-fat milk medium. 4 liters of milk were pasteurized at 85 ° C for 10 min and cooled to fermentation temperature (42 ° C). 400 μL of GODO lactase enzyme was added to the milk. 400 ml LB 1936 acid was inoculated into the Kas-15 medium with non-fat milk medium (Valio). The milk was allowed to acidify at 42 ° C for 24 hours with the pH maintained at 5.0 for the first 20 hours by the addition of 50% KOH solution. The pH was then freely lowered. During acidification, the medium was agitated at 200 rpm. Figure 3 shows the pH curve during acidification. Whey was centrifuged at 4000 rpm for 15 min from the prepared milk and analyzed for VPP and IPP peptides, lactose, glucose, galactose and lactic acid.

In the butter prepared according to Example 2, the pH curve was as in Figure 3 and the end pH was 4.95. At the end of the acidification in the milk, the concentration of live maize-25 acid was 1.3 * 10 9 CFU / g. The whey prepared according to Example 2 had a VPP + IPP concentration of 120 mg / l. The chemical composition of whey is shown in Table 2.

Table 2. Chemical composition of whey after fermentation by lactase up-regulation with pH control Sample__Lactose f% 1 Glucose [%] Gaucose i%] Lactic acid [%]

Hera 24 h lo, QQ 0.00 | o, 01 4.16 30 13

Example 3

Femnation using Lactase and pH Adjustment LB 1936 Acidate: Prepared by inoculating a 1% production strain with 10 ml of sterilized 9% skimmed milk powder and raising at 5 ° C for 17 h. The strain was repeated twice, always using 1% seed size. The final fermentation inoculum was prepared: at 110 ° C for 10 min in a heat-treated 9% skimmed milk by incubation for 22 h and at 37 ° C.

Fermentation with LB 1936 strain was performed on non-fat milk meal 10. 4 liters of milk were pasteurized at 85 ° C for 10 min and cooled to an acidification temperature (42 ° C). 400 µg of GODG lactase enzyme was added to the milk. The medium was inoculated with 400 ml of LB 1936 acid on a non-greasy medium (Valio). The milk was allowed to acidify at 42 ° C for 24 hours with the pH being maintained at 5.2 for the first 20 hours by the addition of a 50% solution of 15 KOH. The pH was then freely lowered. During acid turbulence, the medium was agitated at 200 rpm. Figure 4 shows the pH curve during acid turbulence. Whey was centrifuged at 4000 rpm for 15 min from the prepared milk and analyzed for VPP and IPP peptides, lactose, glucose, galactose and lactic acid.

In the butter prepared according to Example 3, the pH curve was as in Figure 4 and the end pH was 5.15. The concentration of live lactic acid bacteria in the diatomaceous earth was 2.0 x 10 9 CFU / g. The whey VPP + IFP content of the whey prepared according to Example 3 was 136 mg / L. The chemical composition of the whey is shown in Table 3.

25 Table 3. Chemical composition of whey after fermentation by addition of lactate and pH adjustment Sample Lactose 1% 1 Glucose f%] Glycose [%] Lactic acid f% l Hera 24 h 0, QQ 10.00 0.01 13.33

Example 4

Fermentation using pH adjustment without lactase addition 30 LB 1936 acid was prepared by inoculating a 1% production strain from an ampoule at 10 m! sterilized 9% skimmed milk powder and incubated at 42 ° C for 17 h. The strain was repeated twice in the same manner, 14 always using 1% inoculum. The final fermentation inoculum was prepared at 110 ° C for 10 min in heat-treated 9% skimmed milk by growing for 22 h and at 37 ° C.

Fermentation of LB 1936 strains was performed on non-fat milk medium as described in the example above except that no lactase was added to the medium. Figure 5 shows the pH-clade during acidification. The whey was separated from the prepared milk by centrifugation at 4000 rpm for 15 min and the whey was analyzed for VPP and (PR peptides, lactose, glucose, galactose and lactic acid.

In the butter prepared according to Example 4, the pH curve was as in Figure 5 and the final pH was 4.59. At the end of the acidification in the milk, the concentration of live lactic acid bacteria was 2.0 * 10 9 CFU / g. The whey prepared according to the example had a VPP-HPF content of 86 mg / L

Table 4. Chemical composition of whey after fermentation pH adjusted without addition of lactase Sample__Lactose [%] Glucose [%] Glucose [%] Lactic acid [%]

Hera 24h: 0.72 0.00 0.1 3.51 15

Example 5

Use of the product to make the final product

The diatomaceous earth prepared according to Example 2 was separated and the resulting whey was nanofiltered. The nanofiltered retentate was evaporated. The tag-like concentrate was used to prepare milk containing bioactive peptides by adding n, 1% concentrate to the milk.

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Claims (11)

A process for the preparation of a therapeutically useful peptide-containing product, in particular antihypertensives, with a lactic acid bacterium, characterized in that the lactic acid bacterium is a therapeutically useful peptide-producing lactic acid bacterium, and in particular an anti-hypertensive agent. Method according to Claim 1, characterized in that the source of the milk protein is milk or milk-based product. Method according to claim 2, characterized in that the source of the milk protein is casein. Process according to one of Claims 1 to 3, characterized in that a mixture of lactic acid bacteria and other microbes is used in the fermentation. Process according to any one of claims 1 to 4, characterized in that the fermentation process uses the microorganism Lactobacillus helveticus. A method according to claim 5, characterized in that the microorganism is Lactobacillus helveticus strain LB 1936, DSM 17754. Process according to one of Claims 1 to 6, characterized in that the fermentation is carried out by adjusting the pH. Process according to claim 7, characterized in that the pH is maintained at about 4.6-7, preferably at about 4.8-6, more preferably at about 5-5.5 and most preferably at about 5.1-5 , 3. Process according to one of Claims 1 to 8, characterized in that the fermentation produces a mixture of short-chain peptides. Method according to claim 9, characterized in that the peptides are di- and tripeptides, in particular Ile-Pro-Pro and / or Val-Pro-Pro. 11. Lactobacillus helveticus strain LB 1936, DSM 17754.