NEW ISOLATED PEPTIDES, THE PROTEIN HYDROLYSATE CONTAINING THESE ISOLATED PEPTIDES AND USE OF THEM

Abstract

The invention relates to an isolated peptide with an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19); Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20);and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21), a protein hydrolysate comprising the isolated peptides, and its use as a flavor modulating agent in foods and pharmaceutical preparations.;

Description

TECHNICAL FIELD

[0001] The present invention relates to novel isolated peptides, a protein hydrolysate containing said isolated peptides, for use as a taste modulating agent in food and pharmaceutical preparations. BACKGROUND OF THE INVENTION

[0002] The blood of farm animals and poultry is a byproduct obtained from industrial slaughter processes. A large amount of farm animal and poultry blood, such as chicken blood, is produced each year and discarded in many countries around the world despite its valuable proteins and nutrient bioavailability. Therefore, the utilization of this blood is beneficial from both an economic and environmental perspective. To date, the use of farm animal and poultry blood has been limited, regardless of cost-effectiveness or feeding objectives, as the use of whole blood and proteins in the food industry is restricted due to their unpleasant taste or color. To address these problems, some studies have been conducted on the recycling of by-products and waste materials obtained from poultry processing and industry. For example, chicken blood is used to make black pudding, blood tofu, and blood pudding.Blood plasma could be used as a protein supplement, which is an excellent source of trace minerals, or could be used as a substitute for egg white in the baking industry due to its high foaming capacity (Jayathilakan et al., J Food Sci Technol, 2012, 49(3), 378-293).

[0003] To increase the use of chicken blood, the production of taste-active and taste-modulating peptides is proposed. However, existing knowledge about the composition of chicken blood, such as which proteins are present or the exact concentrations of the various components, is limited. Furthermore, no study is available addressing the taste of chicken blood, although many studies have been conducted to identify the taste-active and taste-modulating peptides in all types of food. Due to this limited knowledge and the complex matrix of chicken blood, the identification of taste-active and taste-modulating peptides from farm animal and poultry blood, such as chicken blood, is challenging.

[0004] CN 108047313 A discloses an antioxidant peptide from chicken blood cells and a process for its production by fermentation. The antioxidant peptide has the amino acid sequence Thr-Ser-Phe-Gly-Asp-Ala-Val-Lys-Asn-Leu-Asp-Asn-Ile-Lys (SEQ ID NO: 1). This antioxidant peptide has high radical scavenging activity, high DPPH free radical scavenging activity, and high reducing power. The process comprises the steps of culture activation, preparation of a blood cell medium, inoculation fermentation, separation, and purification.

[0005] CN 108048518 A discloses an antioxidant peptide from chicken blood cells and a process for its production by enzymolysis. The antioxidant peptide has the amino acid sequence Met-Gly-Gln-Lys-Asp-Ser-Tyr-Val-Gly-Asp-Glu-Ala-Gln-Ser-Lys-Arg-Gly-Ile-Leu-Thr (SEQ ID NO: 1), or Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln (SEQ ID NO: 2). These antioxidant peptides have high free radical scavenging activity, high DPPH free radical scavenging activity, superoxide anion free radical scavenging activity, and high reducing capacity. The process comprises the steps of blood cell preparation, hemolysis, proteolysis, and ultrafiltration.

[0006] CN 101843289 A discloses a process for producing blood polypeptide protein powder from livestock and poultry blood. In this process, the high-molecular-weight albumin, which is difficult to absorb and utilize in livestock and poultry blood, is digested into low-molecular-weight albumin, polypeptides, and free amino acids to improve the palatability and nutritional value of dried blood for animal feeding.

[0007] Despite the above-mentioned developments of products and methods for the utilization of farm animal and poultry blood, there is no disclosure of the use of farm animal and poultry blood, in particular peptides derived from farm animal and poultry blood with a specific amino acid sequence, as flavor-active and flavor-modulating agents. SUMMARY OF THE INVENTION

[0008] The present invention has been developed in view of the above-mentioned problems.

[0009] Therefore, it is an object of the present invention to provide the utilization of animal blood, in particular farm animal and poultry blood, which is an industrial waste from the slaughter process, to obtain novel taste-active peptides and a mixture thereof for a taste-modulating agent in food and pharmaceutical preparations.

[0010] It is a further object of the present invention to provide animal blood-derived peptides, particularly peptides derived from farm animal and poultry blood, with specific amino acid sequences. The animal blood-derived peptides of the present invention can be used as flavor-active agents that impart taste-modulating effects, such as salt-reducing, salt-enhancing, umami, and kokumi effects.

[0011] Yet another object of the present invention is to provide a protein hydrolysate and a taste modulating composition containing said animal blood-derived peptides.

[0012] An additional object of the present invention is to provide a method for modulating the taste of food and pharmaceutical preparations using said animal blood-derived peptides, protein hydrolysate and taste-modulating composition.

[0013] In one aspect, the present invention relates to an isolated peptide having an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19); Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20);and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21).;

[0014] In another aspect, the present invention relates to a protein hydrolysate comprising at least one peptide having an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19);Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20); Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21); and a combination thereof.;

[0015] In a further aspect, the present invention relates to a taste-modulating composition comprising said isolated peptides or the protein hydrolysate defined above.

[0016] In yet another aspect, the present invention relates to a food or pharmaceutical preparation comprising said isolated peptides, protein hydrolysate or taste-modulating composition as defined above.

[0017] Furthermore, the present invention relates to a method for adjusting the taste of a food or a pharmaceutical preparation, comprising the application of an effective amount of said isolated peptides, protein hydrolysate or taste-modulating composition as defined hereinbefore in the food or pharmaceutical preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The features of the present invention will become more apparent upon reading the following description of an exemplary embodiment. This example is provided for illustrative purposes and is not limiting. Fig.Figure 1 shows the sensory profiles of: (a) low molecular weight (LMW) fractions of hydrolyzed chicken blood cells in a model broth (MB) at a concentration of 10 g / L; and (b) LMW fractions of hydrolyzed chicken plasma in model broth at a concentration of 10 g / L. Fig. Figure 2 shows an RP18 MPLC ELSD chromatogram and a comparative taste dilution analysis of MPLC fractions F2-F7 in water (a); in model broth (MB) (b); and in water or model broth (c). DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise stated, any aspects shown herein are intended to include application to other aspects of the present invention as well.

[0020] Unless otherwise specified, the technical and scientific terms used herein have the definitions as understood by a person skilled in the art.

[0021] Throughout this document, the term "about" is used to indicate that any values ​​shown or illustrated herein may vary or deviate. Such variation or deviation may be the result of error in the equipment or methods used to determine the values.

[0022] The expressions "consists of" and their variations, such as "consisting of" and "consisted of," "comprises" and their variations, such as "comprising" and "comprised," "has," "includes" and their variations, such as "including" and "included," are open-ended verbs. For example, any method that "consists of," "comprises," "has," or "includes" one or more components or steps is not limited to just the one or more components or steps, but may also cover unstated components or steps.

[0023] The terms “a”, “an”, “the”, when used in reference to a singular form are, unless otherwise specified, meant to include the plural of that noun.

[0024] The term "taste modulating" as used herein is synonymous with the terms "taste-enhancing," "taste-adjusting," "taste-reducing," "taste-enhancing," and "taste-modifying," which may be used interchangeably. According to the present invention, the terms "taste-modulating agent" and "taste-modulating composition" refer to an agent and a composition capable of imparting one or more flavors, e.g., salty, sweet, kokumi, umami, astringent, bitter, sour, etc., to a food or pharmaceutical preparation, and capable of modifying the taste of the food or pharmaceutical preparation as desired. Furthermore, taste adjustment can be achieved by reducing or enhancing one or more of these flavors.

[0025] The term "food preparation" as used herein refers to all edible food and beverage products, compositions, and formulations for humans and animals, including, for example, meals, beverages, dietary supplements, snacks, desserts, and sweets. Similarly, the term "pharmaceutical preparation" as used herein refers to all edible pharmaceutical products, compositions, and formulations. The food and pharmaceutical preparations may be in the form of, for example, solids, e.g., powders, tablets, granules, spheres, capsules, etc., semi-solids, gels, and liquids.

[0026] Unless otherwise stated, all tools, equipment, methods, materials, or chemicals mentioned herein mean the tools, equipment, methods, materials, or chemicals commonly used or practiced by a person skilled in the art.

[0027] All disclosed chemicals, compounds, materials, components and / or methods and the claims of the present invention are intended to cover the aspects of the invention obtained by any acts, practices, modifications or changes performed on the factors without performing an experiment substantially departing from the present invention, and an object having properties, benefits and effects similar to the aspects of the present invention in the opinion of a person of ordinary skill in the art is obtained, even if not expressly recited in the claims.Therefore, the subject matter equivalent or similar to the aspects of the present invention, including any minor modification or change obvious to a person of ordinary skill in the art, should also be considered to be within the spirit, scope and concept of the present invention.

[0028] The present invention will be described in more detail below.

[0029] According to the first aspect of the present invention, the isolated peptides have an amino acid sequence selected from the group comprising: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19); Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20);and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21).;

[0030] The isolated peptides of the present invention can mediate at least one of the following effects: salt-reducing, salt-enhancing, umami and kokumi effects.

[0031] In a preferred aspect, the isolated peptides conferring the salt-reducing effect have an amino acid sequence selected from the group comprising: Pro-Val-Leu-Lys (SEQ ID NO: 1); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); and Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13).

[0032] In a further preferred aspect, the isolated peptides conferring the salt-enhancing effect have an amino acid sequence selected from the group comprising: Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Val-Ala-Glu (SEQ ID NO: 16); and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21).

[0033] According to a further preferred aspect, the isolated peptides conferring the umami effect have an amino acid sequence selected from the group comprising: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19); Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20); and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21).

[0034] In yet another preferred aspect, the isolated peptides conferring the Kokumi effect have an amino acid sequence selected from the group comprising: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19); Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20); and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21).

[0035] The isolated peptides of the present invention are obtained or derived by enzymatic hydrolysis or fermentation. Specifically, the isolated peptides are obtained or derived by the enzymatic hydrolysis of animal blood, including, but not limited to, pigs, cows, and poultry, such as chickens, birds, turkeys, etc. In a preferred aspect, the animal blood useful in the present invention is poultry blood, particularly preferably chicken blood.

[0036] In an exemplary embodiment, the enzymatic hydrolysis to obtain the isolated peptides of the present invention is performed using serine protease. Preferably, the serine protease is subtilisin.

[0037] The present invention also provides the use of the isolated peptides as defined above as a taste modulating agent for a food or pharmaceutical preparation.

[0038] The second aspect of the present invention relates to a protein hydrolysate which contains at least one peptide having an amino acid sequence selected from the group comprising: Pro-Val-Leu-Lys (SEQ ID NO: 1); Pro-Leu-Pro-Arg (SEQ ID NO: 2); Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4); Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7); Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8); Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9); Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12); Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); Pro-Thr-Asp-Leu (SEQ ID NO: 15); Pro-Val-Ala-Glu (SEQ ID NO: 16); Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17); Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18); Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19);Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20); Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21); and a combination thereof.;

[0039] In a preferred embodiment, the protein hydrolysate of the present invention comprises at least the peptides having the amino acid sequence of: Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3); Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6); Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11); Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14); and Pro-Thr-Asp-Leu (SEQ ID NO: 15).

[0040] The protein hydrolysate of the present invention can be obtained or derived by enzymatic hydrolysis. Specifically, the protein hydrolysate is obtained by the enzymatic hydrolysis of animal blood, including, but not limited to, that of pigs, cows, and poultry, such as chickens, birds, turkeys, etc. In a preferred aspect, the animal blood is poultry blood. It is further preferred that the poultry blood is chicken blood.

[0041] The enzymatic hydrolysis to obtain the protein hydrolysate of the present invention is carried out using serine protease. Preferably, the serine protease is subtilisin.

[0042] The invention also provides the use of the protein hydrolysate defined hereinabove as a taste modulating agent for a food or pharmaceutical preparation.

[0043] The third aspect of the present invention relates to a taste-modulating composition comprising said isolated peptides or the protein hydrolysate defined above. The invention also provides the use of the taste-modulating composition in a food or pharmaceutical preparation. Therefore, the present invention provides a food or pharmaceutical preparation comprising the isolated peptides, the protein hydrolysate, or the taste-modulating composition as defined above.

[0044] In addition, the present invention provides a method for adjusting the taste of a food preparation, comprising applying an effective amount of said isolated peptides, the protein hydrolysate or the taste-modulating composition containing an effective amount of the isolated peptide to the food or pharmaceutical preparation. Example

[0045] The present invention will now be described in more detail by the following non-limiting examples. Example 1: Preparation and identification of peptides1. Preparation of peptides by enzymatic digestion

[0046] All samples were prepared using chicken blood from a slaughterhouse. First, the chicken blood was centrifuged at 4000 rpm for 8 minutes and then digested with the enzyme subtilisin (Protin SD-AY10) using 2M NaOH to adjust the initial pH to 9. The enzyme-digested samples were incubated at 55°C for 18 hours at 120 rpm. Afterward, enzyme activity was stopped by heating in a water bath at 100°C for 15 minutes and cooling to ambient temperature. The resulting solutions were freeze-dried and stored at -20°C until further use. 2. Fractionation of the peptide mixture through ultrafiltration membrane

[0047] 5 g of the chicken blood plasma powder was dissolved in 300 ml of deionized water in an ultrasonic bath. The resulting solution was transferred to an ultrafiltration system (8400 Millipore Amicon, EMD Millipore) equipped with a separation membrane of 1, 3, or 5 kDa (Ultacel® The membrane was filtered with a 76 mm diameter chemically regenerated cellulose (RC) filter (EMD Millipore Corporation, Billerica, USA). The high molecular weight (HMW) and low molecular weight (LMW) fractions from each separation were washed with 50 mL of deionized water, freeze-dried, and stored at -20 °C until further analysis. The yields of the different membrane separations are summarized in Table 1 and underline that the LMW fractions from each separation had the highest compound contents. Table 1 Separation membrane sample Yield (%) 1 kDa LMW blood cell 60,5 HMW blood cell 13,6 LMW plasma 75,2 HMW plasma 10,1 3 kDa LMW blood cell 61,1 HMW blood cell 34,7 LMW plasma 75,8 HMW plasma 20,1 5 kDa LMW blood cell 61,8 HMW blood cell 29,1 LMW plasma 78,4 HMW plasma 12,1

[0048] To determine which LMW fractions contain the highest levels of taste-active and taste-modulating peptides, prior to sensory evaluation, a sodium and chloride analysis was performed for all LMW fractions from blood cells and plasma, as well as liquid chromatography with tandem mass spectrometry (LC-MS / MS). Quantitative analysis by ion chromatography showed that chicken blood does not contain high levels of sodium and chloride, as none of the samples contained more than 10% sodium and chloride (Table 2). The plasma samples contained more sodium and chloride than blood cells, and the sodium and chloride levels of 1 kDa LMW plasma were the highest among all samples. Table 2 sample Sodium content (%) Chloride content (%) 1 kDa LMW blood cell 1,5 0,9 1 kDa LMW plasma 5,1 6,4 3 kDa LMW blood cell 1,1 0,5 3 kDa LMW plasma 4,2 6,7 5 kDa LMW blood cell 1,5 0,3 5 kDa LMW plasma 4,3 6,2

[0049] The sensory evaluation was designed to obtain a clear and comprehensive understanding of the taste characteristics of the samples. The HMW fraction was not used for sensory experiments for safety reasons. The sensory samples were prepared by dissolving each LMW fraction of blood cell and plasma in model broth to a final concentration of 10 g / L and adjusting the pH to 6 by adding 1% formic acid in water. The subjects were asked to rate six taste qualities, including umami, kokumi, sweet, salty, sour, and bitter, on a scale of 0 (not recognizable) to 5 (strongly recognizable), as described in Fig. 1(a) and (b).

[0050] The experimental results showed that LMW of the 3 kDa hydrolyzed chicken blood cell fractions exhibited significantly (P < 0.01) more taste-active and taste-modulating properties than 1 kDa with respect to kokumi (3 kDa: 1.6), sweetness (3 kDa: 0.6), and bitterness (3 kDa: 2.0). Saltiness and sweetness were opposite. Umami enhancement was comparable for both 1 kDa and 3 kDa fractions. Additionally, the LMW fractions of the 3 kDa and 5 kDa fractions were compared using duo-trio tests for both blood cells and plasma. The sensory test was conducted in two series, AAB and BBA (the order was random). Subjects were asked to select the sample that differed from the other two samples in each series.The duo-trio test comparing the 3 kDa LMW fractions of hydrolyzed blood cells and 3 kDa plasma was designed to select the sample with a more pronounced taste compared to the other two samples. According to the experimental results, there was no significant difference (P = 0.1), suggesting that peptides with more than 3 kDa do not exhibit significant taste-activating and taste-modulating activity. Compared to the LMW fraction of blood cells, 3 kDa LMW plasma exhibited more pronounced sensations of umami, kokumi, and saltiness. Therefore, the 3 kDa separated LMW plasma fraction was selected for further investigation. 3. Fractionation of 3 kDa LMW plasma by medium-pressure liquid chromatography (MPLC)

[0051] 2 g of the 3 kDa LMW plasma powder was dissolved in 20 mL of deionized water and fractionated by reversed-phase (RP) MPLC (Büchi, Flawil, Switzerland) on a 150 × 40 mm polypropylene cartridge using 25-40 µm LiChroPrep RP18 bulk (Merck, Darmstadt, Germany) as the stationary phase and a gradient of 1% aqueous formic acid (solvent A) and methanol (solvent B) as the mobile phase. The separation was monitored using an evaporative light scattering detector (ELSD) (SEDERE, Alfortville Cedex, France), and chromatography (flow rate of 40 mL / min) was performed with 100% solvent A for 10 min, followed by increasing solvent B to 30% within 15 min and to 100% within another 15 min, followed by elution with 100% solvent B for 15 min.Finally, seven fractions (F1-F7) were collected, separated from the solvent under vacuum, and freeze-dried twice before performing taste dilution analysis (TDA), comparative taste dilution analysis (cTDA), and chemical analysis ( ). Fig. 2(a), (b) and (c)).

[0052] TDA was performed to identify the most intense taste-active fraction, and cTDA was performed to identify the taste-modulating fraction. For TDA, serial 1:2 dilutions of each fraction or subfraction were prepared in water and then presented to sensory participants in order of increasing concentrations. Each dilution was sensorially evaluated using the duo-trio test. The dilution at which a taste difference could just be detected between the diluted fraction and two blank samples (water) was defined as the taste dilution (TD) factor. The TD factor, assessed by five different participants, was averaged. The TD factor between individuals and three separate sessions differed by no more than one dilution step. Eight dilution steps were performed (Oliver Frank, Jezussek, & Hofmann, 2003).For cTDA, the procedure was the same as for TDA, except that the fractions were dissolved in model broth, and the model broth was used as a blank (Dunkel et al., 2007; Salger et al., 2019). The comparative taste dilution (cTD) factor is defined as the dilution at which a taste difference could just be detected between the diluted fraction and two blank samples (model broth).

[0053] According to the TDA and cTDA results, fraction F3 exhibited the highest taste-active and taste-modulating properties. In water, fraction F2 exhibited the highest intrinsic umami taste with a TD factor of 16, followed by fraction F3 (TD factor = 8) and fraction F4 (TD factor = 4), as shown in Fig.2(a). For the salty taste, fractions F2 and F3 have identical TD factors of 4. Fraction F6 exhibited the highest bitter and astringent taste (TD factor = 4). In model broth, fraction F3 showed the highest umami-enhancing activity (cTD factor = 16), followed by fraction F2 (cTD factor = 8) and fraction F4 (cTD factor = 4), as shown in Fig. 2(b). The highest kokumi taste was found for fraction F4. None of the fractions elicited a bitter taste in model broth, which underlines the potential of fractions F2-F5 as flavor-enhancing fractions without any unpleasant taste of their own ( Fig. 2(c)). Since fractions F2, F3, and F4 had the highest TD and cTD factors, further analysis focused on these fractions. 4. Subfractionation of fractions F2, F3, and F4 by high performance liquid chromatography (HPLC)

[0054] To identify the taste-active and taste-modulating peptides in fractions F2, F3 and F4, these fractions were further separated by preparative HPLC using a gradient of 1% aqueous formic acid solution and acetonitrile.

[0055] To separate fraction F2, 10 g of fraction F2 was dissolved in 1 L of deionized water and membrane filtered (0.45 µm). 0.5 mL of this solution was injected into the HPLC system (Jasco, Groß-Umstadt, Germany). The HPLC separation was performed under the following conditions. column Luna HILIC (200 A, 250×21.2 mm, 5 µm) flow rate 18 mL / min detector ELSD and UV (254 nm)

[0056] To separate fraction F3, 10 g of fraction F3 was dissolved in 1 L of deionized water and membrane filtered (0.45 µm). 1 mL of this solution was injected into the HPLC system (Jasco, Groß-Umstadt, Germany). The HPLC separation was performed under the following conditions. column Luna PFP(2) (100 A, 250×21.2 mm, 5 µm) flow rate 21 mL / min detector ELSD and UV (254 nm)

[0057] To separate fraction F4, 20 g of fraction F4 was dissolved in 1 L of deionized water and membrane filtered (0.45 µm). 1 mL of this solution was injected into the HPLC system (Jasco, Groß-Umstadt, Germany). The HPLC separation was performed under the following conditions. column Nucleodur C18 pyramid (100 A, 250×21.2 mm, 5 µm) flow rate 20 mL / min detector ELSD and UV (254 nm)

[0058] The subfractions of F2, F3, and F4 were analyzed by liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) using a C8 column, and the peptides were identified using PEAKS software.

[0059] From the experimental results, fractions F3 and F4 were selected for large-scale separation to identify novel taste-active and taste-modulating peptides, since fraction F2 contained a high concentration of already known taste-active and taste-modulating molecules (e.g., cations, amino acids, and glutamyl dipeptides). Example 2: Identification and evaluation of the taste-active and taste-modulating effects of peptides1. Identification of the peptides

[0060] After analyzing the HPLC subfractions of MPLC fractions F2, F3, and F4, the potentially taste-active and taste-modulating peptides present in fractions F3 and F4 were identified using a combination of proteomics and sensomic techniques (the so-called sensoproteomics approach), adapted from the method presented by Sebald et al., J. Agric. Food Chem. 2018, 66, 11092-11104. Both targeted and untargeted proteomics were applied to improve the productivity of the present invention.

[0061] Using targeted proteomics methods, all theoretically possible peptides from chicken serum albumin and corticosteroid-binding globulin were screened, and 767 peptides were identified. Untargeted proteomics using Maxquant and PEAKS software was used to identify peptides in LC-TOF-MS data from subfractions of MPLC fractions F2, F3, and F4. All peptides identified in fractions F3 and F4 using de novo sequencing and a PEAKS database search with an ALC (average local confidence) > 80 and with an N-terminal E, P, or R were selected. Using Maxquant, taste-active and taste-modulating fractions were first screened, and then the identified peptides were sorted according to an N-terminal E, P, or R and peak areas. 11 peptides from fraction F3 and 44 peptides from fraction F4 were identified by MS 2 -Data further confirmed.

[0062] After peptide identification, followed by several filtering steps, peptide candidates were selected for validation. Final peptide candidates identified in MPLC fractions F3 and F4 are shown in Table 3 below. Table 3 fraction Peptide sequence RT (min) Production confidence identified by Area (PEAKS) or intensity (MaxQuant) ALC (PEAKS) or score (MaxQuant) F4 Pro-Val-Leu-Lys (SEQ ID NO: 1) 11,82 5 de novo 2,19×10 5 97 F4 Pro-Leu-Pro-Arg (SEQ ID NO:2) 9,07 5 de novo 1,73×10 4 92 F4 Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3) 4,65 5 database 2048,6 80,317 F3 and F4 Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4) 0.98(F3), 1.23(F4) 5 de novo 3,35×10 4 (F3), 3,57×10 4 (F4) 98 F3 Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5) 1,03 5 de novo 2,86×10 3 99 F4 Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6) 4,18 5 Database and de novo 1,49×10 5 95 F4 Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7) 10,979 5 de novo 1,84×10 4 98 F4 Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8) 4,18 4 de novo 7,13×10 4 80 F4 Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9) 1,23 4 de novo 1,33×10 4 87 F3 Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10) 1 4 Database and de novo 1,06×10 2 - F4 Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11) 4,18 5 de novo 1,49×10 5 94 F4 Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12) 1,23 5 Database and de novo 1,70×10 4 98 F4 Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13) 4,12 5 database 3787,1 108,73 F4 Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14) 2,08 5 database 32311 107,73 F4 Pro-Thr-Asp-Leu (SEQ ID NO: 15) 4,49 5 database 906800 115,49 F4 Pro-Val-Ala-Glu (SEQ ID NO: 16) 2,15 4 database 375570 109,97 F3 Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17) 0,87 4 de novo 3,77×10 3 99 F4 Arg-Ala-Glu-Asp-Thr-Ala- 13,5 5 database 5940 - Thr-Tyr-Tyr (SEQ ID NO: 18) F4 Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19) 3,2 4 database 64600 - F4 Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20) 1,23 4 de novo 1,45×10 4 87 F4 Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO:21) 5,92 4 de novo 3,44×10 4 80

[0063] The 21 peptide candidates were synthesized and their purities were determined by quantitative 1 H-NMR was evaluated in the range of 62% to 100%. The presence of the peptides in the samples was verified by LC-MS / MS using Skyline. By comparing the MRM transitions detected in MPLC fractions F3 or F4 with the synthesized peptides, it was confirmed that the peptide candidates existed in the hydrolyzed chicken plasma according to the present invention. 2. Determination of taste thresholds of peptides

[0064] The threshold concentrations of 21 newly identified peptides were determined in water for intrinsic taste and in model broth (pH 6.0, adjusted with 1% formic acid) for taste-modulating properties (Table 4). The peptides were dissolved, serially diluted 1:2, and presented to the panel using duo-trio tests with ascending concentrations, as described in the literature. The geometric mean of the last and penultimate concentrations was calculated and used as the individual detection threshold. The sensory panel threshold was approximated by averaging the individuals' thresholds in two independent sessions. Cp=Co×C1 where C p : the threshold of a subject is C1: the first correctly assessed concentration is C0: the first incorrectly assessed concentration is Cs=∏i=1nCpjn where C s : the thresholds of all subjects are, C p : the threshold of a subject is n: the number of committee members is Table 4 Peptide sequence Threshold value in model broth (µmol / L) Taste properties in model broth Threshold value in water (µmol / L) Taste properties in water Pro-Val-Leu-Lys (SEQ ID NO: 1) 296 kokumi, salty, umami, astringent tasteless - Pro-Leu-Pro-Arg (SEQ ID NO: 2) 112 umami, kokumi 296 astringent Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3) 95 kokumi, salty, umami 116 astringent, bitter Pro-Glu-Arg-Asn-Glu (SEQ ID NO:4) 231 umami, kokumi, sweet, bitter, sour 100 astringent, sweet Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5) 133 umami, kokumi, astringent 705 astringent, bitter Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6) 94 Mouthfeel, sweet, umami, astringent tasteless - Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7) 236 kokumi, umami, bitter, mouthfeel 565 astringent Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8) 351 kokumi, salty tasteless - Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9) 36 umami, kokumi 283 astringent, sour Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10) 34 umami, kokumi 233 bitter, astringent Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp 67 kokumi, umami, less salty 865 bitter (SEQ ID NO: 11) Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12) 137 kokumi, umami 907 bitter, umami Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13) 151 kokumi, salty 1024 bitter, astringent Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14) 26 umami, salty, mouth-filling tasteless - Pro-Thr-Asp-Leu (SEQ ID NO: 15) 97 kokumi, mouthfeel tasteless - Pro-Val-Ala-Glu (SEQ ID NO: 16) 243 kokumi, salty 688 astringent, sour Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17) 101 kokumi, umami tasteless - Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18) 247 kokumi, umami 610 sour, astringent Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19) 212 umami, kokumi tasteless - Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20) 61 umami, kokumi 243 astringent, bitter Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21) 96 umami, sweet, kokumi 894 sour, bitter

[0065] According to the results, the taste threshold concentrations ranged from 100 to 1024 µmol / L in water. Seven of the 21 peptides showed no intrinsic taste up to concentrations of 2 mmol / L (namely, the peptides with SEQ ID NOs: 1, 6, 8, 14, 15, 17, and 19).

[0066] Furthermore, all 21 peptides exhibited taste-modulating properties ranging from 26 µmol / L to 351 µmol / L in model broth. The peptide with SEQ ID NO: 14 has the lowest threshold for umami and kokumi at 26 µmol / L. The peptide with SEQ ID NO: 8 exhibits kokumi and salt-enhancing taste properties with the highest threshold of 351 µmol / L. The peptides with SEQ ID NO: 3, 7, and 20 exhibit kokumi, umami, and saltiness-modulating taste properties with a respective threshold of 236, 95, and 61 µmol / L. The peptide with SEQ ID NO: 12, 17, 18, 19 and 20 shows kokumi- and umami-enhancing properties with thresholds of 137, 101, 247, 212 and 61 µmol / L.

[0067] In addition, the peptides with SEQ ID No: 1, 3, 6, 8, 11, 13, 14, 16 and 21, which exhibited salt taste modulating properties, were selected for salt isointensity tests to evaluate their salt enhancing or reducing properties, as further described. 3. Salt isointensity test of peptides

[0068] To investigate the effects of the peptides on perceived saltiness, a salt isointensity test was conducted according to the literature. In the salt isointensity test, the peptides (1 mmol / L) were added to a model broth containing 50 mM NaCl, and the subjects were asked to rate the saltiness intensity compared to the following NaCl concentrations of 30, 40, 50, 60, 70, and 80 mmol / L. The results are shown in Table 5. Table 5 Peptide sequence average perceived salt isointensity (mmol / L NaCl) RSD (%) Increase or decrease (%) Pro-Val-Leu-Lys (SEQ ID NO: 1) 42,9 4,5 14 Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3) 43,0 6,9 14 Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6) 60,0 3,5 20 Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8) 66,7 5,8 33 Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11) 62,1 8,0 24 Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13) 43,8 4,1 13 Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14) 63,6 11,3 27 Pro-Val-Ala-Glu (SEQ ID NO: 16) 62,0 3,3 24 Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21) 59,8 3,6 20

[0069] The perceived salt isointensity of all peptides ranged from 42.9 to 66.7 mmol / L NaCl in model broth. The peptide with SEQ ID NOs: 6, 8, 11, 13, 14, 16, and 21 exhibited salt taste-enhancing properties, whereas the peptides with SEQ ID NOs: 1, 3, and 13 exhibited salt taste-reducing properties. The strongest salt enhancement effect was observed for the peptide with SEQ ID NO: 8 with 33% salt enhancement (from 50 to 66.7 mmol / L perceived salt isointensity), and the smallest salt enhancement effect was observed for the peptide with SEQ ID NO: 21 with 20% salt enhancement (from 50 to 59.8 mmol / L perceived salt isointensity). Furthermore, the salt-reducing effects were observed for the peptide with SEQ ID NO: 1, 3 and 13 with 14% and 13%, respectively. 4. Identification of the essential taste-active and taste-modulating peptides

[0070] To identify those peptides that play a key role in taste-active and taste-modulating effects, not only the 21 newly identified peptides but also basic taste compounds known from the literature (e.g., amino acids, γ-glutamyl peptides, cations, anions, etc.) were quantified in the hydrolyzed chicken plasma sample. Focusing on the 21 newly identified peptides, the taste quality, taste thresholds, dose-above-threshold factors (DoT), and concentrations of the peptides in the hydrolyzed chicken plasma sample are shown in Table 6. The taste thresholds determined for the newly identified peptides (those listed in Table 6) were used to calculate the DoT factors, which are defined as the ratio between the concentration of a substance and its corresponding taste threshold concentration.The DoT factors allow a first estimation of the taste contribution, since substances with DoT factors ≥ 1 most likely contribute directly to the perceived taste. Table 6 Peptidsequenz Geschmacksschwelle in Modellbrühe (µmol / L) Peptidkonzentration in der Probe (µmol / kg) DoT-Faktor Pro-Val-Leu-Lys (SEQ ID NO: 1) 296 2,1 0,0 Pro-Leu-Pro-Arg (SEQ ID NO: 2) 112 1,6 0,0 Glu- Ala- Glu- Phe- Asp (SEQ ID NO:3) 95 123,8 1,3 Pro- Glu- Arg- Asn- Glu (SEQ ID NO: 4) 231 18,6 0,1 Pro- Gln- Pro- Glu- Arg (SEQ ID NO: 5) 133 0,2 0,0 Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6) 94 194,1 2,1 Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7) 236 13,2 0,1 Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8) 351 3,6 0,0 Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9) 36 5,3 0,1 Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10) 34 3,2 0,1 Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11) 67 221,1 3,3 Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12) 137 20,8 0,2 Glu- Asp- Met- Ala- Pro (SEQ ID NO: 13) 151 27,5 0,2 Pro- Ile- Asn- Asp- Asn (SEQ ID NO: 14) 26 102,6 3,9 Pro-Thr-Asp-Leu (SEQ ID NO: 15) 97 995,6 10,3 Pro-Val-Ala-Glu (SEQ ID NO: 16) 243 171,7 0,7 Arg- Asn- Gly- Pro- Arg (SEQ ID NO: 17) 101 0,6 0,0 Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18) 247 6,7 0,0 Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19) 212 47,5 0,2

[0071] According to the results, five peptides with DoT factors ≥ 1 (SEQ ID NO: 3, 6, 11, 14, and 15) played a key role in taste-active and taste-modulating effects in the plasma sample. Example 3: The taste-modulating effects of the identified peptides in food products

[0072] To investigate the taste-modulating performance of the identified peptides in food products, commercial chicken soup and the model broth were used for further sensory experiments. According to the sensory experiment, the peptides Pro-Val-Leu-Lys (SEQ ID NO: 1), Pro-Leu-Pro-Arg (SEQ ID NO: 2), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5), and Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7) were added to the commercial chicken soup (Netto Marken-Discount AG&CO. KG, Maxhütte-Haidhof) and the model broth. For the experiments, 3 g of the commercial chicken soup powder was dissolved in 150 mL of boiling water. The peptides (2 mmol / L each) were added to a 20 mL solution of this solution. The taste threshold was then determined using duo-trio tests as previously described (using chicken soup as a reference).Table 7 shows the sensory evaluation of the identified peptides in the commercial chicken product and model broth. Table 7 Peptidsequenz Taste threshold in food products (µmol / L) commercial chicken soup Model broth Pro-Val-Leu-Lys (SEQ ID NO: 1) 1500 296 Pro-Leu-Pro-Arg (SEQ ID NO: 2) 418 112 Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3) 1000 95 Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5) no effect 133 Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7) 1500 236

[0073] According to the results, the peptide with SEQ ID NOs: 1, 3, and 7 can influence the flavor profile of commercial chicken soup at approximately 1500 µmol / L. In the case of the peptide with SEQ ID NO: 5, the enriched sample could not be distinguished (the maximum tested concentration is 2 mmol / L). The peptide with SEQ ID NO: 2 could be perceived at a concentration of 418 µmol / L in commercial chicken soup, whereas in model broth the threshold was 4 times lower (112 µmol / L). Based on these experimental results, it can be concluded that the peptides with the specific amino acid sequences according to the present invention can enhance the flavor profile of commercial food products, although higher concentrations are required compared to model broth.

[0074] Additionally, other commercial food products with different matrices purchased from the local supermarket, such as chicken broth (Netto Marken-Discount AG&CO. KG, Maxhütte-Haidhof), vegetable broth (Hügli Nahrungsmittel GmbH, Radolfzell), and pepper sauce (Unilever Germany, Hamburg), were used to evaluate the taste-modulating effects of the novel identified peptides of the invention, e.g., Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), and Pro-Thr-Asp-Leu (SEQ ID NO: 15). The food samples were prepared according to the detailed instructions listed on the various products. For chicken broth and vegetable broth, 3 g of the sample was dissolved in 150 mL of boiling water and cooled to room temperature in an ice bath. For pepper sauce, 4 g of the sample was dissolved in 150 mL of boiling water.Subsequently, 200 µmol / L of the peptides were added to 20 mL of broth or sauce. 3-AFC tests were designed to test the flavor properties of the peptides in each commercial product: two samples contained only 20 mL of the commercial product, and one sample contained 20 mL of the commercial product enriched with the peptides. The panel was asked to choose the alternative sample. Table 8 shows the results of the sensory tests for three commercial food products spiked with the five exemplary peptides. Table 8 Food sample Number of subjects who noticed a difference in taste out of a total of 9 subjects Is there a significant difference? Chicken broth 7 Yes vegetable broth 7 Yes pepper sauce 8 Yes

[0075] The results clearly show that the new identified peptides according to the present invention can be used as the taste modulating agents in edible products, especially foods or even pharmaceutical products. Example 4: Effect of enzyme type on peptide sequences

[0076] To investigate the effect of enzyme types on the amino acid sequence of the peptides present in the hydrolyzed plasma sample, the chicken plasma was digested with various enzymes, such as Flavourzyme and Thermolysin.

[0077] For Flavourzyme digestion, 0.0504 g of chicken plasma was extracted three times with methanol and the sample was dried under nitrogen. The sample was redissolved in 1 M TEAB, and urea was added to a final concentration of 8 M. Subsequently, 100 mM DTT was added and kept at 56°C for 45 minutes, followed by the addition of 0.55 M IAA and incubation at room temperature in the dark for 1 hour. The sample was then digested with Flavourzyme for 15 or 30 minutes at 37°C. Digestion was stopped by the addition of concentrated formic acid, and the peptide mixture was purified by SPE on C18 cartridges.

[0078] For thermolysin digestion, a plasma sample was first prepared by adjusting the pH to 8, then incubated with thermolysin at 55 °C for 18 hours. The reaction was stopped by heating in a water bath at 100 °C for 15 min. The solutions were also dried before analysis.

[0079] Peptide identification was performed by de novo sequencing of LC-TOF-MS data using PEAKS, as previously described for the screening of the 21 peptides (SEQ ID NOs: 1-21, Table 3) in the hydrolyzed chicken plasma samples. Due to the high activity of Flavourzyme, the chicken plasma was digested with Flavourzyme for either 15 or 30 minutes.

[0080] Table 9 shows the exemplary peptides identified in chicken plasma after Flavourzyme digestion and Table 10 shows the exemplary peptides identified in chicken plasma after Thermolysin digestion. Table 9 Digestion time (min) Peptide sequence Mass (m / z) RT (min) 15 Gly-Gly-His-Thr-Asp-Ser-Val-Glu-Ala-Gly-Pro-Gly-Ile-Asn-Asp-Asp-Gly-Ser-Gly-Ile-Ile-Ser-Asn 2168,0 29,0 Asp-Ser-Gly-Val-Asn-Val-Asp-His-Glu-Glu-Pro-His-Glu-Glu-Gly-Arg 1588,7 22,7 Gly-Ile-Asn-Asp-Pro-Thr-Glu-Gly-Pro-Arg-Thr-Val-Ile-Ser-Asp-Pro-Trp-Asp-Ser-Ser-Ala-Ser 2300,1 36,3 Glu- Val- His- Ala- Ile- Gly- Ala- Val- Trp- Ala- Asn- Val-Leu-Tyr-Glu-Leu-Leu-Trp-Asn-Leu-Ile-Asp-Lys-His-Gly-Lys-Asn 3101,6 43,9 Gly-Ile-Asn-Asp-Pro-Thr-Glu-Gly-Pro-Arg-Thr-Val-Ile-Ser-Asp-Pro-Trp-Asp-Ser-Ser-Ala-Ser-Ala 2371,1 36,8 Asp-Ser-Gly-Val-Asn-Val-Asp-His-Glu-Glu-Pro-His-Glu-Glu-Gly-Arg-Ala-Ser 1746,7 22,9 Val-Asp-Ser-Gly-Val-Asn-Val-Asp-His-Glu-Glu-Pro-His-Glu-Glu-Gly-Arg-Ala-Ser 1845,8 23,9 Val-Asp-Ser-Gly-Val-Asn-Val-Asp-His-Glu-Glu-Pro-His-Glu-Glu-Gly-Arg 1687,7 23,8 Ser-Asp-Leu-Asp-Tyr-Arg-Leu-Pro-Val-Ser-Glu-Gly-Leu-Ser 1549,8 36,5 Val-Glu-Ala-Gly-Pro-Gly-Ile-Asn-Asp-Asp-Gly-Ser-Gly-Ile-Ile-Ser-Asn 1613,8 29,7 Phe-Glu- Asp-Tyr- Tyr- Asp-Ser-Ile- Asp-Leu-Pro-His-Ile-Pro-Thr 1823,8 42,5 30 Asp-Thr-Val-Asn-Tyr-Leu-Tyr-Glu 1015,5 30,2 Gly-Ala-His-Gln-Asp-Ser-Ile-Asn-Leu-Asp-Ser-Pro-Ser-Glu-Gly-Arg-Ala-Pro-Gly-Ala-Asp-Asp-Asp-Gly-Ser-Gly-Val-Val-Thr 2823,3 28,5 Trp-Asp-Thr-Asn-Pro-Pro-Ser-Gly-Pro-Ser-Ala-Ser-Pro-Ser 1258,5 24,0 Ala-Leu-Val-Ser-Pro-Asp-Glu-Phe-Pro-Glu-Asp 1217,5 33,8 Asp-Tyr-Arg-Leu-Pro-Val-Ser-Glu-Gly-Leu-Ser 1234,6 33,0 Ala- His- Gln- Asp- Ser-Ile- Asn- Leu- Asp- Ser- Pro- Ser-Glu-Gly-Arg-Ala-Pro-Gly-Ala-Asp-Asp-Asp-Gly-Ser-Gly-Val-Val-Thr-Ile-Leu 2992,4 38,2 Phe- Glu- Ser-Ala-Phe- Gly-Asp-Asp- Ser- Pro-Tyr-Ile-His-Ser-Ala-Asp 1756,7 33,3 Asn-Pro- Pro- Ser- Gly- Pro- Ser-Ala- Ser- Pro- Ser- Trp-Thr-Ile-His 1393,6 28,5 Tyr-Lys-Gln-Pro-Gln-Val-His 898,5 16,4 Glu-Phe-Pro-Glu-Asp-Ile-Gln-Leu-Glu 1118,5 37,6 Val-Pro-Asp-Ser-Ser-Lys-Thr-Tyr 895,4 6,4

[0081] In the case of Flavourzyme digestion, it was found that the length of the peptides identified during a 15-minute digestion was longer than during a 30-minute digestion. With a longer digestion time, digestion is more thorough, resulting in a higher number of shorter peptides. Table 10 Peptidsequenz Masse (m / z) RT (min) Leu-Pro-Glu-Asp-Arg-Arg 784,4191 2,08 Leu-Pro-Glu-Asp-Arg 628,318 2,26 Thr-Pro-Asp-Asn-Val-Leu-Leu-Lys 898,5124 23,48 Glu-THR-GLY-LYS-LYS 561,3122 0,84 Val-Gly-Thr-Glu-Glu-Asp-Lys 776,3552 1,46

[0082] Furthermore, targeted LC-MS / MS analysis of the 21 peptides, which are the novel identified peptides according to the present invention (SEQ ID NO: 1-21, Table 3), in the Flavourzyme and Thermolysin digestions revealed that none of these peptides were present due to the different behavior of the enzymes. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] CN 108047313 A

[0004] CN 108048518 A

[0005] CN 101843289 A

[0006] Cited non-patent literature

[0000] Jayathilakan et al., J Food Sci Technol, 2012, 49(3), 378-293

[0002] Oliver Frank, Jezussek, & Hofmann, 2003

[0052] by Sebald et al., J. Agric. Food Chem. 2018, 66, 11092-11104

[0060]

Claims

[1] Isolated peptide having an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1), Pro-Leu-Pro-Arg (SEQ ID NO: 2), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4), Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5), Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7), Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8), Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9), Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12), Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), Pro-Thr-Asp-Leu (SEQ ID NO: 15), Pro-Val-Ala-Glu (SEQ ID NO: 16), Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17), Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18), Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19), Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20), and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21). [2] The isolated peptide according to claim 1, which imparts at least one of the following effects: salt reducing, salt enhancing, umami and kokumi effects. [3] An isolated peptide according to claim 1 or 2, which imparts the salt-reducing effect and has an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), and Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13). [4] An isolated peptide according to claim 1 or 2, which imparts the salt-enhancing effect and has an amino acid sequence selected from the group consisting of: Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), Pro-Val-Ala-Glu (SEQ ID NO: 16), and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21). [5] An isolated peptide according to claim 1 or 2 which imparts the umami effect and has an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1), Pro-Leu-Pro-Arg (SEQ ID NO: 2), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4), Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5), Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7), Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9), Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17), Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18), Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19), Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20), and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO:21). [6] An isolated peptide according to claim 1 or 2 which imparts the Kokumi effect and has an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1), Pro-Leu-Pro-Arg (SEQ ID NO: 2), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4), Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5), Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7), Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8), Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9), Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12), Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13), Pro-Thr-Asp-Leu (SEQ ID NO: 15), Pro-Val-Ala-Glu (SEQ ID NO: 16), Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17), Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18), Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19), Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20), and Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21). [7] An isolated peptide according to any one of claims 1-6, which was obtained by enzymatic hydrolysis or fermentation. [8] An isolated peptide according to claim 7, which was obtained by the enzymatic hydrolysis of animal blood. [9] The isolated peptide of claim 8, wherein the animal blood is poultry blood. [10] The isolated peptide according to claim 8, wherein the enzymatic hydrolysis is carried out using serine protease. [11] The isolated peptide of claim 10, wherein the serine protease is subtilisin. [12] Use of the isolated peptide as defined in any one of claims 1-11 as a taste modulating agent for a food or pharmaceutical preparation. [13] Protein hydrolysate comprising at least one peptide having an amino acid sequence selected from the group consisting of: Pro-Val-Leu-Lys (SEQ ID NO: 1), Pro-Leu-Pro-Arg (SEQ ID NO: 2), Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Pro-Glu-Arg-Asn-Glu (SEQ ID NO: 4), Pro-Gln-Pro-Glu-Arg (SEQ ID NO: 5), Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Asp-Ala-Pro-Val-Leu-His (SEQ ID NO: 7), Glu-Phe-Asp-Ala-Arg-Pro-Thr (SEQ ID NO: 8), Glu-Glu-Thr-Pro-Ser-His-Asp (SEQ ID NO: 9), Glu-Glu-Val-Glu-Glu-Glu-Glu-Val-Glu (SEQ ID NO: 10), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Glu-Asn-Ala-Pro-Asp-Gln-Lys (SEQ ID NO: 12), Glu-Asp-Met-Ala-Pro (SEQ ID NO: 13), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), Pro-Thr-Asp-Leu (SEQ ID NO: 15), Pro-Val-Ala-Glu (SEQ ID NO: 16), Arg-Asn-Gly-Pro-Arg (SEQ ID NO: 17), Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr (SEQ ID NO: 18), Glu-Gly-Asp-Ser-Ala-Ala-Ile (SEQ ID NO: 19), Glu-Pro-Glu-Gly-Asp-Val-His-Gln (SEQ ID NO: 20), Glu-Asp-Glu-Val-Leu-Ala-Thr-Pro (SEQ ID NO: 21), and a combination thereof. [14] Protein hydrolysate according to claim 13, which comprises at least the peptide having the amino acid sequence: Glu-Ala-Glu-Phe-Asp (SEQ ID NO: 3), Glu-Phe-Asp-Glu-Lys-Pro-Ala-Asp (SEQ ID NO: 6), Glu-Phe-Asp-Glu-Lys-Ala-Pro-Asp (SEQ ID NO: 11), Pro-Ile-Asn-Asp-Asn (SEQ ID NO: 14), and Pro-Thr-Asp-Leu (SEQ ID NO: 15). [15] Protein hydrolysate according to one of claims 13 or 14, which was obtained by enzymatic hydrolysis. [16] Protein hydrolysate according to claim 15, which was obtained by the enzymatic hydrolysis of animal blood. [17] Protein hydrolysate according to claim 16, wherein the animal blood is poultry blood. [18] Protein hydrolysate according to claim 16, wherein the enzymatic hydrolysis is carried out using serine protease. [19] Protein hydrolysate according to claim 18, wherein the serine protease is subtilisin. [20] Use of the protein hydrolysate as defined in any one of claims 13-19 as a taste modulating agent for a food or pharmaceutical preparation. [21] A taste modulating composition comprising the isolated peptide as defined in any one of claims 1-11. [22] A taste modulating composition comprising the protein hydrolysate as defined in any one of claims 13-19. [23] Use of the taste-modulating composition according to claim 21 or 22 in a food or pharmaceutical preparation. [24] A food preparation comprising the isolated peptide as defined in any one of claims 1-11. [25] A food preparation comprising the protein hydrolysate as defined in any one of claims 13-19. [26] A food preparation comprising the taste-modulating composition defined in claim 21 or 22. [27] A pharmaceutical preparation comprising the isolated peptide as defined in any one of claims 1-11. [28] A pharmaceutical preparation comprising the protein hydrolysate as defined in any one of claims 13-19. [29] A pharmaceutical preparation comprising the taste-modulating composition defined in claim 21 or 22. [30] A method for adjusting the taste of a food preparation, comprising using an effective amount of the isolated peptide as defined in any one of claims 1-11 in the food preparation. [31] A method for adjusting the taste of a food preparation, comprising using the protein hydrolysate as defined in any one of claims 13-19, which contains an effective amount of the isolated peptide, in the food preparation. [32] A method for adjusting the taste of a food preparation, comprising using the taste-modulating composition as defined in claim 21 or 22, which includes an effective amount of the isolated peptide, in the food preparation. [33] A method for adjusting the taste of a pharmaceutical preparation, comprising using an effective amount of the isolated peptide as defined in any one of claims 1-11 in the pharmaceutical preparation. [34] A method for adjusting the taste of a pharmaceutical preparation, comprising using the protein hydrolysate as defined in any one of claims 13-19, which contains an effective amount of the isolated peptide, in the pharmaceutical preparation. [35] A method for adjusting a taste of a pharmaceutical preparation, comprising using the taste-modulating composition as defined in claim 21 or 22, which includes an effective amount of the isolated peptide, in the pharmaceutical preparation.

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