Bitter masking of peptides

Abstract

Disclosed are: a) conjugated peptides conjugated with at least one reducing sugar, having a degree of conjugation of at least 10 percent, preferably in a solid form such as a powder; b) a method for preparing a non-bitter conjugated protein hydrolysate, the method comprising hydrolyzing a protein with at least one enzyme, mixing the resulting protein hydrolysate and at least one reducing sugar, heating the mixture to cause a Maillard reaction, and drying the composition; c) a method for masking the bitter taste of a peptide, the method comprising mixing the peptide and a reducing sugar, heating the mixture to cause a Maillard reaction, and drying to obtain a powder; and d) uses of the conjugated peptides for food, dietary supplement, pharmaceutical, and animal feed products.

Description

Background Art

[0001] Description Peptides that occur naturally, are produced synthetically, or are obtained by hydrolysis of acidic, alkaline or enzymatic proteins are widely used in the fields of nutrition and medicine. Some peptides are bitter, which reduces the acceptability of the product. Bitterness is produced by specific amino acid sequences that bind to specific bitter receptors on the tongue. Therefore, not all peptides have bitterness, and the degree of bitterness depends on different protein sources and different ways of hydrolyzing proteins (Bumberger E. and Belitz H.-D. 1993). Peptides have become very versatile, especially as functional components and as bioactive substances (Hartmann R. and Meisel H. 2007). However, currently, their use is limited due to their impact on the flavor of the products they are in or the products that contain them.

[0002] Numerous approaches have been taken to reduce bitterness, such as using alpha-cyclodextrin. However, although a large excess of alpha-cyclodextrin is required, the peptide is too large to be covered by alpha-cyclodextrin, so the effect of bitterness reduction was very limited (Tamura M. et al. 1990). Since the peptide can be incorporated into the starch structure and thus can avoid acting on bitter receptors on the tongue, it has been suggested that starch can also mask bitterness. This approach can reduce bitterness, but the method of allowing the peptide to penetrate into the starch core requires heating overnight at 100 °C, so this method cannot be easily scaled up (Tamura M. et al. 1990). Acetylation has also been used to reduce bitterness, but such a method requires another regulatory registration, produces a new component (acetylated peptide) considered as an additional compound, and is not easily acceptable as a health-functional food due to the change in the clear ingredient label. The acetylated peptide only goes in another direction of the additional chemical ingredient label. In addition to the acceptance and efficiency of bitterness masking, some known methods also change the activities of the masked peptides in such a way that they no longer exhibit the desired biological activity or lose other desired properties for their respective uses.

Summary of the Invention

[0003] Therefore, an object of the present invention was to provide a masked peptide with reduced bitterness and without loss of peptide activity.

[0004] Surprisingly, this object is conjugated with at least one reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose and mannose, and has a conjugation degree of at least 10%, and it has been found that it is solved by a peptide with a Gardener value increase of the conjugate protein of less than 6. The conjugation degree is defined as follows:

[0005] Conjugation degree = [(OPA-N start / Nitrogen start ) - (OPA-N end / Nitrogen end )] / (OPA-N start / Nitrogen start )

[0006] OPA-N start is the OPA-N value of hydrolyzed casein without a conjugation reaction, and OPA-N end is the OPA-N value after the conjugation reaction. Nitrogen start is the total nitrogen content in the sample without a conjugation reaction, while nitrogen end is the total nitrogen content after the conjugation reaction. The OPA-N value is the value divided by the total amount of nitrogen, that is, the value obtained by dividing the nitrogen of free amino groups by the total amount of nitrogen from all amino acids. Then, the degree of decrease (%) of this ratio after conjugation is calculated. This ratio is used to take into account the dilution effect that occurs when both the total nitrogen and OPA-N directly decrease due to dilution when the sugar is added to the system. By using this ratio, only the absolute decrease of free amino groups is calculated, and thus the dilution effect of the sugar is excluded.

[0007] The conjugate peptide had a lower bitterness than the combination of the peptide and sugar when the conjugation process was not initiated. Thus, the taste masking is caused not by the sweetness of the sugar, but by a specific conjugation reaction. The degree of conjugation was measured as the ratio of the conjugated amine groups to the free amine groups on the peptide chain.

[0008] According to the present invention, a process called the Maillard reaction, which occurs during food preparation such as baking and frying, was applied. The advantage is that reducing sugars are commonly used in many food preparations, and the Maillard reaction does not change the peptide in a manner that affects its activity and does not change the chemical structure in a manner that requires new regulatory registration or declaration. The Maillard reaction is initiated by the condensation of an amino group on the peptide and a carbonyl group on the reducing sugar, resulting in Schiff base formation and transfer to Amadori and Heyns products (Lund M. N. and Ray C. A. 2017). Reducing sugars are any sugars that can act as reducing agents because they have a free aldehyde group or a free ketone group (Pratt C. W. and Cornely K. 2013). The process can be controlled, for example, by controlling the pH, temperature, and reaction time (Lund M. N. and Ray C. A. 2017). The Maillard reaction can be carried out in solution / dispersion or in the dry state (Lund M. N. and Ray C. A. 2017). The best strategy is always to use sugars and peptides with a very high concentration of reducing ends. According to the present invention, only the first stage of the Maillard reaction is achieved, and the process is stopped before the strong brown color and flavor typical of the Maillard reaction occur.

[0009] The degree of conjugation and the degree of the Maillard reaction are not the same. The degree of conjugation indicates the number of amine groups of the peptide reacting with the sugar, while the degree of the Maillard reaction indicates the stage of the Maillard reaction that ultimately results in the generation of brown color and various flavors. According to the present invention, these secondary flavors are not desirable. On the other hand, the initial stage of the Maillard reaction results in a colorless and flavorless conjugation of sugar and peptide. The degree of the Maillard reaction can be measured using the Gardner value (refer to the method part) that determines the presence and degree of the generation of brown color. Since some peptides already have a natural color, the increase in the Gardner value before and after the Maillard reaction is used to measure the degree of the Maillard reaction.

[0010] According to the present invention, the reducing sugar is selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose, and mannose. It is also possible to apply a mixture obtained from starch hydrolysis such as maltodextrin containing different molecules from monosaccharides, disaccharides to oligosaccharides of reducing sugar, particularly glucose.

[0011] In one embodiment, only a slight increase in the Gardner value (Gardner increase) is acceptable, and the Gardner increase is less than 4, preferably less than 3. This means that according to this embodiment, there is no typical Maillard browning of the conjugate mostly associated with the Maillard reaction. It is a conjugation that does not result in browning and roasted flavor.

[0012] In a preferred embodiment, the degree of conjugation is at least 15%, preferably at least 20%, 25%, 30%, 35% or 40%. The higher the degree of conjugation, the higher the possibility of reducing bitterness. The required and desired degree of bitterness reduction depends on the individual bitterness of the peptide.

[0013] Non-conjugated peptides can be any of those that are naturally occurring, produced synthetically, or obtained by hydrolysis of acidic, alkaline, or enzyme proteins. Preferably, the peptide is a protein hydrolysis product derived from at least one selected from the group consisting of plant or animal proteins, particularly wheat, soybean, rice, potato, pea, sunflower, rapeseed, lupinus, and milk proteins, preferably selected from casein and pea protein. The bitterness of each hydrolysis product also depends on the mode of hydrolysis and the degree of hydrolysis or the MW of the hydrolysis product.

[0014] Preferably, the peptide results from a protein hydrolyzed by an enzyme, particularly an endopeptidase, preferably an alkaline protease. The mode of hydrolysis, such as enzymatic hydrolysis or chemical hydrolysis, results in different properties of the hydrolysis products, for example, different MWs, or different peptide properties for hydrolysis products of the same MW. Generally, chemical hydrolysis results in peptides with larger MWs, and enzymatic hydrolysis results in more characteristic and shorter MW peptides.

[0015] According to certain embodiments, the protein hydrolysis product is not filtered after hydrolysis and / or the pH is neutralized with an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid, and sulfuric acid.

[0016] According to one embodiment, the degree of conjugation ranges from 10% to 90%, preferably from 15% to 70%, more preferably from 20% to 50%, particularly from 25% to 40%. If the degree of conjugation is too high, there is a risk of a darker color or a burnt or caramel flavor of the conjugate. The preferred maximum degree of conjugation depends on the individual combination of sugar and peptide.

[0017] In certain embodiments, the maximum molecular weight of the peptide is 2300 Da, preferably 2000, 1500, 1200, 1000, or 900 Da.

[0018] In another embodiment, the minimum molecular weight of the peptide is 650 Da, preferably 660, 670, 680, 690, 700, 710, 720, 750 or 800 Da.

[0019] Preferably, the molecular weight of the peptide is from 650 to 2400 Da, preferably from 650 to 1000 Da.

[0020] The MW according to the present invention is the apparent average MW determined by measuring OPA-N as described below in the method part (Frister H. et al. 1988).

[0021] Preferably, the molar ratio of reducing sugar to peptide is from 0.5 to 20, preferably from 1.1 to 1.7. Regarding glucose as the reducing sugar, the weight ratio of peptide to reducing sugar is from 90:10 to 60:40, preferably from 80:20 to 70:30. The more the amount of sugar, the more groups causing more bitterness can react with the reducing sugar, so the bitterness of the conjugate peptide is reduced. Therefore, the amount of sugar is more for peptides with more bitterness than for peptides with less bitterness and is adjusted according to individual bitterness.

[0022] Preferably, the conjugate peptide is in solid particle form, preferably in powder form, especially in spray-dried form or freeze-dried form.

[0023] Preferably, the solubility of the conjugate peptide is at least 90%, preferably at least 91, 92, 93, 94, 95, 95, 97, 98 or 99%, especially 100%. Since only the dissolved peptide can exhibit its properties in the applied composition, the solubility of the peptide is an essential feature for many applications.

[0024] The present invention relates to a method for masking the bitterness of a peptide, comprising the steps of mixing a peptide and a reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose and mannose, preferably in a solution or dispersion; heating the mixture to effect a Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C, preferably 60 to 85 °C, particularly 65 °C ± 3 °C, for a period of 20 to 120 minutes, preferably 30 to 60 minutes, and after that time, stopping the Maillard reaction by spray-drying the composition to obtain a powder. Preferably, the peptide is a hydrolyzed protein.

[0025] In another embodiment of the present invention, the conjugation is carried out in the dry state, and a mixture consisting of powders of a peptide and a reducing sugar is stored at a temperature of 25 to 60 °C and a high relative humidity of 60 to 80% for several days to several weeks.

[0026] The preferred reaction parameter ranges can be summarized such that the reaction can be controlled so that it does not proceed to the final product, i.e., does not show brown color and does not have a caramel or burnt taste (Lund M. N. and Ray C. A. 2017). Brown and caramel flavors can be desirable in some specific foods, but they are limiting and not preferred for most applications. As can be seen from Table 1, for the Maillard reaction to obtain a sufficient reduction in bitterness, the higher the temperature, the shorter the time, and the lower the temperature, the longer the time.

[0027] According to the method of the present invention, preferably only the first stage of the Maillard reaction is achieved and the process is stopped before a strong brown color occurs.

[0028] The method according to the present invention is different from the Maillard reaction that occurs during food preparation such as direct baking, because, for example, the concentrations of peptides and reducing sugars in food applications such as bakery products are not high enough for a wide and well-controlled reaction during peptide conjugation and to result in a sufficient degree of conjugation of at least 10%.

[0029] Preferably, the Maillard reaction is carried out until a degree of conjugation of at least 10%, preferably at least 15%, 20%, 25%, 30%, 35% or 40% is obtained. The degree of conjugation required to sufficiently mask bitterness depends on the bitterness of the individual peptides.

[0030] The degree of conjugation and the degree of the Maillard reaction are not the same. The degree of conjugation indicates the number of amine groups of the peptide reacting with the sugar, while the degree of the Maillard reaction indicates the stage of the Maillard reaction that ultimately results in the development of brown color and various flavors. According to the present invention, these secondary flavors are not desirable, while the initial stage of the Maillard reaction results in a colorless and flavorless conjugation of sugar and peptide. The degree of the Maillard reaction can be measured using the Gardner value (see part of the method) that determines the presence and degree of the development of brown color. Since some peptides already have a natural color, the increase in the Gardner value before and after the Maillard reaction is used to measure the degree of the Maillard reaction. According to the present invention, only a slight increase in the Gardner value (Garner increase) is acceptable. In one embodiment, the Gardner increase is less than 6, preferably less than 4, particularly less than 3.

[0031] In a preferred embodiment, the molar ratio of reducing sugar to peptide is 0.5 to 2.0, preferably 1.1 to 1.7. Regarding glucose as the reducing sugar, the weight ratio of peptide to reducing sugar is 90:10 to 60:40, preferably 80:20 to 70:30. The greater the amount of sugar, the more groups that cause bitterness can react with the reducing sugar, so the bitterness of the conjugate peptide is reduced. Therefore, the amount of sugar is greater for peptides with more bitterness than for peptides with less bitterness and is adjusted according to the individual bitterness.

[0032] Preferably, the Maillard reaction is carried out at a pH of 7 to 9, preferably 8 to 8.5.

[0033] In a preferred embodiment, the Maillard reaction is carried out in a solution containing a high concentration of peptide and a sugar having a reducing end, and then the material is spray-dried to stop the reaction and form a powder.

[0034] Preferably, the peptide is a hydrolyzed protein, where the hydrolysis is carried out by an enzyme, in particular an endopeptidase, preferably alkaline protease. The mode of hydrolysis, such as enzymatic hydrolysis or chemical hydrolysis, results in different properties of the hydrolysis products even when the peptides contain the same amino acid sequence.

[0035] According to a particular embodiment, the protein hydrolysis product is not filtered after hydrolysis and / or the pH is neutralized with an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid and sulfuric acid.

[0036] The present invention relates to a method for preparing a non-bitter conjugate protein hydrolysate, comprising the steps of hydrolyzing at least one protein with at least one enzyme, obtaining the resulting protein hydrolysate, and mixing at least one reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose and mannose, preferably in a solution or dispersion, heating the mixture to carry out the Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C, preferably 60 to 85 °C, particularly 65 °C ± 3 °C for 20 to 120 minutes, preferably 30 to 60 minutes, and after that time, stopping the Maillard reaction by spray drying the composition to obtain a non-bitter powder of the protein hydrolysate.

[0037] The preferred reaction parameter ranges can be summarized such that the reaction can be controlled so that it does not proceed to the final product, i.e., it does not show a brown color and does not have a caramel or burnt taste (Lund M. N. and Ray C. A. 2017). Brown and caramel flavors can be desirable in some specific foods, but they are limiting and not preferred for most applications. As can be seen from Table 1, for the Maillard reaction to obtain a sufficient reduction in bitterness, the higher the temperature, the shorter the time, and the lower the temperature, the longer the time. According to the present invention, only the first stage of the Maillard reaction is achieved and the process is stopped before the strong brown color and flavor typical of the Maillard reaction occur.

[0038] Preferably, the protein is hydrolyzed with an endopeptidase, preferably alkaline protease. The mode of hydrolysis, such as enzymatic hydrolysis or chemical hydrolysis, results in different properties of the hydrolysate even when the resulting peptides contain the same amino acid sequence.

[0039] According to certain embodiments, the protein hydrolysate is used without being filtered after hydrolysis, and / or the pH is neutralized by adding an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid, and sulfuric acid to the hydrolysate.

[0040] Preferably, the Maillard reaction is carried out for a sufficient time until at least 10%, preferably at least 15%, 20%, 25%, 30%, 35% or 40% degree of conjugation is obtained. The degree of conjugation required to sufficiently mask bitterness, as well as the time and temperature required therefor, depend on the bitterness of the individual peptides.

[0041] Preferably, the Maillard reaction is carried out at a pH of 7 to 9, preferably 8 to 8.5.

[0042] The degree of conjugation and the degree of the Maillard reaction are not the same. The degree of conjugation indicates the number of amine groups of the peptide reacting with the sugar, whereas the degree of the Maillard reaction indicates the stage of the Maillard reaction that ultimately results in the development of brown color and various flavors. According to the present invention, these secondary flavors are not desirable, while the initial stage of the Maillard reaction results in a colorless and flavorless conjugation of sugar and peptide. The degree of the Maillard reaction can be measured using the Gardner value (see part of the method) that determines the presence and degree of the development of brown color. Since some peptides already have a natural color, the increase in the Gardner value before and after the Maillard reaction is used to measure the degree of the Maillard reaction. According to the present invention, only a slight increase in the Gardner value (Gardner increase) is acceptable. In one embodiment, the Gardner increase is less than 6, preferably less than 4, particularly less than 3.

[0043] In a preferred embodiment, the molar ratio of reducing sugar to peptide is 0.5 to 2.0, preferably 1.1 to 1.7. Regarding glucose as the reducing sugar, the weight ratio of peptide to reducing sugar is 90:10 to 60:40, preferably 80:20 to 70:30. The more the amount of sugar, the more groups causing more bitterness can react with the reducing sugar, so the bitterness of the conjugate peptide is reduced. Therefore, the amount of sugar is more for peptides with more bitterness than for peptides with less bitterness and is adjusted according to individual bitterness.

[0044] In a preferred embodiment, the Maillard reaction is carried out in a solution containing a high concentration of peptide and a sugar having a reducing end, and then the material is spray-dried to stop the reaction and form a powder.

[0045] The present invention further provides the use of the conjugate peptide described in the first embodiment of the present invention or the conjugate protein hydrolysate obtained by the process described in the third above-mentioned embodiment of the present invention for foods, preferably beverages, baking products, chewing gums, sports nutrition foods, dietary supplements, confectioneries, desserts, foam foods or prebiotics, pharmaceuticals, bioactive substances, animal feed products.

Examples

[0046] The results of Tests C1 to C6 and P1 show that bitterness can be significantly reduced by conjugating a peptide with a reducing sugar. There is an obvious correlation between bitterness and the degree of conjugation. The more the amount of sugar, the less bitterness is brought about, and the higher the reaction temperature, the less bitterness is brought about.

[0047]

Table 1

[0048] Protein Hydrolysate 21.15 kg of tap water are heated to 60 °C and the temperature is maintained for the entire hydrolysis time. 182 g of NaOH are added as a 20% NaOH solution. 6.93 kg of casein are dispersed in the warm water and the pH is adjusted to 9.0 using a 20% NaOH solution. 87 g of Alcalase are added and the material is stirred for 30 minutes while gradually adding 10.42 g of casein, maintaining the pH at 9.0. 87 g of Alcalase are added and the pH is kept constant at pH 8.5 for 60 minutes using a 20% NaOH solution. The last 60 minutes are stirred for 60 minutes without keeping the pH constant, resulting in a final pH of about pH 7.9. The enzymatic reaction is stopped by heating to 80-84 °C and this temperature is maintained for 15 minutes. The solution is spray-dried to form a casein hydrolysate powder, which was applied for conjugation.

[0049] Pea protein hydrolysates were purchased commercially.

[0050] Peptide Conjugation 70-90 g of protein hydrolysate is dissolved in 86-110 g of water, 10-30 g of glucose is added to the solution at 65 or 85 °C, and the pH is adjusted to 8 or 8.5 using NaOH. The system is stirred while the pH is kept constant using NaOH. After 30 or 60 minutes, the system is spray-dried to form a powder.

[0051] Measurement of the Degree of Maillard Reaction (Increase in Gardner Value) A 5% (w / w) aqueous solution of peptide and conjugated peptide is prepared and the solution is filtered through a 0.2 μm Whatman filter (red rim syringe filter) to remove any turbidity that may interfere with the measurement. The Gardner value is measured using a LICO 500 (Hach Lange, Rheineck, Switzerland) equipped with an 11 mm cuvette. The measurement is performed according to the method of ISO4630:2015. The Gardner value increase is the difference between the Gardner value before and after the Maillard reaction.

[0052] Gardner Initial -Gardner Maillard = Increased Gardner score

[0053] Degree of Conjugation The OPA-N value is the value divided by the total amount of nitrogen, that is, the value obtained by dividing the nitrogen of the free amino group by the total amount of nitrogen from all amino acids. Subsequently, the degree of decrease (%) of this ratio after conjugation is calculated.

[0054] Degree of conjugation = [(OPA-N start / nitrogen start ) - (OPA-N end / nitrogen end )] / (OPA-N start / nitrogen start )

[0055] OPA-N start is the OPA-N value of hydrolyzed casein without the conjugation reaction, and OPA-N end is the OPA-N value after the conjugation reaction. Nitrogen start is the total nitrogen content in the sample without the conjugation reaction, while nitrogen end is the total nitrogen content after the conjugation reaction. This ratio is used considering the dilution effect that occurs when sugar is added to the system and thus both the total nitrogen and OPA-N are directly decreased by dilution. By using this ratio, only the absolute decrease of the free amino group is calculated, and for this reason, the dilution effect of sugar is excluded.

[0056] To measure the degree of conjugation in samples where the system before conjugation cannot be measured, the HPLC method from Davidek T. et al. (2003) can be utilized.

[0057] Nitrogen Content (Dumas) The protein concentration is analyzed according to the ISO standard method (ISO 16634). The sample is converted into a gas by heating it in a combustion tube that gasifies the sample. Interfering components are removed from the resulting gas mixture. Nitrogen compounds in the gas mixture or their representative parts are converted into molecular nitrogen, which is quantified by a thermal conductivity detector. The nitrogen content is calculated by a microprocessor. The following coefficients were used to estimate the protein content based on nitrogen: 6.25 for casein and soybean.

[0058] OPA-N The OPA-N value was measured using the method developed by Frister H. et al. in 1988.

[0059] Sensory Evaluation of Bitterness Five trained sensory evaluators are used to test the sample as a 1% peptide solution in water at room temperature. To eliminate dilution effects, all casein hydrolysate samples are adjusted to contain only 1% peptide regardless of the amount of sugar added. The evaluators are provided with a reference (standard) (non-conjugated hydrolysate) for comparison, and the bitterness of that reference is set to 3. If any difference in bitterness can be detected, the evaluator assigns a low score if the bitterness is less and a high score if the bitterness is more. Thus, a low "bitterness score" means that the system has less bitterness.

[0060] Since the bitterness of casein is more than that of soybean peptides, the casein reference was administered at 1% peptide and the soybean peptides were administered at 5%. The conjugated peptides were then administered to contain the same weight of peptide as the reference (for this reason, the conjugated peptides were administered at a higher concentration considering the dilution effect of the sugar). Thus, the results should be evaluated as showing a reduction in bitterness compared to the same material without conjugation.

[0061] Average Molecular Weight The apparent average MW value was measured by measuring OPA-N (Frister H. et al. 1988). OPA-N does not indicate a direct index of MW, but only indicates the amount of final amine groups per sample. The apparent MW value can be obtained by dividing the total amount of nitrogen recognized (the total amount of nitrogen is measured by the above Dumas method) by the OPA-N value using the following formula: (Total N / OPA-N)*100 = Apparent MW

[0062] Materials The following materials were used: NaOH, Sigma-Aldrich (St. Louis, Missouri, USA), Casein (Acid Casein 741, Fonterra Ltd, Auckland, New Zealand), Hydrolyzed Pea Protein, (Peptipea, Triballat ingredients, Cedex, France), Glucose (Dextrose Monohydrat, Roquette, Lestrem, France), Alcalase 2.4 L FG, Novozymes (Novozymes A / S, Bagsvaerd, Denmark), Syringe Filter (0.2μm Whatman Red Rim Syringe Filter, Maidstone, UK)

[0063] References TIFF0007695189000002.tif86167 This specification includes the following embodiments. [1] A conjugated peptide, wherein the peptide is conjugated with at least one reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose, and mannose, has a degree of conjugation of at least 10%, and an increase in Gardner value of the conjugated peptide is less than 6. [2] The conjugated peptide according to [1], wherein the increase in Gardner value of the conjugated peptide is less than 4, preferably less than 3. [3] The conjugated peptide according to any one of [1] to [2], wherein the degree of conjugation is at least 15%, preferably at least 20%, 25%, 30%, 35% or 40%. [4] The conjugated peptide according to any one of [1] to [3], wherein the peptide is preferably a protein hydrolyzate derived from at least one selected from the group consisting of plant or animal proteins, particularly wheat, soybean, rice, potato, pea, sunflower, rapeseed, lupinus, and milk proteins, preferably selected from casein and pea protein. [5] The conjugated peptide according to [4], wherein the protein hydrolyzate is not filtered after hydrolysis and / or the pH is neutralized with an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid, and sulfuric acid. [6] The conjugated peptide according to [4] or [5], wherein the peptide is an enzymatically hydrolyzed protein hydrolyzate, preferably hydrolyzed by an endopeptidase, particularly alkaline protease. [7] The conjugated peptide according to any one of [1] to [6], wherein the degree of conjugation is 10 to 90%, preferably 15 to 70%, more preferably 20 to 50%, particularly 25 to 40%. [8] The conjugated peptide according to any one of [1] to [7], wherein the maximum molecular weight of the peptide is 2300 Da, preferably 2000, 1500, 1200, 1000 or 900 Da. [9] The conjugate peptide according to any one of [1] to [8], wherein the minimum molecular weight of the peptide is 650 Da, preferably 660, 670, 680, 690, 700, 710, 720, 750 or 800 Da.

[10] The conjugate peptide according to any one of [1] to [9], wherein the molecular weight of the peptide is 650 to 2400 Da, preferably 650 to 1000 Da.

[11] The conjugate peptide according to any one of [1] to

[10] , wherein the molar ratio of the reducing sugar to the peptide is 0.5 to 2.0, preferably 1.1 to 1.7.

[12] The conjugate peptide according to any one of [1] to

[11] , wherein the conjugate exists in a solid form, preferably a powder form, particularly a spray-dried form or a freeze-dried form.

[13] The conjugate peptide according to any one of [1] to

[12] , wherein the solubility of the conjugate peptide is at least 90%, preferably at least 91, 92, 93, 94, 95, 95, 97, 98 or 99%, particularly 100%.

[14] A method for masking the bitterness of a peptide, - mixing a peptide, preferably a hydrolyzed protein, and a reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose and mannose, preferably in a solution or dispersion; - heating the mixture to cause a Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C, preferably 60 to 85 °C, particularly 65 °C ± 3 °C, for 20 to 120 minutes, preferably 30 to 60 minutes; - spray-drying the composition to obtain a powder The method comprising the above steps.

[15] The method according to

[14] , wherein the mixture is heated until the Gardner value increase is less than 6, preferably less than 4, most preferably less than 3.

[16] The method according to

[14] or

[15] , wherein the molar ratio of the reducing sugar to the peptide is 0.5 to 2.0, preferably 1.1 to 1.7.

[17] The method according to any one of

[14] to

[16] , wherein the Maillard reaction is carried out at a pH of 7 to 9, preferably 8 to 8.5.

[18] The method according to any one of

[14] to

[17] , wherein the Maillard reaction conditions are applied until a degree of conjugation of at least 10%, preferably at least 15%, 20%, 25%, 30%, 35% or 40% is achieved.

[19] A method for preparing a non-bitter conjugate protein hydrolysate, comprising: - hydrolyzing the protein with at least one enzyme; - mixing the obtained protein hydrolysate and at least one reducing sugar selected from the group consisting of glucose, fructose, maltose, lactose, galactose, cellobiose, glyceraldehyde, ribose, xylose and mannose, preferably in a solution or dispersion; - heating the mixture to cause a Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C, preferably 60 to 85 °C, particularly 65 °C ± 3 °C for 20 to 120 minutes, preferably 30 to 60 minutes; - spray-drying the composition. A method comprising the above steps.

[20] The method according to

[19] , wherein the mixture is heated until the Gardner value increase is less than 6, preferably less than 4, most preferably less than 3.

[21] The method according to

[19] or

[20] , wherein the enzyme is an endopeptidase, preferably alkaline protease.

[22] The method according to any one of

[19] to

[21] , wherein the protein hydrolysate is used without filtration after hydrolysis and / or the pH is neutralized by adding an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid and sulfuric acid.

[23] The method according to any one of

[19] to

[22] , wherein heating is applied until a degree of conjugation of at least 10%, preferably at least 15%, 20%, 25%, 30%, 35% or 40% is achieved.

[24] The method according to any one of

[19] to

[23] , wherein the Maillard reaction is carried out at a pH of 7 to 9, preferably 8 to 8.5.

[25] The method according to any one of

[19] to

[24] , wherein the molar ratio of the reducing sugar to the peptide is 0.5 to 2.0, preferably 1.1 to 1.7.

[26] Use of the conjugate peptide according to any one of [1] to

[13] or the conjugate protein hydrolysate obtained by the method according to any one of

[19] to

[25] for food, preferably beverages, baking products, chewing gum, sports nutrition foods, dietary supplements, confectionery, desserts, foam foods or prebiotics, pharmaceuticals, bioactive substances, animal feed products.

Claims

1. A method for masking the bitterness of a protein hydrolysate, comprising: - Mixing a protein hydrolysate and a reducing sugar, wherein the protein hydrolysate is a casein hydrolysate, the reducing sugar is glucose, and the molar ratio of the reducing sugar to the protein hydrolysate is 1.1 to 1.7; - Heating the mixture to cause a Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C for 20 to 120 minutes, and the mixture is heated until the increase in Gardner value is less than 6, and the heating is applied until at least 10% degree of conjugation is achieved; - Spray-drying the composition to obtain a powder. A method comprising the above steps.

2. The method according to claim 1, wherein the mixture is heated until the increase in Gardner value is less than 4.

3. The method according to claim 1 or 2, wherein the Maillard reaction is carried out at a pH of 7 to 9.

4. The method according to any one of claims 1 to 3, wherein the Maillard reaction is applied until a degree of conjugation of 10 to 90% is achieved.

5. The method according to any one of claims 1 to 4, wherein the Maillard reaction conditions are applied until at least 15% degree of conjugation is achieved.

6. The method according to any one of claims 1 to 5, wherein the average molecular weight of the protein hydrolysate is 2300 Da or less.

7. The method according to any one of claims 1 to 6, wherein the average molecular weight of the protein hydrolysate is 650 Da or more.

8. The method according to any one of claims 1 to 7, wherein the average molecular weight of the protein hydrolysate is 650 to 2400 Da.

9. A method for preparing a conjugate protein hydrolysis product without bitterness, comprising: - hydrolyzing casein protein with at least one enzyme; - mixing the obtained casein protein hydrolysis product and glucose so that the molar ratio of reducing sugar to casein protein hydrolysis product is 1.1 to 1.7; - heating the mixture to cause a Maillard reaction between the two components, wherein the Maillard reaction is carried out at a temperature of 55 to 90 °C for 20 to 120 minutes, and the mixture is heated until the increase in Gardner value is less than 6, and the heating is applied until at least 10% of the conjugation degree is achieved; - spray-drying the composition A method comprising the above steps. **Claim 10** The method according to claim 9, wherein the mixture is heated until the increase in Gardner value is less than 4. **Claim 11** The method according to claim 9 or 10, wherein the enzyme is an endopeptidase. **Claim 12** The method according to any one of claims 9 to 11, wherein the protein hydrolysis product is used without filtration after hydrolysis and / or the pH is neutralized by adding an acid selected from the group consisting of citric acid, phosphoric acid, hydrochloric acid, lactic acid and sulfuric acid. **Claim 13** The method according to any one of claims 9 to 12, wherein the heating is applied until at least 15% of the conjugation degree is achieved. **Claim 14** The method according to any one of claims 9 to 13, wherein the Maillard reaction is carried out at a pH of 7 to 9.

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