Method for preparing protein peptide

Abstract

Provided in the present invention is a method for preparing a protein peptide. The method comprises: hydrolyzing a whey protein raw material under the simultaneous action of glutaminase and an ultrasonic treatment, wherein it is assumed that if the total hydrolysis time of the whey protein raw material is T, and the action time of the glutaminase and ultrasonic treatment is t, then T≤5t, and the temperature of hydrolysis is 65°C or less.

Description

Methods for preparing protein peptides Technical Field

[0001] This invention belongs to the food field, and more specifically, relates to a method for preparing protein hydrolysates, particularly a method for preparing a nutritional product rich in oligopeptides. Background Technology

[0002] Protein is an essential nutrient for the human body, playing a vital role in life activities. Infants and young children have underdeveloped digestive systems, making adequate and high-quality protein intake crucial for their growth, development, and physiological functions. Similarly, other groups with immature digestive systems face the same challenge.

[0003] Amino acids are the building blocks of proteins, and their composition and content have a profound impact on protein utilization. There are more than 20 amino acids that make up human proteins, of which nine—isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, and histidine—cannot be synthesized by the human body or their synthesis rate cannot meet the body's needs and must be obtained from food; these are called essential amino acids. In addition, there is tyrosine, which is a semi-essential amino acid.

[0004] Furthermore, undigested proteins are not easily absorbed. Generally, food proteins are better digested and absorbed after being hydrolyzed into amino acids and short peptides. Since saliva does not contain enzymes that hydrolyze proteins, the digestion of food proteins generally begins in the stomach, but mainly occurs in the small intestine. The amino acids or short peptides composed of 2-3 amino acids formed after digestion are absorbed in the small intestine. After being absorbed into the bloodstream, these amino acids mainly participate in the synthesis and breakdown of proteins in the body, along with the amino acids produced from the breakdown of proteins in tissues.

[0005] Clinical enteral nutrition has confirmed that oligopeptides can improve nitrogen absorption in the small intestine. When using them, patients with digestive and absorptive disorders tend to use this formula instead of monomeric amino acid formulas or whole protein formulas.

[0006] Reference 1 discloses a compound short peptide powder formulation and its preparation method, which includes the following raw materials: hydrolyzed whey protein powder, soybean peptide powder, wheat oligopeptides, crystalline fructose, citric acid, lemon flavor, potassium citrate, and sucralose. Its amino acid profile is close to the FAO / WHO model, exhibiting good absorption and suitability for individuals with digestive and absorptive disorders and those requiring protein supplementation. The total protein content of peptides with a molecular weight of 1000 Daltons or less reaches over 87.5%, and the total protein content of peptides with a molecular weight of 180 Daltons or less reaches over 15%.

[0007] Reference 2 provides a method for hydrolyzing whey protein using a complex enzyme. This method utilizes the complex enzyme to hydrolyze whey protein and subsequently produces peptone products from the hydrolysate. The method includes the following steps: preparing a whey protein aqueous solution of a certain concentration, centrifuging to defatt the protein, heating and holding the solution in a hot water bath for a period of time, adjusting the pH and temperature of the solution, adding a certain amount of endo- and exo-proteases for hydrolysis for a period of time, heating the hydrolysate to approximately 90°C, holding it at this temperature for about 10 minutes to inactivate the enzymes, then performing ultrafiltration and reverse osmosis concentration, and finally freeze-drying to obtain whey protein peptone powder. The peptone product produced according to this invention has a degree of hydrolysis of over 60%, is easily soluble in water, has a peptone yield of over 50%, and is of good quality, with an oligopeptide content of over 90%, making it easily digestible and absorbable.

[0008] Although some research has been conducted on protein peptide products, there is still room for further exploration to obtain products with even higher nutritional value.

[0009] References:

[0010] Reference 1: CN108685100A

[0011] Reference 2: CN103074404A Summary of the Invention

[0012] The problem the invention aims to solve

[0013] Existing oligopeptide nutritional compositions can generally be obtained through the following two methods:

[0014] One approach involves directly adding oligopeptide raw materials, as described in reference 1. However, this method is highly dependent on the raw materials and cannot control the state or composition of the oligopeptide raw materials. For example, when adding raw materials directly, the content of free amino acids or those with a molecular weight of 180 Da or less is relatively high; a molecular weight of 180 Da or less will significantly increase the osmotic pressure of the product.

[0015] Another approach is based on the hydrolysis of whey protein, as illustrated in reference 2. This route involves hydrolysis in the presence of enzymes to obtain small molecules, which are then further processed through membrane filtration and concentration to obtain the final product. However, membrane filtration can lead to the loss of some amino acids, resulting in a decrease in the amino acid score. Furthermore, for the same substance content, the viscosity increases, which may affect subsequent production processes.

[0016] Furthermore, to address the aforementioned problems, this invention primarily provides a novel hydrolysis route. This route uses whey protein as the target for hydrolysis, employing specific enzymes and hydrolysis conditions to obtain hydrolysate products with high low molecular weight peptide content. This eliminates the need for membrane filtration or other methods (to separate high molecular weight components) to obtain products rich in oligopeptides. Moreover, the products obtained under the aforementioned process conditions also possess suitable osmotic pressure.

[0017] Solution for solving the problem

[0018] It has been found that the above-mentioned technical problems can be solved by implementing the following technical solutions:

[0019] [1]. A method for preparing a protein peptide, wherein the method comprises:

[0020] This allows the whey protein raw material to be hydrolyzed simultaneously under the action of glutaminase and ultrasonic treatment.

[0021] Wherein, let:

[0022] The total hydrolysis time of the whey protein raw material is T; the reaction time for glutaminase and ultrasonic treatment is t.

[0023] but:

[0024] T≤5t

[0025] Furthermore, the hydrolysis temperature is below 65°C.

[0026] [2]. According to the method of [1], wherein the whey protein raw material is derived from one or more animal milks.

[0027] [3]. The method according to [1] or [2], wherein the amount of the glutaminase is 0.5% to 3% by weight of the whey protein on a dry weight basis.

[0028] [4]. The method according to any one of [1] to [3], wherein the ultrasonic frequency in the ultrasonic treatment is greater than 20 kHz to 30 kHz and the power is 400 to 600 W.

[0029] [5]. The method according to any one of [1] to [4], wherein, in addition to glutaminase, one or more of other endonucleases and exonucleases are optionally used in the hydrolysis.

[0030] [6]. The method according to any one of [1] to [5], wherein the entire hydrolysis process is carried out under the simultaneous action of glutaminase and ultrasonic treatment.

[0031] [7]. The method according to any one of [1] to [6], wherein the hydrolysis ends at the same time as the glutaminase and ultrasonic treatment are completed.

[0032] [8]. The method according to any one of [1] to [7], wherein T is no more than 120 min.

[0033] [9]. The method according to any one of [1] to [8], wherein after the hydrolysis is completed, the method further includes an enzyme inactivation step.

[0034]

[0010] . The method according to any one of [1] to [8], wherein the content of peptides with a molecular weight of 500 to 2000 Da in the protein peptide product obtained by hydrolysis is more than 80% by mass on a dry weight basis.

[0035] The effects of the invention

[0036] By implementing the above technical solution, the present invention can achieve the following technical effects:

[0037] 1) This invention creatively combines a specific enzyme with ultrasonic treatment in the hydrolysis of whey protein, thereby improving the efficiency of whey protein hydrolysis;

[0038] 2) The processing method of the present invention makes the distribution of the hydrolyzed protein peptides / oligopeptides more concentrated in the range of 500 to 2000 Da;

[0039] 3) From the hydrolysis results, the hydrolysis of the present invention, through the use of the above-mentioned control methods, yields hydrolysates with molecular weights that are neither too low nor too high. Therefore, even without membrane concentration and filtration, it can promote human absorption without causing an increase in osmotic pressure or loss of nutrients.

[0040] 4) In some preferred embodiments, in addition to glutaminase, one or more other endonucleases and exonucleases can be used, which not only takes into account the above effects but also the flavor of the protein peptide. Detailed Implementation

[0041] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. Numerous specific details are set forth in the following detailed description to better illustrate the invention. Those skilled in the art will understand that the invention can be practiced without certain specific details.

[0042] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0043] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0045] In this invention, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0046] In this invention, the terms "optional" or "optionally" are used to indicate the use or non-use of certain substances, components, execution steps, application conditions, etc.

[0047] In this invention, “Da” is used to represent the unit of molecular weight, “Dalton”.

[0048] In this invention, unless otherwise specified, "room temperature" generally refers to a temperature of 23±2℃.

[0049] In this invention, unless otherwise stated, "%" refers to weight or mass percentage.

[0050] In this instruction manual, "infants and toddlers" refers to the human group under the age of 3 years.

[0051] In this instruction manual, "ash" and "salt" have the same meaning. They refer to the series of physical and chemical changes that occur when food is burned at high temperatures. In the end, the organic components volatilize and dissipate, while the inorganic components (mainly inorganic salts and oxides) remain. These residues are called ash, which includes soluble ash and insoluble ash.

[0052] In this specification, the terms "about," "substantially," or "essentially" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in the present invention are modified with "about." Here, "about" generally means that the standard deviation of the actual value from the theoretical model or theoretical data is within 3%, preferably 2%, more preferably 1%.

[0053] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0054] This invention mainly provides a method for preparing protein peptide products by hydrolysis of whey protein. This invention is mainly based on the following insights:

[0055] While existing methods for controlling whey protein hydrolysis have been explored, several issues have arisen. Firstly, while extensive hydrolysis yields more small peptides, it also leads to an undesirable increase in the osmotic pressure of the final product. Secondly, from the perspective of reducing osmotic pressure burden, the hydrolyzed products have a wide molecular weight distribution, necessitating membrane treatment to remove large molecules and resulting in nutrient loss. This invention, through research on hydrolysis control methods, unexpectedly discovered that combining glutaminase and ultrasonic treatment resulted in a suitable molecular weight distribution for the hydrolysate, with the majority of the hydrolysate molecules concentrated in the 500–2000 Da range. Consequently, even without membrane separation or other methods, hydrolyzed protein peptide products that are easily absorbed by the human body and have a low osmotic pressure burden can be obtained.

[0056] (Whey protein raw material)

[0057] The method for preparing the protein peptides of the present invention uses whey protein as a raw material.

[0058] In principle, there are no particular restrictions on the source of whey protein raw materials used in this invention. For example, it can be whey protein derived from various animal milks, such as cow's milk, sheep's milk, horse's milk, camel's milk, etc. Preferably, it can be derived from cow's milk.

[0059] Furthermore, there are no particular limitations on the methods for separating or purifying whey from the aforementioned animal milk. In some specific embodiments, the separation or purification methods include defatting to separate the fat components from the animal milk raw material. There are no particular limitations on the defatting method in principle; it can be carried out by methods such as centrifugation. Through defatting, at least 90% by mass, preferably 92% by mass, and more preferably 95% by mass, of the total fat in the animal milk raw material is separated.

[0060] In some specific implementations, the aforementioned separation or purification also includes protein separation from the (defatted) raw milk. Protein separation primarily involves separating casein. There are no particular limitations on the method for separating casein. For example, casein can be separated by adjusting the pH of the raw milk, adding an acidic substance to cause the casein to coagulate and precipitate near its isoelectric point (acid whey); or, whey components can be separated simultaneously with cheese formation by optionally adding coagulants, fermentation agents, or other ingredients to the raw milk (sweet whey). Alternatively, membrane filtration can be used, employing a filter membrane with a suitable pore size to retain and separate protein components of different molecular weights, thereby obtaining a component containing whey protein.

[0061] In some specific embodiments, the separation or purification described above further includes desalting and optionally concentration steps. Preferably, the whey protein-enriched component can be desalted and optionally concentrated after defatting and protein separation. There are no particular limitations on the desalting process in principle; for example, it can be performed using membrane filtration (nanofiltration (NF) and / or electrodialysis). In some preferred embodiments, desalting can remove more than 80% by mass of inorganic salts, preferably more than 90% by mass, and more preferably more than 95% by mass of inorganic salts. The concentration step can be performed simultaneously with or after desalting, typically using ultrafiltration, washing filtration, or reverse osmosis to obtain, for example, a whey protein feedstock with a higher protein content.

[0062] The whey protein-containing components obtained from the various possible processes described above can be further dried to obtain whey protein solids. Typically, spray drying or similar methods can be used to obtain solid whey protein powder.

[0063] Furthermore, the whey protein raw material of this invention can be prepared in-house using the methods described above, or it can be purchased commercially, such as various commercially available concentrated whey protein powders, whey protein isolates, whey protein liquids, and other high-whey protein raw materials. Preferably, the whey protein raw material of this invention can be concentrated whey protein powder.

[0064] Regarding the whey protein content in the whey protein raw material of the present invention, it is related to the preparation method of the whey protein raw material described above. In some specific embodiments, the whey protein content in the whey protein raw material (e.g., based on the total dry weight of the whey protein raw material) can be 70% by mass or more, preferably 75-95% by mass, for example, 80-92% by mass, 85-90% by mass, etc.

[0065] Furthermore, the whey protein of the present invention can be an unhydrolyzed whole protein, or a whey protein that has undergone some degree of hydrolysis but still needs or can still be further hydrolyzed.

[0066] (Hydrolysis system)

[0067] The hydrolysis system of the present invention can be a system in which the above-mentioned whey protein raw material is mixed with water. The content of whey protein raw material in this mixture is typically 8% to 12% by mass, preferably 9% to 11% by mass.

[0068] There are no particular restrictions on the temperature of the hydrolysis system. The above mixing or dissolution can be carried out at room temperature and then left for use. In some specific implementation schemes, the temperature for preparing the above hydrolysis system does not exceed 65°C, preferably not exceeding 60°C.

[0069] (Hydrolysis control)

[0070] In this invention, glutaminase and ultrasonic treatment are used in combination during hydrolysis, and both are applied substantially simultaneously or concurrently to the hydrolysis system. Furthermore, there are no particular limitations on the hydrolysis temperature; it is typically below 65°C, preferably 50°C to 60°C, and examples include 52°C, 55°C, and 62°C.

[0071] In some specific embodiments, the amount of glutaminase used can be 0.5% to 3.0% by mass of the whey protein raw material (on dry weight), preferably 0.8% to 2.5% by mass, and more preferably 1.0% to 1.5% by mass.

[0072] In some other specific embodiments, the frequency of the ultrasound waves used in the ultrasonic treatment is not particularly limited in principle, and can typically be greater than 20 kHz to 30 kHz, preferably 21 kHz to 27 kHz. There are also no particular limitations on the instruments used for the ultrasonic treatment; readily available commercial instruments can be used to provide continuous ultrasound waves.

[0073] There are no particular restrictions on the application of glutaminase and sonication, as long as the duration of the combined treatment is guaranteed.

[0074] Specifically, for the total hydrolysis time T of the whey protein raw material of the present invention, the time t for applying glutaminase and ultrasonic treatment satisfy the following relationship:

[0075] T≤5t

[0076] That is, the duration of t is more than 20% of the duration of T.

[0077] In a further preferred embodiment, the relationship between T and t can be T≤4t, T≤3t, and T≤2t, etc.

[0078] There is no particular limitation on the specific duration of T in principle. However, in some preferred embodiments, from an efficiency perspective, T can be no more than 120 minutes, preferably 60 to 100 minutes, such as 70 minutes, 80 minutes, or 90 minutes. Correspondingly, t can be more than 12 minutes, preferably more than 20 minutes, and more preferably more than 30 minutes. It should be noted that t in this invention refers only to the time during which glutaminase and ultrasonic treatment act simultaneously.

[0079] Furthermore, in some specific embodiments, the application of the glutaminase and sonication is performed immediately upon the commencement of whey protein hydrolysis; in other specific embodiments, the application of the glutaminase and sonication is performed some time after the commencement of whey protein hydrolysis; in still other specific embodiments, the entire hydrolysis process ends when the action of the glutaminase and sonication ends.

[0080] In the hydrolysis process of this invention, in addition to the glutaminase described above, other types of enzymes may be used without limitation, primarily for controlling the taste of the final product. In some specific embodiments, these other enzymes may be used in combination of multiple enzymes.

[0081] There are no particular restrictions on the use of these other enzymes in principle; they can be added at any time during hydrolysis, as long as sufficient efficacy is guaranteed. In some preferred embodiments of the present invention, these other enzymes can be added at the initial stage of hydrolysis, especially at the beginning of hydrolysis.

[0082] Other enzymes that can be used in this invention may include endopeptides and exopeptides. In some specific embodiments, the endopeptide may be one or more of alkaline proteases, trypsin, pepsin, acidic proteases, and neutral proteases; the exopeptide may be one or more of protein deamidases and flavor proteases. In some preferred embodiments, considering hydrolysis efficiency, suppression of undesirable flavor and texture, and mitigation of the impact of high-temperature enzyme inactivation on protein components, the enzymes used in the hydrolysis step of this invention may be a combination of alkaline proteases and flavor proteases in a certain proportion. It has been found that such an enzyme combination not only achieves the aforementioned hydrolysis efficiency but also maintains the aforementioned flavor and texture, while also making it relatively easy to obtain the hydrolysate (protein peptide components) with the molecular weight distribution desired by this invention. More preferably, the ratio of the alkaline protease to the flavor protease may be 1:1 to 3, for example, 2:3, 1:2, 2:5, 3:7, 1:3, etc.

[0083] Furthermore, there is no particular limitation on the total amount of the enzymes used, and it can be determined based on the conventional dosage corresponding to the characteristics of each enzyme in the art. In some preferred embodiments, the total amount of the enzymes used can be 1 to 3% by mass of the whey protein raw material (dry weight), for example, 2 to 2.5% by mass.

[0084] There are no special requirements for the other conditions of hydrolysis control described above in this invention. For example, commonly used pH adjusters and buffers in the art can be used to make the hydrolysis system be under acidic or alkaline conditions suitable for enzyme activity.

[0085] In some preferred hydrolysis control methods of the present invention:

[0086] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, then glutaminase is added and ultrasonic treatment is initiated until hydrolysis is completed; or

[0087] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, then glutaminase is added and ultrasonic treatment is initiated. Simultaneously, a complex enzyme containing endo- and exo-proteases is added for hydrolysis until the hydrolysis is complete; or

[0088] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, and a complex enzyme containing endo- and exo-proteases is added to initiate hydrolysis. After hydrolysis has proceeded for a period of time, glutaminase is added and ultrasonic treatment is initiated until hydrolysis is completed; or

[0089] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, and a complex enzyme containing endopeptidase and exopeptidase is added to initiate hydrolysis. After hydrolysis has proceeded for a period of time, glutaminase is added and ultrasonic treatment is initiated. The ultrasonic treatment is then stopped, and hydrolysis continues until it is completed; or

[0090] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, and a complex enzyme containing endopeptidase and exopeptidase is added to initiate hydrolysis, while simultaneously activating ultrasound. After hydrolysis has proceeded for a period of time, glutaminase is added to continue hydrolysis until it is complete; or

[0091] The hydrolysis system of the present invention is placed in a suitable temperature and pH value, and a complex enzyme containing endopeptidase and exopeptidase and glutaminase is added to start the hydrolysis process. After hydrolysis has been carried out for a period of time, sonication is turned on to continue the hydrolysis until the hydrolysis is completed.

[0092] (Post-processing)

[0093] There are no particular restrictions on the post-treatment after the above hydrolysis, which can mainly include enzyme inactivation treatment, drying treatment, etc.

[0094] After the hydrolysis step, enzyme inactivation treatment can usually be performed to terminate the hydrolysis reaction and obtain hydrolyzed whey protein solution.

[0095] Specifically, in the enzyme inactivation step, there are no particular requirements for the inactivation temperature and time; they can be determined by referring to the characteristics of enzymes in the art. In some specific embodiments of the present invention, the enzyme inactivation temperature can be 90–120°C, preferably 105–110°C, and the enzyme inactivation time can be 10–60 seconds, preferably 15–30 seconds.

[0096] For drying, it can be carried out after enzyme inactivation. This process may optionally include concentrating the system for subsequent spray drying.

[0097] There are no particular restrictions on the concentration step in principle. Preferably, evaporation concentration can be used. In some specific implementations, a triple-effect evaporator can be used to concentrate the enzyme-inactivated system.

[0098] For the drying step, spray drying is typically used to obtain hydrolyzed whey protein peptides in solid powder form. There are no particular limitations on the temperature, pressure, and other conditions for spray drying; standard operating procedures in the field can be followed.

[0099] (Properties of protein peptide products)

[0100] The protein peptide products obtained through the above-described hydrolysis process of this invention have excellent molecular weight distribution.

[0101] Specifically, the protein peptides obtained by the method of the present invention have a molecular weight distribution concentrated between 500 and 2000 Da. In some preferred embodiments, the content of protein peptides with a molecular weight of 500 to 2000 Da can reach more than 70% by mass, more preferably more than 75% by mass, and even more than 80% by mass, based on the dry weight of the obtained protein peptides.

[0102] In a further preferred embodiment, the content of protein peptides with a molecular weight of 1000-2000 Da, based on the dry weight of the obtained protein peptides, can reach 40% by mass or more, more preferably 42% by mass or more, or even 45% by mass or more.

[0103] (Food manufacturing)

[0104] Based on the above-mentioned method for preparing protein peptides, the present invention also provides a food preparation method that includes the above-mentioned method for preparing protein peptides.

[0105] Such food can exist in liquid, semi-solid, or solid form at room temperature.

[0106] For example, the food includes liquid milk containing hydrolyzed whey protein, powder containing hydrolyzed whey protein, or solid food containing hydrolyzed whey protein.

[0107] In principle, such food is suitable for all people, especially for special groups such as infants, children, pregnant women, and the elderly. More specifically, the food includes one or more of the following: infant formula milk powder, infant complementary food, milk powder for middle-aged and elderly people, and food for special medical purposes.

[0108] Example

[0109] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0110] Example 1:

[0111] The whey protein content was 85% by dry weight; the whey protein was dissolved at a solution temperature of 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Simultaneously, glutaminase (enzyme activity 500 U / g, addition amount 1%) was added, and hydrolysis began (start of timing). At the start of hydrolysis, the solution was ultrasonically treated (ultrasonic frequency 28 kHz, power 600 W). The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes. The enzyme inactivation temperature was 110℃, and the time was 21 seconds.

[0112] Example 2:

[0113] The whey protein content was 85% by dry weight; the solution temperature during whey protein dissolution was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting time). After 30 minutes of hydrolysis, glutaminase (enzyme activity 520 U / g, addition amount 1.28%) was added. Simultaneously, the solution was ultrasonicated (ultrasonic frequency 25 kHz, power 470 W). The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 111℃, and the time was 22 seconds.

[0114] Example 3:

[0115] The whey protein content was 85% by dry weight; the whey protein was dissolved at a solution temperature of 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting time). After 60 minutes of hydrolysis, glutaminase (enzyme activity 600 U / g, addition amount 1.5%) was added. Simultaneously, the solution was ultrasonicated (ultrasonic frequency 22 kHz, power 400 W). The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 112℃, and the time was 20 seconds.

[0116] Control group:

[0117] The whey protein content was 85% by dry weight; the solution temperature for dissolving the whey protein was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2% of the solution concentration, and the ratio of the two enzymes was 3:7). Hydrolysis began (starting the timer) at 55.1℃. After 90 minutes of hydrolysis, the enzymes were inactivated. The enzyme inactivation temperature was 110℃, and the inactivation time was 20 seconds.

[0118] Comparative Example 1:

[0119] The whey protein content was 85% by dry weight; the solution temperature for dissolving the whey protein was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Simultaneously, glutaminase (enzyme activity 500 U / g, addition amount 1.1%) was added, and hydrolysis began (starting time). The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 108℃, and the time was 21 seconds.

[0120] Comparative Example 2:

[0121] The whey protein content was 85% by dry weight; the solution temperature for dissolving the whey protein was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting time). After 30 minutes of hydrolysis, glutaminase (enzyme activity 520 U / g, addition amount 1.28%) was added. The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 110℃, and the time was 21 seconds.

[0122] Comparative Example 3:

[0123] The whey protein content was 85% by dry weight; the solution temperature for whey protein dissolution was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting time). After 60 minutes of hydrolysis, glutaminase (enzyme activity 600 U / g, addition amount 1.5%) was added. The hydrolysis temperature was 55.1℃, and the enzyme was inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 109℃, and the time was 22 seconds.

[0124] Comparative Example 4:

[0125] The whey protein content was 85% by dry weight; the solution temperature during whey protein dissolution was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (start of timing). After hydrolysis started, the equipment was turned on to sonicate the solution (ultrasonic frequency 28kHz, power 600W). The hydrolysis temperature was 55.1℃, and the enzymes were inactivated after 90 minutes of hydrolysis. The enzyme inactivation temperature was 110℃, and the time was 19 seconds.

[0126] Comparative Example 5:

[0127] The whey protein content was 85% by dry weight; the solution temperature during whey protein dissolution was 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting the timer). After 30 minutes of hydrolysis, the solution was ultrasonically treated (ultrasonic frequency 25 kHz, power 470 W) at a temperature of 55.1℃. After 90 minutes of hydrolysis, the enzymes were inactivated at 110℃ for 20 seconds.

[0128] Comparative Example 6:

[0129] The whey protein content was 85% by dry weight; the whey protein was dissolved at a solution temperature of 58℃, and the solution concentration was 10.5%. Potassium hydroxide was added to adjust the pH to 6.8. Alkaline protease and flavor protease were added (the enzyme addition ratio was 2.2% of the protein concentration in the solution, with a ratio of 3:7). Hydrolysis began (starting time). After 60 minutes of hydrolysis, the solution was ultrasonically treated (ultrasonic frequency 22 kHz, power 400 W) at a hydrolysis temperature of 55.1℃. After 90 minutes of hydrolysis, the enzymes were inactivated at 112℃ for 20 seconds.

[0130] <Molecular weight distribution>

[0131] Each of the above experiments was repeated three times, and the results are expressed as mean ± standard deviation.

[0132] The molecular weight distribution of the obtained protein hydrolysates is shown in Table 1 below:

[0133] Using glutaminase combined with ultrasound, the proportion of 500-2000 Da peptides in the hydrolysis products reached more than 80%, of which 1000-2000 Da accounted for more than 45%.

[0134] Table 1

[0135] <Osmotic pressure and taste>

[0136] Osmolarity is a commonly used medical indicator and is closely related to human health. Infants' kidneys are not fully developed and their physiological functions are immature; long-term feeding of formula with high osmolarity increases the probability of kidney damage. The osmolarity of breast milk is approximately 292–300 mOsm / kg H2O. Generally speaking, liquid formula with an osmolarity >300 mOsm / kg H2O is called high-osmolarity formula; formula with an osmolarity between 280 and 300 mOsm / kg H2O is called isotonic formula (osmolarity is measured using an osmometer, with each sample measured three times).

[0137] On the other hand, during the hydrolysis of protein raw materials, their structure undergoes a series of changes, specifically the cleavage of peptide chains to generate polypeptides with different molecular weights. At the same time, some hydrophobic amino acids that were originally contained inside the protein are exposed. Therefore, most hydrolyzed proteins have a certain bitter taste (quinine was prepared into solutions with concentrations of 12.5 and 62.5 μmol / L, and bitterness was defined as 1 and 10, respectively. The bitterness value of the samples was tested. Each sample was evaluated by 10 people, and the average value was taken. Each sample was repeated 3 times).

[0138] Using maltodextrin / lactose and blended edible vegetable oil as the main raw materials, the protein hydrolysate from control group 1 (accounting for 27% of the energy supply ratio of the formula) and other ingredients were added. After being mixed evenly, homogenized, and sterilized, the mixture was concentrated and spray-dried to produce sample A. The main process parameters were: homogenization 25 MPa; sterilization (88℃~90℃, 15s); spray drying (inlet air temperature 160℃~180℃, outlet air temperature 85℃~90℃).

[0139] All other raw materials and processes were the same. The protein hydrolysate was derived from Comparative Example 1 and used to prepare Sample B. The remaining samples were prepared in the same manner.

[0140] The osmotic pressure and taste of the milk powder produced using the methods described in Examples 1 and 2 were also greatly improved, as shown in Table 2 below:

[0141] Table 2

[0142] <In vitro simulated gastrointestinal digestion experiment>

[0143] 1. Materials and Methods

[0144] 1.1 Main Reagents

[0145] NaCl, HCl, KCl, NaOH, trichloroacetic acid, potassium dihydrogen phosphate, sodium hydrogen phosphate, magnesium chloride, ammonium carbonate, pepsin, and trypsin were all sourced from Sigma-Aldrich.

[0146] 1.2 Main Instruments and Equipment

[0147] High-speed centrifuge, oscillating constant temperature water bath, magnetic stirrer (IKA RH-A Basic magnetic stirrer).

[0148] 1.3 Experimental Procedure

[0149] (1) Preparation of gastric simulated solution: Weigh 0.2g NaCl, add 1.3mL of 1mol / L HCl, dissolve in 80mL of pure water, add 0.1mol / L HCl to adjust the pH of the solution to 4.0, and finally bring the volume to 100mL with pure water, labeling it solution a. Weigh 5mg of gastric lipase and 0.5mg of pepsin and add to 5mL of solution a, stir magnetically at room temperature for 10min to obtain simulated gastric juice.

[0150] (2) Preparation of intestinal simulation solution: Weigh 8.78g NaCl, 0.37g KCl, and 6.60g CaCl2, dissolve them in pure water, add 0.2mol / L NaOH to adjust the pH of the solution to 6.5, and finally make up to 1L with pure water, labeling it solution b. Weigh 5mg bile salts and 10mg pancreatic enzymes and dissolve them in 10mL of solution b. Stir magnetically at 37℃ for 10min to obtain the simulated small intestinal digestive fluid.

[0151] (3) Simulated gastric digestion: Prepare a sample with a protein content of 4% in an Erlenmeyer flask, add the sample solution to SGF, adjust the pH to 3.0 with hydrochloric acid solution, add CaCl2(H2O)2 solution, add pepsin solution to make the pepsin activity in the final digestion solution reach 2000U / mL, then add water to make up the volume so that the volume ratio of sample solution to SGF is 1:1, place the sample in a shaking water bath at 37℃, and take samples at 0, 5, 10, 30, 60 and 120 min respectively, and adjust the pH to 7.0 to inactivate enzymes.

[0152] (4) Simulated intestinal digestion: After 120 min of simulated gastric digestion, the pH was adjusted to 7.0 with NaOH solution to inactivate the enzyme. Then, SIF, CaCl2(H2O)2 solution, and trypsin solution were added to make the trypsin activity in the final digestion solution reach 100 U / mL. Water was then added to make up the volume so that the volume ratio of sample solution to SIF was 1:1. The sample was placed in a shaking water bath at 37℃ and samples were taken at 60 and 120 min of reaction, respectively. The enzyme was inactivated by boiling water bath for 5 min.

[0153] 1.4 Sample Preparation

[0154] After the sample was removed, an equal volume of 24% trichloroacetic acid (TCA) solution was added to precipitate it. The sample was then centrifuged at 9000 r / min for 20 min, and the supernatant was taken to detect the protein content.

[0155] 1.5 Statistical Analysis and Calculation

[0156] Each sample underwent at least three simulated digestion experiments, and the protein digestibility was measured and the average value was taken. The results are shown in Table 3 below.

[0157] Protein digestibility = (M1 - M0) ÷ M2 × 100%

[0158] M0 represents the total protein content in the supernatant of the blank control (g); M1 represents the total protein content in the supernatant of the sample (g); and M2 represents the total protein content in the sample (g).

[0159] Table 3

[0160] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0161] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a protein peptide, characterized in that, The method includes: This allows the whey protein raw material to be hydrolyzed simultaneously under the action of glutaminase and ultrasonic treatment. Wherein, let: The total hydrolysis time of the whey protein raw material is T; the reaction time for glutaminase and ultrasonic treatment is t. but: T≤5t Furthermore, the hydrolysis temperature is below 65°C.

2. The method according to claim 1, characterized in that, The whey protein raw material is derived from one or more animal milks.

3. The method according to claim 1 or 2, characterized in that, The amount of glutaminase used is 0.5% to 3% by weight of the whey protein on a dry weight basis.

4. The method according to any one of claims 1 to 3, characterized in that, The ultrasonic frequency in the ultrasonic treatment is greater than 20kHz to 30kHz, and the power is 400 to 600W.

5. The method according to any one of claims 1 to 4, characterized in that, In the hydrolysis, in addition to glutaminase, optionally one or more other endonucleases or exonucleases may be used.

6. The method according to any one of claims 1 to 5, characterized in that, The entire hydrolysis process was carried out simultaneously under the action of glutaminase and ultrasonic treatment.

7. The method according to any one of claims 1 to 6, characterized in that, The hydrolysis ends simultaneously with the termination of the glutaminase and ultrasonic treatment.

8. The method according to any one of claims 1 to 7, characterized in that, The time T is no more than 120 minutes.

9. The method according to any one of claims 1 to 8, characterized in that, After the hydrolysis is completed, the step of inactivating enzymes is also included.

10. The method according to any one of claims 1 to 8, characterized in that, The protein peptide product obtained by hydrolysis has a peptide content of 80% by mass with a molecular weight of 500 to 2000 Da, based on dry weight.

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