Preparation method of protein peptide

Abstract

The invention provides a preparation method of protein peptide, which comprises the following steps: hydrolyzing a whey protein raw material under the simultaneous action of glutaminase and ultrasonic treatment, and setting the total hydrolysis time of the whey protein raw material to be T; when the action time of glutaminase and ultrasonic treatment is t, T is less than or equal to 5t, and the hydrolysis temperature is 65 DEG C or below.

Description

Technical Field

[0001] The invention belongs to the field of food, and more specifically, relates to a method for preparing a protein hydrolysate, in particular to a method for preparing a nutritional product rich in oligopeptides. Background Art

[0002] Protein is an essential nutrient for the human body and plays an important role in life activities. The digestive system of infants and young children is not fully developed, and adequate and high-quality protein intake is crucial to their growth, development and physiological functions. Similarly, the same problem exists for other people with imperfect digestive systems.

[0003] Amino acids are the building blocks of proteins, and the composition and content of amino acids have a profound impact on the utilization of proteins. There are more than 20 kinds of amino acids that make up human proteins, of which 9, namely isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and histidine, cannot be synthesized by the human body or the synthesis speed cannot meet the needs of the body, and they must be provided by food. They are called essential amino acids; in addition, there is tyrosine, which is a semi-essential amino acid.

[0004] In addition, protein is not easily absorbed without digestion. Generally, food protein can be better digested and absorbed after being hydrolyzed into amino acids and short peptides. Since saliva does not contain enzymes that hydrolyze protein, the digestion of food protein generally starts in the stomach, but mainly in the small intestine; the amino acids or short peptides composed of 2 to 3 amino acids formed after digestion are absorbed in the small intestine. After being absorbed into the blood, the amino acids mainly participate in the synthesis and decomposition of proteins in the body together with the amino acids produced by the decomposition of tissue proteins in the body.

[0005] Clinical enteral nutrition has confirmed that oligopeptides can improve the absorption of nitrogen in the small intestine. When used, they tend to be used to replace monomeric amino acid formulas or whole protein formulas for patients with digestive and absorption disorders.

[0006] Reference 1 discloses a composite short peptide powder formula and preparation method thereof, which includes the following raw materials: hydrolyzed whey protein powder, soy peptide powder, wheat oligopeptide, crystalline fructose, citric acid, lemon essence, potassium citrate, sucralose. Its amino acid pattern is close to the FAO / WHO pattern, has good absorption effect, and is suitable for people with digestive and absorption disorders and those who need protein supplementation. The content of peptides with a molecular weight of less than or equal to 1000 Daltons in the total protein reaches more than 87.5%, and the content of peptides with a molecular weight of less than or equal to 180 Daltons in the total protein reaches more than 15%.

[0007] Reference 2 provides a method for hydrolyzing whey protein with a composite enzyme, which utilizes the composite enzyme to hydrolyze whey protein and then produces a peptone product with the hydrolyzate. The method comprises the following steps: preparing a whey protein aqueous solution of a certain concentration, centrifugation and defatting, heating and keeping warm in a hot water bath for a period of time, adjusting the pH value and temperature of the feed solution, adding a certain amount of endo- and exo-proteases for composite hydrolysis for a period of time, heating the hydrolyzate to about 90°C, keeping warm for about 10 minutes to inactivate the enzyme activity, then filtering by ultrafiltration and concentrating by reverse osmosis, and finally freeze-drying to obtain a whey peptone powder. The peptone product produced according to the present invention has a hydrolysis degree of more than 60%, is easily soluble in water, has a peptone yield of more than 50%, and has good quality, wherein the oligopeptide content is more than 90%, and is easy to digest and absorb.

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

[0009] References:

[0010] Reference 1: CN108685100A

[0011] Reference 2: CN103074404A Summary of the invention

[0012] Problem that the invention aims to solve

[0013] Existing oligopeptide nutritional compositions can usually be obtained in the following two ways:

[0014] One is to directly add oligopeptide raw materials, such as in Reference 1, but this scheme is highly dependent on the raw materials, and the state or composition of the oligopeptide raw materials cannot be controlled. For example, when the raw materials are directly added, the content of free amino acids or less than or equal to 180Da is high; molecular weight of 180Da and below will significantly increase the osmotic pressure of the product;

[0015] The other is a hydrolysis route based on whey protein, such as cited in Reference 2, which obtains small molecular substances through hydrolysis in the presence of enzymes, and further obtains products through membrane filtration and concentration. However, membrane filtration products will cause some amino acids to be lost and the amino acid score to decrease. On the other hand, under the same substance content, the viscosity increases, which may affect the subsequent production process.

[0016] Further, in view of the above problems, the present invention mainly provides a new hydrolysis route, which uses whey protein as the hydrolysis object, uses specific enzymes and hydrolysis conditions to obtain a hydrolyzate with a high content of low molecular weight peptides, and can also obtain a product rich in oligopeptides without the need for membrane filtration or other methods (separation of high molecular weight components). In addition, the product obtained under the above process conditions also has a suitable osmotic pressure.

[0017] Solutions for solving problems

[0018] It has been found that the above 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] The whey protein raw material is hydrolyzed under the simultaneous action of glutaminase and ultrasonic treatment.

[0021] Among them, suppose:

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

[0023] but:

[0024] T≤5t

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

[0026] [2] The method according to [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 glutaminase used 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 other endonucleases or 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 ends.

[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 comprises an enzyme inactivation step.

[0034]

[10] 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 80% by weight or more on a dry weight basis.

[0035] Effects of the Invention

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

[0037] 1) The present invention creatively combines specific enzymes with ultrasonic treatment in whey protein hydrolysis, thereby improving the efficiency of whey protein hydrolysis;

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

[0039] 3) From the hydrolysis results, the hydrolysis of the present invention uses the above control means to obtain a hydrolyzate with a molecular weight that is neither too low nor too high. Therefore, even without using 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, thereby taking into account not only the above-mentioned effects but also the flavor of the protein peptide. DETAILED DESCRIPTION

[0041] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details.

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

[0043] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0044] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0045] In the present invention, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0046] In the present invention, the use of "optional" or "optionally" indicates that certain substances, components, execution steps, applied conditions and other factors are used or not used.

[0047] In the present invention, "Da" is used to represent the unit of molecular weight "Dalton", that is, "Dalton".

[0048] In the present invention, unless otherwise specified, the "normal temperature" used generally refers to a temperature of 23±2°C.

[0049] In the present invention, unless otherwise stated, "%" used herein means weight or mass percentage.

[0050] In this specification, "infants" are used to refer to a human group under the age of 3 years old.

[0051] In this specification, "ash" and "salt" have the same meaning, which refers to a series of physical and chemical changes that occur in food when burned at high temperature, and finally the organic components evaporate 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 term "about" or "substantially" or "essentially" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value essentially inevitably contains the standard deviation caused by the aforementioned testing device or method. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally means that the standard deviation of the actual value and the theoretical model or theoretical data is within 3%, preferably 2%, and more preferably 1%.

[0053] In the present invention, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0054] The present invention mainly provides a method for preparing a protein peptide product by hydrolyzing whey protein, and the present invention is mainly based on the following insights:

[0055] Although various control methods have been tried for the existing whey protein hydrolysis, on the one hand, although deep hydrolysis can obtain more small molecule peptides, the result will also lead to an undesirable increase in the osmotic pressure of the final product; on the other hand, from the perspective of reducing the osmotic pressure burden, the molecular weight distribution of the product obtained by hydrolysis is relatively wide, so it is necessary to remove the macromolecular components by membrane treatment methods to cause the loss of nutrients. The present invention, through the study of hydrolysis control methods, unexpectedly found that by combining glutaminase and ultrasonic treatment, the hydrolyzate obtains a suitable molecular weight distribution, that is, the molecular weight of most of the hydrolyzate is more concentrated in the range of 500 to 2000Da. As a result, even without using membrane separation and other means, a hydrolyzed protein peptide product that is easy for human body absorption and has a small osmotic pressure burden can be obtained.

[0056] (Whey protein raw materials)

[0057] The preparation method of the protein peptide of the invention uses whey protein as raw material.

[0058] In principle, there is no particular limitation on the source of the whey protein raw material in the present invention. For example, the whey protein can be derived from various animal milks, such as cow's milk, goat's milk, horse's milk, camel's milk, etc. Preferably, it can be derived from cow's milk.

[0059] There is no particular limitation on the method for separating or purifying whey from the animal milk. In some specific embodiments, the separation or purification means includes a defatting process to separate the fat component from the animal milk raw material. In principle, there is no particular limitation on the defatting method, which can be performed by centrifugation or the like. By the defatting process, 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 embodiments, the above separation or purification also includes separating proteins from the raw milk (which has been defatted). The separation of proteins is mainly to separate casein. There is no particular limitation on the method of separating casein. For example, the pH of the raw milk can be adjusted by adding acidic substances to cause casein to coagulate and precipitate near its isoelectric point, thereby separating (acid whey); or, by optionally adding coagulants, leavening agents and other ingredients to the raw milk, the whey component (sweet whey) is separated while the cheese is formed. In addition, it is also possible to obtain a component containing whey protein by using a filter membrane with a suitable pore size to intercept and separate protein components of different molecular weights by membrane filtration.

[0061] In some specific embodiments, the above-mentioned separation or purification also includes desalting and optional concentrating steps. Preferably, after defatting and protein separation, the composition enriched with whey protein is desalted and optionally concentrated. For the processing of desalting, there is no particular restriction in principle, for example, modes such as membrane filtration (nanofiltration (NF) and / or electrodialysis) can be used to carry out. In some preferred embodiments, by desalting, inorganic salts more than 80 mass %, preferably more than 90 mass %, more preferably more than 95 mass % can be removed. For the step of concentrating, it can be carried out while or after desalting, and typically ultrafiltration, filter wash or reverse osmosis can be used to obtain a whey protein raw material with a higher protein content.

[0062] The whey protein-containing components obtained by the above-mentioned possible various process treatments can be further dried to obtain whey protein solids. Typically, solid whey protein powder can be obtained by spray drying or the like.

[0063] In addition, the whey protein raw material of the present invention can be prepared by the above method or purchased through commercial channels, such as various commercially available concentrated whey protein powders, isolated whey protein powders, whey protein liquids and other high whey protein content raw materials. Preferably, the whey protein raw material of the present invention can use concentrated whey protein powder.

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

[0065] Furthermore, the whey protein of the present invention may be whole protein that has not been hydrolyzed, or may be whey protein that has been hydrolyzed to a certain extent but still needs or can be further hydrolyzed.

[0066] (Hydrolysis system)

[0067] The hydrolysis system of the present invention may be a system in which the whey protein raw material is mixed with water. The content of the whey protein raw material in the mixed system may generally be 8% to 12% by mass, preferably 9% to 11% by mass.

[0068] In principle, there is no particular restriction on the temperature of the hydrolysis system. The mixing or dissolving can be performed at room temperature and then set aside. In some specific embodiments, the temperature of the hydrolysis system does not exceed 65°C, preferably does not exceed 60°C.

[0069] (Hydrolysis Control)

[0070] The hydrolysis of the present invention uses glutaminase and ultrasonic treatment in combination, and the two are substantially synchronously or simultaneously applied to the hydrolysis system. In addition, there is no particular limitation on the hydrolysis temperature, which can usually be below 65°C, preferably 50°C to 60°C, including 52°C, 55°C, 62°C, etc.

[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 (based 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 ultrasonic wave in the ultrasonic treatment is not particularly limited in principle, and can generally be greater than 20kHz to 30kHz, and preferably can be 21kHz to 27kHz. For the instrument used for the ultrasonic treatment, there is no particular limitation in principle, and a commercially available instrument can be used to provide continuous ultrasonic waves.

[0073] Regarding the manner of applying glutaminase and ultrasonic treatment, there is no particular limitation in principle as long as the time for the combined treatment is ensured.

[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 satisfies 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 may be T≤4t, T≤3t, T≤2t, and the like.

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

[0079] Further, in some specific embodiments, the glutaminase and ultrasonic treatment are applied immediately from the beginning of the hydrolysis of whey protein; in other specific embodiments, the glutaminase and ultrasonic treatment are applied some time after the hydrolysis of whey protein begins; in other specific embodiments, when the glutaminase and ultrasonic treatment are finished, the entire hydrolysis process is also finished.

[0080] In the whole hydrolysis process of the present invention, in addition to the above-mentioned glutaminase, other types of enzymes can also be used without limitation. These other types of enzymes are mainly used to control the taste of the final product. In some specific embodiments, these other enzymes can be used in a composite form of multiple enzymes.

[0081] In principle, there is no particular limitation on the use of these other enzymes, and they can be added at any time during the hydrolysis process as long as sufficient efficacy can be ensured. In some preferred embodiments of the present invention, these other enzymes can be added at the early stage of the hydrolysis process, especially at the beginning of the hydrolysis process.

[0082] Other enzymes that can be used in the present invention may include endoproteases and exoproteases. In some specific embodiments, the endoproteases may be one or more of alkaline proteases, trypsin, pepsin, acidic proteases and neutral proteases; the exoproteases may be one or more of protein deamidase and flavor proteases. In some preferred embodiments, from the perspective of hydrolysis efficiency and inhibition of bad flavor and taste, and mitigation of the effect of enzyme inactivation high temperature treatment on protein components, the enzymes used in the hydrolysis step of the present invention may be a combination of alkaline proteases and flavor proteases in a certain ratio, and it has been found that such a combination of enzymes not only takes into account the above-mentioned hydrolysis efficiency, but also takes into account the above-mentioned flavor and taste, and can also more easily obtain the hydrolyzate (protein peptide component) with the desired molecular weight distribution of the present invention. More preferably, the ratio of alkaline protease to flavor protease may be 1:1-3, for example, 2:3, 1:2, 2:5, 3:7, 1:3, etc.

[0083] Furthermore, the total amount of the above enzymes is not particularly limited in principle and can be determined according to the conventional amount corresponding to the characteristics of each enzyme in the art. In some preferred embodiments, the total amount of the above enzymes can be used at 1 to 3% by mass of the whey protein raw material mass (dry weight), for example, 2 to 2.5% by mass.

[0084] In principle, there are no special conditions for the above-mentioned hydrolysis control of the present invention. For example, pH regulators and buffer substances commonly used in the art can be used to make the hydrolysis system under acid-base conditions suitable for enzyme activity.

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

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

[0087] The hydrolysis system of the present invention is placed at a suitable temperature and pH value, and then glutaminase is added and ultrasonic treatment is started, and a composite enzyme containing endo- and exo-proteases is added at the same time to perform hydrolysis until the hydrolysis is completed; or

[0088] The hydrolysis system of the present invention is placed at a suitable temperature and pH value, a composite enzyme comprising endo- and exo-proteases is added to start hydrolysis, and after the hydrolysis is carried out for a period of time, glutaminase is added and ultrasonic treatment is started until the hydrolysis is completed; or

[0089] The hydrolysis system of the present invention is placed at a suitable temperature and pH value, a composite enzyme comprising endo- and exo-proteases is added to start hydrolysis, and after hydrolysis is carried out for a period of time, glutaminase is added and ultrasonic treatment is started, the ultrasonic treatment is stopped, and the hydrolysis is continued until the hydrolysis is completed; or

[0090] The hydrolysis system of the present invention is placed at a suitable temperature and pH value, a composite enzyme comprising endo- and exo-proteases is added to start hydrolysis and ultrasound is simultaneously turned on, after hydrolysis is performed for a period of time, glutaminase is added, and hydrolysis is continued until the hydrolysis is completed; or

[0091] The hydrolysis system of the present invention is placed at a suitable temperature and pH value, and a composite enzyme comprising endo- and exo-proteases and glutaminase are added to start hydrolysis. After the hydrolysis is carried out for a period of time, ultrasound is turned on to continue the hydrolysis until the hydrolysis is completed.

[0092] (Post-processing)

[0093] There is no particular limitation on the post-treatment after the hydrolysis in principle, and the post-treatment may mainly include enzyme inactivation treatment, drying treatment, etc.

[0094] After the hydrolysis step, the whey protein may be inactivated to terminate the hydrolysis reaction and obtain a hydrolyzed whey protein solution.

[0095] Specifically, in the step of inactivating the enzyme, there is no special requirement for the inactivation temperature and time in principle, and it can be performed with reference to the characteristics of the enzyme in the art. In some specific embodiments of the present invention, the inactivation temperature can be 90-120°C, preferably 105-110°C, and the inactivation time can be 10-60s, preferably 15-30s.

[0096] The drying process can be carried out after the enzyme is inactivated, and the process can optionally include concentrating the system for subsequent spray drying.

[0097] In principle, there is no particular limitation on the concentration step. Preferably, an evaporation concentration method can be used. In some specific embodiments, a triple-effect evaporator can be used to concentrate the system after the enzyme is inactivated.

[0098] For the drying step, spray drying can be typically used to obtain the hydrolyzed whey protein peptide in a solid powder form. There is no particular limitation on the temperature, pressure and other conditions of spray drying, and the conventional operating specifications in the art can be referred to.

[0099] (Protein peptide product properties)

[0100] The protein peptide product obtained through the hydrolysis treatment step of the present invention has an excellent molecular weight distribution state.

[0101] Specifically, the molecular weight distribution of the protein peptides obtained by the method of the present invention is concentrated between 500 and 2000 Da. In some preferred embodiments, based on the dry weight of the obtained protein peptides, 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, or even more than 80% by mass.

[0102] In a further preferred embodiment, based on the dry weight of the obtained protein peptide, the content of the protein peptide with a molecular weight of 1000-2000 Da 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 the protein peptide, the present invention also provides a food method, which includes the above-mentioned method for preparing the protein peptide.

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

[0106] Exemplarily, 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 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 in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0110] Embodiment 1:

[0111] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the addition ratio of the enzyme is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7). At the same time, glutaminase (enzyme activity is 500U / g, and the addition amount is 1%) is added, and hydrolysis begins (timing starting point). At the beginning of hydrolysis, the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 28KHz, power is 600W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after 90 minutes. The enzyme inactivation temperature is 110°C and the time is 21s.

[0112] Embodiment 2:

[0113] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the addition ratio of the enzyme is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 30 minutes, glutaminase (enzyme activity is 520U / g, and the addition amount is 1.28%) is added, and at the same time, the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 25KHz, power is 470W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 111°C and the time is 22s.

[0114] Embodiment 3:

[0115] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the addition ratio of the enzyme is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 60 minutes, glutaminase (enzyme activity is 600U / g, and the addition amount is 1.5%) is added, and the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 22KHz, power is 400W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 112°C and the time is 20s.

[0116] Control group:

[0117] The whey protein content is 85% by dry weight; the liquid temperature is 58°C and the liquid concentration is 10.5% when the whey protein is dissolved. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2% of the liquid concentration, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). The hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 110°C and the time is 20 seconds.

[0118] Comparative Example 1:

[0119] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed temperature is 58°C and the feed concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2.2% of the feed protein concentration, and the ratio of the two enzymes is 3:7). At the same time, glutaminase (enzyme activity is 500U / g, and the addition amount is 1.1%) is added to start hydrolysis (timing starting point), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 108°C and the time is 21s.

[0120] Comparative Example 2:

[0121] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2.2% of the feed liquid protein concentration, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 30 minutes, glutaminase (enzyme activity is 520U / g, and the addition amount is 1.28%) is added, the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 110°C and the time is 21 seconds.

[0122] Comparative Example 3:

[0123] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed temperature is 58°C and the feed concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the addition ratio of the enzyme is 2.2% of the protein concentration of the feed, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 60 minutes, glutaminase (enzyme activity is 600U / g, and the addition amount is 1.5%) is added, the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 109°C and the time is 22 seconds.

[0124] Comparative Example 4:

[0125] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis begins, the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 28KHz, power is 600W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 110°C and the time is 19s.

[0126] Comparative Example 5:

[0127] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 30 minutes, the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 25KHz, power is 470W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 110°C and the time is 20 seconds.

[0128] Comparative Example 6:

[0129] The whey protein content is 85% by dry weight; when the whey protein is dissolved, the feed liquid temperature is 58°C and the feed liquid concentration is 10.5%. Potassium hydroxide is added to adjust the pH value to 6.8, and alkaline protease and flavor protease are added (the enzyme addition ratio is 2.2% of the protein concentration of the feed liquid, and the ratio of the two enzymes is 3:7), and hydrolysis begins (timing starting point). After the hydrolysis reaches 60 minutes, the equipment is turned on to perform ultrasound on the feed liquid (ultrasonic frequency is 22KHz, power is 400W), the hydrolysis temperature is 55.1°C, and the enzyme is inactivated after the hydrolysis reaches 90 minutes. The enzyme inactivation temperature is 112°C and the time is 20s.

[0130] <Molecular weight distribution>

[0131] The above experiments were repeated three times, and the results were expressed as mean ± standard deviation.

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

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

[0134] Table 1

[0135]

[0136] <Osmotic pressure and taste>

[0137] Osmotic pressure is a commonly used medical indicator with a high correlation with human health. Infants have incomplete kidney development and imperfect physiological functions. Long-term feeding of high-osmotic pressure formula foods increases the probability of kidney damage. The osmotic pressure of breast milk is about 292-300mOsm / kg H2O. Generally speaking, liquid formula milk with an osmotic pressure of >300mOsm / kg H2O is called high-osmotic pressure formula milk; formula milk with an osmotic pressure of 280-300mOsm / kg H2O is called isotonic pressure formula milk (osmotic pressure is tested using an osmometer, and each sample is tested 3 times).

[0138] On the other hand, when protein raw materials are hydrolyzed, their structure will undergo a series of changes. Specifically, the peptide chains are cut off to generate polypeptides of different molecular weights. At the same time, some hydrophobic amino acids originally contained in the protein are exposed. Therefore, most hydrolyzed proteins have a certain bitterness (quinine was prepared into solutions with concentrations of 12.5 and 62.5 μmol / L, respectively, and bitterness was defined as 1 and 10, respectively. The bitterness values ​​of the tested samples were evaluated by 10 people for each sample, and the results were averaged. Each sample was repeated 3 times).

[0139] With maltodextrin / lactose and edible vegetable blended oil as the main raw materials, the protein hydrolyzate solution of the above control group 1 (the added amount accounts for 27% of the energy supply ratio of the formula) and other ingredients are added, mixed evenly, sterilized after homogenization, and then concentrated and spray-dried to prepare sample A. The main process parameters are: homogenization 25MPa; sterilization (88℃~90℃, 15s); spray drying (inlet air temperature 160℃~180℃, outlet air temperature 85℃~90℃).

[0140] The other raw materials and processes were the same, and the protein hydrolyzate solution was from Comparative Example 1 to prepare Sample B. The rest of the samples were prepared in the same manner.

[0141] The osmotic pressure and taste of the milk powder prepared by the process of the embodiment and the embodiment are also greatly improved. The specific results are shown in Table 2 below:

[0142] Table 2

[0143]

[0144] <In vitro simulated gastrointestinal digestion experiment>

[0145] 1. Materials and Methods

[0146] 1.1 Main Reagents

[0147] NaCl, HCl, KCl, NaOH, trichloroacetic acid, potassium dihydrogen phosphate, sodium hydrogen phosphate, magnesium chloride, ammonium carbonate, pepsin, and trypsin were all from Sigma-Aldrich

[0148] 1.2 Main instruments and equipment

[0149] High-speed centrifuge, shaking constant temperature water bath, magnetic stirring (RH-A Basic magnetic stirrer from IKA).

[0150] 1.3 Experimental procedures

[0151] (1) Preparation of gastric simulation fluid: Weigh 0.2 g NaCl, add 1.3 mL 1 mol / L HCl, dissolve in 80 mL pure water, add 0.1 mol / L HCl to adjust the pH of the solution to 4.0, and finally dilute to 100 mL with pure water, marked as solution a. Weigh 5 mg gastric lipase and 0.5 mg pepsin, add 5 mL solution a, and stir magnetically at room temperature for 10 min to obtain simulated gastric fluid.

[0152] (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 value of the solution to 6.5, and finally use pure water to make up to 1L, marked as solution b. Weigh 5mg bile salt and 10mg pancreatic enzyme and dissolve them in 10mL solution b. Stir magnetically at 37℃ for 10min to obtain the simulated small intestinal digestive fluid.

[0153] (3) Simulated gastric digestion: Prepare a sample with a protein content of 4% in a conical 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 the volume ratio of sample solution to SGF 1:1, place the sample in a shaking water bath at 37°C, take samples at 0, 5, 10, 30, 60 and 120 min, and adjust the pH to 7.0 to inactivate the enzyme.

[0154] (4) Simulated intestinal digestion: After 120 min of simulated gastric digestion reaction, adjust the pH to 7.0 with NaOH solution to inactivate the enzyme, then add SIF, add CaCl2(H2O)2 solution, and add trypsin solution to make the trypsin activity in the final digestion solution reach 100 U / mL. Then add water to make the volume ratio of sample solution to SIF 1:1. Place the sample in a shaking water bath at 37°C, take samples at 60 and 120 min of reaction, and inactivate the enzyme in a boiling water bath for 5 min.

[0155] 1.4 Sample processing

[0156] After the sample was taken out, an equal volume of 24% trichloroacetic acid (TCA) solution was added for precipitation, and the sample was centrifuged at 9000 r / min for 20 min. The supernatant was taken to detect the protein content.

[0157] 1.5 Statistical analysis and calculation

[0158] Each sample was subjected to at least 3 simulated digestion tests, and the protein digestibility was determined and the average value was taken. The results are shown in Table 3 below.

[0159] Protein digestibility = (M1-M0) ÷ M2 × 100%

[0160] M0 is the total amount of protein in the blank control supernatant, g; M1 is the total amount of protein in the sample supernatant, g; M2 is the total amount of protein in the sample, g.

[0161] Table 3

[0162]

[0163] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0164] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a protein peptide, characterized in that: The method comprises: The whey protein raw material is hydrolyzed under the simultaneous action of glutaminase and ultrasonic treatment. Among them, suppose: The total hydrolysis time of the whey protein raw material is T; the action time of 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 material is derived from the milk of one or more animals.

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

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

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

6. The method according to any one of claims 1 to 5, characterized in that: The whole process of hydrolysis is carried out under the simultaneous action of glutaminase and ultrasonic treatment.

7. The method according to any one of claims 1 to 6, characterized in that: The hydrolysis ends at the same time as the glutaminase and ultrasonic treatment actions end.

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

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

10. The method according to any one of claims 1 to 8, characterized in that: In the protein peptide product obtained by the hydrolysis, the content of peptides with a molecular weight of 500 to 2000 Da is 80% by weight or more based on dry weight.

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