Lactalbumin moderate hydrolysate with good digestive absorbability and palatability as well as preparation method and application of whey protein moderate hydrolysate

Abstract

The invention provides a moderate whey protein hydrolysate with good digestive absorbability and palatability as well as a preparation method and application of the moderate whey protein hydrolysate. The preparation method of the whey protein hydrolysate provided by the invention is realized by sequentially carrying out first enzymolysis and second enzymolysis on a whey protein raw material by using neutral protease and glutamyltransferase. According to the preparation method, the whey protein hydrolysate with digestive absorbability and palatability can be obtained, the quality of the whey protein hydrolysate is effectively improved, the uniformity of the whey protein hydrolysate is enhanced, and the wall hanging phenomenon of the whey protein hydrolysate after brewing is relieved.

Description

TECHNICAL FIELD

[0001] The present application relates to biotechnology, in particular to a whey protein hydrolysate with good digestive absorption and palatability, and a preparation method and application thereof. BACKGROUND

[0002] Whey protein is a kind of protein obtained by concentrating and refining whey through a special process, accounting for about 18%-20% of milk protein, and has the characteristics of high nutritional value, easy digestion and absorption, and containing various active ingredients, etc. It is recognized as one of the high-quality protein supplements for human body. After whey protein is hydrolyzed by a specific protease, the active peptide segments distributed in the primary structure of whey protein, i.e. the amino acid sequences with biological activity, are released, and then whey protein hydrolysate is generated. Compared with whey protein, whey protein hydrolysate not only has better solubility, but also has better acid and heat resistance, and is more excellent in terms of body digestion and absorption and low allergenicity.

[0003] The application fields of whey protein hydrolysate in the domestic market mainly include sports and performance nutrition, infant formula milk powder, functional food, functional beverage, etc. With the increasing attention of people to health and nutrition, whey protein hydrolysate, as a high-quality protein supplement, is favored by more and more consumers. However, with the progress of protein hydrolysis, the exposure of hydrophobic amino acids or hydrophobic polypeptide chains will lead to the increase of bitterness and the deterioration of taste of whey protein hydrolysate. Therefore, how to ensure the taste of whey protein hydrolysate while improving the digestive absorption under sufficient hydrolysis is a problem to be solved by those skilled in the art. SUMMARY

[0004] The present application provides a whey protein hydrolysate and a preparation method, which has the advantages of high preparation efficiency, fast speed and good taste.

[0005] The present application provides the application of the whey protein hydrolysate prepared by the above preparation method or the above whey protein hydrolysate in a nutritional functional ingredient.

[0006] The present application provides a preparation method of a whey protein hydrolysate, which comprises the following steps:

[0007] The whey protein raw material is subjected to first enzymatic treatment using neutral protease to obtain a first enzymatic product;

[0008] The first enzymatic product is subjected to second enzymatic treatment using glutamine transaminase to obtain a whey protein hydrolysate.

[0009] The preparation method described above, wherein the enzyme activity of the neutral protease is 28000-55000 U / g; and / or,

[0010] The neutral protease is added in an amount of 5-15 mg per gram of whey protein raw material; and / or,

[0011] The enzyme activity of the glutamyl transferase is 100-1500 U / g; and / or,

[0012] The glutamyl transferase is added in an amount of 1-5 mg per gram of whey protein raw material.

[0013] The preparation method as described above, wherein the conditions of the first enzymatic treatment include a temperature of 45-55℃, a time of 2-4 h, and a pH value of 6.0-7.0; and / or,

[0014] The conditions of the second enzymatic treatment include a temperature of 30-60℃, a time of 0.5-1.5 h, and a pH value of 5.8-7.0; and / or,

[0015] The whey protein raw material has a protein content of ≥45 wt%; and / or,

[0016] The whey protein raw material is obtained by mixing whey protein and water in a mass ratio of 1: (25-60).

[0017] The preparation method as described above, wherein the preparation method further comprises, after the second enzymatic treatment of the first enzymatic product using the glutamyl transferase, performing concentration treatment, sterilization treatment, spray drying treatment, and sieving treatment on the second enzymatic product to obtain the whey protein hydrolysate.

[0018] The preparation method as described above, wherein the concentration treatment includes a concentration temperature of 70-80℃, and the second enzymatic product is concentrated to 15-25 Baume; and / or,

[0019] The sterilization treatment includes sterilization at 135-145℃ for 1-5 s; and / or,

[0020] The spray drying treatment includes an inlet air temperature of 160-180℃ and an outlet air temperature of 80-90℃.

[0021] The sieving treatment includes a mesh size of 30-50 mesh.

[0022] The present application provides a whey protein hydrolysate, wherein the whey protein hydrolysate is prepared by the preparation method described above.

[0023] The whey protein hydrolysate as described above, wherein the composition of the whey protein hydrolysate includes peptide segments PP, PX, and PI; the content of the peptide segment PP is 0.0040%-0.0085%, the content of the peptide segment PX is 0.0030%-0.0065%, and the content of the peptide segment PI is 0.0050%-0.0080%, based on the mass of the whey protein hydrolysate.

[0024] The whey protein hydrolysate as described above, wherein the mass content of the peptides with a molecular weight less than 10,000 Da in the whey protein hydrolysate is ≥ 86%.

[0025] The whey protein hydrolysate as described above, wherein the content of the amino acids in the whey protein hydrolysate is that the mass of the hydrophobic amino acids is ≤ 40% based on the total mass of the amino acids.

[0026] The present application provides the whey protein hydrolysate prepared by the preparation method and the application of the whey protein hydrolysate in a nutritional functional ingredient.

[0027] The present application provides a preparation method of a whey protein hydrolysate, by sequentially using a neutral protease and a transglutaminase for first enzymolysis and second enzymolysis on a whey protein raw material, a whey protein hydrolysate with both digestibility and palatability can be obtained, and the following benefits exist:

[0028] (1) The preparation method of the present application can improve the quality of the obtained whey protein hydrolysate, enhance its uniformity, and reduce its wall-hanging phenomenon after brewing;

[0029] (2) The preparation method of the present application can effectively reduce the molecular weight of the obtained whey protein hydrolysate, and at the same time, help to improve the digestibility of the obtained whey protein hydrolysate, and promote its rapid digestion and absorption in the gastrointestinal tract;

[0030] (3) The preparation method of the present application can effectively reduce the content of bitter peptides and hydrophobic amino acids in the obtained whey protein hydrolysate, and at the same time, help to reduce the bitterness degree of the obtained whey protein hydrolysate, and improve its palatability;

[0031] (4) The preparation method provided by the present application has the advantages of high preparation efficiency, fast speed, and sufficient hydrolysis. The whey protein hydrolysate obtained by the preparation method can realize the effects of rich nutrition, easy absorption by the human body, and low bitterness degree, and is easy to be accepted by people with poor digestion or people who need to quickly supplement protein, and can be used as a nutritional functional ingredient. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is a molecular weight diagram of the mixed whey protein powder and whey protein hydrolysate 1-13 in the test example during artificial simulation of gastric digestion;

[0033] Figure 2 Figure 2 is a molecular weight diagram of the mixed whey protein powder and whey protein hydrolysate 1-13 in the test example during artificial simulation of intestinal digestion;

[0034] Figure 3 Figure 3 is a molecular weight diagram of the mixed whey protein powder and whey protein hydrolysate 1-13 in the test example during artificial simulation of gastric and intestinal digestion. DETAILED DESCRIPTION

[0035] In order to better understand the scheme of the present application, the present application is further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are used to explain the present application, but not to limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] In order to solve the problem that it is difficult to simultaneously ensure the digestibility and palatability of whey protein hydrolysate in the prior art, the present application provides a preparation method of whey protein hydrolysate, comprising the following steps:

[0037] using a neutral protease to perform first enzymatic treatment on the whey protein raw material to obtain a first enzymatic product;

[0038] using a glutamine transferase to perform second enzymatic treatment on the first enzymatic product to obtain the whey protein hydrolysate.

[0039] The neutral protease used in the present application is a protease with the highest activity under neutral conditions (pH range between 6.0 and 8.0), and its main function is to decompose proteins in organisms, which can degrade proteins into small molecular peptides, amino acids and other biological molecules. The glutamine transferase (GGT) used in the present application mainly exists on hepatocyte membranes and microsomes, and is involved in the metabolism of glutathione.

[0040] It is found in the present application that first using a neutral protease to perform first enzymatic treatment on the whey protein raw material can promote the loose structure of whey protein, release specific peptide segments, and promote the exposure of specific amino acid sequences at the C-terminal and N-terminal of specific peptide segments; then, adding a glutamine transferase to perform second enzymatic treatment can transfer free γ-glutamine to the peptide segments, thereby avoiding the exposure of hydrophobic amino acids, and reducing the bitterness of the product. It is verified by experiments that using the preparation method of the present application, i.e., first using a neutral protease to perform first enzymatic treatment on the whey protein raw material, and then using a glutamine transferase to perform second enzymatic treatment, can obtain whey protein hydrolysate with both digestibility and palatability.

[0041] Specifically, using the preparation method of the present application has the following benefits:

[0042] (1) Using the preparation method of the present application can improve the quality of the obtained whey protein hydrolysate, enhance its uniformity, and reduce the wall-hanging phenomenon after brewing;

[0043] (2) The preparation method of the present application can effectively reduce the molecular weight of the obtained whey protein hydrolysate, and help to improve the digestion and absorption of the obtained whey protein hydrolysate, and promote its rapid digestion and absorption in the gastrointestinal tract;

[0044] (3) The preparation method of the present application can effectively reduce the content of bitter peptides and hydrophobic amino acids in the obtained whey protein hydrolysate, and help to reduce the bitterness of the obtained whey protein hydrolysate and improve its palatability;

[0045] (4) The preparation method provided by the present application has the advantages of high preparation efficiency, fast speed and sufficient hydrolysis. The whey protein hydrolysate obtained by the preparation method has the functions of rich nutrition, easy absorption by the human body, low bitterness and easy digestion, and can be used as a nutritional functional ingredient.

[0046] In the above technical solution, the enzyme activity may affect the degree of hydrolysis of the whey protein raw material. By limiting the enzyme activity, the degradation degree of the whey protein can be accurately controlled, so as to obtain a whey protein hydrolysate with specific molecular weight distribution and functional characteristics.

[0047] Experiments have found that when the enzyme activity of neutral protease is 28000-55000 U / g or the enzyme activity of glutamine transaminase is 100-1500 U / g, it helps to promote the good matching effect of neutral protease, glutamine transaminase and whey protein raw material, and then play a good catalytic effect, and promote the enzymatic treatment towards the direction of generating whey protein hydrolysate with low bitterness and good digestion and absorption.

[0048] In the above technical solution, the enzyme addition amount can adjust the enzyme hydrolysis reaction rate and affect the degree of hydrolysis of the whey protein raw material. Higher enzyme addition amount usually accelerates the hydrolysis process and produces higher degree of hydrolysis, thereby affecting the molecular weight distribution and functional characteristics of the whey protein hydrolysate.

[0049] Experiments have found that based on per gram of whey protein raw material, the addition amount of neutral protease is 5-15 mg; or the addition amount of glutamine transaminase is 1-5 mg, which helps to further promote the good matching effect of neutral protease, glutamine transaminase and whey protein raw material, so as to promote the enzymatic treatment towards the direction of generating whey protein hydrolysate with low bitterness and good digestion and absorption.

[0050] In the above technical solution, the enzyme hydrolysis temperature, time and pH value are regulated to help accurately control the degree of hydrolysis of the whey protein raw material, thereby promoting the enzyme hydrolysis process to obtain the target whey protein hydrolysate with low bitterness and good digestibility. Specifically, the conditions of the first enzyme hydrolysis process can include: temperature 45-55°C, time 2-4 h, and pH value 6.0-7.0; the conditions of the second enzyme hydrolysis process can include: temperature 30-60°C, time 0.5-1.5 h, and pH value 5.8-7.0.

[0051] It can be understood that the actual enzyme hydrolysis temperature, time and pH value can be selected by those skilled in the art within the above ranges according to actual conditions. For example, the temperature of the first enzyme hydrolysis process can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, and any value within the range between any two of the above values; the time of the first enzyme hydrolysis process can be 2 h, 2.5 h, 3 h, 3.5 h, 4 h, and any value within the range between any two of the above values; the pH value of the first enzyme hydrolysis process can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and any value within the range between any two of the above values. For example, the temperature of the second enzyme hydrolysis process can be 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and any value within the range between any two of the above values; the time of the second enzyme hydrolysis process can be 0.5 h, 1.0 h, 1.5 h, and any value within the range between any two of the above values; the pH value of the second enzyme hydrolysis process can be 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and any value within the range between any two of the above values.

[0052] In the above technical solution, the protein content in the whey protein raw material is ≥45wt%, which helps to obtain the target whey protein hydrolysate with low bitterness and good digestibility. In some embodiments of the present application, concentrated whey protein (WPC) is selected as the raw material. Since the stable concentrated whey protein supplied on the market is generally WPC80 type (i.e. protein content 80wt%), WPC35 type (i.e. protein content 35wt%) and the like, WPC80 type and WPC35 type concentrated whey proteins can be mixed in different mass ratios to obtain a mixed whey protein that meets the above protein content range. Specifically, whey protein with a protein content of 80wt% and whey protein with a protein content of 35wt% can be mixed in a mass ratio of 1:1 to obtain a mixed whey protein with a protein content of 57.5wt%, and the mixed whey protein is used as the raw material.

[0053] In the technical solution, the whey protein raw material is obtained by mixing whey protein and water at a mass ratio of 1: (25-60). Compared with solid whey protein, the whey protein raw material has a certain flowability, a larger surface area, and is more conducive to subsequent enzymolysis, which helps to obtain the target whey protein hydrolysate with low bitterness and good digestibility. If too little water is added, the flowability of the whey protein raw material will be poor, which is not conducive to the action of the enzyme preparation and is likely to reduce the enzymolysis efficiency. If too much water is added, the reaction volume will be too large during the enzymolysis process, which may increase the subsequent operation load and increase the processing cost accordingly.

[0054] In the technical solution, the preparation method further comprises, after the second enzymolysis treatment of the first enzymolysis product by using the glutamine transferase, performing concentration treatment, sterilization treatment, spray drying treatment, and screening treatment on the second enzymolysis product to obtain the whey protein hydrolysate, so as to obtain the whey protein hydrolysate with low bitterness, good digestibility, better quality, and better storage suitability.

[0055] In addition, after the second enzymolysis treatment of the first enzymolysis product by using the glutamine transferase, the enzyme can be inactivated first to lose the catalytic activity. The present application does not limit the method of enzyme inactivation, and the enzyme inactivation can be performed by using the conventional enzyme inactivation means in the art, for example, heating to 95℃ for 30 min.

[0056] After the enzyme inactivation treatment, the second enzymolysis product is subjected to concentration treatment, sterilization treatment, spray drying treatment, and screening treatment.

[0057] Specifically, the concentration treatment can increase the concentration of the whey protein hydrolysate product and improve the preservation of the product, which comprises: the concentration temperature is 70-80℃, and the second enzymolysis product is concentrated to 15-25 Baume.

[0058] The sterilization treatment can avoid the influence of excessive microorganisms on the shelf life of the product, and can be realized by using ultra-high temperature instantaneous sterilization (UHT), and the conditions include: 135-145℃ sterilization for 1-5 s.

[0059] The spray drying treatment can obtain the whey protein hydrolysate in powder form, which is convenient for storage and transportation, and the spray drying treatment comprises: the inlet air temperature is 160-180℃, and the outlet air temperature is 80-90℃.

[0060] Finally, the screening treatment can be performed, wherein the mesh size of the screen is 30-50 mesh, that is, the fineness of the obtained whey protein hydrolysate is 30-50 mesh, which is convenient for sub-packaging and subsequent brewing and consumption.

[0061] Based on the above preparation method, the second aspect of the present application provides a whey protein hydrolysate, which is prepared by the above preparation method.

[0062] It is determined through experiments that the composition of the whey protein hydrolysate includes a dipeptide peptide segment (Pro-Pro, PP) composed of proline and proline in sequence from N-terminal to C-terminal, a dipeptide peptide segment (Pro-Hyp, PX) composed of proline and hydroxyproline in sequence from N-terminal to C-terminal, and a dipeptide peptide segment (Pro-Ile, PI) composed of proline and isoleucine in sequence from N-terminal to C-terminal. Among them, proline and isoleucine are hydrophobic amino acids, and the peptide segment PP, the peptide segment PX and the peptide segment PI are dipeptides containing hydrophobic amino acid terminals, which are hydrophobic dipeptides. Moreover, the content of the peptide segment PP, the peptide segment PX and the peptide segment PI in the whey protein hydrolysate is higher than that of other peptide segments containing hydrophobic amino acid terminals, and therefore, the peptide segment PP, the peptide segment PX and the peptide segment PI have a key influence on the bitterness of the whey protein hydrolysate.

[0063] The whey protein hydrolysate prepared by the preparation method provided in the first aspect of the present application can effectively reduce the content of the peptide segment PP, PX and PI in its composition, and further effectively reduce the degree of bitterness. Specifically, based on the mass of the whey protein hydrolysate, the content of the peptide segment PP is 0.0040%-0.0085%, the content of the peptide segment PX is 0.0030%-0.0065%, and the content of the peptide segment PI is 0.0050%-0.0080%.

[0064] The whey protein hydrolysate described above also has a relatively low molecular weight, wherein the mass content of components with a molecular weight less than 10000 Da is ≥86%. Peptides with a small molecular weight are more easily directly absorbed and utilized by the body, and therefore, the whey protein hydrolysate has good digestibility and high bioavailability. Moreover, peptides with a small molecular weight generally have lower allergenicity and higher stability than macromolecular proteins, and therefore, the whey protein hydrolysate also has the advantages of low immunogenicity and high stability.

[0065] The whey protein hydrolysate described above has an amino acid content of 45-60 g / 100 g. Since amino acids have osmotic ability, if the amino acid content in the whey protein hydrolysate is too high, it is easy to cause the whey protein hydrolysate to exude or leak into the subcutaneous tissue, resulting in an increase in local osmotic pressure, causing water in the blood vessels to permeate into the subcutaneous tissue, causing temporary ischemia and hypoxia of local blood vessels and nerves, and possibly eventually causing necrosis of the subcutaneous tissue. If the amino acid content in the whey protein hydrolysate is too low, it indicates that the degree of hydrolysis of the whey protein hydrolysate is low, and its composition is mainly in the form of macromolecules, which will result in poor digestibility and high allergenicity. Therefore, limiting the content of amino acids in the whey protein hydrolysate of the present application can not only maximize the nutritional value and good digestibility of the whey protein hydrolysate, but also avoid the biological toxicity caused by too high an amino acid content.

[0066] The whey protein hydrolysate has a mass of hydrophobic amino acids ≤40% of the total mass of amino acids. If the content of hydrophobic amino acids is too high, the solubility of the whey protein hydrolysate in water may be affected, thereby affecting the quality and digestibility of the whey protein hydrolysate. Meanwhile, a high content of hydrophobic amino acids may also cause the whey protein hydrolysate to have enhanced bitterness, thereby affecting the taste. Therefore, limiting the proportion of the mass of hydrophobic amino acids in the total mass of amino acids in the whey protein hydrolysate of the present application can not only ensure the taste of the whey protein hydrolysate, but also maximize the hydrolysis of the whey protein raw material.

[0067] Based on the above research, the third aspect of the present application provides a whey protein hydrolysate prepared by the above preparation method or an application of the whey protein hydrolysate in a nutritional functional ingredient.

[0068] The whey protein hydrolysate of the present application can be applied not only to food and health products in the conventional sense, but also to the preparation of medicines, which helps to expand the application range of the whey protein hydrolysate. In detail, the food, health products and medicines, etc. to which the whey protein hydrolysate is applied as a nutritional functional ingredient can include infant formula milk powder, sports nutrition food, elderly nutrition food, protein supplements for people with poor digestion, etc. In particular, the whey protein hydrolysate of the present application has good digestibility and is particularly suitable for people who need to quickly supplement protein, such as athletes. The whey protein hydrolysate of the present application also has very low bitterness and good palatability, and is easily accepted by people, further expanding its application in food, health products and medicines.

[0069] In the following, the technical solutions of the present application will be further explained and described in combination with specific examples. The experimental methods in the following examples, if no specific conditions are noted, are usually carried out under conventional conditions or under the conditions recommended by the manufacturers. The reagents used, if not specifically stated, are commercially available or can be obtained from public channels.

[0070] Example 1

[0071] The present embodiment provides a preparation method of a whey protein hydrolysate, comprising the following steps:

[0072] The concentrated whey protein powder WPC80 and the concentrated whey protein powder WPC35 are mixed according to a mass ratio of 1:1 to obtain a mixed whey protein powder, wherein the protein content of the concentrated whey protein powder WPC80 is 80wt%, the protein content of the concentrated whey protein powder WPC35 is 35wt%, that is, the protein content of the mixed whey protein powder is 57.5wt%. Water is added to the mixed whey protein powder according to a mass ratio of 1:55 to obtain a mixed whey protein solution. Neutral protease is added to the mixed whey protein solution to obtain a first enzyme hydrolyzate through first enzyme hydrolysis treatment, wherein the enzyme activity of the neutral protease is 55000 U / g, the addition amount is 1% of the mass of the mixed whey protein powder, the first enzyme hydrolysis temperature is 50℃, the first enzyme hydrolysis time is 3h, and the first enzyme hydrolysis pH is 6.5. The transglutaminase is first mixed with deionized water according to a mass ratio of 1:5, and ultrasonic vibration is performed at 30℃ for 10min to obtain an enzyme premix. Then, the enzyme premix is added to the first enzyme hydrolyzate to obtain a second enzyme hydrolyzate through second enzyme hydrolysis treatment, wherein the enzyme activity of the transglutaminase is 550 U / g, the addition amount is 0.15% of the mass of the mixed whey protein powder, the second enzyme hydrolysis temperature is 50℃, the second enzyme hydrolysis time is 1h, and the second enzyme hydrolysis pH is 6.0. After the second enzyme hydrolyzate is subjected to enzyme inactivation treatment at 95℃ for 30min, it is concentrated to 20 Baume degrees at 75℃, and then subjected to ultra-high temperature instantaneous sterilization at 137℃ for 4s. Subsequently, spray drying is performed, the inlet air temperature is 170℃, the outlet air temperature is 80-90℃, and the product is sieved through a 40-mesh sieve to obtain whey protein hydrolysate No. 1.

[0073] Example 2

[0074] The embodiment provides a preparation method of a whey protein hydrolysate, and the specific steps can be referred to those in Example 1, and the only difference is that the protein content of the mixed whey protein powder in the embodiment is 45wt%. The whey protein hydrolysate No. 2 is finally obtained in the embodiment.

[0075] Example 3

[0076] The embodiment provides a preparation method of a whey protein hydrolysate, and the specific steps can be referred to those in Example 1, and the only difference is that water is added to the mixed whey protein powder according to a mass ratio of 1:25 to obtain a mixed whey protein solution in the embodiment. The whey protein hydrolysate No. 3 is finally obtained in the embodiment.

[0077] Example 4

[0078] The embodiment provides a preparation method of a whey protein hydrolysate, and the specific steps can be referred to those in Example 1, and the only difference is that the enzyme activity of the neutral protease is 28000 U / g in the embodiment. The whey protein hydrolysate No. 4 is finally obtained in the embodiment.

[0079] Example 5

[0080] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the addition amount of the neutral protease in the embodiment is 1.5% of the mass of the mixed whey protein powder. The embodiment finally obtains whey protein hydrolysate No. 5.

[0081] Embodiment 6

[0082] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the first enzymolysis temperature in the embodiment is 45 DEG C. The embodiment finally obtains whey protein hydrolysate No. 6.

[0083] Embodiment 7

[0084] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the enzyme activity of the glutamine transaminase in the embodiment is 1500 U / g. The embodiment finally obtains whey protein hydrolysate No. 7.

[0085] Embodiment 8

[0086] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the addition amount of the glutamine transaminase in the embodiment is 0.5% of the mass of the mixed whey protein powder. The embodiment finally obtains whey protein hydrolysate No. 8.

[0087] Embodiment 9

[0088] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the second enzymolysis temperature in the embodiment is 60 DEG C. The embodiment finally obtains whey protein hydrolysate No. 9.

[0089] Embodiment 10

[0090] The embodiment provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the second enzymolysis pH in the embodiment is 7.0. The embodiment finally obtains whey protein hydrolysate No. 10.

[0091] Comparative Example 1

[0092] The comparative example provides a preparation method of a whey protein hydrolysate, and specific steps can refer to those in Embodiment 1, and the only difference is that the neutral protease is not used in the comparative example. The comparative example finally obtains whey protein hydrolysate No. 11.

[0093] Comparative Example 2

[0094] The present comparative example provides a preparation method of whey protein hydrolysate, and the specific steps can refer to Example 1, and the only difference is that the present comparative example does not use transglutaminase. The present comparative example finally obtains whey protein hydrolysate No. 12.

[0095] Comparative Example 3

[0096] The present comparative example selects a whey protein hydrolysate produced and sold by Company A as a control, which is named whey protein hydrolysate No. 13.

[0097] Test Example

[0098] 1. Characterization of whey protein hydrolysate

[0099] (1) Identification of core peptide segments of whey protein hydrolysate

[0100] The whey protein hydrolysates Nos. 1-13 obtained in the above examples and comparative examples were respectively added with distilled water to prepare a whey protein hydrolysate solution of 20 μg / mL, and then centrifuged at 10000 r / min for 10 min. The supernatant was taken and filtered with a 0.22 μm nylon filter membrane to obtain test samples Nos. 1-13. The test samples Nos. 1-13 were respectively and sequentially subjected to Q3 scanning, PIS scanning, multiple reaction monitoring (MRM) optimization and peptide sequence quantification using LCMS-8060. According to Q3 scanning and PIS scanning, three core peptide segment structures were identified, which were Pro-Pro (PP), Pro-Hyp (PX) and Pro-Ile (PI). According to MRM optimization and peptide sequence quantification, the contents of the above three core peptide segments were obtained, which can be seen in Table 1, and the MRM parameters can be seen in Table 2.

[0101] Table 1

[0102]

[0103] Table 2

[0104]

[0105] The liquid chromatography conditions are as follows: the injection volume is 10 μL, the chromatographic column is Inertsil ODS-3 (the particle size is 5 μm, the inner diameter is 2.1 mm, and the column length is 250 mm), the mobile phase A is pure water (containing 0.1 % (v / v) formic acid), the mobile phase B is 100 % acetonitrile (containing 0.1 % (v / v) formic acid), and the flow rate is 0.2 mL / min. The gradient elution program specifically includes: the first gradient elution time is 0 min-15 min, the mobile phase B accounts for 0 %-50 %; the second gradient elution time is 15 min-20 min, the mobile phase B accounts for 50 %-100 %; the third gradient elution time is 20 min-25 min, the mobile phase B accounts for 100 %; and the fourth gradient elution time is 25.1 min-35 min, the mobile phase B accounts for 0 %. The column oven is 40 ℃.

[0106] The mass spectrometry conditions are as follows: the ionization mode is electrospray ionization (ESI), the positive ion mode; the ion spray voltage is +4.5 kV, the flow rate of atomization gas (nitrogen) is 3.0 L / min, the flow rate of heating gas (nitrogen) is 10 L / min, the flow rate of dry gas (nitrogen) is 10 L / min, the desolvation tube temperature is 250 ℃, the heating module temperature is 400 ℃, and the ion source temperature is 300 ℃; the scanning mode is MRM, the residence time is 100 ms, and the delay time is 3 ms.

[0107] In Table 1, the peptide segment PP is a dipeptide segment (Pro-Pro) composed of proline and proline from N-terminal to C-terminal, the peptide segment PX is a dipeptide segment (Pro-Hyp) composed of proline and hydroxyproline from N-terminal to C-terminal, and the peptide segment PI is a dipeptide segment (Pro-Ile) composed of proline and isoleucine from N-terminal to C-terminal. Among them, proline and isoleucine are hydrophobic amino acids, so the peptide segment PP, the peptide segment PX and the peptide segment PI are dipeptides containing hydrophobic amino acid terminals, which are hydrophobic dipeptides; and the content of the peptide segment PP, the peptide segment PX and the peptide segment PI in the whey protein hydrolysate is higher than that of other peptide segments containing hydrophobic amino acid terminals, therefore, the peptide segment PP, the peptide segment PX and the peptide segment PI have a key influence on the bitterness of the whey protein hydrolysate.

[0108] According to the data in Table 1, the bitter peptide content of the whey protein hydrolysate No. 1 is the lowest, followed by the whey protein hydrolysates No. 2, No. 4 and No. 11, followed by the whey protein hydrolysates No. 8, No. 9 and No. 10, the bitter peptide content of the whey protein hydrolysates No. 3, No. 6 and No. 7 is higher, the bitter peptide content of the whey protein hydrolysates No. 5 and No. 13 is further increased, and the bitter peptide content of the whey protein hydrolysate No. 12 is the highest.

[0109] (2) Detection of the molecular weight of the whey protein hydrolysate

[0110] The whey protein hydrolysates No. 1-13 obtained in the above examples and comparative examples were respectively prepared into a 1 mg / mL whey protein hydrolysate solution with distilled water, ultrasonicated for 20 min, centrifuged at 9000 r / min for 10 min, and the supernatant was taken and filtered with a 0.22 μm nylon filter membrane to obtain the test samples No. 1-13. The test samples No. 1-13 were subjected to molecular weight detection, and the specific detection method can refer to GB / T 22729 Marine Fish Oligopeptide. The molecular weight less than 10000 Da accounted for (%) was taken as the evaluation index, and the results are shown in Table 3:

[0111] Table 3

[0112]

[0113] Generally speaking, the smaller the molecular weight of the whey protein hydrolysate, the easier it is to be absorbed and utilized by the body, and thus the whey protein hydrolysate with smaller molecular weight generally has better digestibility, lower allergenicity and higher stability. According to Table 3, the molecular weight of whey protein hydrolysates No. 1, 2, 5, 7, 8 and 10 accounted for ≥90%, and the molecular weight was relatively small; the molecular weight of whey protein hydrolysates No. 3, 4, 6, 9, 12 and 13 accounted for ≥80%, and the molecular weight was relatively high; and the molecular weight of whey protein hydrolysate No. 11 accounted for 65.25%, and the molecular weight was the highest.

[0114] (3) Amino acid detection of whey protein hydrolysate

[0115] The whey protein hydrolysates No. 1-13 obtained in the above examples and comparative examples were subjected to amino acid detection according to the method of GB 5009.124-2016 Food Safety National Standard Determination of Amino Acids in Food. The detection results are shown in Table 4.

[0116] Table 4

[0117]

[0118] Generally speaking, if the content of amino acids in the whey protein hydrolysate is too high, it is easy to cause the leakage of amino acids into the subcutaneous tissue, increase the local osmotic pressure, promote the water in the blood vessels to penetrate into the subcutaneous tissue, cause temporary ischemia and hypoxia of local blood vessels and nerves, and eventually cause necrosis of subcutaneous tissue; if the content of amino acids in the whey protein hydrolysate is too low, it indicates that the degree of hydrolysis of the whey protein hydrolysate is low, and the composition is mainly in the form of macromolecules, which will lead to poor digestibility and high allergenicity; therefore, the content of amino acids in the whey protein hydrolysate is generally 45-60 g / 100 g. According to Table 4, only whey protein hydrolysate No. 11 is not within the above range.

[0119] In addition, too high content of hydrophobic amino acids can affect the solubility of whey protein hydrolysate in water, thereby affecting its quality and digestibility, and too high content of hydrophobic amino acids can also cause the bitterness of whey protein hydrolysate to increase, thereby affecting the taste. As can be seen from Table 4, whey protein hydrolysates No. 12 and No. 13 have a higher proportion of hydrophobic amino acids in the total amount of amino acids, which can result in a higher degree of bitterness.

[0120] 2. Product evaluation of whey protein hydrolysate

[0121] (1) Bitterness evaluation of whey protein hydrolysate

[0122] The whey protein hydrolysates No. 1-13 obtained in the above examples and comparative examples were respectively added with water to prepare whey protein hydrolysate solutions with a concentration of 3% (m / v) to obtain test samples No. 1-13. 20 evaluators were trained to score according to the bitterness degree of the 6 kinds of caffeine solutions in Table 5, with a score range of 0-60, and the first decimal place can be read. When the 20 evaluators have been trained and the error rate of scoring the 6 kinds of caffeine solutions is not more than 20%, the formal test can be carried out, and the test samples are tasted and scored, and the results are shown in Table 6. The scoring results in Table 6 are the average values of the scores of the 20 evaluators, and the first decimal place is obtained.

[0123] Table 5

[0124]

[0125] Table 6

[0126]

[0127] As can be seen from Table 6, the whey protein hydrolysate No. 1 has the lowest degree of bitterness, the whey protein hydrolysates No. 7, No. 8 and No. 11 have a lower degree of bitterness, the whey protein hydrolysates No. 2, No. 3, No. 4, No. 6, No. 9 and No. 10 have a moderate degree of bitterness, and the whey protein hydrolysates No. 5, No. 12 and No. 13 have a higher degree of bitterness.

[0128] (2) Evaluation of whey protein hydrolysate wall hanging phenomenon

[0129] The whey protein hydrolysates No. 1-13 obtained in the above examples and comparative examples were respectively added with water to brew, and 5 wt% whey protein hydrolysate solutions were prepared. The brewing conditions were kept consistent, the wall sticking phenomenon was evaluated by direct observation, and the wall sticking phenomenon was graded according to 1-10 levels, wherein the wall sticking of pure water was taken as level 1, and the wall sticking of the most obvious wall sticking (comparative example 3) was taken as level 10. The evaluation can be seen in Table 7. The wall sticking phenomenon refers to the fact that the whey protein hydrolysate solution is in the form of water droplets and slides down or sticks to the side wall of the container. The wall sticking phenomenon is generally related to protein structure and fat content, and can easily lead to poor overall uniformity and appearance of the final product.

[0130] Table 7

[0131]

[0132] According to Table 7, the wall sticking phenomenon of whey protein hydrolysate No. 1 is the lightest, the wall sticking phenomenon of whey protein hydrolysates No. 2, 5, 6, 7, 8, 10, 12 is lighter, the wall sticking phenomenon of whey protein hydrolysates No. 4, 9, 11 is less, the wall sticking phenomenon of whey protein hydrolysate No. 3 is more serious, and the wall sticking phenomenon of whey protein hydrolysate No. 13 is the most serious.

[0133] 3. In vitro artificial simulation digestion experiment of whey protein hydrolysate

[0134] (1) Artificial simulation of gastric digestion experiment:

[0135] Reference INFOGEST in vitro simulation digestion system to prepare corresponding gastric juice, and compare the digestion experiment of mixed whey protein powder (raw material) and whey protein hydrolysates No. 1-13 (samples 1-13). 5 g of mixed whey protein powder, whey protein hydrolysates No. 1-13 were taken, 50 mL of gastric juice was added, the pH value was adjusted to 2.0, and pepsin was added to make the enzyme concentration 2000 U / mL. Under the condition of 37℃ water bath, constant temperature digestion for 2 h, enzyme inactivation by 100℃ water bath for 10 min, cooling to room temperature, adjusting the pH value to 7.5 to obtain the detection liquid, taking 3 mL of the detection liquid and detecting according to the molecular weight of the whey protein hydrolysate, using Origin 8.5 for data analysis, and the results are shown in Table 8 and Figure 1

[0136] Table 8

[0137]

[0138] According to Table 8 and Figure 1 ​It can be seen that the gastric digestion effects of whey protein hydrolysate No. 1, No. 2, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10 are better, the gastric digestion effects of whey protein hydrolysate No. 12, No. 3 are second, and the gastric digestion effects of whey protein hydrolysate No. 13 and No. 11 are poorer.

[0139] (2) Artificial simulation of intestinal digestion experiment:

[0140] Reference INFOGEST in vitro simulation digestion system to prepare the corresponding intestinal fluid, and mix whey protein powder (raw material) and whey protein hydrolysate No. 1-13 (sample 1-13) for digestion experiment comparison. Take 5 g of mixed whey protein powder, whey protein hydrolysate No. 1-13, add 100 mL of intestinal fluid, adjust the pH value to 7.0 with 1 mol / L NaOH aqueous solution or 1 mol / L HCl aqueous solution, then add trypsin to make the enzyme concentration 100 U / mL, and digest at 37℃ water bath for 2 h, then 100℃ water bath for 10 min to inactivate the enzyme, cool to room temperature, adjust the pH value to 7.5 to obtain the detection liquid, take 3 mL of the detection liquid and refer to the molecular weight detection method of whey protein hydrolysate for detection, use Origin 8.5 for data analysis, and the results are shown in Table 2 and Figure 2

[0141] Table 9

[0142]

[0143] According to Table 9 and Figure 2 It can be seen that the intestinal digestion effects of whey protein hydrolysate No. 1, No. 2, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10, No. 12 are better, the intestinal digestion effect of whey protein hydrolysate No. 2 is second, and the intestinal digestion effects of whey protein hydrolysate No. 13 and No. 11 are poorer.

[0144] (3) Artificial simulation of gastric and intestinal digestion experiment:

[0145] ​Reference INFOGEST in vitro simulation of the digestive system to prepare the corresponding gastric juice and intestinal juice, mixed whey protein powder (raw material) and whey protein hydrolysate 1-13 (sample 1-13) were compared. Take 5 g of mixed whey protein powder, whey protein hydrolysate 1-13, add 50 mL of gastric juice, adjust the pH value to 2.0, add pepsin to make the enzyme concentration 2000 U / mL, and digest for 2 h at 37℃ water bath. After gastric digestion, add 50 mL of intestinal juice, adjust the pH value to 7.5, add trypsin to make the enzyme concentration 100 U / mL, and digest for 2 h at 37℃ water bath, then inactivate the enzyme by 100℃ water bath for 10 min, cool to room temperature, adjust the pH value to 7.5 to obtain the detection solution, take 3 mL of the detection solution and detect according to the molecular weight detection method of whey protein hydrolysate, use Origin 8.5 for data analysis, and the results are shown in Table 10 and Figure 3

[0146] Table 10

[0147]

[0148] According to Table 10 and Figure 3 , it can be known that the gastrointestinal digestion effect of whey protein hydrolysate 1, 2, 5 and 8 is good, the gastrointestinal digestion effect of whey protein hydrolysate 4, 6, 7, 9, 10 and 12 is secondary, and the gastrointestinal digestion effect of whey protein hydrolysate 3, 11 and 13 is poor.

[0149] ​From the comprehensive test example data results, the bitterness degree of the whey protein hydrolysate No. 1 provided by Example 1 is the lightest, the molecular weight is the smallest, the digestion and absorption performance is the best, and the wall hanging phenomenon is the lightest; the bitterness degree of the whey protein hydrolysate No. 2 provided by Example 2 is moderate, the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the reduction of the protein content in the raw material may affect the bitterness to some extent, and has certain influence on the molecular weight and the digestion and absorption performance; the bitterness degree of the whey protein hydrolysate No. 3 provided by Example 3 is moderate, the molecular weight is larger, and the digestion and absorption performance is general, indicating that the low ratio of the raw material and deionized water will affect the molecular weight, which is not conducive to the protein hydrolysis and digestion and absorption, and has certain influence on the bitterness; the bitterness degree of the whey protein hydrolysate No. 4 provided by Example 4 is moderate, the molecular weight is moderate, and the digestion and absorption performance is better, indicating that the enzyme activity of the neutral protease will have a more obvious influence on the bitterness; the bitterness degree of the whey protein hydrolysate No. 5 provided by Example 5 is more serious, but the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the addition amount of the neutral protease will have a more obvious influence on the bitterness; the bitterness degree of the whey protein hydrolysate No. 6 provided by Example 6 is moderate, but the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the temperature of the first enzymolysis will have a more obvious influence on the bitterness; the bitterness degree of the whey protein hydrolysate No. 7 provided by Example 7 is lighter, the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the enzyme activity of the glutamine transaminase has a smaller influence on the bitterness and the molecular weight; the bitterness degree of the whey protein hydrolysate No. 8 provided by Example 8 is lighter, the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the addition amount of the glutamine transaminase has a smaller influence on the bitterness and the molecular weight; the bitterness degree of the whey protein hydrolysate No. 9 provided by Example 9 is moderate, but the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the temperature of the second enzymolysis has a more obvious influence on the bitterness; the bitterness degree of the whey protein hydrolysate No. 10 provided by Example 10 is moderate, the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the pH value of the second enzymolysis has a more obvious influence on the bitterness.

[0150] The bitterness degree of the whey protein hydrolysate No. 11 provided by Comparative Example 1 is lighter, but the molecular weight is larger, and the digestion and absorption performance is general, indicating that the influence of not using the neutral protease on the molecular weight is very obvious; the bitterness degree of the whey protein hydrolysate No. 12 provided by Comparative Example 2 is moderate, but the molecular weight is smaller, and the digestion and absorption performance is better, indicating that the influence of not using the glutamine transaminase on the bitterness is very obvious; the bitterness degree of the whey protein hydrolysate No. 13 provided by Comparative Example 3 is serious, the molecular weight is larger, and the digestion and absorption performance is poorer, indicating that the commercially available whey protein hydrolysate currently has difficulty in solving the problems of serious bitterness and poor digestion and absorption.

[0151] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a whey protein hydrolysate, characterized in that, The method comprises the following steps: a first enzymatic treatment of a whey protein raw material using a neutral protease to obtain a first enzymatic product; a second enzymatic treatment of the first enzymatic product using a transglutaminase to obtain the whey protein hydrolysate.

2. The production method according to claim 1, characterized by, The enzyme activity of the neutral protease is 28000-55000 U / g; and / or, The addition amount of the neutral protease is 5-15 mg per gram of the whey protein raw material; and / or, The enzyme activity of the transglutaminase is 100-1500 U / g; and / or, The addition amount of the transglutaminase is 1-5 mg per gram of the whey protein raw material.

3. The production method according to claim 1 or 2, characterized by, The conditions of the first enzymatic treatment include: a temperature of 45-55℃, a time of 2-4 h, and a pH value of 6.0-7.0; and / or, The conditions of the second enzymatic treatment include: a temperature of 30-60℃, a time of 0.5-1.5 h, and a pH value of 5.8-7.0; and / or, The protein content in the whey protein raw material is ≥45 wt%; and / or, The whey protein raw material is obtained by mixing whey protein and water at a mass ratio of 1:(25-60).

4. The production method according to claim 1 or 2, characterized by, The preparation method further comprises, after the second enzymatic treatment of the first enzymatic product using the transglutaminase, a concentration treatment, a sterilization treatment, a spray drying treatment, and a sieving treatment of the second enzymatic product to obtain the whey protein hydrolysate.

5. The preparation method according to claim 4, characterized in that, The concentration treatment includes: a concentration temperature of 70-80℃, and a concentration to 15-25 Baume of the second enzymatic product; and / or, The sterilization treatment includes: 135-145℃ sterilization for 1-5 s; and / or, The spray drying treatment includes: an inlet air temperature of 160-180℃, and an outlet air temperature of 80-90℃; The sieving treatment includes: a mesh size of 30-50 mesh.

6. A whey protein hydrolysate characterized in that, The whey protein hydrolysate is prepared by the preparation method of any one of claims 1-5.

7. The whey protein hydrolysate according to claim 6, characterized in that, The composition of the whey protein hydrolysate comprises peptide segments PP, PX, and PI; the content of the peptide segment PP is 0.0040%-0.0085%, the content of the peptide segment PX is 0.0030%-0.0065%, and the content of the peptide segment PI is 0.0050%-0.0080%, based on the mass of the whey protein hydrolysate.

8. The whey protein hydrolysate according to claim 6 or 7, characterized in that, The mass content of peptides with a molecular weight less than 10000 Da in the whey protein hydrolysate is ≥86%.

9. The whey protein hydrolysate according to claim 6 or 7, characterized in that, The mass of hydrophobic amino acids is ≤40% based on the total mass of amino acids in the whey protein hydrolysate.

10. Application of the whey protein hydrolysate prepared by the preparation method of any one of claims 1-5 or the whey protein hydrolysate of any one of claims 6-9 in a nutrient functional ingredient.

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