Anti-vomiting hydrolyzed whey protein and preparation method thereof

Abstract

The invention discloses anti-vomiting hydrolyzed whey protein and a preparation method thereof, and belongs to the technical field of food processing. The product provided by the invention reduces the digestion burden of protein and relieves the symptom of vomiting. According to the method disclosed by the invention, protein sequences are mutually crosslinked by virtue of a treatment mode of first fibration, second polymerization and final hydrolysis, and a hydrolyzed polypeptide sequence is changed, so that the abundance of the polypeptide SLAMAASDISLA in a hydrolysate is improved. The polypeptide has the characteristics of being easy to recognize by intestinal peptidase and relatively high in PepT1 affinity, the bioavailability of protein is improved, and the deamination proportion of unutilized amino acid is reduced, so that substances generating nitrogen-containing toxoid are reduced, the stimulation of toxin accumulation on gastrointestinal mucosa is relieved, and the symptoms of nausea, vomiting and the like of a user are avoided.

Description

Technical Field

[0001] This invention relates to an anti-nausea hydrolyzed whey protein and its preparation method, belonging to the field of food processing technology. Background Technology

[0002] Whey protein is a rare, high-quality protein found in milk, primarily composed of α-lactalbumin and β-lactoglobulin. Its nutritional value is extremely high, often referred to as the "king of proteins," and it is widely used in sports nutrition, clinical nutrition, and infant formula. Although whey protein is rich in nutrients, its molecular weight is relatively large in its natural state, making it relatively slow to be digested and absorbed by gastrointestinal enzymes.

[0003] Existing hydrolyzed proteins have the following technical challenges: (1) The burden of protein metabolism is heavy, especially in patients with chronic kidney disease who cannot effectively excrete nitrogenous toxins such as urea and creatinine produced by protein metabolism. The accumulation of these toxins will irritate the gastrointestinal mucosa, leading to symptoms such as nausea and vomiting.

[0004] (2) Low digestion and absorption efficiency. The main component of whey protein, β-lactoglobulin, has a dense structure and is resistant to digestion by pepsin, resulting in low digestibility.

[0005] (3) Hydrolyzed whey protein contains free amino acids, and its high osmotic pressure can irritate the gastrointestinal tract. Excessive pursuit of low molecular weight and high degree of hydrolysis in hydrolyzed whey protein will produce a large amount of free amino acids, which will have excessively high osmotic pressure and irritate the gastrointestinal tract after ingestion.

[0006] (4) Strong bitterness and poor palatability. Hydrolyzed protein will have a large number of hydrophobic ends exposed, releasing bitter peptides, which have poor palatability and may also cause vomiting in the user.

[0007] Therefore, there is an urgent need for an anti-vomiting hydrolyzed whey protein that can minimize the allergenicity and viscosity of protein while ensuring nutritional value, and effectively solve the vomiting problem caused by hydrolyzed protein. Summary of the Invention

[0008] To address the drawbacks of existing protein treatments, such as high metabolic burden, low absorption rate, high osmotic pressure, strong gastrointestinal irritation, and pronounced bitterness leading to poor palatability, this invention aims to provide a hydrolyzed protein rich in specific peptides, with low gastrointestinal irritation, high absorption rate, low metabolic burden, and the ability to prevent vomiting. The process involves first modifying the protein through fibrosis, then cross-linking it, and finally hydrolyzing it to obtain a medium- to long-term peptide, SLAMAASDISLA, which is easily digestible, has low gastrointestinal irritation, low bitterness, and rapid metabolism.

[0009] The present invention provides an antiemetic hydrolyzed protein peptide, wherein the antiemetic hydrolyzed protein peptide is SLAMAASDISLA ((Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile-Ser-Leu-Ala)), and the amino acid sequence is shown in SEQ ID NO.1.

[0010] This invention provides a method for preventing vomiting with hydrolyzed whey protein, wherein the hydrolyzed whey protein for preventing vomiting contains SLAMAASDISLA protein peptide, and the method includes the following steps: (1) Raw material pretreatment and preparation of whey protein solution.

[0011] Using whey protein as raw material, an initial whey protein solution with a mass concentration of 3-6% was prepared; the pH value of the initial whey protein solution was adjusted to 2.0-2.5 to obtain the whey protein solution to be fibroinated. (2) Ultrasonic-assisted whey protein fibrinolysis The whey protein solution to be fibrosed obtained in step (1) is subjected to an ultrasonic power of 150-200W and an ultrasonic frequency of 40-60 kHz at a temperature of 55-75℃, using a pulse mode of "ultrasonic 3-5 s / intermittent 6-10 s" for 2.5-3.5 h to obtain a fibrosed whey protein dispersion. (3) Laccase cross-linked fibrin whey protein Add a phenolic adjuvant with a final concentration of 0.3-0.5 mmol / L to the fibrinized whey protein dispersion obtained in step (2); adjust the pH of the system to 4.5-5.0, then add laccase from Trametes versicolor, wherein the amount of laccase is 50-60 U / g whey protein, and continue the reaction for 3-5 h at a temperature of 35-40℃, a stirring speed of 120-150 rpm, and a dissolved oxygen concentration ≥5 mg / L; after the reaction is completed, inactivate the enzyme to obtain a laccase-crosslinked fibrinized whey protein solution; preferably, the phenolic adjuvant is ferulic acid; (4) Preparation of cross-linked whey protein hydrolysate and anti-emetic hydrolysate. Add flavor protease to the laccase-crosslinked fibrinized whey protein solution obtained in step (3), wherein the amount of flavor protease is 300~400 U / g whey protein; adjust the pH of the system to 6.5~7.0, and react for 2~3 h at a temperature of 40~50℃ and a stirring speed of 80~120 rpm; after the reaction is completed, inactivate the enzyme to obtain a solution, and dry the obtained solution to obtain the anti-vomiting hydrolyzed whey protein product. Preferably, the method includes the following steps: (1) Raw material pretreatment and preparation of whey protein solution. Whey protein was used as raw material. Deionized water was added and the solution was dissolved by magnetic stirring at 80-120 rpm to prepare an initial whey protein solution with a mass concentration of 3-6%. The pH value of the initial whey protein solution was adjusted to 2.0-2.5 with 1 mol / L food-grade citric acid solution. Then, the solution was stirred at a constant temperature of 20-25℃ for 20-30 min to fully dissolve and disperse the whey protein evenly, thus obtaining the whey protein solution to be fibroinated.

[0012] (2) Ultrasonic-assisted whey protein fibrillation treatment. The whey protein solution to be fibrillated obtained in step 1 was transferred to an ultrasonic reactor. The reaction temperature was set to 55-75℃, the ultrasonic power to 150-200 W, and the ultrasonic frequency to 40-60 kHz. The pulse mode of "ultrasonic 3-5 s / intermittent 6-10 s" was used for continuous treatment for 2.5-3.5 h to carry out the mild fibrillation reaction of whey protein. The fibrillated whey protein solution after the reaction was kept at a constant temperature of 35℃ for 10 min to eliminate residual bubbles from the ultrasound. Then it was naturally cooled to room temperature (25℃) to obtain a uniformly dispersed fibrillated whey protein dispersion for later use. (3) Laccase crosslinking of fibrinized whey protein. Phenolic adjuvant (ferulic acid) was added to the fibrinized whey protein dispersion obtained in step 2 to make the final concentration of ferulic acid in the system 0.3 mmol / L, and stirred for 5 min to dissolve. The pH of the system was adjusted to 4.8 with 1 mol / L potassium hydroxide solution, and then laccase (enzyme activity ≥1000 U / g) was added, with an enzyme dosage of 50-60 U / g whey protein. The system was transferred to a ventilated reactor, the reaction temperature was set to 35-40℃, the stirring speed was 120-150 rpm, and sterile air was introduced at the same time to control the dissolved oxygen concentration ≥5 mg / L. The reaction was continued for 4 h. After the reaction was completed, the system was heated to 80℃ and kept at 10 min to inactivate laccase and terminate the crosslinking reaction. After cooling to room temperature, the laccase crosslinked fibrinized whey protein solution was obtained.

[0013] (4) Hydrolysis of cross-linked whey protein. Add flavor protease (enzyme activity ≥5000 U / g) to the laccase cross-linked fibrinized whey protein solution obtained in step 3, with an enzyme dosage of 300-400 U / g whey protein; adjust the pH of the system to 6.8, set the reaction temperature to 50℃, and the stirring speed to 100 rpm to carry out the hydrolysis reaction; when the degree of hydrolysis reaches 18-20%, raise the temperature of the system to 85-90℃, keep it at 15-20 min to inactivate the protease, and terminate the hydrolysis reaction; (5) Spray drying and finished product collection. The hydrolyzed whey protein obtained in step 4 is passed into a spray dryer for drying. The process parameters are set as follows: inlet air temperature 170-180℃, outlet air temperature 80-85℃, feed rate 8-10 mL / min, and atomization pressure 0.3-0.5 MPa. The dry powder product is collected, which is the hydrolyzed whey protein product for preventing vomiting. It is stored in a cool and dry place.

[0014] The present invention also provides hydrolyzed whey protein for preventing vomiting prepared by the above method.

[0015] The present invention also provides a food, medicine, health product or nutritional product containing the above-mentioned anti-vomiting hydrolyzed protein peptide or the above-mentioned anti-vomiting hydrolyzed whey protein.

[0016] In one embodiment of the present invention, the food, medicine, health product or nutritional product may also contain derivatives of antiemetic hydrolyzed protein peptides. The derivatives of antiemetic hydrolyzed protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the antiemetic hydrolyzed protein peptide.

[0017] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0018] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0019] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0020] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0021] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0022] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0023] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages.

[0024] In one embodiment of the present invention, the food also includes special dietary foods; the health products also contain acceptable excipients.

[0025] The present invention also provides an expression vector or recombinant microorganism expressing the above-mentioned antiemetic hydrolysate peptide SLAMAASDISLA.

[0026] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0027] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0028] The present invention also provides the application of the above-mentioned anti-vomiting hydrolyzed protein peptide SLAMAASDISLA or the above-mentioned anti-vomiting hydrolyzed whey protein in the preparation of food, pharmaceuticals, health products or nutritional products.

[0029] In one embodiment of the present invention, the food, medicine, health product or nutritional product may also contain derivatives of antiemetic hydrolyzed protein peptides. The derivatives of antiemetic hydrolyzed protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the antiemetic hydrolyzed protein peptide.

[0030] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0031] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0032] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0033] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0034] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0035] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0036] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages.

[0037] In one embodiment of the present invention, the food also includes special dietary foods.

[0038] In one embodiment of the present invention, the nutritional products include medical nutritional products, nutritional products formulated for special populations, and nutritional supplements.

[0039] The present invention also provides the application of the above-mentioned anti-vomiting hydrolyzed protein peptide in the preparation of DPP-IV inhibitors, or hypoglycemic drugs or health products, or drugs or health products for the treatment of type II diabetes.

[0040] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0041] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0042] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0043] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0044] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0045] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0046] Beneficial effects (1) The product of this invention reduces the digestive burden of protein and alleviates vomiting symptoms. This invention uses a process of first fibrinization, then polymerization, and finally hydrolysis to cross-link the protein sequences, thereby altering the hydrolyzed peptide sequences and increasing the abundance of the peptide SLAMAASDISLA (Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile-Ser-Leu-Ala) in the hydrolysate. This peptide is easily recognized by intestinal peptidases and has high affinity for PepT1, improving protein bioavailability, reducing the proportion of unutilized amino acid deamination, thereby reducing the production of nitrogenous toxins, alleviating the irritation of toxin accumulation on the gastrointestinal mucosa, and thus avoiding symptoms such as nausea and vomiting in users.

[0047] (2) Low osmotic pressure, close to that of the human body. The special combination of polymerization and hydrolysis in this invention increases the abundance of the polypeptide SLAMAASDISLA, placing it in a low osmotic pressure range; at the same time, it reduces the proportion of free amino acids in the hydrolyzed protein, making the hydrolyzed protein have a low osmotic pressure and avoiding gastrointestinal irritation caused by high osmotic pressure.

[0048] (3) The pre-digestion properties of hydrolyzed protein can improve protein digestibility. The protein of the present invention is pre-hydrolyzed, eliminating the need for complex digestion and decomposition in the body, reducing the difficulty of protein digestion, and making it more suitable for people with weak gastrointestinal digestive capacity.

[0049] (4) The protein hydrolysate has a low content of bitter peptides and is easy to drink. The hydrolysate of the present invention has a low content of bitter peptides, which makes it palatable and avoids nausea or even vomiting caused by the strong bitterness of protein. Attached Figure Description

[0050] Figure 1 Osmotic pressure determination in hydrolyzed protein samples.

[0051] Figure 2 : Relative abundance of SLAMAASDISLA peptide in hydrolyzed protein samples.

[0052] Figure 3 : The content of free amino acids in hydrolyzed protein samples.

[0053] Figure 4 Protein digestibility in hydrolyzed protein samples.

[0054] Figure 5 Content of bitter peptides in hydrolyzed protein samples.

[0055] Figure 6 Ingestion of hydrolyzed protein samples led to the release of neuropeptides from the stomachs of mice.

[0056] Figure 7 Ingestion of hydrolyzed protein samples led to an increase in blood ammonia levels in mice.

[0057] Figure 8 Ingestion of hydrolyzed protein samples led to an increase in urinary nitrogen levels in mice.

[0058] Figure 9 The ability of intestinal peptidases to recognize and degrade medium- and long peptides.

[0059] Figure 10 PepT1 affinity of medium-length peptides.

[0060] Figure 11 Antioxidant capacity of medium- and long peptides. Detailed Implementation

[0061] The raw material information involved in the following examples is as follows: The whey protein was purchased from Fonterra, New Zealand, and was model WPC 80. The Yunzhi laccase was purchased from: Merck Group Darmstadt, Germany, product number 38429; Flavor protease was purchased from Novozymes (China) Biotechnology Co., Ltd., product name: Flavorzyme; The alkaline protease was purchased from Novozymes (China) Biotechnology Co., Ltd., model Alcalase 2.4L.

[0062] The detection methods involved in the following embodiments osmotic pressure detection Using a freezing point osmometer, the hydrolyzed protein sample to be tested is added to the sample tube, the instrument automatically freezes and measures the freezing point, and the osmotic pressure value is read directly.

[0063] Detection of peptide abundance The samples were desalted and purified, and the relative abundance of specific peptides, i.e., the response value of the liquid chromatography-mass spectrometry, was identified using an ultra-high performance liquid chromatography-tandem mass spectrometer and PEAKS Studio.

[0064] Detection of free amino acid ratio Add 20% trichloroacetic acid to the hydrolyzed protein sample, vortex for 2 min, centrifuge at 15000 rpm for 20 min, collect the supernatant, and analyze it with an amino acid analyzer to obtain the free amino acid content. Calculate the free amino acid ratio using the following formula.

[0065] Free amino acid percentage (%) = Total free amino acids / Total amino acids * 100%.

[0066] Detection of protein digestibility The in vitro digestion model INFOOGEST 2.0 was used to digest the hydrolyzed protein samples, and the digestibility was determined by Kjeldahl nitrogen determination.

[0067] Detection of bitter peptide content Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect bitter hydrophobic peptides, and the relative content of bitter peptides was indirectly characterized by calculating the integral area of ​​the hydrophobic region in the elution curve.

[0068] Detection of neuropeptide content After mice were administered samples by gavage, their gastric tissues were dissected and homogenized to detect the content of vomiting neuropeptides.

[0069] Blood ammonia level test Mice were administered the samples via gavage for one hour. Blood samples were then collected and immediately placed in ice water for centrifugation to separate the plasma. The ammonia content was determined using a blood ammonia assay kit.

[0070] Urine nitrogen content test The total nitrogen content in urine was directly determined using an Elementar elemental analyzer via a high-temperature combustion-thermal conductivity detector.

[0071] Example 1: Preparation of hydrolyzed whey protein to prevent vomiting (1) Raw material pretreatment and preparation of whey protein solution.

[0072] Using whey protein as raw material, deionized water was added and the solution was dissolved by magnetic stirring at 80 rpm to prepare an initial whey protein solution with a mass concentration of 4% (w / w). The pH value of the initial whey protein solution was adjusted to 2.0 using 1 mol / L food-grade citric acid solution. Then, the solution was stirred at a constant temperature of 20℃ for 20 min (speed: 120 rpm) to ensure that the whey protein was fully dissolved and evenly dispersed, thus obtaining a whey protein solution to be fibroinated.

[0073] (2) Ultrasonic-assisted whey protein fibrinolysis The whey protein solution to be fibrosed obtained in step (1) was transferred to an ultrasonic reactor. The reaction temperature was set to 65℃, the ultrasonic power to be 180 W, and the ultrasonic frequency to be 50 kHz. The pulse mode of "ultrasound 5 s / intermittent 10 s" was used and the treatment was continued for 3 h to carry out the mild fibrosis reaction of whey protein. The fibrosed whey protein solution after the reaction was kept at a constant temperature of 35℃ for 10 min to eliminate residual bubbles from the ultrasound. Then it was naturally cooled to room temperature to obtain a uniformly dispersed fibrosed whey protein dispersion for later use. (3) Laccase cross-linked fibrin whey protein Add ferulic acid, a phenolic adjuvant, to the fibrinized whey protein dispersion obtained in step (2) to make the final concentration of ferulic acid in the system 0.3 mmol / L, and stir to dissolve for 5 min (speed: 120 rpm); adjust the pH of the system to 4.8 with 1 mol / L potassium hydroxide solution, and then add laccase (enzyme activity ≥1000 U / g) with an enzyme dosage of 55 U / g whey protein; transfer the system to a ventilated reactor, set the reaction temperature to 35℃, the stirring speed to 120 rpm, and simultaneously introduce sterile air to control the dissolved oxygen concentration ≥5 mg / L, and continue the reaction for 4 h; after the reaction is completed, raise the system to 80℃, keep it at 10 min to inactivate laccase, terminate the cross-linking reaction, and cool to room temperature to obtain laccase-crosslinked fibrinized whey protein solution.

[0074] (4) Cross-linked whey protein hydrolysis Add flavor protease (enzyme activity ≥ 5000 U / g) to the laccase cross-linked fibrinized whey protein solution obtained in step (3), with an enzyme dosage of 300 U / g whey protein; adjust the pH of the system to 6.8, set the reaction temperature to 50℃, and the stirring speed to 100 rpm to carry out the hydrolysis reaction; when the degree of hydrolysis reaches 18% (at this time, the hydrolysis time is 3 h), raise the system temperature to 85-90℃, keep it at 15 min to inactivate the protease, and terminate the hydrolysis reaction; (5) Spray drying and finished product collection The reaction system obtained in step (4) is passed into a spray dryer for drying. The process parameters are set as follows: inlet air temperature 170℃, outlet air temperature 85℃, feed rate 8 mL / min, and atomization pressure 0.3 MPa. The dry powder product is collected, which is the hydrolyzed whey protein product for preventing vomiting. It is stored in a cool and dry place.

[0075] Comparative Example 1: Specifically, the method is the same as in Example 1, except that step (2) is omitted, and the hydrolyzed whey protein product is obtained according to the method in Example 1.

[0076] Comparative Example 2: Specifically, the method is the same as in Example 1, except that step (3) is omitted, and the hydrolyzed whey protein product is obtained according to the method in Example 1.

[0077] Comparative Example 3: Specifically, the method is the same as in Example 1, except that the flavor protease in step (4) is replaced with alkaline protease, and the hydrolyzed whey protein product is obtained according to the method in Example 1.

[0078] Comparative Example 4: Specifically, the method is the same as in Example 1, except that step (4) is omitted, and the whey protein product is obtained according to the method in Example 1.

[0079] Example 2: Experimental Results The effects of the hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were tested respectively. The protein was modified by fiberization using the method of the present invention, then cross-linked, and finally hydrolyzed. The final hydrolyzed whey protein product was analyzed by peptide fragmentation. The results showed that the hydrolyzed whey protein product was rich in the medium- and long peptide SLAMAASDISLA, which is easily digestible, has low gastrointestinal irritation, low bitterness, and fast metabolism.

[0080] (1) The osmotic pressure of the hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 was measured using an ice-point osmometer. (2) The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were used to identify the relative abundance of SLAMAASDISLA peptides, i.e., the response value of liquid chromatography-mass spectrometry, using PEAKS Studio. (3) The proportion of free amino acids in the hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were tested respectively; (4) The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were tested for protein digestibility using the INFOGEST 2.0 in vitro digestion model combined with Kjeldahl nitrogen determination. (5) The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were used to detect the bitter hydrophobic peptides by reversed-phase high-performance liquid chromatography. (6) Detection of neuropeptide content: The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were dissolved in sterile deionized water to obtain sample solutions with a concentration of 100 mg / mL. The sample solutions were then administered to C57 / B6 mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) by gavage at a dose of 10 μL per gram of mouse body weight. After gavage, the mice were fed normally for 1 hour and then sacrificed. The stomach tissue of the mice was dissected and homogenized in PBS on ice. The content of vomiting neuropeptides was detected using an ELISA kit from Shanghai Keaibo Biotechnology Co., Ltd. (7) Blood ammonia test: The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were dissolved in sterile deionized water to obtain sample solutions with a concentration of 100 mg / mL. These sample solutions were then administered to C57 / B6 mice (purchased from Jicui Pharmaceutical Technology Co., Ltd.) via gavage at a dose of 10 μL per gram of mouse body weight. After gavage, the mice were fed normally for 2 hours, and blood samples were collected and immediately incubated at 4°C for 1 hour. Plasma was then separated by centrifugation at 3000 rpm for 15 minutes. The ammonia content was determined using a blood ammonia assay kit purchased from Nanjing Jiancheng Bioengineering Institute. (8) Detection of total nitrogen content: The hydrolyzed whey protein products obtained in Example 1 and Comparative Examples 1-4 were dissolved in sterile deionized water to obtain sample solutions with a concentration of 100 mg / mL. The sample solutions were then administered to C57 / B6 mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) by gavage at a dose of 10 μL of sample solution per gram of mouse body weight. After the mice were fed normally for 4 hours, urine was collected, and the total nitrogen content in the urine was directly determined using an Elementar elemental analyzer with a high-temperature combustion-thermal conductivity detector. The specific results are shown in Table 1 below: Table 1: Effects of different methods on hydrolyzed whey protein products

[0081] The results show: (1) In Comparative Example 1, the protein did not expand, the cross-linking effect was poor, the abundance of specific polypeptides after hydrolysis was low, the osmotic pressure was high, the bitterness was strong, the content of neuropeptides was high, resulting in strong gastrointestinal irritation, and the metabolic burden was heavy, resulting in high blood ammonia and urine nitrogen content.

[0082] (2) In Comparative Example 2, the proteins in the hydrolysate were not cross-linked. After hydrolysis, the abundance of specific polypeptides was low, the osmotic pressure was high, the bitterness was strong, and the content of neuropeptides was high, resulting in strong gastrointestinal irritation. The heavy metabolic burden led to high blood ammonia and urine nitrogen content.

[0083] (3) In Comparative Example 3, the osmotic pressure was the highest and the bitter peptide content was the highest. The high neuropeptide content led to strong gastrointestinal irritation, and the heavy metabolic burden led to high blood ammonia and urine nitrogen content.

[0084] (4) In Comparative Example 4, the protein was not hydrolyzed, the protein digestibility was low, and the metabolic burden was heavy, resulting in high blood ammonia and urine nitrogen levels.

[0085] Example 3: Study on the performance of the medium-length peptide SLAMAASDISLA The protein of this invention contains the medium-length peptide SLAMAASDISLA. The purified SLAMAASDISLA peptide was chemically synthesized, and its performance was tested. The specific method is as follows: 1. Easily recognized by intestinal peptidase The brush border membrane sacs of the mouse small intestine were extracted and prepared into a suspension (concentration: 0.5 mg / mL, based on total protein). 1 mg / mL of SLAMAASDISLA peptide aqueous solution was added, and the suspension was incubated at 37℃ for 15 min. The degradation rate of the peptide was detected by liquid chromatography-mass spectrometry.

[0086] The results show that ( Figure 9 ): The medium-length peptides of this invention are 80% converted into dipeptides and tripeptides within 15 minutes (degradation rate of 80%), indicating that they are readily digested by intestinal enzymes.

[0087] 2. PepT1 affinity Caco-2 cells were cultured on Transwell plates to form a dense monolayer. The medium-length peptide SLAMAASDISLA was hydrolyzed by intestinal peptidase according to the method in step 1. Glycyl-sarcosine (Gly-Sar) was added to the hydrolysate at an amount of 0.8 mM. The peptide concentration required to inhibit 50% Gly-Sar transport was then determined.

[0088] The results show that ( Figure 10 ): The IC50 of the medium-long peptide SLAMAASDISLA prepared in this invention is 0.45 mM, indicating that it can occupy the Gly-Sar transport channel at low concentrations, demonstrating strong affinity and rapid absorption.

[0089] 3. Antioxidant function The antioxidant capacity of medium- and long peptides was determined using DPPH and ABTS kits purchased from Nanjing Jiancheng Biotechnology Research Institute.

[0090] The specific method is as follows: The medium-long peptide SLAMAASDISLA was prepared into test solutions with concentration gradients of 0.1, 0.2, 0.4, 0.8, 1.0, 1.5, and 2.0 mg / mL using deionized water. Whey protein served as the control group, and test solutions were prepared at concentrations of 1.0, 5.0, 10.0, 15.0, 20.0, and 25.0 mg / mL. The tests were performed according to the instructions for the DPPH and ABTS kits, and concentration-scavenging curves were plotted. The IC50 value was calculated using nonlinear regression. The results show that ( Figure 11 ): The medium-length peptides prepared in this invention have IC50 values ​​of 0.8 mg / mL and 0.6 mg / mL against DPPH and ABTS, respectively, which are superior to unhydrolyzed whey protein (initial raw material), with IC50 values ​​greater than 20 mg / mL for both.

[0091] 4. Effects of SLAMAASDISLA peptides on neuropeptides The methods for detecting neuropeptide content are as follows: The medium-length peptide SLAMAASDISLA was dissolved in sterile deionized water to obtain a sample solution with a concentration of 100 mg / mL. The sample solution was then administered to C57 / B6 mice purchased from Jicui Pharmaceutical Technology Co., Ltd. via gavage at a dose of 10 μL of sample solution per gram of mouse body weight. After gavage, the mice were fed normally for 1 hour and then sacrificed. The stomach tissue of the mice was obtained by dissection, homogenized in PBS on ice, and the content of vomiting neuropeptides was detected using an ELISA kit from Shanghai Keaibo Biotechnology Co., Ltd. The results show: The neuropeptide content of the medium-length peptides prepared in this invention is only 39 pg / mL, which is much lower than the data of other examples.

[0092] 5. Effects of SLAMAASDISLA peptide on blood ammonia The methods for detecting blood ammonia are as follows: The medium-length peptide SLAMAASDISLA was dissolved in sterile deionized water to obtain a sample solution with a concentration of 100 mg / mL. The sample solution was then administered via gavage to C57 / B6 mice purchased from Jicui Pharmaceutical Technology Co., Ltd. at a dose of 10 μL per gram of mouse body weight. After 2 hours of normal feeding following gavage, blood samples were collected and immediately incubated at 4℃ for 1 hour. Plasma was then separated by centrifugation at 3000 rpm for 15 minutes. The ammonia content was determined using a blood ammonia assay kit purchased from Nanjing Jiancheng Bioengineering Institute. The results show: Ingesting the medium-length peptide prepared in this invention resulted in a blood ammonia level of only 56 μmol / L, which is far lower than the data from other embodiments.

[0093] 6. Effect of SLAMAASDISLA peptide on urinary nitrogen content The method for detecting total nitrogen content is as follows: The medium-length peptide SLAMAASDISLA was dissolved in sterile deionized water to obtain a sample solution with a concentration of 100 mg / mL. The sample solution was then administered to C57 / B6 mice purchased from Jicui Pharmaceutical Technology Co., Ltd. via gavage at a dose of 10 μL of sample solution per gram of mouse body weight. After the mice were fed normally for 4 hours, urine was collected, and the total nitrogen content in the urine was directly determined using an Elementar elemental analyzer with a high-temperature combustion-thermal conductivity detector. The results show: Ingestion of the medium-length peptide prepared in this invention resulted in a urinary nitrogen content of only 17 mg / 24h, which is far lower than the data from other embodiments.

[0094] 7. Inhibitory effect of SLAMAASDISLA on DPP-IV The DPP-IV kit purchased from Jianglai Biotechnology Co., Ltd. was used for detection.

[0095] The specific method is as follows: The medium-long peptide SLAMAASDISLA was prepared into test solutions with concentration gradients of 0.1, 0.2, 0.4, 0.8, 1.0, 1.5, and 2.0 mg / mL using Tris-HCl buffer. Whey protein served as the control group, and test solutions were prepared at concentrations of 1.0, 5.0, 10.0, 15.0, 20.0, and 25.0 mg / mL. Perform the test according to the DPP-IV kit instructions, plot the concentration-sweep curve, and calculate the IC50 value using nonlinear regression.

[0096] The results show: The medium-length peptide prepared in this invention exhibits a significant dose-dependent inhibitory effect on DPP-IV, with a half-maximal inhibitory concentration (IC50) of 1.25 mg / mL.

[0097] In contrast, the inhibition rate of unhydrolyzed whey protein raw material was still less than 30% at a concentration of 20 mg / mL.

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A hydrolyzed protein peptide for preventing vomiting, characterized in that, The anti-vomiting hydrolyzed protein peptide is SLAMAASDISLA, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing hydrolyzed whey protein to prevent vomiting, wherein the hydrolyzed whey protein to prevent vomiting contains the protein peptide of claim 1, characterized in that, The method includes the following steps: (1) Raw material pretreatment and preparation of whey protein solution Using whey protein as raw material, an initial whey protein solution with a mass concentration of 3-6% was prepared; the pH value of the initial whey protein solution was adjusted to 2.0-2.5 to obtain the whey protein solution to be fibroinated. (2) Ultrasonic-assisted whey protein fibrinolysis The whey protein solution to be fibrosed obtained in step (1) is subjected to an ultrasonic power of 150-200 W and an ultrasonic frequency of 40-60 kHz at a temperature of 55-75℃, using a pulse mode of "ultrasonic 3-5 s / intermittent 6-10 s" for 2.5-3.5 h to obtain a fibrosed whey protein dispersion. (3) Laccase cross-linked fibrin whey protein Add a phenolic adjuvant with a final concentration of 0.3-0.5 mmol / L to the fibrinized whey protein dispersion obtained in step (2); adjust the pH of the system to 4.5-5.0, then add laccase from Trametes versicolor, wherein the amount of laccase is 50-60 U / g whey protein, and continue the reaction for 3-5 h at a temperature of 35-40℃, a stirring speed of 120-150 rpm, and a dissolved oxygen concentration ≥5 mg / L; after the reaction is completed, inactivate the enzyme to obtain a laccase-crosslinked fibrinized whey protein solution; preferably, the phenolic adjuvant is ferulic acid; (4) Preparation of cross-linked whey protein hydrolysate and anti-emetic hydrolysate. Add flavor protease to the laccase cross-linked fibrinized whey protein solution obtained in step (3), wherein the amount of flavor protease is 300~400 U / g whey protein; adjust the pH of the system to 6.5~7.0, and react for 2~3 h at a temperature of 40~50℃ and a stirring speed of 80~120 rpm; after the reaction is completed, inactivate the enzyme to obtain a solution, and dry the obtained solution to obtain the hydrolyzed whey protein product for preventing vomiting.

3. The anti-vomiting hydrolyzed whey protein prepared by the method of claim 2.

4. A food, medicine, health product, or nutritional product, characterized in that, The food, medicine, health product or nutritional product contains the anti-vomiting hydrolyzed protein peptide as described in claim 1 or the anti-vomiting hydrolyzed whey protein as described in claim 3; Optionally, the food, medicine, health product or nutritional product may also contain derivatives of anti-emetic hydrolyzed protein peptides. The derivatives of anti-emetic hydrolyzed protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the anti-emetic hydrolyzed protein peptide. Optionally, the drug may further contain pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field. Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition. Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form; Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection; Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers; Optionally, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; The food products also include special dietary foods; the health products also contain acceptable excipients.

5. An expression vector or recombinant microorganism, characterized in that, The expression vector or recombinant microorganism expresses the anti-vomiting hydrolyzed protein peptide of claim 1; Preferably, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors; Preferably, the recombinant microorganism is a bacterium or a fungus.

6. The use of the anti-emetic hydrolyzed protein peptide of claim 1 or the anti-emetic hydrolyzed whey protein of claim 3 in the preparation of food, pharmaceuticals, health products or nutritional products.

7. The application according to claim 6, characterized in that, The food, medicine, health product or nutritional product may also contain derivatives of anti-vomiting hydrolyzed protein peptides. The derivatives of anti-vomiting hydrolyzed protein peptides refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the anti-vomiting hydrolyzed protein peptide.

8. The application according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field. Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition. Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form; Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection; Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

9. The application according to claim 6, characterized in that, The food products include, but are not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages. The food products mentioned also include special dietary foods; Preferably, the nutritional products include medical nutritional products, nutritional products formulated for special populations, and nutritional supplements.

10. The use of the anti-vomiting hydrolyzed protein peptide according to claim 1 in the preparation of DPP-IV inhibitors, or hypoglycemic drugs or health products, or drugs or health products for treating type II diabetes.

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