Application of millet protein polypeptide and medicine-food homologous composition in improving mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythropenia caused by radiotherapy and chemotherapy

Abstract

The present invention belongs to the technical field of nutritious food and protein hydrolysate, and specifically relates to a millet protein hydrolysate, a nutritional composition and its application in improving mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukopenia and erythropenia caused by tumors and radiotherapy and chemotherapy. The bioactive peptides contained in the millet protein hydrolysate of the present invention all have potential immunomodulatory effects. On this basis, the present invention also provides a nutritional composition containing the above-mentioned millet protein hydrolysate, and the millet protein hydrolysate is combined and used with Poria cocos, donkey-hide gelatin, Euryale ferox, roasted malt, chicken gizzard lining and tangerine peel, which can effectively improve mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukopenia and erythropenia caused by tumors and / or radiotherapy and chemotherapy, and improve the body's white blood cell count and hematopoietic ability.

Description

Technical Field

[0001] The invention belongs to the technical field of nutritional compositions and protein hydrolysates, and specifically relates to the application of millet protein polypeptides and a medicine-food homologous composition in improving mental depression, anorexia, nausea and vomiting, gastrointestinal damage, and leukocytopenia and erythropenia caused by radiotherapy and chemotherapy. Background Art

[0002] Cancer is a major medical and public health problem that seriously threatens human health and life. Chemotherapy is one of the more commonly used treatments for tumors. However, due to the lack of specificity of anti-tumor drugs, this method has a large toxic and side effect. While killing tumor cells, chemotherapy drugs also cause serious damage to normal cells, especially the proliferating bone marrow hematopoietic cells, which is specifically manifested as a decrease in the number of white blood cells and blood cells. Clinically, severe infections secondary to leukopenia often affect the smooth progress of chemotherapy, resulting in reduced clinical efficacy and decreased quality of life of patients. At present, clinically, white blood cell-raising drugs and hematopoietic stimulating factors are mainly used to enhance the body's hematopoietic ability, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or hormones, shark liver alcohol, and leucine. However, the improvement of hematopoietic ability by this type of drug is temporary, the improvement of cell function is relatively insufficient, and it is expensive and accompanied by certain toxic and side effects, so there are certain limitations in clinical application.

[0003] In addition, the incidence of moderate to severe malnutrition in patients with malignant tumors is as high as 58%, especially for patients with gastric cancer, esophageal cancer, pancreatic cancer, liver cancer, laryngeal cancer, lung cancer, intestinal cancer, etc., who have difficulty eating due to organ lesions. 50% to 80% of tumor patients will further develop cachexia, and 20% of patients die directly from malnutrition and cachexia. The main cause of occurrence is the strong proliferation ability of tumor cells, which seizes and consumes most of the nutrients required for normal metabolism of the body. At the same time, some metabolites released by tumor cell growth can easily cause nausea, vomiting, abnormal taste and smell, and anorexia in patients. Energy intake and utilization rate are significantly reduced, causing malnutrition, especially for patients in the middle and late stages, whose condition develops rapidly and their body consumption is greater. Providing patients with reasonable nutritional support can significantly reduce the incidence of complications and disease mortality, improve tolerance to chemotherapy, shorten hospitalization time, and improve quality of life.

[0004] Millet is a plant of the Gramineae family. It is drought-resistant, salt-resistant, and has strong vitality. It is mainly distributed in arid and semi-arid areas and is one of China's traditional crops. Studies have shown that the protein content in millet is 12.3%, and the amino acids are complete and balanced in proportion. It can be used as a high-quality source of plant protein. At the same time, millet protein also has the characteristics of low allergenicity and easy digestion. Millet protein contains 10.20% aromatic amino acids, 19.89% branched-chain amino acids, and 10.15% proline. This shows that millet protein may have unique physical and chemical properties and physiological functions. The essential amino acids in millet protein account for about 44.7% of the total amino acid content, and the content of each amino acid is higher than the recommended value of FAO / WHO, reaching the standard of complete protein. In addition, Chinese medicine believes that millet is sweet, salty, and cool in nature. It enters the kidney, spleen, and stomach meridians. The Compendium of Materia Medica records that millet "treats stomach nausea and dysentery, cooks porridge, benefits Dantian, replenishes deficiency, and opens the stomach." However, no study has reported on the intervention effect and mechanism of millet protein on nausea and vomiting associated with tumors and the decrease of white blood cells and red blood cells associated with chemotherapy. Summary of the invention

[0005] The purpose of the present invention is to provide the use of millet protein polypeptides and a medicine-food composition for improving mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythropenia caused by radiotherapy and chemotherapy. The millet protein hydrolyzate and a nutritional composition containing the millet protein hydrolyzate have a good improvement effect on mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythropenia caused by radiotherapy and chemotherapy.

[0006] The invention provides a nutritional composition, comprising the following raw materials in parts by weight: 2-10 parts of millet protein hydrolysate, 1-5 parts of Poria cocos, 5-15 parts of donkey-hide gelatin, 5-15 parts of Euryale ferox, 2-7 parts of roasted malt, 2-7 parts of chicken gizzard lining and 5-15 parts of tangerine peel; the millet protein hydrolysate comprises bioactive peptides, and the bioactive peptides comprise one or more of 10 polypeptides whose amino acid sequences are shown as SEQ ID NO.1-SEQ ID NO.10.

[0007] Preferably, the method for preparing the millet protein hydrolysate comprises the following steps:

[0008] Grinding and defatting millet to obtain defatted millet flour;

[0009] The aqueous solution of the defatted millet flour is mixed with α-amylase and then subjected to a first enzymolysis to obtain a millet enzymolysis mixture;

[0010] The millet enzymatic hydrolysis mixture is subjected to solid-liquid separation, and a precipitate is collected to obtain a millet protein extract;

[0011] The aqueous solution of the millet protein extract is mixed with pepsin for a second enzymolysis to obtain a pepsin enzymolysis mixture;

[0012] The pepsin enzymatic hydrolysis mixture is mixed with trypsin for a third enzymatic hydrolysis to obtain a trypsin enzymatic hydrolysis mixture;

[0013] The trypsin hydrolysis mixture is subjected to solid-liquid separation again, and the obtained supernatant contains the millet protein hydrolysate.

[0014] Preferably, the volume ratio of the mass of the α-amylase to the aqueous solution of defatted millet flour is 1 g:10000 mL, the enzyme activity of the α-amylase is 30 U / g; the temperature of the first enzymatic hydrolysis is 50°C, the time is 2 h, and the pH value is 7.0.

[0015] Preferably, the mass ratio of the aqueous solution of pepsin to the millet protein extract is 1:70, the enzymatic activity of the pepsin is 500 U / mg; the temperature of the second enzymolysis is 37° C., the time is 2 h, and the pH value is 2.0.

[0016] Preferably, the mass ratio of the trypsin to pepsin enzymatic hydrolysis mixture is 1:10, the enzymatic activity of the trypsin is 4×USP; the temperature of the third enzymatic hydrolysis is 37° C., the time is 2 h, and the pH value is 7.5.

[0017] The present invention also provides the use of the above-mentioned nutritional composition in the preparation of a drug for improving one or more of mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythrocytopenia caused by tumors and / or radiotherapy and chemotherapy.

[0018] Beneficial effects:

[0019] The present invention also provides a nutritional composition containing the millet protein hydrolysate, wherein the bioactive peptides contained in the millet protein hydrolysate all have potential immunoregulatory effects, and the millet protein hydrolysate is combined and used in combination with Poria cocos, donkey-hide gelatin, Euryale ferox, roasted malt, chicken gizzard lining and dried orange peel, which can effectively improve mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythrocytopenia caused by tumors and / or radiotherapy and chemotherapy, and increase the body's leukocyte count and hematopoietic ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The weight changes of mice in each group in Example 4;

[0022] Figure 2The change of food intake of each group of mice in Example 4;

[0023] Figure 3 The changes in kaolin intake of each group of mice in Example 4;

[0024] Figure 4 The white blood cell counts of each group of mice in Example 5;

[0025] Figure 5 The neutrophil counts of each group of mice in Example 5;

[0026] Figure 6 The lymphocyte counts of each group of mice in Example 5 are shown below;

[0027] Figure 7 The red blood cell counts of mice in each group in Example 6;

[0028] Figure 8 The hemoglobin concentration of each group of mice in Example 6;

[0029] Fig. 9 The hematocrit of each group of mice in Example 6;

[0030] Fig.10 The mean corpuscular hemoglobin concentration of each group of mice in Example 6;

[0031] Fig.11 The coefficient of variation of red blood cell distribution width of each group of mice in Example 6;

[0032] Fig.12 The 5-HT content in the brain of each group of mice in Example 7;

[0033] Fig.13 The dopamine content in the brain of each group of mice in Example 7;

[0034] Fig.14 H&E stained colon pathological sections of each group of mice in Example 8;

[0035] Fig.15 The H&E stained pathological sections of gastric mucosa of each group of mice in Example 9;

[0036] exist Figures 1 to 3 The “**” in the table indicates significant difference P Less than 0.01, "*" indicates significant difference P Less than 0.05; Figures 4 to 13 “**” indicates significant difference P Less than 0.01, "*" indicates significant difference P Less than 0.05. DETAILED DESCRIPTION

[0037] The invention provides a nutritional composition, comprising the following raw materials in parts by weight: 2-10 parts of millet protein hydrolysate, 1-5 parts of Poria cocos, 5-15 parts of donkey-hide gelatin, 5-15 parts of Euryale ferox, 2-7 parts of roasted malt, 2-7 parts of chicken gizzard lining and 5-15 parts of tangerine peel; the millet protein hydrolysate comprises bioactive peptides, and the bioactive peptides comprise one or more of 10 polypeptides whose amino acid sequences are shown as SEQ ID NO.1-SEQ ID NO.10.

[0038] The millet protein hydrolyzate of the present invention includes bioactive peptides, and the bioactive peptides include one or more of the 10 polypeptides whose amino acid sequences are shown as SEQ ID NO.1 to SEQ ID NO.10, preferably 10 of the above 10 polypeptides.

[0039] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.10 of the present invention are as follows: SEQ ID NO.1: YGGGEGGEGYLPFPPR; SEQ ID NO.2: YGGGEGGEGYLPFPP; SEQ ID NO.3: HQQPPYGGVP; SEQ ID NO.4: GMHQQPPYGGVP; SEQ ID NO.5: GGIYGGTHGQGVGGGGRPV; SEQ ID NO.6: QMVQQQQPPYGGGVQG; SEQ ID NO.7: ALGYGNPIVQPY; SEQ ID NO.8: LGYGNPIVQPY; SEQ ID NO.9: ALGYGNPIVQP; SEQ ID NO.10: LGYGNPIVQP. The bioactive peptides of the present invention have potential immune activity.

[0040] The method for preparing the millet protein hydrolysate of the present invention preferably comprises the following steps:

[0041] Grinding and defatting millet to obtain defatted millet flour;

[0042] The aqueous solution of the defatted millet flour is mixed with α-amylase and then subjected to a first enzymolysis to obtain a millet enzymolysis mixture;

[0043] The millet enzymatic hydrolysis mixture is subjected to solid-liquid separation, and a precipitate is collected to obtain a millet protein extract;

[0044] The aqueous solution of the millet protein extract is mixed with pepsin for a second enzymolysis to obtain a pepsin enzymolysis mixture;

[0045] The pepsin enzymatic hydrolysis mixture is mixed with trypsin for a third enzymatic hydrolysis to obtain a trypsin enzymatic hydrolysis mixture;

[0046] The trypsin hydrolysis mixture is subjected to solid-liquid separation again, and the obtained supernatant contains the millet protein hydrolysate.

[0047] The present invention preferably grinds and defats the millet to obtain defatted millet flour. The present invention preferably grinds the millet flour and passes it through a 60-mesh sieve, and takes the sieve under to obtain the millet flour.

[0048] In the present invention, the defatting step preferably includes: mixing the millet powder with n-hexane and then ultrasonically extracting it, centrifuging the obtained ultrasonic extraction mixture to obtain an extraction residue; mixing the extraction residue with n-hexane and then ultrasonically extracting it again, air-drying the obtained extraction residue to obtain the defatted millet powder. The mass ratio of the millet powder to the volume of n-hexane in the present invention is preferably 1 g: 5-50 mL, more preferably 1 g: 10 mL; the time of the ultrasonic extraction is preferably 1 h; other parameters of the ultrasonic extraction are not particularly limited, and the parameters of conventional ultrasonic extraction in the field can be used. The mass ratio of the extraction residue to the volume of n-hexane in the present invention is preferably 1 g: 10 mL; the conditions for the second ultrasonic extraction are preferably the same as those in the above-mentioned technical solution, which will not be repeated. The air-drying in the present invention is preferably natural air-drying.

[0049] After obtaining the defatted millet flour, the present invention preferably mixes the defatted millet flour with water and adjusts the pH value to obtain an aqueous solution of defatted millet flour. The mass ratio of the defatted millet flour of the present invention to water is preferably 1g:6mL; the water is preferably ultrapure water. The present invention preferably adjusts the pH value to 7.0. The present invention preferably uses a NaOH solution to adjust the pH value, and the concentration of the NaOH solution is preferably 1 mol / L.

[0050] After obtaining the aqueous solution of defatted millet flour, the present invention preferably mixes the aqueous solution of defatted millet flour with α-amylase and performs a first enzymolysis to obtain a millet enzymolysis mixture. The volume ratio of the mass of the α-amylase of the present invention to the aqueous solution of defatted millet flour is preferably 1g:10000 mL, and the enzyme activity of the α-amylase is preferably 30 U / g; the temperature of the first enzymolysis is preferably 50°C, and the time is preferably 2 h.

[0051] After obtaining the millet enzymatic hydrolysis mixture, the present invention preferably performs enzyme inactivation treatment on the millet enzymatic hydrolysis mixture to obtain the millet enzymatic hydrolysis mixture after enzyme inactivation treatment. The enzyme inactivation treatment conditions of the present invention are preferably boiling water bath enzyme inactivation for 20 minutes.

[0052] After obtaining the millet hydrolysis mixture after the enzyme inactivation treatment, the present invention preferably performs solid-liquid separation on the millet hydrolysis mixture after the enzyme inactivation treatment to obtain a precipitate. The solid-liquid separation method of the present invention is preferably centrifugation, the centrifugal speed is preferably 2000 g, and the time is preferably 10 min.

[0053] After obtaining the precipitate, the present invention preferably rinses the precipitate with water and performs solid-liquid separation, and rinses the obtained precipitate again and performs solid-liquid separation to obtain a millet protein extract. The water of the present invention is preferably pure water; the method of solid-liquid separation is preferably the same as the above technical solution, which will not be repeated; the number of solid-liquid separation is preferably 2 to 4 times.

[0054] After obtaining the millet protein extract, the present invention preferably mixes the millet protein extract with water and adjusts the pH value to obtain an aqueous solution of the millet protein extract. The mass ratio of the millet protein extract of the present invention to the volume of water is preferably 1 g: 50 mL. The present invention preferably uses hydrochloric acid to adjust the pH value, the concentration of the hydrochloric acid is preferably 1 mol / L, and the pH value is preferably 2.0.

[0055] After obtaining the aqueous solution of the millet protein extract, the present invention preferably mixes the aqueous solution of the millet protein extract with pepsin for a second enzymolysis to obtain a pepsin enzymolysis mixture. The mass ratio of the pepsin to the aqueous solution of the millet protein extract of the present invention is preferably 1:70, and the enzymatic activity of the pepsin is preferably 500 U / mg; the temperature of the second enzymolysis is preferably 37°C, and the time is preferably 2 h.

[0056] After obtaining the pepsin hydrolysis mixture, the present invention preferably performs enzyme inactivation treatment on the pepsin hydrolysis mixture to obtain an enzyme inactivated pepsin hydrolysis mixture. The enzyme inactivation treatment method of the present invention is preferably the same as the above technical solution, and will not be described in detail.

[0057] After obtaining the inactivated pepsin hydrolysis mixture, the present invention preferably adjusts the pH value of the inactivated pepsin hydrolysis mixture. The present invention preferably uses a NaOH solution to adjust the pH value, and the concentration of the NaOH solution is preferably 1 mol / L; the pH value is preferably 7.5.

[0058] After adjusting the pH value, the present invention preferably mixes the pepsin hydrolysis mixture after the enzyme inactivation treatment after the pH value is adjusted with trypsin for a third enzymatic hydrolysis to obtain a trypsin hydrolysis mixture. The mass ratio of trypsin to pepsin hydrolysis mixture of the present invention is preferably 1:10, and the enzyme activity of the trypsin is preferably 4×USP; the temperature of the third enzymatic hydrolysis is preferably 37°C, and the time is preferably 2 h.

[0059] After obtaining the trypsin hydrolysis mixture, the present invention preferably performs enzyme inactivation treatment on the trypsin hydrolysis mixture to obtain the trypsin hydrolysis mixture after enzyme inactivation treatment. The enzyme inactivation treatment method of the present invention is preferably the same as the above technical solution, and will not be repeated here.

[0060] After obtaining the trypsin hydrolysis mixture after the enzyme inactivation treatment, the present invention preferably performs solid-liquid separation on the trypsin hydrolysis mixture again to obtain a supernatant. The method of solid-liquid separation in the present invention is preferably the same as the above technical solution, which will not be repeated.

[0061] After obtaining the supernatant, the present invention preferably freeze-dries the supernatant to obtain the millet protein hydrolyzate. The freeze-drying parameters of the present invention are not particularly limited, and conventional freeze-drying parameters in the art can be used.

[0062] The millet protein hydrolysate prepared by the above preparation method has a protein purity of 83.8%±5.95% and a hydrolysis degree of 31.4%±1.44%; and the millet protein hydrolysate contains the bioactive peptides described in the above technical solution, has high potential immune activity, and can assist in improving the body's immunity and assist in increasing the body's white blood cell and red blood cell counts.

[0063] In parts by mass, the raw materials of the nutritional composition of the present invention include 2 to 10 parts of millet protein hydrolysate, preferably 4 to 9 parts, and more preferably 5 to 7 parts; the raw materials of the nutritional composition include 1 to 5 parts of Poria cocos, preferably 2 to 3 parts; the raw materials of the nutritional composition include 5 to 15 parts of donkey-hide gelatin, preferably 7 to 10 parts; the raw materials of the nutritional composition include 5 to 15 parts of Euryale ferox, preferably 7 to 10 parts; the raw materials of the nutritional composition include 2 to 7 parts of roasted malt, preferably 3 to 5 parts; the raw materials of the nutritional composition include 2 to 7 parts of chicken gizzard lining, preferably 3 to 5 parts; the raw materials of the nutritional composition include 5 to 15 parts of tangerine peel powder, preferably 7 to 10 parts.

[0064] The method for preparing the nutritional composition of the present invention preferably comprises: drying and crushing Poria cocos, donkey-hide gelatin, Euryale ferox, roasted malt, chicken gizzard lining and dried orange peel, and then mixing with the millet protein hydrolyzate to obtain the nutritional composition. The nutritional composition of the present invention is preferably in powder form, and the particle size is preferably 200-800 mesh, and more preferably 300-500 mesh.

[0065] The present invention also provides the use of the nutritional composition described in the above technical scheme in the preparation of a drug for improving one or more of mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythrocytopenia caused by tumors and / or chemotherapy and radiotherapy, preferably the use of the nutritional composition in the preparation of a drug for improving mental depression, anorexia, nausea and vomiting, gastrointestinal damage, leukocytopenia and erythrocytopenia caused by tumors and / or chemotherapy and radiotherapy.

[0066] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1

[0068] 1. Preparation of Millet Protein

[0069] (1) First, grind and defatted the millet: grind the millet with an ultrafine grinder, pass the obtained millet powder through a 60-mesh sieve, weigh 100 g of millet powder and mix it with n-hexane at a solid-liquid ratio of 1:10 (w / v, g / mL), perform ultrasonic extraction for 1 h, centrifuge at 2000 g, discard the supernatant, and repeat the above operation once for the remaining residue. The collected residue was placed in a fume hood to dry naturally to obtain defatted millet powder.

[0070] (2) Weigh the defatted millet flour obtained by the method described in (1) and mix it with ultrapure water at a solid-liquid ratio of 1:6 (w / v, g / mL) and stir it thoroughly. Adjust the pH to 7.0 with 1 mol / L NaOH, add 30 U / g of α-amylase at a ratio of enzyme to mixed liquid of 1:10000 (w / v, g / mL), and perform enzymolysis in a 50°C water bath for 2 h. Stir continuously during the process to make the system uniform. After the reaction is completed, inactivate the enzyme in a boiling water bath for 20 min, cool naturally to room temperature, centrifuge at 2000g for 10 min to remove the supernatant, rinse with pure water and repeat centrifugation 2 to 4 times, and freeze-dry the collected solid precipitate to obtain an extract rich in millet protein.

[0071] 2. Determination of protein purity

[0072] The invention adopts the Folin phenol method to determine the protein purity of the extract rich in millet protein.

[0073] Solution A: Prepare 4% Na 2 CO 3 Solution B: Prepare 1% CuSO 4 ·5H 2 O solution and 2% potassium sodium tartrate solution, mix 1:1 before use. Solution A: Mix solution A and solution B in a ratio of 50:1 to obtain solution A. Solution B: Folin phenol reagent.

[0074] Add 40 μL of extract solution (diluted to an appropriate concentration with ultrapure water in advance) to 200 μL of solution A, mix well and react at 30°C for 10 min, then add 20 μL of solution B, mix well and react at 30°C for 30 min, and use bovine serum albumin (BSA) as the standard to measure the absorbance at 500 nm. The protein content of the extract was measured by the Folin phenol method and was 81.8%~94.6%, indicating that the extract was rich in millet protein.

[0075] Example 2

[0076] Preparation of millet protein hydrolysate

[0077] (1) The millet protein extract prepared in Example 1 was weighed and mixed with ultrapure water at a material-liquid ratio of 1:50 (w / v).

[0078] (2) Adjust the pH to 2.0 with 1 mol / L HCl.

[0079] (3) Add pepsin (500 U / mg) at an enzyme / substrate ratio of 1:70 (w / w).

[0080] (4) Enzymatic hydrolysis at 37°C in a water bath for 2 h.

[0081] (5) Inactivate the enzyme in a boiling water bath for 20 min and allow to cool naturally to room temperature.

[0082] (6) Adjust the pH to 7.5 with 1 mol / L NaOH and add trypsin (4× USP) at an enzyme / substrate ratio of 1:10 (w / w).

[0083] (7) Incubate the cells in a 37°C water bath for 2 h.

[0084] (8) Inactivate the enzyme in a boiling water bath for 20 min and then cool naturally to room temperature.

[0085] (9) Centrifuge the hydrolyzed sample at 8000g for 20 min and collect the supernatant.

[0086] (10) Freeze-drying to obtain millet protein hydrolysate, i.e., millet protein hydrolysate powder (FMPH).

[0087] Determination of peptide purity

[0088] The present invention adopts the Folin phenol method to determine the purity of polypeptides in millet protein hydrolysate.

[0089] Solution A: Prepare 4% Na 2 CO 3 Solution B: Prepare 1% CuSO 4 ·5H2 O solution and 2% potassium sodium tartrate solution, mix 1:1 before use. Solution A: Mix solution A and solution B at a ratio of 50:1 to obtain solution A. Solution B: Folin phenol reagent.

[0090] Establishment of standard curve: Prepare BSA standard solution with concentration gradient of 0, 50, 100, 150, 200, 250, 300 μg / mL, add 40 μL sample solution to 200 μL solution A, mix well and react at 30℃ for 10 min, then add 20 μL solution B, mix well and react at 30℃ for 30 min, and measure absorbance at 500 nm. Set three parallels for each concentration, and make a standard curve with BSA concentration as the vertical axis and absorbance as the horizontal axis.

[0091] Determination of peptide purity in millet protein hydrolysate: Add 40 μL millet protein hydrolysate sample solution (diluted to an appropriate concentration in advance) to 200 μL solution A, mix well and react at 30°C for 10 min, then add 20 μL solution B, mix well and react at 30°C for 30 min, and measure the absorbance at 500 nm. Set up three parallels for each sample, and substitute the absorbance of the sample into the BSA standard curve for calculation.

[0092] Determination of the degree of hydrolysis of millet protein hydrolysate

[0093] The degree of hydrolysis (DH) refers to the percentage of the number of hydrolyzed peptide bonds. The present invention adopts the o-phthalaldehyde (OPA) method to measure the degree of hydrolysis of millet protein hydrolysate.

[0094] OPA reagent: Solution A: Dissolve 1.905 g of sodium tetraborate and 50 mg of sodium dodecyl sulfate (SDS) in 40 mL of Milli-Q water and ultrasonicate for 1 h to obtain Solution A. Solution B: Dissolve 40 mg of o-phthalaldehyde (OPA) in 1 mL of anhydrous ethanol and transfer all of it to Solution A after it is completely dissolved. Add 44 mg of dithiothreitol (DTT) to Solution A and finally dilute to 50 mL with Milli-Q water.

[0095] 32 μL of Milli-Q water, serine standard solution and sample solution were added to a 96-well plate, and then 240 μL of OPA solution was added. After mixing, the mixture was reacted at 37°C in the dark for 2 min, and the absorbance was measured at 340 nm.

[0096] Serine standard solution: Weigh 10 mg of serine and dissolve it in Milli-Q water to prepare a 0.9516 mM serine standard solution.

[0097] Add 32 μL of Milli-Q water, serine standard solution and sample solution to a 96-well plate, then add 240 μL of OPA solution, mix well, and react at 37°C in the dark for 2 min. Measure the absorbance at 340 nm, and set up three replicates for each sample.

[0098] Calculation of Degree of Hydrolysis (DH)

[0099] ;

[0100] h tot Depends on the type of raw material. For most food proteins, the average molecular weight of amino acids is 125 g / mol, so h tot It is about 8, which means that each gram of protein contains about 8 mmol of peptide bonds.

[0101] h is a function of the number of millimoles of serine amino groups.

[0102]

[0103] ;

[0104] Where 0.9516 is the concentration of serine in the standard sample, i.e. 0.9516 mmol / L, and C is the protein concentration of the sample (mg / mL).

[0105] The protein purity of millet protein hydrolysate was 83.8%±5.95% as measured by Folin phenol method, and the hydrolysis degree of millet protein was 31.4%±1.44% as measured by OPA method.

[0106] Example 3

[0107] Protein source and peptide sequence identification of millet protein hydrolysate

[0108] UPLC-MS / MS analysis was performed using an Orbitrap Fusion-Lumos. A C18 column (3 µm, 75 µm × 15 cm) was used at a flow rate of 300 μL / min. Mobile phase A was 0.1% formic acid in H 2 O, mobile phase B was 80% acetonitrile / H2O containing 0.1% acetic acid 2 O. The elution conditions were as follows: 0-4 min, 5%-10% B; 4-40 min, 10%-28% B; 40-47 min, 28%-38% B; 47-48 min, 38%-100% B; 48-60 min, 100% B. A cutoff value of -10lgP>15 was used to eliminate biased peptide identifications, and the sequences were manually inspected and aligned with the protein coding information of the Uniprot database to determine the source protein.

[0109] According to the UPLC-MS / MS results, a total of 2620 peptide sequences were identified in millet protein hydrolysates. These peptides were produced by 643 proteins. The PeptideRanker score and BIOPEP protein database were used to predict key peptides with potential immunomodulatory effects in millet protein hydrolysates. The relative peak area was also used as a screening criterion. According to the screening results, a total of 137 peptides were screened out with the peak area relative abundance > 0.1% and PeptideRanker score > 0.2 as the screening criteria. The lengths of these peptides ranged from 8 to 23. According to the BIOPEP search results, a total of 10 peptides had immunoenhancing effects (Table 1), and their key active sequences were YG, LGY, and YGG.

[0110] Table 1 Bioactive peptides in millet protein hydrolysate

[0111] Source Protein Peptide sequence Relative peak area (%) Retention time (min) Exact molecular weight Error(ppm) PeptideRanker Scoring Potentially active sequences Biological Activity K3ZA42 YGGGEGGEGYLPFPPR 0.25 47.2 826.8888 0.1 0.76 YG, YGG immunostimulating K3ZA42 YGGGEGGEGYLPFPP 0.13 48.0 1495.6619 6.5 0.67 YG, YGG immunostimulating K3XUT9 HQQPPYGGVP 0.75 42.4 1078.5195 0 0.65 YG, YGG immunostimulating K3XUT9 GMHQQPPYGGVP 0.36 46.2 1266.5815 -0.2 0.62 YG, YGG immunostimulating K3XRC5 GGIYGGTHGQGVGGGGRPV 0.11 64.2 1681.8284 1 0.47 YG, YGG immunostimulating A0A0Q3VL29 QMVQQQQPPYGGGVQG 0.20 19.3 1700.7941 0 0.28 YG, YGG immunostimulating K3ZJP2 ALGYGNPIVQPY 0.18 34.4 1290.6608 0.5 0.27 LGY, YG immunostimulating A0A4U6TMG4 LGYGNPIVQPY 0.16 29.0 1219.6237 -0.5 0.26 LGY, YG immunostimulating A0A4U6TMG4 ALGYGNPIVQP 0.20 26.9 1127.5974 0.6 0.25 LGY, YG immunostimulating A0A4U6TMG4 LGYGNPIVQP 0.60 40.6 1056.5603 0.1 0.23 LGY, YG immunostimulating

[0112] Example 4

[0113] Animal experiments

[0114] The millet protein hydrolysate and the medicinal and edible substances are mixed in proportion to prepare a nutritional composition: 7 parts of the millet protein hydrolysate powder prepared in Example 2, 3 parts of Poria powder, 10 parts of donkey-hide gelatin powder, 10 parts of Euryale powder, 5 parts of roasted malt powder, 5 parts of chicken gizzard powder, and 10 parts of tangerine peel powder.

[0115] The medicinal and edible substances in the formula are dried and crushed to obtain a powder with a fineness of 300 meshes, and the powder is mixed with the millet protein hydrolyzate.

[0116] Mice were randomly divided into a normal control group (Blank), a cisplatin model group (Model), a low-dose nutritional composition group (LD), a high-dose nutritional composition group (HD), and a positive drug ondansetron control group (Positive, Pos) according to similar body weight. There were 12 mice in each group and 4 mice in each cage. After 3 days of adaptive feeding, kaolin particles were placed to adapt the mice to the presence of kaolin. After the mice basically stopped eating kaolin, the nutritional composition intervention and modeling were carried out. The nutritional composition was suspended in 0.5% CMC-Na and gavaged for 7 consecutive days at 0.5 mL / 200g rat body weight. The modeling method of the chemotherapy-induced mouse pica model was to inject 3 mg / kg cisplatin intraperitoneally once a day for 5 consecutive days, and ondansetron was used as a positive drug. The nutritional composition intervention continued during the 5-day modeling period, and the kaolin intake, food intake and body weight changes of the rats were recorded daily.

[0117] Grouping:

[0118] Normal control group (Blank): ig 0.5% CMC-Na+ ip normal saline;

[0119] Cisplatin model group: ig 0.5% CMC-Na+ ip cisplatin (3 mg / kg);

[0120] Low-dose group: ig nutritional composition (1 g / kg) + ip cisplatin (3 mg / kg).

[0121] High-dose group: ig nutritional composition (4 g / kg) + ip cisplatin (3 mg / kg).

[0122] Positive control group: ig ondansetron (10 mg / kg) + ip cisplatin (3 mg / kg).

[0123] exist Figures 1 to 15 In the figure, normal group, model group, low-dose group, high-dose group and positive control represent normal control group, cisplatin model group, low-dose group, high-dose group and positive control group, respectively.

[0124] The changes in mouse body weight Figure 1 As shown, there was no significant difference in the body weight of mice in each group before modeling. The intraperitoneal injection of cisplatin for 5 days continuously reduced the body weight of mice in the model group (Model), which was significantly different from that of mice in the normal control group (Blank). There was no significant difference between the low-dose nutritional composition group (LD) and the model group, and the body weight of mice in the positive drug (Pos) and high-dose nutritional composition groups (HD) was significantly increased compared with the model group, indicating that the intervention of the nutritional composition can significantly improve the weight loss and malnutrition of mice induced by chemotherapy drugs, and the effect is close to that of the positive drug ondansetron.

[0125] Intraperitoneal injection of cisplatin for 5 days significantly reduced the food intake of the model group mice ( Figure 2 ), increased kaolin intake ( Figure 3 ), indicating that the mouse model of nausea, vomiting and pica was successfully induced. Figure 2 It can also be seen that compared with the model group, the high-dose nutritional composition significantly increased the food intake of mice, and its effect was comparable to that of the positive control drug ondansetron. Both high- and low-dose nutritional compositions significantly reduced the intake of kaolin by mice, indicating that they had a certain improvement effect on nausea, vomiting and pica.

[0126] Example 5

[0127] Effect of the nutritional composition prepared in Example 4 on the number of white blood cells

[0128] After the animal experiment, the mice were anesthetized and killed, and whole blood samples were taken. The total number of white blood cells was determined after dilution in Turk solution using a Newbauer chamber. The typing and number of white blood cells were determined using a fully automatic blood cell analyzer.

[0129] White blood cell count (WBC) refers to the number of white blood cells contained in a unit volume of blood. It is an important part of the body's defense system. A low count indicates aplastic anemia, acute granulocytosis and other diseases. Figure 4 It can be concluded that: compared with the normal control group, the WBC content of mice in the model group was significantly reduced, and both the high-dose nutritional composition and the positive drug could significantly increase WBC, indicating that the nutritional composition has a certain effect of increasing white blood cells.

[0130] The absolute value of neutrophils (Neu) refers to the content of neutrophils in the white blood cell count. Neutrophils are an important type of white blood cell in the human immune system, mainly responsible for fighting against the invasion of bacteria and other foreign pathogens. Neutropenia is common in chemotherapy, infection, autoimmune diseases, etc., which can cause low immunity. Figure 5 It can be concluded that oral administration of cisplatin can significantly reduce the number of neutrophils in the blood of mice. Both positive drugs and high-dose nutritional compositions can restore this change, indicating that nutritional composition intervention can enhance the low immunity induced by chemotherapy drugs.

[0131] The absolute value of lymphocytes (Lym) refers to the content of lymphocytes in the white blood cell count, which reflects the changes in the number of lymphocytes in the human body and can also reflect the changes in the hematopoietic capacity of the bone marrow to a certain extent. Since lymphocytes are an important part of the human immune system, a low absolute value of lymphocytes is often considered to be caused by exposure to radiation, drug effects, immune diseases, etc. Figure 6 It can be concluded that oral administration of cisplatin can significantly reduce the number of lymphocytes in the blood of mice. High-dose nutritional composition can restore this change, and the effect is better than that of positive drugs, indicating that nutritional composition intervention can enhance the weakened hematopoietic function induced by chemotherapy drugs.

[0132] Example 6

[0133] Effect of the nutritional composition prepared in Example 4 on erythrocyte hematopoietic function

[0134] After the animal experiment, the mice were anesthetized and killed, and whole blood samples were taken. The red blood cell count, hemoglobin concentration, and hematocrit were measured using a fully automatic blood cell analyzer.

[0135] The red blood cell count (RBC) is the number of red blood cells per liter of blood. It can monitor whether the production and apoptosis of red blood cells are normal. It is an indicator in a routine blood test. A decrease in the content often indicates various types of anemia. It is often used in the diagnosis of hemolytic anemia, iron deficiency anemia and other diseases and in the evaluation of nutritional status. Figure 7 The results showed that the chemotherapy drug cisplatin significantly reduced the number of red blood cells in mice, which may be related to the nausea, vomiting and loss of appetite in mice. Compared with the model group, the intervention of the nutritional composition significantly increased the number of red blood cells, and the effect was better than that of the positive control drug, indicating that the nutritional composition can improve the nutritional status of chemotherapy mice.

[0136] Hemoglobin concentration (HGB) refers to the amount of hemoglobin contained in a unit volume of blood. Hemoglobin, also known as hemoglobin, is a pigment-containing binding protein and the main component of red blood cells. It can bind to oxygen and transport oxygen and carbon dioxide. It can be used clinically to diagnose certain metastatic hemoglobin disorders and blood system diseases. The clinical significance of its increase or decrease is basically the same as that of the increase or decrease of red blood cells, but hemoglobin can better reflect the degree of anemia. Figure 8 The results showed that cisplatin can induce a decrease in hemoglobin concentration ( Figure 8 ), while high-dose nutritional composition can restore this change, and the effect is better than the positive control drug, suggesting that nutritional composition intervention can improve the nutritional status of chemotherapy.

[0137] In addition, cisplatin significantly reduced the hematocrit (HCT) of the model group ( Fig. 9 ), the high-dose nutritional composition alleviated it, and the effect was better than the positive control group. The high-dose nutritional composition intervention significantly increased the mean corpuscular hemoglobin concentration (MCHC) ( Fig.10 )、Red blood cell distribution width variation coefficient (RDW-CV) ( Fig.11 ), indicating that it has an improving effect on bone marrow hematopoietic disorders.

[0138] Example 7

[0139] Effect of the nutritional composition prepared in Example 4 on mental depression

[0140] After the animal experiment, the mice were anesthetized and killed, the brain tissue was taken, the blood film was removed, the whole brain was peeled, and the brain tissue was weighed after the cerebellum was removed. The mixture was transferred to a homogenizer containing a small amount of ice-cold n-butanol (containing 50 μM acetic acid) and homogenized in an ice water bath. The homogenate was transferred to a stoppered graduated centrifuge tube and supplemented to 4 mL with acidified n-butanol. After mechanical oscillation for 5 min (120 times / min), centrifugation for 5 min (3000 rpm), 2.5 mL of the supernatant was taken, 5.0 mL of n-heptane and 1.2 mL of 0.1 N hydrochloric acid were added, oscillated for 5 min, and centrifuged for 5 min. At this time, the solution was divided into upper and lower layers. 0.5 mL was taken from the above aqueous layer respectively. The concentrations of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the sample were determined by ELISA.

[0141] 5-HT is a central neurotransmitter distributed in the pineal gland and hypothalamus. It is a metabolite of tryptophan and regulates many physiological functions such as pain, sleep, mood and memory. Its level is also related to the disease states of anxiety, depression and schizophrenia. DA plays an important role in maintaining chemical balance in the brain and is an intermediate product of tyrosine metabolism. Blocking or depleting the synthesis of DA can induce or deepen depressive symptoms in individuals whose depression has been relieved.

[0142] Figure 12~Figure 13 The results showed that compared with the normal control group, cisplatin could significantly induce a decrease in the levels of 5-HT and DA in the mouse brain, which may be due to malnutrition in the chemotherapy mouse model and reduced amino acid absorption. The high-dose nutritional composition intervention group could significantly increase the 5-HT content in the brain, and its effect was better than that of the positive control group. The high-dose nutritional composition intervention group could significantly increase the DA content in the brain, and there was no significant difference with the positive control group.

[0143] Example 8

[0144] Effect of the nutritional composition prepared in Example 4 on intestinal damage

[0145] After the animal experiment, the mice were anesthetized and killed, and the colon tissue was taken. After being fixed in 10% formalin solution for 24 h, the colon tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Subsequently, the degree of colon damage was evaluated under a microscope at a magnification of 50 times and a scale bar of 500 μM. The results are shown in Figure 2. Fig.14 shown.

[0146] Depend on Fig.14 The results showed that there was no inflammatory infiltration in the colon of mice in the normal control group, intraperitoneal injection of cisplatin led to inflammatory infiltration in the colon of mice in the cisplatin model group (red arrow), and no relief was observed in the low-dose nutritional composition group. The inflammatory infiltration in the colon of mice in the high-dose nutritional composition group was significantly relieved and slightly better than that in the positive control group, indicating that the nutritional composition can alleviate intestinal damage induced by chemotherapy drugs.

[0147] Example 9

[0148] Effect of the nutritional composition prepared in Example 4 on gastric damage

[0149] After the animal experiment, the mice were anesthetized and killed, and the gastric tissue was taken. After formalin fixation, 4 μm paraffin sections were made and stained with hematoxylin-eosin (H&E). The gastric mucosal stained sections were observed under a microscope and photographed at 100 times the magnification. The scale bar is 200 μM. The results are shown in Figure 2. Fig.15 shown.

[0150] Depend on Fig.15The results showed that there was no obvious gastric pathological change in the gastric mucosa of mice in the normal control group, and intraperitoneal injection of cisplatin led to glandular disorder (red arrow) and submucosal edema (blue arrow) in mice in the cisplatin model group. The low-dose nutritional composition group, high-dose nutritional composition group and positive control group all showed relief, indicating that the nutritional composition can alleviate gastric damage induced by chemotherapy drugs.

[0151] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nutritional composition containing millet protein polypeptide, characterized in that: The invention is composed of the following raw materials in parts by weight: 2-10 parts of millet protein hydrolysate, 1-5 parts of Poria cocos, 5-15 parts of donkey-hide gelatin, 5-15 parts of Euryale ferox, 2-7 parts of roasted malt, 2-7 parts of chicken gizzard lining and 5-15 parts of dried tangerine peel; the millet protein hydrolysate comprises bioactive peptides, and the bioactive peptides comprise 10 polypeptides whose amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.10; The preparation method of the millet protein hydrolysate comprises the following steps: Grinding and defatting millet to obtain defatted millet flour; The aqueous solution of the defatted millet flour is mixed with α-amylase and then subjected to a first enzymolysis to obtain a millet enzymolysis mixture; The millet enzymatic hydrolysis mixture is subjected to solid-liquid separation, and a precipitate is collected to obtain a millet protein extract; The aqueous solution of the millet protein extract is mixed with pepsin for a second enzymolysis to obtain a pepsin enzymolysis mixture; The pepsin enzymatic hydrolysis mixture is mixed with trypsin for a third enzymatic hydrolysis to obtain a trypsin enzymatic hydrolysis mixture; The trypsin hydrolysis mixture is subjected to solid-liquid separation again, and the obtained supernatant contains the millet protein hydrolysate.

2. The nutritional composition according to claim 1, characterized in that The volume ratio of the mass of the α-amylase to the aqueous solution of the defatted millet flour is 1 g:10000 mL, and the enzyme activity of the α-amylase is 30 U / g; the temperature of the first enzymatic hydrolysis is 50° C., the time is 2 h, and the pH value is 7.

0.

3. The nutritional composition according to claim 1, characterized in that The mass ratio of the pepsin to the aqueous solution of the millet protein extract is 1:70, and the enzymatic activity of the pepsin is 500 U / mg; the temperature of the second enzymolysis is 37° C., the time is 2 h, and the pH value is 2.

0.

4. The nutritional composition according to claim 1, characterized in that The mass ratio of the trypsin to pepsin enzymatic hydrolysis mixture is 1:10, and the enzymatic activity of the trypsin is 4×USP; the temperature of the third enzymatic hydrolysis is 37° C., the time is 2 h, and the pH value is 7.

5.

5. Use of the nutritional composition according to any one of claims 1 to 4 in the preparation of a medicament for improving depression, anorexia, nausea and vomiting, and gastrointestinal damage caused by tumor chemotherapy.

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