A preparation method of wheat polypeptide chelated calcium

Abstract

The present invention discloses a preparation method of wheat polypeptide chelated calcium, belonging to the technical field of the preparation of wheat active peptides and calcium supplements. First, protease is added to gluten for enzymatic hydrolysis, and then inactivated. The centrifuged supernatant is the enzymatic hydrolysate solution. The enzymatic hydrolysate solution is separated by an ultrafiltration membrane to obtain wheat polypeptides with a molecular weight less than 500 Da, 500 - 3000 Da, or less than 3000 Da. The above three polypeptides are dissolved in deionized water, and then a calcium salt solution is added, and the pH is adjusted to 7. The chelation reaction is carried out at a constant temperature. The reaction mixture is centrifuged, and the supernatant is taken. After ethanol precipitation and centrifugation, the precipitate is calcium chelated peptide. The wheat polypeptide chelated calcium formed from the mixture of wheat polypeptides with a molecular weight less than 3000 Da separated and purified from the enzymatic hydrolysate of gluten has a high chelation rate, good stability in intestinal simulation, a certain degree of anti-digestibility, good acid and alkali resistance, is easily absorbed by the body, and has good temperature tolerance, and is a good source of the fourth-generation calcium supplement.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of wheat active peptides and calcium supplements, and relates to a method for preparing gluten active peptides and a method for preparing wheat polypeptide chelated calcium (calcium chelated peptides). Background Art

[0002] Wheat (Triiccum Aestivum L.) belongs to the genus Triticum in the family Poaceae and is a biennial (winter wheat) or annual (spring wheat) herbaceous plant. Wheat is a food crop with an extremely wide distribution range and planting area and an extremely high total output in the world. According to the prediction of the Food and Agriculture Organization of the United Nations (FAO), the global grain output in the 2023 / 24 year will be 2.841 billion tons, the wheat output will be 788 million tons, and the global wheat consumption will be 795 million tons. According to the data released by the National Bureau of Statistics, the total grain output in China in 2023 was 1390.82 billion jin, the grain sown area was 1.499 billion mu, of which the wheat sown area was 354 million mu, an increase of 1.632 million mu over the previous year, a growth of 0.5%. The caryopsis of wheat becomes flour after grinding, which can be used to make foods such as steamed buns and cakes, and can be fermented to obtain beverages or seasonings such as alcohol, and can also be used for feeds, biofuels, etc., with a wide range of uses. Wheat protein, commonly known as gluten, is a natural protein extracted from wheat flour and is a by-product of wheat starch production. Wheat peptides prepared by enzymatic hydrolysis of gluten have various biological activities and are a research hotspot at home and abroad.

[0003] Normal biological processes in the human body involve 22 inorganic elements, which can be supplemented through daily diet. Calcium is an essential nutrient for the human body, accounting for 1.5%-2.2% of body weight. Calcium plays an important role in various physiological and metabolic activities, such as muscle contraction, blood clotting, nerve transmission, and bone development. Insufficient calcium intake often leads to metabolic bone diseases, such as rickets, osteomalacia, osteoarthritis, and osteoporosis. The intake of calcium supplements and calcium-fortified foods can relieve calcium deficiency and thus has attracted much attention.

[0004] The development of calcium supplements has gone through four stages: inorganic calcium salts, organic acid calcium salts, amino acid chelated calcium, and peptide chelated calcium. The first-generation inorganic calcium salts are mainly composed of calcium carbonate, calcium phosphate, and calcium chloride. Calcium exists in the form of ions and is prone to form precipitates during gastrointestinal digestion. There are obvious defects in calcium absorption and bioavailability, and long-term use may produce side effects and increase the burden on the human body. The second-generation organic calcium mainly includes calcium lactate, calcium citrate, and calcium gluconate. Although it is more soluble, it still has the disadvantages of easy formation of precipitates and low efficiency of calcium supplements. The third-generation amino acid chelated calcium overcomes the main limitation that ionic calcium has low bioavailability but is prone to precipitate in the gastrointestinal environment, but the reaction implementation cost is extremely high, and unwanted color reactions and lipid oxidation reactions may occur. The fourth-generation calcium chelated peptide has many advantages, such as low energy consumption in the reaction and can form soluble stable chelates with calcium ions. At present, researchers have isolated peptides with calcium chelating activity from many different food sources, such as bovine bone collagen, Pacific cod skin protein, egg white protein, and peanut protein. There are also studies on isolating polypeptides with calcium chelating activity from wheat protein. However, generally, the wheat polypeptide chelated calcium obtained by directly chelating the polypeptide mixture obtained by enzymatic hydrolysis with calcium ions has a low chelating rate. In addition, the enzymatic hydrolysis conditions are the key factors affecting the production of wheat polypeptides from wheat protein. The enzymatic hydrolysis conditions in the existing technology are often not the optimal enzymatic hydrolysis conditions, which affect the yield of polypeptides and ultimately reduce the chelating rate of wheat polypeptide chelated calcium. Therefore, the present invention provides a preparation method of wheat polypeptide chelated calcium with a higher chelating rate. This method determines the enzymatic hydrolysis conditions for the enzymatic hydrolysis of gluten by the response surface method, improves the yield of polypeptides, and prepares wheat polypeptide chelated calcium with a higher chelating rate, good stability in the human digestive pH environment, and good digestion and absorption by separating and screening the best wheat polypeptides. Most importantly, in the existing technology, the most suitable wheat polypeptides for preparing wheat polypeptide chelated calcium have not been screened from aspects such as hydrophobicity and activity, providing a basis for the research and design of polypeptide chelated calcium. Summary of the Invention

[0005] The object of the present invention is a preparation method of wheat polypeptide chelated calcium, which solves the problems that the chelating rate of the polypeptide mixture directly obtained by enzymatic hydrolysis of existing wheat protein with calcium salts is low, the existing enzymatic hydrolysis conditions are not the optimal conditions, and the structure of the most suitable wheat polypeptide for preparing wheat polypeptide chelated calcium is unknown.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A preparation method of wheat polypeptide chelated calcium specifically includes the following steps:

[0008] (1) Weigh gluten and add protease for enzymatic hydrolysis. The protease is composed of alkaline protease and flavor protease;

[0009] (2) After the enzymatic hydrolysis is completed, inactivate the enzymes, centrifuge and take the supernatant, which is the enzymatic hydrolysate solution;

[0010] (3) Use an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da to filter and separate the enzymatic hydrolysate solution. The molecular weight of wheat polypeptides in the first filtrate is less than 10,000 kDa. Then, use an ultrafiltration membrane with a molecular weight cut-off of 3,000 Da to filter and separate the first filtrate, obtaining wheat polypeptides with a molecular weight less than 3,000 Da in the second filtrate. Finally, use an ultrafiltration membrane with a molecular weight cut-off of 500 Da for filtration and separation. The third filtrate and the concentrate obtained from the three filtrations are respectively freeze-dried to obtain wheat polypeptides with a molecular weight less than 500 Da and wheat polypeptides with a molecular weight of 500 - 3,000 Da;

[0011] (4) Dissolve wheat polypeptides with a molecular weight less than 500 Da, 500 - 3,000 Da, or less than 3,000 Da in deionized water respectively, then add a calcium salt solution, adjust the pH = 7, and carry out a chelation reaction at a constant temperature. Centrifuge the reaction mixture, take the supernatant, precipitate with ethanol and then centrifuge, and the precipitate is the calcium chelated peptide.

[0012] It should be noted that the enzymatic hydrolysis conditions in step (1) are as follows: the enzymatic hydrolysis temperature is 45 - 65 °C, the enzymatic hydrolysis pH is 5 - 10, the enzymatic hydrolysis time is 3 - 5 h, the mass ratio of alkaline protease to flavor protease is 1:3 - 3:1, the liquid-to-solid ratio of deionized water to wheat gluten is 13:1 - 22.5:1, and the protease dosage is 5,000 u / g.

[0013] Preferably, the optimal enzymatic hydrolysis conditions in step (1) are: the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis pH is 9.0, the enzymatic hydrolysis time is 4 h, the mass ratio of alkaline protease to flavor protease is 3:1, the liquid-to-solid ratio of deionized water to wheat gluten is 17.5:1, and the enzyme addition amount is 5,000 u / g.

[0014] Preferably, the optimal enzymatic hydrolysis conditions in step (1) are: the enzymatic hydrolysis temperature is 55 °C, the enzymatic hydrolysis pH is 8.5, the mass ratio of alkaline protease to flavor protease is 2.63:1, and the liquid-to-solid ratio of deionized water to wheat gluten is 20:1.

[0015] The determination process of the optimal enzymatic hydrolysis conditions in step (1) is as follows:

[0016] (101) First, through single-factor experiments, conduct experiments on the enzymatic hydrolysis temperature, enzymatic hydrolysis time, enzymatic hydrolysis pH, enzyme addition amount, mass ratio of alkaline protease to flavor protease, and enzymatic hydrolysis liquid-to-solid ratio respectively to determine the optimal enzymatic hydrolysis conditions: the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis pH is 9.0, the mass ratio of alkaline protease to flavor protease is 3:1, the liquid-to-solid ratio is 17.5:1, the enzymatic hydrolysis time is 4 h, and the enzyme addition amount is 5,000 u / g;

[0017] (102) Then, based on the optimal enzymatic hydrolysis conditions, the enzymatic hydrolysis temperature, enzymatic hydrolysis pH, ratio of alkaline protease to flavor protease, and liquid-to-material ratio were selected. Using the chelation rate of enzymatic hydrolysate with metal ion calcium as an index, a response surface experimental design with four factors and three levels was carried out to obtain the response surface factor coding and level parameter table.

[0018] (103) The response surface factor coding and level parameter table were input into the software Design Expert 13 to design and generate the 15-column experimental conditions in Table 2. After conducting the experiments according to the experimental conditions, the measured chelation rates were input into the software, and the software automatically analyzed to obtain the optimal enzymatic hydrolysis conditions: the enzymatic hydrolysis temperature was 55 °C, the enzymatic hydrolysis pH was 8.5, the mass ratio of alkaline protease to flavor protease was 2.63:1, and the liquid-to-material ratio was 20:1.

[0019] It should be noted that after the enzymatic hydrolysis reaction in step (2) ended, the enzyme was inactivated by water bath heating at 90 °C for 10 min, cooled to room temperature, centrifuged at 8000 r / min for 10 min, and the supernatant was collected.

[0020] It should be noted that in step (3), after the enzymatic hydrolysate solution was preliminarily removed of impurities through a pre-washed fiber membrane, ultrafiltration separation was carried out.

[0021] It should be noted that in step (4), the concentration of the wheat polypeptide solution was 13.5%, the calcium source for the chelation reaction was a 2 mol / L CaCl2 solution, the chelation reaction temperature was 38 °C, the shaker conditions were set at a rotation speed of 200 r / min, the chelation reaction time was 12 min, the supernatant was centrifuged after adding 9 times the volume of absolute ethanol, centrifuged again after standing for 35 min to complete alcohol precipitation, and the precipitate was taken, washed twice with alcohol, and then dried in an oven at 42 °C.

[0022] It should be noted that in step (4), the volume ratio of the polypeptide solution to the CaCl2 solution was 5:1, and the two centrifugation conditions were a rotation speed of 8000 r / min for 10 min.

[0023] It should be noted that LC-MS / MS was used to analyze the secondary filtrate in step (3) to obtain 1224 polypeptide sequences. Toxinpred was used to predict whether the polypeptides were toxic, and PeptideRanker was used to predict the polypeptide activity. The polypeptides were screened based on toxicity, the proportion of favorable chelating amino acids, activity, and hydrophobicity. According to the non-toxic principle, 1200 non-toxic sequences were screened out. According to the polypeptide sequences containing favorable amino acids, 782 sequences were obtained. According to the activity screening, 41 polypeptides were obtained. According to the hydrophobicity screening, 21 polypeptides were obtained. Finally, the following four sequences were screened out from these sequences according to the activity level:

[0024] SEQ ID NO1: Val Tyr Ile Pro Pro Tyr Cys;

[0025] SEQ ID NO2: Tyr Ser Met Leu Asp Ser Met Tyr Lys Glu His Gly;

[0026] SEQ ID NO3: Cys Asn Val Asn Val Pro Leu Tyr Glu;

[0027] SEQ ID NO4: Cys Asn Val Asn Val Pro Leu Tyr Glu.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The wheat polypeptide chelated calcium formed by the wheat polypeptide mixture with a molecular weight less than 3000 Da isolated and purified from the gluten hydrolysate of the present invention has a high chelation rate, good stability in intestinal simulation, a certain degree of anti-digestibility, good acid and alkali resistance, is easily absorbed by the body, and has good temperature tolerance, and is a good source of the fourth-generation calcium supplement; (2) Four polypeptide sequences with non-toxicity, good activity and high chelation rate are screened out from the wheat polypeptide mixture, providing a basis for the research and design of polypeptides. Description of the Drawings

[0029] Figure 1 It is the response surface diagram of the interaction between pH and temperature.

[0030] Figure 2 It is the response surface diagram of the interaction between enzyme ratio and temperature.

[0031] Figure 3 It is the response surface diagram of the interaction between liquid-to-material ratio and temperature.

[0032] Figure 4 It is the response surface diagram of the interaction between pH and enzyme ratio.

[0033] Figure 5 It is the response surface diagram of the interaction between pH and liquid-to-material ratio.

[0034] Figure 6 It is the response surface diagram of the interaction between liquid-to-material ratio and enzyme ratio.

[0035] Figure 7The figure shows the effect of gastric digestion on the calcium retention rate of calcium chelated peptides. Among them, A is the change curve of the calcium retention rate in the samples of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight of 500-3000 Da after simulated gastric juice digestion for 0.5 h, 1 h, 1.5 h, and 2 h. B in the figure is the change curve of the calcium retention rate in the samples of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight <500 after simulated gastric juice digestion for 0.5 h, 1 h, 1.5 h, and 2 h.

[0036] Figure 8 The figure shows the effect of intestinal digestion on the calcium retention rate of calcium chelated peptides. Among them, A is the change curve of the calcium retention rate in the samples of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight of 500-3000 Da after simulated gastric juice digestion for 0.5 h, 1 h, 1.5 h, and 2 h. B in the figure is the change curve of the calcium retention rate in the samples of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight <500 after simulated gastric juice digestion for 0.5 h, 1 h, 1.5 h, and 2 h.

[0037] Figure 9 The figure shows the effect of pH on the calcium retention rate of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight of 500-3000 Da.

[0038] Figure 10 The figure shows the effect of temperature on the calcium retention rate of calcium chelated wheat peptides prepared from wheat polypeptides with a molecular weight of 500-3000 Da.

[0039] Figure 11 The figure is the molecular docking simulation diagram of sequence (1).

[0040] Figure 12 The figure is the molecular docking simulation diagram of sequence (2).

[0041] Figure 13 The figure is the molecular docking simulation diagram of sequence (3).

[0042] Figure 14 The figure is the molecular docking simulation diagram of sequence (4).

[0043] Figure 15 The figure is the scanning electron micrograph of wheat polypeptide V.YIPPY.C.

[0044] Figure 16 The figure is the scanning electron micrograph of calcium chelated wheat peptide corresponding to wheat polypeptide V.YIPPY.C. Detailed implementation mode

[0045] The present invention will be further described below through specific examples and drawings.

[0046] Example 1

[0047] 1: Preparation of wheat polypeptide

[0048] ① Sample preparation: Weigh 5 g of gluten.

[0049] ② Enzymolysis: Add a total of 4000 u / g of alkaline protease and flavor protease in proportion, add distilled water, adjust the pH, and carry out a constant-temperature enzymolysis reaction at an appropriate temperature.

[0050] ③ Inactivation: After the enzymolysis reaction, heat in a 90 °C constant-temperature water bath to inactivate the enzyme for 10 min, and then cool to room temperature after completion.

[0051] ④ Centrifugation: Put the enzymolyzed sample solution into a centrifuge tube, and use a high-speed centrifuge at a speed of 8000 r / min for 10 min.

[0052] ⑤ Freeze-drying and storage: Collect and store the supernatant, put it into a freeze dryer for freeze-drying, and then store it in a refrigerator at 20 °C for later use.

[0053] 2: Preparation of wheat polypeptide chelated calcium

[0054] ① Sample preparation: Weigh 13.5 g of the enzymolyzed product freeze-dried powder (wheat polypeptide powder) and dissolve it in 100 ml of deionized water.

[0055] ② Chelation reaction: Mix the sample solution with a 2 mol / L CaCl2 solution at a volume ratio of 5:1, and place the reaction in a shaker at 38 °C and a rotation speed of 200 r / min for 12 min.

[0056] ③ Centrifugation: Put the sample solution into a centrifuge tube, and use a high-speed centrifuge at a speed of 8000 r / min for 10 min.

[0057] ④ Alcohol precipitation: Add 9 times the volume of absolute ethanol to the collected centrifuged supernatant, let it stand for 35 min, and separate the calcium chelated peptide from the supernatant.

[0058] ⑤ Secondary centrifugation: After alcohol precipitation, use a high-speed centrifuge at a speed of 8000 r / min for 10 min.

[0059] ⑥ Precipitate treatment: Take the centrifuged precipitate, wash it 2 times with absolute ethanol, and then dry it in an oven at 42 °C to obtain wheat polypeptide chelated calcium powder.

[0060] 3: Screening and comparison of enzymolysis conditions

[0061] Single-factor experiments were carried out to investigate the enzymolysis temperature, enzymolysis time, enzymolysis pH, enzyme dosage, enzyme ratio (alkaline protease: flavor protease), and enzymolysis liquid-to-material ratio respectively.

[0062] (1) Weigh 5 g of gluten. At an enzymatic hydrolysis time of 4 h, a pH of 7, an enzyme dosage of 4000 u / g, an enzyme ratio of 1:1, and a solid-liquid ratio (mass ratio of gluten to water) of 1:17.5, investigate the effects of temperatures 45, 50, 55, 60, and 65 °C on the hydrolysis degree of the enzymatic hydrolysate.

[0063] (2) Weigh 5 g of gluten. At an enzymatic hydrolysis temperature of 50 °C, a pH of 7, an enzyme dosage of 4000 u / g, an enzyme ratio of 1:1, and a solid-liquid ratio of 1:17.5, investigate the effects of enzymatic hydrolysis times of 3, 3.5, 4, 4.5, and 5 h on the hydrolysis degree of the enzymatic hydrolysate.

[0064] (3) Weigh 5 g of gluten. At an enzymatic hydrolysis time of 4 h, an enzymatic hydrolysis temperature of 50 °C, an enzyme dosage of 4000 u / g, an enzyme ratio of 1:1, and a solid-liquid ratio of 1:17.5, investigate the effects of enzymatic hydrolysis pH values of 5, 6, 7, 8, 9, and 10 on the hydrolysis degree of the enzymatic hydrolysate.

[0065] (4) Weigh 5 g of gluten. At an enzymatic hydrolysis temperature of 50 °C, an enzymatic hydrolysis time of 4 h, a pH of 7, an enzyme ratio of 1:1, and a solid-liquid ratio of 1:17.5, investigate the effects of enzyme dosages of 3000, 3500, 4000, 4500, 5000, and 5500 u / g on the hydrolysis degree of the enzymatic hydrolysate.

[0066] (5) Weigh 5 g of gluten. At an enzymatic hydrolysis temperature of 50 °C, an enzymatic hydrolysis time of 4 h, a pH of 7, an enzyme dosage of 4000 u / g, and a solid-liquid ratio of 1:17.5, investigate the effects of enzyme ratios (alkaline protease: flavor protease) of 1:3, 3:5, 1:1, 5:3, and 3:1 on the hydrolysis degree of the enzymatic hydrolysate.

[0067] (6) Weigh 5 g of gluten. At an enzymatic hydrolysis temperature of 50 °C, an enzymatic hydrolysis time of 4 h, a pH of 7, an enzyme dosage of 4000 u / g, and a solid-liquid ratio of 1:17.5, with an enzyme ratio of 1:1, investigate the effects of solid-liquid ratios of 13:1, 15:1, 17.5:1, 20:3, and 22.5:1 on the hydrolysis degree of the enzymatic hydrolysate.

[0068] Among them, the measurement method for the hydrolysis degree of the enzymatic hydrolysate is to determine the NH₂ group content by the ninhydrin method: Prepare a ninhydrin color reagent. Dilute the gluten enzymatic hydrolysate 400 times as the test solution. Pipette 2.00 ml of the test solution into a test tube, add 1.00 ml of the ninhydrin color reagent, mix well, stopper, and keep it in a constant temperature water bath at 80 °C for 30 min. Cool it with cold water, add 5.00 ml of deionized water, mix well, let it stand for 10 min, and measure the absorbance value at 570 nm. Use deionized water instead of the sample solution as the blank group, use leucine as the standard product to draw a standard curve, and calculate the NH₂ group content.

[0069] The results of single-factor experiments showed that the optimal conditions for enzymatic hydrolysis were a temperature of 50 °C, an enzymatic hydrolysis time of 4 h, an enzyme dosage of 5000 u / g, an enzyme ratio of 3:1, a pH of 9.0, and a liquid-to-solid ratio of 1:17.5.

[0070] Response surface design experiments were conducted to obtain the optimal enzymatic hydrolysis conditions. Based on the results of single-factor experiments, the enzymatic hydrolysis temperature (A), enzymatic hydrolysis pH (B), ratio of alkaline protease to flavor protease (C), and liquid-to-solid ratio (D) were selected, and a response surface experiment design with four factors and three levels was carried out using the chelation rate of enzymatic hydrolysate with metal ion calcium as the index.

[0071] The determination of calcium chelation rate referred to GB / T 5009.92—2016 "Determination of Calcium in Foods" and was determined by the EDTA titration method.

[0072] The coding and levels of response surface factors are shown in Table 1, and the specific experimental design and results are shown in Table 2.

[0073] Table 1 Coding and Levels of Response Surface Factors

[0074]

[0075] Table 2 Response Surface Factor Level Design and Chelation Rate Results

[0076]

[0077]

[0078] Table 3 Variance Analysis and Significance Test of Regression Model

[0079]

[0080]

[0081] Note: "**" indicates highly significant difference, p < 0.01; "*" indicates significant difference, p < 0.05

[0082] The experimental data were regressively fitted to establish a mathematical model, and the regression equation was obtained: R =

[0083] 1042.27778 + 14.34015A + 100.70593B + 77.05407C + 15.55733D - 0.426667AB - 0.528889AC

[0084] - 0.042667AD - 1.68889BC - 0.663333BD - 0.816667CD - 0.075230A 2 - 3.59741B 2 - 2.95296C 2 - 0.124267D2 。

[0085] As can be seen from Table 3, a P-value < 0.001 for the regression model indicates a highly significant difference; a P-value < 0.01 indicates a highly significant difference; a P-value < 0.05 indicates a significant difference. The p-value of the model is significant and the P-value of the lack-of-fit term of the model > 0.05 is not significant, which is beneficial to the model. Analyzing the P-values of each coefficient term, it can be obtained that A, AC, CD, A 2 , B 2 , C 2 have a highly significant or significant impact on the chelating ability of the peptides obtained by hydrolyzing gluten. The F-value represents the degree of influence. The larger the F-value, the greater the degree of influence. Therefore, the degree of influence of single factors on the chelating ability of the peptides obtained by hydrolyzing gluten is: temperature > enzyme ratio > pH > liquid-to-material ratio.

[0086] According to the analysis results of the regression equation, response surface plots and contour plots are made, and the results are shown in Figures 1-6 。

[0087] The slope of the response surface reflects the degree of influence. The steeper the slope, the greater the influence of the interaction between the two factors on the response value; the shape of the contour plot reflects the interaction between the factors. The closer the contour plot is to an ellipse, the more significant the interaction between the two factors. Comparing Figures 1-6 the surface and contour morphologies of each figure in, it can be obtained that the interaction between the two factors of temperature and enzyme ratio is the most significant, and the degree of influence on the chelating ability of the peptides obtained by hydrolyzing gluten is temperature > enzyme ratio > pH > liquid-to-material ratio.

[0088] Analysis was carried out using the software Design Expert 13 to obtain the optimal hydrolysis conditions: temperature 55 °C, pH 8.5, enzyme ratio (alkaline protease: flavor protease) 2.63:1, liquid-to-material ratio 20:1. Under the optimal conditions for hydrolysis, the chelation rate of the peptides obtained by hydrolyzing gluten was 40.1%.

[0089] 4: Polypeptide fractionation and purification

[0090] ① Sample preparation: 20 g of the freeze-dried gluten hydrolysate prepared under the above optimal hydrolysis conditions was redissolved in deionized water,

[0091] to obtain a 2 L sample solution of 0.01 g / mL;

[0092] ② Pretreatment: Impurities were preliminarily removed through a pre-washed fiber membrane;

[0093] ③ Ultrafiltration membrane separation: Dissolve the enzymolysate in deionized water, and perform filtration separation using an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da. The molecular weight of wheat polypeptides in the first filtrate is less than 10,000 kDa. Then, use an ultrafiltration membrane with a molecular weight cut-off of 3,000 Da to filter and separate the first filtrate, and the molecular weight of wheat polypeptides in the second filtrate is 3,000 Da. Finally, use an ultrafiltration membrane with a molecular weight cut-off of 500 Da for filtration separation. The third filtrate and the concentrated solution obtained from the three filtrations are separately freeze-dried to obtain wheat polypeptides with a molecular weight less than 500 Da and wheat polypeptides with a molecular weight of 500 - 3,000 Da. During the process, the pressure is adjusted to 65 MPa, and reflux ultrafiltration is carried out at 25°C. Collect polypeptide components with molecular weights >10,000 Da, 3,000 - 10,000 Da, 500 - 3,000 Da, and <500 Da in stages. After freeze-drying different component samples, store them at 20°C.

[0094] ④ Dissolve the freeze-dried enzymolysate with different molecular weights in water respectively, and then mix it with a 2 mol / L CaCl2 solution at a volume ratio of 5:1. React and place it in a shaker at 38°C and a rotation speed of 200 r / min for 12 min. After centrifugation and secondary centrifugation, wheat polypeptide chelated calcium is obtained.

[0095] ⑤ Determination of chelation rate: Titrate the wheat polypeptide chelated calcium formed by polypeptide components with different molecular weights by the EDTA titration method to determine the chelating ability of each component peptide. The results are shown in the following table. It can be seen that the chelation rates of the components with molecular weights of 500 - 3,000 Da and <500 Da are relatively high. Among them, the component with a molecular weight of <500 Da has the highest chelation rate.

[0096] Table 4 Chelation rates of polypeptides in each ultrafiltration component

[0097]

[0098] 5: In vitro digestion stability of wheat polypeptide chelated calcium

[0099] ① Simulated gastric juice digestion: Take 2 g of wheat polypeptide chelated calcium powder prepared from polypeptides with molecular weights <500 Da and 500 - 3,000 Da respectively, and mix them evenly with 1 L of distilled water to prepare a solution with a concentration of 2 mg / mL. Adjust the pH of this solution to 2 with sulfuric acid, add pepsin, and conduct a simulated gastric digestion experiment in a 37°C water bath. Digest for different times, take samples every 30 min, and measure the stability of wheat polypeptide chelated calcium. The stability is represented by the calcium retention rate.

[0100] ② Simulated intestinal juice digestion: Adjust the pH of the chelate solution after simulated gastric digestion to 7, add trypsin, and conduct a simulated intestinal digestion experiment under water bath conditions at 50°C for 120 min. Take samples every 30 min, and measure the stability of wheat polypeptide chelated calcium. The stability is represented by the calcium retention rate.

[0101] The calcium retention rate formula is as follows:

[0102] Calcium retention rate:

[0103] In this implementation method, the in vitro digestion stability of wheat polypeptide chelated calcium prepared from polypeptides with different molecular weights is different. Specifically, see Figure 7 and 8 . When the molecular weight is 500 - 3000 Da, its stability in simulated gastric juice is slightly stronger than that in simulated intestinal fluid. When the molecular weight is < 500 Da, its stability in simulated intestinal fluid is stronger than that in simulated gastric juice. During digestion in gastric juice, the retention rate first increases and then decreases with the increase of time. When the time reaches 2 h, the retention rates of both are lower than 50%. This may be because the pH in the gastric juice environment is relatively low, showing strong acidity, and hydrogen ions will compete for the binding sites of calcium ions, resulting in a reduction in the binding sites of calcium ions and a decrease in the retention rate. During the digestion process in simulated intestinal fluid, the retention rate of wheat polypeptide chelated calcium mostly remains above 60% when the molecular weight is < 500 Da. This indicates that when the molecular weight is < 500 Da, the stability of wheat polypeptide chelated calcium in the intestinal simulation is good. It can be concluded that wheat polypeptide chelated calcium has good stability when entering the intestine for absorption and has a certain degree of anti-digestibility.

[0104] 6: Acid-base stability of wheat polypeptide chelated calcium

[0105] The wheat polypeptide chelated calcium powder prepared from polypeptides with a molecular weight of 500 - 3000 Da obtained by freeze-drying is formulated into a solution with a concentration of 0.2%. The pH value of the solution is adjusted to 2.0, 4.0, 6.0, 8.0, 10.0 respectively using certain concentrations of HCl and NaOH solutions, and shaken in a constant temperature water bath shaker at 37 °C for 2 h. The free calcium ion content in the system is measured using a calcium ion selective electrode. The acid-base stability of wheat polypeptide chelated calcium is expressed by the calcium retention rate.

[0106] In this implementation method, the acid-base stability of wheat polypeptide chelated calcium is specifically shown in Figure 9。The calcium retention rate of wheat polypeptide chelated calcium remains stable within the pH range of 6 - 10, and the calcium retention rate can reach over 40%. However, when the pH value gradually decreases, the calcium retention rate is relatively low, about 30%. In a strongly acidic environment with a low pH, excessive hydrogen ions may compete with calcium ions for negatively charged groups, affecting the binding of calcium to wheat polypeptides. When the pH of wheat polypeptide chelated calcium is 10, a small amount of precipitation appears in the solution. In an alkaline state, hydroxide ions react with calcium ions dissociated from wheat polypeptide chelated calcium to form hydroxide precipitation. The human intestinal environment is approximately pH = 7.2. At this time, the calcium retention rate of wheat polypeptide chelated calcium is in a relatively high state and has relatively strong stability, so it can be well absorbed by the body. It is concluded that when wheat polypeptide chelated calcium is added to food as a calcium nutritional fortifier, treatment with too strong acidity or alkalinity should be avoided.

[0107] 7: Temperature stability test of wheat polypeptide chelated calcium

[0108] The wheat polypeptide chelated calcium powder prepared from polypeptides with a molecular weight of 500 - 3000 Da obtained by freeze-drying was formulated into a 0.2% solution, which was respectively heated at 55°C, 65°C, 75°C, 85°C, 95°C, and 105°C for 1 h. After cooling to room temperature, the content of free calcium in the system was measured using a calcium ion selective electrode. The thermal stability of wheat polypeptide chelated calcium is expressed by the calcium retention rate.

[0109] In this implementation method, the temperature stability of wheat polypeptide chelated calcium is specifically shown in Figure 10 。Within the range of 55 - 100°C, as the heat treatment temperature of wheat polypeptide chelated calcium increases, the calcium retention rate shows a fluctuating state and mostly stabilizes at 52%. Within a certain temperature range, the structure of wheat polypeptide chelated calcium is relatively stable, and the calcium retention rate basically remains unchanged, indicating that it has a certain tolerance to temperature. This may be because calcium ions and wheat peptides form a dense structure through coordination binding, so its structure is more stable.

[0110] Example 2

[0111] 1: Prediction and screening of polypeptide activity

[0112] ① 1224 polypeptide sequences were obtained by analyzing the polypeptides filtered through an ultrafiltration membrane with a molecular weight less than 3000 Da in step ③ of the polypeptide fractionation and purification in Part 4 of Example 1 using LC MS / MS;

[0113] ② Toxinpred (http: / / crdd.osdd.net / raghava / toxinpred / ) was used to predict whether the polypeptides are toxic;

[0114] ③ PeptideRanker ( http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict polypeptide activity;

[0115] ④The hydrophobicity of the polypeptide was predicted using the Expasy ProtParam tool (https: / / web.expasy.org / protparam / ).

[0116] The polypeptides were screened based on toxicity, the proportion of favorable chelating amino acids, activity, and hydrophobicity. According to the non-toxic principle, 1200 non-toxic sequences were obtained. 782 sequences were obtained based on the polypeptide sequences containing favorable amino acids. 41 polypeptides were obtained by screening according to activity, and 21 polypeptides were obtained by screening according to hydrophobicity. Finally, four sequences were obtained by screening these sequences according to the activity level, as shown in Table 5.

[0117] Table 5 Functional information of the polypeptides after screening

[0118]

[0119]

[0120] ⑤Molecular docking

[0121] Calcium ions were constructed using ChemOffice2020, selected as the ligand in the AutoDock software, dehydrated and hydrogenated, the charges were adjusted, and saved in the pdbgt format for molecular docking.

[0122] The polypeptide sequences obtained by screening through the online tool were drawn by ChemDraw and a stable structure was constructed by energy minimization in the Chem3D software. AutoDock Tools 1.5.7 was used to select it as the receptor, dehydrated and hydrogenated, the charges were calculated, the atom types were added, and saved in the pdbgt format for molecular docking.

[0123] Using AutoDock Tools 1.5.7, open the conformation files of the ligand small molecule and the receptor macromolecule, set the docking Box and save it. Open the saved file to run Autogrid, and set the docking parameters and operation method to run AutoDock. The docking results were analyzed and displayed using the Protein Ligand Interaction Profiler and PyMOL software. The docking information is shown in Table 6, as Figures 11-14 。

[0124] Table 6 Docking result information

[0125]

[0126] It can be obtained from the molecular docking simulation analysis that all four polypeptide sequences have the ability to chelate with the metal ion calcium.

[0127] Four polypeptide sequences with higher activity were screened for synthesis, and their chelation rates were measured, as shown in Table

[0128] Table 7 Chelation rate of synthetic polypeptide

[0129]

[0130]

[0131] 2: Scanning electron microscopy analysis

[0132] Appropriately take the synthetic polypeptide lyophilized powder (V.YIPPY.C) and the corresponding wheat polypeptide chelated calcium lyophilized powder sample, shake them onto the sample tray, and after gold sputtering coating treatment, put them into the scanning electron microscope to evacuate. The electron microscope scanning conditions are set as high voltage 2.00 kV, beam current 6.9x10^2 mA, and working distance 10 mm, and obtain scanning images at 1000 magnification.

[0133] This implementation method uses a scanning electron microscope to analyze wheat polypeptide and the prepared wheat polypeptide chelated calcium. The results are as Figures 15-16 shown. Compared with wheat polypeptide, the appearance of wheat polypeptide chelated calcium has changed significantly. The surface of wheat polypeptide is smooth and sheet-like. After binding with calcium ions, it becomes an irregular polyhedron, and its structure is loose and porous, which is beneficial to dissolution and absorption. The structure of wheat protein peptide substances is loose and the surface is smooth. After chelation with calcium ions, the surface becomes rough and the internal structure becomes more compact. This is because the coordination bond between the polypeptide and calcium causes the folding of the internal structure, thus generating small granular substances, and these particles will attract each other and aggregate.

[0134] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A wheat polypeptide with the ability to chelate metal ion calcium, characterized in that, The sequence of the wheat polypeptide is as follows: SEQ ID NO1: Val Tyr Ile Pro Pro Tyr Cys; SEQ ID NO2: Tyr Ser Met Leu Asp Ser Met Tyr Lys Glu His Gly.

2. Use of the wheat polypeptide having the ability to chelate with metal ion calcium according to claim 1 in the preparation of wheat polypeptide chelated calcium.

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