Coix seed bran polypeptide capable of chelating inorganic calcium, calcium chelate thereof, preparation method and application

Abstract

The invention discloses coix seed bran polypeptide capable of chelating inorganic calcium, a calcium chelate thereof, a preparation method and application, and belongs to the technical field of food processing. The amino acid sequence of the coix seed bran polypeptide comprises KGITDLKPVL, MSAAV, RSGSGG, NGSSGR, TEAEPANLDKFVHHLSENHFFPNRV, KDIFVDTLC, GLDWARLLKL, GSHVL, LHHIGPQTHISL, IEYASSKEGSACKEGV or TIVGFQRHGLHF, and the coix seed bran polypeptide prepared by mixing the coix seed bran protein extract and trypsin is mixed with a calcium source to prepare the coix seed bran polypeptide-calcium chelate. The pearl barley bran is used as the raw material, the process is simple and convenient, the cost is low, the calcium ion release rate of the product chelate is high, the heat stability is high, the absorption efficiency of calcium can be improved, and the pearl barley bran chelate has good functional food and health care product application prospects.

Description

Technical Field

[0001] The invention provides a coix seed bran polypeptide capable of chelating inorganic calcium and a calcium chelate thereof, a preparation method and application thereof, and relates to the technical field of food processing. Background Art

[0002] Calcium is one of the most abundant inorganic minerals in the human body, accounting for about 1.5% to 2.2% of the total body weight. Most of it (99%) is deposited in teeth and bones, while the remaining 1% exists mainly in the form of ions in extracellular fluid and blood, playing a vital role in maintaining the dynamic balance of bone calcium and blood calcium. According to the data of the Chinese Residents' Nutrition Survey, the current daily dietary calcium intake of residents is only 389 mg, which is only half of the recommended daily intake, highlighting the problem of insufficient calcium intake. Calcium deficiency is an important factor in causing a variety of metabolic bone diseases, such as osteoporosis, rickets, hypertension and osteomalacia. The calcium reserve in human bones depends entirely on the calcium intake in the diet, and is digested and absorbed by the intestines to ensure that the serum calcium level is maintained within the normal range. However, the absorption efficiency of calcium is affected by many factors, including the naturally occurring chemical components in food (such as phytic acid and oxalic acid) as well as the individual's nutritional status, metabolic level and physiological conditions.

[0003] Job's tears, as a cereal crop that has not been fully developed and utilized, has a wide range of application value in the field of traditional Chinese medicine. It is known as a "green food" because it is rich in unsaturated fatty acids, has no toxic residues and has an extremely low content of heavy metals. In addition, Job's tears also show auxiliary therapeutic effects on a variety of chronic diseases, including anti-allergy, anti-cancer, anti-diabetes, anti-inflammatory, antioxidant and gastric protection. my country has now become the largest producer and exporter of Job's tears. For a long time, the main processing method of Job's tears has been to refine it into Job's tears rice for sale, and a large amount of by-products will be produced in this process - Job's tears bran, that is, the seed coat of Job's tears, which accounts for about 6% to 10% of the total weight of Job's tears, but is often regarded as waste and discarded. It is worth noting that the protein in Job's tears bran can be hydrolyzed by trypsin to produce a bioactive peptide with broad development prospects - Job's tears bran protein peptide (TCBP). However, to date, there has been no report on the use of coix bran as a raw material to prepare coix bran polypeptide-calcium chelate. Therefore, the development of coix bran protein peptide (TCBP) with high calcium chelating activity from coix bran not only has important academic research value, but also has huge market potential.

[0004] Although there are many kinds of calcium supplement products on the market, they generally have problems such as low calcium absorption rate by the human body, poor bioavailability of calcium ions, obvious gastrointestinal side effects, unstable product quality and safety risks. Therefore, it is urgent to develop new biological calcium supplement preparations to improve the stability, safety and bioavailability of calcium supplement products. Summary of the invention

[0005] To solve the above problems, the present invention provides a coix seed bran polypeptide that can chelate inorganic calcium, and proposes a method for preparing a coix seed bran polypeptide-calcium chelate that has a simple preparation process and is easy to implement. The coix seed bran polypeptide-calcium chelate prepared by this method can be used to prepare a calcium supplement, functional food or health product that has the effect of promoting calcium absorption. The specific scheme is as follows:

[0006] First, the present invention provides a coix seed bran polypeptide capable of chelating inorganic calcium, wherein the amino acid sequence of the peptide comprises one or more of KGITDLKPVL, MSAAV, RSGSGG, NGSSGR, TEAEPANLDKFVHHLSENHFFPNRV, KDIFVDTLC, GLDWARLLKL, GSHVL, LHHIGPQTHISL, IEYASSKEGSACKEGV or TIVGFQRHGLHF.

[0007] Second, the present invention provides a coix seed bran polypeptide-calcium chelate based on the above-mentioned coix seed bran polypeptide, which includes chelated calcium ions and a polypeptide chain skeleton, and the calcium ions are bound to the polypeptide chain skeleton by forming coordination bonds with the coordinating atoms present on the side chains of the polypeptide chain skeleton, wherein the amino acid sequence of the polypeptide chain skeleton includes one or more of KGITDLKPVL, MSAAV, RSGSGG, NGSSGR, TEAEPANLDKFVHHLSENHFFPNRV, KDIFVDTLC, GLDWARLLKL, GSHVL, LHHIGP QTHISL, IEYASSKEGSACKEGV or TIVGFQRHGLHF.

[0008] Preferably, the calcium ion is bound to one or more of the coordinating atoms.

[0009] Preferably, the calcium ion is located in a coordination cavity formed by a plurality of coordination atoms, and is tightly connected to the plurality of coordination atoms via coordination bonds.

[0010] Thirdly, the present invention provides a method for preparing the above-mentioned coix bran polypeptide-calcium chelate, comprising the following steps:

[0011] (1) preparing the coix seed bran protein extract into an aqueous solution, adjusting the pH value, adding trypsin for incubation, killing the enzyme after the incubation, cooling to room temperature, and separating the product to obtain coix seed bran polypeptide powder;

[0012] (2) dissolving the obtained coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution;

[0013] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide obtained in step (2) to mix to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH value and stirring to uniformly perform a chelation reaction to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0014] (4) adding anhydrous ethanol to the coix seed bran polypeptide-calcium chelate reaction solution obtained in step (3), allowing to stand, and separating the product to obtain coix seed bran peptide-calcium chelate powder.

[0015] Preferably, the aqueous solution of the coix bran protein extract in step (1) has a concentration of 2-3% (w / v), and the pH value is adjusted to 8.

[0016] Preferably, the amount of trypsin added in step (1) is 2000-3000 U / g, the incubation temperature is 37° C., and the incubation time is 3-4 h.

[0017] Preferably, the enzyme inactivation in step (1) comprises heating the incubation product to above 85° C. and maintaining the temperature for 15 to 20 minutes.

[0018] Preferably, the separation of the product in step (1) comprises centrifugation at 3000-5000 rpm for 15-20 min, and concentrating, freezing and drying the supernatant.

[0019] Preferably, in the original solution of coix bran polypeptide in step (2), the concentration of coix bran polypeptide is 1% to 5% (w / v), preferably the concentration of coix bran polypeptide is 3% to 5% (w / v), and more preferably the concentration of coix bran polypeptide is 4% (w / v).

[0020] Preferably, in step (3), the mass ratio of calcium chloride to the coix bran polypeptide in the original solution of coix bran polypeptide is 1:(1-8) to 2:1; and the pH value is adjusted to 3-8, and the pH value is more preferably 6.

[0021] Preferably, the chelating reaction time in step (3) is 20 to 100 min, preferably 60 min; the chelating temperature is 15 to 75°C, preferably 30 to 60°C, and more preferably 45°C.

[0022] Preferably, in step (4), the volume ratio of the coix bran polypeptide-calcium chelation reaction solution to anhydrous ethanol is (10-15):1, and the standing is carried out at room temperature for 30-120 min.

[0023] Preferably, the separation of the product in step (4) comprises centrifuging the product obtained by standing, taking the precipitate and drying it; wherein the centrifugal speed is 3000-5000 rpm, the centrifugal time is 20-30 min, the drying temperature is 40-50° C., and the drying time is 6-8 h.

[0024] Preferably, in step (3), after anhydrous calcium chloride is added to the original solution of coix seed bran polypeptide and mixed, tartaric acid is added, stirred and then allowed to stand, the pH value is adjusted, stirred evenly and then heated for chelation reaction to obtain a stabilized coix seed bran polypeptide-calcium chelate reaction solution.

[0025] Preferably, the concentration of tartaric acid added in the above step is 0.3-0.5wt%, the standing temperature is room temperature, and the standing time is 10-15min; the chelating reaction temperature is 50-75°C, and the chelating reaction time is 30-50min.

[0026] Finally, the present invention provides the use of the coix seed bran polypeptide-calcium chelate in the preparation of a calcium supplement, functional food or health product with the effect of promoting calcium absorption.

[0027] Beneficial effects of the present invention:

[0028] 1. The coix bran polypeptide-calcium chelate prepared by the method of the present invention has a high calcium ion release rate and strong thermal stability, and has good application prospects in functional foods and health products.

[0029] 2. The coix bran polypeptide-calcium chelate prepared by the method of the present invention increases the Ca 2+ Improve bioavailability and reduce gastrointestinal side effects.

[0030] 3. The coix seed bran polypeptide-calcium chelate prepared by the method of the present invention can improve the absorption of calcium in the human body and can be used to develop high-value coix seed bran polypeptide-calcium supplements.

[0031] 4. The preparation method provided by the present invention has a simple operation process, does not require complex equipment, and is low-cost.

[0032] The invention increases the added value of coix seed processing by-products, fully utilizes coix seed resources and reduces resource waste, enriches the market of new calcium supplements and further increases their economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention provides a graph showing the effects of chelation pH, chelation time, chelation peptide / calcium ratio, chelation temperature and chelation polypeptide concentration on the yield of calcium ions chelated by coix bran polypeptide. (Y axis is translated into calcium binding capacity in English; X axis represents pH in Figure A, chelation time in Figure B, peptide-calcium ratio in Figure C, chelation temperature in Figure D and polypeptide concentration in Figure E).

[0034] Figure 2 The response surface diagram of the interaction of various factors provided by the present invention on the calcium loading of coix bran polypeptide-calcium chelate. (Figure ABCDEF is translated into English as: Calcium binding capacity: calcium binding capacity; Polypeptide concentration: polypeptide concentration; Mass ratio of peptide calcium: peptide calcium ratio; chelating time: chelating time; chelating temperature: chelating temperature.)

[0035] Figure 3 The electron microscopy, elemental composition, particle size and Zeta potential composition analysis of the coix seed bran polypeptide-calcium chelate provided by the present invention. (Figure AB is: electron microscopy of coix seed bran polypeptide (TCBP) and coix seed bran peptide calcium chelate (TCBP-Ca); Figure CD is: elemental composition of coix seed bran polypeptide and coix seed bran peptide calcium chelate, counts per second on the Y axis; kiloelectron volts on the X axis (three-line table in the figure: element weight, C carbon atom, O oxygen atom, N nitrogen atom, Ca calcium atom); Figure E: spectral intensity on the Y axis; particle size on the X axis; Figure F: potential on the Y axis).

[0036] Figure 4 The test results of the coix seed bran polypeptide-calcium chelate and inorganic calcium salt provided by the present invention at different temperatures, pH and gastrointestinal digestion stability are shown in Figure 1. (Y axis: calcium release rate; X axis: temperature in Figure A, pH in Figure B, time in Figure C; icons: CaCl2: calcium chloride; TCBP-Ca: coix seed bran peptide-calcium chelate). DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0040] Embodiment 1:

[0041] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0042] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 4%;

[0043] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 2:1 to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 6, the chelation time to 60 min, and the chelation temperature to 45° C. to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0044] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0045] Embodiment 2:

[0046] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0047] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 1%;

[0048] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 8:1 to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 3, the chelation time to 20 min, and the chelation temperature to 15° C. to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0049] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0050] Embodiment 3:

[0051] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0052] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 2%;

[0053] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 6:1 to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 4, the chelation time to 40 min, and the chelation temperature to 30° C. to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0054] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0055] Embodiment 4:

[0056] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0057] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 3%;

[0058] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 4:1 to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 5, the chelation time to 50 min, and the chelation temperature to 60° C. to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0059] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0060] Embodiment 5:

[0061] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0062] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 5%;

[0063] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 1:1 and mixing to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 7, the chelation time to 100 min, and the chelation temperature to 75° C. to obtain a coix seed bran polypeptide-calcium chelate reaction solution;

[0064] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0065] Embodiment 6 (based on embodiment 4):

[0066] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0067] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 3%;

[0068] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 4:1, and mixing, and continuing to add 0.3wt% of tartaric acid, adjusting the pH to 5, stirring, standing at room temperature for 10 minutes, heating to 60°C for chelation reaction for 50 minutes, and obtaining a stabilized coix seed bran polypeptide-calcium reaction solution;

[0069] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0070] Embodiment 7 (based on embodiment 4):

[0071] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0072] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 3%;

[0073] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 4:1, and mixing, and continuing to add 0.5wt% of tartaric acid, adjusting the pH to 5, stirring, standing at room temperature for 15 minutes, and heating to 75°C for chelation reaction for 30 minutes to obtain a stabilized coix seed bran polypeptide-calcium reaction solution;

[0074] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0075] Comparative Example 1 (adding tartaric acid after chelation is completed based on Example 7):

[0076] (1) The coix seed bran protein extract is prepared into a 2% (w / v) aqueous solution, the pH value of the solution is adjusted to 8, and trypsin (3000 U / g) is added, the mixture is incubated in a 37° C. water bath for 3 h, the enzyme is inactivated in a boiling water bath, cooled to room temperature, centrifuged at 5000 r / min for 20 min to precipitate macromolecular proteins, and the supernatant is concentrated, frozen, and dried to obtain a powder, which is the coix seed bran polypeptide;

[0077] (2) dissolving the coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution having a coix seed bran polypeptide concentration of 3%;

[0078] (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide in step (2) at a mass ratio of 4:1 and mixing to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH to 5, the chelation time to 50 min, the chelation temperature to 60° C., to obtain a coix seed bran polypeptide-calcium chelate reaction solution, adding 0.5 wt % tartaric acid to the obtained coix seed bran polypeptide-calcium chelate reaction solution to adjust the pH to 5, stirring and standing at room temperature for 15 min, heating to 75° C. for reaction for 30 min, and naturally cooling to room temperature;

[0079] (4) adding anhydrous ethanol in a volume ratio of 10:1 to step (3), allowing to stand and then centrifuging for 20 min (5000 r / min), precipitating and drying at 40° C. for 6-8 h to obtain coix seed bran peptide-calcium chelate powder.

[0080] Experimental example:

[0081] The inventors have conducted a large number of experimental studies on the properties and stability of the coix bran polypeptide-calcium chelate of the present invention, some of which are excerpted as follows:

[0082] 1. Single factor experimental investigation of preparation method

[0083] The coix seed bran protein extract was prepared into a 2% (w / v) aqueous solution, the pH value of the solution was adjusted to 8, and trypsin (3000U / g) was added, the mixture was incubated in a 37°C water bath for 3h, the enzyme was inactivated in a boiling water bath and cooled to room temperature, and the macromolecular protein was precipitated by centrifugation at 5000r / min for 20min, the supernatant was concentrated, frozen, and dried to obtain the powder, which was the coix seed bran polypeptide. The prepared coix seed bran polypeptide was subjected to the following single factor experimental investigation:

[0084] 1.1 Effect of peptide concentration on Ca 2+ Effect of chelation rate

[0085] Under the conditions of pH=6 and peptide-calcium ratio of 4:1, 1%, 2%, 3%, 4%, and 5% of coix seed bran peptide were added to the reaction system, and the reaction was carried out at 45°C for 60 minutes and then centrifuged. The supernatant was taken to determine its Ca 2+ Loading capacity.

[0086] 1.2 pH to Ca 2+ Effect of chelation rate

[0087] Under the conditions of 2% peptide concentration and 4:1 peptide-calcium ratio, the pH of the reaction system was adjusted to 3, 4, 5, 6, 7, and 8 respectively. After reacting at 45°C for 60 min, the supernatant was centrifuged and its Ca was determined. 2+ Loading capacity.

[0088] 1.3 Peptide calcium versus Ca 2+ Effect of chelation rate

[0089] Under the conditions of 2% peptide concentration and pH 6, the peptide calcium mass ratio (coix bran peptide / CaCl2) was adjusted to 8:1, 6:1, 4:1, 2:1, and 1:1, respectively. After reacting at 45°C for 60 min, the supernatant was centrifuged and its Ca was determined. 2+ Loading capacity.

[0090] 1.4 Effect of chelation temperature on Ca 2+ Effect of chelation rate

[0091] Under the conditions of 2% peptide concentration, pH 6, and peptide-calcium ratio of 4:1, the reaction temperature was adjusted to 15°C, 30°C, 45°C, 60°C, and 75°C, respectively. After 60 minutes of reaction, the supernatant was centrifuged and its Ca was determined. 2+ Loading capacity.

[0092] 1.5 Effect of chelation time on Ca 2+ Effect of chelation rate

[0093] Under the conditions of 2% peptide concentration, pH 6, peptide-calcium ratio 4:1, and reaction temperature 45°C, the reaction was carried out for 20 min, 40 min, 60 min, 80 min, and 100 min, and the supernatant was centrifuged and the Ca 2+ Loading capacity.

[0094] Through single factor experiments, the influence of each factor on Ca 2+ The effect of chelation rate was evaluated, e.g. Figure 1 As shown in Figure 2, it is shown that under appropriate conditions of pH, time, peptide-calcium ratio, temperature and peptide concentration, Ca 2+ The chelation rate is increased, thereby increasing the calcium loading of coix bran peptide-calcium chelate.

[0095] 1.6 Response surface optimization experiment

[0096] Based on experiments 1.1-1.5, Ca 2+ The loading capacity was the response variable, and the peptide-calcium mass ratio, peptide concentration, chelation time and chelation temperature were selected to conduct a four-factor three-level response surface experiment. The optimal preparation process of coix bran peptide-calcium chelate was obtained and verified.

[0097] like Figure 2 As shown in the figure, the 3D interaction diagram of the response surface shows the interaction between the two factors. The model shows that the interaction between peptide concentration, chelation time, chelation temperature and peptide calcium ratio is not significant, and the factors do not have a secondary effect on the response value; the interaction between chelation temperature, peptide concentration and chelation time is significant near the test point, indicating that the interaction of factors also has a certain effect on calcium loading.

[0098] The results of the response surface experiment are shown in Table 1. The four factors were analyzed by quadratic polynomial regression fitting, and the second-order polynomial regression equation for calcium loading was obtained as follows: Y = 50.64-13.32A+1.94B+2.08C-1.46D+0.4169AB+0.9457AC-0.8622AD+0.2656BC+1.26BD+1.24CD+0.517A 2 -7.77B 2 -2.72C 2 -3.96D 2The model was subjected to variance analysis, and the results are shown in Table 2. The p value of the regression equation model is lower than 0.01, indicating that the equation fits well. The lack of fit value is 0.1431, which is not significant compared with the pure error, indicating that the model is a reasonable choice. The correlation coefficient (R 2 =0.9453) is relatively reliable. Therefore, this model can be used to analyze and predict the chelation effect between coix bran protein peptide (TCBP) and anhydrous calcium chloride.

[0099] According to the results of the response surface model, the optimal conditions for preparing TCBP-Ca were predicted to be: peptide / calcium mass ratio of 2.029:1; peptide concentration of 4.189%; chelation time of 59.857 min; temperature of 44.965 °C, and the predicted value of chelate calcium loading was 64.148 mg / g. In order to verify the reliability of the response surface prediction results and consider the convenience of actual operation, the experimental conditions were adjusted: TCBP / Ca 2+ is 2:1 (w / w); polypeptide concentration is 4.2% (w / w); chelation time is 60 min; chelation temperature is 45°C; chelation pH is 6, and the final prepared TCBP-Ca chelate has a calcium loading of 64.524 mg / g, which is close to the predicted value (64.148 mg / g), proving that the response surface methodology is effective in optimizing the regression model of coix bran peptide calcium chelate.

[0100] Table 1: Response surface experiment results

[0101]

[0102] Table 2: Regression model coefficients and significance test results

[0103]

[0104]

[0105] Note: * represents P<0.05 significant level, *** represents P<0.001 extremely significant level.

[0106] 2. According to the preparation method of Example 1, the prepared coix bran polypeptide-calcium chelate was subjected to the following structural characterization analysis:

[0107] 2.1 Analysis of amino acid composition before and after coix seed bran polypeptide-calcium chelate

[0108] The amino acid composition of coix bran polypeptide and its chelate was analyzed using a fully automatic amino acid analyzer. 80 mg of sample powder was added to 10 mL of HCl (6 mol / L, containing 0.1% phenol), hydrolyzed at 110°C for 22 h, filtered using a 0.45 μm filter membrane and diluted to 50 mL, and its amino acid composition was determined. The results are shown in Table 3.

[0109] The results showed that the relative content of amino acids changed before and after chelation. The acidic amino acids and basic amino groups of TCBP accounted for 23.12% and 21.53%, respectively. Compared with TCBP, the acidic amino acids and basic amino acids of TCBP-Ca chelate increased by 13.18% and 8.25%, respectively, indicating that this was related to the addition of calcium to TCBP, and the carboxyl groups of acidic amino acids (Glu and Asp) may be the main binding sites for calcium ions.

[0110] Table 3: Amino acid composition analysis of coix seed bran polypeptide-calcium chelate

[0111]

[0112]

[0113] a Contains Phe and Tyr; b Contains Glu and Asp; c Contains Lys, Arg, and His.

[0114] 2.2 Identification of amino acid composition of coix bran polypeptide-calcium chelate peptide

[0115] The coix seed bran polypeptide-calcium chelate was prepared into a 1 mg / ml solution, and the amino acid composition of the coix seed bran polypeptide was further analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) and Byonic software.

[0116] The results showed that, as shown in Table 4, 11 peptides were found in the TCBP-Ca chelate, which have not been detected in other studies. The molecular weights of these peptides are mainly distributed between 1000-3000Da, which confirms that low molecular weight peptides have good metal chelating activity. Among them, 11 peptides contain one or more acidic amino acids or basic amino acids, which also proves that acidic amino acids and basic amino acids have a close relationship with calcium ion chelation. The same result is reflected in Ca 2+ The results of the changes in the amino acid content of TCBP are being analyzed.

[0117] Table 4: Peptide identification of coix bran peptide-calcium chelate

[0118]

[0119] 2.3 Electron microscopic analysis of coix seed bran polypeptide-calcium chelate before and after chelation

[0120] Before imaging, the coix seed bran polypeptide-calcium chelate powder was fixed on a glass slide and sprayed with a gold film coating. The microscopic morphology was observed and recorded using an electron scanning microscope at a voltage of 30 kV.

[0121] The results showed that the coix seed bran polypeptide had a plate-like structure, which was relatively flat and had many cracks ( Figure 3 A), when coix bran polypeptide (TCBP) and Ca 2+ When combined to form a chelate, the surface of the compound is sponge-like with loose gaps ( Figure 3 B), the change of the morphology and structure of coix bran polypeptide (TCBP) strongly proved the coix bran polypeptide (TCBP) and Ca 2+ Chelation occurs.

[0122] 2.4 Analysis of elemental composition of coix seed bran polypeptide-calcium chelate before and after chelation

[0123] The elemental composition of coix seed bran peptide and coix seed bran peptide-calcium chelate was determined using energy dispersive X-ray spectrometry.

[0124] The results showed that after calcium ions participated in chelation, the original composition elements of coix bran peptide changed, including C element (63.66%), O element (25.89%), N element (10.45%). The C element content after chelation decreased by 30.58%, while the O element and N element content increased by 7.03% and 6.24%, respectively. In addition, compared with coix bran peptide, coix bran peptide-calcium chelate showed two calcium ion peaks (17.31%) that were completely different from coix bran peptide. The results are as follows Figure 3 C. Figure 3 D display.

[0125] 2.5 Particle size and Zeta analysis of coix seed bran polypeptide-calcium chelate before and after chelation

[0126] The coix seed bran polypeptide and the coix seed bran polypeptide-calcium chelate were respectively dissolved in deionized water to prepare a 1 mg / ml solution, and the particle size and Zeta potential state of the coix seed bran polypeptide and the coix seed bran polypeptide-calcium chelate were determined using a laser particle size analyzer.

[0127] The results show that: Figure 3 E. Figure 3 As shown in Figure F, the particle size of coix seed bran peptide-calcium chelate (295.3±4.17nm) is significantly larger than that of coix seed bran polypeptide (255.03±8.12nm). The polydispersity index (PDI) of coix seed bran polypeptide and coix seed bran peptide-calcium chelate is 0.455 and 0.238, respectively, indicating that their uniformity is within an acceptable range. Zeta potential can reflect the change in surface charge of polypeptide particles after the addition of metal ions. After chelation, the Zeta potential of coix seed bran polypeptide increased from -13.81±0.33mV to -11.16±0.98mV, which may be due to the charge of -COO- being replaced by Ca in the chelation process. 2+ Neutralize and increase the potential.

[0128] 2.6 Temperature stability of coix seed bran polypeptide-calcium chelate, particle size and Zeta analysis of coix seed bran polypeptide-calcium chelate before and after chelation

[0129] 100mL Coix Bran Peptide-Calcium Chelate Solution, Ca 2+ The concentration was 10 μg / mL and the samples were divided into 5 equal volumes. The samples were incubated in a constant temperature water bath for 60 min at different temperatures (25°C, 40°C, 60°C, 80°C and 95°C) to evaluate the temperature tolerance of the coix seed bran peptide-calcium chelate, and anhydrous calcium chloride was used as a blank reference. After the heat treatment, the sample solution was centrifuged at 4000 r / min for 20 min. Subsequently, the Ca in the supernatant was determined. 2+ concentration.

[0130] The results show that: Figure 4 As shown in A, when the temperature is between 25-95°C, the calcium release rate of the chelate at different temperatures does not change much, and the calcium release rate remains above 90%. On the contrary, although the calcium release rate of CaCl2 does not change significantly in the temperature range of 40-95°C, it is much lower than that of coix seed bran peptide-calcium chelate. This shows that coix seed bran peptide-calcium chelate has good thermal stability, which means that its calcium release rate is less affected by temperature.

[0131] 2.7 pH stability of coix seed bran polypeptide-calcium chelate

[0132] Coix seed bran peptide-calcium chelate samples and CaCl2 were mixed into 100 ml of deionized water at 6 different pH values ​​(1, 3, 5, 7, 9 and 11) adjusted with NaOH (1 M / L) or HCl (1 M / L). 2+ The concentration was 10 μg / ml and incubated in a constant temperature water bath for 60 minutes to evaluate the acid-base tolerance of coix seed bran peptide-calcium chelate. CaCl2 was used as a positive control. After the treatment, the sample solution was centrifuged at 4000r / min for 20 minutes to determine the Ca in the supernatant obtained by centrifugation. 2+ concentration.

[0133] The results show that: Figure 4 As shown in B, there is no significant difference in the calcium release rate of calcium chloride and coix seed bran peptide-calcium chelate within the pH range of ﹤3, and both remain above 90%. In the pH range of 3-11, although the calcium release rate of coix seed bran peptide-calcium chelate gradually decreases in a pH-dependent manner, it is significantly higher than calcium chloride and shows good calcium release capacity. Compared with the traditional calcium supplement calcium chloride, coix seed bran peptide-calcium chelate has better pH adaptability, especially under alkaline conditions. The addition of coix seed bran peptide binds to calcium ions through coordination bonds, inducing the spatial structure of the peptide to fold, making Ca 2+ is protected from Ca 2+ Direct contact with hydroxide prevents calcium ions from forming precipitation in the human gastrointestinal tract to enhance its absorption capacity.

[0134] 2.8 Stability of coix bran polypeptide-calcium chelate in vitro gastrointestinal simulated digestion

[0135] Coix seed bran peptide-calcium chelate was dissolved in 50 ml of deionized water with a calcium ion content of 0.1 mg / mL. The pH was adjusted to 2.0 with HCl (6.0 mol / L), pepsin powder (1 mg) was added, and the mixture was incubated at 37°C for 30, 60, 90 and 120 min. At the end of digestion, the enzyme was inactivated in a boiling water bath for 5 min. After cooling to room temperature, it was centrifuged at 4000 r / min for 10 min. At the same time, 20 ml of gastric digesta were taken and the pH was adjusted to 7.4 with NaOH solution (0.2 mol / L), and 12 mg of trypsin powder was added to simulate pancreatic juice digestion for 150, 180, 210 and 240 min. After the simulated pancreatic juice digestion was completed, the mixture was heated in a 95°C water bath for 5 min to inactivate the enzyme and centrifuged at 4000 r / min for 20 min. The supernatant was collected for analysis of Ca 2+ concentration.

[0136] The results show that: Figure 4 As shown in Figure 3, in vitro digestion simulates dietary nutrients that usually reach the intestine through the stomach and are digested, absorbed and utilized in the intestine. Studies have shown that pepsin in the stomach and trypsin in the intestine can hydrolyze polypeptides into amino acids, resulting in adverse changes in the biological activity of peptides. Therefore, the tolerance of coix seed bran peptide-calcium chelate to gastrointestinal enzymes and its stability in the digestive environment were evaluated using an in vitro simulated digestion system. Under the gastric digestion environment (0-120min), the calcium release rate of coix seed bran peptide-calcium chelate was significantly higher than that of calcium chloride, and its calcium release rate remained above 98%, while the calcium release rate of calcium chloride was only maintained in the range of 85%-86%. Subsequently, under the intestinal digestion environment (120-240min), the calcium release rate of the chelate dropped sharply to 21.82% and remained stable until the end of intestinal digestion. Compared with coix seed bran peptide-calcium chelate, the calcium release rate of calcium chloride finally dropped to 16.57%. Throughout the gastrointestinal digestion process, the calcium release rate of coix seed bran peptide-calcium chelate was significantly higher than that of calcium chloride. In conclusion, coix bran peptide-calcium chelate can significantly increase the calcium ion release rate, showing good oral bioavailability potential, and is expected to become the raw material for high-efficiency calcium supplements in the future.

[0137] 3. According to the preparation methods of Examples 6 and 7, the prepared coix bran polypeptide-calcium chelate was subjected to the following experiments:

[0138] 3.1 Effect of tartaric acid on the digestive stability of coix bran polypeptide-calcium chelate

[0139] The coix seed bran peptide-calcium chelate prepared in Examples 6, 7 and Comparative Example 1 was dissolved in 50 ml of deionized water, and its calcium ion content was 0.1 mg / mL. The pH was adjusted to 2.0 with HCl (6.0 mol / L), pepsin powder (1 mg) was added, and the mixture was incubated at 37°C for 30, 60, 90 and 120 min. At the end of digestion, the enzyme was inactivated in a boiling water bath for 5 min. After cooling to room temperature, centrifuged at 4000 r / min for 10 min. At the same time, 20 ml of gastric digesta were taken and the pH was adjusted to 7.4 with NaOH solution (0.2 mol / L), and 12 mg of trypsin powder was added to simulate pancreatic juice digestion for 150, 180, 210 and 240 min. After the simulated pancreatic juice digestion was completed, the mixture was heated in a 95°C water bath for 5 min to inactivate the enzyme, and centrifuged at 4000 r / min for 20 min. The supernatant was collected for analysis of Ca 2+ concentration.

[0140] As above, the tolerance of coix seed bran peptide-calcium chelates prepared in Examples 6, 7 and Comparative Example 1 to gastrointestinal enzymes and their stability in the digestive environment were evaluated using an in vitro simulated digestive system. Under the gastric digestive environment (0-120 min), the calcium release rate of coix seed bran peptide-calcium chelates prepared in Examples 6 and 7 was maintained at 64%-71%. Subsequently, under the intestinal digestive environment (120-240 min), the calcium release rate of the chelate was 37%-44%, and remained basically stable until the end of intestinal digestion. The performance of Comparative Example 1 was similar to that of the sample in Example 1. The stomach has relatively limited absorption of calcium ions because the physiological environment and function of the stomach are not conducive to the absorption of a large amount of calcium ions. The intestine (especially the small intestine) is the main place for calcium ion absorption, and its surface has a large number of wrinkles and tiny villi, which greatly increases the absorption area. In the upper part of the small intestine, calcium ions enter the cells through the channels on the intestinal cell membrane and, with the help of vitamin D, are transported into the blood circulation through the calcium pump. Absorption efficiency: The small intestine has a much higher absorption efficiency for calcium ions than the stomach. According to information from professional medical websites, the amount of calcium that the human body can absorb every day is about 30% of the calcium intake, and this calcium is mainly absorbed in the small intestine. In addition, factors such as the pH of the small intestine, the concentration of calcium ions, and the presence of vitamin D will affect the absorption efficiency of calcium ions. There are many substances in the stomach, such as gastric acid, food residues, digestive enzymes, etc. These substances may react chemically with calcium ions to form compounds that are difficult to absorb. Calcium ions may also combine with substances such as phosphates, oxalates, and phytates in the stomach to form precipitates or difficult-to-dissolve salts. These compounds are relatively stable in the acidic environment of the stomach and are not easily decomposed and absorbed. Based on the above reasons, the present invention prepares a specific digestion-stable coix seed bran peptide-calcium chelate, which reduces its release in the stomach and increases its release in the intestine. Tartaric acid is usually used as the following roles in previous technologies: Acidulant: The sourness of tartaric acid is 1.2-1.3 times that of citric acid, and it has a unique flavor and is one of the commonly used acidulants in food. It can be used in beverages, canned foods, jams, candies and other foods to provide sourness and taste. Stabilizer: Tartaric acid can help foods increase stability, moisture retention and fluffiness. In foods, it can be used as a stabilizer to keep foods stable, color and avoid hydrolysis. Antioxidant synergist: Tartaric acid has certain antioxidant properties and can be used as an antioxidant synergist to extend the shelf life of foods. Flavoring agent, fast-acting leavening agent: In some cases, tartaric acid can also be used as a flavoring agent and a fast-acting leavening agent to improve the quality and taste of foods. Among them, the original principle of tartaric acid as a stabilizer may include: Lowering pH: Tartaric acid is an organic acid that can lower the pH value of food. In an acidic environment, the growth and reproduction of many microorganisms will be inhibited, thereby extending the shelf life of food. In addition, an acidic environment can also slow down the oxidation reaction of food and prevent food from spoiling. Chelation: Tartaric acid has multiple carboxyl groups, which can form chelates with metal ions (such as calcium, iron, etc.).This chelation can stabilize the metal ions in the food and prevent them from reacting adversely with other ingredients, thereby maintaining the stability of the food. Increase the ionic strength of the food: Tartaric acid dissociates ions in water, increasing the ionic strength of the food. The increase in ionic strength can stabilize the colloidal system in the food, prevent the aggregation and precipitation of the colloidal particles, and thus maintain the uniformity and stability of the food. Moisture retention: Tartaric acid also has a certain moisture retention, which can absorb and retain moisture in the food. This helps to prevent the food from drying and hardening due to water loss during storage and transportation, thereby maintaining the softness and taste of the food. Therefore, from the perspective of the prior art, there is no revelation of using tartaric acid to regulate the digestion stability or pH stability of coix seed bran peptide-calcium chelate. The method provided by the present invention can improve the absorption efficiency: by avoiding the release of calcium ions in the stomach, it can ensure that more calcium ions reach the small intestine, which is the main place for calcium ion absorption. In this way, the concentration of calcium ions in the small intestine will be higher, thereby increasing the chance of it being absorbed into the blood circulation and improving the overall absorption efficiency. Reduce stomach discomfort: Calcium ions released in the stomach may react with gastric acid and cause discomfort, such as stomach bloating, belching, etc. By controlling the release of calcium ions, these adverse reactions can be reduced and patient compliance can be improved.

[0141] 3.2 Tartaric acid promotes the stability of chelates under different pH conditions:

[0142] The coix seed bran peptide-calcium chelate samples prepared in Example 4, Example 7, and Comparative Example 1, and the solution of 10% tartaric acid added to the sample in Example 4 and CaCl2 were mixed into 100 ml of deionized water at 6 different pH values ​​(1, 3, 5, 7, 9 and 11) adjusted with NaOH (1 M / L) or HCl (1 M / L). 2+ The concentration was 10 μg / ml to explore the effect of tartaric acid on the stability of the chelate under different pH conditions. The mixture was incubated in a constant temperature water bath for 60 min to evaluate the acid-base tolerance of coix seed bran peptide-calcium chelate. CaCl2 was used as a positive control. After the treatment, the sample solution was centrifuged at 4000 r / min for 20 min to determine the Ca content in the supernatant obtained by centrifugation. 2+ The results show that after adding tartaric acid in Example 7, the CaCl2 and TCBP-Ca chelate 2+ The release rate was effectively reduced under the conditions of pH 1, 3 and 5, from the original Example 4 CaCl2 (100%, 93.05%, 56.17%), TCBP-Ca chelate (100%, 97.90%, 92.41%) Ca 2+The release rate changes are as follows: CaCl2 (100%, 93.03%, 55.95%) and TCBP-Ca chelate (77.90%, 70.40%, 78.21%) in Example 7. The release rate results of the samples of Comparative Example 1 and Example 4 doped with tartaric acid are similar to those of the samples of Example 4. In addition, due to the participation of tartaric acid in protecting Ca 2+ The release in low pH environment further showed an increase in the release rate in alkaline environment. In solutions with pH of 7, 9, and 11, the CaCl2 and TCBP-Ca chelate groups of Example 4 were significantly higher in the TCBP-Ca chelate group with tartaric acid added than in the CaCl2 and TCBP-Ca chelate groups of Example 4. 2+ The release rate increased TCBP-Ca chelate (22.10%, 27.50%, 14.20%). Therefore, the tartaric acid added in the chelation step can make the chelate more stable at the bottom pH. The possible reason is that there is a synergistic effect between coix bran peptide and tartaric acid, especially the presence of tartaric acid affects the spatial conformation of coix bran peptide, the polypeptide chain undergoes specific bending or folding, thereby forming a specific three-dimensional structure, or affects the charge distribution of the peptide chain, or forms a chelate with the metal on the peptide chain to affect the chelation of the peptide chain with calcium ions, these effects jointly stabilize calcium ions and regulate their release. This synergistic effect may cause a significant change in the release characteristics of the chelated product in the stomach and intestines.

[0143] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0144] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A coix seed bran polypeptide capable of chelating inorganic calcium, characterized in that: The amino acid sequence of the coix bran polypeptide includes one or more of KG ITDLKPVL, MSAAV, RSGSGG, NGSSGR, TEAEPANLDKFVHHLSENHFFPNRV, KDIF VDTLC, GLDWARLLKL, GSHVL, LHHIGPQTHISL, IEYASSKEGSACKEGV or TIVGFQR HGLHF.

2. A coix bran polypeptide-calcium chelate, characterized in that: It includes chelated calcium ions and a polypeptide chain skeleton, wherein the calcium ions are bound to the polypeptide chain skeleton by forming coordination bonds with the coordinating atoms on the side chains of the polypeptide chain skeleton, wherein the polypeptide chain skeleton is derived from the coix bran polypeptide according to claim 1, and the amino acid sequence of the polypeptide chain skeleton includes one or more of KGITDLKPVL, MSAAV, RSGSGG, NGSSGR, TEAEPANLDKFVHHLSENHFFPNRV, KDIFVDTLC, GLDW ARLLKL, GSHVL, LHHIGPQTHISL, IEYASSKEGSACKEGV or TIVGFQRHGLHF.

3. A method for preparing the coix bran polypeptide-calcium chelate according to claim 2, characterized in that: The steps include: (1) preparing the coix seed bran protein extract into an aqueous solution, adjusting the pH value, adding trypsin for incubation, killing the enzyme after the incubation, cooling to room temperature, and separating the product to obtain coix seed bran polypeptide powder; (2) dissolving the obtained coix seed bran polypeptide powder in deionized water to obtain a coix seed bran polypeptide original solution; (3) adding anhydrous calcium chloride to the original solution of the coix seed bran polypeptide obtained in step (2) to mix to obtain a coix seed bran polypeptide-calcium reaction solution, adjusting the pH value and stirring to uniformly perform a chelation reaction to obtain a coix seed bran polypeptide-calcium chelate reaction solution; (4) adding anhydrous ethanol to the coix seed bran polypeptide-calcium chelate reaction solution obtained in step (3), allowing to stand, and separating the product to obtain coix seed bran peptide-calcium chelate powder.

4. The method for preparing the coix seed bran polypeptide-calcium chelate according to claim 3, characterized in that: The aqueous solution of the coix bran protein extract in step (1) has a concentration of 2-3% (w / v), and the pH value is adjusted to 8; the amount of trypsin added in step (1) is 2000-3000 U / g, the incubation temperature is 37° C., and the incubation time is 3-4 hours; the enzyme inactivation in step (1) comprises heating the incubation product to above 85° C. and maintaining it for 15-20 minutes; the product separation in step (1) comprises centrifuging at 3000-5000 rpm for 15-20 minutes, taking the supernatant, concentrating, freezing, and drying.

5. The method for preparing the coix bran polypeptide-calcium chelate according to claim 3, characterized in that: In the coix seed bran polypeptide original solution of step (2), the coix seed bran polypeptide concentration is 1% to 5% (w / v).

6. The method for preparing the coix seed bran polypeptide-calcium chelate according to claim 3, characterized in that: The mass ratio of the calcium chloride in step (3) to the coix seed bran polypeptide in the original solution of coix seed bran polypeptide is 1:(1-8); the pH value is adjusted to 3-8; the chelation reaction time in step (3) is 20-100 minutes; and the chelation temperature is 15-75°C.

7. The method for preparing the coix seed bran polypeptide-calcium chelate according to claim 3, characterized in that: The volume ratio of the coix bran polypeptide-calcium chelating reaction liquid to anhydrous ethanol in step (4) is (10-15):1, and the standing is carried out at room temperature for a standing time of 30-120 min. The product separation in step (4) comprises centrifuging the product obtained by standing, and drying the precipitate; wherein the centrifugal speed is 3000-5000 rpm, the centrifugal time is 20-30 min, the drying temperature is 40-50° C., and the drying time is 6-8 h.

8. The method for preparing the coix seed bran polypeptide-calcium chelate according to claim 3, characterized in that: Step (3) After anhydrous calcium chloride is added to the original solution of coix seed bran polypeptide and mixed, tartaric acid is added, stirred and then allowed to stand, the pH value is adjusted, stirred evenly and then heated for chelation reaction to obtain a stabilized coix seed bran polypeptide-calcium chelate reaction solution.

9. The method for preparing the coix bran polypeptide-calcium chelate according to claim 8, characterized in that: The concentration of tartaric acid added in the above step is 0.3-0.5wt%, the standing temperature is room temperature, and the standing time is 10-15min; the chelating reaction temperature is 50-75°C, and the chelating reaction time is 30-50min.

10. Use of the coix seed bran polypeptide-calcium chelate according to claim 2 in the preparation of a calcium supplement, functional food or health product having a calcium absorption promoting effect.

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