阅读说明：本技术 一种超声辅助矿物质螯合杏仁肽及其制备方法和应用 (Ultrasonic-assisted mineral chelated almond peptide and preparation method and application thereof ) 是由 叶勇 黄传庆 于 2020-12-31 设计创作，主要内容包括：本发明属于医药的技术领域,公开了一种超声辅助矿物质螯合杏仁肽及其制备方法和应用。所述方法：1)在磷酸缓冲溶液中,采用蛋白酶在超声的条件下酶解杏仁蛋白,灭酶,离心,取上清液,冷冻干燥,获得杏仁多肽；2)在溶剂中,将杏仁多肽与矿物质在超声的条件下进行螯合,离心,取上清液,冷冻干燥,获得超声辅助矿物质螯合杏仁肽；矿物质为水溶性亚铁盐、水溶性钙盐和水溶性锌盐；溶剂为水和/或磷酸缓冲液。本发明通过超声辅助和不同矿物质螯合,得到含有多种矿物质螯合杏仁肽,其得率高,抗氧化性能好,同时提供多种矿物质营养。本发明的方法简单。所制备的超声辅助矿物质螯合杏仁肽用作抗氧化肽,并且用于食品、保健品和/或药品的领域。(The invention belongs to the technical field of medicines, and discloses an ultrasonic-assisted mineral substance chelated almond peptide and a preparation method and application thereof. The method comprises the following steps: 1) performing enzymolysis on almond protein in a phosphoric acid buffer solution by adopting protease under the ultrasonic condition, inactivating enzyme, centrifuging, taking supernate, and freeze-drying to obtain almond polypeptide; 2) chelating almond polypeptide and mineral substances in a solvent under the ultrasonic condition, centrifuging, taking supernatant, and freeze-drying to obtain ultrasonic-assisted mineral substance chelated almond peptide; the mineral substance is water-soluble ferrous salt, water-soluble calcium salt and water-soluble zinc salt; the solvent is water and/or phosphate buffer. The invention obtains the almond peptide containing various mineral substances through ultrasonic assistance and chelation of different mineral substances, has high yield and good oxidation resistance, and simultaneously provides various mineral substance nutrition. The method of the invention is simple. The prepared ultrasonic-assisted mineral chelated almond peptide is used as an antioxidant peptide and is used in the fields of food, health care products and/or medicines.)

1. A preparation method of ultrasonic-assisted mineral chelated almond peptide is characterized by comprising the following steps: the method comprises the following steps:

1) performing enzymolysis on almond protein in a phosphoric acid buffer solution by adopting protease under the ultrasonic condition, inactivating enzyme, centrifuging, taking supernate, and freeze-drying to obtain almond polypeptide; the average molecular weight of the almond polypeptide is 300-600 Da;

2) chelating almond polypeptide and mineral substances in a solvent under the ultrasonic condition, centrifuging, taking supernatant, and freeze-drying to obtain ultrasonic-assisted mineral substance chelated almond peptide; the mineral substances are water-soluble ferrous salt, water-soluble calcium salt and water-soluble zinc salt; the solvent is water and/or phosphate buffer.

2. The method for preparing the ultrasound-assisted mineral chelated almond peptide of claim 1, wherein: in the step 2), the molar ratio of the water-soluble ferrous salt to the water-soluble calcium salt to the water-soluble zinc salt in the mineral substance is (1-10): (50-500): (1-10);

in the step 2), the mass molar ratio of the almond polypeptide to the water-soluble ferrous salt in the mineral substance is 10 g: (0.001-1) mmol;

the ultrasonic condition in the step 2) is that the frequency is 20-40 KHz and the power is 50-100W; the ultrasonic time is 0.5-3 h, namely the chelation time.

3. The method for preparing the ultrasound-assisted mineral chelated almond peptide of claim 1, wherein: the specific steps of the step 2) are

S1, preparing the almond polypeptide into a solution by adopting water to obtain an almond polypeptide solution;

s2, preparing the mineral substances into solutions by respectively adopting phosphate buffer solutions to obtain a ferrous salt solution, a calcium salt solution and a zinc salt solution; mixing ferrous salt solution, calcium salt solution and zinc salt solution to obtain mineral solution;

s3, chelating the almond polypeptide solution and the mineral substance solution under the ultrasonic condition, centrifuging, taking supernatant, and freeze-drying to obtain the ultrasonic-assisted mineral substance chelated almond peptide.

4. The method for preparing the ultrasound-assisted mineral chelated almond peptide as claimed in claim 3, wherein: in the step S1, the mass-to-volume ratio of the almond polypeptide to the water is 1g: (10-20) mL;

s2, wherein the respective concentrations of the ferrous salt solution, the calcium salt solution and the zinc salt solution are 10-20 mM;

when mixing, the volume ratio of the ferrous salt solution to the calcium salt solution to the zinc salt solution is (1-5) to (50-250) to (1-5);

the phosphate buffer solution is a phosphate buffer solution with the pH value of 7-8;

the volume ratio of the almond polypeptide solution to the mineral substance solution is 1: (1-5).

5. The method for preparing the ultrasound-assisted mineral chelated almond peptide of claim 1, wherein:

the protease in the step 1) is more than one of alkaline protease, flavourzyme, neutral protease, trypsin and papain;

the dosage of the protease in the step 1) is 1-10% of the mass of the almond protein;

the ultrasonic condition in the step 1) is that the frequency is 30-80 KHz and the power is 50-500W; the ultrasonic time is 10-30 min; the time of ultrasonic treatment is the time of enzymolysis.

6. The method for preparing the ultrasound-assisted mineral chelated almond peptide of claim 1, wherein:

the water-soluble ferrous salt in the step 2) is ferrous chloride, ferrous sulfate or ferrous nitrate; the water-soluble calcium salt is calcium chloride or calcium nitrate; the water-soluble zinc salt is zinc chloride or zinc sulfate;

the phosphate buffer solution in the step 1) is a phosphate buffer solution with the pH value of 7-8.

7. The method for preparing the ultrasound-assisted mineral chelated almond peptide of claim 1, wherein: the mass-to-volume ratio of the almond protein to the phosphate buffer solution in the step 1) is 1g: 10-20 mL;

the enzyme deactivation in the step 1) is high-temperature deactivation;

the centrifugation condition in the step 1) is 5000-10000 rpm for 10-30 min;

the centrifugation condition in the step 2) is 5000-10000 rpm for 10-30 min.

8. An ultrasound-assisted mineral chelated almond peptide obtained by the preparation method of any one of claims 1-7.

9. The use of ultrasound assisted mineral chelated almond peptide as claimed in claim 8, wherein: the ultrasonic-assisted mineral chelated almond peptide is used for preparing antioxidant peptide.

10. The use of ultrasound assisted mineral chelated almond peptide as claimed in claim 8, wherein: the ultrasonic-assisted mineral chelated almond peptide is applied to the fields of food, health-care products and/or medicines.

Technical Field

The invention belongs to the field of medicines, and particularly relates to ultrasonic-assisted mineral chelated almond peptide, a preparation method thereof and application thereof as antioxidant peptide.

Background

Minerals exert various biological functions on human health, and for example, calcium is considered as one of the most abundant inorganic elements in the body, and is considered as one of the essential nutrient minerals of the human body, and has important physiological effects on intracellular metabolism, bone growth, blood coagulation, nerve conduction, muscle contraction, cardiac function, and the like. Insufficient calcium intake can lead to metabolic bone diseases such as rickets in children and osteoporosis in the elderly. Zinc, as a catalytic component of a series of enzymes, plays a structural and biological role in many proteins, peptides, hormones, cytokines, transcription factors and growth factors. Iron is an essential substance for oxygen transport in hemoglobin and is also an important raw material for hemoglobin production, and adequate iron storage is necessary to achieve and maintain adequate hemoglobin levels. Mineral deficiency, which is caused by a deficiency of minerals in the diet or a limited bioavailability of minerals, may cause functional disorders of important organs of the body, resulting in various diseases. Therefore, it is very important to ingest a proper amount of mineral elements.

Metal salts and various mineral supplements have been used in the food industry to overcome mineral deficiencies, but they are prone to calcium phosphate precipitation in the alkaline environment of the intestinal tract in vivo. The mineral chelated peptide has the capacity of combining with metal ions or enhancing the mineral absorption of the gastrointestinal tract, is an effective functional component for improving the bioavailability of dietary minerals, and is widely applied to foods such as oat, biscuits, milk powder and the like. Mineral-chelating peptides have attracted considerable attention as additives to foods of mineral elements. Chinese patent CN104232719B discloses a calcium chelating peptide prepared from collagen, CN107936113A discloses a mullet fish scale iron chelating peptide, CN103626847A discloses a wheat germ protein source zinc chelating peptide, which indicates that the mineral chelating peptide is used as a nutritional supplement product, is feasible and has a wide application potential. However, the above patent only combines a single polypeptide with a metal, and cannot combine multiple metals at the same time, so that the supplemented mineral is single, and unbalanced mineral is easily caused, which is not beneficial to human health.

Ultrasonic treatment mainly utilizes power and cavitation to change or accelerate physical, chemical and biological properties or states of substances; in addition, because of its cavitation-collapse effect, effective cavitation energy and significant mixing effect are generated, accelerating the catalytic reaction. Ultrasonic treatment, as an auxiliary treatment, can increase the substrate surface area and the frequency of action of the catalyst, thereby reducing mass transfer limitations. However, the ultrasonic technology is mostly applied to the extraction of active ingredients and the catalysis of chemical reactions, and is not applied to the preparation of mineral chelated peptides.

The almond has the effects of eliminating phlegm, relieving cough, relieving asthma, moistening intestines and the like, contains a large amount of almond protein, has amino acid composition close to an international reference mode, is good vegetable protein, and can promote absorption of polypeptide generated by degradation, so that the almond has the effects of supplementing nutrition, beautifying, resisting aging and the like. However, at present, no mineral substance chelated almond peptide product and process exist, and no report of preparing almond polypeptide by ultrasonic treatment is seen.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the ultrasonic-assisted mineral chelated almond peptide and the preparation method thereof.

The invention also aims to provide application of the ultrasonic-assisted mineral chelated almond peptide. The ultrasound-assisted mineral chelated almond peptide is used as an antioxidant peptide. The ultrasonic-assisted mineral chelated almond peptide is applied to the fields of food, health-care products and/or medicines.

The purpose of the invention is realized by the following technical scheme:

a preparation method of ultrasonic-assisted mineral chelated almond peptide comprises the following steps:

1) performing enzymolysis on almond protein in a phosphoric acid buffer solution by adopting protease under the ultrasonic condition, inactivating enzyme, centrifuging, taking supernate, and freeze-drying to obtain almond polypeptide; the average molecular weight of the almond polypeptide is 300-600 Da;

2) chelating almond polypeptide and mineral substances in a solvent under the ultrasonic condition, centrifuging, taking supernatant, and freeze-drying to obtain ultrasonic-assisted mineral substance chelated almond peptide; the mineral substances are water-soluble ferrous salt, water-soluble calcium salt and water-soluble zinc salt; the solvent is water and/or phosphate buffer.

The phosphate buffer solution in the step 1) is a phosphate buffer solution with the pH value of 7-8;

the dosage of the protease is 1-10% of the mass of the almond protein.

The ultrasonic condition is that the frequency is 30-80 KHz and the power is 50-500W; the ultrasonic time is 10-30 min; the time of ultrasonic treatment is the time of enzymolysis.

The mass volume ratio of the almond protein to the phosphoric acid buffer solution is 1g: 10-20 mL.

The enzyme deactivation is high-temperature enzyme deactivation, namely boiling for 15-20 min; cooling after enzyme deactivation.

The centrifugation condition is 5000-10000 rpm centrifugation for 10-30 min.

In the step 2), the molar ratio of the water-soluble ferrous salt to the water-soluble calcium salt to the water-soluble zinc salt in the mineral substance is (1-10): (50-500): (1-10);

the mass molar ratio of the almond polypeptide to the water-soluble ferrous salt in the mineral substance is 10 g: (0.001 to 1) mmol.

The ultrasonic condition in the step 2) is that the frequency is 20-40 KHz and the power is 50-100W; the ultrasonic time is 0.5-3 h, namely the chelation time.

The centrifugation condition is 5000-10000 rpm centrifugation for 10-30 min.

The specific steps of the step 2) are

S1, preparing the almond polypeptide into a solution by adopting water to obtain an almond polypeptide solution; s2, preparing the mineral substances into solutions by respectively adopting phosphate buffer solutions to obtain a ferrous salt solution, a calcium salt solution and a zinc salt solution; mixing ferrous salt solution, calcium salt solution and zinc salt solution to obtain mineral solution; s3, chelating the almond polypeptide solution and the mineral substance solution under the ultrasonic condition, centrifuging, taking supernatant, and freeze-drying to obtain the ultrasonic-assisted mineral substance chelated almond peptide.

In the step S1, the mass-to-volume ratio of the almond polypeptide to the water is 1g: (10-20) mL;

and S2, wherein the respective concentrations of the ferrous salt solution, the calcium salt solution and the zinc salt solution are 10-20 mM.

When mixing, the volume ratio of the ferrous salt solution, the calcium salt solution and the zinc salt solution is (1-5) to (50-250) to (1-5).

The phosphate buffer solution is a phosphate buffer solution with pH of 7-8.

The volume ratio of the almond polypeptide solution to the mineral substance solution is 1: (1-5).

The water-soluble ferrous salt is ferrous chloride, ferrous sulfate or ferrous nitrate; the water-soluble calcium salt is calcium chloride or calcium nitrate; the water-soluble zinc salt is zinc chloride, zinc sulfate and the like.

The protease in the step 1) is more than one of alkaline protease, flavourzyme, neutral protease, trypsin and papain.

The mineral chelated almond peptide prepared by the method has the particle size of 50-100 nm, is used as antioxidant peptide, and has good antioxidant capacity.

The principle of the invention is as follows: in the process of decomposing the almond protein into the almond protein polypeptide by protease, ultrasonic treatment is carried out, the size of oligomer is reduced, and the surface area of the substrate and the action frequency of a catalyst are increased, so that the limit of mass transfer is reduced; on the other hand, due to the cavitation-collapse effect of the ultrasonic waves, effective cavitation energy and a remarkable mixing effect are generated, catalytic reaction is accelerated, and the almond polypeptide with small and uniform structure size is obtained. In the process of synthesizing the metal-chelating peptide, the ultrasonic treatment can accelerate the movement rate of metal ions and the almond polypeptide and increase the probability of collision of the metal ions and the active site of the almond polypeptide, thereby improving the yield of the synthesized mineral chelating almond peptide. Through the combination of different minerals, the almond peptide containing various minerals is obtained, can provide various mineral nutrients at the same time, and improves the antioxidant performance of the product.

Compared with the prior art, the invention has the following advantages and effects:

(1) after ultrasonic wave auxiliary treatment of the almond protein and the protease, the solubility and the reaction performance in a solvent are improved, and the almond polypeptide with smaller and uniform structure size can be obtained.

(2) After the ultrasonic-assisted treatment of the metal and the almond protein polypeptide, the invention improves the metal chelation rate, shortens the reaction time, saves the energy, has simple preparation process and is convenient for industrial production.

Drawings

FIG. 1 is a graph of the clearance of hydroxyl radicals by the mineral chelated amygdalin peptide prepared in example 1;

FIG. 2 is a graph of the scavenging rate of superoxide anion radicals by the mineral chelated almond peptide prepared in example 1.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The almond protein is obtained by the following method: removing fat-soluble impurities in almond powder, mixing with water, adjusting the pH to be alkaline, centrifuging, taking supernatant, adjusting the pH of the supernatant to be 4.5-6.5, centrifuging, and freeze-drying precipitates to obtain almond protein. The pH value is 8-12. The fat-soluble impurity removal refers to mixing the almond powder with a fat-soluble solvent and processing for 1-2 hours at 20-50 ℃. The fat-soluble solvent is more than one of n-hexane or petroleum ether. The mass volume ratio of the almond powder to the fat-soluble solvent is 1g (5-10) mL; the mass volume ratio of the filter residue without fat-soluble impurities to water is 1 (10-20) (g/mL). The pH adjustment to be alkaline is to adjust the pH by adopting 1-3 mol/L NaOH aqueous solution. The centrifugation (after the pH is adjusted to be alkaline) condition is 3000-6000 rpm centrifugation for 10-30 min; adjusting the pH of the supernatant fluid means adjusting by using 1-3 mol/L HCl; after the pH value of the supernatant is adjusted to be 4.5-6.5, centrifuging for 10-30 min under the condition of 5000-10000 rpm.

Example 1

(1) Mixing 100g of 80-mesh almond powder with 1000mL of normal hexane, reacting at 50 ℃ for 1h, filtering, mixing 50g of filter residue with 500mL of deionized water, adjusting the pH to 8.0 by using 1mol/L NaOH aqueous solution, and centrifuging at 3000rpm for 30min to obtain supernatant; adjusting the pH of the supernatant to 4.5 with 1mol/L HCl, centrifuging at 5000rpm for 30min to obtain precipitate, and freeze drying to obtain almond protein.

(2) Adding 50g of almond protein into 500mL of phosphate buffer with pH value of 7, adding 0.5g of alkaline protease, simultaneously starting ultrasonic treatment, carrying out frequency of 30KHz and power of 500W ultrasonic treatment for 10min, boiling for 15min after the ultrasonic treatment is finished, cooling to room temperature, centrifuging at 5000rpm for 30min to obtain supernatant, and freeze-drying to obtain the almond polypeptide. The average molecular weight of the almond polypeptide of this example is 310 Da.

(3) Dissolving 10g of almond polypeptide into 100mL of deionized water to obtain an almond polypeptide aqueous solution; dissolving ferrous chloride, calcium chloride and zinc chloride in a phosphate buffer solution with the pH value of 7 respectively to prepare a buffer solution with the concentration of 10mM, and mixing the buffer solutions according to the volume ratio of 1:50:1 to form a mineral solution; mixing the almond polypeptide solution and the mineral substance solution in a volume ratio of 1:5, starting ultrasound at a frequency of 20KHz and a power of 100W for 0.5h, centrifuging at 5000rpm for 30min to obtain a supernatant, and freeze-drying to obtain the mineral substance chelated almond peptide. The yield of mineral chelated amygdalin in this example was 87%.

Example 2

(1) Mixing 100g of 200-mesh almond powder with 500mL of n-hexane, reacting at 20 ℃ for 2h, filtering, mixing 50g of filter residue with 1000mL of deionized water, adjusting the pH to 9.0 by using 3mol/L NaOH aqueous solution, and centrifuging at 6000rpm for 10min to obtain supernatant; adjusting the pH of the supernatant to 6.5 with 3mol/L HCl, centrifuging at 10000rpm for 10min to obtain precipitate, and freeze-drying to obtain almond protein.

(2) Adding 50g of almond protein into 1000mL of phosphate buffer with pH value of 8, adding 0.5g of flavourzyme and 0.5g of neutral protease, simultaneously starting ultrasonic treatment, carrying out ultrasonic treatment at the frequency of 80KHz and the power of 50W for 30min, boiling for 20min after the ultrasonic treatment is finished, cooling to room temperature, centrifuging at 10000rpm for 10min to obtain supernatant, and carrying out freeze drying to obtain the almond polypeptide. The average molecular weight of the almond polypeptide of this example is 580 Da.

(3) Dissolving 10g of almond polypeptide into 200mL of deionized water to obtain an almond polypeptide aqueous solution; dissolving ferrous chloride, calcium chloride and zinc chloride in phosphate buffer solution with the pH value of 8 respectively to prepare buffer solution with the concentration of 20mM, and mixing the buffer solution with the volume ratio of 5:50:5 to form mineral solution; mixing the almond polypeptide solution and the mineral substance solution in a volume ratio of 1:1, starting ultrasound at a frequency of 40KHz and a power of 50W for 3h, centrifuging at 10000rpm for 10min to obtain a supernatant, and freeze-drying to obtain the mineral substance chelated almond peptide. The yield of mineral chelated amygdalin in this example was 94%.

Example 3

(1) Mixing 100g of 100-mesh almond powder with 800mL of normal hexane, reacting at 30 ℃ for 1.5h, filtering, mixing 50g of filter residue with 800mL of deionized water, adjusting the pH value to 8.5 by using 2mol/L NaOH aqueous solution, and centrifuging at 4000rpm for 20min to obtain supernatant; adjusting the pH of the supernatant to 5 with 2mol/L HCl, centrifuging at 8000rpm for 20min to obtain precipitate, and freeze drying to obtain almond protein.

(2) Adding 50g of almond protein into 800mL of phosphate buffer with pH value of 7.5, adding 0.5g of trypsin and 0.5g of papain, simultaneously starting ultrasonic treatment, performing ultrasonic treatment at the frequency of 50KHz and the power of 200W for 20min, boiling for 18min after the ultrasonic treatment is finished, cooling to room temperature, centrifuging at 8000rpm for 20min to obtain supernatant, and freeze-drying to obtain the almond polypeptide. The average molecular weight of the almond polypeptide of this example is 400 Da.

(3) Dissolving 10g of almond polypeptide into 150mL of deionized water to obtain an almond polypeptide aqueous solution; dissolving ferrous chloride, calcium chloride and zinc chloride in a phosphate buffer solution with the pH value of 7.5 to prepare a buffer solution with the concentration of 15mM respectively, and mixing the buffer solutions according to the volume ratio of 1:250:2 to form a mineral solution; mixing the almond polypeptide solution and the mineral substance solution in a volume ratio of 1:3, starting ultrasound at a frequency of 30KHz and a power of 80W for 2h, centrifuging at 8000rpm for 20min to obtain a supernatant, and freeze-drying to obtain the mineral substance chelated almond peptide. The yield of mineral chelated almond peptide of this example was 91%.

Comparative example 1

The ultrasonic agitation was replaced by stirring at 1500rpm, and the other conditions were the same as in example 1. The obtained almond polypeptide has molecular weight of 2000Da and larger molecular weight; the yield of the mineral chelated almond peptide is 36 percent and is obviously lower than that of the mineral chelated almond peptide prepared in the example 1. The antioxidant performance of the mineral chelated almond peptide is measured, and the result shows that the highest clearance rates of the mineral chelated almond peptide on p-hydroxyl free radicals and superoxide anion free radicals are 65% and 40%, respectively, and are obviously lower than the antioxidant performance of the mineral chelated almond peptide prepared in example 1.

Comparative example 2

Commercially available soybean polypeptide chelated iron, soybean polypeptide chelated zinc and fishbone collagen polypeptide chelated calcium are adopted, and the iron content, the zinc content and the calcium content are respectively 10%, 8% and 12%. The results of measuring the antioxidant performance show that the highest clearance rates of the peptide on the p-hydroxyl free radical are respectively 74%, 70% and 39%, and the highest clearance rates of the peptide on the superoxide anion free radical are respectively 58%, 49% and 28%, which are obviously lower than the antioxidant performance of the mineral chelated almond peptide prepared in examples 1-3.

Comparative example 3

The almond polypeptide prepared in the embodiment 1 is taken, and the oxidation resistance of the almond polypeptide is measured, and the result shows that the highest clearance rates of the almond polypeptide to p-hydroxyl free radicals and superoxide anion free radicals are 82 percent and 70 percent respectively, which are obviously superior to the oxidation resistance of the almond polypeptide sold in the market, and shows that the almond polypeptide prepared by the method has smaller molecular weight and more superior oxidation resistance.

And (3) performance testing:

(1) particle size measurement of mineral chelated Almond peptide obtained in examples 1-3

The method comprises the following steps: weighing 1g of the mineral chelated almond peptide prepared in the embodiment 1-3, adding into 50mL of deionized water, stirring uniformly, placing in a Malvern particle sizer, and measuring the particle size distribution.

As a result: the mineral chelated almond peptide prepared in the embodiment 1-3 has the particle size of 50-100 nm, is uniformly distributed and has a small size, and the ultrasonic-assisted treatment is proved to reduce and homogenize the almond polypeptide structure.

The particle size of the chelating peptide in the embodiment 1 is 50-85 nm; the particle size of the chelating peptide of the embodiment 2 is 70-100 nm; the particle size of the chelating peptide of example 3 is 60 to 90 nm.

(2) Determination of mineral content of mineral chelated Almond peptide obtained in examples 1-3

The method comprises the following steps: weighing 0.100g of the mineral chelated almond peptide prepared in the examples 1-3, adding 20mL of aqua regia (concentrated hydrochloric acid: concentrated nitric acid/v: v ═ 3:1) prepared in situ, sealing and placing in a microwave digestion instrument for digestion at 200 ℃ for 2h, naturally cooling and taking out, taking out 1mL of solution to dilute to 50-300ppm, taking standard metal ion liquid as a reference, and measuring the content of metal elements in inductively coupled plasma emission spectrum after a sample liquid filtering membrane.

As a result: the mineral chelated almond peptide prepared in the embodiment 1-3 has a calcium content of 3-8% (mass percentage), an iron content of 0.1-0.5%, and a zinc content of 0.1-0.6%. The proportion of the calcium, iron and zinc nutrient solution meets the proportion of the human body to the calcium, iron and zinc, so that balanced calcium, iron and zinc nutrition can be provided.

(3) Antioxidant Property measurement of mineral chelated Almond peptide obtained in example 1

The method comprises the following steps: hydroxyl radical scavenging ability test: 1ml of 9mmol/L FeSO₄1mL of a 9mmol/L solution of salicylic acid in ethanol and 1mL of 8.8mmol/L H₂O₂Added to 2mL of sample. After 30min of reaction at 37 ℃, the absorbance of the mixture was measured at 510 nm. Commercial almond polypeptide was used as a control.

Superoxide anion radical scavenging capacity determination: 4.5mL of Tris-HCl buffer (50mmol/L, pH 8.2) was placed in a water bath (25 ℃) for 20 min. Then 1mL of the sample and 0.4mL of 25mmol/L pyrogallol were added to Tris-HCl buffer. After 5min reaction in a water bath, the reaction was stopped by adding 8mmol/L HCl. The absorbance was immediately measured at 325nm and the almond polypeptide was used as a control.

As a result: as shown in fig. 1 and 2. FIG. 1 is a graph of the clearance of hydroxyl radicals by the mineral chelated amygdalin peptide prepared in example 1; FIG. 2 is a graph of the scavenging rate of superoxide anion radicals by the mineral chelated almond peptide prepared in example 1.

The mineral chelated almond peptide prepared in the embodiment 1 has stronger capacity of eliminating hydroxyl free radicals and superoxide anion free radicals, and the effect is superior to that of commercial almond polypeptide (molecular weight is 1000Da), which shows that the product of the invention has better oxidation resistance.

The mineral chelated amygdalin of example 1 had the highest clearance of hydroxyl radical and superoxide anion radical of 93% and 80%, respectively, while the commercial amygdalin peptide had only 73% and 45%.

In addition, the mineral chelated amygdalin of example 2 had the highest clearance of hydroxyl radicals and superoxide anion radicals of 95% and 83%, respectively.

The mineral chelated amygdalin of example 3 had the highest clearance of hydroxyl radicals and superoxide anion radicals of 92% and 80%, respectively.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.