Sea-buckthorn oat peptide as well as preparation and application thereof

Abstract

The invention provides hippophae rhamnoides oat peptide as well as preparation and application thereof. According to the invention, three active peptides are separated and purified from oats for the first time, and on the basis of the active peptides, wall-broken oat powder, sea-buckthorn powder, Chinese yam powder and three prebiotics are combined to obtain the sea-buckthorn oat peptide. The hippophae rhamnoides oat peptide can restore the blood sugar storage and transfer functions by reducing the blood sugar content of diabetic patients and increasing the insulin content of the diabetic patients, and provides reference significance for treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of adjuvant therapy for diabetes, and more specifically, to a seabuckthorn oat peptide and a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar levels. High blood sugar levels are caused by defects in insulin secretion or its impaired biological effects, or both. Long-term high blood sugar levels lead to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. Currently, diabetes is one of the four major non-communicable diseases in the world, and conventional drug treatments often lead to huge medical expenses, psychological pain, and physical suffering. People have made great efforts to find new therapeutic targets and develop new drugs to treat diabetes, but new discoveries and treatments are still needed.

[0003] Oats are a low-sugar, high-nutrition, high-energy food. Oats are sweet and flat in nature, and can nourish the spleen and heart, and astringe sweat. Oats are rich in dietary fiber, can promote gastrointestinal motility, and facilitate defecation. Oats are recognized as functional foods, with the effects of lowering cholesterol, stabilizing blood sugar, and maintaining metabolism. Seabuckthorn is a deciduous shrub of the Elaeagnaceae family and the genus Seabuckthorn. The vitamin C content in seabuckthorn fruit is high, and it is known as the king of vitamin C. Seabuckthorn fruit is rich in nutrients and bioactive substances, and has the effects of promoting regeneration, skin care, and delaying aging. The use of medicine and food homology methods for intervention treatment of diabetes is also a development trend in this field, but the blood sugar-lowering effective ingredients in the existing medicine and food homology methods are difficult to identify, resulting in poor blood sugar-lowering effects. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a seabuckthorn oat peptide and its preparation and application. The present invention separates and purifies three active peptides from oats for the first time, and based on the active peptides, combines broken oatmeal powder, seabuckthorn powder, yam powder, and three prebiotics to obtain seabuckthorn oat peptide. The seabuckthorn oat peptide can reduce the blood sugar content of diabetic patients, increase their insulin content, and restore their blood sugar storage and conversion functions, providing a reference for the treatment of diabetes.

[0005] Specifically, the present invention first provides a method for preparing oat peptides, comprising the following steps:

[0006] 4) Physically grind oats, pass through a 50-200 mesh sieve, and prepare oatmeal powder for later use;

[0007] 5) Mix oatmeal powder and deionized water at a mass volume ratio of 1: (15-30) g / mL, adjust the pH to 8.5-11.5, and stir in a 65°C water bath for 60-120 min;

[0008] 6) adding 200-300 U / mL composite protease to the reaction system of step 2), continuously stirring for enzymolysis, and then centrifuging and separating the supernatant to obtain oat peptide enzymolysis solution;

[0009] 4) filtering the oat peptide hydrolysate in step 3) with an ultrafiltration membrane to cut off polypeptide molecules with a molecular weight of 1000-3500 Da; vacuum concentrating the filtered oat peptide to obtain an oat peptide concentrate;

[0010] 5) Identification of oat peptides: The oat peptide concentrate from step 4) was subjected to mass spectrometry sequencing;

[0011] 6) Purity identification of oat peptides: The purity of oat peptides was analyzed by high performance liquid chromatography;

[0012] 7) The oat peptide concentrate with qualified purity is transferred to a spray dryer and concentrated to a moisture content of 3-9% of the oat peptide dry powder, and the dry powder is collected to obtain the oat peptide.

[0013] Preferably, the composite protease in step 3) is a combination of alkaline protease and papain, and the mass ratio of the two is (2-5):1.

[0014] Preferably, the enzymolysis conditions in step 3) are continuous stirring at a temperature of 30-45°C for 2-4 hours.

[0015] Preferably, step 3) further comprises heating the enzymatic hydrolysate to 90-100° C. and maintaining the temperature for 10-30 min to inactivate the enzyme.

[0016] Preferably, the vacuum concentration in step 4) is performed to a concentration of 10-18° Bé.

[0017] Preferably, the amino acid sequences of the oat peptides identified in step 5) are shown in SEQ ID NOs. 1-3.

[0018] Preferably, in step 7), the inlet air temperature of the spray dryer is 80-100°C, and the outlet air temperature is 60-80°C.

[0019] In another aspect of the present invention, a sea buckthorn oat peptide is provided, which includes the following components and their mass percentages: 60%-70% broken oatmeal powder, 5%-10% sea buckthorn powder, 5%-10% yam powder, 10%-20% oat peptide, 1%-5% fructooligosaccharide, 1%-5% inulin, and 1%-5% xylo-oligosaccharide.

[0020] Preferably, the method for preparing seabuckthorn oat peptide comprises the following steps:

[0021] 4) Weigh the broken oatmeal powder, seabuckthorn powder, yam powder and oat peptide in order according to the mass percentage, mix them evenly and set aside;

[0022] 5) Weigh oligofructose, inulin and oligoxylose in order according to their mass percentages, mix them evenly and set aside;

[0023] 6) The mixture obtained in step 2) and the mixture in step 1) are put into a mixer, mixed for 20 minutes, and then packaged.

[0024] Preferably, the sea buckthorn oat peptide comprises the following components and their mass percentages: 60% of broken oatmeal powder, 6% of sea buckthorn powder, 6% of yam powder, 19% of oat peptide, 3% of oligofructose, 3% of inulin and 3% of oligoxylose.

[0025] Preferably, the sea buckthorn oat peptide comprises the following components and their mass percentages: 69% of broken oatmeal powder, 5% of sea buckthorn powder, 5% of yam powder, 15% of oat peptide, 2% of oligofructose, 2% of inulin and 2% of oligoxylose.

[0026] Preferably, the sea buckthorn oat peptide comprises the following components and their mass percentages: 65% of broken oatmeal powder, 5% of sea buckthorn powder, 5% of yam powder, 10% of oat peptide, 5% of oligofructose, 5% of inulin and 5% of oligoxylose.

[0027] Another object of the present invention is to provide the use of seabuckthorn oat peptide in preparing medicine for preventing and treating diabetes.

[0028] Preferably, the use is to achieve therapeutic purposes by controlling blood sugar and insulin.

[0029] The advantages of the present invention are as follows: the present invention separates and purifies three active peptides from oats for the first time, and based on the active peptides, combines wall-broken oatmeal powder, seabuckthorn powder, yam powder, and three prebiotics to obtain seabuckthorn oat peptides. The seabuckthorn oat peptides can reduce the blood sugar content of diabetic patients, increase their insulin content, and restore their blood sugar storage and conversion functions, providing a reference for the treatment of diabetes. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0031] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0032] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0033] Example 1 Preparation, separation and identification of oat peptides

[0034] The preparation method of oat peptide comprises the following steps:

[0035] 1) Physically grind oats, pass through a 100-mesh sieve, and prepare oatmeal powder for later use;

[0036] 2) Mix oatmeal powder and deionized water at a mass volume ratio of 1:15 g / mL, adjust the pH value to 10.5, and stir in a 65°C water bath for 60 minutes;

[0037] 3) Add 200 U / mL of composite protease to the reaction system of step 2), continue to stir and hydrolyze, and heat to 95°C for 15 minutes to inactivate the enzyme, then centrifuge and separate the supernatant to obtain oat peptide hydrolysate; wherein the composite protease is a combination of alkaline protease and papain, and the mass ratio of the two is 2:1; the enzymatic hydrolysis conditions are continuous stirring and hydrolysis at a temperature of 37°C for 2 hours;

[0038] 4) Filter the oat peptide hydrolysate in step 3) with an ultrafiltration membrane to cut off polypeptide molecules with a molecular weight of 1000-3500 Da; vacuum concentrate the filtered oat peptide to a concentration of 15° Bé to obtain an oat peptide concentrate;

[0039] 5) Identification of oat peptides: The oat peptide concentrate from step 4) was sequenced by Ob itrap Fusion Lumos mass spectrometer;

[0040] 6) Purity identification of oat peptide: The oat peptide concentrate was transferred to a spray dryer with an inlet air temperature of 100°C and an outlet air temperature of 85°C to concentrate the oat peptide dry powder to a moisture content of 4.01%. The dry powder was collected to obtain oat peptide.

[0041] The results showed that three active peptides were obtained through sequencing and identification analysis, and their sequences were shown in SEQ ID NO.1-3. After purity determination, the purities of the active peptides corresponding to SEQ ID NO.1-3 were 87.6%, 89.5% and 91.2% respectively.

[0042] Example 2 Preparation of Broken Oatmeal Powder, Seabuckthorn Powder and Chinese Yam Powder

[0043] 2.1 The preparation method of broken oatmeal powder comprises the following steps:

[0044] 1) After the oats are screened, cleaned, and awn-removed, they are sent to a high-frequency oscillator with positive and negative charged particles for wall breaking;

[0045] 2) crushing and sieving the broken oats into broken oatmeal powder with a particle size of 80-120 meshes;

[0046] 3) drying the cracked oatmeal powder by microwave drying until the moisture content is 4% by weight, and setting aside;

[0047] 2.2 The preparation method of seabuckthorn powder comprises the following steps:

[0048] 1) After the fresh seabuckthorn fruit is washed and homogenized, it is sent to a spray dryer for drying;

[0049] 2) crushing and sieving the dried seabuckthorn into seabuckthorn powder with a particle size of 80-120 meshes for later use;

[0050] 2.3 The preparation method of yam powder comprises the following steps:

[0051] 1) After the yam is washed, sliced ​​and air-dried, it is sent to a spray dryer for drying;

[0052] 2) Crush the dried yam and sieve it into broken yam powder with a particle size of 80-120 meshes for later use.

[0053] The raw materials prepared in the above examples were used to prepare seabuckthorn oat peptide.

[0054] Example 3 Seabuckthorn oat peptide and its preparation

[0055] A seabuckthorn oat peptide comprises the following components and their mass percentages: 60% of broken oatmeal powder, 6% of seabuckthorn powder, 6% of yam powder, 19% of oat peptide, 3% of oligofructose, 3% of inulin and 3% of oligoxylose.

[0056] The preparation method of seabuckthorn oat peptide comprises the following steps:

[0057] 1) Weigh the broken oatmeal powder, seabuckthorn powder, yam powder and oat peptide in order according to the mass percentage, mix them evenly and set aside;

[0058] 2) Weigh oligofructose, inulin and oligoxylose in order according to their mass percentages, mix them evenly and set aside;

[0059] 3) The mixture obtained in step 2) and the mixture in step 1) are put into a mixer, mixed for 20 minutes, and then packaged.

[0060] Example 4 Seabuckthorn oat peptide and its preparation

[0061] A seabuckthorn oat peptide comprises the following components and their mass percentages: 69% of broken oatmeal powder, 5% of seabuckthorn powder, 5% of yam powder, 15% of oat peptide, 2% of oligofructose, 2% of inulin and 2% of oligoxylose.

[0062] The preparation method of seabuckthorn oat peptide comprises the following steps:

[0063] 1) Weigh the broken oatmeal powder, seabuckthorn powder, yam powder and oat peptide in order according to the mass percentage, mix them evenly and set aside;

[0064] 2) Weigh oligofructose, inulin and oligoxylose in order according to their mass percentages, mix them evenly and set aside;

[0065] 3) The mixture obtained in step 2) and the mixture in step 1) are put into a mixer, mixed for 20 minutes, and then packaged.

[0066] Example 5 Seabuckthorn oat peptide and its preparation

[0067] A seabuckthorn oat peptide comprises the following components and their mass percentages: 65% of broken oatmeal powder, 5% of seabuckthorn powder, 5% of yam powder, 10% of oat peptide, 5% of oligofructose, 5% of inulin and 5% of oligoxylose.

[0068] The preparation method of seabuckthorn oat peptide comprises the following steps:

[0069] 1) Weigh the broken oatmeal powder, seabuckthorn powder, yam powder and oat peptide in order according to the mass percentage, mix them evenly and set aside;

[0070] 2) Weigh oligofructose, inulin and oligoxylose in order according to their mass percentages, mix them evenly and set aside;

[0071] The mixture obtained in step 2) and the mixture in step 1) are put into a mixer, mixed for 20 minutes, and then packaged.

[0072] Comparative Example

[0073] A seabuckthorn oat peptide comprises the following components and their mass percentages: 65% of broken oatmeal powder, 10% of seabuckthorn powder, 10% of yam powder, 5% of oligofructose, 5% of inulin and 5% of oligoxylose.

[0074] The preparation method is the same as Example 3.

[0075] Example 6 Analysis of the efficacy of seabuckthorn oat peptide

[0076] To further analyze the effect of seabuckthorn oat peptide on fasting blood glucose and insulin content in mice: 40 SPF-grade healthy male ICR mice were selected and divided into an experimental group, a control group and a blank group, wherein the mice in the experimental group were fed with basic feed + 15 mg / kg of any of the seabuckthorn oat peptides described in Examples 3-5, the control group was fed with basic feed + 15 mg / kg of the seabuckthorn oat peptide described in the comparative example, and the mice in the blank group were fed only with basic feed. After one week of feeding, the mice in each group were fasted for 6 hours but not water, and the tails were cut to collect blood to detect the fasting blood glucose level; then the eyeballs were removed to collect blood, and the fasting serum insulin level was detected by radioimmunoassay.

[0077] The statistical results are shown in the following table: Compared with the control group, adding the seabuckthorn oat peptides described in Examples 3-5 to the basic feed can cause a sharp decrease in the blood sugar content of mice, and a significant increase in the insulin content. This further proves that the seabuckthorn oat peptides are involved in the production and metabolism of blood sugar, and further proves that diabetes can be treated by taking seabuckthorn oat peptides.

[0078] Table 1. Analysis of the effects of seabuckthorn oat peptide on fasting blood glucose and insulin content in mice

[0079] Fasting blood glucose (mmol / L) Fasting insulin (μIU) Example 3 9.56±0.63 60.59±2.36 Example 4 8.89±0.59 66.58±3.25 Example 5 10.98±0.85 58.67±2.46 Control group 35.98±1.57 25.58±3.24 Blank Group 42.36±2.89 10.25±1.98

[0080] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing oat peptide, characterized in that: The method comprises the following steps: 1) Physically grind oats, pass through a 50-200 mesh sieve, and prepare oatmeal powder for later use; 2) Mix oatmeal powder and deionized water at a mass volume ratio of 1: (15-30) g / mL, adjust the pH to 8.5-11.5, and stir in a 65°C water bath for 60-120 min; 3) adding 200-300 U / mL composite protease to the reaction system of step 2), continuously stirring for enzymolysis, and then centrifuging and separating the supernatant to obtain oat peptide enzymolysis solution; 4) filtering the oat peptide hydrolysate in step 3) with an ultrafiltration membrane to cut off polypeptide molecules with a molecular weight of 1000-3500 Da; vacuum concentrating the filtered oat peptide to obtain an oat peptide concentrate; 5) Identification of oat peptides: The oat peptide concentrate from step 4) was subjected to mass spectrometry sequencing; 6) Purity identification of oat peptides: The purity of oat peptides was analyzed by high performance liquid chromatography; 7) The oat peptide concentrate with qualified purity is transferred to a spray dryer and concentrated to a moisture content of 3-9% of the oat peptide dry powder, and the dry powder is collected to obtain the oat peptide.

2. The method according to claim 1, characterized in that The composite protease in step 3) is a compound of alkaline protease and papain, and the mass ratio of the two is (2-5):1; the enzymatic hydrolysis condition of step 3) is continuous stirring and enzymatic hydrolysis at a temperature of 30-45°C for 2-4h.

3. The method according to claim 1, characterized in that Step 3) further includes heating the enzymatic solution to 90-100° C. and maintaining the temperature for 10-30 minutes to inactivate the enzyme.

4. The method according to claim 1, characterized in that The vacuum concentration in step 4) is carried out to a concentration of 10-18° Bé.

5. The method according to claim 1, characterized in that The amino acid sequences of the oat peptides identified in step 5) are shown in SEQ ID NO. 1-3.

6. The method according to claim 1, characterized in that In step 7), the inlet air temperature of the spray dryer is 80-100°C, and the outlet air temperature is 60-80°C.

7. A seabuckthorn oat peptide, characterized in that: The invention comprises the following components measured in percentage by mass: 60%-70% of broken oatmeal powder, 5%-10% of sea buckthorn powder, 5%-10% of yam powder, 10%-20% of oat peptides, 1%-5% of oligofructose, 1%-5% of inulin and 1%-5% of oligoxylose; wherein the oat peptides are obtained from the preparation method of the oat peptides according to any one of claims 1 to 6.

8. The method for preparing seabuckthorn oat peptide according to claim 7, characterized in that: The method comprises the following steps: 1) Weigh cracked oatmeal powder, seabuckthorn powder, yam powder and oat peptide in sequence, mix them evenly and set aside; 2) weighing oligofructose, inulin and oligoxylose in sequence, and mixing them for later use; 3) The mixture obtained in step 2) and the mixture in step 1) are put into a mixer, mixed evenly, and then packaged to obtain the product.

9. Use of the seabuckthorn oat peptide according to claim 7 in the preparation of drugs for preventing and treating diabetes.

10. The use according to claim 9, characterized in that The use is to achieve the therapeutic purpose by controlling blood sugar and insulin.