Bioactive peptide powder, antihypertensive and antioxidative goat milk powder and preparation method of antihypertensive and antioxidative goat milk powder

Abstract

The invention provides bioactive peptide powder, antihypertensive and antioxidative goat milk powder and a preparation method thereof, goat milk is used as a raw material, firstly, compound protease is adopted to hydrolyze the goat milk to obtain goat milk enzymatic hydrolysate containing bioactive peptide, then nutrient supplements required by middle-aged and elderly people are added, the mixture is stirred uniformly, then the mixture is concentrated to 45%-52% of solids, and the bioactive peptide powder is obtained. And carrying out spray drying to obtain the antihypertensive and antioxidative goat milk powder. Or performing centrifugal degreasing on the goat milk subjected to enzymolysis, performing ultrafiltration and preparative chromatography to obtain a component with the strongest ACE inhibitory activity and oxidation resistance, performing freeze drying on the component to obtain bioactive peptide powder, and finally mixing the bioactive peptide powder with goat milk powder and other nutrient supplements required by middle-aged and elderly people by adopting a dry method to prepare the antihypertensive and antioxidative goat milk powder.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular belongs to a bioactive peptide powder, a blood pressure reducing and antioxidant goat milk powder and a preparation method thereof. Background Art

[0002] Hypertension is one of the common chronic diseases among the elderly. With the aging of the population, its incidence continues to rise. Statistics show that the prevalence of hypertension is quite high among adults, and it often exceeds half among the elderly. Hypertension not only significantly increases the risk of cardiovascular and cerebrovascular diseases such as stroke and myocardial infarction, but may also cause serious health problems such as renal failure.

[0003] In the treatment of hypertension, commonly used drugs include angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor antagonists (ARB), calcium channel blockers (CCB) and diuretics, etc. However, these drugs may cause some side effects during use, such as electrolyte imbalance and renal damage.

[0004] In recent years, studies have found that some natural bioactive peptides have significant antihypertensive and antioxidant effects, especially antihypertensive peptides and antioxidant peptides. Antihypertensive peptides inhibit the activity of angiotensin converting enzyme and reduce the production of angiotensin II, thereby achieving the purpose of lowering blood pressure. Antioxidant peptides, on the other hand, play a positive role in maintaining vascular health by removing excess free radicals in the body and slowing down the damage caused by oxidative stress. Compared with traditional antihypertensive drugs, antihypertensive peptides and antioxidant peptides have the advantages of natural sources, no toxic side effects, and better biocompatibility. Therefore, the development of foods containing antihypertensive peptides and antioxidant peptides for the prevention and relief of hypertension has important practical significance and broad application prospects. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a bioactive peptide powder, a blood pressure reducing and antioxidant goat milk powder and a preparation method thereof. Goat milk is used as a raw material, and firstly a composite protease is used to hydrolyze the goat milk to obtain a goat milk enzymatic hydrolysate containing bioactive peptides, and then a nutritional enhancer required by the middle-aged and elderly is added and stirred evenly, and then the powder is concentrated to 45-52% of solids, and then spray-dried to obtain the blood pressure reducing and antioxidant goat milk powder; or the enzymatically hydrolyzed goat milk is first defatted by centrifugation, and then ultrafiltration and preparative chromatography are performed to obtain a component with the strongest ACE inhibitory activity and antioxidant activity, and then the powder is freeze-dried to obtain the bioactive peptide powder, and finally the powder is dry-mixed with goat milk powder and other nutritional enhancers to prepare the blood pressure reducing and antioxidant goat milk powder.

[0006] To achieve the above object, the present invention provides the following technical solution: a bioactive peptide powder, the ACE inhibitory activity IC 50The IC value of DPPH free radical scavenging rate was 0.28±0.012 mg / mL. 50 The value is 0.36±0.015 mg / mL. The peptide powder includes 13 bioactive peptides with antihypertensive and antioxidant effects. The amino acid sequences of the 13 bioactive peptides are shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0007] The present invention also provides a method for preparing bioactive peptide powder, the specific steps of which are as follows: Calculated by mass fraction, 2‰~4‰ of the composite protease was added to the sterilized and cooled goat milk; The goat milk with the compound protease added is subjected to constant temperature enzymolysis at 50°C to 60°C for 2.5h to 3.5h, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain a goat milk enzymolysis solution; The goat milk enzymatic hydrolysate with a molecular weight Mw < 2k Da was freeze-dried to obtain a bioactive peptide powder with antihypertensive and antioxidant effects.

[0008] Furthermore, the composite protease includes protease P and protease N, the addition ratio of protease P and protease N is 1:1~1:3, protease P is Bacillus subtilis protease, and the enzyme activity is 400,000 U / g; protease N is a neutral protease, and the enzyme activity is 0.8AU / g.

[0009] Furthermore, the ACE inhibition rate of the goat milk enzymatic hydrolysate was 76.79±2.19%~89.28±1.42% and the DPPH free radical scavenging rate was 65.90±1.18%~82.68±1.96%.

[0010] Further, in the step of adding the composite protease to the goat milk and subjecting it to constant temperature enzymolysis at 50°C to 60°C for 2.5h to 3.5h, inactivating the enzyme, and centrifuging to obtain the supernatant to obtain the goat milk enzymolysate: The enzyme is inactivated by keeping it in a boiling water bath for 10-15 minutes; The centrifugation speed is 8000r / min~10000r / min for 10min~15min.

[0011] The present invention also provides a bioactive peptide powder prepared by the above-mentioned preparation method of a bioactive peptide powder, wherein the ACE inhibitory activity IC 50 The IC value of DPPH free radical scavenging rate was 0.28±0.012 mg / mL. 50 The value is 0.36±0.015 mg / mL. The peptide powder includes 13 bioactive peptides with antihypertensive and antioxidant effects. The amino acid sequences of the 13 bioactive peptides are shown in SEQ ID NO: 1 to SEQ ID NO: 13.

[0012] The present invention also provides a blood pressure reducing and antioxidant goat milk powder, the total amount of which is 1kg, the raw materials of which include 0.5g-1.0g of bioactive peptide powder, 2g-10g of phytosterol ester, 0.5g-1.5g of phosphatidylserine, 0.2g-1.5g of probiotic powder, 10g-45g of prebiotics, and the rest is goat milk powder, and the above raw materials are mixed to obtain the blood pressure reducing and antioxidant goat milk powder; Wherein, the bioactive peptide powder is the above-mentioned bioactive peptide powder.

[0013] Furthermore, the probiotics are one or more of Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus acidophilus, Bifidobacterium animalis subspecies lactis, Streptococcus salivarius thermophilus subspecies, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus helveticus, and the number of viable bacteria is ≥1.0×10 11 CFU / g; The prebiotics are one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin.

[0014] The present invention also provides a blood pressure reducing and antioxidant goat milk powder, the raw materials of which include 7.55kg-7.90kg goat milk enzymatic hydrolysate, 2g-10g phytosterol ester, 0.5g-1.5g phosphatidylserine, and 10g-45g prebiotics, and the raw materials are mixed and concentrated to a solid content of 45%-52%, and then spray-dried to obtain 1kg of blood pressure reducing and antioxidant goat milk powder; The goat milk enzymolysate is the above-mentioned goat milk enzymolysate, and the solid content of the goat milk enzymolysate is 12.5g / 100g.

[0015] Furthermore, the prebiotics are one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylo-oligosaccharides, stachyose and inulin; the spray drying conditions are 130°C~150°C, the outlet air temperature is 75°C~85°C, and the liquid flow rate is 4mL / min~5mL / min.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention prepares a blood pressure lowering and antioxidant bioactive peptide powder, which uses goat milk as a raw material, utilizes the composite protease proposed in the present invention to hydrolyze the protein in the goat milk, and simultaneously obtains blood pressure lowering peptides and antioxidant peptides with good activity, and adopts ultrafiltration and preparative chromatography for separation. The bioactive peptide powder is safe and healthy, has no side effects, can be added to various foods as a functional ingredient, can assist in lowering blood pressure and anti-oxidation for consumers with moderate hypertension after consumption, and has no side effects for consumers with normal blood pressure.

[0017] The present invention applies the prepared bioactive peptide powder to goat milk powder, and according to the IC values ​​of the ACE inhibitory activity and DPPH free radical scavenging rate of the bioactive peptide powder, 50value, determined the appropriate amount of addition, and added the nutritional enhancers needed by the middle-aged and elderly, developed blood pressure lowering and antioxidant goat milk powder, increased the added value and functionality of goat milk powder, and improved consumers' blood pressure and antioxidant properties.

[0018] The present invention also provides a method of preparing antihypertensive and antioxidant goat milk powder by directly adding nutritional enhancers required by the middle-aged and elderly after enzymatic hydrolysis of goat milk with a composite protease, and then concentrating and spray-drying. There is no need to separate and purify antihypertensive peptides and antioxidant peptides, thus reducing the equipment and related costs of separation and purification, and the technology has strong competitiveness.

[0019] The composite protease of the invention comprises protease P and protease N. When the protease P and protease N are added in a ratio of 1:1 to 1:3, they can act synergistically to improve the hydrolysis efficiency, obtain more bioactive peptides, and further improve the ACE inhibitory activity and DPPH free radical scavenging rate of the bioactive peptide powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of different proteases on DPPH free radical scavenging rate and ACE inhibition rate in goat milk; Figure 2 Effects of different proteases on Fe 2+ Influence of chelation capacity and hydroxyl radical scavenging rate; Figure 3 Effects of protease combination on DPPH free radical scavenging rate and ACE inhibition rate in goat milk; Figure 4 Protease combination against Fe 2+ Influence of chelation capacity and hydroxyl radical scavenging rate; Figure 5 Effect of compound protease ratio on DPPH free radical scavenging rate and ACE inhibition rate of goat milk; Figure 6 Effect of enzymatic hydrolysis temperature on DPPH free radical scavenging rate and ACE inhibition rate in goat milk; Figure 7 Effect of enzymatic hydrolysis time on DPPH free radical scavenging rate and ACE inhibition rate in goat milk; Figure 8 Effect of enzyme addition on DPPH free radical scavenging rate and ACE inhibition rate in goat milk; Fig. 9 Preparative chromatogram of the fraction less than 2K from ultrafiltrated goat milk; Fig.10 Secondary mass spectrum of the bioactive peptide PINIF; Fig.11 Secondary mass spectrum of the bioactive peptide ERFFDDK; Fig.12Secondary mass spectrum of the bioactive peptide FPKYPVEP; Fig.13 Secondary mass spectrum of the bioactive peptide FYQKFPQY; Fig.14 Secondary mass spectrum of the bioactive peptide HLPLP; Fig.15 MS / MS of the bioactive peptide LAYFYPQLFRQ; Fig.16 Secondary mass spectrum of the bioactive peptide LDIQKVAGTW; Fig.17 Secondary mass spectrum of the bioactive peptide LHLPLPLV; Fig.18 Secondary mass spectrum of the bioactive peptide LPLPLVQS; Fig.19 MS / MS spectrum of the bioactive peptide QYPYQGPIV; Fig. 20 Secondary mass spectrum of the bioactive peptide VLPVPQ; Fig.21 MS / MS spectrum of the bioactive peptide VTQTMKGLDIQK; Fig. 22 Secondary mass spectrum of the bioactive peptide YQKFPQY. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0022] The present invention provides a bioactive peptide powder, which uses a composite protease to hydrolyze the protein in goat milk, and then obtains a freeze-dried peptide powder through ultrafiltration, preparative chromatography separation and freeze drying. The ACE inhibitory activity IC 50 The IC value of DPPH free radical scavenging rate was 0.28±0.012mg / mL. 50 The value is 0.36±0.015mg / mL.

[0023] The above-mentioned bioactive peptide powder includes 13 bioactive peptides with antihypertensive and antioxidant effects. The amino acid sequences of the 13 bioactive peptides are shown in SEQ ID NO: 1 to SEQ ID NO: 13, specifically: EPINIF, ERFFDDK, FPKYPVEP, FYQKFPQY, HLPLP, LAYFYPQLFRQ, LDIQKVAGTW, LHLPLPLV, LPLPLVQS, QYPYQGPIV, VLPVPQ, VTQTMKGLDIQK and YQKFPQY.

[0024] The preparation method of the above-mentioned bioactive peptide powder is as follows: 1) Hydrolyze goat milk with compound protease By mass fraction, add 2‰ - 5‰ of compound protease to the sterilized and cooled goat milk, and carry out constant-temperature enzymatic hydrolysis at 40°C - 60°C for 2h - 4h. Inactivate the enzyme and centrifuge to obtain the supernatant, which is the goat milk enzymatic hydrolysate containing bioactive peptides. Its ACE inhibition rate is 76.79±2.19% - 89.28±1.42% and the DPPH free radical scavenging rate is 65.90±1.18% - 82.68±1.96%.

[0025] The compound protease includes protease P and protease N, and the ratio of the two is 1:1 - 1:3. Protease P is Bacillus subtilis protease with an enzyme activity of 400,000 U / g; protease N is neutral protease with an enzyme activity of 0.8 AU / g.

[0026] 2) Preparation of bioactive peptides with antihypertensive and antioxidant activities in the bioactive peptide powder and peptide composition Ultrafilter the goat milk enzymatic hydrolysate with ultrafiltration membranes with cut-off molecular weights of 5K Da and 2K Da respectively to obtain three components with molecular weights Mw>5k Da, 2k Da < Mw < 5k Da and Mw < 2k Da. Select the component with the strongest ACE inhibitory activity and antioxidant property, and then carry out preparative chromatography separation and purification to obtain each component. Select the component with the highest ACE inhibition rate and DPPH free radical scavenging rate, that is, the component with Mw < 2k Da, and obtain the bioactive peptide powder by freeze-drying; Preferably, in view of the needs of middle-aged and elderly people to improve cardiovascular health, regulate the balance of intestinal flora, prevent Alzheimer's disease and constipation, the present invention also provides a antihypertensive and antioxidant goat milk powder, which adds phytosterol esters for improving cardiovascular health, probiotics for regulating the balance of intestinal flora, phosphatidylserine for improving memory and preventing Alzheimer's disease, and prebiotics for preventing constipation to the goat milk powder formula. Its goat milk powder formula (1kg) is: 1g - 20g of phytosterol esters, 0.5g - 2g of phosphatidylserine, 0.1g - 1g of probiotic powder, 5g - 25g of prebiotics, 0.5g - 1.5g of the above-mentioned bioactive peptide powder, and the balance is goat milk powder; Preferably, the probiotics are one or more of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Streptococcus salivarius subsp. thermophilus, Lactiplantibacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, and their viable cell counts are all ≥1.0×10 11 CFU / g; Preferably, the prebiotics are one or more of inulin, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose and inulin; Preferably, the present invention further provides a blood pressure reducing and antioxidant goat milk powder, the raw materials comprising 7.55kg-7.90kg goat milk enzymatic hydrolysate, 2g-10g phytosterol ester, 0.5g-1.5g phosphatidylserine, 10g-45g prebiotics, the above raw materials are mixed and concentrated to a solid content of 45%-52%, and then spray-dried to obtain 1kg of blood pressure reducing and antioxidant goat milk powder; The goat milk enzymolysate is the above-mentioned goat milk enzymolysate, and the solid content of the goat milk enzymolysate is 12.5g / 100g.

[0027] The specific steps of the preparation method of the blood pressure reducing and antioxidant goat milk powder are as follows: 1) Compound protease enzymatic hydrolysis of goat milk In terms of mass fraction, 2‰~4‰ of the composite protease was added to the sterilized and cooled goat milk, and the enzymatic hydrolysis was carried out at a constant temperature of 50℃~60℃ for 2.5~3.5h. The enzyme was inactivated and centrifuged to obtain the supernatant, which was the goat milk enzymatic hydrolysate containing bioactive peptides. The ACE inhibition rate was 76.79±2.19%~89.28±1.42%, and the DPPH free radical scavenging rate was 65.90±1.18%~82.68±1.96%.

[0028] The composite protease includes protease P and protease N, the ratio of the two is 1:1~1:3, protease P is Bacillus subtilis protease, and the enzyme activity is 400,000 U / g; protease N is a neutral protease, and the enzyme activity is 0.8AU / g.

[0029] 2) Preparation of antihypertensive and antioxidant goat milk powder Nutritional enhancers required by middle-aged and elderly people are added to the enzymatic hydrolysate of goat milk, and the nutritional enhancers are plant sterol esters, phosphatidylserine and prebiotics. The hydrolysate is then vacuum concentrated by a rotary evaporator to a solid content of 45% to 52%, and then spray-dried (130°C to 150°C, air outlet temperature of 75°C to 85°C, and liquid flow rate of 4ml / min to 5ml / min) to obtain the antihypertensive antioxidant goat milk powder.

[0030] Preferably, the prebiotic is one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin; Obtaining the optimal parameters of the above-mentioned method for preparing a bioactive peptide powder: 1. Process optimization of compound protease hydrolysis of goat milk to prepare bioactive peptides with antihypertensive and antioxidant activities 1.1 Single enzyme screening Bromelain (A), protease P (B), protease X (C), papain (D), alkaline protease (E), protease C (F), flavor protease (G) and protease N (H) in Table 1 were added to the enzyme-inactivated and cooled goat milk at an addition amount of 2‰ (w / v), and the milk was enzymatically hydrolyzed at 50℃ for 3h. After boiling and enzyme inactivation, the samples were centrifuged and the antioxidant and ACE inhibition rates were determined. The results are shown in Table 1. Figure 1~Figure 2 As shown: Table 1 Sources and optimal conditions of different proteases

[0031] Depend on Figure 1~Figure 2 It can be seen that there are great differences in the DPPH free radical scavenging rate, ferrous ion chelating rate, hydroxyl free radical scavenging rate and ACE inhibition rate after different proteases hydrolyzed goat milk. The DPPH free radical scavenging rate ranged from 37.05% to 54.77%, with protease N being the highest, followed by protease P, and alkaline protease being the lowest; the ACE inhibition rate ranged from 41.53% to 74.62%, with protease N being the highest, followed by protease P, and flavor protease being the lowest; the ferrous ion chelating rate scavenging rate ranged from 54.76% to 81.93%, with protease P being the highest, followed by protease X, and protease N being the lowest; the hydroxyl free radical scavenging rate ranged from 55.42% to 80.77%, with flavor protease being the highest, followed by protease P, and papain being the lowest. The DPPH radical scavenging rate of goat milk hydrolysate of 8 proteases was not high, and DPPH radical scavenging rate is the main indicator of antioxidant activity. Therefore, based on the DPPH radical scavenging rate and ACE inhibition rate, protease X (B), protease P (D), protease C (F) and protease N (H) were selected for compounding to improve the antioxidant capacity and ACE inhibition rate of goat milk hydrolysate.

[0032] 2.2 Composite protease screening and ratio optimization 2.2.1 Effects of protease combination on antioxidant capacity and ACE inhibition rate of goat milk enzymatic hydrolysate Protease X (B), Protease P (D), Protease C (F) and Protease N (H) were compounded, with the total addition amount of 2‰ (w / v). When two enzymes were compounded, each accounted for 1‰ (w / v), when three enzymes were compounded, each accounted for 1 / 3, and when four enzymes were compounded, each accounted for 0.5‰ (w / v). The enzymes were hydrolyzed at 50℃ for 3h, then boiled to inactivate the enzymes and centrifuged to measure the antioxidant capacity and ACE inhibition rate. The results are as follows: Figure 3~Figure 4 shown.

[0033] Depend on Figure 3It can be seen that the DPPH free radical scavenging rate of goat milk hydrolyzed by the combination of four proteases ranged from 36.38% to 65.87%, and the maximum value was significantly higher than that of goat milk hydrolyzed by a single enzyme, indicating that the use of a composite protease can improve the DPPH free radical scavenging rate of goat milk hydrolysate. Among them, the goat milk hydrolysate of the combination of protease P and protease N had the highest DPPH, while the goat milk hydrolysate of the combination of three enzymes and four enzymes was lower than that of the single enzyme, indicating that the more enzyme types are not necessarily better when the protease is compounded. Too many enzyme types may cause the active peptides to be over-hydrolyzed and lose their activity. The ACE inhibition rate of goat milk hydrolyzed by the composite protease ranged from 64.3% to 81.33%. The most active protease combination was protease X (B), protease C (F) and protease N (H), followed by the combination of protease P and protease N. Depend on Figure 4 It can be seen that the ferrous ion chelation rate of goat milk hydrolyzed by the four proteases ranged from 79.50% to 88.35%, and the hydroxyl free radical scavenging rate ranged from 57.66% to 81.93%.

[0034] Taking into account the antioxidant properties and ACE inhibition rate, the proteinase P and proteinase N combination enzymes were selected for subsequent experiments.

[0035] 2.2.2 Effect of the ratio of protease P and protease N on DPPH free radical scavenging rate and ACE inhibition rate of goat milk enzymatic hydrolysate Protease P (D) and protease N (H) were compounded, and the total addition amount was 2‰ (w / v). The effects of the two enzyme ratios of 3:1, 2:1, 1:1, 1:2 and 1:3 on the DPPH free radical scavenging rate and ACE inhibition rate of goat milk enzymatic hydrolysate were studied. The enzymatic hydrolysis was carried out at 50℃ for 3h, and then the enzyme was boiled and inactivated, and the antioxidant and ACE inhibition rates were determined by centrifugation. The results are shown in Table 1. Figure 5 shown.

[0036] Depend on Figure 5 It can be seen that the ratio of protease P (D) and protease N (H) has a significant effect on the DPPH free radical scavenging rate and ACE inhibition rate of goat milk. The DPPH free radical scavenging rate gradually increased from 46.34% to 77.99%, while the ACE inhibition rate changed little, indicating that protease N is more conducive to providing DPPH free radical scavenging rate. With the goal of DPPH free radical scavenging rate ≥ 65% and ACE inhibition rate ≥ 75%, the appropriate ratio of protease P and protease N is 1:1~1:3.

[0037] 2.3 Effects of enzymatic hydrolysis conditions on DPPH free radical scavenging rate and ACE inhibitory activity of goat milk enzymatic hydrolysate The ratio of protease P to protease N was 1:3, and the effects of enzymatic hydrolysis temperature (40℃, 45℃, 50℃, 55℃ and 60℃), time (2h, 2.5h, 3.0h, 3.5h, 4h) and enzyme addition amount (1‰, 2‰, 3‰, 4‰ and 5‰) on the ACE inhibition rate and anti-DPPH free radical scavenging rate of goat milk enzymatic hydrolysate were studied. Figures 6 to 8 shown.

[0038] Depend on Figure 6 It can be seen that with the increase of enzymatic hydrolysis temperature, the DPPH free radical scavenging rate and ACE inhibition rate of goat milk hydrolysate first increased and then decreased, both reaching the maximum value at 55℃, indicating that the optimal temperature for the enzymatic hydrolysis of the composite protease in goat milk is 55℃. Too high temperature will cause enzyme denaturation and inactivation, thereby reducing the enzymatic hydrolysis efficiency. With the goal of DPPH free radical scavenging rate ≥65% and ACE inhibition rate ≥75%, the appropriate enzymatic hydrolysis temperature is 50℃~60℃.

[0039] Depend on Figure 7 It can be seen that with the extension of enzymatic hydrolysis time, the DPPH free radical scavenging rate and ACE inhibition of goat milk enzymatic hydrolysate first increased and then tended to remain unchanged. The activity increased rapidly in the first 3.5 hours and then increased slowly. With the DPPH free radical scavenging rate ≥65% and the ACE inhibition rate ≥75% as the goal, the appropriate time is 2.5~3.5 hours.

[0040] Depend on Figure 8 It can be seen that with the increase of compound protease dosage, the DPPH free radical scavenging rate and ACE inhibition of goat milk enzymatic hydrolysate first increased and then tended to remain unchanged. If the enzyme dosage is too small, the protein in the goat milk will not be bound by the enzyme, resulting in low activity. If the enzyme dosage is too large, the binding sites on the protein in the goat milk will be saturated, which will not help to improve its activity. With the goal of DPPH free radical scavenging rate ≥65% and ACE inhibition rate ≥75%, and considering the cost, the appropriate enzyme addition amount is selected to be 2.0‰~4‰.

[0041] 3. Preparation of bioactive peptide powder and peptide spectrum identification 4‰ composite protease (protease P and protease N ratio of 1:3) was added to sterilized and cooled goat milk, and enzymolysis was carried out at 55℃ for 3.5h. Then, the goat milk enzymolysis solution was ultrafiltrated and separated with ultrafiltration membranes with molecular weight cutoffs of 5K and 2K to obtain three components (I:>5k Da, II:2~5k Da and III:<2k Da). As shown in Table 2, the ACE inhibition rate and DPPH free radical scavenging rate of the peptides were closely related to their relative molecular weight. The ACE inhibition rate and DPPH scavenging rate increased with the decrease of molecular weight. Component III had the best antihypertensive activity and antioxidant activity, with an ACE inhibition rate of 89.75±1.79% and a DPPH free radical scavenging rate of 84.65±1.82%. The above results showed that the activity of the <2k Da peptide component was improved after ultrafiltration, and it had strong ACE inhibition activity and antioxidant activity, so the enzymolysis solution component less than 2k Da was selected for further separation.

[0042] Table 2 ACE inhibitory activity and antioxidant activity of each ultrafiltration fraction

[0043] Fraction III was further separated and purified by preparative chromatography ( Fig. 9 ), four components were obtained (I was the collected solution from 12min to 32min, II was the collected solution from 32min to 41min, III was the collected solution from 41min to 49min, and IV was the collected solution from 49min to 58min), which were freeze-dried and dissolved in pure water to prepare different concentrations, and their IC 50 The results show that component II has the strongest ACE inhibitory activity and antioxidant activity, and its ACE inhibitory activity IC 50 The IC value of DPPH free radical scavenging rate is 0.28mg / mL. 50 The value is 0.36mg / mL.

[0044] Table 3 IC values ​​of ACE inhibition rate and DPPH free radical scavenging rate of each component collected by preparative chromatography 50 value

[0045] The peptide solution obtained by dissolving the bioactive peptide powder in pure water was identified by HPLC-MS / MS, and 13 peptides were obtained as shown in Table 4. Figures 10 to 22 shown.

[0046] Table 4 Amino acid sequence of bioactive peptide powder and its position in goat milk protein

[0047] From Table 4, Figures 10 to 22It can be seen that the bioactive peptide powder of component II contains 13 peptides, the number of amino acids ranges from 6 to 12, and the molecular weight ranges from 575.34Da to 1444.75Da, which mainly come from β-lactoglobulin, αs1-casein, αs2-casein, β-casein and κ-casein in goat milk.

[0048] 4. Preparation of antihypertensive and antioxidant goat milk powder 4.1 Dry preparation The component II obtained by preparative chromatography separation is freeze-dried to obtain 0.5-1.0g of bioactive peptide powder, which is then mixed with 2g-10g of plant sterol ester, 0.5g-1.5g of phosphatidylserine, 0.2g-1.5g of probiotic powder, and 10g-45g of prebiotics, a nutritional enhancer required by middle-aged and elderly people (calculated based on 1kg of antihypertensive and antioxidant goat milk powder), and then added to the goat milk powder, and mixed evenly with a three-dimensional mixer to obtain antihypertensive and antioxidant goat milk powder; 4.2 Wet preparation To obtain 1kg of blood pressure lowering and antioxidant goat milk powder, it is necessary to add 2g~10g of plant sterol esters, 0.5g~1.5g of phosphatidylserine, and 10g~45g of prebiotics to 7.55kg~7.90kg of goat milk enzymatic hydrolysate, and then concentrate it to a solid content of 45%~52% and then spray dry it (spray drying conditions are: 130℃~150℃, air outlet temperature 75℃~85℃, and liquid flow rate 4mL / min~5mL / min) to obtain the blood pressure lowering and antioxidant goat milk powder.

[0049] The calculation method of the relevant parameters of the preparation method of the above-mentioned bioactive peptide powder is as follows: 1. Antioxidant activity - DPPH free radical scavenging rate determination Dissolve DPPH in 95% ethanol to prepare a solution with a concentration of 0.1 mmol / L. Mix 1 mL of the sample solution to be tested with 5 mL of DPPH free radical solution as the test group, 1 mL of the ethanol solution with 5 mL of DPPH free radical solution as the control group, and 1 mL of the sample solution to be tested with 5 mL of ethanol solution as the blank group. Keep each group away from light for 30 min, measure the absorbance at a wavelength of 517 nm, and calculate the DPPH scavenging rate according to the following formula.

[0050] (1) Where: A i For the experimental group, A j A0 was the blank group and A1 was the control group.

[0051] 2. Antioxidant activity - determination of ferrous ion chelation rate 0.5 mL of the sample solution to be tested was placed in a test tube, and 1 mL of 0.02 mmol / L ferrous chloride solution and 0.5 mmol / L phenanthroline solution were added in sequence. After being fully mixed on a vortex mixer, the mixture was placed at room temperature for 10 min, and the absorbance value A1 was measured at a wavelength of 562 nm. Distilled water was used as a control group, and the absorbance value measured was A2. The calculation formula is as follows: Chelation rate (%) = (2) Where: A1 is the absorbance value of the sample group; A2 is the absorbance value of the control group.

[0052] 3. Antioxidant activity - determination of hydroxyl radical scavenging rate Mix 0.5 mL of sample solution with 1 mL of 9 mmol / L FeSO4 solution and 1 mL of 10 mmol / L H2O2 solution, place in a 37℃ water bath for 10 min, then mix with 1 mL of 9 mmol / L salicylic acid solution, place in a 37℃ water bath for 30 min, and measure the absorbance at 510 nm. Calculate as follows: (3) Where: A i is the absorbance value of the control group; A j is the absorbance value of the sample solution group.

[0053] 4. In vitro determination of ACE inhibitory activity Table 5 ACE inhibition rate detection method

[0054] The sample addition reaction was carried out in the order shown in Table 5. Finally, 1.7 mL of ethyl acetate was added and shaken for 15 seconds. After stratification, 1 mL of the ethyl acetate layer was taken into a test tube and evaporated in an oven at 100°C. After cooling to room temperature, 3 mL of deionized water was added. After sufficient dissolution, the absorbance was measured at a wavelength of 228 nm. The ACE inhibition rate was calculated according to the following formula: ACE inhibition rate (%) = (4) Where: A is the reaction of sample with ACE and HHL at the same time; B is the reaction of ACE with HHL; C is the blank control.

Claims

1. A bioactive peptide powder, characterized in that: The ACE inhibitory activity IC of the peptide powder 50 The IC value of DPPH free radical scavenging rate was 0.28±0.012 mg / mL. 50 The value is 0.36±0.015 mg / mL. The peptide powder includes 13 bioactive peptides with antihypertensive and antioxidant effects. The amino acid sequences of the 13 bioactive peptides are shown in SEQ ID NO: 1 to SEQ ID NO:

13.

2. A method for preparing a bioactive peptide powder, characterized in that: The specific steps are as follows: Calculated by mass fraction, 2‰~4‰ of the composite protease was added to the sterilized and cooled goat milk; The goat milk with the compound protease added is subjected to constant temperature enzymolysis at 50°C to 60°C for 2.5h to 3.5h, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain the goat milk enzymolysis solution; The goat milk enzymatic hydrolysate with a molecular weight Mw < 2k Da was freeze-dried to obtain a bioactive peptide powder with antihypertensive and antioxidant effects.

3. The method for preparing a bioactive peptide powder according to claim 2, characterized in that: The composite protease comprises protease P and protease N, the addition ratio of protease P and protease N is 1:1-1:3, protease P is Bacillus subtilis protease with an enzyme activity of 400,000 U / g; protease N is a neutral protease with an enzyme activity of 0.8 AU / g.

4. The method for preparing a bioactive peptide powder according to claim 2, characterized in that: The ACE inhibition rate of the goat milk enzymatic hydrolysate is 76.79±2.19%~89.28±1.42% and the DPPH free radical scavenging rate is 65.90±1.18%~82.68±1.96%.

5. The method for preparing a bioactive peptide powder according to claim 2, characterized in that: In the step of adding composite protease to goat milk and performing enzymolysis at 40°C to 60°C for 2.5h to 3.5h, inactivating the enzyme, centrifuging and taking the supernatant to obtain goat milk enzymolyzate: The enzyme is inactivated by keeping it in a boiling water bath for 10-15 minutes; The centrifugation speed is 8000r / min~10000r / min for 10min~15min.

6. The bioactive peptide powder prepared by the method for preparing a bioactive peptide powder according to any one of claims 2 to 5, characterized in that: The ACE inhibitory activity IC of the peptide powder 50 The concentration of 1 μg / mL of the peptide powder was 0.28±0.012 mg / mL, and the IC50 value of the DPPH free radical scavenging rate was 0.36±0.015 mg / mL. The peptide powder included 13 bioactive peptides with antihypertensive and antioxidant effects, and the amino acid sequences of the 13 bioactive peptides were shown in SEQ ID NO: 1 to SEQ ID NO:

13.

7. A blood pressure lowering and antioxidant goat milk powder, characterized in that: The total amount is 1 kg, and the raw materials include 0.5 g to 1.0 g of bioactive peptide powder, 2 g to 10 g of phytosterol esters, 0.5 g to 1.5 g of phosphatidylserine, 0.2 g to 1.5 g of probiotic powder, 10 g to 45 g of prebiotics, and the rest is goat milk powder. The above raw materials are mixed to obtain antihypertensive and antioxidant goat milk powder; Wherein, the bioactive peptide powder is the bioactive peptide powder according to claim 1 or the bioactive peptide powder according to claim 6.

8. The blood pressure reducing and antioxidant goat milk powder according to claim 7, characterized in that: The probiotics are one or more of Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus acidophilus, Bifidobacterium animalis subspecies lactis, Streptococcus salivarius thermophilus subspecies, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus helveticus, and the number of viable bacteria is ≥1.0×10 11 CFU / g; Prebiotics are one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin.

9. A blood pressure lowering and antioxidant goat milk powder, characterized in that: The raw materials include 7.55kg-7.90kg goat milk enzymatic hydrolysate, 2g-10g phytosterol ester, 0.5g-1.5g phosphatidylserine, and 10g-45g prebiotics. The raw materials are mixed and concentrated to a solid content of 45%-52%, and then spray-dried to obtain 1kg of antihypertensive and antioxidant goat milk powder; The goat milk enzymolysate is the goat milk enzymolysate described in claim 2, and the solid content of the goat milk enzymolysate is 12.5g / 100g.

10. The blood pressure reducing and antioxidant goat milk powder according to claim 9, characterized in that: Prebiotics are one or more of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose and inulin; The spray drying conditions are 130° C. to 150° C., an air outlet temperature of 75° C. to 85° C., and a liquid flow rate of 4 mL / min to 5 mL / min.

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