CN108410938A - A method of preparing low bitter taste lactalbumin antioxidant peptide powder - Google Patents

Abstract

The invention discloses a kind of method preparing low bitter taste lactalbumin antioxidant peptide powder, this method includes：Aqueous whey protein solution is prepared, after high-temperature denatured, neutral proteinase is added and carries out primary enzymolysis, add ProteAX protease and carry out secondary enzymolysis, after enzymolysis, high temperature enzyme deactivation obtains lactalbumin enzymolysis liquid；By lactalbumin enzymolysis liquid successively through centrifuging with after hyperfiltration treatment, freeze-drying obtains lactalbumin antioxidant peptide powder.The present invention carries out two-stage enzymolysis using neutral proteinase and ProteAX protease to lactalbumin, obtains the lactalbumin antioxidant peptide powder that nitrogen recovery is high, molecular weight is small, antioxidant activity is strong and bitter taste is low.

Description

A method for preparing low-bitter whey protein antioxidant peptide powder

technical field

The invention relates to the technical field of dairy products, in particular to a method for preparing low-bitter whey protein antioxidant peptide powder.

Background technique

Milk and dairy products are one of the foods with the longest history in human diet. The rich protein they contain can not only provide rich nutrition, but also an important source of bioactive peptides. Whey protein is a general term for various protein components separated from cow milk after precipitation of casein. It used to be a by-product of industrial production of cheese and casein, accounting for about 20% of the total protein content of milk.

In recent years, more and more people have paid attention to the physiologically active peptides contained in milk and dairy products. The transport, absorption and utilization of small molecular peptides in the intestine are superior to monomeric amino acids, and their different amino acid arrangements can have different physiological activities, such as participating in immune regulation, scavenging free radicals, and regulating blood pressure balance in the body , antithrombotic and strengthen bone health. Among them, antioxidant active peptides are a relatively extensive research direction. Different antioxidant fragments have been identified in various milks and dairy products. Whey protein is rich in hydrophobic amino acids and basic amino acids. High-quality raw material of antioxidant peptides.

In recent years, more and more people pay attention to the physiologically active peptides contained in bovine whey protein. The transport, absorption and utilization of small molecular peptides in the intestine are superior to monomeric amino acids, and their different amino acid arrangements can have different physiological activities, such as participating in immune regulation, scavenging free radicals, and regulating blood pressure balance in the body , antithrombotic and strengthen bone health. Among them, antioxidant active peptides are a relatively extensive research direction. Different antioxidant fragments have been identified in various milks and dairy products. Whey protein is rich in hydrophobic amino acids and basic amino acids. High-quality raw material of antioxidant peptides.

Enzymatic hydrolysis is currently the most commonly used method for industrially preparing active peptides. This method has mild conditions, strong controllability, and stable products. However, during the enzymatic hydrolysis process, hydrophobic amino acids are exposed when they are broken from proteins into peptides, which often makes the peptides with hydrophobic amino acids at the C-terminus have a bitter taste, resulting in a decrease in the sensory quality of peptide products. In the industry, the addition of adsorbents or embedding agents is often used to remove or combine hydrophobic amino acids to reduce the bitterness, but these methods will cause the loss of essential amino acids, reduce the nutritional value of milk itself, and improve the finished product. The coordinated hydrolysis of endopeptidase and telopeptidase (aminopeptidase and carboxypeptidase) has a significant effect on reducing the bitterness of polypeptide products. Firstly, use the endoprotease whose cleavage site is a hydrophobic amino acid to cut the protein, release the small molecular peptide with a hydrophobic amino acid at the end, and then use aminopeptidase to cut off the hydrophobic amino acid at the C-terminal of the bitter peptide or use carboxypeptidase to cut off the bitter taste The hydrophobic amino acid at the N-terminal of the peptide achieves the effect of reducing bitterness. However, in the process of synergistic hydrolysis of endopeptidases, different proteases are suitable for different conditions, and the acid-base and temperature need to be constantly adjusted, and the desalination process needs to be added in actual production.

Therefore, it is very important for the active peptide industry to find a depicroprotease with strong hydrolysis ability and matching hydrolysis environment.

Contents of the invention

The invention provides a method for preparing low-bitter whey protein antioxidant peptide powder. The whey protein short peptide prepared by the method not only has high nitrogen recovery rate, low molecular weight, strong antioxidant activity, and is effective against DPPH free radicals and ABTS The free radical scavenging rate is high, and can significantly reduce the bitterness of the product, which is beneficial to the promotion of the product.

The specific technical scheme is as follows:

A method for preparing low-bitter whey protein antioxidant peptide powder, comprising:

(1) Prepare whey protein aqueous solution, after high temperature denaturation, add neutral protease to carry out enzymatic hydrolysis once, then add ProteAX protease to carry out second enzymatic hydrolysis, after enzymatic hydrolysis, high temperature inactivates the enzyme to obtain whey protein enzymatic hydrolyzate;

(2) The whey protein enzymatic hydrolyzate is subjected to centrifugation and ultrafiltration in sequence, and then freeze-dried to obtain whey protein antioxidant peptide powder.

The neutral protease used in the present invention is purchased from Guangbo Nanning Pangbo Biological Engineering Co., Ltd., derived from Bacillus subtilis, and the neutral protease activity is 200,000U/g; the used ProteAX protease is purchased from Japan Amano Enzyme Company, which is a kind of protease derived from Aminopeptidase extracted from Aspergillus oryzae by fermentation, the activity of exopeptidase is 1400U/g.

The whey protein used in the present invention is concentrated whey protein powder (WPC), the whey protein content is more than 80%, purchased from Fonterra Company of New Zealand; WPC90, WPC75 or WPC35 can also be used in specific operations.

Appropriate protein concentration can improve the efficiency of enzymatic hydrolysis and increase nitrogen recovery rate. Too low protein concentration will increase the production cost, and too high protein concentration is not conducive to efficient enzymatic hydrolysis by protease. Preferably, in the whey protein aqueous solution, the mass fraction of whey protein is 2-8%.

In order to ensure that the whey protein is fully dissolved in water, it is necessary to place the whey protein in water at 90°C to 100°C and stir for 10 to 20 minutes. This dissolution process is also a denaturation process. Preferably, in step (1), the temperature of the high-temperature denaturation is 90-100° C., and the time is 10-20 minutes. High temperature can destroy the three-dimensional structure of the protein and expose the peptide chain, which is conducive to protease digestion and significantly improves the efficiency of enzyme digestion.

Neutral protease is suitable for the natural pH value of the whey protein solution, and there is no need to adjust the pH of the solution with acid or alkali. The amount of protease added had an effect on the nitrogen recovery rate, the molecular weight and antioxidant properties of whey protein antioxidant peptides. Preferably, in step (1), the mass ratio of the neutral protease to whey protein is 1-3:100.

Different enzymatic hydrolysis conditions will obtain enzymatic hydrolyzates with different final pH, which will affect the effectiveness of the subsequent ProteAX protease. Therefore, suitable enzymatic hydrolysis conditions can obtain the most suitable enzymatic hydrolyzate for the subsequent ProteAX protease debittering process. Preferably, in step (1), the temperature of the primary enzymatic hydrolysis is 40-60° C., and the time is 2-5 hours. The enzymatic hydrolysis conditions can efficiently exert the protease activity and improve the yield of the polypeptide.

The amount of ProteAX protease added will affect the bitterness of whey protein antioxidant peptides. At the same time, ProteAX has a certain endopeptidase activity, which can further cut proteins and peptides, affecting nitrogen recovery rate, peptide molecular weight and antioxidant capacity. Preferably, in step (1), the mass ratio of ProteAX protease to whey protein is 1-3:100.

Preferably, in step (1), the temperature of the second enzymatic hydrolysis is 40-60° C., and the time is 2-5 hours. The enzymatic hydrolysis conditions can efficiently exert the protease activity, increase the yield of the polypeptide, and reduce the bitter taste of the polypeptide.

Preferably, in step (2), the centrifugation speed is 6000-8000r/min, and the centrifugation time is 10-20min; the polypeptide and insoluble protein can be separated as much as possible, and the yield of the polypeptide can be increased.

Preferably, in step (2), the cut-off of the ultrafiltration is 6000-10000 Da, and the pressure is 0.4-0.6 MPa. Ultrafiltration can remove a small amount of residual lipids and macromolecular proteins and peptides, effectively remove impurities, obtain clear and pure peptide liquid, and improve the antioxidant performance of the product.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention uses neutral protease and ProteAX protease to carry out two-stage enzymatic hydrolysis of whey protein to obtain whey protein antioxidant peptide powder with high nitrogen recovery rate, small molecular weight, strong antioxidant activity and low bitter taste.

(2) The method of the present invention does not need to repeatedly adjust pH and temperature, and only needs to kill enzymes in one step, which is not only green and energy-saving, but also reduces subsequent desalination steps, and is easy for industrial continuous scale production.

(3) The enzyme used in the method of the present invention has strong hydrolysis ability, high nitrogen recovery rate, small molecular weight of peptide powder, strong anti-oxidation ability and good sensory effect, and can be used alone as a functional food or as an additive in other foods.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the following enumerations are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

The assay method of DPPH free radical scavenging rate, ABTS free radical scavenging rate, nitrogen recovery rate and molecular weight distribution involved in the following examples is as follows:

(1) Determination of DPPH free radical scavenging rate:

①Put 0.1mL pure water and 1.9mL DPPH absolute ethanol solution (1mmoL/L) into a 5mL centrifuge tube respectively, shake well and react in the dark for 30min, then use a UV spectrophotometer to detect the absorbance value A _{c at} 515nm ;

②Put 0.1mL of the polypeptide solution to be tested (2mg/mL) and 1.9mL of DPPH absolute ethanol solution (1mmoL/L) into a 5mL centrifuge tube, shake well, react in the dark for 30min, and then use a UV spectrophotometer to detect its concentration. Absorbance value A _i at 515nm;

③The DPPH clearance rate is calculated by the following formula:

(2) Determination of ABTS free radical scavenging rate:

① Preparation of ABTS free radicals: Add 440 μL of potassium persulfate solution with a concentration of 140 mmoL/L to 25 mL of ABTS solution with a concentration of 7 mmol/L, mix well and react in the dark for 12 hours, then dilute to 100 mL to obtain an ABTS free radical reaction solution.

②Add 0.1mL pure water and 1.9mL ABTS reaction solution (2mmoL/L) into a 5mL centrifuge tube respectively, mix and shake for 10min, and then measure the absorbance value A ₀ at 734nm with a UV spectrophotometer;

③Add 0.1mL of the polypeptide solution to be tested (2mg/mL) and 1.9mL of the ABTS reaction solution to a 5mL centrifuge tube, mix well and react for 10min, then use a UV spectrophotometer to detect the absorbance value _A1 at 734nm;

④ ABTS clearance rate is calculated by the following formula:

(3) Determination of nitrogen recovery rate: N ₁ and N ₂ were determined for nitrogen percentages in whey protein and peptide powder respectively by Kjeldahl method.

Nitrogen recovery is calculated by the following formula:

(4) Determination of small molecular weight peptide content: use GE AKTA pure protein purification instrument, equipped with Superdex Peptide10/300GL chromatographic column, mobile phase is 30% (V/V) acetonitrile solution containing 0.1% (V/V) TFA, flow rate 0.5 mL/min, the detection wavelength is 215nm. Prepare a 2 mg/mL peptide solution, pass through a 0.22 μm water filter membrane and inject 100 μL of the sample. Molecular weight standards were selected from aprotinin (6511Da), bacitracin (1422Da), reduced glutathione (614Da) and tyrosine (181Da). The molecular weight distribution of peptide powder is in the range of 200-1500Da, which is a small molecular peptide, and the proportion of small molecular weight peptides is determined by using different molecular weight standards.

(5) Sensory evaluation: Peptide powder and 10 mg/mL peptide solution were used for sensory evaluation. Eight assessors (4 males and 4 females, aged 22-25 years old) with sensory evaluation basis were selected to judge the bitterness, color and solubility of peptide powder and peptide liquid according to the scoring table, in which the bitterness value was measured by different concentrations of tannins The solution is the evaluation standard, the 50mmol/L tannin solution is weakly bitter, the 100mmol/L tannin solution is obviously bitter, and the 200mmol/L tannin solution is strongly bitter. Summarize 8 ratings, remove the highest and lowest scores for each item and take the average to get the product score.

Table 1 Sensory Evaluation Form

Example 1

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein is 2:100, and the constant temperature enzymatic hydrolysis is 3.5h. After the first enzymatic hydrolysis, the enzyme will not be extinguished. Continue to add ProteAX protease. The mass ratio of ProteAX protease to whey protein is 1.5:100. The hydrolysis was carried out for 3.5 hours, and after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10 minutes to obtain the whey protein enzymatic hydrolysis solution.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Example 2

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 2%, stir at 90°C for 10 minutes to dissolve the whey protein, and when the temperature is lowered to 55°C, place it in a magnetic constant temperature enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein is 0.5:100, and the enzyme is hydrolyzed at constant temperature for 3 hours. 3h, after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10min to obtain the whey protein enzymatic hydrolyzate.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Example 3

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease hydrolysis: Take whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein is 1.5:100, and the enzyme is hydrolyzed at constant temperature for 4 hours. After the first enzymolysis, the enzyme is not extinguished, and ProteAX protease is added. The mass ratio of ProteAX protease to whey protein is 1:100, and the enzyme is hydrolyzed at constant temperature. 3h, after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10min to obtain the whey protein enzymatic hydrolyzate.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Example 4

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: Take an aqueous solution of whey protein with a mass fraction of 8%, stir at 90°C for 15 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein is 2.5:100, and the constant temperature enzymatic hydrolysis is 2.5h. After the first enzymatic hydrolysis, the enzyme will not be extinguished. Continue to add ProteAX protease. The mass ratio of ProteAX protease to whey protein is 1.5:100. After the hydrolysis for 2.5 hours, the enzymatic hydrolysis solution of whey protein was obtained by boiling at 95°C for 10 minutes after the two enzymatic hydrolysis.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 1

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein was 2:100, and the enzyme was hydrolyzed at a constant temperature for 3 hours. After the enzymolysis was completed, the enzyme was boiled at 95°C for 10 minutes to obtain a whey protein hydrolyzate.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 2

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add ProteAX protease, ProteAX protease The mass ratio of whey protein to whey protein is 2:100, and the enzyme is hydrolyzed at constant temperature for 3 hours. After the enzymolysis is completed, the enzyme is boiled at 95°C for 10 minutes to obtain whey protein hydrolyzate.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 3

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take whey protein aqueous solution with a mass fraction of 4%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add neutral protease, and The mass ratio of protease to whey protein is 1.5:100, and the enzyme is hydrolyzed at constant temperature for 3 hours. 3h, after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10min to obtain the whey protein enzymatic hydrolyzate. The flavor protease used was purchased from Guangbo Nanning Pangbo Bioengineering Co., Ltd., derived from the fermentation of Aspergillus oryzae, and contains two activities of endo-protease and exo-peptidase.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 4

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease hydrolysis: Take whey protein aqueous solution with a mass fraction of 4%, boil it at 90°C for 10 minutes, cool it to 50°C, put it in a magnetic constant temperature water bath, add 0.015g per 1g of whey protein The proportion of protease Add neutral protease, constant temperature enzymatic hydrolysis for 3 hours, do not extinguish the enzyme after one enzymatic hydrolysis, lower the temperature to 40°C, add Peptidase R protease at a ratio of 0.015g protease per 1g whey protein, keep 40°C constant temperature enzymatic hydrolysis After 3 hours, after the two enzymatic hydrolysis, the enzyme was boiled to obtain the whey protein enzymatic hydrolyzate. The Peptidase R protease used was purchased from Amano Enzyme Co., Ltd., Japan. It is an aminopeptidase derived from the fermentation and extraction of Rhizopus oryzae. The exopeptidase activity is 420 U/g.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 5

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: Take whey protein aqueous solution with a mass fraction of 4%, stir at 90°C for 10 minutes to dissolve whey protein, cool to 37°C, put it in a magnetic constant temperature water bath device, add whey protein per 1g Add trypsin at a ratio of 0.015g protease, and perform enzymatic hydrolysis at a constant temperature for 3 hours. After the first enzymatic hydrolysis, the enzyme will not be extinguished, and continue to add ProteAX protease at a ratio of 0.010g protease per 1g of whey protein, and keep the enzymatic hydrolysis at 37°C for 3 hours, twice. After the enzymolysis is completed, the enzyme is boiled to obtain a whey protein enzymatic hydrolysis solution. The trypsin used was purchased from Guangbo Nanning Pangbo Bioengineering Co., Ltd. and extracted and purified from porcine pancreas.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 6

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 4%, stir at 90°C for 10 minutes to dissolve the whey protein, and when the temperature is lowered to 50°C, place it in a magnetic constant temperature enzyme reactor, add alkaline protease, alkali The mass ratio of protease to whey protein is 1.5:100, and the enzyme is hydrolyzed at constant temperature for 3 hours. After the first enzymolysis, the enzyme is not extinguished, and ProteAX protease is added. The mass ratio of ProteAX protease to whey protein is 1.5:100, and the enzyme is hydrolyzed at constant temperature 3h, after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10min to obtain the whey protein enzymatic hydrolyzate. The alkaline protease used was purchased from Guangbo Nanning Pangbo Bioengineering Co., Ltd., and was fermented from Bacillus licheniformis 2709 bred by bacterial protoplast mutagenesis. The protease activity was 800,000 U/g.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 7

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: Take a whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic constant temperature enzyme reactor when the temperature is lowered to 50°C, and add neutral protease and ProteAX protease, the mass ratio of neutral protease and whey protein is 1.5:100, the mass ratio of ProteAX protease and whey protein is also 1.5:100, constant temperature enzymolysis for 4h, after enzymolysis, boil at 95°C for 10min to obtain Whey protein hydrolyzate.

(2) Preparation of peptide powder: After the enzymatic solution was centrifuged at 7000r/min for 15min, the supernatant was collected as a crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

Comparative example 8

A method for preparing low-bitter whey protein antioxidant peptide powder, the specific content is as follows:

(1) Protease enzymatic hydrolysis: take a whey protein aqueous solution with a mass fraction of 5%, stir at 90°C for 10 minutes to dissolve the whey protein, put it in a magnetic thermostatic enzyme reactor, add ProteAX protease, ProteAX protease The mass ratio of neutral protease to whey protein is 1.5:100, and the enzyme is hydrolyzed at constant temperature for 3 hours. 3h, after the two enzymatic hydrolysis, the enzyme was boiled at 95°C for 10min to obtain the whey protein enzymatic hydrolyzate.

(2) Preparation of peptide powder: After centrifuging the enzymatic solution at 7000r/min for 15min, the supernatant was collected as crude peptide solution, and the crude peptide solution was passed through an ultrafiltration membrane with a cut-off of 10kDa at a pressure of 0.5MPa to remove lipids , macromolecular proteins and peptides, and freeze-dried to obtain whey protein peptide powder.

The whey protein peptide powder products prepared in the above-mentioned Examples 1-4 and Comparative Examples 1-8 were tested for nitrogen recovery rate, small molecule peptide content, DPPH free radical scavenging rate and ABTS free radical scavenging rate, as well as bitterness, color and luster And solubility evaluation, the results are shown in Table 2.

The result contrast of table 2 different embodiment 1～4 and comparative example 1～8

Claims (9)

1. A method for preparing low-bitter whey protein antioxidant peptide powder, characterized in that, comprising: (1) Prepare whey protein aqueous solution, after denaturing at high temperature, add neutral protease for primary enzymolysis, then add ProteAX protease for secondary enzymatic hydrolysis, after enzymatic hydrolysis, high temperature inactivate the enzyme to obtain whey protein enzymatic hydrolysis; (2) The whey protein enzymatic hydrolyzate is subjected to centrifugation and ultrafiltration in sequence, and then freeze-dried to obtain whey protein antioxidant peptide powder. 2. The method according to claim 1, characterized in that, in the whey protein aqueous solution, the mass fraction of whey protein is 2-8%. 3. The method according to claim 1, characterized in that, in step (1), the mass ratio of the neutral protease to whey protein is 1-3:100. 4. The method according to claim 1, characterized in that, in step (1), the temperature for high-temperature enzyme inactivation is 90-100° C., and the time is 10-15 minutes. 5 . The method according to claim 1 , characterized in that, in step (1), the temperature of the first enzymolysis is 40-60° C. and the time is 2-5 hours. 6. The method according to claim 1, characterized in that, in step (1), the mass ratio of the ProteAX protease to whey protein is 1-3:100. 7. The method according to claim 1, characterized in that, in step (1), the temperature of the second enzymatic hydrolysis is 40-60° C., and the time is 2-5 hours. 8. The method according to claim 1, characterized in that, in step (2), the rotational speed of the centrifuge is 6000-8000r/min, and the time is 10-20min. 9. The method according to claim 1, characterized in that, in step (2), the cut-off of the ultrafiltration is 6000-10000 Da, and the pressure is 0.4-0.6 MPa.