CN112075529A - Method for preparing antioxidant peptide powder by enzymolysis of whey protein powder and application of antioxidant peptide powder in milk tea powder - Google Patents

Method for preparing antioxidant peptide powder by enzymolysis of whey protein powder and application of antioxidant peptide powder in milk tea powder Download PDF

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CN112075529A
CN112075529A CN202010965757.XA CN202010965757A CN112075529A CN 112075529 A CN112075529 A CN 112075529A CN 202010965757 A CN202010965757 A CN 202010965757A CN 112075529 A CN112075529 A CN 112075529A
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powder
whey protein
enzymolysis
antioxidant peptide
antioxidant
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皇甫洁
邵志芳
裴疆森
栾春光
姜欣
王德良
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China National Research Institute of Food and Fermentation Industries
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/14—Tea preparations, e.g. using additives

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Abstract

The invention discloses a method for preparing antioxidant peptide powder by enzymolysis of whey protein powder and application of the antioxidant peptide powder in milk tea powder, wherein the method comprises the following steps: preparing a whey protein powder (WPC-80) aqueous solution, performing high-temperature denaturation, adding trypsin and bromelain for enzymolysis, and performing high-temperature enzyme deactivation after the enzymolysis is finished to obtain a whey protein powder enzymolysis solution; freeze-drying the lactalbumin hydrolysate to obtain lactalbumin antioxidant peptide powder, and applying the obtained lactalbumin antioxidant peptide powder to milk tea powder. According to the invention, the lactalbumin powder is subjected to compound enzymolysis by adopting trypsin and bromelain to obtain lactalbumin antioxidant peptide powder with small molecular weight and strong antioxidant activity, and the lactalbumin antioxidant peptide powder can improve the antioxidant activity of the milk tea powder.

Description

Method for preparing antioxidant peptide powder by enzymolysis of whey protein powder and application of antioxidant peptide powder in milk tea powder
Technical Field
The invention relates to a method for preparing antioxidant peptide powder by enzymolysis of whey protein powder and application thereof, and relates to the technical field of food.
Background
Whey generally refers to a by-product of cheese and casein production from cow milk, Whey Protein (WP) is one of the main components of whey, and WP is rich in various amino acids required by human body, is easy to digest and absorb by the body, has higher biological value, protein efficiency ratio and utilization ratio, and is one of the most comprehensive natural proteins available at present.
The WP product mainly includes Whey Protein Concentrate (WPC), Whey Protein Isolate (WPI), and the like. The WPC is prepared from whey by pretreating, ultrafiltering, and spray drying, and is divided into concentrated whey protein-50 (WPC-50), concentrated whey protein-80 (WPC-80), etc.
The milk-derived antioxidant is a natural antioxidant, has the function of scavenging free radicals, is related to the functions of body defense, immunoregulation, mineral absorption promotion and the like, is used as a natural milk-containing product, has extremely high nutritional value, and accords with the health concept of modern people when being applied to food. Based on this, the research on milk-derived antioxidants is receiving attention from researchers.
The most common method for preparing milk-derived antioxidant peptides is to carry out enzymolysis on milk-based raw materials by using one or more proteases in cooperation. Protease commonly used includes digestive tract protease, microbial protease, etc. The characteristics of high efficiency, mild reaction conditions, wide protease source and easy control of the reaction process are that the enzymolysis method is superior to other preparation methods, so the enzymolysis method is most widely applied when preparing the milk-derived antioxidant peptide.
The antioxidant active peptide has the function of scavenging free radicals, and can maintain the balance of free radicals in the organism when being taken properly, thereby reducing diseases caused by excessive free radicals. In addition, the milk-derived antioxidant can be absorbed by the body through the reverse gradient effect, the amino acid content is rich and balanced, the safety is high, no toxic or side effect exists, the animal-derived milk-based raw material for preparing the milk-derived antioxidant is wide in source and high in cost performance, the milk-derived antioxidant has great commercial value when being used for preparing health-care food with the antioxidant function, and meanwhile, the health-care food is very prospective.
Disclosure of Invention
The whey protein antioxidant peptide powder prepared by the method has low molecular weight and high clearance rate for DPPH free radicals and hydroxyl free radicals, and can improve the antioxidant activity of the traditional Mongolian milk tea powder.
The specific technical scheme is as follows:
a method for preparing antioxidant peptide powder by enzymolysis of whey protein powder comprises the following steps:
step (1): preparing a whey protein powder aqueous solution, performing high-temperature denaturation, adding trypsin and bromelain for enzymolysis, and performing high-temperature enzyme deactivation after the enzymolysis is finished to obtain a whey protein powder enzymolysis solution.
Step (2): and (4) carrying out vacuum freeze drying on the lactalbumin hydrolysate to obtain lactalbumin antioxidant peptide powder.
And (3): the lactalbumin antioxidant peptide powder is applied to milk tea powder.
The whey protein powder in the step (1) is concentrated whey protein powder with the mass fraction of 3g/100 mL, the preparation method of the aqueous solution comprises the steps of placing the whey protein powder in deionized water for homogenization for 30 s, and the high-temperature denaturation temperature is 95 ℃ and the time is 5 min.
In the step (1): the enzyme activity ranges of the trypsin and the bromelain are as follows: the activity of trypsin enzyme is more than or equal to 4000U/g, and the activity of bromelain enzyme is more than or equal to 1200000U/g.
Trypsin and bromelain have their optimum pH, and the spatial configuration of the protease is changed under the conditions of acid-base incompatibility, thereby leading to enzyme reduction or even inactivation. Preferably, in step (1), the enzymatic reaction pH is selected to be 6.0.
Each enzyme has an optimal temperature for exerting the activity, and when the temperature is lower than the optimal temperature, the enzyme activity is inhibited and cannot exert the activity; at temperatures above their optimum, the enzyme structure is destroyed and loses activity. Preferably, the enzymolysis reaction temperature in the step (1) is 50 ℃.
Preferably, the enzyme base ratio of the enzymatic reaction in the step (1) is selected to be 0.3%.
Preferably, the temperature of the whey protein powder enzymolysis liquid in the step (2) is selected to be-45 ℃.
Preferably, the ratio of the whey protein antioxidant peptide powder to the milk tea powder in the step (3) is 0.2: 1000.
Drawings
FIG. 1: influence of whey protein antioxidant peptide powder on sensory quality of milk tea powder
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1
A method for preparing antioxidant peptide powder by enzymolysis of whey protein powder comprises the following specific contents:
carrying out enzymolysis on whey protein powder by using compound protease: weighing a proper amount of whey protein powder, dissolving in deionized water to prepare a solution with a certain substrate concentration of 3% (W/V), cooling to 50 ℃ (keeping the temperature change of the solution not to exceed +/-1 ℃ in the whole enzymolysis process) after a water bath at 95 ℃ is carried out for 5 min, placing on a constant-temperature electromagnetic stirrer, adjusting the pH of the solution to 6.0 by using 0.5 mol/L HCL solution, adding trypsin and bromelain according to an enzyme-substrate ratio (W/W) of 0.25%, carrying out enzymolysis for 2.5 h, and adding 0.5 mol/L NaOH solution into the enzymolysis solution every 10 min in the reaction process to ensure that the pH change is not to exceed +/-0.05. And after enzymolysis is finished, putting the whey protein powder into boiling water for 10 min to inactivate enzyme, and cooling to room temperature to obtain the whey protein powder enzymolysis liquid.
And (3) carrying out vacuum freeze drying on the whey protein powder enzymolysis liquid to obtain whey protein antioxidant peptide powder: wiping with 75% alcohol, placing into tray and immersing in the enzymolysis solution, closing the hatch, and starting vacuum freeze drying process. And after the vacuum freeze drying process is finished, freeze-drying the enzymatic hydrolysate to be powder, closing the vacuum pump, opening the cabin door after the air pressure inside and outside the cabin is consistent, taking out the tray, filling the lactalbumin antioxidant peptide powder into the self-sealing bag, and storing the self-sealing bag at the temperature of minus 20 ℃ in a dark place.
The method for detecting the molecular weight distribution of the lactalbumin antioxidant peptide powder comprises the following specific contents:
high performance liquid gel chromatography (HPLC) was used: the column was TSKgel G2000 SWXL (300X 7.8 mm); the mobile phase composition is acetonitrile: water: trifluoroacetic acid = 45: 55: 0.1 (V/V); the flow rate is 0.5 mL/min; sampling amount is 10 muL; the detection wavelength is 220 nm; the column temperature was set at 30 ℃. Dissolving the sample in a mobile phase, passing through a membrane, performing gel filtration by using a high performance liquid chromatograph, detecting by using an ultraviolet detector, and processing data by using GPC software. With four peptide standards: and (3) making a relative molecular mass standard curve by using the amino acid.
The method for detecting the pH stability of the lactalbumin antioxidant peptide powder comprises the following specific contents: diluting whey protein antioxidant peptide powder with deionized water to a certain concentration, adjusting pH to 4.0, 6.0, 8.0, 10.0, 12.0 with 0.5M HCl or NaOH solution, water bathing at 40 deg.C for 0min, 30min, 60min, 90min, 120 min, 150min, and measuring antioxidant index (DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, total antioxidant capacity).
The method for detecting the temperature stability of the lactalbumin antioxidant peptide powder comprises the following specific contents: diluting whey protein antioxidant peptide powder with deionized water to a certain concentration, adjusting pH to 6.0, respectively placing in water bath at 4 deg.C, 25 deg.C, 40 deg.C, 60 deg.C, 80 deg.C, and measuring antioxidant index after 0min, 30min, 60min, 90min, 120 min, and 150 min.
The determination method of DPPH free radical clearance, hydroxyl free radical clearance, lipid oxidation inhibition rate and molecular weight distribution related to the lactalbumin antioxidant peptide powder comprises the following steps:
(1) determination of DPPH radical scavenging Rate:
Figure DEST_PATH_IMAGE001
mixing 2 mL sample with equal volume of DPPH anhydrous ethanol solution (0.1 mmol/L), vigorously shaking to mix well, reacting at room temperature in the dark for 30min, and detecting its light absorption value A at 517 nm with ultraviolet spectrophotometeri。
Figure 292248DEST_PATH_IMAGE002
Mixing 2 mL of deionized water with an equal volume of DPPH anhydrous ethanol solution (0.1 mmol/L), vigorously shaking to mix uniformly, reacting at room temperature in the dark for 30min, and detecting the light absorption value A at 517 nm with an ultraviolet spectrophotometer0。
Figure DEST_PATH_IMAGE003
Mixing 2 mL of sample with anhydrous ethanol of the same volume, vigorously shaking to mix uniformly, reacting at room temperature in the dark for 30min, and detecting the light absorption value A at 517 nm with an ultraviolet spectrophotometerj。
Figure 148078DEST_PATH_IMAGE004
DPPH clearance was calculated using the following formula:
DPPH clearance (%) =
Figure DEST_PATH_IMAGE005
(2) Determination of hydroxyl radical clearance:
Figure 141441DEST_PATH_IMAGE001
2 mL of sample was taken and added to 2 mL of 9 mmol/L FeSO4Solution and 2 mL of H with a concentration of 10 mmol/L2O2Shaking the water solution, standing at 37 deg.C for 10 min, adding 2 mL salicylic acid solution with concentration of 9 mmol/L, mixing, standing at 37 deg.C for 30min, and detecting its light absorption value A at 510 nm with ultraviolet spectrophotometeri。
Figure 895771DEST_PATH_IMAGE002
2 mL of deionized water was added to 2 mL of 9 mmol/L FeSO4Solution and 2 mL of H with a concentration of 10 mmol/L2O2Shaking the water solution, standing at 37 deg.C for 10 min, adding 2 mL salicylic acid solution with concentration of 9 mmol/L, mixing, standing at 37 deg.C for 30min, and detecting its light absorption value A at 510 nm with ultraviolet spectrophotometer0。
Figure 570466DEST_PATH_IMAGE003
2 mL of sample was taken and added to 2 mL of 9 mmol/L FeSO4Solution and 2 mL of H with a concentration of 10 mmol/L2O2Shaking the water solution, standing at 37 deg.C for 10 min, adding 2 mL deionized water, mixing, standing at 37 deg.C for 30min, and detecting its light absorption value A at 510 nm with ultraviolet spectrophotometerj。
Figure 93851DEST_PATH_IMAGE004
The hydroxyl radical clearance was calculated using the following formula:
hydroxyl radical clearance (%) =
Figure 523695DEST_PATH_IMAGE005
(3) Lipid oxidation inhibition assay:
Figure 765321DEST_PATH_IMAGE001
250 mg of soybean lecithin and 250 mg of Tween 20 were weighed and dissolved in a 50 ml PBS (10 mmol/L, pH 7.4) solution, and sonication was carried out for 40 min to obtain a homogeneous lipid dispersion. The pipette accurately pipetted 2 mL of lipid dispersion into the tube and 1 mL of FeCl was added2Adding 2 mL of whey protein lyophilized powder water solution with a certain concentration into the solution (0.04 mol/L), placing the test tube in a water bath kettle at 37 deg.C, reacting in the dark for 1 h, taking out the test tube, adding 2 mL of 10% TCA solution, standing for 10 min, centrifuging at 4000 g for 10 min, and sucking 4 mL of supernatantAdding 2 mL of 0.375% TBA solution, water bathing at 100 deg.C for 15 min, rapidly cooling, and detecting the absorbance A at 532 nm with ultraviolet spectrophotometer1。
Figure 40444DEST_PATH_IMAGE002
250 mg of soybean lecithin and 250 mg of Tween 20 were weighed and dissolved in a 50 ml PBS (10 mmol/L, pH 7.4) solution, and sonication was carried out for 40 min to obtain a homogeneous lipid dispersion. The pipette accurately pipetted 2 mL of lipid dispersion into the tube and 1 mL of FeCl was added2Adding 2 mL of deionized water into the solution (0.04 mol/L), placing the test tube in a water bath kettle at 37 ℃, keeping out of the sun for reaction for 1 h, taking out the test tube, adding 2 mL of 10% TCA solution, standing for 10 min, centrifuging for 10 min at 4000 g, sucking 4 mL of supernatant, adding 2 mL of 0.375% TBA solution, carrying out water bath at 100 ℃ for 15 min, rapidly cooling, and detecting the light absorption value A under 532 nm by using an ultraviolet spectrophotometer0。
Lipid oxidation inhibition rate =
Figure 870866DEST_PATH_IMAGE006
%
(4) And (3) detecting the molecular weight distribution:
high performance liquid gel chromatography (HPLC) was used. The column was TSKgel G2000 SWXL (300X 7.8 mm); the mobile phase composition is acetonitrile: water: trifluoroacetic acid = 45: 55: 0.1 (V/V); the flow rate is 0.5 mL/min; sampling amount is 10 muL; the detection wavelength is 220 nm; the column temperature was set at 30 ℃. Dissolving the sample in a mobile phase, passing through a membrane, performing gel filtration by using a high performance liquid chromatograph, detecting by using an ultraviolet detector, and processing data by using GPC software. With four peptide standards: and (3) making a relative molecular mass standard curve by using the amino acid.
(5) And (3) detecting the pH stability of the whey protein antioxidant peptide powder:
diluting whey protein antioxidant peptide powder with deionized water to a certain concentration, adjusting pH to 4.0, 6.0, 8.0, 10.0, 12.0 with 0.5M HCl or NaOH solution, water bathing at 40 deg.C for 0min, 30min, 60min, 90min, 120 min, 150min, and measuring antioxidant index.
(6) And (3) detecting the temperature stability of the whey protein antioxidant peptide powder:
diluting whey protein lyophilized powder solution with deionized water to a certain concentration, adjusting pH to 6.0, respectively placing in water bath kettle at 4 deg.C, 25 deg.C, 40 deg.C, 60 deg.C, and 80 deg.C for 0min, 30min, 60min, 90min, 120 min, and 150min, and measuring antioxidant index.
The results of measurement of each index of the antioxidant peptide powder (concentration: 40 mg/mL) are shown in Table 1.
TABLE 1 detection results of various oxidation resistance indexes
Index of oxidation resistance DPPH radical clearance rate Hydroxyl radical scavenging rate Inhibition of lipid oxidation
Numerical value 94.14±1.23% 83.61±1.04 36.21±0.48%
The molecular weight detection results of the obtained antioxidant peptide powder (concentration: 40 mg/mL) small molecular peptide are shown in Table 2.
TABLE 2 molecular weight of whey protein antioxidant peptide powder
Molecular weight (Da) 10000 or more 5000-10000 3000-5000 2000-3000 1000-2000 150-1000 150 below
Ratio (%) 7.15±0.52 4.11±0.33 5.35±0.49 7.14±0.77 16.80±0.64 56.25±0.98 3.18±0.28
The obtained antioxidant peptide powder has the most stable antioxidant activity at pH 6.0. The pH value of the product is 8.0-12.0 in alkaline environment, and the antioxidant activity is gradually reduced along with the increase of the pH value. The antioxidant activity of the whey protein freeze-dried powder is obviously changed at different temperatures. Antioxidant activity of whey protein antioxidant peptide powder at 4 ℃ and 25 ℃Stable and its oxidation resistance does not change significantly at 40: (P>0.05), which shows that the antioxidant activity of the whey protein antioxidant peptide powder is stable at 40 ℃ and below 40 ℃.
Example 2
The whey protein antioxidant peptide powder prepared by the method in the embodiment 1 is applied to milk tea powder, and the specific contents are as follows:
adding whey protein antioxidant peptide powder into milk tea powder at a ratio of 0.2 g/kg (W/W), placing 5 g of the milk tea powder to be detected on parchment paper under sufficient sunlight or incandescent light, and observing the tissue state of the milk tea powder. According to the requirements of the packaging bag, adding 75 ℃ of warm water: milk tea powder =8:1, rinsed with clear water at room temperature and in the dark at 50 ℃, then smelled with nases, finally drunk once (about 5 mL), carefully tasted and swallowed.
Adding whey protein antioxidant peptide powder into milk tea powder at a ratio of 0.2 g/kg (W/W), accurately weighing 1.0 g of milk tea powder into a conical flask, adding 99 mL of deionized water, performing ultrasonic treatment until the milk tea powder is completely dissolved, performing centrifugation at 4000 r/min for 15 min, and detecting the antioxidant activity index of the supernatant.
The lactalbumin antioxidant peptide powder can be added into the milk tea powder to improve the antioxidant activity of the milk tea powder, wherein the DPPH free radical clearance rate is increased by 33.34%, and the hydroxyl free radical clearance rate is increased by 35.72%.
The sensory evaluation results of the milk tea powder before and after the whey protein antioxidant peptide powder is added are shown in table 3 and figure 1, and the results show that the milk flavor of the milk tea powder is more intense, the rice flavor is more pure and the taste is overall more mellow after the whey protein lyophilized powder is added.
TABLE 3 sensory evaluation Table
Figure DEST_PATH_IMAGE008A

Claims (10)

1. A method for preparing antioxidant peptide powder by enzymolysis of whey protein powder comprises the following steps:
(1) preparing a whey protein powder aqueous solution, performing high-temperature denaturation, adding trypsin and bromelain for enzymolysis, and performing high-temperature enzyme deactivation after the enzymolysis is finished to obtain a whey protein powder enzymolysis solution;
(2) carrying out vacuum freeze drying on the lactalbumin hydrolysate to obtain lactalbumin antioxidant peptide powder;
(3) the whey protein antioxidant peptide powder is applied to Mongolian traditional milk tea powder.
2. The method of claim 1 wherein the aqueous whey protein powder solution comprises 3% whey protein powder by weight.
3. The method according to claim 1, wherein in step (1), the mass ratio of trypsin to bromelain is 1: 1.
4. The method according to claim 1, wherein in the step (1), the mass ratio of the protease to the whey protein powder is 0.25: 100.
5. The method according to claim 1, wherein in the step (1), the temperature of the high-temperature denaturation is 95 ℃ and the time is 5 min.
6. The method of claim 1, wherein in step (1), the temperature of the enzymatic hydrolysis is 50 ℃.
7. The method of claim 1, wherein in step (1), the enzymatic hydrolysis is performed at a pH of 6.0 for a period of 2.5 hours.
8. The method of claim 1, wherein in step (2), the vacuum freeze-drying procedure is performed at a temperature of-45 ℃.
9. The method according to claim 1, wherein in the step (3), the mass ratio of the whey protein antioxidant peptide powder to the milk tea powder is 0.2: 1000.
10. Use of the method of claim 1 in milk tea powder.
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CN114958950A (en) * 2022-06-22 2022-08-30 内蒙古大学 Donkey milk antioxidant peptide and preparation method thereof

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Publication number Priority date Publication date Assignee Title
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CN114958950A (en) * 2022-06-22 2022-08-30 内蒙古大学 Donkey milk antioxidant peptide and preparation method thereof

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Application publication date: 20201215