CN110923284A - Walnut nut flavor peptide and preparation method thereof - Google Patents
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Abstract
The invention relates to a walnut nut flavor peptide and a preparation method thereof. The method takes walnut meal as a raw material, and the walnut peptide is obtained by extracting walnut protein, preparing walnut polypeptide through enzymolysis, performing ultrafiltration and gel chromatography separation and purification, and freeze-drying. The amino acid sequence of the walnut nut flavor peptide is SKHEETTPR, QQRPGEHGQQ, EEAVILPSPK, SVETWLPLPR, LAGNPDDEFRQE. The walnut nut flavor peptide prepared by the method has obvious dose-effect relationship, generates obvious nut flavor by being added into walnut milk in a proportion of 0.04 percent, and fills up the blank of the research on the walnut nut flavor peptide in the prior art.
Description
Technical Field
The invention belongs to the technical field of food processing, relates to a food flavor peptide and a preparation method thereof, and particularly relates to a walnut nut flavor peptide and a preparation method and application thereof.
Background
The walnut is one of four nuts, is widely distributed in China, and the yield of the dry fruit reaches 384.55 ten thousand tons at present. The walnut has the nutritive value that the walnut is rich in high-quality walnut oil and antioxidant polyphenol, 18 kinds of amino acids are complete in protein amino acid composition, and the walnut has rich glutamic acid, aspartic acid and arginine with health care effects. The walnut meal is a byproduct of walnut oil extraction, and is usually treated as feed or low-value material, so that the great waste of the nutritional value is caused. The protein content of the walnut meal is more than 50 percent, the walnut meal is good walnut protein powder and a good raw material for preparing walnut protein polypeptide, the bioactive peptide and the flavor peptide are the research hotspots of protein peptide in recent years, in the study of the walnut protein peptide, the activity study is mainly performed, and the walnut nut flavor peptide is reported rarely. Research shows that the walnut protein and the polypeptide have certain functions of reducing blood sugar, resisting fatigue, reducing blood pressure, resisting tumor, improving exercise and cognitive ability and the like. Currently, in the study of walnut protein peptides, the study of activity is mainly carried out, and the study of walnut flavor peptides is rarely reported.
The flavor peptides are polypeptides with molecular weight less than 5000Da, and are classified into flavor peptides and flavor peptides according to flavor development difference. The flavor peptide is smelled flavor peptide, the taste peptide is tasted flavor peptide, and the flavor peptide can generate special flavor for food, improve the sensory quality of products and increase the purchasing desire of consumers. Although the walnut milk product has high nutritive value, the taste and flavor of the walnut milk product are flat and not favored by consumers.
Therefore, how to improve the sensory quality of the walnut milk product and enable more people to enjoy the nutrition of the walnut product by increasing the flavor of the walnut milk becomes a problem to be solved urgently by manufacturers.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a preparation method of walnut kernel flavor peptide, which takes walnut dregs as a raw material to prepare the walnut kernel flavor peptide, on one hand, the walnut dregs can be fully utilized, so that the development and utilization values of the walnut dregs are optimal and maximized, on the other hand, the flavor of walnut milk products can be improved, and the consumption confidence of consumers is improved.
The invention also aims to provide the walnut nut flavor peptide.
The third purpose of the invention is to provide a preparation method of walnut milk added with the walnut nut flavor peptide.
The fourth purpose of the invention is to provide the walnut milk with the nut flavor.
In order to achieve the purpose, the invention provides a preparation method of walnut nut flavor peptide, which comprises the following steps:
(1) extraction of walnut protein
a. Primary alkali extraction: adding water into the walnut meal after oil extraction according to the mass-volume ratio g/mL of 1:6-10 to prepare a suspension I, adjusting the pH to 8.0-8.5 by using a sodium carbonate solution with the mass percent of 10%, stirring and leaching for 150min at the water bath temperature of 50-60 ℃, and centrifuging for 10-15min at 4000r/min to separate a precipitate and a supernatant I;
b. secondary alkali extraction: adding water into the precipitate according to the mass-volume ratio g/mL of 1:4 to prepare a suspension II, adjusting the pH to 8.0-8.5 by using a sodium carbonate solution with the mass percent of 10%, stirring and leaching at the water bath temperature of 50-60 ℃ for 20-60min, centrifuging at 4000r/min for 10-15min, and taking a supernatant II;
most of protein is dissolved in the supernatant by the primary alkali extraction, but a small amount of residue still remains in the walnut meal precipitate, so the amount of the residual protein is small when the secondary alkali extraction is carried out, the secondary alkali extraction time is shorter than the primary alkali extraction, and the protein in the walnut meal is extracted as completely as possible by the two alkali extractions.
c. Mixing the supernatant I and the supernatant II, adding 10% hydrochloric acid solution to adjust pH to 5.0-5.5, standing in 50-60 deg.C water bath for 90-150min, centrifuging at 4000r/min for 10-15min, collecting precipitate, washing with water to neutrality, collecting the precipitate as walnut protein, and storing at-20 deg.C.
The pH value is adjusted to reach the isoelectric point of the walnut protein, the solubility of the walnut protein is low, and the walnut protein can be obtained by centrifugal precipitation.
(2) Preparation of walnut nut flavor peptide
Weighing walnut protein, preparing an aqueous solution with the mass volume concentration (g/mL) of 12%, adding compound protease with the mass of 0.67% of the walnut protein, adjusting the pH to 8.2 by using a 10% sodium carbonate solution, carrying out enzymolysis for 5.3h (5.30h is 318min), heating to 100 ℃ after the enzymolysis is finished, inactivating the enzyme for 20min, and refrigerating for later use.
(3) Separation and purification of walnut nut flavor peptide
Performing molecular interception on the walnut protein polypeptide hydrolysate by adopting ultrafiltration membranes with intercepted molecular weights of 5KDa and 1KDa to obtain components with molecular weights of 1-5KDa, separating the components with the molecular weights of 1-5KDa by adopting gel chromatography, collecting the components with elution time of 135-ion 163min, and obtaining the walnut nut flavor peptide containing SKHEETTPR, QQRPGEHGQQ, EEAVILPSPK, SVETWLPLPR, LAGNPDDEFRQE by using a high performance liquid mass spectrometry.
Further, the compound protease in the step (2) is papain, neutral protease and alkaline protease according to the following steps: neutral protease: alkaline protease ═ 1: 1:1, and the weight ratio is prepared.
Further, the molecular interception method in step (3) is: separating the walnut protein polypeptide enzymolysis liquid by using a 5KDa ultrafiltration membrane to obtain two components of 5KDa and 5KDa, and then carrying out ultrafiltration on the component of 5KDa by using a 1KDa ultrafiltration membrane to intercept the component with the molecular weight of 1-5 KDa.
The invention also provides the walnut nut flavor peptide prepared by the preparation method, and the amino acid sequence of the peptide is SKHEETTPR, QQRPGEHGQQ, EEAVILPSPK, SVETWLPLPR, LAGNPDDEFRQE.
The invention also provides a preparation method of the walnut milk with nut flavor, which comprises the steps of adding the walnut nut flavor peptide into walnut milk primary pulp in a mass volume ratio of 0.04%, simultaneously adding 1.3% of glucose and 2.7% of cane sugar in a mass volume ratio, homogenizing for 10min under 30MPa, heating to 121 ℃ by using a high-pressure sterilization pot, keeping for 1min, and naturally cooling to room temperature to obtain the walnut milk with nut flavor.
Furthermore, the preparation method of the walnut milk primary pulp comprises the following steps: soaking semen Juglandis for 6 hr, and peeling; pulping the peeled walnut kernels and water according to the mass ratio of 1:25 until no residue exists.
The invention also provides walnut milk with nut flavor, which is prepared by adopting the preparation method.
The invention is characterized in that:
the peach nut flavor peptide provided by the invention is subjected to single-factor screening on each enzymolysis condition such as walnut protein concentration, enzyme addition amount, enzymolysis temperature, enzymolysis pH, enzymolysis time and the like, and then the key enzymolysis process parameters are obtained through response surface design and optimization of enzymolysis process parameters on the basis. Because the peptide segments obtained by different enzymolysis conditions are different, the specific peach nut flavor peptide can be obtained by adopting the specific preparation method of the invention.
The invention has the beneficial effects that:
the invention provides walnut nut flavor peptide and a preparation method thereof. The walnut nut flavor peptide is prepared by using the compound protease, the optimal enzymolysis process is determined by taking the prepared walnut peptide milk flavor sensory score as a response value, and the obtained enzymolysis conditions can be more accurately used for preparing the walnut nut flavor peptide. The walnut nut flavor peptide is prepared by adopting ultrafiltration membranes with molecular weight cut-off of 5KDa and 1KDa, and convenient conditions are provided for obtaining purer walnut nut flavor peptide by next chromatographic separation.
Drawings
FIG. 1 is a diagram of sensory evaluation results of ultra-filtration separation components of walnut nut flavor peptides prepared by the preparation method of walnut nut flavor peptides provided by the invention.
FIG. 2 is a Sephadex G-25 gel chromatography separation and purification diagram of a component with the molecular weight of 1-5KDa of the walnut nut flavor peptide prepared by the preparation method of the walnut nut flavor peptide provided by the invention.
FIG. 3 is a diagram of sensory evaluation results of Sephadex G-25 separation fractions F1, F2, F3 and F4 of a 1-5kDa component of walnut nut flavor peptide prepared by the preparation method of walnut nut flavor peptide provided by the invention.
Detailed Description
The following detailed and complete description of the embodiments of the present invention is provided to enable those skilled in the art to more easily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.
Example 1 preparation of walnut nut flavor peptides
(1) Extracting walnut protein: the extraction of walnut protein adopts an alkali dissolution and acid precipitation method.
a. Primary alkali extraction: preparing the walnut meal powder after oil extraction into a suspension I according to the material-liquid ratio g/mL of 1:8, adjusting the pH to 8.0 by using a sodium carbonate solution with the mass percent of 10%, stirring and leaching for 120min at the water bath temperature of 55 ℃, and centrifuging for 10min at 4000r/min to separate a precipitate and a supernatant I.
b. Secondary alkali extraction: preparing a precipitate into a suspension II according to the material-liquid ratio g/mL of 1:4, adjusting the pH to 8.0 by using a sodium carbonate solution with the mass percent of 10%, stirring and leaching for 30min at the water bath temperature of 55 ℃, and centrifuging for 10min at 4000r/min to obtain a supernatant.
c. Mixing the supernatant I and the supernatant II, adding 10% hydrochloric acid solution to adjust pH to 5.2, standing in 55 deg.C water bath for 120min, centrifuging at 4000r/min for 10min, collecting precipitate, washing with water to neutrality to obtain walnut protein, and storing at-20 deg.C;
(2) preparation of walnut nut flavor peptide
Weighing walnut protein, preparing into an aqueous solution with the mass concentration of 12%, adding compound protease with the mass of 0.67% of the walnut protein, adjusting the pH to 8.2 by using 10% sodium carbonate solution, performing enzymolysis for 5.3h, heating to 100 ℃ after the enzymolysis is finished, inactivating the enzyme for 20min to obtain walnut protein polypeptide hydrolysate, and refrigerating for later use;
wherein, the compound protease is papain, neutral protease and alkaline protease according to the following ratio: neutral protease: alkaline protease ═ 1: 1:1, and the weight ratio is prepared.
(3) Separation and purification of walnut nut flavor peptide
Preparing walnut nut flavor peptide by using ultrafiltration membranes with cut-off molecular weights of 5KDa and 1KDa, separating the walnut protein polypeptide enzymolysis liquid by using the ultrafiltration membranes with 5KDa under the conditions of 25 ℃ and 0.3MPa to obtain two components of 5KDa and 5KDa, performing ultrafiltration on the component of 5KDa by using the ultrafiltration membranes with 1KDa, cutting off the components with molecular weights of 1KDa to 5KDa and 1KDa, and finally dividing the walnut protein polypeptide enzymolysis liquid into three components of 1KDa, 1KDa to 5KDa and 5 KDa.
Freeze drying the above components to obtain peptide powder, and storing at-20 deg.C.
(4) Walnut nut flavor peptide sensory evaluation
In order to evaluate the flavor characteristics of different components of the walnut protein polypeptide enzymatic hydrolysate, prepared peptide powder with different components is added into walnut milk, and the walnut milk is subjected to sensory evaluation.
The walnut milk process comprises the following steps: soaking semen Juglandis for 6 hr, and peeling; pulping the peeled walnut kernels and water according to the mass ratio of 1:25 until no residue exists to prepare the walnut milk protoplasm.
The flavor peptide is a peptide segment obtained by enzymolysis of walnut protein through plant protein hydrolase, and the directly pulped protein in the walnut kernel is not subjected to enzymolysis and does not contain walnut nut flavor peptide. Only adding flavor peptide obtained by enzymolysis into walnut milk, adding sweetener according to the formula shown in table 1, homogenizing under 30MPa for 10min, heating at 121 deg.C for 1min, naturally cooling to room temperature, and performing sensory evaluation. The walnut milk added with the flavor peptide has nut fragrance, and the walnut milk not added with the flavor peptide has no nut fragrance.
TABLE 1 walnut milk formula
Note: the addition amount (%) was calculated as 100% of 100mL of walnut milk.
And sensory evaluation is carried out by adopting a grading method, and the color, the flavor, the tissue state and the taste of the walnut milk with the nut flavor added with the peptide components with different sizes are taken as evaluation indexes. Please refer to 10 food professionals with relevant expertise and experience and trained in sensory evaluation to form a sensory evaluation panel (male-female ratio 1:1), score according to the fine rules of sensory score, and take the average value as the final sensory score (percent).
TABLE 2 sensory evaluation method for flavor of walnut milk and nuts
The final sensory evaluation results are shown in fig. 1, and it can be seen that the sensory evaluation score of the 1-5KDa component is the highest, indicating that the component with the strongest nut flavor is the 1-5KDa component.
(5) Separating the fraction with molecular weight of 1-5KDa by gel chromatography:
the method comprises the following steps: preparing a sample with the concentration of 40mg/mL by using purified water for peptide powder with the molecular weight of 1-5KDa component, and performing gel chromatography separation by using Sephadex G-25, wherein a glass chromatographic column is 26mm multiplied by 50cm, a chromatographic filler Sephadex G-25, mobile phase pure water is used, the sample loading amount is 2.0mL, and the flow rate of eluent deionized water is 1.0 mL/min. Four components F1, F2, F3 and F4 are obtained by elution, and the results are shown in figure 2.
Purifying the four components, freeze-drying to obtain peptide powder, preparing into walnut milk respectively containing F1, F2, F3 and F4 according to the mixture ratio of the table 1 and the preparation method of the walnut milk in the step (4), and carrying out sensory evaluation according to the sensory evaluation standards of the table 2, wherein the evaluation result is shown in a figure 3. As can be seen from fig. 3, among the four components F1, F2, F3 and F4, the component F3 with the elution time in the period of 135-163min scored the highest, indicating that the component with the strongest nut flavor was the component F3.
(6) And (3) carrying out high performance liquid mass spectrometry on the F3 component to determine the sequence.
The polypeptide structure was determined using a liquid high performance liquid mass spectrometer (LC-MS/MS) instrument from Thermo corporation, USA. Methanol and water are used as mobile phases of a system, a F3 component polypeptide powder sample is dissolved in ultrapure water according to the concentration required by the specification of equipment, and the solution is filtered through a 0.22 mu m microporous filter membrane and then is loaded with 2.0 mu L. Mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: 80% acetonitrile and 0.1% formic acid; flow rate: 0.3 uL/min. The gradient elution conditions are shown in table 3.
TABLE 3 gradient elution conditions
Mass spectrum conditions: the Q active mass spectrometer is operated in a data-dependent acquisition technology mode by using Xcalibur 2.1.2 software, and the mass spectrum full-scan range is as follows: 300-1800 m/z, the resolution is 70000, 20 times of MS/MS related data are collected after full scanning, and the standardized collision energy (HCD) is 27%.
A group of peptides with high amino acid sequence matching degree in the F3 component can be obtained by high performance liquid mass spectrometry combined determination of the sequence, and the polypeptide sequence structures are respectively: SKHEETTPR (Seq ID No.1), QQRPGEHGQQ (Seq ID No.1), EEAVILPSPK (Seq ID No.3), SVETWLPLPR (Seq ID No.4), LAGNPDDEFRQE (Seq ID No. 5). The small molecular peptide consisting of the 5 amino acids of 7-12 brings the most intense nut flavor to the walnut milk, and the walnut nut flavor peptide is formed.
When the walnut nut flavor peptide is added into walnut milk in a proportion of 0.04%, the walnut nut flavor peptide can generate obvious nut flavor and is used for improving the flavor and taste of the walnut milk.
The embodiments can show that the walnut kernel flavor peptide prepared by the method can utilize the nutritional value of oil-extracted walnut meal to improve the development and utilization value of the walnut meal, improve the mouthfeel of walnut milk products, enrich the product taste and improve the consumption desire of consumers.
SEQUENCE LISTING
<110> Beijing Union university
<120> walnut nut flavor peptide and preparation method thereof
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Ser Lys His Glu Glu Thr Thr Pro Arg
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Leu Ala Gly Asn Pro Asp Asp Glu Phe Arg Gln Glu
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Claims (7)
1. A preparation method of walnut and nut flavor peptide is characterized by comprising the following steps:
(1) extraction of walnut protein
a. Primary alkali extraction: adding water into the walnut meal powder after oil extraction according to the mass volume ratio g/mL of 1:6-10 to prepare a suspension I, adjusting the pH to 8.0-8.5 by using a 10% sodium carbonate solution, carrying out stirring extraction at the water bath temperature of 50-60 ℃ for 150min, and centrifuging at 4000r/min for 10-15min to obtain a precipitate and a supernatant I;
b. secondary alkali extraction: adding water into the precipitate according to the mass-volume ratio of 1:4 g/mL to prepare a suspension II, adjusting the pH to 8.0-8.5 by using a 10% sodium carbonate solution, stirring and leaching at the water bath temperature of 50-60 ℃ for 20-60min, and centrifuging at 4000r/min for 10-15min to obtain a supernatant II;
c. mixing the supernatant I and the supernatant II, adjusting pH to 5.0-5.5 with 10% hydrochloric acid solution, standing in 50-60 deg.C water bath for 90-150min, centrifuging at 4000r/min for 10-15min, collecting precipitate, washing with water to neutrality, collecting the precipitate as walnut protein, and storing at-20 deg.C;
(2) preparation of walnut nut flavor peptide
Weighing walnut protein, preparing into an aqueous solution with the mass concentration of 12%, adding compound protease with the mass of 0.67% of the walnut protein, adjusting the pH to 8.0-8.5 by using 10% sodium carbonate solution, carrying out enzymolysis for 5.3h, heating to 100 ℃ after the enzymolysis is finished, inactivating enzyme for 20min to obtain walnut protein polypeptide hydrolysate, and refrigerating for later use;
(3) separation and purification of walnut nut flavor peptide
Performing molecular interception on the walnut protein polypeptide hydrolysate by adopting ultrafiltration membranes with intercepted molecular weights of 5KDa and 1KDa to obtain components with molecular weights of 1-5KDa, separating the components with the molecular weights of 1-5KDa by adopting gel chromatography, collecting the components with elution time of 135-ion and 163min, obtaining the walnut nut flavor peptide containing SKHEETTPR, QQRPGEHGQQ, EEAVILPSPK, SVETWLPLPR, LAGNPDDEFRQE by using high performance liquid mass spectrometry, and performing vacuum concentration at 60 ℃ and freeze drying to obtain peptide powder.
2. The method for preparing walnut nut flavor peptide according to claim 1, wherein the compound protease in the step (2) is papain, neutral protease and alkaline protease according to the following steps: neutral protease: alkaline protease ═ 1: 1:1, and the weight ratio is prepared.
3. The method for preparing walnut nut flavor peptide according to claim 1, wherein the molecular entrapment method in step (3) is: separating the walnut protein polypeptide enzymolysis liquid by using a 5KDa ultrafiltration membrane to obtain two components of 5KDa and 5KDa, and then carrying out ultrafiltration on the component of 5KDa by using a 1KDa ultrafiltration membrane to intercept the component with the molecular weight of 1-5 KDa.
4. A walnut flavor peptide produced by the production method according to any one of claims 1 to 3, characterized in that: the amino acid sequence of the peptide is SKHEETTPR, QQRPGEHGQQ, EEAVILPSPK, SVETWLPLPR, LAGNPDDEFRQE.
5. A preparation method of walnut milk with nut flavor is characterized in that the walnut nut flavor peptide of any one of claims 1 to 4 is added into walnut milk primary pulp in a mass volume ratio of 0.04%, and simultaneously added with glucose in a mass volume ratio of 1.3% and sucrose in a mass volume ratio of 2.7%, homogenized for 10min under 30MPa, heated to 121 ℃ by using an autoclave, kept for 1min, and naturally cooled to room temperature to obtain the walnut milk with nut flavor.
6. The nut-flavored walnut milk according to claim 5, wherein the preparation method of the walnut milk puree comprises the following steps: soaking semen Juglandis for 6 hr, and peeling; pulping the peeled walnut kernels and water according to the mass ratio of 1:25 until no residue exists.
7. Walnut milk with a nut flavor, characterized in that it is produced by the method of preparation according to claim 5 or 6.
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| CN114606283B (en) * | 2021-12-17 | 2023-08-15 | 陕西师范大学 | Walnut polypeptide with good emulsifying property and preparation method thereof |
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