CN112106994A - Walnut polypeptide nutrient solution and preparation method and application thereof - Google Patents

Walnut polypeptide nutrient solution and preparation method and application thereof Download PDF

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CN112106994A
CN112106994A CN202010961904.6A CN202010961904A CN112106994A CN 112106994 A CN112106994 A CN 112106994A CN 202010961904 A CN202010961904 A CN 202010961904A CN 112106994 A CN112106994 A CN 112106994A
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walnut
polypeptide
solution
nutrient solution
protein
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李玉锋
兰林
任昱至
张庆
钱叶迁
李宇航
谢玲
汪晟羽
张晋森
周冠伟
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Xihua University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • A23J3/14—Vegetable proteins
    • 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
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A23L29/37—Sugar alcohols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses a walnut polypeptide nutrient solution and a preparation method and application thereof, and the walnut polypeptide nutrient solution comprises: the walnut polypeptide powder, the walnut essence, the citric acid, the xylitol and the water are mixed according to a feed-liquid ratio of 5%, the addition amount of the walnut essence is 0.08%, the addition amount of the citric acid is 0.12% and the addition amount of the xylitol is 9%; the walnut polypeptide powder comprises the following polypeptide sequences: the amino acid sequence shown as SEQ ID NO. 1-16. The walnut polypeptide nutrient solution is obtained by processing the degreased walnut dregs and utilizing the walnut polypeptide powder, and has antithrombin activity and antioxidant activity.

Description

Walnut polypeptide nutrient solution and preparation method and application thereof
Technical Field
The invention relates to a polypeptide nutrient solution, in particular to a walnut polypeptide nutrient solution and a preparation method and application thereof.
Background
In order to solve the problem of heart-blood diseases at home and abroad, a lot of research and effort are made, research and development on antithrombotic medicaments are rapidly developed, a large amount of medicaments continuously emerge in the market, and a solid foundation is provided for prevention and treatment of the disease thrombus. In a study on hirudin by yebin, it was found that the antithrombin activity after acetone extraction is greater than that of non-and non-extracted hirudins. The first anticoagulant polypeptide in China, named leech polypeptide, is firstly separated and purified from the whitmania pigra. Qianz. y found that glycopeptides derived from k-casein in sheep could potentiate the ability to reduce platelet aggregation with increasing amounts. The method comprises the steps of preparing a hydrolysate of fresh buffalo milk by a complex enzymatic hydrolysis method by the Miao Yangli et al, and then measuring the anticoagulant activity of the hydrolysate to be up to 52.0 ATU/mL. Most of the existing researches mainly adopt anticoagulant polypeptide from blood sucking animals, but few reports are found on anticoagulant polypeptide from plants, and further intensive researches are needed to develop new anticoagulant polypeptide sources and whether the anticoagulant polypeptide activity from plants has greater advantages.
The walnut contains rich nutrient substance matrixes such as protein, fat, carbohydrate, vitamins, minerals and other active ingredients, the utilization research of the walnut and byproducts thereof is one of the hot spots of the current generation, and the problems that the utilization rate is still low and how to improve the food source utilization value of the walnut and increase the value and recycle of the byproducts are urgently needed to be solved are that the walnut meal after walnut oil extraction is used for feed and fertilizer and even is directly discarded at present.
Disclosure of Invention
The invention aims to provide a walnut polypeptide nutrient solution and a preparation method and application thereof, which solve the problem of low utilization rate of walnut meal, treat defatted walnut meal and obtain the polypeptide nutrient solution by using walnut polypeptide powder, and have antithrombin activity and antioxidant activity.
In order to achieve the above object, the present invention provides a walnut polypeptide nutrient solution, comprising: the walnut polypeptide powder, the walnut essence, the citric acid, the xylitol and the water are mixed according to a feed-liquid ratio of 5%, the addition amount of the walnut essence is 0.08%, the addition amount of the citric acid is 0.12% and the addition amount of the xylitol is 9%; the walnut polypeptide powder comprises the following polypeptide sequences: the amino acid sequence shown as SEQ ID NO. 1-16.
Preferably, the walnut polypeptide powder is obtained by enzymolysis of walnut protein by alkaline protease; the enzymolysis is carried out, wherein the concentration of the alkaline protease is 2%, the enzymolysis temperature is 50 ℃, and the pH is 7.
Preferably, the walnut protein is obtained by stirring an aqueous solution of defatted walnut meal in a water bath at the temperature of 55 ℃ and the pH of 8-9, centrifuging to obtain a supernatant, stirring the supernatant at the pH of 4-5, centrifuging to obtain a precipitate, adjusting the pH to be neutral, and drying.
The invention also aims to provide a preparation method of the walnut polypeptide nutrient solution, which comprises the steps of mixing and uniformly blending walnut polypeptide powder, walnut essence, citric acid and xylitol in water, filling, sterilizing at 115 ℃ and 0.1MPa, and cooling after sterilization to obtain the walnut polypeptide nutrient solution.
Preferably, the preparation of the walnut polypeptide powder comprises the following steps: extracting walnut oil from walnuts by using subcritical butane to obtain degreased walnut meal; dispersing the degreased walnut meal in water, stirring in water bath at 55 ℃ by adjusting the pH to 8-9, centrifuging at a low temperature of 8000r/min, and keeping a supernatant; adjusting the pH value of the supernatant to 4-5, stirring, centrifuging at a low temperature of 8000r/min, adjusting the pH value of the precipitate to be neutral, and freeze-drying to obtain walnut protein; adding water into the walnut protein to prepare a protein solution, adjusting the pH to 7, adding alkaline protease to enable the concentration of the protein solution to be 2%, hydrolyzing at the temperature of 50 ℃, maintaining the pH to 7 during hydrolysis, inactivating the enzyme after enzymolysis is finished to stop reaction, centrifuging at the low temperature of 8000r/min, and keeping supernatant; the low-temperature centrifugation temperature is 3-5 ℃, and protein denaturation caused by overhigh temperature is avoided.
The invention also aims to provide application of the walnut polypeptide nutrient solution, and the walnut polypeptide nutrient solution has antioxidant activity and antithrombin activity.
The walnut polypeptide nutrient solution and the preparation method and application thereof solve the problem of low utilization rate of walnut meal, and have the following advantages:
according to the walnut polypeptide nutrient solution, the defatted walnut protein meal is treated by alkaline protease to obtain the walnut polypeptide powder with high-activity antioxidant and antithrombin polypeptides, and the obtained walnut polypeptide nutrient solution has high sensory score through reasonable matching with walnut essence, citric acid and xylitol.
Drawings
FIG. 1 shows the results of sensory scores of comparative examples 1 to 5 of the present invention.
FIG. 2 shows the results of sensory scores of comparative examples 6 to 10 according to the present invention.
FIG. 3 is the results of sensory scores of comparative examples 11 to 15 of the present invention.
FIG. 4 shows the results of sensory scores of comparative examples 16 to 20 according to the present invention.
FIG. 5 is a contour line of the interaction between the walnut essence and the ratio of liquid to solid.
FIG. 6 is a contour line showing the interaction between citric acid and the feed-liquid ratio according to the present invention.
FIG. 7 is a contour line showing the interaction of xylitol and feed-liquor ratio according to the present invention.
Fig. 8 is a contour line of the interaction of citric acid and walnut essence of the present invention.
FIG. 9 is a contour line of the interaction of xylitol and walnut essence of the invention.
FIG. 10 is a contour line of the interaction of xylitol and citric acid according to the present invention.
FIG. 11 shows the results of the chromatography of the Sephadex according to the invention.
FIG. 12 is a chromatogram of a chromatographic separation of a peptide fragment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A preparation method of walnut polypeptide nutrient solution comprises the following steps:
mixing semen Juglandis polypeptide powder, semen Juglandis essence, citric acid, and xylitol in water, bottling, sterilizing at 115 deg.C and 0.1Mpa for 15min, and cooling to obtain semen Juglandis polypeptide nutritional liquid.
Wherein the feed liquid ratio is 5% (volume ratio of walnut polypeptide powder to water), the walnut essence addition amount is 0.08% (mass percent of walnut essence in walnut polypeptide nutrient solution), the citric acid addition amount is 0.12% (mass percent of citric acid in walnut polypeptide nutrient solution), and the xylitol addition amount is 9% (mass percent of xylitol in walnut polypeptide nutrient solution).
The preparation method of the walnut polypeptide powder comprises the following specific steps:
firstly, drying fresh walnuts at 55 ℃, then crushing, extracting walnut oil by using subcritical butane, and repeatedly extracting for three times until the walnut oil is completely extracted, thereby obtaining the degreased walnut dregs. Storing at 4 deg.C for low temperature refrigeration;
secondly, dispersing the dried degreased walnut meal in distilled water according to a certain feed-liquid ratio to prepare a walnut protein solution with the mass fraction of 5%, adjusting the pH value to 8.5 by using 1mol/L NaOH solution, stirring in a water bath at 55 ℃ for 2h, centrifuging at a low temperature of 8000r/min (3-5 ℃) for 20min, removing an oil layer and precipitates, and keeping a supernatant;
then, regulating the pH of the supernatant to be 4.5 by using 1mol/LHCl solution, stirring for 1h, centrifuging at a low temperature of 8000r/min for 20min, washing the precipitate to be neutral by using distilled water, regulating the pH to be neutral, and freeze-drying for 24h to obtain walnut protein;
finally, adding distilled water into walnut protein to prepare protein solution, pretreating in a water bath at a certain temperature for a certain time, adjusting the pH to 7, adding alkaline protease to make the concentration of the alkaline protease 2%, hydrolyzing at 50 ℃ for 4h, maintaining the pH with 1.0mol/L NaOH during the hydrolysis, placing in a 90 ℃ water bath for 10min after the enzymolysis is finished, inactivating the enzyme to terminate the reaction, centrifuging at 8000r/min for 20min, drying the precipitate, and drying in a dryer at 60 ℃ for 2 h. Separating the walnut polypeptide, namely separating the walnut polypeptide by adopting macroporous adsorption resin, ultrafiltering to obtain a hydrolysate with the molecular weight of less than 3kD, and separating the hydrolysate with the molecular weight of less than 3kD by using sephadex chromatography to obtain a component A with the strongest antithrombin activity and antioxidant activity, wherein the component A is walnut polypeptide powder adopted in the walnut polypeptide nutrient solution. The walnut polypeptide powder comprises the following polypeptide sequences: the amino acid sequence shown as SEQ ID NO. 1-16.
The degree of hydrolysis of alkaline protease varies under different conditions of enzymatic hydrolysis, and is the best under the conditions of enzymatic hydrolysis of the present invention.
Comparative examples 1 to 5
A preparation method of walnut polypeptide nutrient solution is basically the same as that of the embodiment 1, and is characterized in that: the feed-to-liquid ratios of comparative examples 1 to 4 were 5%, 10%, 15%, 20% and 25%, respectively, when the addition amount was 0.08%, the addition amount of citric acid (food grade, available from Xiamen, south Broussonetia technology Co., Ltd.) was 0.12%, and the addition amount of xylitol (food grade, available from Xiamen, south Broussonetia technology Co., Ltd.) was 8%.
Comparative examples 6 to 10
A preparation method of walnut polypeptide nutrient solution is basically the same as that of the embodiment 1, and is characterized in that: when the feed-liquid ratio is 10%, the addition amount of citric acid is 0.12% and the addition amount of xylitol is 8%, the addition amounts of the walnut essence in the comparative examples 6-10 are 0, 0.04%, 0.08%, 0.12% and 0.16%, respectively.
Comparative examples 11 to 15
A preparation method of walnut polypeptide nutrient solution is basically the same as that of the embodiment 1, and is characterized in that: when the feed-liquid ratio is 10%, the addition amount of walnut essence is 0.08%, and the addition amount of xylitol is 8%, the addition amounts of citric acid in comparative examples 11-15 are 0.04%, 0.08%, 0.12%, 0.16%, and 0.2%, respectively.
Comparative examples 16 to 20
A preparation method of walnut polypeptide nutrient solution is basically the same as that of the embodiment 1, and is characterized in that: when the feed-liquid ratio is 10%, the addition amount of walnut essence is 0.08%, and the addition amount of citric acid is 0.12%, the addition amounts of xylitol in comparative examples 16-20 are 2%, 4%, 6%, 8%, and 10%, respectively.
Comparative examples 21 to 22
A preparation method of walnut polypeptide nutrient solution is basically the same as that of the embodiment 1, and is characterized in that: when the walnut polypeptide powder is prepared, papain and trypsin are respectively adopted for enzymolysis in comparative examples 21-22, and the hydrolysis degree and the content of the walnut polypeptide in the obtained walnut polypeptide powder are lower than those in example 1, wherein the hydrolysis degree DH of the alkaline protease in example 1 is 19.56%, and the content of the polypeptide is 1.210 mg/mL; the hydrolysis degree DH of the papain is 9.24 percent, and the content of the polypeptide is 0.812 mg/mL; the degree of hydrolysis DH of trypsin was 11.70% and the polypeptide content was 0.936 mg/mL.
Experimental example 1 sensory evaluation experiment
Sensory evaluation experiments are carried out on the walnut polypeptide nutrient solution of the embodiment 1 and the comparative examples 1-19, and the details are as follows:
the sensory indexes of the walnut polypeptide nutrient solution involved in the test are carried out by referring to the national vegetable protein beverage walnut dew (milk) standard (GB/T31325-2014), the color, taste, smell, sweetness and tissue state of the walnut are evaluated, and the specific evaluation score standard is shown in Table 1.
And establishing a corresponding function relation by taking the feed liquid ratio (A), the walnut essence adding amount (B), the citric acid adding amount (C) and the xylitol adding amount (D) as independent variables and taking the sensory score obtained after proportioning as a target function value. The fitting equation is:
Y＝78-6.83A-11.67B+3.25C-4.75D+18.25AB+15.75AC+12.50AD+3.75BC+1.50BD-14.75CD-17.42A2-7.92B2-10.04C2-8.29D2
r of the above fitting equation20.9173, and the significance values of the model are all less than 0.0001, indicating that the linear relationship performs well and the degree of fitting is good.
TABLE 1 walnut milk sensory evaluation score criteria
Figure BDA0002680824110000051
Figure BDA0002680824110000061
As shown in FIG. 1, the sensory score results of comparative examples 1 to 5 according to the present invention show that the sensory score of comparative examples 1 to 5 increases with the feed-to-liquid ratio, but decreases with the feed-to-liquid ratio of more than 10%, and the sensory score is 83 minutes, which is the highest at 10%.
As shown in fig. 2, the sensory score results of comparative examples 6 to 10 of the present invention show that the sensory scores of comparative examples 6 to 10 increase with the addition amount of the walnut essence, but the sensory score decreases again when the addition amount of the walnut essence is greater than 0.08%, and the sensory score is 86 minutes, which is the highest when the addition amount of the walnut essence is greater than 0.08%.
As shown in FIG. 3, the sensory score results of comparative examples 11 to 15 according to the present invention revealed that the sensory scores of comparative examples 11 to 15 increased with the addition of citric acid, but the sensory score decreased with the addition of citric acid exceeding 0.12%, and the sensory score was the highest at 0.12%, and was 85 points.
As shown in FIG. 4, the sensory scores of comparative examples 16 to 20 according to the present invention were found to increase as the addition amount of xylitol increased, but when the addition amount of xylitol was more than 8%, the sensory score decreased, and when the addition amount of xylitol was more than 8%, the sensory score was the highest at 8%, and was 68 points.
As shown in fig. 5-10, it can be determined whether the interaction is significant according to the difference of the positions of the contour lines, the ellipse of the contour lines indicates that the interaction is significant, the interaction is more significant as the major and minor axes of the ellipse become longer, and the circle indicates that the interaction is not significant. The graph shows that the feed liquid ratio and the addition amount of the walnut essence have obvious influence interaction on the sensory score of the nutrient solution, and the sensory score shows a trend of rising first and then falling down along with the increase of the feed liquid ratio and the addition amount of the essence; the interaction between the feed liquid ratio and the citric acid addition amount has obvious influence on the sensory score of the nutrient solution, and when the feed liquid ratio is too large and the citric acid addition amount is too high, the mouthfeel is opposite. When the ingredients were added in the amount of example 1, the sensory score was 89.42, which was the highest.
Experimental example 2 measurement of indices
1. Example 1 determination of the amount of polypeptide
(1) Determination of the Standard Curve
The accurate amount is taken 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4mg/mLGly-Gly-Tyr-Arg tetrapeptide standard solution prepared by 5 percent TCA (trichloroacetic acid) to be added with water to supplement to a scale mark in a 6-branch 10mL volumetric flask, 6mL of each solution is taken, then 4mL of biuret reagent is added, after full shaking and shaking, the incubation is carried out for 0.5h at room temperature, centrifugation is carried out for 5min at 3000r/min, and the light absorption value is measured at 540 nm. The first blank protein solution was used as a control. And (4) drawing a standard curve by taking the protein concentration as an abscissa and the light absorption value as an ordinate.
(2) Example 1 determination of walnut polypeptide nutrient solution
Accurately measuring 2.5mL of walnut polypeptide nutrient solution in example 1, adding the same amount of 10% TCA aqueous solution, fully and uniformly shaking, incubating at room temperature for 10min, centrifuging at 3000r/min for 15min, metering the volume of the supernatant into a 50mL volumetric flask by using 5% TCA aqueous solution, fully shaking and uniformly shaking, performing enzymolysis under a certain enzymolysis condition, and obtaining the polypeptide content (mg/mL) in the sample solution according to a standard curve, wherein the results are shown in the following table 2.
2. Fat content determination
The fat content of the walnut polypeptide nutrient solution of example 1 is measured according to a second method acid hydrolysis method specified in GB/T5009.6-2003, and the results are shown in the following table 2.
3. Determination of coliform, pathogenic bacteria, mold and yeast
Coliform, pathogenic bacteria, mold and yeast of the walnut polypeptide nutrient solution of example 1 were determined according to the regulations of GB/T4789.21-2008, and the results are shown in Table 2 below.
Table 2 shows the measurement results of the contents of polypeptide, fat and bacteria in the walnut polypeptide nutrient solution of example 1 of the invention
Figure BDA0002680824110000071
Experimental example 3 analysis of walnut polypeptide fragments
1. Walnut polypeptide separation
(1) Separating by macroporous adsorption resin: soaking DA 201-CII type macroporous adsorption resin with 95% ethanol for 24h, washing with deionized water for several times until the solution is clear and not turbid, packing with a wet method, soaking and washing the resin with 70% and 30% ethanol respectively, adding three times of ethanol volume and requiring no white turbid phenomenon, and finally washing with deionized water fully until no ethanol smell exists. Next, the resin layer was passed through with 5% HCl solution and kept rinsed for 4h, followed by deionized water at a flow rate of 2mL/min to neutrality. And then 5% NaOH solution is used for passing through the resin layer, the operation is repeated, and the resin is stored for standby after the operation is finished. And (3) uniformly stirring the pretreated pasty resin, and adding the pasty resin into the chromatographic column at a constant speed in one step to ensure that no bubble or gap exists in the chromatographic column, and the piston at the bottom is in an open state to ensure that water slowly flows out from the bottom.
When the liquid level is reduced to be level with the surface of the adsorption resin, the sample injection is started, the protein hydrolysate passes through the exchange column at the flow rate of 2mL/min, no bubbles exist in the resin layer, the elution is started, the effluent liquid is collected, the light absorption value of the effluent liquid is measured at 220nm, the elution is stopped when the elution reaches the dynamic adsorption saturation end point, and the elution is carried out by using 75% ethanol at the flow rate of 1 mL/min. Keeping the required substances according to the measured absorbance, vacuum freeze-drying for 24h, and refrigerating for later use.
(2) And (3) ultrafiltration: ultrafiltering the walnut protein hydrolysate with macroporous adsorbent resin by ultrafiltration centrifuge tube, and separating with ultrafiltration membrane net with membrane molecule cut-off molecular mass of 10kD and 3kD to obtain product with molecular mass of more than 10kD (A'3)、3～10kD(A’2) And less than 3kD (A'1) Respectively collecting the hydrolysis products with the molecular mass of 3, and measuring the antithrombin activity and the antioxidant activity of the hydrolysis products;
(3) dextran gel chromatography: accurately weighing 4.5G of gel of Sephadex G-25, placing in a beaker, pouring into deionized water, boiling in water bath for 1-2h, discarding the gel particles floating on the upper layer, and subjecting the resin completely swollen to ultrasonic treatment for 30min to remove bubbles. Standing for 12h after the gel is cooled, pouring into a gel column perpendicular to the ground at a constant speed, and placing the filled column in a chromatography cabinet at 4 ℃. Connecting the top of the column with pump, connecting the lower end of the column with detector, ultrafiltering with 0.02M buffer solution (pH 6.8) containing disodium hydrogen phosphate-sodium dihydrogen phosphate to obtain component A with strongest antithrombin activity1Preparing a solution of 30mg/mL, filtering with a 0.22-micrometer filter membrane, separating by Sephadex G-25 Sephadex column (1.6cm multiplied by 30cm), loading 3mL of the solution, taking distilled water as eluent, carrying out detection at a wavelength of 220nm at an elution flow rate of 0.5mL/min, collecting one tube of components every 3min to obtain three peaks (see figure 11) with different peak areas, dividing the collected three peaks into A, B, C from left to right, freeze-drying, evaluating the antioxidant activity and antithrombin activity of the components, and determining the antithrombin activity and the antioxidant activity.
2. Antithrombin activity assay
The fibrinogen solution and the thrombin solution were both prepared with 0.154mol/L NaCl. First, the target temperature of the microplate reader was set to 37 ℃ and the measurement wavelength was 405 nm. Selecting a target small well on the ELISA plate, sequentially adding 40 mu L of Tris-HCl buffer solution and 140 mu L of 1mg/mL (w/v) fibrinogen solution into the small well, fully oscillating and uniformly mixing, and incubating for 10min at room temperature. Subsequently, 10. mu.L of thrombin solution (12U/mL) was added thereto, well mixed, and the absorbance was measured. The samples were assayed using 40. mu.L of sample solution instead of Tris-HCl buffer solution. The blank was Tris-HCl buffer solution instead of thrombin solution. The data obtained were calculated according to the following formula.
Figure BDA0002680824110000091
Wherein Y is the hydrolysate inhibition rate; a. theiThe absorbance value of the added inhibitor is obtained; a. theeThe absorbance value of the inhibitor is not added; a. theoAbsorbance was blank.
3. Determination of Total antioxidant Activity
(1) DPPH radical clearance rate
A0.1 mmol/L DPPH-absolute ethanol solution is prepared and stored in a non-light environment. Accurately measuring 1mL of solution to be detected and 1mL of DPPH solution in the same test tube, oscillating uniformly, and then incubating for 30min in a dark environment. The absorbance at 517nm was measured as A1. The absorbance of the sample was measured at 517nm with 1mL of distilled water as a blank2With VCPreparation of standard koji. The calculation formula is as follows:
Figure BDA0002680824110000092
in the formula, A1The light absorption values of the sample solution to be detected and the DPPH solution are obtained; a. the2The light absorption value of the mixed solution of distilled water and DPPH is shown.
DPPH free radical scavenging results: the greater the scavenging capacity for DPPH free radicals with increasing polypeptide concentration. In the concentration range of 0.0-1.0mg/mL, A'1(＜3KDa)、A’2(3～10KDa)、A’3The activity of the component (> 10KDa) for eliminating DPPH free radicals is in a straight-line rising trend, while the activity for eliminating Vc reaches a maximum plateau value within the range of 0.4-1mg/mL, and the clearance rate is obviously lower than Vc (ascorbic acid) at lower concentration, but the difference is gradually reduced along with the increase of the concentration. It is obvious that the clearance rate of the A component to DPPH free radical is 71.02 percent and is obviously larger than that of the B, C component at the concentration of 1mg/mL, the clearance rate is respectively 26.78 percent and 19.45 percent, and the small molecular weight walnut polypeptide has the effect of quenching DPPH free radicalThe free radical capacity, which is an antioxidant with the capacity of scavenging free radicals, and the activities of walnut polypeptides with different molecular weights are remarkably different.
(2) Hydroxyl radical scavenging Activity
Taking 1.0mL of polypeptide solution with different molecular weights, adding 2.0mL of 9mM FeSO4, 1.0mL of 9mM salicylic acid and 1.0mL of 9mM H2O2, uniformly oscillating, carrying out environmental conservation at 37 ℃ for 30min, and centrifuging at 8000r/min for 10 min. Distilled water is used as a reference, Vc is used for preparing standard curve, and the calculation formula of the absorbance value is as follows at 510 nm:
Figure BDA0002680824110000101
in the formula, A is the light absorption value of distilled water with the same volume; b is the absorbance value measured by the sample.
Hydroxyl radical scavenging results: the walnut polypeptide has a favorable scavenging effect on hydroxyl free radicals, and the scavenging rate liquid is gradually increased along with the increase of the concentration. The clearance rate of the walnut polypeptide component A is separated from the component B, C by a larger difference after the concentration is 1 mg/mL. However, the three components are weaker than Vc, which shows that the walnut polypeptide liquid has certain antioxidant activity, and the component A in the three separated components has the best effect.
(3) Determination of ABTS free radical scavenging Capacity
Distilled water was added to prepare a 7mmol/L stock solution of ABTS and a 2.5mmol/L solution of potassium persulfate in a ratio of 1: 1, standing and preserving for 12-16h under the dark condition to obtain ABTS stock solution, and diluting until the absorbance at 734nm is 0.7 +/-0.01. Mixing 1.0mL of diluted ABTS stock solution with 1mL of solution to be tested, incubating in dark for 10min, and measuring absorbance at 734 nm. Distilled water was used as a control and Trolox-methanol was used to draw a standard curve as a control. Calculating the clearance rate of ABTS free radicals according to the formula:
Figure BDA0002680824110000102
in the formula, A is the light absorption value of mixed liquid of ABTS reagent and blank solvent; b is the light absorption value of the mixed liquid of the ABTS reagent and the sample liquid.
ABTS free radical scavenging results: three components separated from the sephadex have a certain dose-effect relationship on ABTS free radical scavenging capacity, and the clearance rate of the three components is gradually increased along with the increase of the concentration of the polypeptide. The ABTS free radical clearance of the component A is higher than that of the component B and the component C under any concentration, and the clearance value is close to Vc already when the concentration reaches 5 mg/mL. Although the three components do not exceed the scavenging capacity of Vc, they also demonstrate some ABTS free radical scavenging capacity.
(4)Fe2+Determination of chelating Capacity
100mM FeSO was prepared4·7H2O was diluted with distilled water to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM, an equal volume of 0.5mL TPTZ solution and 5.0mL acetic acid-sodium acetate buffer (0.3mol/L, pH 3.6) was added to each gradient, mixed well with shaking, and the absorbance was measured at 593 nm. At different concentrations of FeSO4The solution is represented by the abscissa, the measured light absorption value is represented by the ordinate, and a standard curve is drawn.
According to the volume ratio of 1: 1: acetic acid-sodium acetate solution (0.3mol/L, pH 3.6): 20mmol/L FeCl3: preparing FRAP working solution by 10mmol/L TPTZ. Adding a certain amount of polypeptide sample solution into 4.5mLFRAP working solution, incubating at 37 deg.C for 20min, and detecting its absorbance A at 593nm1The absorbance of the sample was measured as A by using distilled water instead of the sample as a control0。VcPreparing reference standard curve, finding A corresponding to the standard curve1-A0FeSO corresponding to difference4The concentration of the solution.
Fe2+Chelating ability results: three components separated from the sephadex are in Fe2+In chelating ability, the clearance rate of the polypeptide also shows a gradually increasing trend along with the increase of the concentration of the polypeptide. Fe of component A2+The chelating ability is higher than that of the component B and the component C at any concentration, and the clearance value is always close to Vc with the concentration of 5 mg/mL. Although the three components do not exceed Vc, it is also stated that they have some Fe2+Chelating ability。
4. Determination of amino acid content and sequence of high-activity polypeptide
And (4) determining the amino acid composition and content of the finally separated components with high antithrombin activity and high antioxidant activity by adopting an amino acid automatic analyzer.
The molecular weight and the peptide fragment sequence of the high-activity component are detected by liquid phase secondary mass spectrometry (LC-MS/MS), and the result is analyzed by software, and the higher the peptide fragment score is, the higher the possibility that the peptide exists in the detected object is. Chromatographic conditions are as follows: the buffer solution A was 0.1% formic acid aqueous solution, and the solution B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile: 84%). The column was equilibrated with 95% of solution A. Sample was applied to a chromatographic column (2 cm. times.100. mu.m 5. mu.m-C)18) Then passing through an analytical column (75 μm × 100mm 3 μm-C)18) The separation was carried out at a flow rate of 300 nL/min. The relevant liquid phase gradients are as follows: gradient for 2 hours: 0-110 min; the linear gradient of the liquid B is from 0 to 55 percent; 110-115min, the linear gradient of the liquid B is from 55 to 100 percent; 115-120 min; the content of the solution B is maintained at 100%. The peptide fragments were chromatographically separated and then analyzed by mass spectrometry using a mass spectrometer.
High activity polypeptide amino acid content results: 1-440nm is used for detecting relevant proline, and 1-570nm is used for detecting other amino acids except proline, and analysis shows that the detected components have no impurity interference, and the peak-appearing time is matched with the peak-appearing shape, which indicates that the detection separation result is good. The amino acid content of the walnut polypeptide is 17.9g/100g, 10.97g/100g and 8.44g/100g respectively, wherein glutamic acid is the first amino acid in the walnut polypeptide, and arginine and aspartic acid are the next amino acid.
Sequence determination results for highly active polypeptides: and (3) performing three-time repeated parallel polypeptide peptide segment analysis on the walnut polypeptide with high antithrombin activity and high in-vitro antioxidant activity obtained by chromatography by adopting LC-MS-MS. After LC-MS-MS detection is carried out on the three parallel walnut polypeptide samples, a specific peak shape, namely a walnut polypeptide mass spectrometry Basepak map, is formed and is shown in figure 12. And performing simulated protease hydrolysis on the result in a uniprot _ Juglans _ regia protein library, and analyzing by PD-Mascot database analysis software to match with the corresponding source protein. The pattern was analyzed with the results output from the protein library to screen out 16 polypeptides with a polypeptide score higher than twenty (see table 3), with higher peptide score indicating greater activity.
TABLE 3 high antithrombin Activity polypeptide sequences and molecular weights
Figure BDA0002680824110000121
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
<110> university of Western Hua
<120> walnut polypeptide nutrient solution and preparation method and application thereof
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 1
Val Gln Ile Val Asp Asp Asn Gly Asn Asn Val Phe Asp Glu Arg
1 5 10 15
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 2
Val Gln Ile Val Asp Asp Asn Gly Asp Asn Val Phe Asp Glu Arg Val
1 5 10 15
Lys
<210> 3
<211> 29
<212> PRT
<213> Artificial Sequence
<400> 3
Val Gly Ala Thr Ala Ala Val Tyr Ser Ala Ala Ile Leu Glu Tyr Leu
1 5 10 15
Thr Ala Glu Val Leu Glu Leu Ala Gly Asn Ala Ser Lys
20 25
<210> 4
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 4
His Asn Leu Asp Thr Gln Thr Glu Ser Asp Val Phe Ser Arg
1 5 10
<210> 5
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 5
Gly Asn Ala Arg Leu Gln Val Val Gly Glu Asn Gly Gln Asn Val Phe
1 5 10 15
Asp Gly Glu Val Arg
20
<210> 6
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 6
Gly Glu Ala Arg Val Gln Ile Val Asp Asp Asn Gly Asn Asn Val Phe
1 5 10 15
Asp Glu Arg Val Lys
20
<210> 7
<211> 15
<212> PRT
<213> Artificial Sequence
<400> 7
Val Gln Ile Val Asp Asp Asn Gly Asp Asn Val Phe Asp Glu Arg
1 5 10 15
<210> 8
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 8
Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg
1 5 10
<210> 9
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 9
Arg Leu Gln Glu Asp Asp Ser Gln Arg Gly Ile Ile Val Arg
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 10
Phe Ser Arg Gln Glu Thr Phe Leu Ala Arg
1 5 10
<210> 11
<211> 12
<212> PRT
<213> Artificial Sequence
<400> 11
Gly Leu Leu Leu Pro Ser Phe Ser Asn Ala Pro Arg
1 5 10
<210> 12
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 12
Val Gln Ile Val Asp Asp Asn Gly Asn Asn Val Phe Asp Glu Arg Val
1 5 10 15
Lys
<210> 13
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 13
Phe Phe Asp Gln Gln Glu Gln Arg Glu Gly Val Ile Ile Arg
1 5 10
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence
<400> 14
Ala Leu Pro Glu Asp Val Leu Ile Asn Ala Tyr Arg
1 5 10
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence
<400> 15
Arg Glu Ser Trp Pro Gly Ser Arg
1 5
<210> 16
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 16
Arg Thr Ile Glu Pro Asn Gly Leu Leu Leu Pro Gln Tyr Ser Asn Ala
1 5 10 15
Pro Gln Leu Val Tyr Ile Ala Arg
20

Claims (6)

1. The walnut polypeptide nutrient solution is characterized by comprising the following components in parts by weight: the walnut polypeptide powder, the walnut essence, the citric acid, the xylitol and the water are mixed according to a feed-liquid ratio of 5%, the addition amount of the walnut essence is 0.08%, the addition amount of the citric acid is 0.12% and the addition amount of the xylitol is 9%; the walnut polypeptide powder comprises the following polypeptide sequences: the amino acid sequence shown as SEQ ID NO. 1-16.
2. The walnut polypeptide nutrient solution of claim 1, wherein the walnut polypeptide powder is obtained by subjecting walnut protein to enzymatic hydrolysis with an alkaline protease; the enzymolysis is carried out, wherein the concentration of the alkaline protease is 2%, the enzymolysis temperature is 50 ℃, and the pH is 7.
3. The walnut polypeptide nutrient solution as claimed in claim 1, wherein the walnut protein is obtained by stirring an aqueous solution of defatted walnut meal in a water bath at a temperature of 55 ℃ and a pH of 8-9, centrifuging to obtain a supernatant, stirring the supernatant at a pH of 4-5, centrifuging to obtain a precipitate, adjusting the pH of the precipitate to be neutral, and drying.
4. A method for preparing a walnut polypeptide nutrient solution as claimed in any one of claims 1 to 3, which comprises:
mixing semen Juglandis polypeptide powder, semen Juglandis essence, citric acid, and xylitol in water, bottling, sterilizing at 115 deg.C and 0.1Mpa, and cooling to obtain semen Juglandis polypeptide nutritional liquid.
5. The preparation method of the walnut polypeptide nutrient solution as claimed in claim 4, wherein the preparation of the walnut polypeptide powder comprises:
extracting walnut oil from walnuts by using subcritical butane to obtain degreased walnut meal;
dispersing the degreased walnut meal in water, stirring in water bath at 55 ℃ by adjusting the pH to 8-9, centrifuging at a low temperature of 8000r/min, and keeping a supernatant;
adjusting the pH value of the supernatant to 4-5, stirring, centrifuging at a low temperature of 8000r/min, adjusting the pH value of the precipitate to be neutral, and freeze-drying to obtain walnut protein;
adding water into the walnut protein to prepare a protein solution, adjusting the pH to 7, adding alkaline protease to enable the concentration of the protein solution to be 2%, hydrolyzing at the temperature of 50 ℃, maintaining the pH to 7 during hydrolysis, inactivating the enzyme after enzymolysis is finished to stop reaction, centrifuging at the low temperature of 8000r/min, and keeping supernatant;
the low-temperature centrifugation temperature is 3-5 ℃.
6. The use of a walnut polypeptide nutritional solution as claimed in any one of claims 1 to 3 wherein the walnut polypeptide nutritional solution has antioxidant activity and antithrombin activity.
CN202010961904.6A 2020-09-14 2020-09-14 Walnut polypeptide nutrient solution and preparation method and application thereof Pending CN112106994A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106561828A (en) * 2015-10-08 2017-04-19 青岛莹辉达通化工科技有限公司 Low-sugar nutritious milk powder
CN110923284A (en) * 2019-11-22 2020-03-27 北京联合大学 Walnut nut flavor peptide and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106561828A (en) * 2015-10-08 2017-04-19 青岛莹辉达通化工科技有限公司 Low-sugar nutritious milk powder
CN110923284A (en) * 2019-11-22 2020-03-27 北京联合大学 Walnut nut flavor peptide and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陆一敏，等: "脱脂核桃乳饮料配方的研究", 《饮料工业》 *

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