Preparation method of a lycium chinense polypeptide and application of the lycium chinense polypeptide

Abstract

The application discloses a preparation method of a phyllium polypeptide, and comprises the following steps: 1) phyllium kernel pretreatment: phyllium kernels are pretreated to obtain defatted phyllium powder; 2) preparation of phyllium protein: sodium chloride is added into water to prepare a sodium chloride solution with a concentration of 0.05-0.25 mol / L, then the defatted phyllium powder in step 1) is added and mixed to obtain a material liquid, and the phyllium protein is prepared by adopting an alkali extraction and acid precipitation method; 3) phyllium protein enzymolysis: the phyllium protein in step 2) is added into water and mixed, a protease is added for hydrolysis, enzyme inactivation and filtration to obtain the phyllium polypeptide; the dosage ratio of the phyllium protein to water is 1 g:(5-20) mL. Meanwhile, the application provides an application of the phyllium polypeptide in an alcoholism-relieving beverage. In an alkaline environment, the phyllium functional protein is specifically hydrolyzed by alkaline protease, the degree of hydrolysis is high, and the ADH activation rate of the phyllium functional polypeptide fragment obtained is high.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for preparing pecan polypeptides and the application of pecan polypeptides. Background Technology

[0002] Pecans, also known as American hickory nuts, are one of the most common nuts, second only to almonds and walnuts. They are a nutritious and healthy food, with significant benefits such as lowering blood lipids and blood pressure, and promoting beauty and skin health, making them very popular. Pecans originated in northern Mexico, the United States, and Australia. Since their introduction to China in the late 19th century, through continuous exploration and practice, they have been widely cultivated in my country and have become one of the crops with significant economic value.

[0003] Pecans are rich in monounsaturated fatty acids and low in saturated fatty acids. Compared to olive and sunflower seed oils, pecan oil contains more monounsaturated fatty acids, which helps reduce the formation of low-density lipoprotein cholesterol, thus reducing the risk of cardiovascular disease. Pecans offer numerous health benefits, such as boosting energy, preventing anemia, improving eyesight, and preventing neurasthenia. Furthermore, pecans are rich in vitamin E, which acts as an antioxidant, protecting human cells from the effects of external environmental factors and internal metabolic products, and slowing down the aging process. Meanwhile, pecan kernels can be used to make various foods, such as pastries, ice cream, cakes, and candies. Pecan oil can also be used in cooking, and peptides extracted from pecan kernels can be used to make functional products such as beverages and pharmaceuticals. In addition, pecans hold an important place in traditional Chinese medicine, possessing properties such as clearing heat and detoxifying, killing parasites and relieving itching, and can treat conditions such as itchy toes and tinea pedis. Although pecans are highly nutritious and have a good taste, research on their functional substances is still insufficient. Over 90% of pecans are processed into edible kernels. Pecans are usually eaten fresh, processed, or roasted into various products, including pies, cakes, candies, and cookies; there are few commercially available processed pecan products.

[0004] Pecans are an excellent nut product, rich in protein, amino acids, vitamins, and other nutrients, making them highly nutritious. Regular consumption can improve brain function, strengthen the body, and lower blood lipids. However, despite being a premium nut product, there are few reports on the protein properties of pecans, and highly processed pecan products are extremely rare on the market. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing pecan peptides, and another purpose of this invention is to provide applications of the pecan peptides prepared by this method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing pecan nut polypeptide includes the following steps:

[0008] 1) Pecan kernel pretreatment: Pecan kernels are pretreated to obtain defatted pecan powder;

[0009] 2) Preparation of pecan protein: Sodium chloride is added to water to make a sodium chloride solution with a concentration of 0.05-0.25 mol / L. Then defatted pecan powder from step 1) is added, mixed, and the resulting liquid is prepared by alkaline extraction and acid precipitation to obtain pecan protein.

[0010] 3) Pecan protein hydrolysis: Add pecan protein from step 2) to water, mix to obtain pecan protein solution, add protease for hydrolysis, enzyme inactivation, filtration, freeze drying to obtain pecan polypeptide.

[0011] In step 2), the ratio of defatted pecan powder to water in the liquid is 1g:(10-100)mL; the steps of the alkaline extraction and acid precipitation method are as follows: adjust the pH of the liquid to 7-11 with sodium hydroxide solution, extract in a water bath, centrifuge, collect the supernatant, adjust the pH of the supernatant to 3-5 with hydrochloric acid solution, centrifuge, collect the precipitate, freeze dry to obtain pecan protein, and use deionized water.

[0012] In step 2), the pH of the feed solution was adjusted to 8-9 using 2 mol / L sodium hydroxide solution, and the mixture was extracted in a water bath at 40°C for 10-14 h. After centrifugation at 4000 r / min for 10 min, the supernatant was collected. The pH of the supernatant was adjusted to 3.5-4.5 using 6 mol / L hydrochloric acid solution, and the mixture was centrifuged at 4000 r / min for 10 min. The precipitate was collected and freeze-dried to obtain pecan protein.

[0013] In step 3), the ratio of pecan protein to water is 1g:(5-20)mL, and the ratio of protease to pecan protein solution is (1-5)g:100mL. The pH is adjusted to 8-12 using 2mol / L sodium hydroxide solution, and hydrolysis is carried out at 55-85℃ for 2-4h. Enzyme inactivation method: the hydrolyzed enzyme solution is heated to 90-100℃ and kept at that temperature for 10-30min. Filtration is carried out by molecular membrane filtration and freeze drying to obtain pecan polypeptides with a molecular weight of 3-30kDa.

[0014] In step 3), the protease is an alkaline protease, a neutral protease, a flavor protease, or papain.

[0015] In step 3), the protease is an alkaline protease.

[0016] In step 1), the method for pre-treating pecans is as follows: remove impurities from pecan kernels, press them, defatt them with petroleum ether, centrifuge them, take out the sediment, dry them at low temperature, and pulverize them to obtain defatted pecan powder.

[0017] In step 1), petroleum ether defatting: the defatted pecan pulp obtained after pressing is mixed with petroleum ether at a mass ratio of 1:1 and stirred for 1-3 hours to obtain a defatted solid-liquid mixture; centrifugation: the solid-liquid mixture is centrifuged at 800 r / min for 5-20 min and the sediment is collected; low temperature drying: the drying temperature is 30-60℃.

[0018] Application of pecan peptides in hangover remedies.

[0019] The hangover relief beverage is made from the following components by weight percentage: 4-10% pecan peptides, 5-25% white sugar, and the remainder is water.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) This invention optimizes the alkaline extraction process of pecan protein, achieving an extraction rate and purity of over 80%. Under alkaline conditions, the functional pecan protein is specifically hydrolyzed using alkaline protease, resulting in a high degree of hydrolysis and an ADH activation rate of up to 50% for the obtained functional pecan polypeptide fragments. Furthermore, the alkaline protease hydrolysis method offers advantages such as high speed, high efficiency, good specificity, few byproducts, and mild operating conditions.

[0022] 2) The pecan peptides obtained by the method of the present invention are used to make a hangover relief beverage. The beverage has good color, taste, mouthfeel, off-flavor and overall evaluation. The beverage is a white transparent liquid with uniform color. The pecan aroma is suitable, pure and without off-flavor. The sweetness is appropriate, without astringency, with obvious aftertaste. There are no protein particles and no sediment. Attached Figure Description

[0023] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a standard curve of proteins.

[0025] Figure 2 The effect of NaCl concentration on the preparation of pecan protein is shown in the figure.

[0026] Figure 3 Figure showing the effect of pH value of alkaline extract on the preparation of pecan protein;

[0027] Figure 4 Image showing the SDS-PAGE electrophoresis results of pecan protein;

[0028] Figure 5 The effect of five proteases on the activation rate of pecan peptide ADH is shown in the figure.

[0029] Figure 6 Figure 1 shows the effect of enzymatic hydrolysis temperature on the activation rate of ADH peptides in pecan fruit.

[0030] Figure 7 Figure showing the effect of pH value on the activation rate of ADH peptide in pecan fruit;

[0031] Figure 8 The graph shows the effect of alkaline protease on the activation rate of pecan polypeptide ADH.

[0032] Figure 9 The graph shows the effect of the molecular weight of pecan peptides on the ADH activation rate of pecan peptides.

[0033] Figure 10 Figure showing the effect of pecan peptide addition on the sensory properties of hangover relief beverage.

[0034] Figure 11 The graph shows the effect of added white sugar on the sensory properties of hangover remedies. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described in detail below. However, the following embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] The list of reagents and instruments involved in the examples and experimental cases is shown in Tables 1 and 2 below.

[0037] Table 1 List of Experimental Drugs and Reagents

[0038]

[0039] Table 2 Commonly Used Experimental Instruments

[0040]

[0041]

[0042] Example 1

[0043] A method for preparing pecan nut polypeptide includes the following steps:

[0044] 1) Pecan kernel pretreatment: Remove impurities from pecan kernels and press them. Mix the defatted pecan pulp obtained after pressing with petroleum ether at a mass ratio of 1:1 and stir with a magnetic stirrer for 1 hour to obtain a defatted solid-liquid mixture. Then, centrifuge the solid-liquid mixture at 800 r / min for 10 min, remove the sediment, and dry and pulverize it at a low temperature of 50℃ to obtain defatted pecan powder.

[0045] 2) Preparation of pecan protein: Sodium chloride was added to deionized water to prepare a sodium chloride solution with a concentration of 0.15 mol / L. Then, defatted pecan powder from step 1) was added and mixed to obtain a solution. The pH of the solution was adjusted to 8.0 with 2 mol / L sodium hydroxide solution. The solution was extracted in a water bath at 40℃ for 12 h, centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The pH of the supernatant was adjusted to 4 with 6 mol / L hydrochloric acid solution, centrifuged at 4000 r / min for 10 min, and the precipitate was collected. The precipitate was freeze-dried under vacuum at a cold trap temperature of -55℃ and a vacuum degree of 5 Pa for 20 h to obtain pecan protein. The ratio of defatted pecan powder to deionized water in the solution was 1 g: 10 mL.

[0046] 3) Pecan protein hydrolysis: Add pecan protein from step 2) to deionized water at a ratio of 1g:10mL to obtain a pecan protein solution. Add alkaline protease (alkaline protease to pecan protein solution ratio of 2g:100mL) and mix. Adjust the pH to 11 with 2mol / L sodium hydroxide solution and hydrolyze at 75℃ for 3h. Then heat the hydrolysate to 95℃ and incubate for 30min to inactivate the enzyme. Filter by molecular membrane and freeze-dry (cold trap temperature -55℃, vacuum degree 5Pa, vacuum freeze-drying for 20h) to obtain pecan polypeptides with a molecular weight of 3-10kDa.

[0047] The above-prepared pecan peptides are used in hangover remedies. The hangover remedy is made from the following components by weight percentage: 6% pecan peptides, 10% white sugar, and the remainder is water. After homogenizing the pecan peptides, white sugar, and water, the mixture is sterilized at 143°C for 5 seconds and then packaged.

[0048] The ADH activation rate of pecan peptides is greater than 60%. Sensory evaluation of the hangover remedy beverage revealed uniform color, a pleasant and pure pecan aroma with no off-flavors, suitable sweetness, no astringency, a pronounced aftertaste, no protein particles, and no sediment. The score was 78.4.

[0049] Example 2

[0050] A method for preparing pecan nut polypeptide includes the following steps:

[0051] 1) Pecan kernel pretreatment: Remove impurities from pecan kernels and press them. Mix the defatted pecan pulp obtained after pressing with petroleum ether at a mass ratio of 1:1 and stir with a magnetic stirrer for 1 hour to obtain a defatted solid-liquid mixture. Then, centrifuge the solid-liquid mixture at 800 r / min for 10 min, remove the sediment, and dry and pulverize it at a low temperature of 50℃ to obtain defatted pecan powder.

[0052] 2) Preparation of pecan protein: Sodium chloride was added to deionized water to prepare a 0.20 mol / L sodium chloride solution. Then, defatted pecan powder from step 1) was added and mixed to obtain a solution. The pH of the solution was adjusted to 9.0 using a 2 mol / L sodium hydroxide solution. Extraction was performed in a water bath at 40℃ for 12 h, followed by centrifugation at 4000 r / min for 10 min. The supernatant was collected, and the pH of the supernatant was adjusted to 4 using a 6 mol / L hydrochloric acid solution. The precipitate was collected and freeze-dried under vacuum at -55℃ and 5 Pa for 20 h to obtain pecan protein. The ratio of defatted pecan powder to deionized water in the solution was 1 g: 50 mL.

[0053] 3) Pecan protein hydrolysis: Add pecan protein from step 2) to deionized water at a ratio of 1g:10mL to obtain a pecan protein solution. Add alkaline protease (3g:100mL to alkaline protease solution) and mix. Adjust the pH to 9 with 2mol / L sodium hydroxide solution and hydrolyze at 65℃ for 3h. Then heat the hydrolysate to 95℃ and incubate for 30min to inactivate the enzyme. Filter the solution through a molecular membrane and freeze-dry (cold trap temperature -55℃, vacuum degree 5Pa, vacuum freeze-drying for 20h) to obtain pecan polypeptides with a molecular weight of 3-10kDa.

[0054] The above-prepared pecan peptides are used in hangover remedies. These remedies are made from the following components by weight percentage: 8% pecan peptides, 10% white sugar, and the remainder is water. After homogenizing the pecan peptides, white sugar, and water, the mixture is sterilized at 143°C for 5 seconds and then packaged.

[0055] Sensory evaluation of the hangover relief beverage showed that it had a uniform color, a pleasant and pure pecan aroma with no off-flavors, a suitable sweetness, no astringency, a noticeable aftertaste, no protein particles, and no sediment.

[0056] Example 3

[0057] A method for preparing pecan nut polypeptide includes the following steps:

[0058] 1) Pecan kernel pretreatment: Remove impurities from pecan kernels and press them. Mix the defatted pecan pulp obtained after pressing with petroleum ether at a mass ratio of 1:1 and stir with a magnetic stirrer for 1 hour to obtain a defatted solid-liquid mixture. Then, centrifuge the solid-liquid mixture at 800 r / min for 10 min, remove the sediment, and dry and pulverize it at a low temperature of 40℃ to obtain defatted pecan powder.

[0059] 2) Preparation of pecan protein: Sodium chloride was added to deionized water to prepare a 0.10 mol / L sodium chloride solution. Then, defatted pecan powder from step 1) was added and mixed to obtain a solution. The pH of the solution was adjusted to 8 using a 2 mol / L sodium hydroxide solution. Extraction was performed in a water bath at 40℃ for 12 h, followed by centrifugation at 4000 r / min for 10 min. The supernatant was collected, and the pH of the supernatant was adjusted to 4 using a 6 mol / L hydrochloric acid solution. The precipitate was collected and freeze-dried under vacuum at -55℃ and 5 Pa for 20 h to obtain pecan protein. The ratio of defatted pecan powder to deionized water in the solution was 1 g: 10 mL.

[0060] 3) Pecan protein hydrolysis: Add pecan protein from step 2) to deionized water at a ratio of 1g:20mL to obtain a pecan protein solution. Add alkaline protease (alkaline protease to pecan protein solution ratio of 2g:100mL) and mix. Adjust the pH to 11 with 2mol / L sodium hydroxide solution and hydrolyze at 75℃ for 2h. Then heat the hydrolysate to 95℃ and incubate for 30min to inactivate the enzyme. Filter by molecular membrane and freeze-dry (cold trap temperature -55℃, vacuum degree 5Pa, vacuum freeze-drying for 20h) to obtain pecan polypeptides with a molecular weight of 10-30kDa.

[0061] The above-prepared pecan peptides are used in hangover remedies. These remedies are made from the following components by weight percentage: 10% pecan peptides, 10% white sugar, and the remainder water. After homogenizing the pecan peptides, white sugar, and water, the mixture is sterilized at 143°C for 5 seconds and then packaged.

[0062] Sensory evaluation of the hangover relief beverage showed that it had a uniform color, a pleasant and pure pecan aroma with no off-flavors, a suitable sweetness, no astringency, a noticeable aftertaste, no protein particles, and no sediment.

[0063] Example 4: Single-factor experiment

[0064] 1. Preparation of Pecan Protein

[0065] 1.1 Construction of protein standard curve

[0066] Bovine serum albumin solutions with protein concentrations of 0.10, 0.40, 0.80, 1.20, and 1.60 mg / mL were prepared, and the absorbance of the protein solutions at different concentrations was measured at a wavelength of 595 nm. A standard curve was plotted with the standard protein concentration as the x-axis and the absorbance value as the y-axis.

[0067] The absorbance of bovine serum albumin (BSA) at different concentration gradients was measured using a UV spectrophotometer, and a protein standard curve was plotted. The absorbance of BSA solutions at each concentration showed a linear relationship, R0. 2 =0.9927>0.99, indicating a good fit. The results are shown in Table 3 and... Figure 1 As shown.

[0068] Table 3 Protein Standard Curve

[0069]

[0070]

[0071] Depend on Figure 1 It can be seen that the protein solution concentration and the absorbance at a 595nm light source have the following relationship:

[0072] A = 0.348x + 0.0212

[0073] Where A is absorbance and x is protein concentration in mg / L.

[0074] 1.2 Effect of NaCl concentration on the preparation of pecan protein

[0075] Five small beakers were prepared, each containing 5g of pecan powder and 50mL of deionized water, labeled 1-5. A specific mass of NaCl was added to each beaker to achieve concentrations of 0.05, 0.10, 0.15, 0.20, and 0.25 mol / L. The pH was adjusted to 8.0 with 1 mol / L NaOH. The solutions were incubated in a water bath at 40℃ for 12 hours, then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected to obtain the pecan protein solution, which was then used. The absorbance of the supernatant at 595 nm was measured using the Coomassie Brilliant Blue method with an automatic Kjeldahl nitrogen analyzer and a UV spectrophotometer. The protein concentration in the sample solution was determined by comparing with a protein standard curve, thus determining the pecan protein content and purity. The results are as follows: Figure 2 As shown.

[0076] Protein content determination

[0077] The protein content was analyzed using the Kjeldahl method, following the steps described in Wang Yuxian, Qiang Hong, Study on the Determination of Protein in Food by Domestic Kjeldahl Nitrogen Analyzer [J]. Analytical Laboratory, 2008(S1):390-391.

[0078] Calculate according to the following formula:

[0079]

[0080] In the formula:

[0081] ω — the mass fraction of protein;

[0082] c — concentration of the standard solution, mol / L;

[0083] V1—Volume of hydrochloric acid standard solution consumed during titration of the sample absorption solution, in mL;

[0084] V2—Volume of standard hydrochloric acid solution consumed during titration of blank absorbent, in mL;

[0085] m—sample mass, g;

[0086] M—molar mass of nitrogen, 14.01 g / mol;

[0087] F – The coefficient for converting nitrogen to protein.

[0088] The following formula was used to analyze the purity of pecan protein.

[0089]

[0090] η represents protein purity, in %

[0091] M1 represents the protein content in dry matter, in grams.

[0092] M2 is the dry matter mass, in grams.

[0093] Depend on Figure 2 It was found that the extraction rate of pecan protein initially increased and then decreased with increasing NaCl concentration, reaching a maximum of 70.6% at a NaCl concentration of 0.15 mol / L. The purity of pecan protein also initially increased and then decreased with increasing NaCl concentration, reaching a maximum of 79.5% at a NaCl concentration of 0.15 mol / L. During the extraction of pecan protein, the NaCl concentration affects both the extraction rate and purity. When the NaCl concentration is low, the protein is easily soluble in water, and adding NaCl as a salt to the extraction solution can promote protein dissolution, improving the extraction rate and purity of pecan protein. However, when the NaCl concentration is too high, it may cause protein denaturation due to protonation or strong ionic effects, leading to changes in the protein molecule's structure and making extraction or purification difficult.

[0094] 1.3 Effect of pH of alkaline extract on the preparation of pecan protein

[0095] Five small beakers were prepared, each containing 5g of pecan powder and 50mL of deionized water, labeled 1-5. A certain amount of NaCl was added to each beaker to achieve a NaCl concentration of 0.15mol / L. The pH was adjusted using 1mol / L NaOH to achieve pH values ​​of 8.0, 9.0, 10.0, 11.0, and 12.0 for beakers 1-5, respectively. The solutions were incubated in a water bath at 40℃ for 12 hours, then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected as the pecan protein solution. The absorbance of the supernatant at 595nm was measured using the Coomassie Brilliant Blue method. The protein concentration in the sample solution was determined by comparing with a protein standard curve. The results are as follows: Figure 3 As shown.

[0096] Depend on Figure 3 It was found that the pH of the alkaline extraction solution affects both the extraction rate and purity of pecan protein. As the pH of the alkaline extraction solution increases, the extraction rate of pecan protein initially increases and then decreases. Within a certain pH range, the higher the pH of the alkaline extraction solution, the higher the extraction efficiency of pecan protein. However, when the pH exceeds a certain threshold, the extraction rate decreases. The extraction rate of pecan protein reaches its maximum of 85.5% at pH 8.0. Furthermore, the purity of pecan protein also initially increases and then decreases with increasing pH of the alkaline extraction solution. At pH 8.0, this trend reaches a certain extreme, meaning the purity of pecan protein reaches its highest point of 83.8%. This may be because, under this pH condition, impurities and other unwanted proteins are removed more thoroughly during the extraction process.

[0097] Both protein extraction rate and protein yield reached their maximum values ​​at pH 8.0, therefore 8.0 can be selected as the pH for the alkaline extraction solution. 2. Pecan protein SDS-PAGE gel electrophoresis.

[0098] The SDS-PAGE gel electrophoresis of pecan protein was performed according to the method described in Gao Yanli, Yang Siwen, and Fan Kaiqi. Study on protein analysis by SDS-PAGE electrophoresis [J]. Liaoning Chemical Industry, 2007, 249(07): 460-463. The specific operation is shown in the table below:

[0099] Table 4. SDS-PAGE gel preparation methods

[0100]

[0101] Key points of operation:

[0102] (1) 10% ammonium persulfate solution should be prepared fresh before use. Ammonium persulfate is highly hygroscopic and very unstable in aqueous solution, therefore this solution should be freshly prepared before use. Preparation method: Dissolve 1g of ammonium persulfate in 10mL of ultrapure water.

[0103] (2) TEMED is a coagulant and is added last. Immediately after adding TEMED, the gel is poured into the electrophoresis tank.

[0104] (3) Weigh 0.05g of pecan protein and dissolve it in 10mL of water until it is fully dissolved, with a final concentration of approximately 5mg / mL. Take 50uL of the supernatant and mix it with 2x protein loading buffer at a volume ratio of 1:1. Boil the mixture at 95℃ for 5min to ensure thorough mixing.

[0105] (4) When loading the sample, add 10 μL of sample to each well, first set the voltage to 50V and run for 30 minutes, then set the voltage to 110V and run until the electrophoresis is finished. The whole time is about 2.5 hours.

[0106] (5) At the end of electrophoresis, remove the gel, add staining solution, and stain in a shaker for about 40 minutes. If the staining effect is not good, it can be stained overnight. After staining, use destaining solution to destain.

[0107] (6) Preparation of staining solution: 1g Coomassie Brilliant Blue (R-250), 300mL ethanol, 100mL acetic acid, and pure water to a final volume of 1000mL. Stir overnight and then filter.

[0108] (7) Preparation of decolorizing solution: Mix 100mL methanol, 100mL glacial acetic acid and 800mL distilled water.

[0109] The 2x protein loading buffer is a standard reagent with the following components: 100mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.2% bromophenol blue, and 3% DTT.

[0110] The pecan protein obtained by alkaline extraction and acid precipitation was subjected to SDS-PAGE protein electrophoresis to analyze its relative molecular weight. The band results are shown below. Figure 4 As shown.

[0111] Depend on Figure 4 It was found that pecan protein exhibited multiple distinct bands in SDS-PAGE, with five bands located between 22.0 kDa and 43 kDa, indicating that the corresponding pecan alkali-extracted proteins with molecular weights between 22.0 kDa and 43 kDa were the most abundant. This is consistent with previous research showing that alkali-extracted proteins are the main protein component of pecans.

[0112] 3. Preparation of Pecan Peptides

[0113] Pecan protein solution was prepared by using a ratio of 1g:10mL of pecan protein to deionized water. A certain amount of protease was added, the pH was adjusted, and the solution was hydrolyzed for 3 hours at a certain temperature.

[0114] 3.1 Selection of enzyme preparations

[0115] In this invention, alkaline protease, neutral protease, papain, flavor protease, and pepsin were selected for study during the hydrolysis process. The final degree of hydrolysis of the enzymatic hydrolysate and its effect on alcohol dehydrogenase were measured, and the results are as follows: Figure 5 As shown in Table 5, the enzymatic hydrolysis conditions for various enzymes are as follows.

[0116] Table 5 Enzymatic hydrolysis conditions for various enzymes

[0117]

[0118] The method for determining the degree of hydrolysis is as follows (the same applies below):

[0119] The average peptide chain length in protein hydrolysis has a linear relationship with the degree of hydrolysis (DH): peptide chain length = 1 / DH. Therefore, DH is selected as an indicator of the degree of protein hydrolysis. The degree of hydrolysis in pecan protein enzymatic hydrolysis was determined using the pH-state method. The specific operation method is as follows:

[0120] At the start of hydrolysis, the pH of the enzymatic hydrolysate was adjusted to the optimum pH. During the reaction, standard alkali solution was continuously added to maintain the pH value of the solution, and the consumption of standard alkali solution was recorded until the end of the reaction. The total consumption of standard alkali solution was accumulated, and the degree of protein hydrolysis was calculated using the following formula:

[0121]

[0122] Where: V: total consumption of standard alkali (mL); Malkali: molar concentration of standard alkali (mol / L);

[0123] in

[0124] pH is the pH of the reaction system; pK is the average dissociation constant of the protons released under the reaction conditions; m: total amount of substrate protein (g); 7.58: number of peptide bonds in the protein (mmol / g).

[0125] ADH activation rate determination method (hereinafter the same):

[0126] Perform the procedure according to the Waller-Hoch method and ADH kit. Add 1.5 mL of sodium pyrophosphate buffer (pH 8.8), 1.0 mL of 0.027 mol / L oxidized coenzyme I (NAD+), 0.1 mL of pecan protein supernatant, and 0.5 mL of 11.5% ethanol solution to a test tube. Mix well and incubate at 25°C for 5 min. Immediately add 0.1 mL of 0.25 U / mL alcohol dehydrogenase (ADH), mix well, and measure the absorbance at 340 nm, zeroing the chamber with double-distilled water. Observe the change in absorbance at 340 nm after 5 min to determine the amount of reduced coenzyme I (NADH) generated. Use distilled water instead of pecan protein peptide solution as a blank control. Calculate the ADH activation rate using the following formula.

[0127] W ADH =(A 1,420nm -A 0,420nm ) / A 0,420nm ×100%

[0128] W ADH —ADH activation rate, %;

[0129] A 1,420nm —The absorbance of the sample at 420 nm;

[0130] A 0,420nm —Absorbance of the control solution at 420 nm.

[0131] Depend on Figure 5 It was found that among the five enzymes, alkaline protease hydrolyzed the functional peptides with the highest ADH activation rate (34.5%), at which point the degree of hydrolysis was 37.2%. Among the five enzymes—alkaline protease, neutral protease, flavor protease, pepsin, and papain—pepsin hydrolyzed the functional peptides with the highest degree of hydrolysis (64.8%), at which point the ADH activation rate was 8.6%. The pepsin hydrolysis product had the highest degree of hydrolysis, but the peptide functionality was very low. This may be because different proteases have different hydrolytic sites, and pepsin may have destroyed the functional regions of the peptides during hydrolysis. Because proteins have different compositions and functions, even with the same enzyme and substrate, different degrees of hydrolysis result in different ADH activities. The alkaline protease hydrolysis product had a degree of hydrolysis of 37.2%, but the ADH activation rate of the pecan peptides it produced was the highest. Therefore, alkaline protease was chosen as the hydrolytic enzyme for pecan protein in the next stage of the experiment.

[0132] 3.2 Effect of enzymatic hydrolysis temperature on peptide preparation

[0133] The effect of temperature on enzymatic hydrolysis was analyzed using the degree of hydrolysis and ADH activation rate as indicators. Based on the preliminary experimental results, single-factor experiments were conducted at hydrolysis temperatures of 45, 55, 65, 75, and 85℃. The amount of alkaline protease added to 100 mL of pecan protein solution was 2 g, the pH was set to 11, and the hydrolysis time was 3 h. The results are as follows: Figure 6 As shown.

[0134] Depend on Figure 6 It was found that when the enzymatic hydrolysis temperature was 55℃, the ADH activation rate reached its minimum of 29.7%, possibly because the enzyme activity was low at the relatively low temperature, but the reaction was not yet complete, leading to a decrease in the ADH activation rate. At 75℃, the enzyme activity reached its maximum, and the ADH activation rate also reached its maximum of 44.3%. As the temperature continued to rise, the enzyme activity began to decrease, and the ADH activation rate decreased accordingly.

[0135] 3.3 Effect of enzymatic hydrolysis pH on peptide preparation

[0136] The effects of pH on enzymatic hydrolysis efficiency were analyzed using the degree of hydrolysis, DPPH scavenging rate, and ADH activation rate as indicators. Based on preliminary experimental results, single-factor experiments were conducted at pH values ​​of 8.0, 9.0, 10.0, 11.0, and 12.0, with an enzyme dosage of 2g, a temperature of 75℃, and a hydrolysis time of 3 hours. The results are as follows. Figure 7 As shown.

[0137] Depend on Figure 7 It was found that under alkaline conditions, pecan protease hydrolysates can activate ADH, thereby enhancing its catalytic activity. This activation phenomenon exhibits a multi-stage increase and decrease trend at different pH values. Among these trends, pH 9.0 and 11.0 represent the points where the ADH activation rate reaches its maximum. Specifically, the ADH activation rate reaches its highest value of 48.6% at pH 11.0, which may be because the molecular structure and charge properties of the pecan protease hydrolysates are most suitable for interaction with ADH at this pH.

[0138] 3.4 Effect of enzyme dosage on peptide preparation

[0139] The effect of alkaline protease dosage on enzymatic hydrolysis was analyzed using the degree of hydrolysis and ADH activation rate as indicators. Based on the preliminary experimental results, single-factor experiments were conducted with alkaline protease dosages of 1, 2, 3, 4, and 5 g in 100 mL of pecan protein solution, adjusting the pH to 11, the temperature to 75℃, and the hydrolysis time to 3 h. The results are as follows. Figure 8 As shown.

[0140] Depend on Figure 8It was found that, within a certain range, the ADH activation rate of the enzymatic hydrolysis product increased with the increase of the amount of alkaline protease added. When the amount of alkaline protease added was 2g, the ADH activation rate reached its highest value. When the amount of protease added exceeded 2g, the ADH activation rate of the enzymatic hydrolysis product decreased and then did not change significantly.

[0141] 4. Isolation of pecan peptides

[0142] The prepared pecan peptides were prepared into solutions of specified concentrations. These solutions were then ultrafiltered using 3kDa, 10kDa, and 30kDa ultrafiltration tubes to obtain pecan peptide fragments with molecular weights of less than 3kDa, 3–10kDa, 10–30kDa, and greater than 30kDa. The proteolytic hydrolysate of each component was separated into solutions of different molecular weights and freeze-dried to obtain pecan peptides of different molecular weights. The DPPH scavenging capacity and ADH activation rate of each peptide group were measured. Results are as follows: Figure 9 As shown.

[0143] Depend on Figure 9 It was found that when the concentration of pecan peptides was 1 mg / mL, solutions of pecan peptide fragments with molecular weights of 3-10 kDa and 10-30 kDa exhibited strong ADH activation ability, especially the 3-10 kDa fragments. ADH is a cellular enzyme that includes subunits and cofactors. It can promote alcohol metabolism and play a detoxifying role. Therefore, the ADH activation effect of pecan peptide fragments is expected to have health benefits, potentially regulating alcohol metabolism, relieving hangovers, and protecting the liver. This result further demonstrates the potential role of pecan peptides as a natural health food and medicine, and also provides a scientific basis for the development of related health products.

[0144] 5. Single-factor experiment on hangover remedies

[0145] The effects of the amount of pecan peptides with a molecular weight of 3-10 kDa prepared in Example 1 on the sensory evaluation of the beverage were investigated through single-factor experiments to determine the optimal beverage formula.

[0146] Sensory evaluation methods

[0147] Sensory evaluation is conducted based on the sensory characteristics of the beverage raw materials and ingredients themselves, evaluating them from four aspects: color, aroma, taste, and morphological characteristics, accounting for 20, 30, 30, and 20 points respectively, with a total score of 100 points. A sensory evaluation panel of 10 people (5 men and 5 women) with food science backgrounds was organized to conduct the evaluation. The specific evaluation criteria are shown in Table 2.5.

[0148] Table 6 Sensory Evaluation Scoring Criteria

[0149]

[0150] 5.1 Effect of Pecan Peptide Addition on Beverage

[0151] The amount of white sugar added was 5%. Sensory evaluation was used to explore the effect of the amount of pecan peptides added on the sensory properties of the beverage, and to develop a pecan peptide beverage. The results are as follows: Figure 10 As shown.

[0152] Depend on Figure 10 It can be seen that the beverage achieves the highest sensory score when the pecan peptide content is 6%. Within this concentration range, the beverage's color, taste, mouthfeel, off-flavors, and overall evaluation are all relatively good. When the pecan peptide content is 2%, the beverage has almost no pecan aroma. When the pecan peptide content is 4%, the pecan aroma score is relatively low. When the pecan peptide content exceeds 6%, an astringent taste begins to appear in the beverage, leading to a poorer mouthfeel and a lower sensory score.

[0153] 5.2 The effect of added white sugar on beverages

[0154] When the pecan peptide content is 6%, sensory evaluation was conducted to investigate the effect of added white sugar on the sensory properties of the beverage, in order to develop a pecan peptide beverage. The results are as follows: Figure 11 As shown.

[0155] Depend on Figure 11 It can be seen that the beverage achieves the highest sensory score when the added white sugar content is 10%. Within this concentration range, the beverage's color, taste, texture, off-flavors, and overall evaluation are all relatively good. When the added white sugar content is 5%, the beverage has almost no sweetness, and the sensory score decreases. When the added white sugar content exceeds 10%, the beverage becomes too sweet, resulting in a poor taste and a lower sensory score.

Claims

1. The application of pecan peptides in hangover remedies, characterized in that, The preparation method of pecan nut polypeptide includes the following steps: 1) Pecan kernel pretreatment: Pecan kernels are pretreated to obtain defatted pecan powder; 2) Preparation of pecan protein: Sodium chloride is added to water to make a sodium chloride solution with a concentration of 0.05-0.25 mol / L. Then defatted pecan powder from step 1) is added, mixed, and the resulting liquid is prepared by alkaline extraction and acid precipitation to obtain pecan protein. 3) Pecan protein hydrolysis: Add pecan protein from step 2) to water, mix to obtain pecan protein solution, add alkaline protease for hydrolysis, enzyme inactivation, filtration, freeze drying to obtain pecan polypeptides with a molecular weight of 3-30 kDa. In step 3), the ratio of pecan protein to water is 1g:(5-20)mL, and the ratio of alkaline protease to pecan protein solution is (1-5)g:100mL. The pH is adjusted to 8-12 using 2 mol / L sodium hydroxide solution, and hydrolysis is carried out at 55-85℃ for 2-4 hours.

2. The application as described in claim 1, characterized in that, The hangover relief beverage is made from the following ingredients by weight percentage: 4-10% pecan peptides, 5-25% white sugar, and the remainder is water.

3. The application as described in claim 1, characterized in that, In step 2), the ratio of defatted pecan powder to water in the liquid is 1g:(10-100)mL; the steps of the alkaline extraction and acid precipitation method are as follows: adjust the pH of the liquid to 7-11 with sodium hydroxide solution, extract in a water bath, centrifuge, collect the supernatant, adjust the pH of the supernatant to 3-5 with hydrochloric acid solution, centrifuge, collect the precipitate, freeze dry to obtain pecan protein, and use deionized water.

4. The application as described in claim 3, characterized in that, In step 2), the pH of the feed solution was adjusted to 8-9 using 2 mol / L sodium hydroxide solution, and the mixture was extracted in a water bath at 40℃ for 10-14 h. After centrifugation at 4000 r / min for 10 min, the supernatant was collected. The pH of the supernatant was then adjusted to 3.5-4.5 using 6 mol / L hydrochloric acid solution, and the mixture was centrifuged at 4000 r / min for 10 min. The precipitate was collected and freeze-dried to obtain pecan protein.

5. The application as described in claim 4, characterized in that, In step 3), the enzyme inactivation method is as follows: the hydrolyzed enzyme hydrolysate is heated to 90-100℃ and kept at that temperature for 10-30 min; the filtration is carried out by molecular membrane filtration and freeze drying to obtain pecan peptides.

6. The application as described in claim 1, characterized in that, In step 1), the method for pre-treating pecans is as follows: remove impurities from pecan kernels, press them, defatt them with petroleum ether, centrifuge them, take out the sediment, dry them at low temperature, and pulverize them to obtain defatted pecan powder.

7. The application as described in claim 6, characterized in that, In step 1), petroleum ether defatting: the defatted pecan pulp obtained after pressing is mixed with petroleum ether at a mass ratio of 1:1 and stirred for 1-3 hours to obtain a defatted solid-liquid mixture; Centrifugation: Centrifuge the solid-liquid mixture at 800 r / min for 5-20 min and collect the sediment; Low-temperature drying: Dry at 30-60℃.

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