Low-bitter amygdalin photonic walnut meal antioxidant polypeptide and preparation method and application thereof

Abstract

The application belongs to the field of food processing and biotechnology, and specifically discloses a low-bitter amygdalin light walnut meal antioxidant polypeptide as well as a preparation method and application thereof. The light walnut meal antioxidant polypeptide is prepared by using the waste light walnut cold-pressed fruit meal as the only raw material. In view of the dual characteristics of high protein and high amygdalin, an optimized process route of 'ultrasonic-assisted extraction-endogenous enzyme controllable hydrolysis detoxification-stepwise composite enzymatic hydrolysis-flavor modification-membrane separation-macroporous resin deep detoxification-concentration and drying' is designed. The optimal parameters of each step are determined through single-factor experiments combined with orthogonal experiments, so that the efficient dissolution of protein, the synchronous removal of amygdalin, the directional preparation of polypeptide and the improvement of flavor are realized. The light walnut meal is used as the raw material to prepare the antioxidant polypeptide for the first time. Through the two-stage detoxification system of 'endogenous enzyme hydrolysis + macroporous resin adsorption', the residual amount of amygdalin in the product can be reduced, and finally the polypeptide product with high antioxidant activity, low bitterness and high safety is obtained.

Description

Technical Field

[0001] This invention belongs to the field of food processing and biotechnology, and relates to an antioxidant polypeptide of low amygdalin from walnut meal, its preparation method and application. Specifically, it is a polypeptide product with excellent antioxidant activity and low amygdalin residue obtained by extracting and purifying the fruit meal after cold pressing of walnut (Prunus mira Koehne) through an optimized process, as well as its preparation method and application in cosmetics. Background Technology

[0002] The Tibetan walnut (Prunus mira Koehne), also known as the Tibetan peach or wild peach, is mainly distributed in the plateau regions of Tibet, northwestern Yunnan, and southwestern Sichuan in China, at altitudes of 2000–4000 m. It is a plant endemic to China. The kernel of the Tibetan walnut has an oil content as high as 44%–51%, with over 90% of its oil being unsaturated fatty acids, primarily oleic and linoleic acids. It has extremely high nutritional value and has been developed into a high-end edible oil and cosmetic oil. However, in the production of Tibetan walnut oil, a large amount of the pulp (oil meal) left after oil extraction is not utilized for high-value purposes. Traditionally, it is only used as low-value feed or disposed of as waste, resulting in a serious waste of high-quality plant protein resources.

[0003] The walnut (Prunus spp.) belongs to the genus Prunus in the family Rosaceae. Its kernels are similar to peach kernels and bitter almonds, containing a high amount of amygdalin (molecular formula: Prunus spp.). (Molecular weight 457.43 Da). Amygdalin itself has low toxicity, but it can be hydrolyzed by β-glucosidase (endogenous or exogenous enzyme) to produce prunasin, which further hydrolyzes to produce benzaldehyde and highly toxic hydrocyanic acid (HCN). Hydrocyanic acid (HCN) can inhibit cytochrome oxidase and block the respiratory chain in tissues; its lethal dose for adults is approximately 0.5–3.5 mg / kg body weight (approximately 30–210 mg for a 60 kg body weight). The toxicity of amygdalin mainly comes from the hydrocyanic acid released during its hydrolysis. Low-temperature cold pressing oil extraction processes, due to their low temperature (usually <60℃), cannot destroy amygdalin and its hydrolytic enzymes. Furthermore, amygdalin is highly water-soluble and weakly fat-soluble; therefore, the oilseed meal after pressing is rich in amygdalin. If used directly for peptide preparation without detoxification, the product poses a serious food safety risk, and the bitterness of amygdalin severely affects the sensory quality and palatability of the peptide product.

[0004] Current research on walnut-derived peptides mainly focuses on common walnuts. However, common walnuts belong to the genus Juglans of the family Juglandaceae, and their amygdalin content is extremely low, so detoxification is not a concern in peptide processing. Polished walnuts, on the other hand, belong to different families and genera, and their protein composition, amino acid sequence, and functional properties differ significantly from those of common walnuts. Directly applying the peptide preparation process for common walnuts not only fails to solve the detoxification problem of amygdalin in polished walnut meal but also makes it difficult to obtain ideal peptide yields and activities. Therefore, it is necessary to develop a process specifically tailored to the characteristics of polished walnut meal.

[0005] In addition, existing methods for detoxifying amygdalin (long-term soaking, high-temperature boiling, acid-base treatment, and simple enzymatic / fermentation methods) have the following drawbacks:

[0006] (1) Prolonged soaking leads to a large loss of protein (significant increase in protein content in wastewater) and a significant decrease in peptide yield;

[0007] (2) High-temperature boiling destroys the protein structure and consumes a lot of energy, resulting in reduced enzymatic hydrolysis efficiency and loss of activity;

[0008] (3) Acid and alkali treatment introduces chemical residues, affecting the safety of the product;

[0009] (4) Simple enzymatic or fermentation methods require the addition of additional microbial strains / enzymes, which increases costs and makes the process complicated;

[0010] (5) Most existing technologies do not integrate detoxification and peptide preparation into a single design, or the coupling method has certain defects, resulting in lengthy processes, serious protein loss, and poor industrialization feasibility.

[0011] In view of the above-mentioned technical problems, this invention is proposed. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide low-amygin antioxidant peptides from walnut meal, their preparation methods, and applications, specifically addressing the following technical problems: (1) Solve the technical bottleneck that walnut meal cannot be directly used for polypeptide preparation due to the enrichment of amygdalin; (2) A method for preparing walnut meal polypeptides that is simple in process, cost controllable, suitable for industrial production, and can fully retain protein activity and simultaneously remove amygdalin; (3) Solve the problem of severe protein loss and low peptide yield caused by existing detoxification methods; (4) Solve the industrialization problem of detoxification and enzymatic hydrolysis processes being disconnected, complicated steps, and high costs; (5) Prepare a light walnut meal polypeptide product with excellent antioxidant activity, low bitterness, high safety and no risk of amygdalin residue; (6) Expand the application field of this extract and provide a natural and efficient functional raw material for antioxidant cosmetics.

[0013] The primary objective of this invention is to provide a method for preparing low-amygin antioxidant peptides from walnut meal, comprising the following steps:

[0014] S1: Raw material pretreatment

[0015] Select the fruit pulp after cold pressing of walnuts for oil extraction, remove impurities, and dry it in a constant temperature drying oven at 60-70℃ until the moisture content is ≤8%; then crush it using a universal pulverizer, pass it through a 40-100 mesh sieve, collect the sieve-underfilled powder, and seal it for later use.

[0016] S2: Ultrasonic-assisted extraction

[0017] Add walnut meal powder to deionized water at a material-to-liquid ratio of 1:10-1:30 (g / mL), adjust the pH to 6.5-7.5, and sonicate for 15-30 min at an ultrasonic power of 200-600 W, a frequency of 20-40 kHz, and a temperature of 45-55℃ to obtain an ultrasonically treated solution. The ultrasonic cavitation effect can disrupt the compact structure of proteins, increase the protein dissolution rate, and simultaneously promote the transfer of amygdalin to the aqueous phase.

[0018] S3: Endogenous enzyme-controlled hydrolysis and detoxification

[0019] The ultrasonically treated liquid is transferred to a reactor equipped with a sealed stirring device and a negative pressure gas extraction system. It is maintained at a temperature of 40-55℃, pH 6.0-7.0, and a stirring speed of 100-300 r / min for 30-90 min, allowing the endogenous β-glucosidase in the walnut meal to hydrolyze amygdalin into benzaldehyde and hydrogen cyanide. The hydrogen cyanide (HCN) gas generated during hydrolysis is continuously evacuated through the reactor headspace and introduced into an alkaline absorption tower (such as NaOH solution) for neutralization and absorption, achieving safe discharge. Subsequently, the temperature is raised to 80-90℃ and maintained for 10-15 min to inactivate the endogenous enzyme. The solution is then cooled to 50-55℃ to obtain the detoxified liquid.

[0020] S4: Stepwise complex enzymatic hydrolysis

[0021] S41: Alkaline protease hydrolysis

[0022] Add alkaline protease to the detoxification solution at a concentration of 0.5%-2.0% of the mass of the walnut meal powder. Adjust and maintain the pH at 7.5-8.5 and the temperature at 50-55℃, and allow the enzyme to hydrolyze for 1.5-2.5 hours.

[0023] S42: Neutral protease hydrolysis

[0024] Adjust the pH of the system in step S41 to 6.5-7.5, add neutral protease at a concentration of 0.5%-1.5% of the mass of the walnut meal powder, and continue enzymatic hydrolysis at 45-55℃ for 1-2 hours. Stir and monitor the pH every 30 minutes during this period. If necessary, use 1 mol / L NaOH or 1 mol / L HCl to maintain pH stability to obtain the first enzymatic hydrolysate.

[0025] The total amount of alkaline protease and neutral protease added is 1.0%-3.0% of the mass of the walnut meal powder, and the mass ratio of the two is 1:1-3:1.

[0026] S5: Flavor Modification

[0027] The pH of the primary enzymatic hydrolysate was adjusted to 6.0-7.0, and flavor protease was added at a concentration of 0.5%-1.5% of the mass of the walnut meal powder. Enzymatic hydrolysis was carried out at 45-50℃ for 1-2 hours to obtain the secondary enzymatic hydrolysate. The flavor protease can cleave the hydrophobic amino acids at the ends of peptide chains, significantly reducing the bitterness of the product and improving its sensory quality.

[0028] S6: Enzyme inactivation

[0029] Heat the secondary enzymatic hydrolysate to 90-95℃ and hold for 10-15 minutes to inactivate the enzyme, then quickly cool to room temperature.

[0030] S7: Separation and Purification

[0031] Centrifuge the enzyme-inactivated hydrolysate at 4000-8000 r / min for 15-20 min to remove the precipitate; filter the supernatant through a 0.22-0.45 μm microfiltration membrane, and then perform ultrafiltration fractionation through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da at an operating pressure of 0.1-0.3 MPa and a temperature of 25-30℃. Collect the permeate to obtain the ultrafiltration permeate.

[0032] S8: Deep Detoxification of Macroporous Resin

[0033] The ultrafiltration permeate is passed through a chromatography column packed with macroporous adsorption resin at a flow rate of 1-3 BV / h, and the effluent is collected. The macroporous adsorption resin is a weakly polar styrene-type macroporous adsorption resin, specifically D101, AB-8, or HPD100.

[0034] In this step, the macroporous adsorption resin is activated with 95% ethanol and washed with deionized water until there is no alcohol odor before use; the column diameter-to-height ratio is 1:4-1:8, and the sample loading amount is 5%-15% of the volume of the macroporous adsorption resin, calculated as peptides.

[0035] AB-8 type weakly polar macroporous adsorption resin exhibits selective adsorption capacity for amygdalin. The benzene ring in the amygdalin molecule interacts π-π with the styrene skeleton of the resin, and multiple hydroxyl groups of the sugar unit form hydrogen bonds with polar groups on the resin surface, leading to its adsorption and retention by the resin. In contrast, oligopeptides with a pH << 1000 Da have a high degree of ionization and strong hydration under these pH conditions, exhibiting weak affinity for the resin and preferentially eluting with the solution. Therefore, an "elution" mode is employed, and the collected effluent is the detoxified peptide solution, with amygdalin removed by resin adsorption.

[0036] S9: Concentrated and dried

[0037] The effluent is concentrated under vacuum to a solid content of 15%-25%, and then freeze-dried or spray-dried to obtain low-bitter amygdalin-containing walnut meal antioxidant polypeptide powder.

[0038] Preferably, in step S1, the material is pulverized through a 60-80 mesh sieve.

[0039] Preferably, in step S2, the material-to-liquid ratio is 1:15-1:20, pH=7, ultrasonic power is 300-500 W, frequency is 28 kHz, temperature is 50℃, and ultrasonic treatment is performed for 20 min.

[0040] Preferably, step S3 is as follows: the ultrasonic treatment liquid is transferred to a reaction vessel equipped with a closed stirring device and a negative pressure gas exhaust system, and kept at a temperature of 45-50℃, pH 6.5-7.0, and stirring speed of 150-250 r / min for 45-60 min, then heated to 85℃ and kept for 12 min to inactivate endogenous enzymes, and then cooled to 50-55℃ to obtain a detoxified treatment liquid.

[0041] Preferably, in step S3, before inactivating the endogenous enzyme by heating, a centrifugation separation step is also included: the hydrolyzed treatment solution is centrifuged at 3000-5000 r / min for 10-15 min, the precipitate is collected, the precipitate is washed with deionized water 1-2 times, and the washing liquid and supernatant are combined for the recovery of benzaldehyde (amygin has been fully hydrolyzed by the endogenous enzyme, and the supernatant mainly contains benzaldehyde and a very small amount of unhydrolyzed amygin), and the washed precipitate is used for the stepwise compound enzymatic hydrolysis in step S4.

[0042] Preferably, in step S41, the amount of enzyme added is 1.3% of the mass of the walnut meal powder, and the pH is adjusted and maintained at 8.0 and the temperature at 50°C for 2 hours of enzymatic hydrolysis.

[0043] Preferably, in step S42, the pH is adjusted to 7.0, the amount of enzyme added is 0.7% of the mass of the walnut meal powder, the temperature is 50℃, and the enzymatic hydrolysis is carried out for 1 h.

[0044] Preferably, the total amount of alkaline protease and neutral protease added is 2.0% of the mass of the walnut meal powder, and the mass ratio of the two is 2:1.

[0045] Preferably, in step S5, the pH is adjusted to 6.5, the amount of enzyme added is 1.0% of the mass of the walnut meal powder, and the enzymatic hydrolysis is carried out at 48℃ for 1.5 h.

[0046] Preferably, in step S6, the secondary enzymatic hydrolysate is heated to 95°C and maintained for 10 min.

[0047] Preferably, step S7 is as follows: the enzyme hydrolysate after enzyme inactivation is centrifuged at 6000 r / min for 15-20 min to remove the precipitate; the supernatant is filtered through a 0.45 μm microfiltration membrane and then ultrafiltration fractionated through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, with an operating pressure of 0.2 MPa and a temperature of 25-30℃, and the permeate is collected to obtain the ultrafiltration permeate.

[0048] Preferably, the flow rate in step S8 is 2 BV / h, and the macroporous adsorption resin is type AB-8.

[0049] Preferably, the macroporous adsorption resin is activated with 95% ethanol and washed with deionized water until there is no alcohol odor before use; the column diameter-to-height ratio is 1:6, and the sample loading amount is 10% of the volume of the macroporous adsorption resin based on peptides.

[0050] Preferably, the macroporous resin effluent obtained in step S8 can also be subjected to the following operation: passing the macroporous resin effluent through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da at an operating pressure of 0.5-1.5 MPa to remove inorganic salts and small molecule bitter substances (such as free benzaldehyde and short-chain bitter peptides).

[0051] Using the above technical solution, this invention uses waste cold-pressed walnut meal as the sole raw material. Targeting its dual characteristics of high protein and high amygdalin content, it optimizes the process route as follows: "ultrasound-assisted extraction - controlled hydrolysis and detoxification with endogenous enzymes - stepwise complex enzymatic hydrolysis - flavor modification - membrane separation - deep detoxification with macroporous resin - concentration and drying." Optimal parameters for each step are determined through single-factor experiments combined with orthogonal experiments, achieving efficient protein dissolution, simultaneous removal of amygdalin, targeted peptide preparation, and flavor improvement. This invention is the first to prepare antioxidant peptides from walnut meal. Through a two-stage detoxification system of "endogenous enzyme hydrolysis + macroporous resin adsorption," the residual amygdalin content in the product can be reduced, while simultaneously recovering byproducts (benzaldehyde), ultimately yielding a peptide product with high antioxidant activity, low bitterness, and high safety.

[0052] This invention uses the pulp from cold-pressed walnuts as the sole raw material. The walnut pulp is preferably dried pulp obtained after low-temperature cold pressing (temperature ≤60℃) and is pretreated before being used as a raw material for enzymatic hydrolysis. The main active ingredient of the polypeptide product is small molecule peptides, of which oligopeptides with a molecular weight <1000 Da account for 68±2% (HPGPC method), and oligopeptides with a molecular weight <500 Da account for 35±3%, with a total polypeptide content of 82.5±1.2% (biuret method). The product obtained by this invention is a white to pale yellow powder, odorless, with good flowability, a moisture content of 3.8±0.3%, an ash content of 2.5±0.2%, a pH value (1% aqueous solution) of 6.5±0.3, amygdalin residue of 3.2±0.5 mg / kg, hydrocyanic acid content of 0.6±0.1 mg / kg, and benzaldehyde residue of 5±0.8 mg / kg. The antioxidant activity index is the DPPH free radical scavenging rate IC50. 50 ≤2.5 mg / mL; Heavy metal content: lead <0.5 mg / kg, arsenic <0.3 mg / kg, meeting the requirements of the "Cosmetic Safety Technical Specifications" and GB 2762. The peptide yield is expressed as the percentage of crude peptide powder mass to the mass of walnut meal powder, or as the pure peptide yield (crude peptide powder yield × peptide content).

[0053] The second objective of this invention is to provide a low-amyglucan antioxidant polypeptide from walnut meal prepared by the above method, wherein the proportion of oligopeptides with a molecular weight <1000 Da is ≥60% (HPGPC method), the total polypeptide content is ≥80% (biuret method), and the antioxidant activity index is DPPH free radical scavenging rate IC50 ≤2.5 mg / mL.

[0054] A third objective of this invention is to provide the application of the above-mentioned low-amyglucan-containing walnut meal antioxidant polypeptide in cosmetics.

[0055] Preferably, the low-amyglucan-containing walnut meal antioxidant polypeptide is used in the preparation of antioxidant cosmetics.

[0056] The beneficial effects of this invention are:

[0057] (1) Advantages of resource utilization: This invention uses the cold-pressed fruit meal of walnut, which is discarded or used at low value in traditional development, as raw material, and realizes the high-value utilization of the whole fruit of walnut plant resources (oil pressing + peptide production + benzaldehyde recovery), reducing resource waste and environmental burden. At the same time, the raw materials are widely available and inexpensive.

[0058] (2) Process advantages: The optimized process of "ultrasound-assisted extraction - endogenous enzyme controllable hydrolysis and detoxification - stepwise compound enzymatic hydrolysis - flavor modification - membrane separation - deep detoxification with macroporous resin - drying" is simple in steps, clear in parameters, and highly controllable, making it suitable for large-scale industrial production. By revealing the optimal cavitation window of 400 W ultrasound, the detoxification-retention balance point of 50℃ / 60 min, the synergistic ratio of alkaline protease: neutral protease = 2:1, and the synergistic separation mechanism of "membrane separation + resin adsorption", the coupling of efficient protein dissolution and simultaneous removal of amygdalin was achieved.

[0059] (3) Safety advantages: Through the two-stage detoxification system of "endogenous enzyme hydrolysis + macroporous resin adsorption", the residual amount of amygdalin in the product is ≤5 mg / kg and HCN is ≤1.0 mg / kg, which meets the safety requirements of cosmetic raw materials. After multiple skin irritation tests, acute eye irritation tests, guinea pig skin allergy tests and human patch tests, no skin irritation or sensitization was shown under the test conditions.

[0060] (4) Efficacy advantages: The obtained polypeptides have small molecular weight (<1000 Da accounted for ≥60%), excellent antioxidant activity, DPPH free radical scavenging rate IC50≤2.5 mg / mL, ABTS free radical scavenging rate IC50≤1.5 mg / mL, hydroxyl free radical scavenging rate (5mg / mL)>70%, and strong total reducing power. They can be used as natural antioxidants to replace chemically synthesized antioxidants.

[0061] (5) Sensory quality advantages: After modification with flavor protease, the bitterness score of the product drops from 3.5 to below 0.5. It is white in color and odorless, and can be directly applied to cosmetics without additional taste masking treatment.

[0062] (6) Advantages of by-product recovery: By adding a centrifugation separation step after endogenous enzyme hydrolysis and before enzyme inactivation, benzaldehyde by-product can be efficiently recovered from the hydrolysis supernatant (recovery rate >75%) and used in the fragrance industry, realizing the full utilization of raw materials and maximizing economic value.

[0063] (7) Application advantages: This polypeptide has a wide range of applications and can be used directly or in combination with other raw materials to prepare antioxidant cosmetics (serums, creams, masks), filling the application gap of walnut meal polypeptide in related fields. Attached Figure Description

[0064] Figure 1 This is a high-performance liquid chromatogram of the product of Example 1 of the present invention.

[0065] Figure 2 This is a high-performance liquid chromatogram of the product in Example 2 of the present invention.

[0066] Figure 3This is a high-performance liquid chromatogram of the product of Comparative Example 1 of the present invention.

[0067] Figure 4 This is a high-performance liquid chromatogram of the product of Comparative Example 2 of the present invention.

[0068] Figure 5 This is a high-performance liquid chromatogram of the product of Comparative Example 3 of the present invention.

[0069] Figure 6 This is a high-performance liquid chromatogram of the product of Comparative Example 4 of the present invention.

[0070] Figure 7 This is the high-performance liquid chromatogram of amygdalin. Detailed Implementation

[0071] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0072] The technical solution adopted in this invention is as follows:

[0073] A method for preparing a low-amygin antioxidant polypeptide from walnut meal includes the following steps:

[0074] S1: Raw material pretreatment

[0075] Select the fruit pulp after cold pressing of walnuts for oil extraction, remove impurities, and dry it in a constant temperature drying oven at 60-70℃ until the moisture content is ≤8%; then crush it using a universal pulverizer and pass it through a 40-100 mesh sieve (non-limiting examples, such as 40, 50, 60, 80, 90, 100 mesh, etc.), collect the sieve-passed powder, and seal it for later use.

[0076] S2: Ultrasonic-assisted extraction

[0077] Walnut meal powder was added to deionized water at a material-to-liquid ratio of 1:10-1:30 (g / mL) (non-limiting examples, such as 1:10, 1:15, 1:20, 1:25, 1:30, etc.), and the pH was adjusted to 6.5-7.5 (non-limiting examples, such as 6.5, 7.0, 7.5, etc.). The mixture was then ultrasonically treated for 15-30 min (non-limiting examples, such as 15, 20, 30, 40 kHz, etc.) at an ultrasonic power of 200-600 W (non-limiting examples, such as 200, 400, 600 W, etc.), a frequency of 20-40 kHz (non-limiting examples, such as 20, 30, 40 kHz, etc.), and a temperature of 45-55℃ (non-limiting examples, such as 45, 50, 55℃, etc.) to obtain an ultrasonically treated solution. The ultrasonic cavitation effect can disrupt the compact structure of proteins, increase protein dissolution rate, and simultaneously promote the transfer of amygdalin to the aqueous phase.

[0078] S3: Endogenous enzyme-controlled hydrolysis and detoxification

[0079] The ultrasonically treated liquid is transferred to a reactor equipped with a sealed stirring device and a negative pressure gas extraction system. It is maintained at a temperature of 40-55℃, pH 6.0-7.0, and a stirring speed of 100-300 r / min for 30-90 min, allowing the endogenous β-glucosidase in the walnut meal to hydrolyze amygdalin into benzaldehyde and hydrogen cyanide. The hydrogen cyanide (HCN) gas generated during hydrolysis is continuously evacuated through the reactor headspace and introduced into an alkaline absorption tower (such as NaOH solution) for neutralization and absorption, achieving safe discharge. Subsequently, the temperature is raised to 80-90℃ and maintained for 10-15 min to inactivate the endogenous enzyme. The solution is then cooled to 50-55℃ to obtain the detoxified liquid.

[0080] S4: Stepwise complex enzymatic hydrolysis

[0081] S41: Alkaline protease hydrolysis

[0082] Add alkaline protease to the detoxification solution at a concentration of 0.5%-2.0% of the mass of the walnut meal powder (non-limiting examples, such as 0.5%, 1.0%, 1.5%, 2.0%, etc.), adjust and maintain the pH at 7.5-8.5 and the temperature at 50-55℃, and perform enzymatic hydrolysis for 1.5-2.5 h.

[0083] S42: Neutral protease hydrolysis

[0084] Adjust the pH of the system in step S41 to 6.5-7.5, add neutral protease, and add 0.5%-1.5% of the mass of the walnut meal powder (non-limiting examples, such as 0.5%, 1.0%, 1.5%, etc.). Continue enzymatic hydrolysis for 1-2 hours at a temperature of 45-55℃, stirring every 30 minutes and monitoring the pH. If necessary, use 1 mol / L NaOH or 1 mol / L HCl to maintain pH stability to obtain the first enzymatic hydrolysate.

[0085] The total amount of alkaline protease and neutral protease added is 1.0%-3.0% of the mass of the walnut meal powder, and the mass ratio of the two is 1:1-3:1.

[0086] S5: Flavor Modification

[0087] The pH of the primary enzymatic hydrolysate was adjusted to 6.0-7.0, and flavor protease was added at a concentration of 0.5%-1.5% of the mass of the walnut meal powder (non-limiting examples, such as 0.5%, 1.0%, 1.5%, etc.). Enzymatic hydrolysis was carried out at 45-50℃ for 1-2 hours to obtain the secondary enzymatic hydrolysate. The flavor protease can cleave the hydrophobic amino acids at the ends of peptide chains, significantly reducing the bitterness of the product and improving its sensory quality.

[0088] S6: Enzyme inactivation

[0089] Heat the secondary enzymatic hydrolysate to 90-95℃ and hold for 10-15 minutes to inactivate the enzyme, then quickly cool to room temperature.

[0090] S7: Separation and Purification

[0091] Centrifuge the enzyme-inactivated hydrolysate at 4000-8000 r / min for 15-20 min to remove the precipitate; filter the supernatant through a 0.22-0.45 μm microfiltration membrane, and then perform ultrafiltration fractionation through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da at an operating pressure of 0.1-0.3 MPa and a temperature of 25-30℃. Collect the permeate to obtain the ultrafiltration permeate.

[0092] S8: Deep Detoxification of Macroporous Resin

[0093] The ultrafiltration permeate is passed through a chromatography column packed with macroporous adsorption resin at a flow rate of 1-3 BV / h, and the effluent is collected; the macroporous adsorption resin is of type D101, AB-8 or HPD100.

[0094] In this step, the macroporous adsorption resin is activated with 95% ethanol and washed with deionized water until there is no alcohol odor before use; the column diameter-to-height ratio is 1:4-1:8, and the sample loading amount is 5%-15% of the volume of the macroporous adsorption resin, calculated as peptides.

[0095] AB-8 type weakly polar macroporous adsorption resin exhibits selective adsorption capacity for amygdalin. The benzene ring in the amygdalin molecule interacts π-π with the styrene skeleton of the resin, and multiple hydroxyl groups of the sugar unit form hydrogen bonds with polar groups on the resin surface, leading to its adsorption and retention by the resin. In contrast, oligopeptides with a pH << 1000 Da have a high degree of ionization and strong hydration under these pH conditions, exhibiting weak affinity for the resin and preferentially eluting with the solution. Therefore, an "elution" mode is employed, and the collected effluent is the detoxified peptide solution, with amygdalin removed by resin adsorption.

[0096] S9: Concentrated and dried

[0097] The effluent is concentrated under vacuum to a solid content of 15%-25%, and then freeze-dried or spray-dried to obtain low-bitter amygdalin-containing walnut meal antioxidant polypeptide powder.

[0098] In some embodiments, the material is pulverized through a 60-80 mesh sieve in step S1 (non-limiting examples, such as 60, 65, 70, 75, 80 mesh, etc.).

[0099] In some embodiments, the material-to-liquid ratio in step S2 is 1:15-1:20 (non-limiting examples, such as 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.), pH=7, ultrasonic power 300-500 W (non-limiting examples, such as 300, 350, 400, 450, 500 W, etc.), frequency 28 kHz, temperature 50℃, and ultrasonic treatment for 20 min.

[0100] In some embodiments, step S3 is as follows: the ultrasonic treatment liquid is transferred to a reactor equipped with a closed stirring device and a negative pressure gas exhaust system, and kept at a temperature of 45-50°C, pH 6.5-7.0, and stirring speed of 150-250 r / min for 45-60 min, then heated to 85°C and kept for 12 min to inactivate endogenous enzymes, and then cooled to 50-55°C to obtain a detoxified treatment liquid.

[0101] In some embodiments, before inactivating the endogenous enzyme by heating in step S3, a centrifugation separation step is further included: the hydrolyzed treatment solution is centrifuged at 3000-5000 r / min for 10-15 min, the precipitate is collected, the precipitate is washed with deionized water 1-2 times, and the washing solution and supernatant are combined for the recovery of benzaldehyde (amyglucosinolate has been fully hydrolyzed by the endogenous enzyme, and the supernatant mainly contains benzaldehyde and a very small amount of unhydrolyzed amyglucosinolate), and the washed precipitate is used for the stepwise compound enzymatic hydrolysis in step S4.

[0102] In some embodiments, in step S41, the amount of enzyme added is 1.3% of the mass of the walnut meal powder, and the pH is adjusted and maintained at 8.0 and the temperature at 50°C for 2 hours of enzymatic hydrolysis.

[0103] In some embodiments, in step S42, the pH is adjusted to 7.0, the amount of enzyme added is 0.7% of the mass of the walnut meal powder, the temperature is 50°C, and the enzymatic hydrolysis is carried out for 1 h.

[0104] In some embodiments, the total amount of alkaline protease and neutral protease added is 2.0% of the mass of the walnut meal powder, and the mass ratio of the two is 2:1.

[0105] In some embodiments, in step S5, the pH is adjusted to 6.5, the amount of enzyme added is 1.0% of the mass of the walnut meal powder, and the enzymatic hydrolysis is carried out at 48°C for 1.5 h.

[0106] In some embodiments, in step S6, the secondary enzymatic hydrolysate is heated to 95°C and held for 10 min.

[0107] In some embodiments, step S7 is as follows: the enzyme hydrolysate after enzyme inactivation is centrifuged at 6000 r / min for 15-20 min to remove the precipitate; the supernatant is filtered through a 0.45 μm microfiltration membrane and then ultrafiltration fractionated through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, with an operating pressure of 0.2 MPa and a temperature of 25-30℃, and the permeate is collected to obtain the ultrafiltration permeate.

[0108] In some embodiments, the flow rate in step S8 is 2 BV / h, and the macroporous adsorption resin is type AB-8.

[0109] In some embodiments, the macroporous adsorption resin is activated with 95% ethanol and washed with deionized water until there is no alcohol odor before use; the column diameter-to-height ratio is 1:6, and the sample loading amount is 10% of the volume of the macroporous adsorption resin based on peptides.

[0110] In some embodiments, the macroporous resin effluent obtained in step S8 may also be subjected to the following operation: passing the macroporous resin effluent through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da at an operating pressure of 0.5-1.5 MPa to remove inorganic salts and small molecule bitter substances (such as free benzaldehyde and short-chain bitter peptides).

[0111] The low-amyglucan antioxidant peptides obtained by the above method have an oligopeptide content of ≥60% (HPGPC method) with a molecular weight <1000 Da and a total peptide content of ≥80% (biuret method); the antioxidant activity index is DPPH free radical scavenging rate IC50 ≤2.5 mg / mL.

[0112] Application of the above-mentioned low-amyglucan-containing antioxidant peptides from walnut meal in cosmetics.

[0113] Example 1

[0114] A method for preparing a low-amygin antioxidant polypeptide from walnut meal includes the following steps:

[0115] S1: Raw material pretreatment

[0116] The fruit meal after cold pressing of walnuts (with amygdalin content of 1.8% on a dry basis) was dried in hot air at 60℃ until the moisture content was ≤8%, then pulverized in a universal pulverizer and passed through a 60-mesh sieve to obtain walnut meal powder.

[0117] S2: Ultrasonic-assisted extraction

[0118] Weigh 100 g of walnut meal powder, add 2000 mL of deionized water at a material-to-liquid ratio of 1:20 (g / mL), adjust the pH to 7.0 with 1 mol / L NaOH solution, place in an ultrasonic device, and ultrasonically treat for 20 min at an ultrasonic power of 400 W, a frequency of 28 kHz, and a temperature of 50℃ to obtain an ultrasonically treated solution.

[0119] S3: Endogenous enzyme-controlled hydrolysis and detoxification

[0120] The ultrasonically treated solution was transferred to a reactor equipped with a closed stirring device and a negative pressure gas extraction system (maintaining a slight negative pressure of -0.02 to -0.05 MPa, gauge pressure), and kept at a temperature of 50℃, pH 6.8, and a stirring speed of 200 r / min for 60 min to allow endogenous β-glucosidase to fully hydrolyze amygdalin. A negative pressure ventilation pipe was connected to the top of the reactor to introduce the volatile HCN into an alkaline absorption tower (NaOH solution) for neutralization and absorption, ensuring that the emissions meet the occupational exposure limits of GBZ 2.1. Subsequently, the temperature was raised to 85℃ and held for 12 min to inactivate the endogenous enzyme, and then cooled to 52℃ to obtain the detoxified treatment solution.

[0121] S4: Stepwise complex enzymatic hydrolysis

[0122] S41: Alkaline protease hydrolysis

[0123] Add 2.4L (1.33g) of alkaline protease to the detoxification solution, adjust and maintain pH 8.0 and temperature 50℃, stir and enzymatically hydrolyze for 2 h, stirring once every 30 min and monitoring pH, and use 1 mol / L NaOH to maintain pH stability if necessary;

[0124] S41: Neutral protease hydrolysis

[0125] Adjust the pH of the above system to 7.0, add 0.8 L or 0.67 g of neutral protease Neutrase, and continue enzymatic hydrolysis at 50 °C for 1 h. Stir every 30 min and monitor the pH. If necessary, use 1 mol / L NaOH or 1 mol / L HCl to maintain pH stability to obtain the first enzymatic hydrolysate.

[0126] S5: Flavor Modification

[0127] The pH of the first enzymatic hydrolysate was adjusted to 6.5 with 1 mol / L HCl, 1.0 g of flavor protease was added, and the hydrolysate was carried out at 48℃ for 1.5 h to obtain the second enzymatic hydrolysate.

[0128] S6: Enzyme inactivation

[0129] The secondary enzymatic hydrolysate was heated to 95°C and held for 10 minutes, then rapidly cooled to room temperature.

[0130] S7: Separation and Purification

[0131] The enzyme-inactivating solution was centrifuged at 6000 r / min for 15 min to remove the precipitate. The supernatant was filtered through a 0.45 μm microfiltration membrane and then ultrafiltration fractionated through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The operating pressure was 0.2 MPa and the temperature was 25℃. The permeate was collected.

[0132] S8: Deep Detoxification of Macroporous Resin

[0133] The ultrafiltration permeate was passed through an AB-8 macroporous adsorption resin column (resin column diameter to height ratio 1:6, resin volume 500 mL, pre-activated with 95% ethanol and washed with deionized water until no alcohol odor was detected) at a flow rate of 2 BV / h, and the eluent was collected.

[0134] S9: Concentrated and dried

[0135] The effluent was concentrated under vacuum to a solid content of 20%, and then freeze-dried to obtain 26.8 g of low-amyglucan-containing walnut meal antioxidant polypeptide powder.

[0136] The resulting product is an off-white powder, odorless, with a moisture content of 3.8%, an ash content of 2.5%, a polypeptide yield of 26.8% (based on walnut meal powder), a polypeptide content of 82.5%, amygdalin residue of 3.2 mg / kg, hydrocyanic acid content of 0.6 mg / kg, a molecular weight <1000 Da component proportion of 68%, a molecular weight <500 Da component proportion of 35%, and a DPPH free radical scavenging rate IC50. 50 2.1 mg / mL, ABTS free radical scavenging rate IC50 50 1.3 mg / mL, hydroxyl radical scavenging rate (5 mg / mL) 72.5%, total reducing power (5 mg / mL, absorbance at 700 nm) 1.25, bitterness sensory score 0.5 (no bitterness, average score of a 10-person group). The product's high-performance liquid chromatogram is shown below. Figure 1 As shown. Figure 7 This is the high-performance liquid chromatogram of amygdalin.

[0137] Example 2

[0138] The difference from Example 1 is that step S9 employs spray drying: the effluent is vacuum concentrated to a solid content of 25%, with inlet air temperature set at 180°C, outlet air temperature at 85°C, feed rate at 12 mL / min, and atomization pressure at 0.25 MPa, to obtain 28.5 g of pale yellow powdered polypeptide. The product exhibits good solubility, a polypeptide yield of 28.5%, amygdalin residue of 3.5 mg / kg, and HCN of 0.7 mg / kg. Other indicators are similar to those in Example 1. The high-performance liquid chromatogram of the product is shown below. Figure 2 As shown.

[0139] Example 3

[0140] The difference from Example 1 is that a nanofiltration step is added between steps S8 and S9: the AB-8 resin effluent is passed through a nanofiltration membrane with a molecular weight cutoff of 200 Da, operating at a pressure of 1.0 MPa and a temperature of 25°C. Results show that: the inorganic salt removal rate is >85%, the conductivity decreases from 1850 μS / cm to 260 μS / cm; the peptide retention rate is >96%; small molecule bitter substances (such as free benzaldehyde and short-chain bitter peptides) are further reduced, and the product flavor is improved (bitterness score decreases from 0.5 to 0.2). This step is particularly suitable for cosmetic raw materials.

[0141] Example 4

[0142] The difference from Example 1 is that a centrifugation step is added before heating to inactivate the endogenous enzyme in step S3: the hydrolyzed solution is centrifuged at 4000 r / min for 15 min, and the precipitate (mainly protein and a small amount of insoluble matter) is collected. The precipitate is washed twice with deionized water (500 mL each time), and the washing liquid and supernatant are combined. The supernatant and washing liquid mainly contain benzaldehyde (amygdalin has been fully hydrolyzed by the endogenous enzyme), which can be recovered by vacuum distillation (boiling point 178℃, which can be lowered under vacuum), with a recovery rate >75%. The recovered benzaldehyde can be used as a fragrance raw material. The washed precipitate is used for the compound enzymatic hydrolysis in step 4. This process realizes a three-in-one production line of "detoxification-peptide production-benzene recovery" for walnut meal, maximizing resource utilization.

[0143] Example 5: Antioxidant Essence Containing Light-Faced Walnut Meal Peptides

[0144] Formula composition (by weight percentage):

[0145] Phase A: Glycerin 5.0%, Butylene Glycol 3.0%, Xanthan Gum 0.2%, Carbomer 0.15%, Sodium Hyaluronate 0.05%, Deionized Water Balance;

[0146] Phase B: Cetyl alcohol 2.0%, squalane 3.0%, polydimethylsiloxane 1.5%, glyceryl stearate / PEG-100 stearate 2.0%;

[0147] Phase C: 2.0% antioxidant polypeptide from the walnut meal obtained in Example 1, 0.2% bisabolol, 0.8% phenoxyethanol / ethylhexylglycerin, and appropriate amount of flavoring.

[0148] Preparation process: (1) Disperse xanthan gum and carbomer in phase A in glycerol and butylene glycol, add deionized water, heat to 80-85℃, and stir until dissolved and homogeneous; (2) Mix the components of phase B and heat to 80-85℃ to dissolve; (3) Add phase B to phase A at 80℃, homogenize and emulsify for 5 min, and then stir and cool. (4) When the temperature drops to 45°C, add phase C (the polypeptide is dissolved in a small amount of deionized water beforehand), continue stirring until room temperature, and discharge the product to obtain the antioxidant essence.

[0149] The resulting essence is a milky white, fine paste with a pH of 5.5-6.0. It does not separate into layers after centrifugation (3000 r / min, 30 min) and has satisfactory cold and heat resistance.

[0150] Comparative Example 1: No detoxification treatment

[0151] Test results: The obtained polypeptide powder had an amygdalin residue of 1560 mg / kg and a hydrocyanic acid content of 312 mg / kg; the bitterness score was 4.0 (extremely bitter, inedible); the polypeptide yield was 29.5%. The high-performance liquid chromatogram of the product is shown below. Figure 3 As shown.

[0152] Comparative Example 2: Traditional High-Temperature Boiling Detoxification

[0153] The difference from Example 1 is that step S3 is replaced by boiling the ultrasonic treatment solution for 30 min and then cooling it to 50°C for enzymatic hydrolysis, while the rest is the same as in Example 1.

[0154] Test results: Amygdalin removal rate >99%, but protein suffered severe thermal denaturation, and peptide yield was only 12.3%, a 54% decrease compared to Example 1; enzymatic hydrolysis efficiency was extremely low (degree of hydrolysis only 18.5%, compared to 35.2% in Example 1); antioxidant activity was significantly reduced (DPPH IC50 increased to 4.8 mg / mL, ABTS IC50 increased to 3.2 mg / mL). The high-performance liquid chromatogram of the product is shown below. Figure 4 As shown.

[0155] Comparative Example 3: Detoxification of endogenous enzymes only, without resin-based deep detoxification.

[0156] The difference from Example 1 is that step S8 is omitted, while the rest is the same as in Example 1.

[0157] Test results: Amygdalin residue was 28 mg / kg, hydrocyanic acid was 5.6 mg / kg, and bitterness score was 2.0. The high-performance liquid chromatogram of the product is shown below. Figure 5 As shown.

[0158] Comparative Example 4: No ultrasonic pretreatment

[0159] The difference from Example 1 is that step S2 is omitted, while the rest is the same as in Example 1.

[0160] Test results: The peptide yield was only 21.3%, a decrease of 20.5% compared to Example 1; the degree of hydrolysis was 26.8%, significantly lower than the 35.2% in Example 1; the amygdalin removal rate decreased to 85.6% (due to insufficient dissolution of amygdalin and insufficient substrate for endogenous enzyme hydrolysis). The high-performance liquid chromatogram of the product is shown below. Figure 6 As shown.

[0161] Comparative Example 5: Single enzymatic digestion (alkaline protease only)

[0162] The difference from Example 1 is that in step S4, only 2.0 g of alkaline protease (Alcalase 2.4L) was added and enzymatically hydrolyzed at pH 8.0 and 50°C for 3 h. Neutral protease was not added and stepwise enzymatic hydrolysis was not performed. The rest is the same as in Example 1.

[0163] The test results showed that the peptide yield was 24.6% and the degree of hydrolysis was 29.5%, both significantly lower than those in Example 1. The molecular weight distribution of the product was biased towards 1000–3000 Da, with the proportion of components <1000 Da being only 48% (68% in Example 1). The content of small molecule antioxidant active peptides was insufficient, and the DPPH IC50 increased to 3.1 mg / mL.

[0164] Comparative Example 6: Flavor enzyme modification omitted

[0165] The difference from Example 1 is that step S5 is omitted, while the rest is the same as in Example 1.

[0166] Test results: The peptide yield was similar to that of Example 1 (28.2%), but the product had a strong bitter taste and a sensory score of 3.5.

[0167] Experimental Example 1

[0168] (1) Ultrasonic power (single-factor preliminary experiment)

[0169] Following the preparation method of Example 1, the ultrasonic time was controlled at 20 min, the material-to-liquid ratio at 1:20, the temperature at 50℃, and the pH at 7.0. Extraction was performed using ultrasonic powers of 200, 300, 400, 500, and 600 W, respectively. The protein dissolution rate, amygdalin dissolution rate, and protein denaturation rate (determined by turbidimetric method) were measured. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0170]

[0171] Note: Protein denaturation rate is based on the turbidity of the protein solution before ultrasound, and is the percentage increase in turbidity after ultrasound.

[0172] Conclusion: At 400 W, both protein dissolution rate and amygdalin dissolution rate reached their peak values, and the protein denaturation rate was controllable. After 500 W, the denaturation rate increased significantly, reaching as high as 15.5% at 600 W, indicating severe damage to the protein structure. This is expected to have an adverse effect on subsequent enzymatic hydrolysis efficiency and product activity.

[0173] (2) Orthogonal experimental verification of ultrasonic extraction process ( )

[0174] Four factors were selected: ultrasonic power (A), ultrasonic time (B), material-to-liquid ratio (C), and ultrasonic temperature (D), with three levels for each factor. Protein dissolution rate (%) and amygdalin dissolution rate (%) were used as evaluation indicators. Orthogonal experiment.

[0175]

[0176] Range analysis results: The order of importance of factors is A (ultrasonic power) > D (ultrasonic temperature) > B (ultrasonic time) > C (material-liquid ratio). The optimal combination is... The optimal conditions were: ultrasonic power 400 W, time 20 min, material-to-liquid ratio 1:20, and temperature 50℃. The protein dissolution rate (78.5%) in the verification experiment was the same as that in experiment 6, while the amygdalin dissolution rate (85.2%) was higher than all the results of the orthogonal experiments, verifying the reproducibility and superiority of the optimized combination.

[0177] Experimental Example 2

[0178] (1) Stepwise compound enzyme ratio (single-factor preliminary experiment)

[0179] Following the preparation method of Example 1, the total enzyme addition was controlled at 2.0% (alkaline protease: neutral protease = 2:1, i.e., 1.3% alkaline protease and 0.7% neutral protease). The alkaline protease stage was conducted at pH 8.0, temperature 50℃, and time 2 h; the neutral protease stage was conducted at pH 7.0, temperature 50℃, and time 1 h. The effects of different mass ratios of alkaline protease (A) and neutral protease (N) on the degree of hydrolysis, peptide yield, proportion of proteins <1000 Da, and bitterness score (after flavor enzyme modification) were investigated. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0180]

[0181] Conclusion: When A:N=2:1, the degree of hydrolysis, peptide yield, and proportion of <1000 Da all reached their peak values, and the bitterness score was the lowest, and the DPPH IC50 was the smallest (with the strongest activity).

[0182] (2) Orthogonal experimental verification of the compound enzymatic hydrolysis process ( )

[0183] Four factors were selected: the mass ratio of alkaline protease to neutral protease (A), the total amount of complex protease added (B), the alkaline protease hydrolysis temperature (C), and the total hydrolysis time (D). Each factor had three levels. The degree of hydrolysis (%) and peptide yield (%) were used as evaluation indicators. Orthogonal experiment.

[0184]

[0185] Range analysis results: The order of importance of factors is B (enzyme dosage) > A (enzyme ratio) > D (total hydrolysis time) > C (alkaline protease hydrolysis temperature). The optimal combination is... The optimal hydrolysis conditions were: alkaline protease:neutral protease ratio of 2:1, total enzyme dosage of 2.0% (1.3% alkaline protease + 0.7% neutral protease), alkaline protease hydrolysis temperature of 50℃, and total hydrolysis time of 3 h (2 h alkaline protease + 1 h neutral protease). Verification experiments showed that under these conditions, the degree of hydrolysis reached 35.8 ± 0.7%, and the peptide yield reached 28.8 ± 0.6%, both higher than the results of other experiments in the orthogonal array.

[0186] Experimental Example 3: Validation of the temperature-time combination for endogenous enzyme hydrolysis

[0187] Referring to the preparation method in Example 1, step S3 was conducted under pH 6.8 and stirring at 200 r / min. A temperature-time combination verification experiment was designed, using amygdalin removal rate and protein retention rate as indicators. Two parallel samples were used in each group, and the average value was taken. The results are shown in the table below:

[0188]

[0189] Conclusion: The 50℃ / 60 min combination achieved the optimal balance between amygdalin removal rate (99.2%) and protein retention rate (96.2%), reducing HCN residue to 0.8 mg / kg. Further heating or extending the time had limited effect on improving the detoxification rate, but significantly increased protein loss.

[0190] Experimental Example 4: Screening of Macroporous Resin Types

[0191] Following the preparation method of Example 1, the same sample loading amount, flow rate, and elution conditions were controlled in step S8. The effects of AB-8, D101, and HPD100 resins on the adsorption selectivity of amygdalin and the peptide retention rate were compared. The results are shown in the table below:

[0192]

[0193] Conclusion: AB-8 resin exhibits the best selective adsorption capacity for amygdalin, reducing the residual amygdalin content to 3.2 mg / kg after purification. Following prior endogenous enzyme hydrolysis and volatilization, the HCN content was already reduced to an extremely low level (0.6 mg / kg). Combined with this resin purification step, the final product fully complies with safety standards.

[0194] Experimental Example 5: Antioxidant Activity

[0195] (1) In vitro antioxidant activity

[0196] Using vitamin C (VC) as a positive control, the in vitro antioxidant activity of the sample from Example 1 was determined, and the results are shown in the table below:

[0197]

[0198] Conclusion: The DPPH and ABTS scavenging abilities of the polypeptide of this invention are significantly better than those of the control groups, and the hydroxyl radical scavenging rate is >70%, which is close to the level of the VC positive control.

[0199] (2) Dose-effect relationship

[0200] The DPPH free radical scavenging rate of the peptide from Example 1 was investigated at concentrations of 0.5, 1.0, 2.0, 3.0, and 5.0 mg / mL. The results showed a clear concentration-dependent effect: the scavenging rate was 28.5 ± 2.0% at 0.5 mg / mL, 45.2 ± 2.5% at 1.0 mg / mL, 62.8 ± 2.0% at 2.0 mg / mL, 78.5 ± 1.8% at 3.0 mg / mL, and 89.2 ± 1.5% at 5.0 mg / mL. IC50 50 It is 2.1 mg / mL.

[0201] (3) Validation of antioxidant activity at the cellular level (HepG2 cell model)

[0202] A H2O2 (400 μmol / L)-induced oxidative damage model was established using HepG2 hepatocytes to detect the protective effect of peptides on cell viability. Blank group, model group, positive control group (VC 50 μg / mL), and sample groups (0.5, 1.0, 2.0 mg / mL) were set up. After 24 h of culture, the CCK-8 assay was used for detection.

[0203]

[0204] Conclusion: The 2.0 mg / mL peptide can restore the survival rate of H2O2-damaged cells from 52.5% to 82.5%, which is close to the level of the VC positive control, demonstrating its significant cellular-level antioxidant protective ability.

[0205] In the HepG2 oxidative damage model described above, intracellular ROS levels were detected using the DCFH-DA probe, and the activities of SOD, CAT, GSH-Px, and MDA content were also measured. The results showed that 2.0 mg / mL of the peptide could scavenge 78.5 ± 3.0% of intracellular ROS and significantly restore antioxidant enzyme activity and MDA levels to near-normal levels.

[0206] Test Example 6: Safety Test

[0207] (1) Repeated skin irritation tests

[0208] Following the repeated skin irritation test method in the "Cosmetic Safety Technical Specifications" (2022 edition), six healthy rabbits weighing 2.0-2.5 kg were selected, and the hair on both sides of the spine on the back was clipped (approximately 3 cm × 3 cm area). The experimental group was treated with 0.5 mL of a 5% polypeptide aqueous solution prepared in Example 1, while the control group was treated with 0.5 mL of physiological saline. The solution was covered with two layers of gauze and one layer of cellophane, and fixed with non-irritating adhesive tape. The treatment was applied for 4 hours daily for 14 consecutive days. Skin reactions were observed 1 hour after each removal of the test substance. The results showed that the experimental group rabbits exhibited no irritation reactions such as erythema or edema, with an average irritation score of 0 (<0.5), indicating no skin irritation.

[0209] (2) Acute eye irritation / corrosion test

[0210] According to the acute eye irritation / corrosiveness test method in the "Cosmetic Safety Technical Specifications" (2022 edition), three healthy rabbits were selected. 0.1 mL of a 5% aqueous solution of the polypeptide prepared in Example 1 was instilled into the conjunctival sac of one eye, with the other eye serving as a self-control. Corneal, iris, and conjunctival reactions were observed and scored at 1, 24, 48, and 72 hours, and on days 4 and 7. The results showed that the score was 0 at all observation time points, indicating no eye irritation.

[0211] (3) Guinea pig skin sensitization test (GPMT)

[0212] According to the skin allergy test method in the "Cosmetic Safety Technical Specifications" (2022 edition), 24 healthy guinea pigs were selected and divided into an experimental group (n=12), a negative control group (n=6, saline), and a positive control group (n=6, 2,4-dinitrochlorobenzene). Induction period (day 0): The experimental group received an intradermal injection of 0.1 mL of 1% polypeptide solution prepared in Example 1 (containing Freund's complete adjuvant FCA); Skin induction (day 7): The experimental group received a skin application of 0.5 mL of 10% polypeptide solution prepared in Example 1, followed by blocking fixation for 48 h; Challenge (day 21): The experimental group received a skin application of 0.5 mL of 5% polypeptide solution prepared in Example 1, followed by blocking fixation for 24 h. Skin reactions were observed 24 h and 48 h after challenge removal. The results showed that the sensitization rate in the experimental group was 0%, with no sensitization reactions such as erythema or papules; all guinea pigs in the positive control group showed positive reactions. This indicates that the polypeptide is not sensitizing.

[0213] (4) Acute oral toxicity test (mice)

[0214] According to GB 15193.3-2014 "National Food Safety Standard - Acute Oral Toxicity Test", 20 healthy Kunming mice (half male and half female, weighing 18-22 g) were selected. The maximum tolerated dose (MTD) method was used for the test, with an oral dose of 5000 mg / kg·bw (an aqueous solution of the polypeptide powder prepared in Example 1). The mice were observed for 14 days, and symptoms of poisoning and mortality were recorded. Results showed that the mice exhibited no abnormal behavior and there were no deaths during the test. The MTD was >5000 mg / kg·bw, classifying them as practically non-toxic according to the toxicity grading standard.

[0215] Stability study data from Experiment Example 7

[0216] (1) Accelerated stability test

[0217] The polypeptide powder obtained in Example 1 was placed in a constant temperature and humidity chamber at 40°C and 75% relative humidity (sealed packaging) for 3 months. Samples were taken monthly to test the polypeptide content, amygdalin residue, DPPH free radical scavenging rate and appearance. Three parallel samples were taken for each group, and the average value was taken.

[0218]

[0219] Conclusion: After 3 months of accelerated stability testing, the peptide content and antioxidant activity remained above 90% of their initial values. Although the contents of amygdalin and HCN increased slightly, they were still far below the safety limits, indicating good stability of the active ingredients. Slight clumping occurred from the third month onwards, suggesting that long-term storage requires sealing and moisture protection.

[0220] (2) Long-term stability test

[0221] The polypeptide powder prepared in Example 1 was sealed and packaged, and then stored at 25°C and 60% relative humidity for 12 months, with samples taken every 3 months for testing. The results showed that after 12 months, the polypeptide content retention rate was 95.5±0.6%, amygdalin was 3.8±0.5 mg / kg, HCN was 0.8±0.1 mg / kg, and DPPH scavenging rate retention rate was 96.0±0.6%. There were no obvious changes in appearance, indicating that the polypeptide has excellent stability under normal storage conditions. Under normal storage conditions, the product stability can support a shelf life of 24 months.

[0222] (3) Stability of formulation application

[0223] The peptides obtained in Example 1 were added at 2.0% to a basic skin care lotion (pH 5.5-6.5, containing 5% glycerin, 3% squalane, 2% cetyl alcohol, 1.5% emulsifier, and 0.8% preservative). The lotions were placed at 25°C (room temperature) and 40°C (accelerated temperature) for 3 months, respectively. The appearance, color, and odor of the products were observed monthly, and the peptide content and antioxidant activity were tested.

[0224]

[0225] Conclusion: The product showed no abnormal phenomena such as stratification, discoloration, or odor within 3 months under both normal temperature and accelerated conditions. The peptide content and antioxidant activity did not decrease significantly, indicating that the peptides have good stability in cosmetic formulations and good compatibility with commonly used cosmetic raw materials.

[0226] The detection methods used in the embodiments, comparative examples, and experimental examples of this invention are as follows:

[0227] (1) Determination of polypeptide content (biuret method)

[0228] Standard curve: Using bovine serum albumin (BSA) as a standard, prepare solutions of concentrations ranging from 0 to 1.0 mg / mL, add biuret reagent, incubate at room temperature for 30 min, and measure the absorbance at 540 nm to plot the standard curve. Sample determination: Prepare peptide samples into solutions of appropriate concentrations and determine their content using the same method. Calculate the peptide content based on the standard curve.

[0229] (2) Determination of degree of hydrolysis (pH-stat method)

[0230] During enzymatic hydrolysis, the pH was maintained constant by titration with 0.1 mol / L NaOH, and the degree of hydrolysis (DH%) was calculated based on the amount of alkali consumed. The formula is: DH% = (B×N_b) / (M_p×h_tot×α)×100%, where B is the amount of alkali consumed (mL), N_b is the alkali equivalent concentration, M_p is the protein mass (g), h_tot is the peptide bond content of the substrate protein (mmol / g, 8.0 mmol / g for walnut protein), and α is the degree of α-amino dissociation (0.97 at pH 8.0 and 50℃). Since this invention employs stepwise enzymatic hydrolysis, the pH conditions for each step are different. When calculating the degree of hydrolysis, the corresponding α value is selected based on the pH and temperature conditions of each step, and the proportion of broken peptide bonds to the total peptide bonds of the substrate is calculated according to the formula. The total degree of hydrolysis is obtained by summing the amounts based on the same protein substrate mass.

[0231] (3) Determination of molecular weight distribution (HPGPC method)

[0232] Chromatographic column: TSK gel G2000SWXL (300 mm × 7.8 mm, 5 μm); mobile phase: acetonitrile / water / trifluoroacetic acid (30:70:0.1, v / v / v); flow rate: 0.5 mL / min; detection wavelength: 220 nm; column temperature: 30℃. Standards: cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), glycine-glycine-glycine (189 Da), glycine (75 Da). A molecular weight standard curve was plotted based on the retention times of the standards, and the proportion of peptides in each molecular weight range in the sample was calculated.

[0233] (4) Determination of amygdalin content (HPLC method)

[0234] Chromatographic column: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile / water (15:85, v / v); flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 30℃. Standard: amygdalin reference standard (purity ≥98%). Sample preparation: Accurately weigh 0.5 g of polypeptide powder, add 20 mL of 50% methanol, ultrasonically extract for 30 min, centrifuge (10000 r / min, 10 min), and filter the supernatant through a 0.22 μm microporous membrane before injection, with an injection volume of 10 μL. Quantification: external standard method. Detection limit ≤0.5 mg / kg. This method shows good linearity in the concentration range of 1.0-100 μg / mL (R0). 2 The limit of quantitation (LOQ) is 1.0 mg / kg, and the limit of detection (LOD) is 0.3 mg / kg.

[0235] (5) Determination of residual hydrocyanic acid (HCN) (pyridine-barbituric acid colorimetric method)

[0236] The sample was distilled under acidic conditions (tartaric acid, pH < 2), and HCN was distilled off with the steam and absorbed into a 1% NaOH solution. Under alkaline conditions, HCN reacted with chloramine T to form cyanogen chloride, which then reacted with pyridine-barbituric acid to form a purple-red dye. The absorbance was measured at 580 nm, and the HCN content was calculated based on the standard curve. Standard curve: A series of HCN standards ranging from 0 to 2.0 μg / mL were prepared.

[0237] (6) Determination of benzaldehyde residue (HPLC method)

[0238] Chromatographic column: C18 column; mobile phase: methanol / water (60:40); detection wavelength: 250 nm; flow rate: 1.0 mL / min. Standard: benzaldehyde reference standard. The sample was directly injected after methanol extraction.

[0239] (7) DPPH free radical scavenging ability

[0240] Reagents: 0.1 mmol / L DPPH ethanol solution (freshly prepared, protected from light). Method: Mix 2.0 mL of sample solution with 2.0 mL of DPPH ethanol solution, react at room temperature in the dark for 30 min, and measure the absorbance A_i at 517 nm; use anhydrous ethanol instead of DPPH to measure A_j; use deionized water instead of sample to measure A_0. Formula: Scavenging rate (%) = [1 - (A_i - A_j) / A_0] × 100%. Results: Calculate IC50. 50 value.

[0241] (8) ABTS free radical scavenging ability

[0242] Reagents: ABTS working solution (7 mmol / L ABTS and 2.45 mmol / L potassium persulfate mixed in equal volumes, reacted in the dark for 12–16 h, diluted with PBS to an absorbance of 0.70 ± 0.02 at 734 nm before use). Method: Mix 0.5 mL of sample solution with 3.0 mL of ABTS working solution, react at room temperature in the dark for 6 min, and measure the absorbance at 734 nm. Formula: Scavenging rate (%) = (A_0 - A_i) / A_0 × 100%. Results: Calculate IC50. 50 value.

[0243] (9) Hydroxyl radical scavenging ability (Fenton reaction method)

[0244] Reagents: 9 mmol / L salicylic acid-ethanol solution, 9 mmol / L FeSO4 solution, 8.8 mmol / L H2O2 solution. Method: Add 1.0 mL of sample solution, 1.0 mL of salicylic acid-ethanol, and 1.0 mL of FeSO4 sequentially, and finally add 1.0 mL of H2O2 to initiate the reaction. Incubate at 37℃ for 30 min, and measure the absorbance at 510 nm. Formula: Scavenging rate (%) = [A_0 - (A_i - A_j)] / A_0 × 100%, where A_j is the absorbance with deionized water instead of H2O2.

[0245] (10) Determination of total reducing power (potassium ferricyanide method)

[0246] Take 1.0 mL of sample solution, add 2.5 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution, react in a 50℃ water bath for 20 min, cool rapidly, add 2.5 mL of 10% trichloroacetic acid, centrifuge at 3000 r / min for 10 min; take 2.5 mL of the supernatant, add 2.5 mL of deionized water and 0.5 mL of 0.1% FeCl3, mix well, and measure the absorbance at 700 nm. The higher the absorbance, the stronger the reducing power.

[0247] (11) Amino acid composition analysis

[0248] Acid hydrolysis method: Accurately weigh 50 mg of peptide powder, add 10 mL of 6 mol / L HCl, seal under nitrogen purging, and hydrolyze at 110℃ for 24 h. Evaporate the hydrolysate to dryness using a rotary evaporator, dissolve in 0.02 mol / L HCl, filter through a 0.22 μm filter membrane, and determine the content of 17 hydrolyzed amino acids using an automated amino acid analyzer or HPLC-pre-column derivatization (OPA / FMOC derivatization).

[0249] (12) Sensory evaluation

[0250] A sensory evaluation team of at least 10 people (trained) was formed to score the bitterness of the samples. Scoring criteria: 0 points = no bitterness, 1 point = slightly bitter, 2 points = moderate bitterness, 3 points = noticeable bitterness, 4 points = strong bitterness, 5 points = extremely bitter and inedible. The average value was taken, and a significance analysis was performed (p < 0.05).

[0251] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0252] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal, comprising: S1: Raw material pretreatment The pulp from cold-pressed walnuts for oil extraction is then cleaned of impurities, dried, and pulverized to obtain walnut meal powder. Its characteristic is that it further includes: S2: Ultrasonic-assisted extraction Add the walnut meal powder to deionized water at a material-to-liquid ratio of 1:10-1:30, adjust the pH to 6.5-7.5, and then perform ultrasonic treatment to obtain an ultrasonic treatment solution. S3: Endogenous enzyme-controlled hydrolysis and detoxification The ultrasonic treatment solution was continuously stirred at a temperature of 40-55℃ and a pH of 6.0-7.0 for 30-90 min, and then the endogenous enzymes were inactivated by heating to obtain the detoxified treatment solution. S4: Stepwise complex enzymatic hydrolysis S41: Alkaline protease hydrolysis Add alkaline protease to the detoxification solution, with the amount of enzyme added being 0.5%-2.0% of the mass of the walnut meal powder. Adjust and maintain the pH at 7.5-8.5 and the temperature at 50-55℃, and perform enzymatic hydrolysis for 1.5-2.5 h. S42: Neutral protease hydrolysis Adjust the pH of the system in step S41 to 6.5-7.5, add neutral protease, the amount of enzyme added is 0.5%-1.5% of the mass of the walnut meal powder, and continue enzymatic hydrolysis for 1-2 hours at a temperature of 45-55℃ to obtain the first enzymatic hydrolysate. S5: Flavor Modification The pH of the primary enzymatic hydrolysate was adjusted to 6.0-7.0, and flavor protease was added at a rate of 0.5%-1.5% of the mass of the walnut meal powder. The hydrolysate was then carried out at a temperature of 45-50℃ for 1-2 hours to obtain the secondary enzymatic hydrolysate. S6: Enzyme inactivation The secondary enzymatic hydrolysate was heated to inactivate the enzyme, and then rapidly cooled to room temperature to obtain an enzyme-inactivated enzymatic hydrolysate. S7: Separation and Purification The enzyme hydrolysate after enzyme inactivation is centrifuged, and the supernatant is filtered through a 0.22-0.45 μm microfiltration membrane and then ultrafiltration fractionated through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain the ultrafiltration permeate. S8: Deep Detoxification of Macroporous Resin The ultrafiltration permeate is passed through a chromatography column packed with macroporous adsorption resin, and the effluent is collected. The macroporous adsorption resin is a weakly polar styrene macroporous adsorption resin such as D101, AB-8, or HPD100. S9: Concentrated and dried The effluent is concentrated under vacuum and then freeze-dried or spray-dried to obtain low-bitter amygdalin-containing walnut meal antioxidant polypeptide powder.

2. The method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal as described in claim 1, characterized in that, Step S2 is as follows: Add the walnut meal powder to deionized water at a material-to-liquid ratio of 1:10-1:30, adjust the pH to 6.5-7.5, and sonicate for 15-30 minutes under ultrasonic power of 200-600 W, frequency of 20-40 kHz, and temperature of 45-55℃ to obtain ultrasonic treatment solution.

3. The method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal as described in claim 1, characterized in that, Step S3 specifically involves: transferring the ultrasonic treatment solution to a reactor equipped with a sealed stirring device and a negative pressure gas extraction system, maintaining it at a temperature of 40-55℃, pH 6.0-7.0, and a stirring speed of 100-300 r / min for 30-90 min, then raising the temperature to 80-90℃ and maintaining it for 10-15 min to inactivate endogenous enzymes, and then cooling it to 50-55℃ to obtain the detoxified treatment solution.

4. The method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal as described in claim 1, characterized in that, Step S42 is as follows: Adjust the pH of the system from step S41 to 6.5-7.5, add neutral protease, the amount of enzyme added is 0.5%-1.5% of the mass of the walnut meal powder, and continue enzymatic hydrolysis for 1-2 hours at a temperature of 45-55℃. During this period, stir once every 30 minutes and monitor the pH. Maintain pH stability with 1 mol / L NaOH or 1 mol / L HCl to obtain a first enzymatic hydrolysate.

5. The method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal as described in claim 4, characterized in that, The total amount of alkaline protease and neutral protease added is 1.0%-3.0% of the mass of the walnut meal powder, and the mass ratio of the two is 1:1-3:

1.

6. The method for preparing a low-amyglucan-containing antioxidant polypeptide from walnut meal as described in claim 1, characterized in that, Step S7 is as follows: Centrifuge the enzyme hydrolysate after enzyme inactivation at 4000-8000 r / min for 15-20 min to remove the precipitate; filter the supernatant through a 0.22-0.45 μm microfiltration membrane, and then perform ultrafiltration fractionation through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, with an operating pressure of 0.1-0.3 MPa and a temperature of 25-30℃, and collect the permeate to obtain the ultrafiltration permeate.

7. A low-amygin, antioxidant polypeptide from walnut meal prepared by the method according to any one of claims 1-6, characterized in that, The proportion of oligopeptides with a molecular weight <1000 Da is ≥60%, and the total content of polypeptides is ≥80%; the antioxidant activity index is DPPH free radical scavenging rate IC50 ≤2.5 mg / mL.

8. The application of the low-amyglucan-containing antioxidant polypeptide in walnut meal as described in claim 7, characterized in that, The application of the low-amyglucan-containing walnut meal antioxidant polypeptide in the preparation of antioxidant cosmetics.

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