Method for enriching active ingredients of walnut kernel skin based on differential pressure blasting puffing and gradient biotransformation

Abstract

The invention discloses a walnut kernel skin active component enrichment method based on differential pressure blasting puffing and gradient biotransformation, and relates to the technical field of agricultural product deep processing and plant active component extraction, and the method comprises the following steps: S1, carrying out differential pressure blasting puffing treatment on dried walnut kernel skin, maintaining the pressure of the material at 0.8-1.5 MPa for 10-60 seconds, and then instantaneously releasing the pressure to normal pressure within 0.05-0.2 seconds, the porosity is increased, cell walls are broken, and the specific surface area is increased; s2, mixing the puffed walnut kernel skin and water according to a solid-liquid mass ratio of 1: 10-1: 30 to form slurry, adding a compound enzyme system containing cellulase, hemicellulase, pectinase, tannase and / or protease, and performing enzymolysis for 1-4 hours under the conditions that the temperature is 45-55 DEG C and the pH is 4.5-6.0, so that cell wall components are partially hydrolyzed and bound phenols are released; and S3, sequentially carrying out first-stage fermentation and second-stage fermentation on the materials subjected to enzymolysis.

Description

Technical Field

[0001] This invention relates to the field of deep processing of agricultural products and extraction of active plant ingredients, and in particular to a method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation. Background Technology

[0002] Walnut kernel skin is rich in polyphenolic active substances such as ellagic acid, proanthocyanidins, and phenolic acids, which have good antioxidant, free radical scavenging, and potential health functions. However, due to the dense cell walls, high lignin content, and the fact that most polyphenols exist in bound form in walnut kernel skin, traditional water extraction, alcohol extraction, or simple fermentation methods are difficult to effectively release polyphenols, resulting in low utilization and limiting the high-value development of walnut by-products.

[0003] Existing research attempts to promote the release of active ingredients in walnut shells through fermentation, enzymatic hydrolysis, or physical pulverization, but the following prominent bottlenecks remain: Insufficient structural disruption: Ordinary pulverization methods cannot significantly improve porosity and specific surface area, making it difficult for enzymes and microorganisms to penetrate deep into the substrate; Low release rate of bound polyphenols: There is a lack of targeted enzymatic hydrolysis strategies tailored to the structural characteristics of walnut shells, and polyphenols are still largely bound by the cell wall matrix; Limited depth of biotransformation: Traditional fermentation strains are unable to effectively convert bound or original polyphenols into more easily absorbed and more active small molecule derivatives; Outdated enrichment methods: Commonly used precipitation or coarse filtration methods have poor selectivity and significant polyphenol loss, making it impossible to achieve high-purity enrichment. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation, addressing the following issues: insufficient cell wall disruption: ordinary pulverization methods cannot significantly improve porosity and specific surface area, making it difficult for enzymes and microorganisms to penetrate deep into the substrate; low release rate of bound polyphenols: lacking targeted enzymatic hydrolysis strategies tailored to the structural characteristics of walnut kernel peel, polyphenols remain largely bound by the cell wall matrix; limited depth of biotransformation: traditional fermentation strains cannot effectively convert bound or original polyphenols into more easily absorbed, higher-activity small molecule derivatives; and outdated enrichment methods: commonly used precipitation or coarse filtration methods suffer from poor selectivity and significant polyphenol loss, failing to achieve high-purity enrichment.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for enriching the active ingredients of walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation, comprising: S1. Differential pressure explosion puffing treatment is carried out on the dried walnut kernel skin, so that the material is held under pressure of 0.8-1.5 MPa for 10-60 seconds and then instantaneously released to atmospheric pressure within 0.05-0.2 seconds to obtain puffed walnut kernel skin with increased porosity, cell wall rupture and increased specific surface area. S2. Mix the puffed walnut kernel skin with water at a solid-liquid mass ratio of 1:10 to 1:30 to form a slurry. Add a complex enzyme system containing cellulase, hemicellulase, pectinase, tannic acidase and / or protease. Enzymatically hydrolyze the mixture for 1 to 4 hours at 45 to 55°C and pH 4.5 to 6.0 to partially hydrolyze the cell wall components and release bound phenols. S3. The enzymatically hydrolyzed material is then subjected to the first and second stage fermentation sequentially: The first stage involves inoculating with one or more microorganisms selected from Aspergillus niger, Aspergillus oryzae, or Bacillus subtilis, and fermenting at 32–38°C for 12–24 hours. The second stage involves inoculating with one or more lactic acid bacteria selected from Lactobacillus plantarum, Lactobacillus acidophilus, or Lactobacillus casei, and fermenting at 32–38°C for 12–24 hours to promote the freeing and stabilization of polyphenols. S4. After fermentation, the material is separated into solid and liquid components to obtain the supernatant. S5. After concentrating the supernatant through a nanofiltration membrane with a molecular weight cutoff of 1-10 kDa, the solution is dried to obtain walnut kernel peel active ingredient powder.

[0007] As a preferred embodiment of the method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein: the differential pressure explosion puffing treatment pressure in step S1 is 1.0 to 1.3 MPa, and the holding time is 20 to 40 seconds.

[0008] Furthermore, the instantaneous decompression time is 0.05–0.1 s.

[0009] As a preferred embodiment of the method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation described in this invention, the porosity of the walnut kernel peel is increased by 2 to 5 times and the water absorption rate is increased by 30% to 80% after the puffing treatment.

[0010] Furthermore, the moisture content of the walnut kernel skin after puffing is 5% to 12%.

[0011] As a preferred embodiment of the method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein: the total amount of the compound enzyme system added in step S2 is 0.1% to 0.5% (w / w) of the dry weight of the puffed walnut kernel peel.

[0012] Furthermore, the enzyme preparation preferably uses a combination of hemicellulase and tannic acidase.

[0013] Furthermore, after enzymatic hydrolysis, the enzyme preparation is inactivated by heating at 85°C for 10 minutes.

[0014] As a preferred embodiment of the method for enriching active ingredients in walnut kernel skin based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein: the microorganisms used in the first stage fermentation in step S3 have cellulase activity ≥10 U / g and polyphenol oxidase activity ≥5 U / g.

[0015] As a preferred embodiment of the method for enriching active ingredients in walnut kernel skin based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein the final pH of the second stage fermentation in step S3 is 3.8 to 4.5.

[0016] Furthermore, the inoculation ratio of lactic acid bacteria to yeast is 3:1 to 1:1; the fermentation time is preferably 24 to 36 hours.

[0017] Furthermore, the preferred fermentation strains are Lactobacillus plantarum, Saccharomyces cerevisiae, or a combination of both.

[0018] As a preferred embodiment of the method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein: the solid-liquid separation in step S4 is performed by centrifugation or plate and frame filtration, and the solid content of the obtained supernatant is ≤0.5% (w / v).

[0019] Furthermore, the resin is preferably AB-8, macroporous D101, or HPD-100 resin.

[0020] Furthermore, the nanofiltration membrane operates at a pressure of 0.5–1.5 MPa and a temperature of 20–35°C; the molecular weight cutoff of the nanofiltration membrane is preferably 3 kDa or 5 kDa.

[0021] As a preferred embodiment of the method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation according to the present invention, wherein: the operating pressure of the nanofiltration membrane in step S5 is 0.3 to 0.8 MPa, and the concentration factor is 5 to 15 times.

[0022] Furthermore, the inlet air temperature of the spray dryer is 150–180°C, and the outlet air temperature is 70–90°C.

[0023] Furthermore, the freeze-drying temperature is -50℃, and the drying time is 12 to 24 hours.

[0024] Secondly, the present invention provides a powder of active ingredients in walnut kernel skin, wherein: the total polyphenol content in the powder is not less than 12 mg GAE / g, the proportion of free polyphenols in the total polyphenols is ≥45%, and the proportion of components with a molecular weight less than 10 kDa is ≥80%.

[0025] As a preferred embodiment of the walnut kernel peel active ingredient powder of the present invention, wherein: the DPPH free radical scavenging rate of the powder is ≥60%, and the ABTS free radical scavenging rate is ≥55%.

[0026] The beneficial effects of this invention are: 1. During the puffing process, the instantaneous release of high pressure causes the cell walls of the walnut kernel skin to burst, expand, and undergo a transformation into a porous structure. This increases the porosity, specific surface area, and internal openness of the kernel, effectively opening up the originally dense and difficult-to-degrade plant tissue. The difference in structure before and after puffing enhances the contact ability of enzymes and microorganisms with the substrate and reduces the cell wall barrier effect, which is the key basis for the overall high efficiency of this invention.

[0027] 2. Through the synergistic action of directed enzyme systems such as hemicellulase, cellulase, and tannic acidase, the cell wall polysaccharide network and polyphenol binding bonds can be effectively destroyed, converting bound polyphenols into soluble free polyphenols. Enzymatic hydrolysis not only increases the release of polyphenols but also enhances the bioavailability of microorganisms, making subsequent fermentation and transformation more complete.

[0028] 3. Lactic acid bacteria and yeast have rich extracellular enzyme systems, which can further transform complex polyphenols into smaller molecule derivatives with simpler structures, stronger antioxidant capacity, and higher absorption rates. By optimizing the combination of strains (lactic acid bacteria: yeast = 3:1 to 1:1), dual functions can be achieved, including β-glucosidase, which can break polyphenol-glycosidic bonds to convert bound phenols into free phenols, and phenolic acid decarboxylase and reductase, which can derivatize polyphenols and improve their antioxidant properties.

[0029] 4. Combining macroporous resin adsorption with 1-10 kDa nanofiltration membranes achieves efficient fractionation and enrichment. The extracts enriched by membrane separation exhibit better solubility, stability, and palatability in functional food applications. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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.

[0031] Figure 1This is a schematic diagram of the process flow for the enrichment method of active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation according to the present invention.

[0032] Figure 2 This is a schematic diagram showing the comparison of porosity, polyphenol exposure, and water absorption of walnut kernel skin before and after differential pressure explosion puffing treatment.

[0033] Figure 3 This is a schematic diagram showing the comparison results of polyphenol release from walnut kernel skin under different processing techniques.

[0034] Figure 4 This diagram illustrates the changes in free polyphenol content in walnut kernel peel fermentation broth under different processing techniques.

[0035] Figure 5 This is a schematic diagram showing the comparison of the content of active ingredients in the membrane separation enrichment solution under different treatment processes.

[0036] Figure 6 This is a schematic diagram showing the comparison of the antioxidant capacity of enriched powders obtained from different processing techniques. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] Example 1: This example provides a method for enriching the active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation. The specific steps are as follows: S0. Raw material pretreatment. Walnut shells produced during the industrial pressing of walnut oil are selected as raw materials. Obvious broken shells and other mechanical impurities are removed, and the shells are dried in hot air at 45-50℃ until the moisture content is ≤10%. The dried walnut shells are then crushed to about 20 mesh using a pulverizer, and excessively coarse or fine particles are removed by sieving. The shells are then placed in sealed bags for later use.

[0041] S1. Differential Pressure Explosive Puffing Process. Add the pre-treated walnut shells to the material hopper of the differential pressure explosive puffing equipment at a rate of 3-5 kg ​​per batch. Close the hopper door and check for a tight seal. Turn on the heating system to ensure the material is heated evenly while rotating and stirring. When the equipment pressure reaches approximately 1.1 MPa and the internal temperature reaches approximately 140-150°C, start timing and maintain the pressure for 40 seconds. After the pressure maintenance, quickly open the pressure relief valve to reduce the internal pressure to atmospheric pressure within 0.1 seconds, completing instantaneous pressure release. Simultaneously, open the discharge port to quickly discharge the puffed walnut shells, spreading them on a stainless steel tray to cool naturally to room temperature. The puffed walnut shells are loose and porous and are ready for use.

[0042] S2. Enzymatic-Directed Decomposition. Take 100 g of puffed walnut kernel peel, add 2000 mL of distilled water to prepare a slurry with a solid-liquid ratio of 1:20 (g:mL), place it in a constant-temperature reactor with stirring function, turn on stirring (150-200 r / min), and adjust the temperature to 50℃. Add a compound enzyme preparation of hemicellulase and tannic acidase to the slurry, wherein the mass ratio of hemicellulase to tannic acidase is 1:1, and the total enzyme dosage is 0.25% of the mass of puffed walnut kernel peel. Adjust the pH of the slurry to 4.5±0.1 using a citrate-sodium citrate buffer solution, and continuously stir and hydrolyze for 2 h at 50℃. During the enzymatic hydrolysis, take one sample every 30 min for subsequent determination of total polyphenols and free polyphenols. After the enzymatic hydrolysis is completed, raise the system to 85℃ and keep it at this temperature for 10 min to inactivate the enzyme system, then cool it to room temperature to obtain the enzymatic hydrolysate.

[0043] S3. Microbial-Assisted Biotransformation. Strain Activation and Preparation: Lactobacillus plantarum and Saccharomyces cerevisiae slant cultures were obtained from preservation tubes and cultured in MRS liquid medium and YPD liquid medium at 37°C with shaking for 18–24 h to obtain logarithmic growth phase cultures. Inoculation and Fermentation: The enzymatic hydrolysate obtained in Example 1 was transferred to a fermenter, with the liquid volume controlled at 70%–80% of the working volume. The system temperature was lowered to approximately 36°C using steam or hot water jacket heating. A composite strain was inoculated at a total inoculation rate of 10⁷ CFU / mL, with lactic acid bacteria and yeast in a 3:1 ratio. The initial pH was adjusted to 4.5 with a small amount of dilute hydrochloric acid or citric acid. During fermentation, the temperature was maintained at 36°C, the stirring speed at 80–100 r / min, and the fermentation time at 24 h. Samples were taken every 6 h during fermentation for subsequent detection of changes in free polyphenols, pH, and soluble solids. After fermentation, the microbial activity was terminated by heating the fermentation broth to 80°C and holding it for 10 minutes, thus obtaining the fermentation broth.

[0044] S4. Solid-Liquid Separation and Active Ingredient Enrichment. Solid-Liquid Separation: Place the fermentation broth in a centrifuge at 6000 rpm for 10 min, and collect the supernatant as the subsequent enrichment stock solution. Alternatively, a plate and frame filter can be used for solid-liquid separation, with the supernatant collected for later use. Macroporous Resin Adsorption and Elution: Select AB-8 macroporous adsorption resin, soak it in 95% ethanol for 12 h to remove impurities, then wash it with a large amount of distilled water until there is no ethanol odor, and pack it into a glass column for later use. Pass the supernatant into the resin column at a flow rate of 1–2 BV / h for 30–60 min, then wash it with distilled water to remove water-soluble sugars and other impurities, and then elute with a 50% (v / v) ethanol solution, collecting the eluent. Membrane Separation and Enrichment: Concentrate the eluent under reduced pressure or use it directly as the feed solution, and send it to a nanofiltration membrane system, selecting a nanofiltration membrane with a molecular weight cutoff of 3 kDa. Control the operating pressure to 0.8–1.2 MPa and the temperature to 25–30°C until the volume concentration reaches the preset value (e.g., 3–5 times). Collect the concentrate for later use.

[0045] S5. Drying to prepare enriched powder. The concentrated solution obtained in S4 is filtered through a 0.45 μm filter membrane to remove any potentially present small suspended particles, and then fed into a spray drying tower. The spray drying conditions are: inlet air temperature 165℃, outlet air temperature 85℃, and feed flow rate controlled between 5 and 15 mL / min according to equipment specifications. After drying, the resulting brownish-yellow or dark brown powder is collected, placed in a sealed aluminum foil bag, and stored under dry and light-protected conditions to obtain the walnut kernel peel active ingredient enriched powder of this embodiment.

[0046] Example 2 is a preferred embodiment of the present invention. Compared with Example 1, it is adjusted only in terms of differential pressure explosion expansion conditions, while the other steps and conditions are the same.

[0047] Specifically, in S1, the differential pressure bursting puffing pressure is set to 0.9 MPa, the holding time is 30 s, and other operating steps are the same as in S1 of Example 1; S2 to S5 are exactly the same as in Example 1. This example can be used to investigate the effect of different puffing intensities on the subsequent release and enrichment of active ingredients.

[0048] Example 3: This example is based on Example 1, but only the enzymatic hydrolysis conditions are adjusted to investigate the effects of enzyme dosage and hydrolysis time on the release of active ingredients.

[0049] Specifically: S1 is the same as in Example 1; in S2, the amount of compound enzyme added is adjusted to 0.15% of the mass of puffed walnut kernel skin, the enzymatic hydrolysis temperature is kept constant at 50℃, the enzymatic hydrolysis time is extended to 3 h, and the enzyme type and pH conditions are the same as in Example 1; the inactivation method after enzymatic hydrolysis is the same as in Example 1; S3 to S5 are the same as in Example 1.

[0050] Example 4: This example is based on Example 1, but only the ratio of fermentation strains and fermentation conditions are adjusted to investigate the effects of different strain ratios and fermentation time on biotransformation.

[0051] Specifically: S1 and S2 are the same as in Example 1; in S3, the inoculation ratio of lactic acid bacteria to yeast is adjusted to 1:1, and the total inoculation amount is maintained at 10. 7 CFU / mL; fermentation temperature was set at 34℃, fermentation time was extended to 36 h, and other pH control and stirring conditions were the same as in Example 1; S4 and S5 were the same as in Example 1.

[0052] Comparative Example 1 (Unexpanded Group): This comparative example was used to verify the necessity and effectiveness of the differential pressure explosion expansion step.

[0053] The specific steps are as follows: After drying and pulverizing, the raw materials are not subjected to differential pressure explosion puffing; 20-mesh walnut kernel skin powder is directly used as the enzymatic hydrolysis raw material. Enzymatic hydrolysis conditions: solid-liquid ratio 1:20; enzyme type, enzyme dosage, hydrolysis temperature, and time are the same as in Example 1, S2. Fermentation conditions: strain combination, inoculum size, fermentation temperature, and time are the same as in Example 1, S3. Solid-liquid separation, resin adsorption, membrane separation, and drying processes (S4, S5) are all the same as in Example 1. Comparative Example 1 does not include the puffing step and can be compared with Examples 1-4 to evaluate the contribution of puffing pretreatment to structural modification and the release of active ingredients.

[0054] Comparative Example 2 (Puffing + Single-Stage Fermentation Group): This comparative example retains the differential pressure explosion puffing and enzymatic hydrolysis steps, but only uses single-strain fermentation and does not use multi-strain gradient biotransformation, in order to evaluate the effect of gradient fermentation.

[0055] The specific steps are as follows: S1: The puffing conditions are the same as in Example 1; S2: The enzymatic hydrolysis conditions are the same as in Example 1; S3: During fermentation, only Lactobacillus plantarum is inoculated as a single strain, and the total inoculation amount is 10. 7 CFU / mL, fermentation temperature 36℃, fermentation time 24 h; no yeast or other strains added; S4, S5: solid-liquid separation, resin adsorption, membrane separation and drying conditions are the same as in Example 1.

[0056] Detection methods The following detection methods are used to evaluate the structural characteristics, polyphenol content, and antioxidant properties of the samples obtained in the examples and comparative examples, providing a basis for subsequent result analysis.

[0057] (1) Porosity and water absorption rate determination. Walnut kernel skin samples were taken before and after puffing, and the volume change was measured by liquid displacement method to calculate porosity; water absorption rate was calculated by recording the mass difference of the sample before and after soaking in deionized water. The porosity and water absorption rate results can be used to compare the effects of different treatments on the structure and water absorption capacity of walnut kernel skin.

[0058] (2) Polyphenol Exposure Determination. The content of reactive polyphenols in the sample was determined by the Folin-Ciocalteu colorimetric method, and the polyphenol exposure was expressed as the relative change in the content of reactive polyphenols per unit mass of sample. The specific method was as follows: a certain amount of sample was weighed, and after appropriate pre-wetting or dispersion treatment, Folin-Ciocalteu reagent was added. The color reaction was carried out under alkaline conditions, and the absorbance was measured at a suitable wavelength after the reaction was completed. A standard curve was plotted using gallic acid as a standard, and the content of reactive polyphenols in the sample was calculated. The polyphenol exposure was expressed as the relative value with respect to the control sample.

[0059] (3) Determination of total polyphenol content. The sample solution was subjected to a colorimetric reaction using Folin-Ciocalteu reagent, and the absorbance was measured at a wavelength of 760 nm. The total polyphenol content was calculated using the gallic acid standard curve. Samples could be taken from the enzymatic hydrolysate, fermentation broth, and membrane enrichment solution to compare the polyphenol release and enrichment at different process stages.

[0060] (4) Determination of free polyphenol content. Take the solution after enzymatic hydrolysis or fermentation of the sample, separate the free polyphenols by an appropriate extraction method (e.g., extraction with ethyl acetate), evaporate the organic phase to dryness, redissolve with methanol or methanol-water, and then determine the absorbance by the Folin-Ciocalteu method to calculate the relative content of free polyphenols.

[0061] (5) Determination of active ingredient content in membrane enrichment solution. Dilute the nanofiltration membrane concentrate to bring its absorbance within the linear range of the instrument, and measure the absorbance at a specific wavelength (such as 280 nm or 320 nm) to estimate the content of small molecule phenolic substances; if necessary, high performance liquid chromatography (HPLC) can be used to perform qualitative or semi-quantitative analysis of representative phenolic substances to compare the enrichment effect under different process conditions.

[0062] (6) Antioxidant performance determination. The antioxidant activity of the samples was evaluated using a DPPH free radical scavenging experiment. DPPH method: The sample was mixed with DPPH ethanol solution and reacted. After a certain period of time, the absorbance was measured at 517 nm, and the free radical scavenging rate was calculated. The antioxidant indices of different embodiments and comparative samples can be used to compare the effectiveness of the process of this invention in improving functionality.

[0063] The specific embodiments and testing methods described above are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make appropriate adjustments to the process conditions and testing methods based on the inventive concept, and such adjustments should be considered to fall within the scope of protection of this invention.

[0064] Results Analysis 1. Effects of puffing treatment on the structural properties of walnut kernel skin Figure 2 The differences between the unexpanded and expanded groups in three key structural indicators: porosity increase factor, protein exposure, and water absorption rate. As shown in the figures, compared to the unexpanded group, the expansion treatment significantly improved the structural accessibility of the walnut kernel skin: the porosity increase factor increased from 1.0 to 3.2, indicating that expansion caused the originally dense and compact tissue to rapidly expand and crack, forming numerous interconnected micropores; protein exposure increased from 100 to 165, indicating that expansion promoted the exposure of phenols originally embedded in the cell wall or bound structure; and water absorption rate increased from 1.2 to 2.8 g / g, indicating that the hydrophilicity of the expanded material was enhanced. These results collectively demonstrate that the expansion technology, through instantaneous pressure release causing structural expansion and cracking, transforms the walnut kernel skin from a low-accessibility substrate into a fermentable material with a high specific surface area and multiple exposed sites. This structural modification provides significant advantages for the release and transformation of macromolecular phenols in subsequent enzymatic hydrolysis and two-stage fermentation.

[0065] 2. Effects of different processes on total polyphenol release capacity Figure 3 The differences in total polyphenol release among Comparative Example 1, Comparative Example 2, and Examples 1-4 are illustrated. The overall trend is: Comparative Example 1 (unexpanded + single-stage fermentation) < Comparative Example 2 (expanded + single-stage fermentation) < Examples 1-4 of this invention (expanded + two-stage gradient fermentation). Specifically, Comparative Example 1 (8.5 mg GAE / g) had the lowest release, because the unexpanded substrate structure is dense, making it difficult for enzymes and cells to enter the interior, thus hindering the release of bound polyphenols. Comparative Example 2 (11.2 mg GAE / g) showed a significant improvement over Comparative Example 1, indicating that expansion can enhance the openness of the substrate structure, but single-stage fermentation is still insufficient to completely release the polyphenols bound in the walnut kernel skin. Examples 1-4 all showed significant improvements (13.8-15.8 mg GAE / g), demonstrating the synergistic advantages of the "complex enzymatic hydrolysis + gradient fermentation" mode of this invention: the complex enzyme stage promotes the initial dissociation of cell wall polysaccharides and protein-bound structures; the first-stage fermentation promotes the release of macromolecular bound phenols; and the second-stage fermentation further converts some phenols into more soluble forms. Among them, Example 3 showed the highest result (15.8 mg GAE / g), indicating that its enzymatic hydrolysis conditions and fermentation parameters were most conducive to the complete release of polyphenols.

[0066] 3. Effects of different processes on the amount of free polyphenols generated Free polyphenols are the main antioxidant active substances in walnut kernel skin, and their content directly affects the bioactivity of the final product. Figure 4 As can be seen, Comparative Example 1 showed only 3.2 mg GAE / g, because the unexpanded structure limited enzyme contact, making it difficult to break down bound phenols. Comparative Example 2 increased to 4.6 mg GAE / g, indicating that expansion can improve substrate solubility, allowing some bound phenols to be released. Examples 1-4 showed a significant increase to 6.8-8.9 mg GAE / g, fully demonstrating the dual effect of "release + biotransformation" brought about by the combined enzymatic hydrolysis and two-stage fermentation. In particular, Example 3 (8.9 mg GAE / g) had the highest value because: the moderate enzymatic hydrolysis intensity allowed for effective dissociation of the polyphenol-bound structure; and the second-stage lactic acid bacteria metabolism converted some phenols into smaller, more polar free phenols, increasing the detection limit. The process of this invention significantly increases the proportion of free polyphenols, laying the foundation for improved antioxidant capacity.

[0067] 4. Effects of different processes on the content of active ingredients in the membrane enrichment solution Figure 5 The concentration of active substances (including phenols, small molecule phenolic glycosides, soluble aromatic acids, etc.) in the membrane enrichment solution was reflected using A280. The trends are as follows: Comparative Example 1 had the lowest concentration (0.42), because most phenols remained in a bound state due to the lack of expansion and could not enter the membrane separation system; Comparative Example 2 (0.58) showed a significant increase, thanks to the structural opening brought about by expansion; Examples 1-4 (0.72-0.93) showed a significant increase, indicating that the process of the present invention can significantly promote the generation and dissolution of small molecule active substances. Among them, Example 3 had the highest concentration (0.93), indicating that its conditions were most favorable: full release of polyphenols; freed phenols entering the membrane enrichment stage; and small molecules more easily permeating the filtration membrane and being enriched in the permeate. The results fully demonstrate that the three processes of compound enzymatic hydrolysis + gradient fermentation + membrane separation have a synergistic effect.

[0068] 5. The impact of different processes on antioxidant capacity Figure 6 The comparison of DPPH free radical scavenging rates is presented. The data shows that Comparative Example 1 had the lowest rate, due to its unmodified structure and low release of active substances; Comparative Example 2 showed a moderate increase, indicating that puffing was a fundamental contribution; Examples 1-4 showed a significant increase to 58%-71%, indicating that the dissociation effect of the complex enzymatic hydrolysis, the biotransformation effect of gradient fermentation, and the enrichment effect of membrane separation collectively improved the antioxidant activity. Example 3 had the highest rate (71%), compared to... Figure 3 , Figure 4 , Figure 5 The consistency demonstrates that its process combination is the most advantageous.

[0069] From structural modification ( Figure 2 ), release of active substances ( Figure 3 ), biotransformation ( Figure 4 ), enrichment efficiency ( Figure 5 From functional activity () Figure 6 All results show a consistent trend: the "expansion pretreatment + compound enzymatic hydrolysis + two-stage fermentation + membrane separation" process of this invention has significant comprehensive advantages in the release of active ingredients and functional enhancement of walnut kernel peel, representing a synergistic innovation in the process chain. In particular, Example 3 shows the best performance in all indicators and is the optimal implementation path of this invention.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enriching the active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation, characterized in that: include: S1. Differential pressure explosion puffing treatment is carried out on the dried walnut kernel skin, so that the material is held under pressure of 0.8-1.5 MPa for 10-60 seconds and then instantaneously released to atmospheric pressure within 0.05-0.2 seconds to obtain puffed walnut kernel skin with increased porosity, cell wall rupture and increased specific surface area. S2. Mix the puffed walnut kernel skin with water at a solid-liquid mass ratio of 1:10 to 1:30 to form a slurry. Add a complex enzyme system containing cellulase, hemicellulase, pectinase, tannic acidase and / or protease. Enzymatically hydrolyze the mixture for 1 to 4 hours at 45 to 55°C and pH 4.5 to 6.0 to partially hydrolyze the cell wall components and release bound phenols. S3. The enzymatically hydrolyzed material is then subjected to the first and second stage fermentation sequentially: The first stage involves inoculating with one or more microorganisms selected from Aspergillus niger, Aspergillus oryzae, or Bacillus subtilis, and fermenting at 32–38°C for 12–24 hours. The second stage involves inoculating with one or more lactic acid bacteria selected from Lactobacillus plantarum, Lactobacillus acidophilus, or Lactobacillus casei, and fermenting at 32–38°C for 12–24 hours to promote the freeing and stabilization of polyphenols. S4. After fermentation, the material is separated into solid and liquid components to obtain the supernatant. S5. After concentrating the supernatant through a nanofiltration membrane with a molecular weight cutoff of 1-10 kDa, the solution is dried to obtain walnut kernel peel active ingredient powder.

2. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 1, characterized in that: The differential pressure bursting expansion treatment pressure in step S1 is 1.0 to 1.3 MPa, and the pressure holding time is 20 to 40 seconds.

3. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 2, characterized in that: The porosity of the walnut kernel skin increases by 2 to 5 times and the water absorption rate increases by 30% to 80% after the puffing treatment.

4. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 3, characterized in that: The total amount of the compound enzyme system added in step S2 is 0.1% to 0.5% (w / w) of the dry weight of the puffed walnut kernel skin.

5. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 4, characterized in that: The microorganisms used in the first stage of fermentation in step S3 have cellulase activity ≥10 U / g and polyphenol oxidase activity ≥5 U / g.

6. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 5, characterized in that: The endpoint pH of the second stage fermentation in step S3 is 3.8 to 4.

5.

7. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 6, characterized in that: In step S4, the solid-liquid separation is performed by centrifugation or plate and frame filtration, and the solid content of the obtained supernatant is ≤0.5% (w / v).

8. The method for enriching active ingredients in walnut kernel peel based on differential pressure explosion puffing and gradient biotransformation as described in claim 7, characterized in that: The nanofiltration membrane in step S5 operates at a pressure of 0.3–0.8 MPa and has a concentration factor of 5–15 times.

9. The walnut kernel peel active ingredient powder obtained by the method according to claim 1, characterized in that: The powder contains a total polyphenol content of not less than 12 mg GAE / g, with free polyphenols accounting for ≥45% of the total polyphenols, and components with a molecular weight less than 10 kDa accounting for ≥80%.

10. The walnut kernel peel active ingredient powder according to claim 9, characterized in that, The powder has a DPPH radical scavenging rate of ≥60% and an ABTS radical scavenging rate of ≥55%.

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