A method for preparing and applying abalone fruit oil, abalone fruit protein, bioactive protein peptides, and their co-production.

Abstract

This application relates to the field of extraction of effective components from woody plants, and particularly to a method and application for the preparation of abalone fruit oil, abalone fruit protein, abalone fruit active protein peptides, and their co-production. This method achieves efficient preparation of the target products: firstly, specific infrared-assisted technology is used to efficiently extract oil from abalone fruit, improving the oil extraction rate and purity; subsequently, a calcium hydroxide-phosphate system is used to directionally separate and extract proteins from the remaining raw materials, ensuring the structural integrity and extraction efficiency of the proteins, and exhibiting good emulsification stability; finally, using the extracted proteins as substrates, directional hydrolysis is performed via enzymatic hydrolysis to prepare selenium / zinc-enriched abalone fruit active peptides with good acetylcholinesterase / lipase inhibitory activity. This preparation process is rational, simple to operate, efficient, and environmentally friendly, achieving comprehensive utilization of abalone fruit resources. The prepared active peptides possess multiple advantages, including zinc / selenium enrichment and bioactivity, and the preparation process is suitable for industrial production.

Description

Technical Field

[0001] This application relates to the field of extraction of effective components from woody plants, and in particular to a method for preparing and using abalone fruit oil, abalone fruit protein, active protein peptides, and their co-production. Background Technology

[0002] Abalone fruit ( Bertholletia excelsa Brazil chestnut (Salix babylonica), also known as Brazilian chestnut, is a tropical nut tree species belonging to the genus Salix babylonica in the family Sterculiaceae. As a nut resource with both ecological and economic value, the kernel of Brazil chestnut is rich in oil, protein, dietary fiber, vitamins, and various minerals. Its oil content is as high as 60%-70%, and its protein content is approximately 15%-25%, with a balanced amino acid composition containing all eight essential amino acids. Unsaturated fatty acids account for over 90%, especially abundant in polyunsaturated fatty acids such as linoleic acid and linolenic acid. It is also rich in bioactive substances such as phytosterols and polyphenols. Due to its excellent nutritional composition, Brazil chestnut has broad application prospects in the fields of food, health products, and cosmetics.

[0003] However, the current development of abalone fruit resources is still mainly focused on primary processing, with most products circulating as shelled nuts, roasted nuts, and other primary processed products. The deep processing links are weak, the extraction efficiency of core nutrients such as oil and protein is low, and the development of high value-added products is insufficient.

[0004] Traditional oil extraction methods mainly involve Soxhlet extraction and mechanical pressing. Soxhlet extraction suffers from drawbacks such as long extraction time (typically 6-12 hours), high solvent consumption, high energy consumption, and the risk of solvent residue. While mechanical pressing is simpler to operate, its oil extraction rate is relatively low, and prolonged high-temperature pressing can easily lead to the degradation of active ingredients, affecting product quality. Furthermore, the defatted cake residue after oil extraction, as a major byproduct, is often discarded directly or used as animal feed, failing to effectively utilize its rich high-quality protein, resulting in serious resource waste and environmental pollution, and hindering the high-value development of the abalone fruit industry chain. As the food industry transforms towards green, efficient, and high-value production, the research and application of new extraction technologies have become crucial to overcoming the shortcomings of traditional processes. How to learn from and combine a novel green extraction technology for abalone fruit oil extraction to improve the extraction effect and efficiency of the target product has not yet been systematically reported, and the optimization of process parameters and extraction mechanisms of novel green extraction technologies applied to abalone fruit oil still require in-depth exploration.

[0005] Meanwhile, abalone fruit protein, as the core nutrient in defatted abalone fruit cake, requires efficient extraction and high-value conversion for full resource utilization. Currently, plant protein extraction methods mainly include alkali extraction and acid precipitation, salt dissolution, and enzymatic extraction. While alkali extraction and acid precipitation are simple and low-cost, excessively high pH levels during extraction can lead to protein denaturation, affecting its functional properties and subsequent processing performance. Salt dissolution offers higher extraction rates, but the subsequent desalination process is complex, increasing production costs. Therefore, further research is needed to explore the feasibility and optimization of processes that offer advantages such as mildness, a narrow pH adjustment range (avoiding excessive protein denaturation), high extraction rates, and no toxic or harmful residues in abalone fruit protein extraction.

[0006] Bioactive peptides, as hydrolysis products of proteins, are small molecule compounds composed of 2 to 10 amino acids. They possess various physiological functions such as antioxidation, lowering blood pressure, lowering blood lipids, immune regulation, and antibacterial activity. They are also easily digested and absorbed by the human body, showing broad application prospects in functional foods, health products, and pharmaceuticals. Enzymatic hydrolysis, as the mainstream technology for preparing bioactive peptides, features mild reaction conditions, controllable hydrolysis products, high safety, and effective preservation of peptide physiological activity. Compared to traditional methods such as acid hydrolysis and alkaline hydrolysis, it better meets the needs of green development in the food industry.

[0007] Currently, various bioactive peptides have been successfully prepared from raw materials such as soybeans, whey, and fish protein both domestically and internationally. Zinc and selenium are essential trace elements for the human body. Given that abalone fruit grows in an environment rich in selenium, its kernels and processing byproducts are naturally enriched with selenium, providing a unique raw material advantage for preparing selenium-enriched functional components. Zinc / selenium-enriched peptides, as a combination of zinc / selenium and bioactive peptides, possess both the physiological functions of zinc / selenium, such as antibacterial and antiviral activity and immune regulation, and the characteristics of peptides, such as easy absorption and strong targeting. Therefore, they have broad application prospects in the fields of functional foods and health products.

[0008] However, research on extracting zinc / selenium-rich bioactive peptides from abalone fruit protein using a specific process remains lacking. Therefore, converting the protein in defatted abalone fruit cake into high-value-added zinc / selenium-rich peptides through enzymatic hydrolysis not only achieves deep utilization of the protein but also extends the abalone fruit industrial chain, increases product added value, and is of great significance for promoting the comprehensive development of abalone fruit resources.

[0009] Based on the aforementioned research background and existing problems, there is an urgent need to establish a green, efficient, and low-cost comprehensive utilization technology for abalone fruit resources. This technology would provide a theoretical basis and technical support for the industrial production of abalone fruit oil, abalone fruit protein, and abalone fruit bioactive peptides, promote the high-value development of the abalone fruit industry chain, and provide a reference for the comprehensive development of other nut resources. Summary of the Invention

[0010] To address the problems mentioned in the prior art as described in the background section, this application provides the following technical solution:

[0011] This application provides a method for the co-production of abalone fruit oil, abalone fruit protein, and abalone fruit active protein peptides, which includes the following steps: S1. Infrared-assisted oil extraction: Abalone kernel powder is mixed with a buffer solution and pretreated under infrared radiation; then an enzyme preparation is added for enzymatic hydrolysis, and solid-liquid separation is performed to obtain abalone kernel oil and defatted solid residue. S2. Calcium hydroxide-phosphate combined extraction of protein: The defatted solid residue is powdered and mixed with water. The pH is adjusted to alkaline using calcium hydroxide. After extraction with pulsed electric field assistance, the supernatant is collected by solid-liquid separation. Then, the pH of the supernatant is adjusted to acidic using phosphate, and centrifugation is performed to obtain acid-precipitated protein. Alternatively, the supernatant is mixed with ethanol to precipitate, and centrifugation is performed to obtain alcohol-precipitated protein. S3. Enzymatic hydrolysis to prepare active protein peptides: Using the acid-precipitated protein and / or alcohol-precipitated protein as substrates, water is added to prepare a protein suspension, and protease is added to carry out enzymatic hydrolysis. After the enzymatic hydrolysis is completed, solid and liquid are separated, the hydrolysate is collected, and dried to obtain abalone fruit protein peptides.

[0012] In some experimental groups, in step S1, abalone kernel powder was mixed with a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.2–6.0, and the weight ratio of abalone kernel powder to buffer solution was 1:10–1:20; the conditions for infrared radiation treatment were: infrared wavelength range of 2.5–15 μm, and infrared power of 1–3 W / cm². 2 The sample distance was 5–20 cm, the treatment method was intermittent, and the total treatment time was 15–60 min.

[0013] In some experimental groups, in S1, the enzyme preparation is selected from one or more combinations of cellulase, hemicellulase, and phospholipase; the enzymatic hydrolysis conditions are: temperature 40-50℃, time 3-6h, and the amount of the enzymatic hydrolysant is 2%-6% of the mass of abalone kernel powder.

[0014] In some experimental groups, the conditions for pulsed electric field treatment in S2 are: electric field strength 5-10 kV / cm, pulse width 40μs, and treatment time 40-60min.

[0015] In some experimental groups, the conditions for infrared radiation treatment in S1 are: sample distance of 5 to 20 cm, intermittent treatment, and total treatment time of 45 to 60 min.

[0016] In some experimental groups, in S1, the enzyme preparation is selected from a combination of cellulase, hemicellulase and phospholipase, and the enzymatic hydrolysis conditions are: temperature 40-50℃, time 4-6h; the amount of the enzymatic hydrolysate is 3%-6% of the mass of abalone kernel powder; and the mass ratio of cellulase, hemicellulase and phospholipase is 1:1:1. In some experimental groups, the conditions for pulsed electric field treatment in S2 are: electric field strength 8-10 kV / cm, pulse width 40 μs, and treatment time 40-60 min.

[0017] In some experimental groups, in S2, calcium hydroxide was used to adjust the pH to 10–12, and phosphoric acid was used to adjust the pH to 4.0–4.7.

[0018] In some experimental groups, based on the protein weight of acid-precipitated and / or alcohol-precipitated proteins, in step S3, 1%–2% of the alkaline protease, and / or 1%–2% of the acidic protease, and / or 1%–2% of the bromelain were added for enzymatic hydrolysis. The total amount of the protease added was 1%–3% based on the protein weight of acid-precipitated and / or alcohol-precipitated proteins. During the enzymatic hydrolysis, calcium hydroxide solution and / or citric acid were used to adjust the pH of the system, and zinc citrate was added during the hydrolysis process to enrich the protein peptides with zinc. The amount of zinc citrate added was 0.5–2.5‰ of the substrate protein weight. In some experimental groups, the protease in S3 is selected from a combination of three of the following: alkaline protease, acidic protease, and bromelain. The enzymatic hydrolysis process is as follows: using the acid-precipitated protein and / or alcohol-precipitated protein as substrates, water is added to prepare a protein suspension; during the enzymatic hydrolysis process, calcium hydroxide solution and / or citric acid are used to adjust the pH of the system; alkaline protease is added to carry out the enzymatic hydrolysis reaction for 90-120 min; then acidic protease and bromelain are added, followed by zinc citrate, and the enzymatic hydrolysis continues for 60-120 min; after the enzymatic hydrolysis is completed, solid-liquid separation is performed, the hydrolysate is collected, and dried to obtain abalone fruit protein peptides; after the enzymatic hydrolysis is completed, carbon dioxide is introduced to adjust the pH of the system to 6.3-6.7, generating calcium carbonate precipitate.

[0019] In some experimental groups, the amount of the protease was 3% based on the protein weight of acid-precipitated protein and / or alcohol-precipitated protein; the weight ratio of the alkaline protease, acidic protease and bromelain was 1:1:1; and the amount of zinc citrate added was 1‰ of the substrate protein weight.

[0020] This application provides an abalone fruit oil, which is prepared by the method described above.

[0021] This application provides an abalone fruit protein, which is prepared by the method described above.

[0022] This application provides an abalone fruit active protein peptide, which is prepared by the method described above.

[0023] This application also provides the application of abalone fruit active protein peptide in the preparation of functional products. The abalone fruit protein peptide is prepared by the preparation method described above. The abalone fruit protein peptide is rich in selenium and / or zinc and has acetylcholinesterase inhibitory activity and / or lipase inhibitory activity.

[0024] Based on the above, compared with the prior art, the solution of this application has the following technical effects: This application provides a method for the co-production of abalone fruit oil, abalone fruit protein, and abalone fruit bioactive peptides. The method achieves efficient preparation of the target products through a series of processes including infrared-assisted extraction, calcium hydroxide-phosphate combined extraction, and enzymatic hydrolysis. First, a specific infrared-assisted extraction technology is used to efficiently extract oil from abalone fruit, improving the extraction rate and purity. The resulting abalone fruit oil has a high content of phytosterols. Subsequently, a calcium hydroxide-phosphate combined system is used to directionally separate and extract proteins from the remaining raw materials, ensuring the structural integrity of the proteins and extraction efficiency, while maintaining good emulsification stability. Finally, using the extracted proteins as substrates, a specific enzymatic hydrolysis method is used to directionally hydrolyze the proteins, resulting in selenium-enriched abalone fruit bioactive peptides with good acetylcholinesterase / lipase inhibitory activity. This application features a reasonable and simple process, achieving comprehensive utilization of abalone fruit resources. The prepared zinc / selenium-enriched bioactive peptides possess multiple advantages, including zinc / selenium elements and bioactivity. The extraction and preparation processes are efficient and environmentally friendly, suitable for industrial production, and have application potential in food fortification and functional food development.

[0025] Other features and beneficial effects of this application will be set forth in the following description and will be apparent in part from the description or may be learned by practicing the application. The objectives and other beneficial effects of this application can be realized and obtained from the description, claims and drawings. Attached Figure Description

[0026] Figure 1 This is a graph showing the activity detection data of different peptide samples; Detailed Implementation To make the purpose, technical solutions, and advantages of the experimental group of this application clearer, the technical solutions in the experimental group of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described experimental group is a part of the experimental group of this application, not all of the experimental group. The technical features designed in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other. All other experimental groups obtained by those skilled in the art based on the experimental group of this application without creative effort are within the scope of protection of this application.

[0027] In the description of this application, it should be noted that all terms used in this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and should not be construed as limiting this application; it should be further understood that the terms used in this application should be understood to have the same meaning as those in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this application.

[0028] This application provides a specific feasible implementation method for preparing abalone fruit oil, abalone fruit protein, and active protein peptides, as follows: The purpose of this application is to provide a method for the co-production of abalone fruit oil, abalone fruit protein, and abalone fruit active protein peptides, and to prepare selenium / zinc-enriched chelate peptides using protein as a raw material. The obtained abalone fruit oil has a high content of phytosterols, and the abalone fruit protein peptides have good acetylcholinesterase / lipase inhibitory activity. The specific steps include: (1) Extraction of oil: Weigh abalone kernels, dry them, and grind them into powder. Add citrate-disodium hydrogen phosphate buffer solution at pH 5.5 at a weight ratio of 1:10 to 1:20. Under stirring, use infrared-assisted intermittent treatment for 15 to 60 min (30 s every 2 min). The wavelength range is 2.5-15 μm, and the infrared power is 1 to 3 W / cm. 2 The sample was kept 5–20 cm away from the infrared light source. Then, an enzyme preparation (one or more combinations of cellulase, hemicellulase, and phospholipase) was added. The enzymatic hydrolysis reaction was carried out at 40–50 °C for 3–6 h with constant stirring. Finally, the enzyme was inactivated at 90 °C for 10–15 min. The mixture was then centrifuged, and the upper abalone fruit oil and the lower solid residue were collected. Specifically, for the infrared-assisted intermittent processing parameters: preferably, the sample distance is 5–20 cm, the processing method is intermittent, and the total processing time is 45–60 min; more preferably, the infrared-assisted intermittent processing is 45–60 min (processing for 30 s every 2 min), and the sample distance is 10 cm. Among these, the infrared-assisted intermittent processing for 45 min (processing for 30 s every 2 min) and the sample distance of 10 cm are the optimal choices.

[0029] Specifically, regarding the enzymatic hydrolysis process parameters: the enzyme preparation is selected from one or more combinations of cellulase, hemicellulase, and phospholipase, and the amount of the hydrolysate is 2% to 6% of the mass of abalone kernel powder; preferably, the enzyme preparation is selected from a combination of cellulase, hemicellulase, and phospholipase, and the amount of the hydrolysate is 3% to 6% of the mass of abalone kernel powder; the mass ratio of cellulase, hemicellulase, and phospholipase is 1:1:1; furthermore, the 1% cellulase + 1% hemicellulase + 1% phospholipase (enzymatic hydrolysis for 6 hours) and 2% cellulase + 2% hemicellulase + 2% phospholipase (enzymatic hydrolysis for 6 hours) schemes have good effects.

[0030] (2) Protein extraction: Dry the lower solid material, pulverize it into powder, mix the sample with deionized water at a material-to-liquid ratio of 1:10 to 1:25 g / mL, adjust the pH of the mixture to 10 to 12 using calcium hydroxide solution, treat with a pulsed electric field of 5 to 10 kV / cm for 40 to 60 min with a pulse width of 40 μs, then adjust the pH of the supernatant to 7.0 using phosphoric acid solution to obtain an alkali-soluble protein extract, adjust the pH to 4.0 to 4.5 using phosphoric acid solution, centrifuge to obtain the precipitate, and obtain acid-precipitated protein. Alternatively, add ethanol to the extract at a volume ratio of 1:4, continue stirring to precipitate, centrifuge, and dry the precipitate to obtain alcohol-precipitated protein. Specifically, the pulsed electric field processing parameters are as follows: preferably, the electric field strength is 8–10 kV / cm, the pulse width is 40 μs, and the processing time is 40–60 min; more preferably, the pulsed electric field processing at 10 kV / cm is 50–60 min. Among these, 60 min of pulsed electric field processing at 10 kV / cm is the optimal choice.

[0031] (3) Enzymatic digestion of proteins: Using the aforementioned abalone fruit acid-precipitated protein / alcohol-precipitated protein as raw materials, a protein suspension with a concentration of 10–20 wt% was prepared by adding water. During enzymatic hydrolysis, the pH of the system was adjusted using a 2 wt% calcium hydroxide solution and / or citric acid. Based on the protein weight of the acid-precipitated protein and / or alcohol-precipitated protein, 1%–2% of the alkaline protease, and / or 1%–2% of the acid protease, and / or 1%–2% of the bromelain were added for enzymatic hydrolysis to obtain protein peptides. At 90–120 min of enzymatic hydrolysis, 0.5–2.5 wt‰ zinc citrate (based on protein weight) was added, and enzymatic hydrolysis continued for 60–120 min. After enzymatic hydrolysis, an appropriate amount of carbon dioxide was introduced to adjust the pH to 6.3–6.7. The solution was centrifuged to obtain the hydrolysate, treated at 90℃ for 10 min, and then spray-dried to obtain protein peptide powder.

[0032] For the proteolytic combination: preferably, the protease is selected from a combination of three of the following: alkaline protease, acidic protease, and bromelain; more preferably, the amount of protease used is 3% based on the protein weight of acid-precipitated protein and / or alcohol-precipitated protein; the weight ratio of alkaline protease, acidic protease, and bromelain is 1:1:1. The combination of 1% alkaline protease + 1% acidic protease + 1% bromelain exhibits the best decomposition effect.

[0033] This application provides the following experiments to verify the effectiveness of the proposed solution: Effect of infrared treatment on oil extraction in experimental group 1 Weigh abalone kernels, dry them, and grind them into powder. Add the powder to a citrate-disodium hydrogen phosphate buffer solution at a mass ratio of 1:15. Under stirring, treat intermittently with infrared-assisted radiation for 0–60 min (30 s every 2 min). The infrared wavelength range is 5 μm, and the infrared power is 2 W / cm². 2 The samples were kept 5–20 cm apart. The infrared light source was turned off, and 1.5% of cellulase and 1.5% of hemicellulase (based on the mass of abalone kernel powder) were added. The enzymatic hydrolysis reaction was carried out at 40°C for 5 hours with constant stirring. Finally, the enzymes were inactivated at 90°C for 15 minutes, centrifuged, and the upper abalone fruit oil was collected. For detailed data on the oil extraction effect, please refer to Table 1.

[0034] Note: Sample distance refers to the vertical straight-line distance between the infrared radiation source (heating lamp / plate) and the surface of the extracted sample.

[0035] Table 1: Effect of infrared treatment time on oil extraction

[0036] analyze: Infrared radiation is an electromagnetic wave between visible light and microwaves. In infrared radiation heating, the radiation from the heat source directly acts on the object being heated, while the air between the objects is mainly composed of oxygen and nitrogen molecules, which do not absorb radiation energy in the infrared band. Compared with traditional convective heat transfer, it saves the energy spent heating the intermediate medium, resulting in extremely high thermal efficiency and thus exhibiting the characteristics of rapid and uniform heating. Table 1 shows that infrared-assisted extraction effectively improves the extraction rate of abalone fruit oil.

[0037] Furthermore, numerous studies both domestically and internationally have shown that phytosterols possess the effects of lowering serum low-density lipoprotein cholesterol, as well as antioxidant, anticancer, anti-inflammatory, and antibacterial properties. The United States has approved phytosterols for sale and use as a new resource food or food ingredient. Natural phytosterols are found in vegetable oils and are relatively abundant. Phytosterols exist in plant cells and are important structural components for stabilizing plant biomembranes.

[0038] This application proposes an infrared-assisted extraction method for extracting abalone fruit oil and phytosterols from abalone fruit oil. The infrared-assisted extraction technology increased the phytosterol content in abalone fruit oil, and it was found that the phytosterol content tended to stabilize after 45 minutes of treatment. The reason for this is likely due to the vibrational excitation of atoms or molecules in the abalone fruit plant material by infrared radiation, generating excess heat. This raises the intracellular temperature of the abalone fruit, causing vaporization of internal fluids and tissue rupture. Simultaneously, the properties of the solvent also change during infrared radiation. For example, the dielectric properties, surface tension, viscosity, and polarity of water decrease after infrared radiation. Therefore, during the extraction of the effective components from abalone fruit, the solubility of certain polar compounds in water increases with increasing temperature, thereby improving the mass transfer of the targeted compounds.

[0039] In summary, the extraction method of this application targets specific abalone fruit oil and phytosterols. The entire process parameters of the infrared-assisted intermittent treatment are designed as follows: infrared-assisted intermittent treatment for 15–60 min (30 s treatment every 2 min), wherein the infrared wavelength range is 5 μm and the infrared power is 2 W / cm². 2 The sample distance was 5–20 cm. Table 1 shows that, compared with the method without infrared-assisted extraction, the infrared-assisted extraction method in this application effectively improved the extraction rate of abalone fruit oil. Simultaneously, it increased the content of phytosterols in the abalone fruit oil.

[0040] Furthermore, to enhance the extraction efficiency of specific abalone fruit oils and phytosterols, the optimized parameters for the infrared-assisted intermittent treatment process were as follows: infrared-assisted intermittent treatment for 45–60 min (30 s per 2 min), with a sample distance of 5–20 cm. Preferably, the infrared-assisted intermittent treatment for 45–60 min (30 s per 2 min), with a sample distance of 10 cm, was selected as the optimal choice.

[0041] Experimental Group 2: Effect of Enzyme Treatment on Oil Extraction Weigh abalone kernels, dry them, and grind them into powder. Add a citrate-disodium hydrogen phosphate buffer solution (pH 5.5) at a mass ratio of 1:15. Under stirring, perform intermittent infrared-assisted treatment for 45 min (30 s every 2 min). The wavelength range is 5 μm, and the infrared power is 2 W / cm². 2 The sample was kept 10 cm away from the infrared light source. Then, 0-2% of cellulase, 0-2% of hemicellulase, and 0-2% of phospholipase (by weight of abalone kernel powder) or one or more combinations thereof were added. The enzymatic hydrolysis reaction was carried out at 40℃ for 3-6 hours with constant stirring. Finally, the enzymes were inactivated at 90℃ for 15 minutes, centrifuged, and the upper abalone fruit oil was collected. For detailed extraction effect data, please refer to Table 2.

[0042] Table 2: Effects of enzyme treatment on oil extraction

[0043] analyze: For the extraction of specific abalone fruit oil and phytosterols, the selection and combination of enzyme preparations significantly affect the extraction efficiency. Enzymes, being proteins, exhibit strict specificity; each enzyme acts on only one substrate. Furthermore, the hydrolysis time is of considerable importance. Table 2 shows that when the three enzymes are mixed in a certain proportion and hydrolyzed for a longer period, such as 1% cellulase + 1% hemicellulase + 1% phospholipase (hydrolysis for 6 hours) or 2% cellulase + 2% hemicellulase + 2% phospholipase (hydrolysis for 6 hours), good results are achieved.

[0044] Effect of pulsed electric field treatment on protein extraction rate in experimental group 3 Oil extraction: Abalone kernels were weighed, dried, and pulverized into powder. A citrate-disodium hydrogen phosphate buffer solution (pH 5.5) was added at a mass ratio of 1:15. The mixture was then subjected to intermittent infrared assisted treatment for 45 min (30 s every 2 min) under stirring conditions. The wavelength range was 5 μm, and the infrared power was 2 W / cm². 2The sample was placed 10 cm away from the infrared light source. Then, 1% of cellulase, 1% of hemicellulase, and 1% of phospholipase (based on the mass of abalone kernel powder) were added. The enzymatic hydrolysis reaction was carried out at 40°C for 6 hours with constant stirring. Finally, the enzymes were inactivated at 90°C for 10 minutes. The mixture was then centrifuged, and the upper abalone kernel oil was collected. Protein extraction: The lower solid layer was dried, pulverized into powder, and mixed with deionized water at a material-to-liquid ratio of 1:15 g / mL. The pH of the mixture was adjusted to 11 using calcium hydroxide solution. The mixture was then treated with a pulsed electric field of 0-10 kV / cm for 30-120 min, with a pulse width of 40 μs. The pH of the supernatant was then adjusted to 7.0 using phosphoric acid solution to obtain an alkali-soluble protein extract. The pH was adjusted to between 4.2 using phosphoric acid or hydrochloric acid solution, and the precipitate was obtained by centrifugation, yielding acid-precipitated protein. Alternatively, the extract was mixed with ethanol at a volume ratio of 1:4, stirred to precipitate, centrifuged, and the precipitate was dried to obtain alcohol-precipitated protein. Detailed data on protein extraction efficiency are shown in Table 3.

[0045] Table 3: Effect of pulsed electric field treatment on protein extraction rate (with calcium hydroxide and phosphate as pH adjusters)

[0046] analyze: Table 3 shows that the proposed method utilizes a pulsed electric field treatment for the extraction of the target product from abalone fruit. Compared to methods without pulsed electric field-assisted extraction, the proposed method effectively improves the extraction rate of the target protein from abalone fruit. Pulsed electric field is a physical field non-thermal processing technology that uses high-intensity, narrow-pulse-width high-frequency electrical pulses to act on cells, promoting cell rupture in abalone fruit and thus enhancing the permeability and extraction rate of abalone fruit proteins. This technology allows for the completion of the extraction process in a relatively short time.

[0047] This application proposes a method for extracting target products from abalone fruit. The entire process parameters for pulsed electric field treatment are designed as follows: 5–10 kV / cm pulsed electric field treatment for 40–60 min, with a pulse width of 40 μs. Further, to improve the extraction efficiency of the specific abalone fruit protein target product, the pulsed electric field treatment parameters are optimized to: 8–10 kV / cm pulsed electric field treatment for 40–60 min, with a pulse width of 40 μs. Preferably, 10 kV / cm pulsed electric field treatment for 50–60 min is used. Among these, 10 kV / cm pulsed electric field treatment for 60 min is the optimal choice.

[0048] Experimental Group 4: Effect of Alkali / Acid Type on Protein Extraction Rate Oil extraction: Abalone kernels were weighed, dried, and pulverized into powder. A citrate-disodium hydrogen phosphate buffer solution (pH 5.5) was added at a mass ratio of 1:15. The mixture was then subjected to intermittent infrared assisted treatment for 45 min (30 s every 2 min) under stirring conditions. The wavelength range was 5 μm, and the infrared power was 2 W / cm². 2 The sample was placed 10 cm away from the infrared light source. Then, 1% of cellulase, 1% of hemicellulase, and 1% of phospholipase (based on the mass of abalone kernel powder) were added. The enzymatic hydrolysis reaction was carried out at 40°C for 6 hours with constant stirring. Finally, the enzymes were inactivated at 90°C for 10 minutes. The mixture was then centrifuged, and the upper abalone kernel oil was collected. Protein extraction: The lower solid layer was dried and pulverized into powder. The sample was mixed with deionized water at a material-to-liquid ratio of 1:15 g / mL. The pH of the mixture was adjusted to 11 using calcium hydroxide or sodium hydroxide solution (variable). The mixture was then treated with a pulsed electric field of 10 kV / cm for 50 min with a pulse width of 40 μs. The pH of the supernatant was then adjusted to 7.0 using phosphoric acid or hydrochloric acid solution (variable) to obtain an alkali-soluble protein extract. The pH was then adjusted to 4.2 using phosphoric acid or hydrochloric acid solution, and the precipitate was obtained by centrifugation, yielding acid-precipitated protein. Alternatively, the extract was mixed with ethanol at a volume ratio of 1:4, stirred to precipitate, centrifuged, and the precipitate was dried to obtain alcohol-precipitated protein. Detailed data on protein extraction efficiency are shown in Table 4.

[0049] Table 4: Effect of pH adjuster on protein extraction rate

[0050] The chemical reactions between the two acids and bases mentioned above are as follows. By controlling the ratio of the two, different products can be obtained. 2H3PO4+ Ca(OH)2= Ca(H2PO4)2+ 2H2O 2H3PO4+ 3Ca(OH)2= Ca3(HPO4)2↓ + 6H2O 2H3PO4+ 3Ca(OH)2= Ca3(PO4)2↓ + 6H2O NaOH + HCl = NaCl + H2O In this application, after obtaining Ca3(HPO4)2 and acid-soluble protein through the reaction 2H3PO4 + 3Ca(OH)2 = Ca3(HPO4)2↓ + 6H2O, H3PO4 is added to obtain Ca(H2PO4)2, which is easily soluble in water, thus separating the acid-soluble protein. However, the currently mainstream NaOH and HCl used as pH adjusters produce soluble NaCl in the process, requiring the sample to be washed and desalted.

[0051] Although different types of alkali and acid showed no significant difference in the extraction rate of abalone fruit protein during the aforementioned alkaline-acid precipitation process, the wastewater generated during the washing process differed. Using calcium hydroxide / phosphate is a more environmentally friendly process. Furthermore, due to the stronger alkalinity of NaOH, the selenium content of the extracted protein is lower compared to that obtained with calcium hydroxide / phosphate.

[0052] Note: The substrates used in verification experiment 4-1 and control experiment 4-1 are the same; the only difference is the type of alkali and acid used. Therefore, the difference in selenium content results between the two experiments is due to the extraction system's ability to retain selenium, not a difference in the background of the raw materials.

[0053] Experimental Group 5: Effects of Enzyme Preparations on Protein Peptides Oil extraction: Abalone kernels were weighed, dried, and pulverized into powder. A citrate-disodium hydrogen phosphate buffer solution (pH 5.5) was added at a mass ratio of 1:15. The mixture was then subjected to intermittent infrared assisted treatment for 45 min (30 s every 2 min) under stirring conditions. The wavelength range was 5 μm, and the infrared power was 2 W / cm². 2 The sample was placed 10 cm away from the infrared light source. Then, 1% of cellulase, 1% of hemicellulase, and 1% of phospholipase (based on the mass of abalone kernel powder) were added. The enzyme was hydrolyzed at 40°C for 6 hours with constant stirring. Finally, the enzymes were inactivated at 90°C for 10 minutes. The mixture was then centrifuged and the upper abalone oil was collected. Protein extraction: The lower solid layer was dried and pulverized into powder. The sample was mixed with deionized water at a solid-liquid ratio of 1:15 g / mL. The pH of the mixture was adjusted to 11 using calcium hydroxide solution. The mixture was then treated with a pulsed electric field of 10 kV / cm for 50 min (pulse width 40 μs). The pH of the supernatant was then adjusted to 7.0 using phosphoric acid or hydrochloric acid solution to obtain an alkali-soluble protein extract. The pH was adjusted to 4.2 using phosphoric acid solution, and the precipitate was obtained by centrifugation, yielding acid-precipitated protein. Alternatively, the extract was mixed with ethanol at a volume ratio of 1:4, stirred to precipitate, centrifuged, and the precipitate was dried to obtain alcohol-precipitated protein. Enzymatic hydrolysis of proteins: Using the above-mentioned abalone protein precipitated with fruit acid or alcohol as raw materials (variable), water was added to prepare a 12.5 wt% protein suspension. The specific enzymatic hydrolysis process is as follows (the enzymatic hydrolysis process is a variable; note: the enzymatic hydrolysis steps (1) and (2) are different in different experiments, and the enzymatic hydrolysis steps are selected according to Table 5-6): (1) During the enzymatic hydrolysis process, use a 2wt% calcium hydroxide solution or citric acid to adjust the pH (the specific pH value should be adjusted according to the pH value of the environment in which the protease is used later). Add 1-2 wt% (based on the protein weight of the precipitated protein) of alkaline protease and hydrolyze for 90-120 min. (2) Add 1-2 wt% acidic protease and / or 1-2 wt% bromelain (based on the protein weight of the precipitated protein), then add 0-1 wt‰ zinc citrate (based on the protein weight of the precipitated protein), and continue enzymatic hydrolysis for 60-120 min; After enzymatic hydrolysis, an appropriate amount of carbon dioxide was introduced, and the mixture was stirred for 10 minutes before centrifugation to obtain the enzymatic hydrolysate. This hydrolysate was then treated at 90℃ for 10 minutes and spray-dried to obtain protein peptide powder. Detailed results are shown in Tables 5-6. Table 5: Effects of enzyme preparations on protein peptides

[0054] Table 6: Effects of enzyme preparations on protein peptides

[0055] in conclusion: As can be seen from Table 5-6: Different enzyme preparations significantly affected the yield of abalone fruit protein peptides in the extraction of specific target products from abalone fruit. Among them, alkaline protease had the most significant effect on the decomposition of abalone fruit protein, while acidic protease or bromelain had relatively weak effects on abalone fruit protein.

[0056] For the extraction of target products from specific abalone fruits, this application specifically designed a zinc citrate addition process combined with enzymatic hydrolysis, which facilitates the exposure of more zinc-binding active sites, allowing the target product to be loaded with more zinc. Furthermore, this application innovatively discovered that the combination of 1% alkaline protease + 1% acidic protease + 1% bromelain achieves the best decomposition effect.

[0057] In addition, carbon dioxide is introduced into the enzymatic hydrolysis process. CO2, as an acidic oxide, reacts with Ca(OH)2 (a strong base) to form calcium carbonate (CaCO3) precipitate and water. The reaction equation is: Ca(OH)2 + CO2 → CaCO3↓ + H2O. Therefore, no soluble salt is formed. The desalination process required to obtain protein peptides with high purity and good flavor requires less washing water.

[0058] Depend on Figure 1 The content indicates that: Note: Figure 1In the experiment, peptide 1 was the protein precipitated by 2% alkaline protease (with 1wt‰ zinc citrate) in experimental group 5; peptide 2 was the protein precipitated by 1% alkaline protease + 1% acidic protease + 1% bromelain (with 1wt‰ zinc citrate) in experimental group 5; peptide 3 was the protein precipitated by 1% acidic protease + 1% bromelain (with 1wt‰ zinc citrate) in experimental group 5; peptide 4 was the protein precipitated by 1% acidic protease + 1% bromelain (with 1wt‰ zinc citrate) in experimental group 6; and peptide 5 was the protein precipitated by 2% alkaline protease (with 1wt‰ zinc citrate) in experimental group 6.

[0059] The active functions of different peptide samples, such as Figure 1 As shown, peptide 3 exhibited the best lipase inhibitory activity, while peptide 2 showed the best acetylcholinesterase (AChE) inhibitory activity. Acetylcholine (ACh), as a key neurotransmitter, participates in regulating the homeostasis of cholinergic signaling pathways related to learning and memory. Its biosynthesis is completed by choline and acetyl-CoA under the catalysis of choline acetyltransferase, while ACh in the synaptic cleft is rapidly hydrolyzed into choline and acetic acid by acetylcholinesterase (AChE). This dynamic balance between synthesis and hydrolysis is an important guarantee for the orderly transmission of memory signals. When the amount of ACh synthesis decreases and the degradation process accelerates, it will lead to insufficient nerve signal transmission, ultimately manifesting as symptoms of memory impairment. Acetylcholinesterase inhibitors can block ACh degradation by inhibiting AChE activity, thereby improving the efficiency of cholinergic neurotransmission and thus playing a role in improving memory. On the other hand, dietary triglycerides are the main form of fat ingested by the human body, and their digestion and absorption depend on the catalytic action of pancreatic lipase (PL). This enzyme can hydrolyze 50% to 70% of dietary fat in the human body and is a key rate-limiting enzyme regulating the fat absorption process. By specifically inhibiting PL activity, it is possible to effectively reduce the uptake of free fatty acids in the intestines and block the fat resynthesis process involving them through blood circulation, thereby achieving an intervention effect on obesity.

[0060] Experimental Group 6: Effects of Sodium Hydroxide and Hydrochloric Acid on Protein Peptides Compared to experimental group 5, the only difference was the pH adjuster used; all other process steps were the same. The process is as follows: Enzymatic hydrolysis of protein: Using the above-mentioned abalone fruit acid / alcohol-precipitated protein as raw materials, water was added to prepare a 12.5 wt% protein suspension. During the enzymatic hydrolysis process, a 2 wt% sodium hydroxide solution was used. 1-2 wt% (based on protein weight) of alkaline protease was added. After hydrolysis for 90-120 min, hydrochloric acid (variable) was added to adjust the pH to 6.0. Then, 1-2 wt% acidic protease and / or bromelain were added, followed by 0.5-2.5 wt‰ zinc citrate (based on protein weight). Enzymatic hydrolysis continued for 60-120 min. After the enzymatic hydrolysis was completed, the hydrolysate was obtained. After treatment at 90℃ for 10 min, it was spray-dried to obtain protein peptide powder.

[0061] Based on the reaction formula NaOH + HCl = NaCl + H2O, it is known that the introduction of soluble NaCl during enzymatic hydrolysis increases the saltiness of the peptides. Therefore, sodium filtration is required to reduce the saltiness, thus increasing the amount of water used. However, the acid-base balance has no significant effect on the peptide yield. Specific comparative results are shown in Table 7-8. Table 7

[0062] Table 8

[0063] In addition, the index measurement method used in this article is as follows: (1) Oil extraction rate The fat content is determined according to the national food safety standard "Determination of Fat in Food" (GB 5009.6-2025), and the weight ratio of the fat content to the raw materials used is the extraction rate.

[0064] (2) Protein extraction rate The protein content was determined according to the national food safety standard "Determination of Protein in Food" (GB 5009.5-2025), and the weight ratio of the protein content to the raw materials used is the extraction rate.

[0065] (3) Content of phytosterols References: The following literature determines the content of phytosterols: Bai Ge. Migration and transformation mechanism of phytosterols during corn oil refining [D]. Henan University of Technology, 2022. (4) Peptide content The peptide content was determined in accordance with the National Food Safety Standard for Plant Protein Peptides for Food Processing (GB 31611-2023). (5) Selenium content The selenium content was determined in accordance with the National Food Safety Standard for Determination of Selenium in Food (GB 5009.93-2017).

[0066] (6) Zinc content The zinc content was determined in accordance with the National Food Safety Standard for Determination of Zinc in Food (GB 5009.14-2017).

[0067] (7) Desalination water consumption refers to the amount of water used to ensure that the conductivity of the protein / peptide solution does not exceed 10 μs / cm.

[0068] (8) Acetylcholinesterase (AChE) inhibitory activity of protein peptides The determination was made with reference to the following literature: Wu Binxia, ​​Hu Xiao, Chen Shengjun, et al. Enzymatic preparation of acetylcholinesterase inhibitory peptide from oval pomfret and its physicochemical properties and bioactivity [J]. Advances in Fisheries Science, 2025, 46(05): 244-254. DOI:10.19663 / j.issn2095-9869.20241021001. (9) Lipase (PL) inhibitory activity of protein peptides The determination was made with reference to the following literature: Zhang Zhenghai, Li Baiyang, Tian Yuan, et al. Preparation, structural characterization and activity screening of hemp protein-derived pancreatic lipase inhibitory peptides [J]. Food Industry Technology, 1-15. Doi.org / 10.13386 / j.issn1002-0306.2025060326. In summary, the proposed solution has at least the following design concepts and beneficial effects: Design concept: This application proposes a tiered utilization technology system using abalone fruit as raw material, integrating "oil extraction, protein extraction, and active peptide preparation." Its core design concept lies in achieving efficient conversion and high-value utilization of all resources through multi-technology synergy and green processes. Specific core design points are as follows: (1) In the oil extraction stage, a novel extraction technology was adopted: infrared-assisted intermittent treatment technology was used to extract abalone fruit oil, and the parameters of infrared-assisted intermittent treatment were optimized. As can be seen from the data in Table 1, compared with the scheme without infrared-assisted extraction, the infrared-assisted extraction of this application effectively improved the extraction rate of abalone fruit oil. At the same time, it increased the content of phytosterols in abalone fruit oil. After optimizing the treatment process, when the infrared treatment lasted for 45 minutes and the sample distance was 10 cm, the oil extraction rate reached 36.5% and the phytosterol content reached 615 mg / 100g, which is nearly doubled compared with the 19.4% and 320 mg / 100g of the untreated group, and the phytosterol content increased by 92%. This design utilizes intermittent infrared radiation with a wavelength of 2.5 to 15 micrometers and a power of 1 to 3 watts per square centimeter to achieve a balance between efficient cell wall disruption and retention of active ingredients. Furthermore, the synergistic effect of the compound enzyme preparation was designed: Table 2 shows that when the three enzymes are mixed in a certain proportion, the enzymatic hydrolysis has a significantly better effect. When the combination of 1% cellulase, 1% hemicellulase and 1% phospholipase is used for 6 hours of enzymatic hydrolysis, the oil extraction rate reaches 33.6% and the sterol content is 617 mg per 100g; while the extraction rate is only 28.1% when treated with cellulase alone. This confirms that the synergistic effect of the three enzymes in a 1:1:1 mass ratio is significantly better than that of a single enzyme, and can simultaneously break down cell wall cellulose, hemicellulose and cell membrane phospholipids, promoting the full release of oil.

[0069] (2) In the protein extraction stage, a specific pulsed electric field-assisted extraction method was designed, and the parameters were optimized. Table 3 shows that, compared with the scheme without pulsed electric field-assisted extraction, the scheme of this application effectively improved the extraction rate of target proteins in abalone fruit by using pulsed electric field-assisted extraction. In the further parameter optimization scheme, under an electric field strength of 10 kV / cm for 60 minutes, the acid precipitation protein extraction rate reached 13.6%, and the alcohol precipitation protein reached 4.1%; while without the application of a pulsed electric field, even after 120 minutes of treatment, the acid precipitation protein extraction rate was only 7.5%, and the alcohol precipitation protein was only 1.4%. This design point utilizes a high-intensity pulsed electric field to disrupt the cell membrane structure, thereby increasing the protein extraction rate by nearly double. The optimal parameters were locked at 8 to 10 kV / cm and 40 to 60 minutes.

[0070] Furthermore, an environmentally friendly design using a calcium hydroxide-phosphate system was adopted. Table 4 shows a comparison: the protein extraction rate of the calcium hydroxide-phosphate system was comparable to that of the conventional sodium hydroxide-hydrochloric acid system (13.5% vs. 13.2%), but wastewater generation was reduced by approximately 50% (235% vs. 450%), and selenium retention was significantly improved (23.1 mg / kg vs. 13.2 mg / kg). This design replaces sodium hydroxide with calcium hydroxide (mild alkalinity, reducing protein denaturation) and hydrochloric acid with phosphoric acid (forming calcium phosphate precipitate for easy separation), achieving the dual goals of green extraction and activity retention. In addition, the separation mechanism using calcium phosphate precipitation, by controlling the ratio of calcium hydroxide to phosphoric acid to form an insoluble precipitate, not only achieves protein separation but also reduces subsequent washing steps.

[0071] (3) In the active peptide preparation stage, the design focuses on the synergistic effect of stepwise enzymatic hydrolysis. Tables 5 and 6 show that different enzyme preparations have a significant impact on the yield of abalone fruit protein peptides in the extraction of specific target products from abalone fruit. Among them, alkaline protease has the most significant effect on the decomposition of abalone fruit protein. Acidic protease or bromelain has a relatively weak effect on abalone fruit protein. In this application, zinc citrate is specially designed to be added to cooperate with the enzymatic hydrolysis process for the extraction of specific target products from abalone fruit, which is beneficial to loading more zinc elements. In addition, the innovative scheme of this application found that the combination of 1% alkaline protease + 1% acidic protease + 1% bromelain has the best decomposition effect: using the combination of 1% alkaline protease, 1% acidic protease and 1% bromelain, the yield of acid precipitation protein reaches 57.7%, and the yield of alcohol precipitation protein reaches 54.9%; while the yields of alkaline protease alone are 54.3% and 52.3%, respectively, and the yields of acidic protease alone are only 34.7% to 35.3%. This design utilizes alkaline protease to initially hydrolyze and expose internal sites, followed by acidic protease and bromelain to further cleave specific peptide bonds. The synergistic effect of these three enzymes significantly improves peptide yield.

[0072] Regarding the timing and dosage design of zinc citrate addition, data shows that adding 1‰ zinc citrate resulted in a zinc content of 39.2 to 43.7 mg / kg, compared to 40.1 to 41.3 mg / kg (baseline) in the group without addition, representing an approximately 8% increase in zinc content. Furthermore, the yield of the three-enzyme combination increased from 54.2% to 57.7%. This design point, adding zinc citrate during the mid-stage of enzymatic hydrolysis, allows zinc ions to chelate with the peptides being generated, successfully preparing zinc-rich peptides.

[0073] In addition, a green process combining carbon dioxide neutralization and protease hydrolysis was designed. Comparing Tables 5-6 and 7-8, it can be seen that when using the carbon dioxide neutralization system for calcium hydroxide, the desalination water consumption is only 1:15 to 1:20; while the conventional sodium hydroxide-hydrochloric acid system requires 1:45 to 1:60, reducing water consumption by more than 60%. This design utilizes the reaction of carbon dioxide with calcium hydroxide to generate calcium carbonate precipitate, replacing acid neutralization, avoiding the formation of soluble salts, and simultaneously improving product flavor.

[0074] (4) In terms of functional orientation, the core design point of this application lies in the utilization of the natural advantages of selenium-rich raw materials. Table 4 shows that the selenium content of the protein extracted by the calcium hydroxide-phosphate system reaches 23.1 mg / kg, which is significantly higher than the 13.2 mg / kg of the sodium hydroxide system. This design point maximizes the retention of the naturally enriched selenium element in the raw materials through a mild extraction system, so that the final peptide product has selenium-rich characteristics and does not require exogenous addition.

[0075] In addition, functional screening was conducted on different bioactive peptides. Figure 1 The results showed that peptide 2, prepared by the combination of three enzymes and alcohol-precipitated protein, exhibited the best acetylcholinesterase inhibitory activity, while peptide 3, prepared by the combination of acidic protease and bromelain and acid-precipitated protein, exhibited the best lipase inhibitory activity. This design point demonstrates that the functional predisposition of products can be regulated by selecting different enzyme combinations and protein types, providing a possibility for the targeted preparation of specific active peptides.

[0076] (5) At the process level, this application realizes a three-stage material closed loop: the defatted residue after oil extraction is used for protein extraction, the calcium hydrogen phosphate precipitate in the protein extraction process can be recycled as a by-product, and the calcium carbonate precipitate generated by carbon dioxide neutralization can also be recycled, realizing the full utilization of abalone fruit and zero waste discharge, which is in line with the concept of circular economy.

[0077] Beneficial effects: As the background technology indicates, the development and application of novel extraction technologies are key to overcoming the drawbacks of traditional abalone fruit oil extraction processes. Infrared-assisted extraction (IR), as a novel green extraction technology, has gained widespread attention in the extraction of natural products, especially important active ingredients, due to its advantages such as high thermal efficiency, strong penetration, and short extraction time. However, research on its application in abalone fruit oil extraction has not yet been systematically reported, and further exploration is needed to optimize its process parameters and extraction mechanisms.

[0078] Meanwhile, protein, as the core nutrient in defatted abalone cake, requires efficient extraction and high-value conversion for optimal utilization of its resources. Currently, plant protein extraction methods suffer from a trade-off between extraction efficiency and cost. Therefore, exploring a process with advantages such as mildness, a narrow pH adjustment range (avoiding excessive protein denaturation), high extraction rate, and no toxic or harmful residues, and its feasibility and optimization for abalone fruit protein extraction, is of practical significance.

[0079] Bioactive peptides, as hydrolysis products of proteins, possess various physiological functions and are easily digested and absorbed by the human body, showing broad application prospects in functional foods, health products, and pharmaceuticals. Given that abalone fruit grows in an environment rich in selenium, its kernels and processing byproducts are naturally enriched with selenium, providing a unique raw material advantage for preparing selenium-enriched functional components. Zinc / selenium-enriched peptides, as a combination of zinc / selenium and bioactive peptides, possess both the antibacterial and antiviral / immune-regulating physiological functions of zinc / selenium and the easily absorbed and highly targeted characteristics of peptides, showing broad application prospects in functional foods and health products. However, research on whether a specific process can be used to obtain zinc / selenium-enriched bioactive peptides from abalone fruit protein remains incomplete.

[0080] Therefore, converting the protein in defatted abalone fruit cake into high-value-added zinc / selenium-enriched peptides through enzymatic hydrolysis not only achieves deep utilization of the protein but also extends the abalone fruit industrial chain, increases product added value, and is of great significance for promoting the comprehensive development of abalone fruit resources. Based on this, this application proposes a comprehensive resource-level utilization technology system using abalone fruit kernels as raw material, encompassing "oil extraction - protein extraction - active peptide preparation." First, infrared-assisted extraction technology is used to extract abalone fruit oil, optimizing key parameters such as extraction temperature, extraction time, and material-to-liquid ratio, and exploring the effects of infrared radiation on abalone fruit cell structure and oil extraction rate. Second, using the defatted cake after oil extraction as a substrate, abalone fruit protein is extracted using a calcium hydroxide combined with phosphoric acid method, clarifying the effects of parameters such as pH value and extraction time on protein extraction rate and functional properties. Finally, a suitable protease is selected to hydrolyze abalone fruit protein, optimizing enzymatic hydrolysis process parameters to prepare selenium-enriched abalone fruit active peptides, and their physiological activity is preliminarily evaluated. This application aims to establish a green, efficient, and low-cost technology for the comprehensive utilization of abalone fruit resources, providing a theoretical basis and technical support for the industrial production of abalone fruit oil, protein, and bioactive peptides, promoting the high-value development of the abalone fruit industry chain, and providing a reference for the comprehensive development of other nut resources.

[0081] It should be noted that: In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0082] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each experimental group or technical solution of this application can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0083] Finally, it should be noted that the above experimental groups are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the aforementioned experimental groups, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned experimental groups, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the experimental groups of this application.

Claims

1. A method for the co-production of abalone fruit oil, abalone fruit protein, and abalone fruit active protein peptides, characterized in that, Includes the following steps: S1. Infrared-assisted oil extraction: Abalone kernel powder is mixed with a buffer solution and pretreated under infrared radiation; then an enzyme preparation is added for enzymatic hydrolysis, and solid-liquid separation is performed to obtain abalone kernel oil and defatted solid residue. S2. Calcium hydroxide-phosphate combined extraction of protein: The defatted solid residue is powdered and mixed with water. The pH is adjusted to alkaline using calcium hydroxide. After extraction with pulsed electric field assistance, the supernatant is collected by solid-liquid separation. Then, the pH of the supernatant is adjusted to acidic using phosphate, and centrifugation is performed to obtain acid-precipitated protein. Alternatively, the supernatant is mixed with ethanol to precipitate, and centrifugation is performed to obtain alcohol-precipitated protein. S3. Enzymatic hydrolysis to prepare active protein peptides: Using the acid-precipitated protein and / or alcohol-precipitated protein as substrates, water is added to prepare a protein suspension, and protease is added to carry out enzymatic hydrolysis. After the enzymatic hydrolysis is completed, solid and liquid are separated, the hydrolysate is collected, and dried to obtain abalone fruit protein peptides.

2. The preparation method according to claim 1, characterized in that, In step S1, abalone kernel powder is mixed with a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.2 to 6.0, and the weight ratio of abalone kernel powder to buffer solution is 1:10 to 1:

20. The conditions for infrared radiation treatment are: infrared wavelength range of 2.5–15 μm, and infrared power of 1–3 W / cm². 2 The sample distance is 5–20 cm, the treatment method is intermittent, and the total treatment time is 15–60 min; in S1, the enzyme preparation is selected from one or more combinations of cellulase, hemicellulase, and phospholipase; the enzymatic hydrolysis conditions are: temperature 40–50℃, time 3–6 h, and the amount of the enzymatic hydrolysant is 2%–6% of the mass of abalone kernel powder; In S2, the conditions for pulsed electric field processing are: electric field strength 5-10 kV / cm, pulse width 40μs, and processing time 40-60min.

3. The preparation method according to claim 1, characterized in that: In S1, the conditions for infrared radiation treatment are: sample distance is 5-20 cm, treatment method is intermittent treatment, and total treatment time is 45-60 min. In step S1, the enzyme preparation is selected from a combination of cellulase, hemicellulase, and phospholipase. The enzymatic hydrolysis conditions are: temperature 40-50℃, time 4-6h; the amount of the enzymatic hydrolysant is 3%-6% of the mass of abalone kernel powder; and the mass ratio of cellulase, hemicellulase, and phospholipase is 1:1:

1. In S2, the conditions for pulsed electric field processing are: electric field strength 8-10 kV / cm, pulse width 40 μs, and processing time 40-60 min. In S2, calcium hydroxide is used to adjust the pH to 10-12, and phosphoric acid is used to adjust the pH to 4.0-4.

7.

4. The preparation method according to claim 1, characterized in that: Based on the protein weight of acid-precipitated protein and / or alcohol-precipitated protein, in step S3, 1% to 2% of the alkaline protease, and / or 1% to 2% of the acidic protease, and / or 1% to 2% of the bromelain are added to carry out enzymatic hydrolysis to obtain protein peptides. The total amount of protease added is 1% to 3% based on the protein weight in acid-precipitated proteins and / or alcohol-precipitated proteins. In the enzymatic hydrolysis process, calcium hydroxide solution and / or citric acid are used to adjust the pH of the system, and zinc citrate is added during the enzymatic hydrolysis process to enrich the zinc element in the protein peptides; the amount of zinc citrate added is 0.5 to 2.5‰ of the weight of the substrate protein.

5. The preparation method according to claim 1, characterized in that: In S3, the protease is selected from a combination of three of the following: alkaline protease, acidic protease, and bromelain. The enzymatic hydrolysis process is as follows: using the acid-precipitated protein and / or alcohol-precipitated protein as substrates, water is added to prepare a protein suspension; during the enzymatic hydrolysis process, calcium hydroxide solution and / or citric acid are used to adjust the pH of the system; Alkaline protease was added for enzymatic hydrolysis for 90–120 min; then acidic protease and bromelain were added, followed by zinc citrate, and enzymatic hydrolysis was continued for 60–120 min. After enzymatic hydrolysis, solid-liquid separation was performed, the hydrolysate was collected, and dried to obtain abalone fruit protein peptides. After enzymatic hydrolysis, carbon dioxide is introduced to adjust the pH of the system to 6.3–6.7, resulting in the formation of calcium carbonate precipitate.

6. The preparation method according to claim 5, characterized in that: The amount of protease used is 3% based on the protein weight of acid-precipitated protein and / or alcohol-precipitated protein; the weight ratio of alkaline protease, acidic protease and bromelain is 1:1:

1. The amount of zinc citrate added is 1‰ of the weight of the substrate protein.

7. An abalone fruit oil, characterized in that: It is prepared by the preparation method described in any one of claims 1-6.

8. An abalone fruit protein, characterized in that: It is prepared by the preparation method described in any one of claims 1-6.

9. An abalone fruit active protein peptide, characterized in that: It is prepared by the preparation method described in any one of claims 1-6.

10. The application of an abalone fruit active protein peptide in the preparation of functional products, characterized in that: The abalone fruit protein peptide is prepared by the preparation method described in any one of claims 1-6; the abalone fruit protein peptide is rich in selenium and / or zinc, and has acetylcholinesterase inhibitory activity and / or lipase inhibitory activity.

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