Method for obtaining function-enhanced isolated protein through oil by-products

Abstract

The invention discloses a method for obtaining function-enhanced isolated protein from oil by-products, which comprises the following steps: mixing the oil by-products with plant extracts rich in phenolic substances and flavones, and carrying out common alkali treatment to prepare a function-enhanced protein compound. Flavonoid compounds have remarkable antioxidant, anti-inflammatory, antibacterial and other biological activities, but are poor in stability and easy to degrade. Through a protein embedding technology, active ingredients of natural plants are combined with plant proteins in the process of separating protein components, so that the stability and bioavailability of flavonoid substances are remarkably improved, and the flavonoid substances are endowed with a slow release characteristic. The method not only realizes efficient utilization of oil by-products, but also improves the functional characteristics of the soybean protein through a simple compound process in extraction, and has remarkable economic and social benefits. Meanwhile, the prepared function-enhanced isolated protein has high nutritional value and wide application prospects, and can be applied to the fields of functional foods, health care products, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of plant protein extraction, and in particular to a method for obtaining functional enhanced isolated protein through oil by-products. Background Art

[0002] As one of the three major nutrients for the human body, protein plays a key role in maintaining life activities and human functioning. Soybean is an important oil crop, and its processing produces a large amount of soybean meal. These by-products are rich in protein, but the traditional utilization method is inefficient, resulting in waste of resources. Peanut meal is the main by-product produced in the peanut oil extraction process. It is rich in protein (about 40%-50%), and its amino acid composition is balanced, with high nutritional value. Peanut protein is rich in amino acids such as arginine and glutamic acid, and has physiological functions such as regulating immunity and promoting cardiovascular health. How to convert these by-products that have been mostly used for low-value feed or fertilizer into high-value functional enhanced proteins and build a more efficient resource recycling system is an urgent problem to be solved.

[0003] Ginkgo biloba is rich in various bioactive ingredients such as flavonoids and terpenes, and has significant antioxidant, anti-inflammatory, and lipid-lowering functions. However, these active ingredients are extremely sensitive to light, heat, and oxygen, and are easily oxidized or decomposed, thereby affecting their biological activity and stability. Therefore, during the extraction, storage, and application of Ginkgo biloba, appropriate protective measures must be taken, such as avoiding light, keeping the ingredients at low temperatures, and sealing them, to maximize the stability and efficacy of their active ingredients. In addition, research and development of new stabilization technologies and formulations, such as microencapsulation and nano-encapsulation, are also effective ways to improve the stability and bioavailability of Ginkgo biloba's bioactive ingredients.

[0004] At present, there are few studies on the use of oil by-products to obtain functionally enhanced isolated proteins, and there is a lack of systematic and effective preparation methods. Existing protein extraction and functional enhancement technologies have problems such as complex processes, large losses of active ingredients, and unsatisfactory product purity and functional properties. Chinese patent CN117603295A provides a green and efficient preparation method for high-quality oil isolated proteins with high yields. Small molecule impurities (such as polyphenols and monosaccharides) are removed by weak base synchronous ultrafiltration technology to avoid the aggregation problem of proteins in traditional alkali dissolution and acid precipitation methods, thereby improving protein yield. Although the use of strong acids and strong bases is avoided, the energy consumption of the ultrafiltration process is high, and the protein product has a single function, which cannot meet the market demand for functional proteins (such as antioxidants and immunomodulators), and the application scenarios are limited.

[0005] Therefore, there is an urgent need in the art to develop a method for obtaining functional enhanced protein isolates from oil by-products that is efficient, environmentally friendly, and can fully retain active ingredients, which has important practical significance and economic value. Summary of the Invention

[0006] The present invention aims to provide a method for obtaining functionally enhanced protein isolates from oil byproducts, aiming to address the problems of low protein extraction efficiency and insufficient functional properties in the above-mentioned prior art. The present invention combines ginkgo biloba extract with oil byproducts (such as soybean meal and peanut meal), utilizing the changes in physical and chemical conditions (such as pH and temperature) during the protein extraction process to simultaneously achieve the compounding and stabilization of the active ingredients. This method can efficiently extract protein and enhance its emulsification, foaming, and antioxidant activity, making it suitable for use in the fields of food, medicine, and so on.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0009] Ginkgo powder, defatted oilseed meal and water are mixed, the pH value of the mixture is adjusted to alkaline, and the mixture is heated and stirred for extraction, and the supernatant is collected; the supernatant is precipitated under acidic conditions to obtain a preliminary extracted protein; the preliminary extracted protein is then washed with water, redissolved in water, the pH value is adjusted to neutral, and freeze-dried to obtain a functional enhanced isolated protein.

[0010] Furthermore, the mass volume ratio of the ginkgo powder, defatted oilseed meal and water is (5-20g):25g:300mL.

[0011] Furthermore, the pH value of the mixed solution is 9.0-10.5.

[0012] Furthermore, the heating and stirring conditions are: stirring at 45-95° C. for 1 hour.

[0013] Preferably, the mass volume ratio of the ginkgo powder, defatted oilseed meal and water is 10g:25g:300mL.

[0014] Preferably, the pH value of the mixed solution is 9.5.

[0015] Preferably, the heating and stirring conditions are: stirring at 95° C. for 1 hour.

[0016] Furthermore, the supernatant is precipitated under acidic conditions with a pH value of 4.5; and the precipitate is centrifuged at 8000 rpm to obtain the preliminarily extracted protein.

[0017] Furthermore, the water re-dissolution time is 1 hour.

[0018] Furthermore, the defatted oilseed meal includes defatted soybean meal or peanut meal.

[0019] The present invention also provides a function-enhanced isolated protein, which is prepared by the above method.

[0020] The present invention also provides the use of the above-mentioned functionally enhanced isolated protein in the preparation of antioxidant functional foods and / or health products and / or medicines.

[0021] The present invention also provides a functional food, health product or medicine with antioxidant activity, which contains the above-mentioned function-enhanced isolated protein.

[0022] The present invention discloses the following technical effects:

[0023] The present invention innovates through the combination of a ginkgo leaf-defatted oilseed meal composite system and chemical-physical synergistic modification. Existing technologies mainly focus on protein extraction from single oil by-products, while the present invention combines ginkgo leaf with defatted oilseed meal for the first time to achieve functional enhancement through chemical-physical synergistic modification. Chemical modification: Ginkgo leaf polyphenols bind to plant proteins through hydrophobic interaction to form a stable complex, which significantly enhances antioxidant properties (such as DPPH clearance ≥ 80%); Physical regulation: High temperature (95°C) treatment promotes protein conformational unfolding, exposes binding sites, and improves polyphenol adsorption efficiency. This multi-component synergistic effect breaks through the limitations of traditional single raw materials and is an innovation in raw material combination, which makes the functional properties of the final product (such as antioxidant properties and emulsification stability) significantly better than traditional single raw material processes.

[0024] This invention achieves green and economical production through dual-path optimization of raw materials and processes. The raw materials, ginkgo biloba leaves and defatted oilseed meal, are both byproducts of oilseed processing, reducing costs by over 40% compared to pure plant raw materials. The process eliminates complex steps such as ultrafiltration and resin chromatography, replacing them with washing and neutralization, reducing equipment investment and energy consumption. The entire process uses an aqueous phase, avoiding organic solvent contamination, and production wastewater can be reused, in line with the principles of green chemistry.

[0025] The present invention uses a staged pH gradient control process to break through the limitations of traditional alkali dissolution and acid precipitation. The existing technology still uses the traditional "alkali dissolution and acid precipitation" method, which has the problem of protein aggregation leading to functional degradation. The present invention optimizes through pH control:

[0026] (1) Pretreatment stage (pH 10): A strong alkaline environment promotes the disintegration of the cell wall of ginkgo leaves and the release of polyphenols, and high temperature (95°C) treatment improves the dissolution efficiency; under weak alkaline conditions, the protein molecules unfold and expose hydrophobic groups, which are pre-bound to the flavonoids released from the ginkgo leaves through hydrophobic interactions, avoiding excessive denaturation of the protein caused by strong alkali (such as pH 12).

[0027] (2) Acid precipitation stage (pH 4.5→7.0): During protein isoelectric precipitation, the phenolic hydroxyl groups of flavonoids form a hydrogen bond network with the polar side chains of the protein, further stabilizing the complex structure. Precisely controlling the endpoint of acid precipitation and then neutralizing reduces irreversible aggregation and maintains the activity of the complex.

[0028] (3) This process achieves a protein yield of over 85%, and its functional properties (such as gel strength and emulsification) are significantly improved compared to traditional methods.

[0029] The present invention adopts the alkali dissolution and acid precipitation method to extract protein. During the alkali treatment of the mixed raw materials, the pH value is slowly raised to 10, which helps to fully release the effective ingredients in the ginkgo leaves at different stages, avoids the destruction of some heat-sensitive components by high temperature and strong alkali at the beginning, and allows the protein in the defatted oilseed meal to be gradually released under milder conditions, thereby improving the extraction rate and quality of the protein. In the acid precipitation method, the pH value of the supernatant is adjusted to 4.5 to precipitate the target protein. Through this two-step acid precipitation method, the target protein can be separated more accurately and the mixing of impurities is reduced.

[0030] This invention breaks away from the traditional "extraction first, modification later" approach by introducing the active ingredients from ginkgo biloba during the protein dissolution stage. Through coordinated pH-temperature regulation, the protein's three-dimensional structure unfolds in a directional manner, exposing hydrophobic regions for binding to flavonoids, forming a functional complex with sustained-release properties. This "integrated extraction-compounding" strategy significantly enhances the stability of the active ingredients and the functional diversity of the protein.

[0031] The present invention not only efficiently extracts protein but also enhances its emulsifying, foaming, and antioxidant properties, making it suitable for use in the food, pharmaceutical, and other fields. Furthermore, the present invention achieves high-value utilization of oil byproducts and ginkgo biloba resources, resulting in significant economic and environmental benefits and providing a new technological approach for the development of functional proteins. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] A method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0039] (1) Alkali treatment of mixed raw materials: Weigh 15 g of dried and pulverized ginkgo powder to ensure uniform particle size and that the active ingredient content meets the requirements (flavonoid glycosides ≥ 24%, terpene lactones ≥ 6%). Mix the 15 g ginkgo powder sample with 25 g defatted soybean meal, add 300 mL of deionized water, adjust the pH to 9.5 with 2 mol / L sodium hydroxide solution, heat to 95°C, and stir for 1 hour. After cooling to room temperature, centrifuge at 8000 rpm for 15 minutes to obtain the supernatant.

[0040] (2) Protein extraction by acid precipitation: The supernatant was adjusted to pH 4.5 using a 2 mol / L hydrochloric acid solution by acid precipitation to precipitate the protein to produce white flocs. The protein was centrifuged at 4°C and 8000 rpm for 10 minutes, and the precipitate was collected, which was the initially extracted protein.

[0041] (3) Washing and freeze-drying of the protein: The protein initially extracted in step (2) was washed three times with deionized water to remove impurities remaining on the surface of the precipitate, redissolved in water for 1 hour, and the pH was adjusted to 7 again. The protein was freeze-dried to obtain a functionally enhanced isolated protein.

[0042] Example 2

[0043] A method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0044] The difference from Example 1 is that the ginkgo powder in step (1) is 5 g, and the other steps and conditions are the same as those in Example 1.

[0045] Example 3

[0046] A method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0047] The difference from Example 1 is that the mass of ginkgo powder in step (1) is 10 g, and the other steps and conditions are the same as those in Example 1.

[0048] Example 4

[0049] A method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0050] The difference from Example 1 is that the mass of ginkgo powder in step (1) is 20 g, and the other steps and conditions are the same as those in Example 1.

[0051] Example 5

[0052] A method for obtaining functional enhanced protein isolate from oil by-products, which differs from Example 1 only in that: step (1) directly takes an appropriate amount of soybean meal, adds deionized water, and has a solid-liquid ratio of 1:10, and uses 2 mol / L sodium hydroxide solution to adjust the pH to 9.0. The other steps and conditions are consistent with Example 1.

[0053] Example 6

[0054] A method for obtaining functional enhanced protein isolate from oil by-products, which differs from Example 1 only in that: in step (1), the supernatant is added to soybean meal at a solid-to-liquid ratio of 1:10, and the pH is adjusted to 8.0 using a 2 mol / L sodium hydroxide solution. The other steps and conditions are consistent with Example 1.

[0055] Example 7

[0056] A method for obtaining functional enhanced protein isolate from oil by-products, which differs from Example 1 only in that: during the acid precipitation in step (2), the pH is adjusted to 5.0 with 2M HCl, and the other steps and conditions are the same as those in Example 1.

[0057] Example 8

[0058] A method for obtaining functional enhanced protein isolate from oil by-products, comprising the following steps:

[0059] The difference from Example 1 is that step (1) is heated to 45° C. and stirred for 1 hour, and the other steps and conditions are consistent with Example 1.

[0060] The functionally enhanced isolated proteins prepared according to Examples 1-8 were tested for protein content, DPPH and ABTS free radical scavenging rates, iron reducing ability of the copolymer, particle size, and emulsifying activity.

[0061] (1) Protein content determination

[0062] The protein content of the examples was determined by the Kjeldahl method, and the nitrogen conversion coefficient was determined according to the national standard GB5009.5-2016.

[0063] The calculation formula for protein yield is:

[0064] Protein extraction rate (%) = (protein mass / cake mass) × 100%

[0065] Protein yield (%) = [(protein mass × protein content) / (meal mass × protein content)] × 100%

[0066] The test results are shown in Table 1.

[0067] Table 1 Protein content determination

[0068] Experimental group H1 H2 H3 H4 H5 H6 H7 H8 Protein yield (%) 25 30 35 32 18 22 24 27

[0069] (2) DPPH and ABTS free radical scavenging rate

[0070] Prepare a 0.8 mmol / L (0.315 mg / mL) DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution in ethanol. Dilute the sample solution five-fold with deionized water. Add 2 mL of the diluted sample solution to the solution, mix, and place in the dark. After reacting at room temperature for 30 minutes, measure the absorbance at 517 nm. Use ethanol as a blank in place of the sample solution.

[0071]

[0072] Where: A Sample A is the absorbance of DPPH solution after adding sample solution, Blank is the absorbance value of DPPH solution when no sample solution is added.

[0073] Potassium persulfate (2.45 mmol / L) and ABTS (7 mol / L) solutions were mixed in a 1:1 volume ratio and stored in the dark for 12-16 hours to generate ABTS free radicals. A 0.5 mg / mL sample solution (prepared by dissolving 0.5 mg of sample in 70% ethanol) was then added to the diluted ABTS solution (2 mL). The mixture was allowed to react in the dark for 1 hour, and the absorbance was measured at 734 nm using a UV-visible spectrophotometer.

[0074]

[0075] Where: As is the absorbance of the ABTS solution after the sample solution is added, and Ac is the absorbance of the ABTS solution without the sample solution.

[0076] The test results are shown in Table 2.

[0077] Table 2 DPPH and ABTS free radical scavenging rates

[0078]

[0079] (3) Iron reducing ability of copolymers

[0080] Preparation of potassium ferrocyanide solution: Accurately weigh 1.00 g of potassium ferrocyanide and dissolve it in an appropriate amount of distilled water and dilute to 100 mL.

[0081] Preparation of ferric chloride (FeCl3) solution: Accurately weigh 0.1665 g of FeCl3·6H2O and dissolve it in an appropriate amount of distilled water and dilute to 100 mL.

[0082] Preparation of trichloroacetic acid (TCA) solution: Accurately weigh 10 g of TCA and dissolve it in an appropriate amount of distilled water and dilute to 100 mL.

[0083] Ferric reducing power determination: 50 μL of sample solution was placed in a 2 mL centrifuge tube and distilled water was added to make a total volume of 300 μL. Subsequently, 300 μL of PBS buffer and K3Fe(CN)6 solution were added to each tube, mixed thoroughly, incubated at 50°C for 20 minutes, and then quickly cooled in an ice-water bath. 300 μL of TCA was added to each tube, mixed thoroughly, and centrifuged at 3500 rpm for 10 minutes. 500 μL of the supernatant from each tube was transferred to a 2 mL centrifuge tube. 500 μL of distilled water and 100 μL of FeCl3 solution were added to each tube, shaken, and allowed to stand for 10 minutes. 300 μL of distilled water was added to replace the sample solution and distilled water combination in the experimental group. All other steps were the same as for the experimental group. The absorbance of each solution at a wavelength of 700 nm was measured using a UV spectrophotometer. The entire procedure was performed in the dark.

[0084] The formula for calculating iron reducing power is as follows:

[0085] Iron reducing power = A1-A0

[0086] Where: A1 is the absorbance of the sample solution to be tested, and A0 is the absorbance of the blank control.

[0087] The test results are shown in Table 3.

[0088] Table 3 Iron reducing ability of copolymers

[0089]

[0090] (4) Particle size

[0091] The average particle size was measured using a Zetasizer Nano ZS90 nanoparticle size analyzer. The sample was diluted 100-fold with deionized water before measurement, and the equilibration time was 10 seconds. The particle size was measured three times at room temperature and the average value was taken.

[0092] The test results are shown in Table 4.

[0093] Table 4 Particle size

[0094] Experimental group H1 H2 H3 H4 H5 H6 H7 H8 Particle size (nm) 150 140 130 135 180 160 145 140

[0095] (5) Emulsifying activity

[0096] Dissolve the protein in deionized water to prepare a 1% (w / v) protein solution. Prepare the emulsion: Mix 10 mL of the protein solution with 10 mL of vegetable oil (e.g., soybean oil) and homogenize using a high-speed homogenizer (10,000 rpm) for 1 minute. Immediately after homogenization, remove a 50 μL sample from the bottom of the emulsion and add 5 mL of 0.1% (w / v) sodium dodecyl sulfate (SDS) solution. Mix thoroughly. Measure the absorbance (A) at 500 nm.

[0097]

[0098] Where: A is the absorbance value of the emulsion measured at a wavelength of 500nm, C is the concentration of the protein solution in the emulsification system, is the volume fraction of the oil phase.

[0099] The test results are shown in Table 5.

[0100] Table 5 Emulsifying activity

[0101] Experimental group H1 H2 H3 H4 H5 H6 H7 H8 Emulsifying activity (EA) 4.745 5.067 5.988 5.527 3.224 4.406 4.643 4.967

[0102] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for obtaining functional enhanced protein isolate from oil by-products, characterized in that: The following steps are involved: Mix ginkgo powder, defatted oilseed meal and water, adjust the pH value of the mixture to alkaline, heat and stir for extraction, and collect the supernatant; The supernatant is precipitated under acidic conditions to obtain a primary extracted protein; The initially extracted protein is washed with water, redissolved in water, the pH value is adjusted to neutral, and freeze-dried to obtain a functionally enhanced isolated protein.

2. The method for obtaining functional enhanced protein isolate from oil by-products according to claim 1, wherein: The mass volume ratio of the ginkgo powder, defatted oil meal and water is (5-20g):25g:300mL; The pH value of the mixed solution is 9.0-10.5; The heating and stirring conditions are: stirring at 45-95° C. for 1 hour.

3. The method for obtaining functional enhanced protein isolate from oil by-products according to claim 2, wherein: The mass volume ratio of the ginkgo powder, defatted oil meal and water is 10g:25g:300mL; The pH value of the mixed solution is 9.5; The heating and stirring conditions are: stirring at 95° C. for 1 hour.

4. The method for obtaining functional enhanced protein isolate from oil by-products according to claim 1, wherein: The supernatant is precipitated under acidic conditions with a pH value of 4.5; The precipitate was centrifuged at 8000 rpm to obtain the preliminarily extracted protein.

5. The method for obtaining functional enhanced protein isolate from oil by-products according to claim 1, wherein: The water reconstitution time is 1 hour.

6. The method for obtaining functional enhanced protein isolate from oil by-products according to claim 1, wherein: The defatted oil meal includes defatted soybean meal or peanut meal.

7. A functional enhanced protein isolate, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. Use of the functionally enhanced isolated protein according to claim 7 in the preparation of antioxidant functional foods and / or health products and / or medicines.

9. A functional food, health product or medicine with antioxidant activity, characterized in that: It comprises the function-enhanced isolated protein according to claim 7.

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