Method for preparing peony seed protein from oil peony byproduct

Abstract

The invention belongs to the technical field of biology, and relates to a method for preparing peony seed protein from an oil peony byproduct. The peony seed protein is obtained by taking oil peony seed meal as a raw material and adopting a separation and purification method of protease enzymolysis, saccharifying enzyme desugaring, centrifugation, microfiltration and ultrafiltration. The peony seed protein disclosed by the invention is low in alkali consumption and dosage, simple to operate and high in solubility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for preparing peony seed protein from by-products of oil peony. Background Technology

[0002] Protein is an essential nutrient, and with global population growth and increased focus on healthy eating, the demand for plant-based protein is constantly rising. The development of plant proteins from legumes, peas, wheat, and other sources has gradually become a research hotspot in the food industry. However, due to limitations in raw material yield, nutrient absorption rate, and cost, plant protein production remains in short supply.

[0003] Oilseed peony specifically refers to two varieties: *Paeonia suffruticosa* and *Paeonia suffruticosa*. Because their seeds contain approximately 20% oil, comparable to common oilseed crops, pressing the seeds produces a large amount of byproducts, namely peony seed meal. Peony seed meal contains 20%-30% protein based on dry weight, and its amino acid composition is well-balanced, making it a high-quality protein source. Currently, peony seed meal is mainly used for composting or discarded, leading to resource waste and environmental pollution. Effectively utilizing the protein in peony seed meal would significantly increase its added value.

[0004] According to reports, the main methods for extracting proteins from plants include alkaline extraction and acid precipitation, protease extraction, and organic solvent extraction. The most common method for preparing peony seed protein is alkaline dissolution and acid precipitation, or extraction using physical methods such as ultrasound and microwaves. Wang Huatian et al. (Wang Huatian, Dong Zhuofan, Li Liang, et al. Optimization of extraction process and study on properties of peony seed meal protein [J]. Food Industry Technology, 2023, 44(19):217-224. DOI:10.13386 / j.issn1002-0306.2022110076.) extracted peony seed protein using alkaline dissolution and acid precipitation, achieving a yield of 79.83%. However, the extraction process involved a large amount of acid and alkali, causing serious environmental pollution. Ding Dongyuan (Ding Dongyuan, Li Haibo, Liang Rui, et al. Optimization of crude protein extraction process from peony seeds [J]. Grains and Oils, 2021, 34(04):96-99.) used peony seed meal as raw material to extract peony seed protein using microwave-assisted alkali dissolution and acid precipitation method. The extraction rate was 86.53% under the condition of pH 9.5. Ultrasonic microwave-assisted alkali dissolution and acid precipitation method, but the alkali consumption is large and the energy consumption is high, which increases the production cost. Li Qiang (Li Qiang. Study on extraction, separation, structural characterization and emulsification properties of salt-soluble protein from peony seeds [D]. Sichuan Agricultural University, 2018.) used salt dissolution method to extract peony seed protein, and the protein extraction rate was 23.49%. The salt content in the extracted protein solution is high, and desalting treatment is usually required, which increases the operation steps and cost. Gao L (Gao L, Li Y, Wang Z, et al. Physicochemical characteristics and functionality of tree peony (Paeonia suffruticosa Andr.) seed protein[J]. Food Chemistry, 2018, 240980-988.) extracted peony seed protein using the alkali dissolution and acid precipitation method, with a solubility of 58.63%.

[0005] The above preparation method consumes a large amount of alkali, is complex in preparation process, causes environmental pollution, and the use of a large amount of acid and alkali in the preparation process can cause the protein to aggregate and affect the physicochemical properties of the protein. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the above-mentioned technologies and provide a separation and purification method combining enzymatic hydrolysis and ultrafiltration. This method simplifies the protein preparation steps, reduces alkali consumption, and improves solubility by establishing a feasible peony seed protein extraction process. The research results can provide a reference for the comprehensive utilization of peony seed meal.

[0007] The peony seed protein extraction rate obtained by this invention is 84.07% by mass, the amount of alkali used is reduced by 16.7%, and the solubility is increased by 39.58%.

[0008] The present invention discloses a method for preparing peony seed protein from by-products of oil peony, comprising the following steps: ① Enzymatic hydrolysis method for extracting protein from peony seeds Add peony seed meal to a buffer solution to a concentration of 35-40 g / L and adjust the initial pH to 7.5-8.5; add neutral protease to a concentration of 3000-4000 U per gram of peony seed protein, hydrolyze at a constant temperature of 50-60℃ for 60 min, and inactivate the enzyme at 100℃ for 10 min to obtain the hydrolysate. The optimized process conditions for the enzymatic hydrolysis are: pH 8.0, enzyme dosage of 3400 U / g, concentration of 37 g / L, temperature of 55℃, and time of 60 min. The peony seed meal is peony seeds that have been dehulled and pressed for oil. The neutral protease is food grade; The buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate solution.

[0009] ② Isolation and purification of peony seed protein The enzymatic hydrolysate prepared in step ① was centrifuged at 3000 r / min for 10 min. The pH of the hydrolysate was adjusted to 5.5-6.5, and saccharifying enzyme was added at 100000-120000 U / L. Enzymatic hydrolysis was carried out at 55-65℃ for 1 h, followed by enzyme inactivation at 100℃ for 10 min. After cooling to room temperature, the hydrolysate was centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to microfiltration (0.45 μm) and ultrafiltration at 10000 Da (molecular weight) membranes. The retentate was concentrated and dried to obtain peony seed protein. The optimized process conditions for the enzymatic hydrolysis are: pH 6.0, enzyme dosage 110,000 U / L, temperature 60°C, and hydrolysis time 1 hour. The microfiltration process used is carried out using a microfiltration membrane from Shanghai Precision Instruments Co., Ltd. The ultrafiltration membrane used is an ultrafiltration tube from Beijing Xubang Membrane Equipment Co., Ltd. Compared with the prior art, the process of the present invention is as follows: (1) Protein was extracted from peony seeds using an enzymatic hydrolysis method; (2) Using peony seed meal as raw material, the amount of alkali used is reduced by enzymatic hydrolysis extraction with neutral protease; (3) The use of saccharifying enzymes to remove impurities improved the purity of peony seed protein; (4) The optimized process conditions for adding neutral protease are pH 8.0, enzyme dosage 3400 U / g, concentration 37 g / L, temperature 55°C, and time 60 min. The enzymatic hydrolysate is centrifuged at 3000 r / min for 10 min. The optimized process conditions for removing impurities from the supernatant are pH 6.0, enzyme dosage 110000 U / L, temperature 60°C, and enzymatic hydrolysis time 1 h. The supernatant is then subjected to microfiltration (0.45 μm) and ultrafiltration with a 10000 Da (molecular weight) membrane. The retentate is concentrated and dried to obtain peony seed protein.

[0010] (5) The peony seed protein obtained in this invention is comparable to that obtained by Wang Huatian, Ding Dongyuan, Li Qiang, Gao L (Wang Huatian, Dong Zhuofan, Li Liang, et al. Optimization of extraction process and study on properties of peony seed meal protein [J]. Food Industry Technology, 2023, 44(19):217-224.DOI:10.13386 / j.issn1002-0306.2022110076. Ding Dongyuan, Li Haibo, Liang Rui, et al. Optimization of crude protein extraction process of peony seeds [J]. Grains and Oils, 2021, 34(04):96-99. Li Qiang. Study on extraction, separation, structural characterization and emulsification properties of salt-soluble protein from peony seeds [D]. Sichuan Agricultural University, 2018. Gao L, Li Y, Wang Z, et al. Physicochemical characteristics and functionality of tree peony (Paeoniasuffruticosa Andr.) seed protein [J]. Food (Chemistry, 2018, 240980-988.) This simplified the protein preparation process, reducing alkali consumption by 16.7% and 39.58%. Detailed Implementation

[0011] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the embodiments. Example

[0012] ① Enzymatic hydrolysis method for extracting protein from peony seeds Peony seed meal was added to a buffer solution to a concentration of 37 g / L, and the initial pH was adjusted to 8.0. Neutral protease was added to a concentration of 3400 U per gram of peony seed protein. The mixture was hydrolyzed at a constant temperature of 55°C for 60 min, and then the enzyme was inactivated at 100°C for 10 min to obtain the hydrolysate. The peony seed meal is peony seeds that have been dehulled and pressed for oil. The buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate solution; The neutral protease is food grade; ② Isolation and purification of peony seed protein The enzymatic hydrolysate prepared in step ① was centrifuged at 3000 r / min for 10 min. The pH of the hydrolysate was adjusted to 6.0, and saccharifying enzyme was added at 110000 U / L. Enzymatic hydrolysis was carried out at 60℃ for 1 h, followed by enzyme inactivation at 100℃ for 10 min. After cooling to room temperature, the hydrolysate was centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to microfiltration (0.45 μm) and ultrafiltration at 10000 Da (molecular weight) membranes. The retentate was concentrated and dried to obtain peony seed protein. The saccharifying enzyme is food grade; The microfiltration process used is carried out using a microfiltration membrane from Shanghai Precision Instruments Co., Ltd. The ultrafiltration membrane used is an ultrafiltration tube from Beijing Xubang Membrane Equipment Co., Ltd. The current invention yields a peony seed protein extraction rate of 84.07% by mass and a solubility of 98.21%. Example

[0013] ① Extraction of peony seed protein using the solution method Add peony seed meal to a buffer solution to a concentration of 35 g / L and adjust the initial pH to 7.5; add neutral protease to a concentration of 3000 U per gram of peony seed protein, hydrolyze at a constant temperature of 50℃ for 60 min, and inactivate the enzyme at 100℃ for 10 min to obtain the hydrolysate. The peony seed meal is peony seeds that have been dehulled and pressed for oil. The buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate solution; The neutral protease is food grade; ② Isolation and purification of peony seed protein The enzymatic hydrolysate obtained in step ① was centrifuged at 3000 r / min for 10 min. The pH of the hydrolysate was adjusted to 5.5, and saccharifying enzyme was added at 100000 U / L. Enzymatic hydrolysis was carried out at 55℃ for 1 h, followed by enzyme inactivation at 100℃ for 10 min. After cooling to room temperature, the hydrolysate was centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to microfiltration (0.45 μm) and ultrafiltration at 10000 Da (molecular weight) membranes. The retentate was concentrated and dried to obtain peony seed protein. The saccharifying enzyme is food grade; The microfiltration process used is carried out using a microfiltration membrane from Shanghai Precision Instruments Co., Ltd. The ultrafiltration membrane used is an ultrafiltration tube from Beijing Xubang Membrane Equipment Co., Ltd. The current invention yields a peony seed protein extraction rate of 81.72% by mass and a solubility of 95.79%. Example

[0014] ① Extraction of peony seed protein using the solution method Peony seed meal was added to a buffer solution to a concentration of 40 g / L, and the initial pH was adjusted to 8.5. Neutral protease was added to a concentration of 4000 U per gram of peony seed protein. The mixture was hydrolyzed at a constant temperature of 60°C for 60 min, and then the enzyme was inactivated at 100°C for 10 min to obtain the hydrolysate. The peony seed meal is peony seeds that have been dehulled and pressed for oil. The buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate solution; The neutral protease is food grade; ② Isolation and purification of peony seed protein The enzymatic hydrolysate prepared in step ① was centrifuged at 3000 r / min for 10 min. The pH of the hydrolysate was adjusted to 6.5, and saccharifying enzyme was added at 120000 U / L. Enzymatic hydrolysis was carried out at 65℃ for 1 h, followed by enzyme inactivation at 100℃ for 10 min. After cooling to room temperature, the hydrolysate was centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to microfiltration (0.45 μm) and ultrafiltration at 10000 Da (molecular weight) membranes. The retentate was concentrated and dried to obtain peony seed protein. The saccharifying enzyme is food grade; The microfiltration process used is carried out using a microfiltration membrane from Shanghai Precision Instruments Co., Ltd. The ultrafiltration membrane used is an ultrafiltration tube from Beijing Xubang Membrane Equipment Co., Ltd. The current invention yields a peony seed protein extraction rate of 82.51% by mass and a solubility of 96.41%.

[0015] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing peony seed protein from by-products of oil peony, comprising the following steps: ① Enzymatic hydrolysis method for extracting protein from peony seeds; ②Isolation and purification of peony seed protein.

2. The method for preparing peony seed protein according to claim 1, characterized in that, Step ① is as follows: Add peony seed meal to a buffer solution to a concentration of 35-40 g / L and adjust the initial pH to 7.5-8.5; add neutral protease to a concentration of 3000-4000 U per gram of peony seed protein, hydrolyze at a constant temperature of 50-60℃ for 60 min, and inactivate the enzyme at 100℃ for 10 min to obtain the hydrolysate. The optimized process conditions for the enzymatic hydrolysis are: pH 8.0, enzyme dosage of 3400 U / g, concentration of 37 g / L, temperature of 55℃, and time of 60 min.

3. The method for preparing peony seed protein according to claim 2, characterized in that, Step ② specifically involves: The enzymatic hydrolysate prepared in step ① was centrifuged at 3000 r / min for 10 min. The pH of the hydrolysate was adjusted to 5.5-6.5, and saccharifying enzyme was added at 100000-120000 U / L. Enzymatic hydrolysis was carried out at 55-65℃ for 1 h, followed by enzyme inactivation at 100℃ for 10 min. After cooling to room temperature, the hydrolysate was centrifuged at 3000 rpm for 10 min. The resulting supernatant was subjected to microfiltration (0.45 μm) and ultrafiltration at 10000 Da (molecular weight) membranes. The retentate was concentrated and dried to obtain peony seed protein.

4. The method for preparing peony seed protein according to claim 3, characterized in that, The peony seed meal is peony seeds after being shelled and pressed for oil.

5. The method for preparing peony seed protein according to claim 3, characterized in that, The neutral protease and glucoamylase mentioned are food-grade proteases.

6. The method for preparing peony seed protein according to claim 3, characterized in that, The enzymatic hydrolysis involves adding a neutral protease to expose the hydrophilic groups of the protein, allowing it to dissolve in water.

7. The method for preparing peony seed protein according to claim 3, characterized in that, The buffer solution is a disodium hydrogen phosphate-sodium dihydrogen phosphate solution.

8. The method for preparing peony seed protein according to claim 3, characterized in that, The peony seed protein is prepared at a temperature of 50-60℃.

9. The method for preparing peony seed protein according to claim 3, characterized in that, The extraction rate of peony seed protein was 84.07% by mass.

10. The method for preparing peony seed protein according to claim 3, characterized in that, The amount of peony seed protein saccharifying enzyme added is 100,000-120,000 U / L.