Extraction method of seabuckthorn seed protein

Abstract

The invention relates to the technical field of extraction and separation of biomacromolecules from plant seeds, and particularly discloses an extraction method of seabuckthorn seed protein. The method comprises the following steps: mixing ammonium sulfate, tertiary butanol, distilled water and sea-buckthorn seed powder, performing ultrasonic treatment to form a three-phase extraction system, and respectively extracting to obtain refined sea-buckthorn seed protein, sea-buckthorn seed oil and refined sea-buckthorn polysaccharide. The method is easy and convenient to operate, economical and environmentally friendly, the extraction efficiency and purity are improved by adopting an ultrasonic treatment and alkali-solution and acid-isolation method, and comprehensive and efficient utilization of sea-buckthorn seed resources is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of extracting and separating biomacromolecules from plant seeds, and in particular to a method for extracting seabuckthorn seed protein. Background Art

[0002] As a processing byproduct, seabuckthorn seeds have the characteristics of high protein content and high amino acid content. In addition, seabuckthorn seed oil and seabuckthorn seed polysaccharides also have high contents, so they have important economic value and application prospects. However, traditional extraction methods have the problems of low extraction efficiency, complicated steps, high cost and environmental pollution. Therefore, it is of great significance to develop an efficient, low-cost and environmentally friendly extraction method for the comprehensive utilization of seabuckthorn seed resources. The extraction method of the present invention not only improves the extraction efficiency of oil, protein and polysaccharide in seabuckthorn seeds, but also simplifies the extraction process, reduces production costs and reduces the impact on the environment. By the extraction method of the present invention, high-purity seabuckthorn seed oil, seabuckthorn seed protein and seabuckthorn seed polysaccharide can be obtained, which provides new possibilities for the further processing and application of seabuckthorn seeds.

[0003] Three-phase extraction technology combined with ultrasonic treatment is an innovative extraction technology that improves extraction efficiency by utilizing the cavitation effect generated by ultrasound. This technology is particularly suitable for complex compounds that are difficult to extract using traditional methods. The high-frequency vibration of ultrasound can break the plant cell walls and release more active ingredients, thereby improving the extraction efficiency and purity of the extract. In addition, three-phase extraction technology further optimizes the extraction process by effectively transferring substances between distilled water, organic solvents and supercritical fluids. This combined method not only shortens the extraction time, but also reduces the use of organic solvents, making it more environmentally friendly.

[0004] The alkali dissolution and acid precipitation method is mainly used to separate and purify certain specific substances from solutions. The basic principle of this method is to use the difference in solubility of substances under different pH conditions for separation. Specifically, the substance to be separated is first dissolved in an alkaline solution to form a soluble protein. Then, by adding an acidic substance and adjusting the pH value of the solution, the solubility of the target substance is reduced, so that it precipitates from the solution in the form of a precipitate. This method has a wide range of applications in industrial production, chemical analysis, and environmental protection. By precisely controlling the reaction conditions, such as temperature, pH value, and reaction time, the separation efficiency and purity can be effectively improved to ensure that a high-quality product is obtained in the end.

[0005] Based on the above content, the present invention provides a method for extracting protein that is simple to operate, economical and environmentally friendly, while improving the extraction efficiency and purity, and realizing the comprehensive and efficient utilization of seabuckthorn seed resources. Summary of the invention

[0006] The purpose of the invention is to provide a simple, efficient, green and economical method for extracting protein, while improving the extraction efficiency and purity of protein, oil and polysaccharide in seabuckthorn seeds.

[0007] The purpose of the invention is to provide a method for extracting seabuckthorn seed protein.

[0008] The invention provides a method for extracting seabuckthorn seed protein. The method combines ultrasonic treatment and three-phase extraction technology, and adopts alkali dissolution and acid precipitation method to extract protein, and can also obtain oil and polysaccharide at the same time.

[0009] To achieve the above object, the technical solution of the present invention is a method for extracting seabuckthorn seed protein, comprising the following steps:

[0010] (1) Raw material pretreatment: The seabuckthorn seeds are crushed and passed through a 100-200 mesh sieve to obtain seabuckthorn seed powder, which is then mixed with distilled water in a certain proportion to prepare seabuckthorn seed slurry.

[0011] (2) Constructing a three-phase extraction system: adding a certain amount of ammonium sulfate to the sea buckthorn seed slurry to adjust the pH of the system to 3-7; then slowly adding tert-butyl alcohol in proportion to form a three-phase system; at a set temperature, using a magnetic stirrer to gently stir the system until it is uniform; subjecting the system to ultrasonic treatment to promote the dispersion of the components; and centrifuging the treated system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification.

[0012] (3) Oil extraction: Collect the upper organic phase in the three-phase system, perform vacuum evaporation, recover tert-butyl alcohol, and obtain oil at the same time, with a content of 11-13%.

[0013] (4) Protein extraction: The middle phase of the three-phase system is dissolved in Tris-HCl buffer and hydrated overnight at 4°C; the hydrated solution is dialyzed in distilled water to remove salt; the alkali dissolution and acid precipitation method is performed, and the precipitate is collected to obtain refined seabuckthorn seed protein with a content of 10-15%.

[0014] (5) Polysaccharide extraction: The lower layer solution in the three-phase system is dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 1.1-1.4%.

[0015] As a further illustration of the present invention, in step (1), the ratio of seabuckthorn seeds to distilled water is 1:10-15.

[0016] As a further illustration of the present invention, the amount of ammonium sulfate added in step (2) is 20-60% (w / v).

[0017] As a further illustration of the present invention, in step (2), the ratio of seabuckthorn seed slurry to tert-butanol is 1:0.5-2.5 (v / v).

[0018] As a further illustration of the present invention, the stirring temperature of the system in step (2) is 30-60°C.

[0019] As a further illustration of the present invention, the stirring time of the system in step (2) is 0.5-2.5h.

[0020] As a further illustration of the present invention, the system in step (2) is subjected to ultrasonic treatment at a frequency of 30-50 kHz and a power of 200-400 W.

[0021] As a further illustration of the present invention, the concentration of the Tris-HCl buffer in step (4) is 50-200 mmol / L, and the pH is 8-9.

[0022] As a further illustration of the present invention, the alkali dissolution and acid precipitation method in step (4) is to first adjust the pH of the supernatant after dialysis to 10-11, centrifuge the supernatant, adjust the pH of the supernatant to 3-4 again, filter, collect the precipitate, and obtain refined seabuckthorn seed protein.

[0023] The beneficial effect is that the extraction method of the present invention is compared with the traditional extraction method as follows:

[0024]

[0025] Note: Each content is the mass ratio of each component to sea buckthorn seeds. DETAILED DESCRIPTION

[0026] Example 1

[0027] A method for extracting seabuckthorn seed protein comprises the following steps:

[0028] (1) Raw material pretreatment: The seabuckthorn seeds were crushed and passed through a 100-mesh sieve to obtain seabuckthorn seed powder, which was mixed with distilled water at a ratio of 1:10 to prepare seabuckthorn seed slurry.

[0029] (2) Constructing a three-phase extraction system: adding 30% (w / v) ammonium sulfate to the sea buckthorn seed slurry to adjust the pH of the system to 3-7; then slowly adding tert-butyl alcohol at a ratio of 1:0.5 (v / v) to form a three-phase system; at a temperature of 30°C, using a magnetic stirrer to gently stir the system for 0.5 h until it is uniform; subjecting the system to ultrasonic treatment at a frequency of 30 kHz and a power of 200 W to promote the dispersion of the components; and centrifuging the treated system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification.

[0030] (3) Oil extraction: The upper organic phase in the three-phase system was collected and subjected to vacuum evaporation to recover tert-butyl alcohol and obtain oil at the same time, with a content of 11.5±0.3%.

[0031] (4) Protein extraction: The middle phase of the three-phase system was dissolved in a Tris-HCl buffer solution with a concentration of 50-200 mmol / L and a pH of 8-9, and hydrated overnight at 4°C; the hydrated solution was dialyzed in distilled water to remove salt, and seabuckthorn seed protein was obtained with a content of 9.8±0.5%.

[0032] (5) Polysaccharide extraction: The lower layer solution in the three-phase system was dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 1.15±0.05%.

[0033] Example 2

[0034] A method for extracting seabuckthorn seed protein comprises the following steps:

[0035] (1) Raw material pretreatment: The seabuckthorn seeds were crushed and passed through a 100-mesh sieve to obtain seabuckthorn seed powder, which was mixed with distilled water at a ratio of 1:10 to prepare seabuckthorn seed slurry.

[0036] (2) Constructing a three-phase extraction system: adding 30% (w / v) ammonium sulfate to the sea buckthorn seed slurry and adjusting the pH of the system to 3-7; then slowly adding tert-butyl alcohol at a ratio of 1:0.5 (v / v) to form a three-phase system; at a temperature of 30°C, using a magnetic stirrer to gently stir the system for 0.5 h until it is uniform; centrifuging the system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification.

[0037] (3) Oil extraction: The upper organic phase in the three-phase system was collected and subjected to vacuum evaporation to recover tert-butyl alcohol and obtain oil at the same time, with a content of 6±0.3%.

[0038] (4) Protein extraction: The middle phase of the three-phase system was dissolved in a Tris-HCl buffer solution with a concentration of 50-200 mmol / L and a pH of 8-9, and hydrated overnight at 4°C; the hydrated solution was dialyzed in distilled water to remove salt, and seabuckthorn seed protein was obtained with a content of 8.3±0.3%.

[0039] (5) Polysaccharide extraction: The lower layer solution in the three-phase system was dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 0.82±0.01%.

[0040] Example 3

[0041] A method for extracting seabuckthorn seed protein comprises the following steps:

[0042] (1) Raw material pretreatment: The seabuckthorn seeds were crushed and passed through a 100-mesh sieve to obtain seabuckthorn seed powder, which was mixed with distilled water at a ratio of 1:10 to prepare seabuckthorn seed slurry.

[0043] (2) Constructing a three-phase extraction system: adding 30% (w / v) ammonium sulfate to the sea buckthorn seed slurry to adjust the pH of the system to 3-7; then slowly adding tert-butyl alcohol at a ratio of 1:0.5 (v / v) to form a three-phase system; at a temperature of 30°C, using a magnetic stirrer to gently stir the system for 0.5 h until it is uniform; subjecting the system to ultrasonic treatment at a frequency of 30 kHz and a power of 200 W to promote the dispersion of the components; and centrifuging the treated system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification.

[0044] (3) Oil extraction: The upper organic phase in the three-phase system was collected and subjected to vacuum evaporation to recover tert-butyl alcohol and obtain oil at the same time, with a content of 11.5±0.3%.

[0045] (4) Protein extraction: The middle phase of the three-phase system was dissolved in a Tris-HCl buffer solution with a concentration of 50-200 mmol / L and a pH of 8-9, and hydrated overnight at 4°C; the hydrated solution was dialyzed in distilled water to remove salt; an alkali-dissolution and acid precipitation method was performed, firstly the pH of the dialyzed supernatant was adjusted to 10, the supernatant was centrifuged, the pH of the supernatant was adjusted to 4 again, and the precipitate was filtered and collected to obtain refined seabuckthorn seed protein with a content of 10.9±0.4%.

[0046] (5) Polysaccharide extraction: The lower layer solution in the three-phase system was dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 1.15±0.05%.

[0047] Example 4

[0048] A method for extracting seabuckthorn seed protein comprises the following steps:

[0049] (1) Raw material pretreatment: The seabuckthorn seeds were crushed and passed through a 100-mesh sieve to obtain seabuckthorn seed powder, which was mixed with distilled water at a ratio of 1:15 to prepare seabuckthorn seed slurry.

[0050] (2) Constructing a three-phase extraction system: adding 50% (w / v) ammonium sulfate to the sea buckthorn seed slurry to adjust the pH of the system to 3-7; then slowly adding tert-butyl alcohol at a ratio of 1:1.5 (v / v) to form a three-phase system; at a temperature of 50°C, using a magnetic stirrer to gently stir the system for 1.5 h until it is uniform; subjecting the system to ultrasonic treatment at a frequency of 40 kHz and a power of 300 W to promote the dispersion of the components; and centrifuging the treated system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification.

[0051] (3) Oil extraction: The upper organic phase in the three-phase system was collected and subjected to vacuum evaporation to recover tert-butyl alcohol and obtain oil at the same time, with a content of 12.6±0.35%.

[0052] (4) Protein extraction: The middle phase of the three-phase system was dissolved in a Tris-HCl buffer solution with a concentration of 50-200 mmol / L and a pH of 8-9, and hydrated overnight at 4°C; the hydrated solution was dialyzed in distilled water to remove salt; an alkali-dissolution and acid precipitation method was performed, firstly the pH of the dialyzed supernatant was adjusted to 10, the supernatant was centrifuged, the pH of the supernatant was adjusted to 4 again, and the precipitate was filtered and collected to obtain refined seabuckthorn seed protein with a content of 14.7±0.2%.

[0053] (5) Polysaccharide extraction: The lower layer solution in the three-phase system was dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 1.35±0.02%.

[0054] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for extracting seabuckthorn seed protein, characterized in that Combining ultrasonic treatment and three-phase extraction technology, and using alkali dissolution and acid precipitation to extract protein, oil and polysaccharide can also be obtained at the same time. The method includes the following steps: (1) Raw material pretreatment: The seabuckthorn seeds are crushed and passed through a 100-200 mesh sieve to obtain seabuckthorn seed powder, which is then mixed with distilled water in a certain proportion to prepare seabuckthorn seed slurry. (2) Constructing a three-phase extraction system: adding a certain amount of ammonium sulfate to the sea buckthorn seed slurry to adjust the pH of the system to 3-7; then slowly adding tert-butyl alcohol in proportion to form a three-phase system; at a set temperature, using a magnetic stirrer to gently stir the system until it is uniform; subjecting the system to ultrasonic treatment to promote the dispersion of the components; and centrifuging the treated system at a speed of 6000 r / min for 15 minutes to obtain a clear three-phase stratification. (3) Oil extraction: Collect the upper organic phase in the three-phase system, perform vacuum evaporation, recover tert-butyl alcohol, and obtain oil at the same time, with a content of 11-13%. (4) Protein extraction: The middle phase of the three-phase system is dissolved in Tris-HCl buffer and hydrated overnight at 4°C; the hydrated solution is dialyzed in distilled water to remove salt; the alkali dissolution and acid precipitation method is performed, and the precipitate is collected to obtain refined seabuckthorn seed protein with a content of 10-15%. (5) Polysaccharide extraction: The lower layer solution in the three-phase system is dialyzed in distilled water, desalted, concentrated, and dried to obtain refined seabuckthorn polysaccharide with a content of 1.1-1.4%.

2. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: In the step (1), the ratio of seabuckthorn seeds to distilled water is 1:10-15.

3. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: The amount of ammonium sulfate added in step (2) is 20-60% (w / v).

4. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: In the step (2), the ratio of seabuckthorn seed slurry to tert-butanol is 1:0.5-2.5 (v / v).

5. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: The stirring temperature of the system in step (2) is 30-60°C.

6. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: The stirring time of the system in step (2) is 0.5-2.5h.

7. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: The system in step (2) is ultrasonically treated at a frequency of 30-50 kHz and a power of 200-400 W.

8. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: The concentration of the Tris-HCl buffer in step (4) is 50-200 mmol / L, and the pH is 8-9.

9. The method for extracting seabuckthorn seed protein according to claim 1, characterized in that: In the step (4), the alkali dissolution and acid precipitation method is to first adjust the pH of the supernatant after dialysis to 10-11, centrifuge the supernatant, adjust the pH of the supernatant to 3-4 again, filter, collect the precipitate, and obtain the refined seabuckthorn seed protein.

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