An ultrasonic-assisted extraction method of sericin

Abstract

The application provides an ultrasonic-assisted sericin extraction method and relates to the technical field of natural protein extraction.The ultrasonic-assisted sericin extraction method comprises the following steps: raw material pretreatment, preparation of a low-salt weak-alkali protective extraction solution, first-stage low-frequency ultrasonic loosening, second-stage moderate-high-frequency gentle ultrasonic stripping, end-point control, solid-liquid separation, membrane grading and desalination, concentration and drying and the like.The application realizes the efficient and selective stripping of sericin from the surface of silk fibroin fibers and the directional grading of the molecular weight of sericin under mild conditions by constructing a low-salt weak-alkali buffer extraction system composed of ammonium bicarbonate and an amino acid stabilizer, combining a segmented ultrasonic method of low-frequency ultrasonic loosening and moderate-high-frequency gentle ultrasonic stripping, and introducing end-point control based on protein release kinetics, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of natural protein extraction technology, and in particular to an ultrasound-assisted method for extracting sericin. Background Technology

[0002] Sericin is a natural globular protein that coats the outer layer of silk fibers, accounting for approximately 20% to 30% of the total weight of silkworm cocoons. Rich in polar amino acids such as serine and aspartic acid, it possesses excellent moisturizing, antioxidant, and biocompatibility properties, showing broad application prospects in biomedicine, functional foods, and high-end cosmetics. However, in traditional silk reeling and cocoon boiling processes, sericin is typically discharged with wastewater, resulting in a waste of high-quality protein resources and increasing the organic load and treatment costs of the wastewater.

[0003] Currently, the main extraction methods for sericin include high-temperature boiling, alkaline degumming, enzymatic hydrolysis, microwave-assisted extraction, ultrasound-assisted extraction, and membrane recovery from silk reeling wastewater. While high-temperature alkaline boiling and microwave-assisted extraction offer high extraction efficiency, the intense thermal effects and alkaline environment easily lead to random breakage of sericin peptide chains, resulting in a wide molecular weight distribution and a low proportion of high-molecular-weight components, making it difficult to guarantee the film-forming properties and bioactivity of the protein. Low-temperature alkaline soaking for extended periods can reduce thermal denaturation to some extent, but the long processing cycle and high alkaline residue, coupled with the large amount of inorganic salts generated during subsequent neutralization, not only increase the purification burden but also make it difficult to achieve targeted enrichment of high-molecular-weight sericin components. Existing ultrasound-assisted extraction technologies primarily aim to increase the overall degumming rate, lacking methods to protect protein molecular weight. Excessive ultrasound treatment can easily lead to reduced protein particle size, damage to secondary structures, and decreased functional properties. While membrane recovery from silk reeling wastewater offers environmental advantages, the raw materials have already undergone pre-treatment with high temperatures or alkaline solutions, compromising the integrity of protein molecules and limiting the product grade and application range. Furthermore, the above methods are difficult to obtain sericin products with different molecular weight ranges simultaneously in a single process.

[0004] Therefore, there is an urgent need to develop a novel extraction method that can rapidly and efficiently extract sericin under mild conditions, while also taking into account molecular weight protection, low salt residue, and molecular weight-oriented fractionation. Summary of the Invention

[0005] In view of this, the present invention provides an ultrasound-assisted method for extracting sericin. This invention constructs a low-salt, weak-base buffer extraction system composed of ammonium bicarbonate and an amino acid stabilizer, combines a segmented ultrasound method of low-frequency ultrasonic loosening and medium-to-high-frequency gentle ultrasonic ablation, and introduces endpoint control based on protein release kinetics. This achieves highly efficient and selective ablation and molecular weight-oriented fractionation of sericin from the surface of silk fibers under mild conditions, and has broad application prospects.

[0006] The ultrasound-assisted method for extracting sericin of the present invention includes the following steps: S1. Raw material pretreatment: Remove pupa debris, dust and mechanical impurities from silkworm cocoons, cocoon shells, cocoon skins, waste cocoons or silk reeling waste, cut them into pieces, and wash them with purified water to obtain pretreated raw materials; S2. Preparation of low-salt and weak-base protective extract: Dissolve ammonium bicarbonate and amino acids in water to obtain a low-salt and weak-base protective extract. The low-salt, weak-alkali protected extract contains ammonium bicarbonate and an amino acid stabilizer, wherein the total concentration of the amino acid stabilizer is 0.01%~0.30% w / v and the pH is 8.0~9.3; S3, First stage of low-frequency ultrasonic loosening: The pretreated raw material is mixed with the low-salt weak-alkali protective extract and subjected to low-frequency ultrasonic treatment at a frequency of 18~28 kHz; S4. Second stage of medium-high frequency gentle ultrasonic peeling: medium-high frequency ultrasonic treatment is performed at a frequency of 35~60 kHz to peel the sericin from the surface of the silk fiber into the extraction solution. S5. Endpoint control: The A280 absorbance is measured during the ultrasonic treatment to determine the ultrasonic endpoint; S6. Solid-liquid separation: The ultrasonically treated system is filtered, centrifuged or microfiltered to obtain a crude extract of sericin. S7. Membrane fractionation and desalting: The crude sericin extract is subjected to membrane fractionation, washing and desalting to obtain sericin components with different molecular weight ranges. S8. Concentration and Drying: The sericin components after membrane fractionation are concentrated and dried to obtain sericin powder.

[0007] Preferably, in step S1, the length of the fragment is 5-30 mm, and the number of times the purified water is washed is 1-3.

[0008] Preferably, in step S3, before mixing with the low-salt, weak-alkali protective extract, the pretreated raw material is pre-soaked in water at 30-45°C for 10-30 min; the mass-to-volume ratio of the pretreated raw material to the low-salt, weak-alkali protective extract is 1 g: 20-60 mL.

[0009] Preferably, in step S2, the concentration of ammonium bicarbonate in the low-salt, weak-base protective extract is 5-80 mmol / L; the amino acid stabilizer is at least one of glycine, arginine, and serine; the low-salt, weak-base protective extract further contains a chelating agent, which is citrate or EDTA-2Na, with a concentration of 0.1-2 mmol / L.

[0010] Preferably, in step S3, the power density of the first low-frequency ultrasonic loosening is 50~300 W / L, the duty cycle is 10%~40%, the processing time is 1~8 min, and the processing temperature is ≤40℃.

[0011] Preferably, in step S4, the power density of the second stage of high-frequency mild ultrasonic ablation is 30~180 W / L, the duty cycle is 20%~60%, the processing time is 5~30 min, and the processing temperature is 25~48℃. During the second stage of high-frequency mild ultrasonic ablation, low-speed stirring is carried out simultaneously at a stirring speed of 50~300 rpm.

[0012] Preferably, in step S5, the absorbance of A280 is detected every 1 to 5 minutes. When the increase of A280 in two consecutive detections is less than 5% to 15% of the average increase in the previous stage, the ultrasonic treatment is stopped.

[0013] Preferably, in step S6, the ultrasonically treated system is cooled to 10~25℃, coarsely filtered through an 80~200 mesh filter, and then centrifuged or microfiltered to obtain a crude extract of sericin protein; the centrifugation speed is 3000~10000 rpm, and the centrifugation time is 5~20 min; the microfiltration uses a 0.22~1.0 μm microfiltration membrane.

[0014] Preferably, in step S7, large particles are removed sequentially using a 100-300 kDa membrane, high molecular weight components are collected using a 30-100 kDa membrane, medium and low molecular weight components are collected using a 3-30 kDa membrane, and desalination is performed by washing with 3-8 times the volume of the filter.

[0015] Preferably, in step S8, the solid content is concentrated under vacuum at 20~45℃ to 2%~15%; the drying is freeze drying, spray drying or low-temperature vacuum drying; the outlet temperature of the spray drying is ≤85℃.

[0016] This invention achieves selective stripping of sericin from the surface of silk fibers at lower temperatures and in a shorter time, while reducing protein chain degradation and salt residue. Through segmented ultrasound and endpoint control, this invention ensures that the extraction process is controlled by the target molecular weight sericin yield, purity, and structural integrity, rather than simply pursuing the maximum degumming rate.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention uses a weak base buffer extraction system composed of ammonium bicarbonate and amino acid stabilizers to replace the traditional strong base system. Ammonium bicarbonate can be volatilized and removed during subsequent concentration and drying, and amino acid stabilizers can protect protein molecules, thereby effectively reducing salt residue in the product, reducing the risk of protein hydrolysis in an alkaline environment, and improving product purity and safety.

[0018] This invention employs a segmented ultrasonic method that combines low-frequency ultrasonic loosening with medium- and high-frequency gentle ultrasonic peeling. The first segment of low-frequency ultrasound utilizes a strong cavitation effect to loosen the sericin binding force between cocoon fibers. The second segment of medium- and high-frequency ultrasound uses gentle micro-jet and vibration effects to selectively peel sericin from the surface of silk fibers. The synergistic effect of the two segments ensures extraction efficiency while avoiding excessive damage to the protein molecular chains caused by single high-intensity ultrasound, which is beneficial to maintaining the high molecular weight and structural integrity of sericin.

[0019] This invention controls the endpoint by monitoring absorbance in real time during ultrasonic processing, which can stop ultrasonic processing in a timely manner and avoid protein degradation and structural changes caused by excessive ultrasonic time, thus achieving precise and controllable extraction process.

[0020] This invention combines sericin extraction with membrane fractionation. By sequentially fractionating the sericin components with ultrafiltration membranes of different molecular weight cutoffs, different molecular weight ranges of sericin components can be obtained in the same process, meeting the differentiated requirements of sericin molecular weight for different application scenarios, rather than obtaining only a single mixed product.

[0021] The entire process of this invention is completed at a lower temperature and in a shorter time, the pretreatment conditions of the raw materials are controllable, and the molecular weight distribution and purity of the product are easier to standardize. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: An ultrasound-assisted method for extracting sericin, comprising the following steps: S1. Raw material pretreatment: Take silkworm cocoons, remove pupa debris, dust and mechanical impurities, cut them into 10 mm segments, wash them twice with purified water to obtain pretreated raw materials; S2. Preparation of low-salt, weak-base protective extract: Dissolve ammonium bicarbonate and glycine in water to obtain a low-salt, weak-base protective extract. The concentration of ammonium bicarbonate is 30 mmol / L, the concentration of glycine is 0.15% w / v, and 0.8 mmol / L of food-grade sodium citrate is added. The pH is adjusted to 8.7. S3, First stage of low-frequency ultrasonic loosening: After pre-soaking the pretreated raw material in 40℃ water for 15 min, it is mixed with the protective extract at a ratio of 1 g: 40 mL and subjected to low-frequency ultrasonic treatment at a frequency of 23 kHz, with a power density of 180 W / L, a duty cycle of 25%, a treatment time of 4 min, and a temperature controlled below 38℃. S4. Second stage of medium-high frequency gentle ultrasonic stripping: medium-high frequency ultrasonic treatment is performed at 48 kHz, with a power density of 110 W / L, a duty cycle of 40%, a treatment time of 18 min, and a temperature controlled at 42℃. During the process, low-speed stirring at 150 rpm is performed simultaneously. S5. Endpoint control: Detect A280 absorbance every 3 minutes. Stop the ultrasonic treatment when the A280 increment is less than 10% of the average increment of the previous stage in two consecutive measurements. S6. Solid-liquid separation: Cool the system to 20°C, filter it through a 120-mesh filter, and centrifuge it at 8000 rpm for 10 min to obtain a crude extract of sericin. S7. Membrane fractionation and desalination: Large particles are removed sequentially using a 200 kDa membrane, high molecular weight components are collected using a 50 kDa membrane, medium and low molecular weight components are collected using a 10 kDa membrane, and desalination is performed by washing with 5 times the volume of the filter. S8. Concentration and drying: Vacuum concentration at 35°C to a solid content of 8%, followed by freeze drying to obtain sericin powder.

[0024] Example 2 The difference from Example 1 is as follows: in step S2, the concentration of ammonium bicarbonate is 15 mmol / L, the amino acid stabilizer is 0.12% w / v arginine, and the pH is 8.4; in step S3, the power density is 150 W / L and the treatment time is 3 min; in step S4, the frequency is 52 kHz, the power density is 90 W / L, and the treatment time is 22 min.

[0025] Example 3 The difference from Example 1 is as follows: Step S1 uses silk reeling waste material with a fragment length of 15 mm and washes it 3 times; Step S3 uses a pre-soaking temperature of 35°C for 20 min and a material-to-liquid ratio of 1 g:50 mL; Step S7 uses a 150 kDa membrane to remove large particles, an 80 kDa membrane to collect high molecular weight components, and a 20 kDa membrane to collect medium and low molecular weight components, and washes and filters 6 times the volume.

[0026] Example 4 The difference from Example 1 is as follows: Step S2 adds 1.2 mmol / L of EDTA-2Na; Step S3 uses a low frequency of 20 kHz, a power density of 220 W / L, a duty cycle of 30%, and a processing time of 5 min; Step S4 uses a medium-high frequency of 45 kHz, a duty cycle of 35%, a processing time of 15 min, and a stirring speed of 200 rpm; Step S5 detects A280 every 2 min.

[0027] Comparative Example 1 The difference from Example 1 is that conventional high-temperature NaOH (0.5%) was used for boiling and degumming for 60 min, and the rest of the post-treatment was the same as in Example 1.

[0028] Comparative Example 2 The difference from Example 1 is that step S3 is omitted, and ultrasonic treatment is performed directly at a single frequency of 45 kHz for 25 min (power density 120 W / L), while other conditions are the same as in Example 1.

[0029] Comparative Example 3 The difference from Example 1 is that step S5 is omitted, the total ultrasound time is fixed at 30 min, and other conditions are the same as in Example 1.

[0030] Comparative Example 4 The difference from Example 1 is that in step S2, a 0.02 mol / L NaOH solution (pH≈12) is used instead of the low-salt weak-base protective extract, and glycine and sodium citrate are not added.

[0031] Comparative Example 5 The difference from Example 1 is that steps S3 and S4 both use continuous ultrasound mode (duty cycle 100%), while other parameters remain unchanged.

[0032] Comparative Example 6 The difference from Example 1 is that: first, 48 kHz mid-high frequency processing is performed for 18 minutes, and then 23 kHz low frequency processing is performed for 4 minutes, while other parameters remain unchanged.

[0033] Comparative Example 7 The difference from Example 1 is that the membrane fractionation in step S7 is omitted, and all proteins are collected by precipitation with 2 times the volume of anhydrous ethanol, followed by centrifugation and drying.

[0034] Table 1. Comparison of sericin extraction performance between different examples and comparative examples (n=3, mean ± standard deviation)

[0035] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for ultrasound-assisted extraction of sericin, characterized in that, The steps include the following: S1. Raw material pretreatment: Remove pupa debris, dust and mechanical impurities from silkworm cocoons, cocoon shells, cocoon skins, waste cocoons or silk reeling waste, cut them into pieces, and wash them with purified water to obtain pretreated raw materials; S2. Preparation of low-salt and weak-base protective extract: Dissolve ammonium bicarbonate and amino acids in water to obtain a low-salt and weak-base protective extract. The low-salt, weak-alkali protected extract contains ammonium bicarbonate and an amino acid stabilizer, wherein the total concentration of the amino acid stabilizer is 0.01%~0.30% w / v and the pH is 8.0~9.3; S3, First stage of low-frequency ultrasonic loosening: The pretreated raw material is mixed with the low-salt weak-alkali protective extract and subjected to low-frequency ultrasonic treatment at a frequency of 18~28 kHz; S4. Second stage of medium-high frequency gentle ultrasonic peeling: medium-high frequency ultrasonic treatment is performed at a frequency of 35~60 kHz to peel the sericin from the surface of the silk fiber into the extraction solution. S5. Endpoint control: The A280 absorbance is measured during the ultrasonic treatment to determine the ultrasonic endpoint; S6. Solid-liquid separation: The ultrasonically treated system is filtered, centrifuged or microfiltered to obtain a crude extract of sericin. S7. Membrane fractionation and desalting: The crude sericin extract is subjected to membrane fractionation, washing and desalting to obtain sericin components with different molecular weight ranges. S8. Concentration and Drying: The sericin components after membrane fractionation are concentrated and dried to obtain sericin powder.

2. The method for ultrasound-assisted extraction of sericin according to claim 1, characterized in that, In step S1, the length of the fragment is 5-30 mm, and the number of times the purified water is washed is 1-3.

3. The ultrasound-assisted method for extracting sericin according to claim 1, characterized in that, In step S3, before mixing with the low-salt, weak-alkali protective extract, the pretreated raw material is pre-soaked in water at 30-45°C for 10-30 minutes; the mass-to-volume ratio of the pretreated raw material to the low-salt, weak-alkali protective extract is 1 g: 20-60 mL.

4. The ultrasound-assisted method for extracting sericin according to claim 1, characterized in that, In step S2, the concentration of ammonium bicarbonate in the low-salt, weak-base protected extract is 5-80 mmol / L; the amino acid stabilizer is at least one of glycine, arginine, and serine; the low-salt, weak-base protected extract also contains a chelating agent, which is citrate or EDTA-2Na, with a concentration of 0.1-2 mmol / L.

5. The ultrasound-assisted method for extracting sericin according to claim 1, characterized in that, In step S3, the power density of the first low-frequency ultrasonic loosening is 50~300 W / L, the duty cycle is 10%~40%, the processing time is 1~8 min, and the processing temperature is ≤40℃.

6. The method for ultrasound-assisted extraction of sericin according to claim 1, characterized in that, In step S4, the power density of the second stage of high-frequency mild ultrasonic ablation is 30~180 W / L, the duty cycle is 20%~60%, the processing time is 5~30 min, and the processing temperature is 25~48℃. During the second stage of high-frequency mild ultrasonic ablation, low-speed stirring is carried out simultaneously at a stirring speed of 50~300 rpm.

7. The ultrasound-assisted method for extracting sericin according to claim 1, characterized in that, In step S5, the absorbance of A280 is detected every 1 to 5 minutes. When the increase of A280 in two consecutive detections is less than 5% to 15% of the average increase in the previous stage, the ultrasonic treatment is stopped.

8. The method for ultrasound-assisted extraction of sericin according to claim 1, characterized in that, In step S6, the ultrasonically treated system is cooled to 10~25℃, coarsely filtered through an 80~200 mesh filter, and then centrifuged or microfiltered to obtain a crude extract of sericin protein; the centrifugation speed is 3000~10000 rpm, and the centrifugation time is 5~20 min; the microfiltration uses a 0.22~1.0 μm microfiltration membrane.

9. The method for ultrasound-assisted extraction of sericin according to claim 1, characterized in that, In step S7, large particles are removed sequentially using a 100-300 kDa membrane, high molecular weight components are collected using a 30-100 kDa membrane, medium and low molecular weight components are collected using a 3-30 kDa membrane, and desalination is performed by washing with 3-8 times the volume of the filter.

10. The method for ultrasound-assisted extraction of sericin according to claim 1, characterized in that, In step S8, the solid content is concentrated under vacuum at 20~45℃ to 2%~15%; the drying is freeze drying, spray drying or low temperature vacuum drying; the outlet temperature of the spray drying is ≤85℃.

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