Extraction process of earthworm protein

Abstract

The invention discloses an extraction process of earthworm protein, which comprises the following steps: (1) raw material preparation: selecting high-quality and mildew-free earthworms, rinsing, drying to constant weight, crushing and sieving; (2) tenderization treatment (enzymolysis): mixing the dried earthworm powder with a protease solution for enzymolysis reaction; (3) ultrasonic-assisted extraction: adding deionized water, and performing ultrasonic extraction in an ultrasonic extraction device; (4) centrifugal separation: centrifuging the mixed solution and collecting supernate; (5) membrane separation and purification: removing suspended particles from the supernate for separation; and (6) drying treatment: drying the concentrated earthworm protein liquid. The invention relates to the field of biotechnology and food science, in particular to an extraction process of earthworm protein, and aims to improve the extraction efficiency and product purity of the earthworm protein, simplify the operation process, reduce the use of chemical reagents and reduce the production cost by creatively integrating biological enzymolysis, physical field-assisted extraction and modern membrane separation technologies. The requirements of green production are met.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and food science, specifically to an extraction process for earthworm protein. Background Technology

[0002] Earthworm (Lumbricus terrestris), a traditional Chinese medicine, is rich in protein, various amino acids, vitamins, and minerals, possessing extremely high nutritional value and a wide range of pharmacological effects, such as antioxidant, anti-inflammatory, and immune-boosting properties. However, existing earthworm protein extraction processes mostly employ traditional acid extraction, alkali extraction, or hot water extraction methods. These methods generally suffer from low extraction efficiency, easy protein denaturation, low purity, cumbersome process steps, and potential environmental pollution from the use of chemical reagents. This severely restricts the application of earthworm protein in high-end food, health products, and pharmaceutical fields. Therefore, there is an urgent need in this field to develop a new, efficient, high-quality, environmentally friendly, and large-scale earthworm protein extraction process. Summary of the Invention

[0003] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide an extraction process for earthworm protein. This process innovatively integrates biological enzymatic hydrolysis, physical field-assisted extraction and modern membrane separation technology, aiming to significantly improve the extraction efficiency and product purity of earthworm protein, while simplifying the operation process, reducing the use of chemical reagents, and meeting the requirements of green production.

[0004] The technical solution adopted in this invention is as follows: An extraction process for earthworm protein according to this invention includes the following steps:

[0005] (1) Raw material preparation: Select high-quality, mold-free earthworms, rinse them with clean water to remove surface mud and impurities, then dry them at 50-60℃ to constant weight, pulverize them and pass them through a 40-60 mesh sieve to obtain earthworm dry powder for later use.

[0006] (2) Tenderization treatment (enzymatic hydrolysis): The earthworm powder is mixed with a certain concentration of protease solution at a material-to-liquid ratio of 1:10-1:15 (g / mL), and the enzymatic hydrolysis reaction is carried out at 30-40℃ and pH 6.5-7.5 for 2-4 hours; the protease is papain or bromelain.

[0007] (3) Ultrasonic-assisted extraction: After the enzymatic hydrolysis is completed, without filtration, add an appropriate amount of deionized water directly to the enzymatic hydrolysis system, place it in an ultrasonic extraction device, and perform ultrasonic extraction for 30-60 minutes at a frequency of 20-40kHz and a power of 200-500W, while controlling the temperature not to exceed 50℃.

[0008] (4) Centrifugation: Centrifuge the mixture after ultrasonic extraction at 8000-12000 rpm for 10-15 minutes, carefully collect the supernatant and discard the precipitate;

[0009] (5) Membrane separation and purification: The obtained supernatant is passed through a microfiltration membrane (pore size 0.1-0.45μm) to remove suspended particles, and then separated by an ultrafiltration membrane with a molecular weight cutoff of 5-10kDa. The retentate is collected to obtain concentrated earthworm protein solution.

[0010] (6) Drying treatment: The concentrated earthworm protein liquid is dried by spray drying (inlet air temperature 180-200℃, outlet air temperature 80-90℃) or freeze drying (pre-frozen to below -40℃, vacuum degree <10Pa in the main drying stage, plate temperature 20-30℃) to finally obtain light yellow to milky white earthworm protein powder.

[0011] After adopting the above technical solution, the beneficial effects of the present invention are as follows: The extraction process of the present invention combines enzymatic tenderization, ultrasonic-assisted extraction, and membrane separation technology, and has the following advantages:

[0012] 1. High extraction efficiency: The enzymatic method effectively destroys the cell structure of earthworms, and combined with the cavitation effect and mechanical action of ultrasound, it greatly promotes the dissolution of proteins, shortens the extraction time, and the extraction rate can reach more than 70%.

[0013] 2. High product purity: The ultrafiltration membrane separation technology can accurately remove small molecule impurities, salts and some pigments, resulting in a significant improvement in the purity of the final product protein.

[0014] 3. Good activity retention: The entire process adopts gentle enzymatic hydrolysis and physical extraction methods, avoiding the damage to protein structure caused by strong acids, strong alkalis or high temperatures, thus better preserving the biological activity of earthworm protein;

[0015] 4. Environmentally friendly process: It mainly uses biological enzymes and physical methods, avoiding the use of a large number of chemical reagents. The amount of wastewater generated is small and easy to treat, which is environmentally friendly. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: Extraction of earthworm protein using papain combined with ultrasound

[0018] Raw material preparation: Take 100g of dried earthworm, wash it, dry it in an oven at 55℃, pulverize it and pass it through a 60-mesh sieve to obtain earthworm powder;

[0019] Tenderization treatment: Mix earthworm powder with 0.4% papain solution (prepared with phosphate buffer, pH 7.0) at a material-to-liquid ratio of 1:12 (g / mL) and place in a 38℃ water bath shaker for 3 hours for enzymatic hydrolysis;

[0020] Ultrasonic-assisted extraction: After enzymatic hydrolysis, the mixture was transferred to an ultrasonic cell disruptor, the ultrasonic frequency was set to 30kHz and the power to 300W, and ultrasonic extraction was performed at 45℃ for 45 minutes.

[0021] Centrifugation: The extract was centrifuged at 10,000 rpm for 12 minutes, and the supernatant was collected;

[0022] Membrane separation and purification: The supernatant is first pre-filtered through a 0.22μm microfiltration membrane, and then concentrated and desalted using an ultrafiltration membrane system with a molecular weight cutoff of 8kDa. The ultrafiltration retentate is collected.

[0023] Drying treatment: The ultrafiltration retentate was spray-dried at an inlet air temperature of 190℃ and an outlet air temperature of 85℃ to obtain 18.5g of milky white earthworm protein powder. The protein content was determined by the Kjeldahl method to be 81.3%, and the extraction rate was 76.8%.

[0024] Example 2: Extraction of earthworm protein from earthworms using bromelain combined with ultrasound

[0025] Raw material preparation: Take 100g of dried earthworm, wash it, dry it in an oven at 55℃, pulverize it and pass it through a 60-mesh sieve to obtain earthworm powder;

[0026] Tenderization treatment: Mix earthworm powder with 0.5% bromelain solution (prepared with phosphate buffer, pH 6.8) at a material-to-liquid ratio of 1:10 (g / mL) and enzymatically hydrolyze at 35℃ for 2.5 hours;

[0027] Ultrasonic-assisted extraction: After enzymatic hydrolysis, ultrasonic extraction was performed at a frequency of 25kHz and a power of 250W for 50 minutes, with the temperature controlled at 40℃.

[0028] Centrifugation: Centrifuge at 9000 rpm for 15 minutes and collect the supernatant;

[0029] Membrane separation and purification: Same membrane treatment steps as in Example 1;

[0030] Drying treatment: Freeze-drying was used. The ultrafiltration retentate was pre-frozen to -45℃, and then freeze-dried for 24 hours under a vacuum of 8 Pa and a plate temperature of 25℃ to obtain 17.8 g of light yellow earthworm protein powder. The protein content was determined to be 83.5%, and the extraction rate was 74.2%.

[0031] Example 3: Optimizing ultrasound time to improve extraction rate

[0032] Raw material preparation: Take 100g of dried earthworm, wash it, dry it in an oven at 55℃, pulverize it and pass it through a 60-mesh sieve to obtain earthworm powder;

[0033] Tenderization treatment: Mix earthworm powder with 0.4% papain solution (prepared with phosphate buffer, pH 7.0) at a material-to-liquid ratio of 1:12 (g / mL) and place in a 38℃ water bath shaker for 3 hours for enzymatic hydrolysis;

[0034] Ultrasonic-assisted extraction: After enzymatic hydrolysis, the mixture was transferred to an ultrasonic cell disruptor, and the ultrasonic frequency was set to 30kHz and the power to 300W. Ultrasonic extraction was performed at 45℃ for 60 minutes.

[0035] Centrifugation: The extract was centrifuged at 10,000 rpm for 12 minutes, and the supernatant was collected;

[0036] Membrane separation and purification: The supernatant is first pre-filtered through a 0.22μm microfiltration membrane, and then concentrated and desalted using an ultrafiltration membrane system with a molecular weight cutoff of 8kDa. The ultrafiltration retentate is collected.

[0037] Drying treatment: The ultrafiltration retentate was spray-dried at an inlet air temperature of 190℃ and an outlet air temperature of 85℃ to obtain 19.2g of milky white earthworm protein powder. The protein content was determined to be 80.5%, and the extraction rate was increased to 79.5%.

[0038] The results showed that appropriately extending the ultrasonic time could help further improve the extraction rate, but it should be noted that excessively long ultrasonic time may lead to increased energy consumption and potential impact on protein activity.

[0039] Comparative example: Traditional hot water extraction method

[0040] To highlight the advantages of this invention, a comparative example was set up: 100g of earthworm powder was taken and added to deionized water at a material-to-liquid ratio of 1:15. The mixture was then extracted in a 90℃ water bath for 2 hours. After centrifugation, the supernatant was concentrated and spray-dried. The final yield was 12.1g of protein powder with a protein content of 68.7%, and an extraction rate of only 41.5%.

[0041] The comparison shows that the extraction process of the present invention is far superior to traditional methods in terms of extraction efficiency and product purity.

[0042] The present invention and its embodiments have been described above. This description is not restrictive, and the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and, without departing from the spirit of the invention, design similar structures and embodiments without creative effort, all such designs should fall within the protection scope of the present invention.

Claims

1. An extraction process of earthworm protein, characterized in that, The method comprises the following steps: (1) raw material preparation: cleaning, drying and crushing the earthworm to obtain earthworm dry powder; (2) tenderization treatment: mixing the earthworm dry powder with a protease solution, and performing enzymolysis at 30-40 DEG C and pH 6.5-7.5 for 2-4 hours; (3) ultrasonic-assisted extraction: adding deionized water to the system after enzymolysis, and performing ultrasonic extraction at a frequency of 20-40 kHz for 30-60 minutes; (4) centrifugal separation: centrifuging the mixture after ultrasonic extraction at a rotation speed of 8000-12000 rpm for 10-15 minutes, and collecting the supernatant; (5) membrane separation and purification: purifying and concentrating the supernatant by microfiltration membrane and ultrafiltration membrane in sequence to obtain earthworm protein concentrate; (6) drying treatment: drying the earthworm protein concentrate to obtain earthworm protein powder.

2. The extraction process of earthworm protein according to claim 1, characterized in that: In the step (1), the drying temperature of the earthworm is 50-60 DEG C, and the crushed earthworm is sieved through a 40-60 mesh sieve.

3. The extraction process of earthworm protein according to claim 1, characterized in that: In the step (2), the protease is papain or bromelain; the solid-liquid ratio of the earthworm dry powder to the protease solution is 1:10-1:15 (g / mL).

4. The extraction process of earthworm protein according to claim 1, characterized in that: The power of the ultrasonic extraction is 200-500 W, and the extraction temperature is controlled at less than or equal to 50 DEG C.

5. The extraction process of earthworm protein according to claim 1, characterized in that: The pore size of the microfiltration membrane is 0.1-0.45 μm, and the molecular weight cut-off of the ultrafiltration membrane is 5-10 kDa.

6. The extraction process of earthworm protein according to claim 1, characterized in that: In the step (6), the drying is one of spray drying and freeze drying; In the spray drying, the inlet air temperature is 180-200 DEG C, and the outlet air temperature is 80-90 DEG C; The freeze drying comprises: pre-freezing the protein concentrate to below-40 DEG C, and then drying under the conditions of a vacuum degree lower than 10 Pa and a plate temperature of 20-30 DEG C.

7. The extraction process of earthworm protein according to any one of claims 1 to 6, characterized in that: The enzymolysis in the step (2) is performed in a water bath shaker.

8. The extraction process of earthworm protein according to any one of claims 1 to 6, characterized in that: The ultrasonic extraction in the step (3) is performed in an ultrasonic cell disruptor.