CN115976143A - Earthworm peptide and preparation method thereof - Google Patents
Earthworm peptide and preparation method thereof Download PDFInfo
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- CN115976143A CN115976143A CN202310049001.4A CN202310049001A CN115976143A CN 115976143 A CN115976143 A CN 115976143A CN 202310049001 A CN202310049001 A CN 202310049001A CN 115976143 A CN115976143 A CN 115976143A
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Abstract
The invention relates to the technical field of health care products, in particular to earthworm peptide and a preparation method thereof.
Description
Technical Field
The invention relates to the technical field of health care products, in particular to earthworm peptide and a preparation method thereof.
Background
Peptides are bioactive substances that are involved in various cellular functions in the body and participate in regulating various complex physiological processes in the human body. The peptide has the characteristics of high activity, strong affinity, high safety and the like, and the peptide molecules are in a nanometer level and are easily absorbed by intestinal tracts of human bodies, or directly enter deep skin through the surface layer of the skin and are quickly absorbed by the human bodies to play a role.
Earthworm (Geosaurus), also known as earthworm, belongs to the Oligochaeta of Annelida, and is a very traditional animal Chinese medicinal material in China. Modern researches have shown that earthworm has the effects of promoting blood circulation to remove blood stasis, dissolving thrombus, reducing blood pressure, resisting tumor, relieving asthma and cough, diminishing inflammation, relieving pain and the like. The earthworm peptide is a general name of small molecular polypeptides in earthworms and has various varieties, wherein natural polypeptides have an anti-tumor effect, and the polypeptides generated by hydrolysis can promote the production performance and the immunocompetence of animals.
The traditional earthworm peptide extraction method comprises a soaking and decocting method, a reflux method and the like. The methods have the defects of long extraction time and low extraction rate, the obtained earthworm peptides have low content and are not suitable for large-scale production, and the traditional earthworm peptide extraction method generally uses distilled water for dissolution, then hydrolyzes protein through protease to obtain a mixture of hydrolyzed polypeptide and natural polypeptide, and can not separate and extract the hydrolyzed polypeptide and the natural polypeptide, so that corresponding products can be prepared for users in a targeted manner.
Therefore, there is a need for a method for preparing earthworm peptides to solve the above technical problems.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of earthworm peptide.
The earthworm peptide prepared by the invention is natural polypeptide and hydrolyzed polypeptide with the molecular weight below 6000 daltons, and the specific method comprises the following steps:
1. raw material feeding: cleaning Lumbricus, drying, weighing with a weighing device, adding into a crusher, adding 60-70% alcohol into the crusher, gradually adding alcohol with volume ratio of 4L/1kg to Lumbricus, and crushing and mixing;
2. flash extraction of raw materials: uniformly mixing alcohol and frozen earthworms, feeding the mixture into a flash extractor through a conveying pipeline, and extracting for 1 minute through the flash extractor;
3. centrifuging the mixed solution: after flash extraction is finished, centrifuging through a low-speed centrifuge, and separating a precipitate in the flash-extracted mixed solution from a supernatant under the condition of 5000r/min for 15min to obtain a mixed solution;
4. filtering the supernatant: separating the supernatant after flash extraction through a separation membrane, and separating the protein and the natural polypeptide in the supernatant through a separation device to obtain a solution a containing the protein and a solution b containing the natural polypeptide;
5. and (3) drying the solution b in vacuum: and after separation is finished, putting the solution b into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the water is completely dried to obtain the natural heat polypeptide.
6. Protein flash extraction: adding distilled water into the solution a for mixing, wherein the mixing ratio is 1. Flash extraction time is 1min;
7. ultrasonic extraction: putting the solution after flash extraction into a constant-temperature ultrasonic extractor for ultrasonic extraction, wherein the temperature is set at 38 ℃, the ultrasonic extraction time is set at 30min, and the ultrasonic power is 800W;
8. low-temperature centrifugation: carrying out high-speed low-temperature centrifugation on the suspension after ultrasonic extraction; centrifuging at-4 deg.C and 11000r/min for 50min;
9. enzymolysis: after low-temperature centrifugation is finished, taking supernatant, adding distilled water, adjusting the pH to 5.5, adding protease accounting for 2% of the mass of the earthworm, and magnetically stirring for 2 hours at 50 ℃;
10. separating enzymolysis liquid: after the magnetic stirring time is finished, immediately separating the enzymatic hydrolysate by using a separation membrane, and separating the protease and the hydrolyzed polypeptide;
11. centrifuging enzymolysis liquid: cooling the boiled enzymolysis liquid, sending the cooled enzymolysis liquid into a centrifugal container, and centrifuging by using a low-speed centrifuge under the condition of 5000r/min for 15min to obtain supernatant liquid as a solution c;
12. and (3) vacuum drying of the enzymolysis liquid: and after the centrifugation is finished, taking the solution c, putting the solution c into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the water is completely dried.
Preferably, the method for cleaning the earthworms in the step (1) is to wash the outer surfaces of the fresh earthworms and put the washed fresh earthworms into water for 48 hours to obtain the earthworm cleaning solution; putting the earthworms into water to discharge and clean the dirt in the digestive tract of the earthworms;
preferably, distilled water is added into the precipitate after the step 3, and the ratio of the volume of the distilled water to the mass of the precipitate is 4ml:1g, then carrying out flash extraction by using a flash extractor, wherein the flash extraction time is 1min, repeating the step 3 and the subsequent operations, carrying out flash extraction on the precipitate obtained after the centrifugation in the step 3 by using the flash extractor, and repeating the step 3 and the subsequent operations, so that the protein and the natural polypeptide which are not extracted in the precipitate can be further extracted, and the waste of raw materials is avoided.
Preferably, the separation membrane in the step 4 and the separation membrane in the step 10 are ultrafiltration membranes or nanofiltration membranes of 10000 daltons. The ultrafiltration membrane or the nanofiltration membrane is used for separation, so that the protein and the natural polypeptide are not damaged, and the subsequent operation is facilitated.
Preferably, after the ultrasonic extraction in the step 6, the suspension can be bottled, put into a low-temperature device for freezing for 12 hours, and then thawed at normal temperature after being frozen, and then the step 7 and the subsequent operations are performed;
preferably, the protease in the step 8 is papain, and the pH is continuously adjusted in the stirring process and is controlled between 5.5 and 6;
preferably, the method for controlling the pH value in the step 8 to be between 5.5 and 6 is to adjust the pH value to be between 5.5 and 6 by using 0.1 to 2.0mol/L hydrochloric acid (HCl);
preferably, the solution b and the solution c obtained in the steps 4 and 11 can be subjected to secondary filtration through nanofiltration membranes with different molecular weights, so as to obtain the earthworm peptides with corresponding size intervals.
Compared with the related art, the preparation method of the earthworm peptide provided by the invention has the following beneficial effects:
1. according to the invention, earthworms and alcohol are simultaneously added into a crusher to be crushed, then, a flash extractor is used for crushing earthworm solution, natural polypeptide and macromolecular protein in the earthworms are dissolved in the alcohol, a centrifugal machine is used for centrifuging, precipitates are separated from the protein and the natural polypeptide, further, the natural polypeptide and the protein are extracted from the earthworms, the natural polypeptide and the protein are separated by a membrane separation technology, and then, the protein is hydrolyzed by protease to form hydrolyzed polypeptide, so that the problem that only a mixture of the hydrolyzed polypeptide and the natural polypeptide can be extracted when the earthworms are extracted in the prior art, and the natural polypeptide and the hydrolyzed polypeptide can not be extracted independently at the same time is solved, and the flash extractor is used for consuming short time, so that the alcohol in an extracting solution can be recycled after extraction, and the utilization rate of raw materials is improved;
2. according to the invention, the precipitate obtained after centrifugation in the step 3 is subjected to flash extraction by using a flash extractor, and the step 3 and the subsequent operations are repeated, so that the protein and the natural polypeptide which are not extracted in the precipitate can be further extracted, and the waste of raw materials is avoided.
3. According to the invention, the natural polypeptide solution b and the hydrolyzed polypeptide solution c are obtained, and secondary filtration can be carried out by combining nanofiltration membranes and ultrafiltration membranes with different molecular weights, so that the earthworm peptides with corresponding size intervals can be obtained, and can be used for preparing subsequent products.
Drawings
FIG. 1 is a schematic flow chart of a process for preparing Lumbricus peptide;
FIG. 2 is a schematic diagram of a process for preparing a natural polypeptide;
FIG. 3 is a schematic diagram of the process for preparing hydrolyzed polypeptides.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Preparation of natural polypeptide:
1. raw material feeding: washing the outer surface of fresh earthworms, putting the fresh earthworms into water for 48 hours to obtain clean earthworms, drying the earthworms, weighing the earthworms by using a weighing device, adding the earthworms into a crusher, adding 66% alcohol by volume into the crusher, gradually adding the alcohol, and starting the crusher to mix the earthworms after adding the alcohol, wherein the ratio of the volume of the added alcohol to the mass of the earthworms is 4L/1 kg;
2. flash extraction of raw materials: uniformly mixing alcohol and frozen earthworms, feeding the mixture into a flash extractor through a conveying pipeline, and extracting for 1 minute through the flash extractor;
3. centrifuging the mixed solution: after flash extraction is finished, centrifuging through a low-speed centrifuge, and separating a precipitate in the flash-extracted mixed solution from a supernatant under the condition of 5000r/min for 15min to obtain a mixed solution;
4. filtering and filtering the supernatant: separating the supernatant subjected to flash extraction by using an ultrafiltration membrane, and separating the protein in the supernatant from the natural polypeptide by using the ultrafiltration membrane to obtain a solution a containing the protein and a solution b containing the natural polypeptide;
5. and (3) drying the solution b in vacuum: and after the separation is finished, putting the solution b into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the moisture is completely dried to obtain the Tianthermi polypeptide.
Preparation of hydrolyzed polypeptides
1. Protein flash extraction: adding distilled water into the solution a for mixing, wherein the mixing ratio is 1. Flash extraction time is 1min;
2. ultrasonic extraction: putting the solution after flash extraction into a constant-temperature ultrasonic extractor for ultrasonic extraction, wherein the temperature is set at 38 ℃, the ultrasonic extraction time is set at 30min, and the ultrasonic power is 800W;
3. freezing treatment: after ultrasonic extraction, bottling the suspension, freezing in a low temperature device for 12 hr, thawing at normal temperature, and performing step 7 and subsequent operations
4. Low-temperature centrifugation: carrying out high-speed low-temperature centrifugation on the suspension after ultrasonic extraction; centrifuging at-4 deg.C at 11000r/min for 50min;
5. enzymolysis: after low-temperature centrifugation is finished, taking supernatant, adding distilled water, adjusting the pH to 5.5, adding papain with the mass of 2% of that of the earthworm, magnetically stirring for 2 hours at 50 ℃, continuously adjusting the pH in the stirring process, and controlling the pH to be between 5.5 and 6 by using 0.1 to 2.0mol/L hydrochloric acid (HCl);
6. separating enzymolysis liquid: after the magnetic stirring time is finished, separating the enzymolysis liquid by a 10000 Dalton nanofiltration membrane, and separating the protease and the hydrolyzed polypeptide;
7. centrifuging enzymolysis liquid: cooling the boiled enzymolysis liquid, sending the cooled enzymolysis liquid into a centrifugal container, and centrifuging by using a low-speed centrifuge under the condition of 5000r/min for 15min to obtain supernatant liquid as a solution c;
8. and (3) vacuum drying of the enzymolysis liquid: and after the centrifugation is finished, taking the solution c, putting the solution c into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the water is completely dried.
The principle of separation of the natural polypeptide and the hydrolyzed polypeptide is as follows:
earthworm and alcohol are simultaneously added into a crusher to be crushed, then earthworm solution is crushed by a flash extractor, the characteristic that protein and polypeptide can be dissolved in alcohol is utilized, natural polypeptide and macromolecular protein in the earthworm are dissolved in the alcohol, then centrifugation is carried out by a centrifuge, precipitates in the alcohol solution are separated from the protein and the natural polypeptide by utilizing different weights, the natural polypeptide and the protein are further extracted from the earthworm, the natural polypeptide and the protein are separated by a membrane separation technology, the protein or the natural polypeptide are prevented from being damaged by other methods, the product quality is influenced, then the protein is hydrolyzed by papain, continuous stirring is carried out in the hydrolysis process, and PH is controlled, the hydrolysis can be carried out to the maximum extent, the problem that when the existing earthworm peptide is extracted, only a mixture of the hydrolyzed polypeptide and the natural polypeptide can be extracted, the natural polypeptide and the hydrolyzed polypeptide can not be simultaneously and independently extracted is solved, the time is consumed by the flash extractor, the alcohol in the extracting solution can be recycled after extraction, and the utilization rate of raw materials is improved.
Isolation of polypeptides
Filtering the solution b by an ultrafiltration membrane with the molecular weight cutoff of less than or equal to 3000 daltons to obtain a solution b1, intercepting the filtrate by a nanofiltration membrane with the molecular weight of 300 daltons to obtain a solution b2 and a solution b3, putting the intercepted solutions b1, b2 and b3 into a vacuum drier after the interception is finished, setting the temperature at 50 ℃, and taking out the solid after the moisture is completely dried to obtain the natural polypeptides with different molecular weights;
filtering the solution c by an ultrafiltration membrane with the molecular weight cutoff of less than or equal to 5000 daltons to obtain a solution c1, intercepting the filtrate by a nanofiltration membrane with the molecular weight of 500 daltons to obtain a solution c2 and a solution c3, putting the intercepted solutions c1, c2 and c3 into a vacuum drier after the interception is finished, setting the temperature at 50 ℃, and taking out the solid after the moisture is completely dried to obtain the hydrolyzed polypeptides in different molecular weight regions.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (9)
1. A earthworm peptide, comprising: the Lumbricus peptide is natural polypeptide and hydrolyzed polypeptide with molecular weight below 6000 daltons.
2. A method for preparing earthworm peptide is characterized in that,
(1) Raw material feeding: cleaning and drying earthworms, weighing the earthworms by using a weighing device, adding the earthworms into a crusher, adding 60-70% alcohol into the crusher, wherein the ratio of the volume of the added alcohol to the mass of the earthworms is 4L/1kg, gradually adding the alcohol, and starting the crusher to crush and mix the earthworms after adding the alcohol;
(2) Flash extraction of raw materials: uniformly mixing alcohol and frozen earthworms, feeding the mixture into a flash extractor through a conveying pipeline, and extracting for 1 minute through the flash extractor;
(3) Centrifuging the mixed solution: after the flash extraction is finished, centrifuging through a low-speed centrifuge, and separating the precipitate and the supernatant in the mixed solution after the flash extraction, wherein the centrifuging condition is 5000r/min, and the time is 15min, so as to obtain the mixed solution;
(4) Filtering the supernatant: separating the supernatant subjected to flash extraction by using a separation membrane, and separating the protein and the natural polypeptide in the supernatant by using the separation membrane to obtain a solution a containing the protein and a solution b containing the natural polypeptide;
(5) And (3) drying the solution b in vacuum: and after separation is finished, putting the solution b into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the water is completely dried to obtain the natural heat polypeptide.
(6) Protein flash extraction: adding distilled water into the solution a for mixing, wherein the mixing ratio is 1. Flash extraction time is 1min;
(7) Ultrasonic extraction: putting the solution after flash extraction into a constant-temperature ultrasonic extractor for ultrasonic extraction, wherein the temperature is set at 38 ℃, the ultrasonic extraction time is set at 30min, and the ultrasonic power is 800W;
(8) Low-temperature centrifugation: carrying out high-speed low-temperature centrifugation on the suspension after ultrasonic extraction; centrifuging at-4 deg.C and 11000r/min for 50min;
(9) Enzymolysis: after low-temperature centrifugation is finished, taking supernatant, adding distilled water, adjusting the pH to 5.5, adding protease accounting for 2% of the mass of the earthworm, and magnetically stirring for 2 hours at 50 ℃;
(10) Separating enzymolysis liquid: after the magnetic stirring time is finished, immediately separating the enzymatic hydrolysate by using a separation membrane, and separating the protease and the hydrolyzed polypeptide;
(11) And (3) centrifuging enzymolysis liquid: cooling the boiled enzymolysis liquid, sending the cooled enzymolysis liquid into a centrifugal container, and centrifuging by using a low-speed centrifuge under the condition of 5000r/min for 15min to obtain supernatant liquid of the solution c;
(12) And (3) vacuum drying of the enzymolysis liquid: and after the centrifugation is finished, taking the solution c, putting the solution c into a vacuum drier, setting the temperature to be 50 ℃, and taking out the solid after the water is completely dried.
3. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: the method for cleaning the earthworms in the step (1) is to wash the outer surfaces of the fresh earthworms and put the washed earthworms in water for 48 hours.
4. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: adding distilled water into the precipitate after the step (3), wherein the ratio of the volume of the distilled water to the mass of the precipitate is 4ml:1g, then carrying out flash extraction by using a flash extractor, wherein the flash extraction time is 1min, and repeating the step (3) and the subsequent operations.
5. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: and (3) separating by using an ultrafiltration membrane or a 10000 Dalton nanofiltration membrane as the separation membrane in the steps (4) and (10).
6. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: after the ultrasonic extraction in the step (6), the suspension can be bottled, and then the suspension is put into a low-temperature device for freezing for 12 hours, and then the suspension is thawed at normal temperature after being frozen, and then the step (7) and the subsequent operation are carried out.
7. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: the protease in the step (8) is papain, and the pH is continuously adjusted in the stirring process and is controlled between 5.5 and 6.
8. The method for preparing the earthworm peptide according to claim 7, wherein the earthworm peptide comprises the following components: the method for controlling the pH value in the step (8) to be between 5.5 and 6 is to adjust the pH value to be between 3 and 5.5 by using 0.1 to 2.0mol/L hydrochloric acid (HCl) solution.
9. The method for preparing the earthworm peptide according to claim 2, wherein the earthworm peptide comprises the following components: and (3) performing secondary filtration on the solution b obtained in the step (4) and the solution b and the solution c obtained in the step (11) through nanofiltration membranes with different molecular weights to obtain the earthworm peptides with corresponding size intervals.
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