CN112176017A - Method for efficiently extracting protein peptide from sea cucumber intestines - Google Patents
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention relates to a method for efficiently extracting protein peptide from sea cucumber intestines, which belongs to the technical field of biological engineering of polypeptide and enzyme processing and comprises the steps of raw material pretreatment, blanching, high-temperature extraction, enzymolysis, deodorization and decoloration, membrane filtration, concentration and drying. The invention makes full use of sea cucumber intestine which is a byproduct of sea cucumber, effectively utilizes sea cucumber intestine waste, can reduce the problem of environmental pollution caused by waste, can improve the resource utilization rate of the sea cucumber, and is beneficial to increasing the added value of the sea cucumber. The method has reasonable process design, can maintain the nutrient components of the sea cucumber to the maximum extent, and has green products and no pollution. The sea cucumber intestine is subjected to enzymolysis and deodorization treatment, and is subjected to graded filtration by adopting a membrane filtration technology, different peptide segments can be retained according to different molecular weights, and the sea cucumber intestine small molecular peptide powder with the molecular weight less than 1000Da is prepared, can be directly absorbed by a human body, and can be used for preparing various functional foods.
Description
Technical Field
The invention relates to the technical field of biological engineering of polypeptide and enzyme processing, in particular to a method for efficiently extracting protein peptide from sea cucumber intestines.
Background
Sea cucumber is a traditional precious sea product and contains abundant and diverse bioactive substances, the processed sea cucumber products at home and abroad mostly take the body wall of the sea cucumber as the main part at present, the research reports on the viscera functionality of the sea cucumber are less, and the sea cucumber is applied to the fields of health care and medicine and is more rare and less.
A large amount of byproducts such as sea cucumber intestines and sea cucumber eggs are generated in the sea cucumber processing process, the nutritional value of the byproducts is high, and resource waste and environmental pollution can be caused if the byproducts are directly discarded.
In addition, the sea cucumber intestines contain rich protein, 8 amino acids and a plurality of trace elements which are necessary for human bodies, have a plurality of biological functions of resisting tumors, resisting blood coagulation, resisting inflammation and the like, and have extremely high medicinal value. However, the sea cucumber intestines do not draw enough attention and are lack of deep research, processing and utilization.
Disclosure of Invention
The invention provides a method for efficiently extracting protein peptide from sea cucumber intestines in order to solve the technical problems.
The invention is realized by the following technical scheme:
a method for efficiently extracting protein peptide from sea cucumber intestines comprises the following steps;
s1, pretreatment of raw materials: peeling and cleaning sea cucumber to obtain sea cucumber intestines;
s2, blanching sea cucumber intestines;
s3, extracting at high temperature to obtain an extracting solution;
s4, enzymolysis: adding an enzyme preparation into the extracting solution to obtain an enzymolysis solution;
s5, cooling the enzymolysis liquid, and performing deodorization and decoloration treatment;
s6, membrane filtration to obtain a purified solution;
s7, concentrating the purified solution to obtain a purified concentrated solution;
s8, drying and purifying the concentrated solution to obtain the sea cucumber intestinal peptide powder.
Further, the S3 specifically includes:
transferring the blanched sea cucumber intestines to an extraction tank, adding purified water with the weight being 3-4 times that of the sea cucumber intestines into the extraction tank, and heating to 95 ℃;
the extraction was continued for 1 hour with continuous stirring during the extraction.
Further, the S4 specifically includes:
adding the extract into an enzymolysis tank, heating to 60 ℃, adjusting to the optimal reaction condition of the enzyme, and then sequentially adding a first enzyme preparation, a second enzyme preparation and a third enzyme preparation;
and after the enzymolysis reaction is finished, heating to inactivate the enzyme to obtain an enzymolysis liquid.
Preferably, the first enzyme preparation and the second enzyme preparation are both endoproteases, and the third enzyme preparation is an exoprotease.
Further preferably, the first enzyme preparation is a neutral protease, the second enzyme preparation is an alkaline protease and the third enzyme preparation is a debittering protease.
Preferably, the weight ratio of the first enzyme preparation to the sea cucumber intestine is 0.2%, the weight ratio of the second enzyme preparation to the sea cucumber intestine is 0.2%, and the weight ratio of the third enzyme preparation to the sea cucumber intestine is 0.05%.
Further preferably, the first enzyme preparation is added, the second enzyme preparation is added after stirring for 15min, and the third enzyme preparation is added for shearing for 2h after the second enzyme preparation is added for shearing for 3 h.
Further, the S6 includes;
s6.1, tube membrane filtration:
s6.2, filtering by a nanofiltration membrane: the separation of 150-1000Dal organic small molecules is carried out.
Further, the S7 utilizes a double-effect concentrator to concentrate the purified solution by reducing pressure and raising temperature.
Further, the drying method of S8 is a centrifugal spray drying method.
Compared with the prior art, the invention has the following beneficial effects:
the method disclosed by the invention has the advantages that the sea cucumber intestines which are waste products of the sea cucumbers are fully utilized, so that the sea cucumber intestine waste is effectively utilized, the problem of environmental pollution caused by waste can be reduced, the resource utilization rate of the sea cucumbers can be improved, and the added value of the sea cucumbers can be increased;
2, the process is reasonable in design, can maintain the nutrient components of the sea cucumber to the maximum extent, and is green and pollution-free;
3, the invention has simple process and low cost;
4, the sea cucumber intestinal peptide obtained by the invention has inherent smell and nutrition of sea cucumber intestines, and small molecules can be directly absorbed by human bodies, so that the sea cucumber intestinal peptide can be used for preparing various functional foods.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention.
FIG. 1 is a flow chart of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
As shown in figure 1, the method for efficiently extracting protein peptide from sea cucumber intestines disclosed by the invention comprises the following steps;
s1, pretreatment of raw materials: peeling and cleaning sea cucumber to obtain sea cucumber intestines;
s2, adding purified water into the sea cucumber intestines, cleaning for three times, desalting, removing sand, and blanching.
S3, extracting at high temperature to obtain an extracting solution. The method utilizes the different heat stability of protein peptide and non-peptide substances to extract the sea cucumber intestines at high temperature. The method specifically comprises the following steps:
transferring the blanched sea cucumber intestines from the deashing tank to an extraction tank, adding purified water with the weight of 3-4 times that of the sea cucumber intestines into the extraction tank, heating to 95 ℃, continuously extracting for 1 hour, and continuously stirring by a stirring paddle in the process.
S4, enzymolysis: adding enzyme preparation into the extract to obtain enzymatic hydrolysate. The method specifically comprises the following steps:
and (3) putting the extracting solution into an enzymolysis tank, heating to 60 ℃, adjusting to the optimal reaction condition of the enzyme, and then sequentially adding a first enzyme preparation, a second enzyme preparation and a third enzyme preparation. After the reaction is finished, heating to 95 ℃, keeping for 30min, and inactivating the enzyme to obtain enzymatic hydrolysate.
In this embodiment, the first enzyme preparation and the second enzyme preparation are endoproteases, and the third enzyme preparation is an exoprotease. Preferably, the first enzyme preparation is selected from neutral protease, the second enzyme preparation is selected from alkaline protease, and the third enzyme preparation is selected from debittering protease.
Wherein the dosage of the enzyme preparation is respectively as follows: the weight ratio of the first enzyme preparation to the sea cucumber intestine is 0.2%, the weight ratio of the second enzyme preparation to the sea cucumber intestine is 0.2%, and the weight ratio of the third enzyme preparation to the sea cucumber intestine is 0.05%.
The enzyme preparation addition time is as follows: adding the first enzyme preparation, stirring for 15min, adding the second enzyme preparation, shearing for 3h, adding the third enzyme preparation, and shearing for 2 h. The stirring is continuously carried out in the enzyme shearing process, so that the enzyme and the materials are uniformly mixed, and the high-efficiency shearing effect is ensured.
S5, cooling the enzymolysis liquid, and removing odor and color. Specifically comprises the following steps of;
cooling the enzymolysis solution to 50-60 deg.C, pre-coating diatomaceous earth on plate and frame filter, adding 3% of active carbon of sea cucumber intestine, mixing and stirring for 0.5 hr, and pressurizing by plate and frame filter until the filtrate is yellow clear transparent liquid.
And S6, membrane filtration to obtain a purified solution. Specifically comprises the following steps of;
s6.1, tube membrane filtration: and (3) separating solute and solvent with certain molecular weight by adopting a composite tubular membrane and other permeable membranes. The membrane inlet pressure is 1.6-1.8/Bar, the membrane outlet pressure is 0.8-1.0 Bar (temperature is 55-60 ℃), the flow rate of the concentrated solution is 400-600LPM, and the flow rate of the clear solution is 80-90 LPM. In the separation process, the product has no phase change, the effective components are completely retained, and the membrane and the pipeline system meet the food sanitation level requirement, thereby greatly improving the product quality. Removing mycelium, pigment and small molecular impurities.
S6.2, filtering by a nanofiltration membrane: a nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules. Because the operation mode of the membrane adopts cross-flow filtration, the interception substances retained on the surface of the membrane pores can be automatically cleaned during working, thereby achieving the purpose of predetermined purification and separation. The membrane inlet pressure is 10-20Bar, the membrane outlet pressure is 10-20Bar, the flow rate of the concentrated solution is 20-40LPM in the first 5 minutes, the flow rate of the clear solution is more than 100LPM, and the pressure of the filter is 0.35 MPA.
And S7, concentrating the purified solution to obtain a purified concentrated solution.
The double-effect concentrator is used for carrying out reduced pressure heating concentration on the purified liquid, the increment of water can be increased, the concentration efficiency is increased, the vacuum degree is 0.8MPA, and the enzymatic hydrolysate can be sprayed and dried when the content of soluble solid reaches 40-60 degrees.
S8, drying and purifying the concentrated solution to obtain the small molecular sea cucumber intestinal peptide powder.
In this embodiment, a centrifugal spray drying method is used. During drying, the temperature of the feeding port is controlled to be 160-190 ℃, the temperature of the air outlet is controlled to be 90-100 ℃, and the feeding speed is controlled to be 180-220 r/min.
The invention makes full use of sea cucumber by-product sea cucumber intestines, carries out enzymolysis and deodorization treatment on the sea cucumber intestines, and adopts a membrane filtration technology to carry out graded filtration, so that different peptide fragments can be cut off according to different molecular weights, and the sea cucumber intestine small molecular peptide powder with the molecular weight less than 1000Da is prepared.
The process according to the invention is described in detail below with reference to specific examples:
example 1
1. Pretreatment of raw materials: cleaning fresh sea cucumber, cutting open to obtain sea cucumber intestine, and weighing 100kg of sea cucumber intestine.
2. Blanching: cleaning sea cucumber intestine, removing sand, desalting, and blanching with hot water.
3. Hot water extraction: the protein peptide is separated from other components in a heating extraction mode by utilizing the different thermal stability of the protein peptide. Adding 300kg hot water, and extracting with 95 deg.C hot water for 1 hr.
4. Liquid phase separation: cooling and standing the extractive solution, and separating liquid phase with centrifuge.
5. Enzymolysis: adding alkaline protease (200 g) 0.2%, neutral protease (200 g) 0.2%, and debittering protease (50 g) 0.05%; enzymolysis is carried out at the temperature of 50-55 ℃. After the enzymolysis is finished, the temperature is raised to 95 ℃, and the temperature is kept for 30 min.
6. And (3) filtering: cooling to 50-60 deg.C, and removing odor and decolorizing; precoating a plate-and-frame filter with diatomite, adding 3kg of activated carbon, mixing and stirring for 0.5h, and pressurizing the mixture through the plate-and-frame filter until the filtrate is yellow clear transparent liquid.
Firstly, a composite tube type membrane equal permeable membrane is adopted for separation, wherein the membrane inlet pressure is 1.6Bar, the membrane outlet pressure is 0.8Bar, the temperature is 55 ℃, the concentrated solution flow is 300LPM, and the clear solution flow is 79 LPM.
Then a nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules: the membrane inlet pressure is 12Bar, the membrane outlet pressure is 10Bar, the concentrated solution flow is 23LPM at the first 5 minutes, the clear solution flow is more than 100LPM, and the filter pressure is 0.35 MPA.
7. And (3) concentrating under reduced pressure: and concentrating the enzymolysis liquid to 50% of solid content by using a double-effect concentrator.
8. Centrifugal spray drying: controlling the temperature of the feed inlet at 175 ℃, the temperature of the air outlet at 95 ℃ and the feeding speed at 200r/min, and spray-drying the concentrated filtrate to obtain the small-molecule sea cucumber intestinal peptide powder.
In the invention, after the sea cucumber intestines are pretreated, blanching treatment is carried out, so that further desalting can be realized; then, extracting the sea cucumber intestines at high temperature by utilizing different heat stability of protein peptides and non-peptide substances, and shearing the peptides by using the extracting solution through different enzyme reagents to obtain uniform enzyme shearing solution; and subsequently, the enzyme shearing liquid is concentrated and filtered by different permeable membranes to obtain small peptides with molecular weights within a certain range, and the small peptides within the molecular weight range account for more than 95% of the total peptides, so that the absorption efficiency can be greatly increased, and a good basis can be provided for the research on the efficacy of peptide products with different molecular weight ranges.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The method for efficiently extracting protein peptide from sea cucumber intestines is characterized by comprising the following steps: comprises the following steps;
s1, pretreatment of raw materials: peeling and cleaning sea cucumber to obtain sea cucumber intestines;
s2, blanching sea cucumber intestines;
s3, extracting at high temperature to obtain an extracting solution;
s4, enzymolysis: adding an enzyme preparation into the extracting solution to obtain an enzymolysis solution;
s5, cooling the enzymolysis liquid, and performing deodorization and decoloration treatment;
s6, membrane filtration to obtain a purified solution;
s7, concentrating the purified solution to obtain a purified concentrated solution;
s8, drying and purifying the concentrated solution to obtain the sea cucumber intestinal peptide powder.
2. The method for efficiently extracting protein peptides from sea cucumber intestines as claimed in claim 1, wherein: the S3 specifically includes:
transferring the blanched sea cucumber intestines to an extraction tank, adding purified water with the weight being 3-4 times that of the sea cucumber intestines into the extraction tank, and heating to 95 ℃;
the extraction was continued for 1 hour with continuous stirring during the extraction.
3. The method for efficiently extracting protein peptides from sea cucumber intestines as claimed in claim 1 or 2, wherein: the S4 specifically includes:
adding the extract into an enzymolysis tank, heating to 60 ℃, adjusting to the optimal reaction condition of the enzyme, and then sequentially adding a first enzyme preparation, a second enzyme preparation and a third enzyme preparation;
and after the enzymolysis reaction is finished, heating to inactivate the enzyme to obtain an enzymolysis liquid.
4. The method for extracting protein peptide from sea cucumber intestine with high efficiency as claimed in claim 3, wherein: the first enzyme preparation and the second enzyme preparation are both endoproteases, and the third enzyme preparation is an exoprotease.
5. The method for extracting protein peptide from sea cucumber intestine with high efficiency as claimed in claim 4, wherein: the first enzyme preparation is neutral protease, the second enzyme preparation is alkaline protease, and the third enzyme preparation is debitterizing protease.
6. The method for efficiently extracting protein peptides from sea cucumber intestines as claimed in claim 4 or 5, wherein: the weight ratio of the first enzyme preparation to the sea cucumber intestines is 0.2%, the weight ratio of the second enzyme preparation to the sea cucumber intestines is 0.2%, and the weight ratio of the third enzyme preparation to the sea cucumber intestines is 0.05%.
7. The method for extracting protein peptide from sea cucumber intestine with high efficiency as claimed in claim 6, wherein: adding the first enzyme preparation, stirring for 15min, adding the second enzyme preparation, shearing for 3h, adding the third enzyme preparation, and shearing for 2 h.
8. The method for efficiently extracting protein peptides from sea cucumber intestines as claimed in claim 1, 4, 5 or 7, wherein: said S6 includes;
s6.1, tube membrane filtration:
s6.2, filtering by a nanofiltration membrane: the separation of 150-1000Dal organic small molecules is carried out.
9. The method for efficiently extracting protein peptides from sea cucumber intestines as claimed in claim 8, wherein: and S7, performing reduced pressure and temperature rise concentration on the purified solution by using a double-effect concentrator.
10. The method for extracting protein peptides from sea cucumber intestines with high efficiency as claimed in claim 1 or 9, wherein: the drying method of the S8 is a centrifugal spray drying method.
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| CN113912640A (en) * | 2021-10-25 | 2022-01-11 | 威海市宇王集团海洋生物工程有限公司 | Method for extracting sea cucumber lecithin from sea cucumber intestines |
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| CN109628540A (en) * | 2019-01-15 | 2019-04-16 | 威海市宇王集团海洋生物工程有限公司 | A method of it is decolourized using membrane filtration technique to sea cucumber Gly-His-Lys |
| CN109943616A (en) * | 2019-04-18 | 2019-06-28 | 威海市宇王集团海洋生物工程有限公司 | A method of the abstraction function albumen from giant salamander |
| CN110195090A (en) * | 2019-05-15 | 2019-09-03 | 池州市月亮湾生物科技有限公司 | The preparation method and gained sea cucumber intestine ovum polypeptide powder of sea cucumber intestine ovum polypeptide powder |
| CN111808185A (en) * | 2020-07-08 | 2020-10-23 | 威海市宇王集团海洋生物工程有限公司 | Method for extracting elastin peptide from bovine cartilage |
| CN111802505A (en) * | 2020-07-28 | 2020-10-23 | 威海市宇王集团海洋生物工程有限公司 | Industrialized abalone peptide extraction method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113912640A (en) * | 2021-10-25 | 2022-01-11 | 威海市宇王集团海洋生物工程有限公司 | Method for extracting sea cucumber lecithin from sea cucumber intestines |
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