CN117205306B - Collagen extract, composition, preparation method and application - Google Patents
Collagen extract, composition, preparation method and application Download PDFInfo
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- CN117205306B CN117205306B CN202310951207.6A CN202310951207A CN117205306B CN 117205306 B CN117205306 B CN 117205306B CN 202310951207 A CN202310951207 A CN 202310951207A CN 117205306 B CN117205306 B CN 117205306B
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Abstract
The invention discloses a collagen extract, a composition, a preparation method and application thereof, and belongs to the technical field of collagen function research. Aiming at the problem that the prior art lacks of using the collagen extract for treating and protecting gastrointestinal diseases, the invention provides a collagen extract, a composition, a preparation method and application; then carrying out enzymolysis on the defatted bovine Achilles tendon to obtain an enzymolysis solution; and then, carrying out centrifugal treatment and dialysis treatment on the obtained enzymolysis liquid to obtain a stock solution containing the collagen extract. The collagen extract of the invention can be used for the regression of gastrointestinal tract inflammation and the auxiliary protection of gastrointestinal mucosa injury.
Description
Technical Field
The invention belongs to the technical field of collagen function research, and particularly relates to a collagen extract, a collagen composition, a collagen preparation method and an application of the collagen extract.
Background
At present, there is a general concern in society that alcohol causes damage to the liver, and in fact, excessive intake of alcohol causes damage not only to the liver but also directly and indirectly to the gastrointestinal tract. The gastrointestinal tract is an important organ in the human digestive system and occupies an irreplaceable important place in the metabolism of body substances. The inner wall of the gastrointestinal tract is provided with a layer of mucosal tissue which is taken as a natural barrier, and has great significance for protecting the gastrointestinal tract. However, the mucous membrane itself is fragile and is easily damaged by external factors such as environment, diet, emotion change, and life rhythm. If damaged for a long time, acute and chronic gastroenteritis, ulcer and even tumor can be caused. Taking alcohol as an example, alcohol can be dissolved in lipid to enter the stomach wall, hydrogen ions in gastric juice permeate back into mucosal epithelial cells, so that histamine, 5-hydroxytryptamine and heparin are released, submucosal artery short circuit is caused, capillary pressure is increased, permeability is increased, and gastric mucosal injuries such as mucosal congestion, red blood cell exudation, gastric mucosal erosion bleeding and the like occur. The effect of alcohol on the small intestine is mainly to cause swelling of the duodenal and jejunal villi, disappearance of surface folds, and decrease of microvilli density of mucosal epithelial cells.
Collagen is the most important component of extracellular matrix, and is a structural protein constituting animal supporting tissues, and is widely distributed in the skin, hair, joints, tendons, cartilage, etc. of human body. Meanwhile, collagen also shows good digestibility, is the most important component in extracellular matrix, is structural protein forming animal supporting tissues, and is widely distributed in the places such as skin, hair, joints, tendons, cartilage and the like of a human body.
Collagen is currently added to commercial products for whitening and anti-aging. Thus, exploring more functions of collagen is a problem that those skilled in the art are urgent to solve.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a collagen extract, a composition, a preparation method and application.
In one aspect, the invention provides the use of a collagen extract in the preparation of a composition for the regression of gastrointestinal inflammation and the assisted protection of gastrointestinal mucosal lesions.
Further, the collagen extract is prepared by the following method, which comprises the following steps:
step A: degreasing the beef achilles tendon to obtain the degreased beef achilles tendon;
And (B) step (B): carrying out enzymolysis on the defatted bovine Achilles tendon to obtain an enzymolysis solution;
step C: centrifuging the obtained enzymolysis liquid;
Step D: and C, dialyzing the enzymatic hydrolysate after the centrifugal treatment obtained in the step C to obtain a collagen extract stock solution.
After the technical scheme is adopted, the type I collagen is the most ubiquitous collagen, accounts for 90 percent of the total amount of human collagen and accounts for approximately 20 percent of the total protein mass, is mainly present in skin, bones and tendons, mainly plays a role in supporting and retaining water, is high in content and pure in beef achilles tendon, is a macromolecule, and is matched with an enzymolysis collagen extraction method, so that the triple helix structure of the collagen is ensured, the protein activity such as the reactivity of side chain groups and the effect between acid and alkali salts is ensured, and when a stimulus contacts the collagen, the stimulus interacts with the collagen, and further consumes and coats the stimulus, so that a protective film is formed on gastric mucosa in such a way as to reduce the protection of gastrointestinal tract and mucosa, and the gastrointestinal tract inflammation is resolved and the gastrointestinal mucosa injury is assisted and protected.
Further, in the step a, the bovine achilles tendon is fresh bovine achilles tendon, and soft tissue on the bovine achilles tendon is removed before degreasing the bovine achilles tendon, and the bovine achilles tendon after removing the soft tissue is cleaned and minced.
Further, in the step A, when the beef achilles tendon is degreased, the beef achilles tendon is firstly soaked in NaHCO 3 solution with the concentration of 1-5% for 10-30 minutes, and purified water is added into the beef achilles tendon after the soaking is finished and the beef achilles tendon is stirred for 10-30 minutes.
Further, during enzymolysis in the step B, acetic acid solution with the concentration of 0.4-0.6 mol/L is firstly used for soaking the bovine achilles tendon for 1-2 hours, then pepsin is added for enzymolysis of the bovine achilles tendon, and the mass ratio of the pepsin to the bovine achilles tendon is 1: 20-1: 10, the enzymolysis time is 1-36h.
After the technical scheme is adopted, the acetic acid solution can cause collagen to unwind, which is beneficial to pepsin to act; meanwhile, the pepsin has the highest activity under the acidic condition, and the condition for extracting the collagen by the pepsin is mild, so that the terminal peptide of the collagen fiber protein can be hydrolyzed, the yield of the collagen protein is improved, the triple helix structure of the collagen protein can not be damaged, and the excellent physical and biochemical properties of the collagen protein can be maintained.
Further, in the step D, dialysis is performed by using a dialysis bag, and the molecular weight cut-off of the dialysis bag is 1K-5kDa
In one aspect, the present invention provides a collagen extract for use in the resolution of gastrointestinal inflammation and the assisted protection of gastrointestinal mucosal lesions.
In one aspect, the present invention provides a method for preparing a collagen extract, comprising the steps of:
step A: degreasing the beef achilles tendon to obtain the degreased beef achilles tendon;
And (B) step (B): carrying out enzymolysis on the defatted bovine Achilles tendon to obtain an enzymolysis solution;
step C: centrifuging the obtained enzymolysis liquid;
Step D: and C, dialyzing the enzymatic hydrolysate after the centrifugal treatment obtained in the step C to obtain a collagen extract stock solution.
Further, during enzymolysis in the step B, acetic acid solution with the concentration of 0.4-0.6 mol/L is firstly used for soaking the bovine achilles tendon for 1-2 hours, then pepsin is added for enzymolysis of the bovine achilles tendon, and the mass ratio of the pepsin to the bovine achilles tendon is 1: 20-1: 10, the enzymolysis time is 1-36h;
And D, dialyzing by using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is 1K-5kDa.
In one aspect, the invention provides a collagen extract composition, which comprises the following components in parts by weight: 1.0-5.0 parts of collagen extract, 0.5-3.5 parts of vitamin C, 0.05-1.0 parts of potassium sorbate, 0.01-0.05 parts of citric acid and the balance of water, wherein the composition is used for the regression of gastrointestinal tract inflammation and the auxiliary protection of gastrointestinal mucosa injury.
After the technical scheme is adopted, the vitamin C is a nutrition enhancer, the citric acid is an acidity regulator, and the potassium sorbate is a preservative.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. according to the invention, the collagen of the bovine Achilles tendon is extracted by adopting a process of combining acetic acid and pepsin, and the collagen extracted by the method can keep a complete triple helix structure.
2. The collagen of the beef achilles tendon prepared by the method has high purity, no fishy smell, uniform liquid body, no precipitate and no bitter taste.
3. The raw materials in the composition are easy to obtain, and the cost is low.
4. The composition has a simple formula and has the effects of eliminating gastrointestinal inflammation and assisting in protecting gastrointestinal mucosa injury.
Drawings
FIG. 1 is a typical graph of the number of neutrophils in the intestinal tract of zebra fish after sample treatment according to the invention, wherein the dashed area is the gastrointestinal tract of the analysis area.
FIG. 2 is a bar graph of the number of neutrophils in the intestinal tract of zebra fish after sample treatment in accordance with the present invention.
Fig. 3 is a schematic diagram showing the analysis of the intestinal lumen area of zebra fish after sample treatment according to the present invention, wherein the dashed area is the gastrointestinal tract of the analysis area.
FIG. 4 is a representative graph of the intestinal lumen area of a zebra fish after sample treatment in accordance with the present invention, wherein the dashed area is the gastrointestinal tract of the analysis area.
FIG. 5 is a bar graph of the intestinal lumen area of zebra fish after sample treatment in accordance with the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments.
Example 1
A method for preparing a collagen extract, comprising the steps of:
And A, removing soft tissues of fresh bovine Achilles tendon, cleaning with purified water, cutting the bovine Achilles tendon into small sections (about 3 cm), starting a meat grinder, and mincing the bovine Achilles tendon one by one. Putting 1% NaHCO 3 solution into a degreasing tank with a filter screen, putting minced beef achilles tendon into a degreasing tank containing 1% NaHCO 3 solution, soaking for 30min, and then opening a degreasing tank drain valve to drain degreasing liquid as much as possible. And pouring the purified water into a degreasing tank, stirring for 30min, and then opening a drainage valve of the degreasing tank to drain the purified water.
Transferring the beef achilles tendon treated in the step A into a reaction kettle, soaking for 1 hour by using 0.5 mol/L acetic acid solution, and then adding the beef achilles tendon into the reaction kettle according to the following formula of 1:15, pepsin is added in proportion, and after complete dissolution, a stirrer is started and slowly stirred for 24 hours.
And C, transferring the enzymolysis liquid into a decanter centrifuge after the enzymolysis reaction in the step B is finished, centrifuging for 30min at 8000r/min, and repeating the operation until the separation of the enzymolysis liquid is finished.
And D, packaging the enzymolysis liquid obtained in the step C into dialysis bags with the molecular weight cut-off of 1K Da, then placing the dialysis bags into a dialysis tank, injecting purified water to the designated liquid level of the dialysis tank, covering a dialysis tank cover, and obtaining the stock solution containing the collagen extract after dialysis is completed.
Example 2
A method for preparing a collagen extract, comprising the steps of:
And A, removing soft tissues of fresh bovine Achilles tendon, cleaning with purified water, cutting the bovine Achilles tendon into small sections (about 3 cm), starting a meat grinder, and mincing the bovine Achilles tendon one by one. Putting 1% NaHCO 3 solution into a degreasing tank with a filter screen, putting minced beef achilles tendon into a degreasing tank containing 1% NaHCO 3 solution, soaking for 30min, and then opening a degreasing tank drain valve to drain degreasing liquid as much as possible. And pouring the purified water into a degreasing tank, stirring for 30min, and then opening a drainage valve of the degreasing tank to drain the purified water.
Transferring the beef achilles tendon treated in the step A into a reaction kettle, soaking for 1 hour by using 0.6 mol/L acetic acid solution, and then adding the beef achilles tendon into the reaction kettle according to the following formula of 1:10, pepsin is added in proportion, and after complete dissolution, a stirrer is started and slowly stirred for 24 hours.
And C, transferring the enzymolysis liquid into a decanter centrifuge after the enzymolysis reaction in the step B is finished, centrifuging for 30min at 8000r/min, and repeating the operation until the separation of the enzymolysis liquid is finished.
And D, packaging the enzymolysis liquid obtained in the step C into dialysis bags with the molecular weight cut-off of 1K Da, then placing the dialysis bags into a dialysis tank, injecting purified water to the designated liquid level of the dialysis tank, covering a dialysis tank cover, and obtaining the stock solution containing the collagen extract after dialysis is completed.
Example 3
A method for preparing a collagen extract, comprising the steps of:
And A, removing soft tissues of fresh bovine Achilles tendon, cleaning with purified water, cutting the bovine Achilles tendon into small sections (about 3 cm), starting a meat grinder, and mincing the bovine Achilles tendon one by one. Putting 1% NaHCO 3 solution into a degreasing tank with a filter screen, putting minced beef achilles tendon into a degreasing tank containing 1% NaHCO 3 solution, soaking for 30min, and then opening a degreasing tank drain valve to drain degreasing liquid as much as possible. And pouring the purified water into a degreasing tank, stirring for 30min, and then opening a drainage valve of the degreasing tank to drain the purified water.
Transferring the beef achilles tendon treated in the step A into a reaction kettle, soaking for 1 hour by using 0.4 mol/L acetic acid solution, and then adding the beef achilles tendon into the reaction kettle according to the following formula of 1:20, and after the pepsin is completely dissolved, starting a stirrer, and slowly stirring for 24 hours.
And C, transferring the enzymolysis liquid into a decanter centrifuge after the enzymolysis reaction in the step B is finished, centrifuging for 30min at 8000r/min, and repeating the operation until the separation of the enzymolysis liquid is finished.
And D, packaging the enzymolysis liquid obtained in the step C into dialysis bags with the molecular weight cut-off of 1K Da, then placing the dialysis bags into a dialysis tank, injecting purified water to the designated liquid level of the dialysis tank, covering a dialysis tank cover, and obtaining the stock solution containing the collagen extract after dialysis is completed.
Example 4
A composition comprising the collagen extract prepared in example 1, the composition comprising the following components in parts by weight: 3 parts of collagen extract, 2 parts of vitamin C, 0.5 part of potassium sorbate, 0.02 part of citric acid and the balance of water;
the preparation method of the composition comprises the following steps:
The collagen stock solution obtained in example 1 was diluted to 10mg/mL, and then the collagen solution, vitamin C, potassium sorbate, citric acid and water were added together into a compounding kettle according to the formulation requirements and stirred to obtain the final composition.
Zebra fish lack individual organ stomachs, but express several digestive enzymes in their bodies that are functionally identical to mammalian gastric markers, including renin, triphosphatase, several cathepsins, and lipase. The intestinal structures of zebra fish are similar to those of mammals and are divided into the foregut, midgut and hindgut. The foregut and midgut are similar to the small intestine, mainly involved in the absorption of nutrients; the hindgut is primarily involved in water absorption, similar to the large intestine. The intestinal tract of zebra fish is divided into mucosal (epithelial, lamina propria), muscular and serosal layers, lacking submucosa in comparison to humans. Intestinal cells of zebra fish, consistent with mammals, exist in epithelial cells, endocrine cells, goblet cells, neuronal cells, and interstitial cells of Caja, but lack paneth cells and end-of-year lymphoid nodules-associated epithelial cells. Therefore, the gastrointestinal tract of zebra fish is highly similar to human beings, and the evaluation of the gastrointestinal tract protection effect developed on zebra fish is highly predictive. Therefore, zebra fish is selected as an experimental animal for evaluating the effect of the composition.
The used wild type AB strain zebra fish or transgenic neutrophil fluorescent zebra fish is fed into water for fish culture at 28 ℃, 200mg of instant sea salt is added into each 1L of reverse osmosis water, the conductivity is 450-550 mu S/cm, the pH is 6.5-8.5, and the hardness is 50-100 mg/L CaCO3.
The transgenic neutrophil green fluorescent MPX strain zebra fish is produced in natural pairing mating propagation mode. Zebra fish aged 3 days after fertilization (3 dpf) were used for maximum detection concentration (MTC) determination of efficacy of auxiliary protection of gastrointestinal mucosal damage of the composition and efficacy evaluation thereof.
MTC assay experiments on the composition prepared in example 2
The experimental procedure was as follows: the 3dpf transgenic neutrophil green fluorescent MPX strain zebra fish was randomly selected in 6-well plates, and 30 zebra fish were treated in each well (experimental group). Each of the remaining experimental groups, except the normal control group, was water-soluble to trinitrobenzenesulfonic acid (TNBS) to establish a zebra fish gastrointestinal mucosal injury model. After 2 days of treatment at 28 ℃, TNBS was removed and the composition was given in water-soluble form (concentrations are shown in table 1), while setting the normal control group and the model control group to a capacity of 3mL per well. After further treatment at 28 ℃ for 2 days, the composition was assayed for MTC in model zebra fish.
Table 1 results of experiments were searched for concentrations of the composition for auxiliary protection against gastrointestinal mucosal injury (n=30)
Under the experimental conditions, the auxiliary protection effect MTC of the gastrointestinal mucosa injury of the composition is 125 mu L/mL.
Evaluation experiment of gastrointestinal inflammation resolution efficacy of the composition prepared in example 2
The experimental procedure was as follows: the 3dpf transgenic neutrophil green fluorescent MPX strain zebra fish was randomly selected in 6-well plates, and 30 zebra fish were treated in each well (experimental group). Each experimental group except the normal control group was given TNBS in a water-soluble manner to establish a zebra fish gastrointestinal mucosa injury model. After 2 days of treatment at 28 ℃, TNBS was removed, the composition was given in water (concentration see table 2), and the positive control prednisone 15.0 μg/mL, while setting the normal control group and the model control group to a capacity of 3mL per well. After the treatment is continued for 2 days at 28 ℃,10 zebra fish are randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data are collected by NIS-ELEMENTS D3.20.20 advanced image processing software, the number of intestinal neutrophils of the zebra fish is analyzed, and the efficacy of eliminating the gastrointestinal inflammation of the composition is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.
Table 2 results of composition gastrointestinal inflammation resolution efficacy evaluation experiment (n=10)
P <0.05, p <0.01, p <0.001 compared to model control group
When the gastrointestinal mucosa is damaged, neutrophils as first-line immune cells are recruited from the circulatory system to the inflammatory sites of the gastrointestinal tract, and clear pathogens, which are manifested as neutrophil increase in the gastrointestinal tract. Samples with a regressive effect on gastrointestinal inflammation will have reduced neutrophils in the gastrointestinal tract, which is manifested by reduced green fluorescent particles in the yellow region. As can be seen from table 2, fig. 1 and fig. 2, the composition has gastrointestinal inflammation resolution efficacy.
Evaluation test of gastrointestinal mucosal injury auxiliary protection efficacy of the composition prepared in example 2
The experimental procedure was as follows: the 3dpf transgenic neutrophil green fluorescent MPX strain zebra fish was randomly selected in 6-well plates, and 30 zebra fish were treated in each well (experimental group). Each experimental group except the normal control group was given TNBS in a water-soluble manner to establish a zebra fish gastrointestinal mucosa injury model. After 2 days of treatment at 28 ℃, TNBS was removed, the composition was given in water (concentration see table 3), the positive control prednisone 15.0 μg/mL, while the normal control and model control were set to a capacity of 3mL per well. After the treatment is continued for 2 days at 28 ℃,10 zebra fish are randomly selected from each experimental group, photographed under an dissecting microscope, analyzed and data are collected by NIS-E LEMENTS D3.20.20 advanced image processing software, the intestinal cavity area of the zebra fish is analyzed, and the auxiliary protection effect of the gastrointestinal mucosa injury of the composition is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.
Table 3 results of the evaluation of the efficacy of the composition for the auxiliary protection against gastrointestinal mucosal lesions (n=10)
P <0.01, p <0.001 compared to model control group
When the gastrointestinal mucosa is damaged, the gastrointestinal myolayer and the intestinal myonerve plexus are involved, the tension of the gastrointestinal wall is reduced, the peristalsis disappears, gastrointestinal contents and gas accumulate, and the gastrointestinal cavity is expanded, so that the gastrointestinal area is increased. Samples with a gastrointestinal mucosal lesion repair effect will reduce the gastrointestinal area, which is shown by the reduced area of the dashed area in the example of fig. 1. As can be seen from table 3, fig. 4 and fig. 5, the composition has an auxiliary protection effect on gastrointestinal mucosa injury, and the collagen prepared in examples 2 to 3 has a similar effect after the effect is detected by the same method as in example 4.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
Claims (3)
1. Use of a collagen extract for the preparation of a composition characterized in that: the composition comprises the following components in parts by weight: 1.0-5.0 parts of collagen extract, 0.5-3.5 parts of vitamin C, 0.05-1.0 parts of potassium sorbate, 0.01-0.05 parts of citric acid and the balance of water, wherein the collagen extract is I-type collagen with a triple helix structure, the composition is used for the regression of intestinal inflammation and the auxiliary protection of intestinal mucosa injury, and the extraction method of the collagen extract comprises the following steps:
Step A: degreasing the beef achilles tendon to obtain the degreased beef achilles tendon; when the beef achilles tendon is degreased, the beef achilles tendon is firstly soaked in NaHCO 3 solution with the concentration of 1-5% for 10-30 minutes, and purified water is added into the beef achilles tendon after the soaking is finished and the beef achilles tendon is stirred for 10-30 minutes;
And (B) step (B): and (3) carrying out enzymolysis on the defatted bovine achilles tendon to obtain an enzymolysis solution, wherein during enzymolysis, acetic acid solution with the concentration of 0.5mol/L is firstly used for soaking the bovine achilles tendon for 1-2h, then pepsin is added for carrying out enzymolysis on the bovine achilles tendon, and the mass ratio of the pepsin to the bovine achilles tendon is 1: 20-1: 10, the enzymolysis time is 1-36h;
step C: centrifuging the obtained enzymolysis liquid;
Step D: and C, dialyzing the enzymatic hydrolysate after the centrifugal treatment obtained in the step C to obtain a collagen extract stock solution.
2. Use of a collagen extract according to claim 1 for the preparation of a composition, characterized in that: in the step A, the bovine achilles tendon is fresh, soft tissues on the bovine achilles tendon are removed before degreasing the bovine achilles tendon, and the bovine achilles tendon after the soft tissues are removed is cleaned and minced.
3. Use of a collagen extract according to claim 1 for the preparation of a composition, characterized in that: and D, dialyzing by using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is 1K-5kDa.
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