阅读说明：本技术 一种鱼鳔胶原蛋白的提取方法 (Method for extracting collagen from swimming bladder ) 是由 郭红星 周尽学 于 2021-04-13 设计创作，主要内容包括：本发明涉及蛋白提取技术领域,尤其涉及一种胶原蛋白的提取方法。该方法通过酸-酶提取法提取鱼鳔胶原蛋白,可有效缩短胶原蛋白的提取时间,以及保证胶原蛋白中与活性相关的结构的完整性,作用条件温和,材料无毒,无污染,采用鱼鳔提取胶原蛋白,鱼鳔胶原蛋白可应用于医学中人工软骨组织凝胶用来修复骨组织的缺损和修复,应用于组织细胞的培养,应用于止血和伤口修复,应用于制药和保健食品,应用于制作纳米发电机,还具有抗疲劳功效。(The invention relates to the technical field of protein extraction, in particular to a collagen extraction method. The method extracts the swimming bladder collagen by the acid-enzyme extraction method, can effectively shorten the extraction time of the collagen, ensures the integrity of the structure related to the activity in the collagen, has mild action condition, non-toxic materials and no pollution, adopts the swimming bladder to extract the collagen, and can be applied to the defect repair and repair of bone tissues by artificial cartilage tissue gel in medicine, the culture of tissue cells, the hemostasis and wound repair, the pharmacy and health-care food, the manufacture of a nano generator and the anti-fatigue effect.)

1. A method for extracting swimming bladder collagen is characterized by comprising the following steps:

extracting swim bladder with 0.1mol/L acetic acid for 1h to obtain extractive solution;

adding 0.1% -0.2% of pepsin or trypsin into the extracting solution for enzymolysis for 1-2 h, inactivating enzyme, centrifuging, and collecting supernatant;

and purifying the supernatant by chromatography to obtain the swimming bladder collagen.

2. The extraction method as claimed in claim 1, wherein the swimming bladder further comprises a pretreatment step before the acetic acid extraction, wherein the pretreatment step comprises decolorization, deproteinization and degreasing treatment.

3. The extraction method according to claim 2, wherein the decolorization and deproteinization treatment is: soaking with 0.1m/L sodium hydroxide for 24 h.

4. The extraction method according to claim 2, wherein the degreasing treatment is: soaking in 10% butanol for 12 hr.

5. The extraction method as claimed in claim 2, wherein the pretreatment further comprises a step of removing impurities inside the swimming bladder by ultrasonic treatment for 30-60 min.

6. The extraction method according to claim 1, wherein the enzymolysis time is 1 h.

7. The extraction process according to claim 1, characterized in that the detection wavelength of the chromatographic purification is 230 nm.

8. Swim bladder collagen obtained by the extraction method of any one of claims 1 to 7.

9. The swimming bladder collagen of claim 8, wherein the glycine content is 1/3 of the total mass of the swimming bladder collagen.

Technical Field

The invention relates to the technical field of protein extraction, in particular to an extraction method of swimming bladder collagen.

Background

The swim bladder has the function of assisting the fish to ascend and descend in water and is used stably, is positioned in the center of the fish body and can keep the center of gravity downward; the fish abdominal cavity can generate enough space to protect internal organs of the fish, and the internal organs are prevented from being damaged due to overlarge water pressure; in the pulmonids and the finfish, swim bladders can be used as auxiliary respiratory organs; in an anoxic environment, the swim bladder can be used as an auxiliary respiratory organ to provide oxygen for the fish. At present, research reports that collagen is extracted from fish byproducts such as fish scales, fish skins, fish bones and viscera and the like and is applied are reported, however, fish scales have fishy smell, fish skins containing more calcium impurities have colors and have lower thermal denaturation temperature, the application of the fish scales is limited to a certain extent, swim bladders become another important substitute raw material rich in collagen, the swim bladders are balance and proportion regulating organs of fish and are used for pressure sensing, sounding and breathing assistance, and are also named as fish glue, fish bubbles, fish white, fish maws and pressed cells, so that the fish maws are famous and precious dishes for cooking, can nourish blood, enrich blood, nourish yin, tonify kidney, eliminate fatigue, resist diseases and the like, and have extremely high edible and medicinal values.

At present, the utilization rate of swimming bladders is low, most of the swimming bladders are discarded in the form of waste, resources are wasted, the environment is polluted, the swimming bladders cannot be utilized to produce better collagen, and different extraction methods have great influence on the activity and the production efficiency of the collagen.

Disclosure of Invention

In view of the above, the invention provides a method for extracting swimming bladder collagen. The swimming bladder collagen prepared by the method provided by the invention can keep the triple helix structure of the collagen to the maximum extent and keep the natural activity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for extracting swimming bladder collagen, which comprises the following steps:

extracting swim bladder with 0.1mol/L acetic acid for 1h to obtain extractive solution;

adding 0.1% -0.2% of pepsin or trypsin into the extracting solution for enzymolysis for 1-2 h, inactivating enzyme, centrifuging, and collecting supernatant;

and purifying the supernatant by chromatography to obtain the swimming bladder collagen.

Preferably, the swimming bladder further comprises a pretreatment step before the acetic acid extraction, wherein the pretreatment step comprises decolorization, deproteinization and degreasing treatment.

Specifically, the decoloring and deproteinizing treatment comprises the following steps: soaking with 0.1m/L sodium hydroxide for 24 h.

The degreasing treatment comprises the following steps: soaking in 10% butanol for 12 hr.

Preferably, the pretreatment further comprises the step of removing impurities in the swim bladder by ultrasonic treatment for 30-60 min.

Preferably, the enzymolysis time is 1 h.

Preferably, the detection wavelength of the chromatographic purification is 230 nm.

The invention also provides the swimming bladder collagen prepared by the extraction method.

In the fish glue protein prepared by the extraction method, the glycine content accounts for 1/3 of the total mass of the fish glue collagen.

The invention provides a method for extracting swimming bladder collagen, which comprises the following steps: extracting swim bladder with 0.1mol/L acetic acid for 1h to obtain extractive solution; adding 0.1% -0.2% of pepsin or trypsin into the extracting solution for enzymolysis for 1-2 h, inactivating enzyme, centrifuging, and collecting supernatant; and purifying the supernatant by chromatography to obtain the swimming bladder collagen. The invention has the following beneficial effects:

1. the swimming bladder collagen is extracted by the acid-enzyme extraction method, and the electropherogram analysis shows that the amide band of the extracted collagen is clear and has no impurity band, so the acid method can keep the triple helix structure of the swimming bladder collagen to the maximum degree and keep the natural activity, and the method is particularly suitable for preparing biomedical materials.

2. According to the method, collagen is extracted from swimming bladder, the content and activity of the collagen are determined, and compared with mammals, the swimming bladder collagen forms high tensile strength through covalent crosslinking effects such as hydrogen bonds of glycine triad, the structural stability of the swimming bladder collagen is maintained, meanwhile, the metastable state of proline provides structural flexibility, the collagen is endowed with certain flexibility, the swimming bladder collagen content is equivalent to that of fish skin, and swimming bladder collagen is obviously higher than collagen fibrils such as fishbone, fish scale, fish fin, fish meat, visceral calf and sturgeon skin, the forming speed is slow, the stability is poor, the swimming bladder collagen is suitable in viscosity, and the swimming bladder collagen has extremely fast fibril forming capability, so that the denaturation temperature is obviously improved.

3. The invention extracts collagen by adopting swim bladder, the swim bladder collagen can be applied to artificial cartilage tissue gel in medicine for repairing the defect and the repair of bone tissue, can be applied to the culture of tissue cells, is applied to hemostasis and wound repair, is applied to pharmacy and health food, is applied to manufacturing a nano generator, and also has the effects of skin care and fatigue resistance.

Drawings

FIG. 1 is a schematic view of the process for extracting collagen from swimming bladder.

Detailed Description

The invention provides a method for extracting swimming bladder collagen. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1

Sp 1: pretreatment of swim bladders: the pretreatment of the swimming bladder comprises the steps of removing pigments, hybrid proteins and lipids in the swimming bladder and impurities in the swimming bladder; soaking in 0.1m/L sodium hydroxide for 24 hr to remove pigment and foreign protein, soaking in 10% butanol for 12 hr to degrease lipid, and removing impurities in air bladder by ultrasonic treatment for 60 min.

Sp 2: extracting collagen in the swim bladder: the acid-enzyme method comprises the following specific steps: soaking the pretreated swimming bladder in 0.1mol/L acetic acid for 1h to obtain an extracting solution; adding 0.2% pepsin into the extract for enzymolysis for 1h, inactivating enzyme, centrifuging, and collecting supernatant;

sp 3: purifying the supernatant by chromatography to obtain swim bladder collagen; wherein the detection wavelength of chromatographic separation is 230 nm.

The result shows that the acid-enzyme method can keep the triple helix structure of the swimming bladder collagen to the maximum extent and keep the natural activity, is particularly suitable for preparing biomedical materials, can effectively shorten the extraction time of the collagen by combining the acid method and the enzyme method, ensures the integrity of the structure related to the activity in the collagen, has mild action condition, and has no toxicity and no pollution.

The characteristic absorption peak of hydroxyproline can be seen at 230nm by adopting infrared spectrum analysis, the glycine content accounts for 1/3 of the total amount, the proline and the histidine are sequentially contained, and the difference between the characteristics of the swimming bladder collagen and the mammal collagen can be analyzed according to the content ratio of each amino acid.

Collagen is extracted through swim bladders, the content and the activity of the collagen are determined, compared with mammals, the swim bladder collagen forms high tensile strength through covalent crosslinking effects such as hydrogen bonds of glycine triad, the structural stability of the swim bladder collagen is maintained, meanwhile, the metastable state of proline provides structural flexibility, the collagen is endowed with certain flexibility, the swim bladder collagen content is equivalent to that of fish skin, swim bladders are obviously higher than collagen fibrils such as fishbone, fish scales, fish fins, fish meat, visceral cowhide, sturgeon skin and the like, the forming speed is slow, the stability is poor, the swim bladder collagen is suitable due to the viscosity, and the swim bladder collagen has extremely fast fibril forming capability, so that the denaturation temperature is obviously improved.

Example 2

Sp 1: pretreatment of swim bladders: the pretreatment of the swimming bladder comprises the steps of removing pigments, hybrid proteins and lipids in the swimming bladder and impurities in the swimming bladder; soaking in 0.1m/L sodium hydroxide for 24 hr to remove pigment and foreign protein, soaking in 10% butanol for 12 hr to degrease lipid, and removing impurities in air bladder by ultrasonic treatment for 30 min.

Sp 2: extracting collagen in the swim bladder: the acid-enzyme method comprises the following specific steps: soaking the pretreated swimming bladder in 0.1mol/L acetic acid for 1h to obtain an extracting solution; adding 0.1% pepsin into the extract for enzymolysis for 1h, inactivating enzyme, centrifuging, and collecting supernatant;

sp 3: purifying the supernatant by chromatography to obtain swim bladder collagen; wherein the detection wavelength of chromatographic separation is 230 nm.

The result shows that the acid-enzyme method can keep the triple helix structure of the swimming bladder collagen to the maximum extent and keep the natural activity, is particularly suitable for preparing biomedical materials, can effectively shorten the extraction time of the collagen by combining the acid method and the enzyme method, ensures the integrity of the structure related to the activity in the collagen, has mild action condition, and has no toxicity and no pollution.

The characteristic absorption peak of hydroxyproline can be seen at 230nm by adopting infrared spectrum analysis, the glycine content accounts for 1/3 of the total amount, the proline and the histidine are sequentially contained, and the difference between the characteristics of the swimming bladder collagen and the mammal collagen can be analyzed according to the content ratio of each amino acid.

Collagen is extracted through swim bladders, the content and the activity of the collagen are determined, compared with mammals, the swim bladder collagen forms high tensile strength through covalent crosslinking effects such as hydrogen bonds of glycine triad, the structural stability of the swim bladder collagen is maintained, meanwhile, the metastable state of proline provides structural flexibility, the collagen is endowed with certain flexibility, the swim bladder collagen content is equivalent to that of fish skin, swim bladders are obviously higher than collagen fibrils such as fishbone, fish scales, fish fins, fish meat, visceral cowhide, sturgeon skin and the like, the forming speed is slow, the stability is poor, the swim bladder collagen is suitable due to the viscosity, and the swim bladder collagen has extremely fast fibril forming capability, so that the denaturation temperature is obviously improved.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.