CN100369929C - Jellyfish collagen and preparation method thereof - Google Patents

Abstract

The invention discloses a jellyfish collagen. It is extracted from the middle gelatinous layer of jellyfish. The collagen is a white fibrous solid or light yellow solid powder with a bulk density of 0.110-0.645g/ml. The collagen is completely dissolved in an acidic solution and becomes a clear and transparent solution; The single-chain molecular weight of the collagen is 100,000-300,000 Daltons, the sugar content is 2.5-5%, and the amino acid content is 80-95%mg/100mg; among them, aspartic acid accounts for 7.9-8.3%, glutamic acid 10.0-12.9%, threonine 3.5-4.0%, valine 3.5-3.9%, isoleucine 2.2-2.5%, lysine 3.4-3.8%, and hydroxylysine 2.0- 2.7%. The preparation method of the jellyfish collagen is as follows: (1) using salted jellyfish or fresh jellyfish; (2) pretreatment of raw materials; (3) separating the middle glue layer; (4) dissociating the middle glue layer; (5) ultrafiltration Separation and concentration; collagen with a molecular weight of 100,000-300,000 Daltons is obtained, and the retention rate is 95%-98%; this method can effectively recover protease, recycle the reaction system, and obtain high-purity collagen products.

Description

Jellyfish collagen and preparation method thereof

technical field

The invention relates to the improvement of marine biochemical raw materials, in particular to a jellyfish collagen and a preparation method thereof. It belongs to the technical field of biomedicine and chemical industry.

Background technique

Jellyfish, also known as jellyfish, is a coelenterate. Jellyfish are carnivorous, feeding on plankton, small crustaceans, polychaetes and even small fish. The body is hemispherical, and the diameter of the umbrella is generally 30 to 45 centimeters, and the largest one can reach 100 centimeters. Between the epidermis and stomach layer of jellyfish is the mesoglea, which is very developed and occupies almost the entire thickness of its body, which contains fibers and a small amount of cells derived from the ectoderm. The middle glue layer is hard and thick, and there are many small fibers in the middle glue layer that can be seen under the electron microscope. The main component in the middle glue layer is water, which contains a very small amount of protein and polysaccharides, including 5% organic matter, and exists in the form of collagen protein, which has strong elasticity and adhesion. Jellyfish is a well-known edible large zooplankton in my country. In recent years, the breeding industry has overcome technical difficulties such as artificial seedling breeding, polyp larvae overwintering and release of juvenile stings, and has a continuous supply of jellyfish resources. The main component of jellyfish skin is protein (collagen), free of cholesterol and fatty acids. There is a U.S. patent (USpatent6,894,029) reporting the use of Stomolophus meleagris of the family Stomolophus meleagris to prepare class II collagen by freeze-drying, alkali washing, pickling, protease digestion, and graded salting-out processes for the treatment of Rheumatoid Arthritis. There is also a Japanese patent (JP2004099513) reporting the use of Aureiia aurita of the Aureiia family, using a buffer to extract collagen, collagen precipitation and separation, collagen dehydration, and freeze-drying processes to prepare collagen. The activity extracted from Aureiia aurita Jellyfish collagen has antibacterial activity, and the microorganism Propionibacterium acnes, Propionibacterium acnes, is a pathogenic bacterium that causes facial pimples. Many domestic literatures report the technology and process of extracting collagen from beef tendon, pig skin, pig cartilage, fish skin and fish scale, all of which use enzymatic hydrolysis and salting out extraction process. The problem with salting out extraction is that protease recovery is difficult , Difficulty in salt recovery and recycling of acidic solution. The jellyfish collagen extraction methods in the prior art all adopt the mixed extraction of the jellyfish ectoderm and mesoderm. The dissolving and dissociation of this endodermal miscellaneous protein affects the extraction of jellyfish collagen. In addition, most of them use the combination of collagenase dissociation and salting-out process for extraction. The disadvantage of the salting-out process is that a large amount of table salt is used, which is harmful to the formation of saline wastewater.

Contents of the invention

The purpose of the present invention is to provide a jellyfish collagen and a preparation method thereof, adopt a new purification process, effectively recover the extraction tool enzyme, and recycle the reaction system, so that the obtained active collagen has high purity and high yield.

The purpose of the present invention is achieved by the following technical solutions, a jellyfish collagen has been developed. The collagen is extracted from the middle gelatinous layer of jellyfish, the collagen is white fibrous solid or light yellow solid powder, the bulk density is 0.110-0.645g/ml, the collagen is completely dissolved in the acidic solution, showing Clear and transparent solution; the single-chain molecular weight of the collagen is 100,000-300,000 Daltons, the sugar content is 2.5-5%, and the amino acid content is 80-95% mg/100mg; among them, aspartic acid accounts for 7.9-8.3% , glutamic acid 10.0-12.9%, threonine 3.5-4.0%, valine 3.5-3.9%, isoleucine 2.2-2.5%, lysine 3.4-3.8%, hydroxylysine Acid accounts for 2.0-2.7%.

A preparation method of the above-mentioned jellyfish collagen, carried out as follows;

(1) The raw material adopted is large jellyfish, using salted jellyfish or fresh jellyfish;

(2) Pretreatment of raw materials;

Wash and soak the salted and dehydrated jellyfish with an alkaline solution with a concentration of 0.1-0.5%; the soaking time is 0.5-2 hours, and the pH of the solution after soaking is 6-8. During alkaline washing, ammonia gas is generated and bubbles are obvious ;The upper and lower cortex of jellyfish skin changes from white opaque to transparent, and it is easy to peel off to remove bad smell, ammonia ions and impurities;

(3) separating the middle rubber layer;

Wash and soak the salted dehydrated jellyfish in alkaline solution, remove the transparent upper and lower skin layers, and the white middle rubber layer can be clearly seen, and grind the white middle rubber layer;

(4) Dissociate the middle glue layer;

Use protease to dissociate the ground medium rubber layer, the enzyme dosage is 0.1%-2%, the dissociation temperature is 10-30°C, and the stirring dissociation time is 10-48h;

(5) ultrafiltration separation and concentration;

Use the ultrafiltration membrane filter to control the membrane inlet pressure at 1-2kg/cm ² , or use an external electric field of 10-70V/cm to cut off and concentrate the molecular weight at 100,000-300,000 Alton's collagen, its retention rate is 95% to 98%;

Then, the remaining dissociation solution containing protease is separated by interception and separation of protease by using cellulose acetate HFA-200 membrane, and the protease is recovered for repeated recycling;

(6) The separated and concentrated collagen is then freeze-dried, that is, slow freezing is adopted, the vacuum degree in the sublimation stage is 10-30Pa, and the cold trap temperature is -50±0.1°C to obtain a white fibrous solid or light yellow solid powder .

The large-scale jellyfish raw material that described (1) step adopts comprises: the jellyfish (Rhopilema esculentum Kishinouye) of the Jellyfish genus of Rhizostomalidae (Rhizostomalidae); Lobonma Smithi of the genus Lobonema.

The quality of the raw material salted jellyfish in the step (1) complies with the national standard, that is, the water content is less than 68%, the salt content is 18-25%, and the alum content is 1.2-2.2%.

The alkaline solution that adopts in the pretreatment of described (2) step raw material is sodium hydroxide solution, sodium carbonate solution, sodium bicarbonate solution.

The protease used in the step (4) to dissociate the middle gel layer is either acid protease or alkaline protease.

After the dissociation of the middle rubber layer in the step (4), the concentrated wet collagen can also be deeply hydrolyzed by collagenase, the dosage of the collagenase is 0.1-2.0%, the dissociation temperature is 20-40°C After 10-48 hours, a hydrolyzed polypeptide is obtained, that is, a structural collagen polypeptide containing about 30 amino acid residues is obtained. The molecular length of the polypeptide is 30-50 nanometers, and the molecular weight is 3000-20000 Daltons.

The ultrafiltration membrane used in the (5) step ultrafiltration separation and concentration, or the polymer membrane Diaflo (XM30), or the polyarylethersulfone membrane, or the hollow cellulose membrane, or the asymmetric ultrafiltration UF Membrane, or adopt asymmetric ultrafiltration FS membrane, or adopt asymmetric ultrafiltration HF membrane, or adopt asymmetric ultrafiltration T membrane.

The advantages of the jellyfish collagen of the present invention and the preparation method thereof are: in (2) step raw material pretreatment, the jellyfish after salting and dehydration is washed and soaked with an alkaline solution with a concentration of 0.1-0.5%; Ammonia gas is produced during washing, and the bubbles are obvious; the purpose of alkali washing pretreatment is to change the upper and lower skin layers of the jellyfish skin (i.e., the epidermis layer and stomach layer) from the original white opaque to transparent, softened and easy to peel off, and remove the colored layer; Remove impurities such as alum in the salting process and decomposed ammonia substances, etc., to restore the activity of the jellyfish protein; make the (3) step separate the middle glue layer, remove the transparent upper and lower skin layers, and successfully obtain the white middle glue layer. White medium layer. Due to the dissociation of the middle rubber layer in step (4), protease is used to dissociate the ground middle rubber layer while retaining the sugar content of the collagen. The phenol-sulfuric acid method (Zhang Weijie, 1999) was used to measure the sugar content in the sample; the fructose was used as the standard, and it was measured at 490nm. Due to the adoption of the ultrafiltration concentration purification process, the protease can be effectively recovered and the reaction system can be recycled. The collagen product obtained by this process is obviously different from the characteristics of collagen extracted from cattle, pigs, fish, etc. The separated dehydrated and deodorized medium rubber layer is used, after being minced, dissociated by protease, ultrafiltration, and freeze-dried to obtain high-purity and active collagen. If the collagen polypeptide is further obtained, the undried collagen can be deeply hydrolyzed by collagenase to obtain the hydrolyzed polypeptide, and the dry powder can be obtained by spray drying to facilitate storage. The extracted protein gel is collagen, which is consistent with the jellyfish collagen reported in the literature, and the purity of the obtained collagen is also high. The jellyfish collagen of the present invention is calculated by taking the raw material of salted jellyfish, and the recovery rate of the collagen is 3-5%.

Detailed ways

Example 1)

Take 120g of salted jellyfish, soak in 100ml of 0.2M sodium hydroxide solution for 2 hours, the pH of the solution after soaking is 6.5, ammonia gas will be generated during alkali washing, and the bubbles will be obvious, which can remove ammonia ions and impurities. The jellyfish skin changes from white opaque to transparent (similar to frozen dehydrated jellyfish), and the middle glue layer can be clearly seen. Remove the upper and lower cortex, slice the white middle layer and store in the freezer.

Take the above middle rubber layer, grind it, add 20ml of 0.1M acetic acid, and add 600mg of pepsin (Huamei, Sina-American Biotec, pepsin 1:3000), stir and dissociate at room temperature for 30h.

Adopt ultrafiltration membrane filter, control the membrane inlet pressure at 1-2kg/cm ² , and apply an external electric field of 10-70V/cm to intercept and concentrate collagen with a molecular weight of 100,000-300,000 Daltons, with a retention rate of 98%; The ultrafiltration membrane used is either a polymer membrane Diaflo (XM30), or a polyarylethersulfone membrane, or a hollow cellulose membrane, or an asymmetric ultrafiltration UF membrane, or an asymmetric ultrafiltration FS membrane, or Use asymmetric ultrafiltration HF membrane, or use asymmetric ultrafiltration T membrane.

The separated and concentrated collagen is then freeze-dried, that is, slow freezing is adopted. The vacuum degree of the sublimation stage is 10-30Pa, and the temperature of the cold trap is -50±0.1°C to obtain 3.851g of fibrous light-colored solid collagen. The yield was 3.21%. The jellyfish collagen of the present invention is calculated with the salted jellyfish raw material, and the calculation formula of the yield is: C%=(m ₁ /m)×100%, wherein, C% is the yield, m ₁ is the collagen protein weight (g), m is the weight (g) of salted jellyfish.

The bulk density of the collagen is 0.110-0.645g/ml; the collagen is completely dissolved in the acidic solution and is a clear and transparent solution; 5%, the amino acid content is 80-95% mg/100mg; among them, aspartic acid accounts for 7.9-8.3%, glutamic acid accounts for 10.0-12.9%, threonine accounts for 3.5-4.0%, valine accounts for 3.5- 3.9%, isoleucine accounts for 2.2-2.5%, lysine accounts for 3.4-3.8%, and hydroxylysine accounts for 2.0-2.7%.

The sugar content of collagen was determined by the phenol-sulfuric acid method (Zhang Weijie, 1999). Measured at 490nm with fructose as the standard. With the absorbance value (A490) as the abscissa and the sugar concentration as the abscissa, draw a standard curve to obtain a linear regression equation: A490=0.004×C-0.0016, R=0.9999. Weigh 0.114g sample, dissolve it with 0.5M acetic acid and make it volume up to 50ml, and measure the sugar content to be 3.09%.

Then, the remaining dissociation solution containing protease was blocked by pepsin (molecular weight: 35000) using cellulose acetate membrane (HFA-200) for recovery and recycling.

Embodiment (1)-(7) concrete process conditions and results are shown in Table 1:

Comparative example:

Take 50g of salted jellyfish, soak in 0.2M sodium hydroxide, 100ml for 2h, take 40g of the middle rubber layer, crush it, add 250ml of 0.5M acetic acid, 250mg of 0.5% protease, stir and incubate at room temperature for 48h at 15°C. After centrifugation, there are a lot of dissociated products. 3.5M salting out, foam generation, 4.5M white solid, 5.5M a large amount of flocculent precipitate, dialysis bag (8000-12000D) for dialysis. Freeze and dry for 24 hours to obtain 0.761g of light flocculent white solid. Yield 1.52%.

Determination of amino acid composition content of jellyfish collagen protein of the present invention:

Weigh 10 mg of dried jellyfish collagen, add 2 mL of hydrochloric acid solution with a concentration of 5.7 mol/L, place it in a 110-degree oven for 24 hours, and measure it with a HITACHI 835 amino acid automatic analyzer. The amino acid content is 92.57 mg/100 mg. The results of amino acid composition and content are shown in Table 2.

Table 2 Results of amino acid composition and content of jellyfish collagen

Amino acid type Jellyfish collagen (mg/100mg) Types of amino acids Jellyfish collagen (mg/100mg) Aspartate Asp 8.31 Valine Val 3.92 Glutamic acid Glu 12.99 Methionine Met 0.62 Serine Ser 3.19 Phenylalanine Phe 1.21 Histidine His 0.25 Isoleucine Iso 2.46 Glycine Gly 18.01 Leucine Leu 3.66 Threonine Thr 4.05 Lysine Lys 3.40 Alanine Ala 7.74 Hydroxylysine Hydroxylys 2.41 Arginine Arg 7.77 Proline Pro 7.42 Cysteine Cys 0 Hydroxyproline Hydroxypro 3.62 Tyrosine Tyr 0.33 Tryptophan Try 0

Those skilled in the art will understand that within the protection scope of the present invention, modifications, additions and substitutions are all possible to the above embodiments, and none of them exceed the protection scope of the present invention.

Claims (6)

1. a preparation method of jellyfish collagen, characterized in that: the preparation method is carried out as follows; (1) The raw material adopted is large jellyfish, using salted jellyfish or fresh jellyfish; (2) Pretreatment of raw materials; Wash and soak the salted and dehydrated jellyfish with an alkaline solution with a concentration of 0.1-0.5%; the soaking time is 0.5-2 hours, and the pH of the solution after soaking is 6-8. During alkaline washing, ammonia gas is generated and bubbles are obvious ;The upper and lower cortex of jellyfish skin changes from white opaque to transparent, and it is easy to peel off to remove bad smell, ammonia ions and impurities; (3) separating the middle rubber layer; Wash and soak the salted dehydrated jellyfish in alkaline solution, remove the transparent upper and lower skin layers, and the white middle rubber layer can be clearly seen, and grind the white middle rubber layer; (4) Dissociate the middle glue layer; Use protease to dissociate the ground medium rubber layer, the enzyme dosage is 0.1%-2%, the dissociation temperature is 10-30°C, and the stirring dissociation time is 10-48h; (5) ultrafiltration separation and concentration; Use the ultrafiltration membrane filter to control the membrane inlet pressure at 1-2kg/cm ² , or use an external electric field of 10-70V/cm to cut off and concentrate the molecular weight at 100,000-300,000 Collagen of Alton, its retention rate is 95%-98%; Then, the remaining dissociation solution containing protease is separated by interception and separation of protease by using cellulose acetate HFA-200 membrane, and the protease is recovered for repeated recycling; (6) The separated and concentrated collagen is then freeze-dried, that is, slow freezing is adopted, the vacuum degree in the sublimation stage is 10-30Pa, and the cold trap temperature is -50±0.1°C to obtain a white fibrous solid or light yellow solid powder . 2. according to the preparation method of the described jellyfish collagen of claim 1, it is characterized in that: the large-scale jellyfish raw material that described (1) step adopts comprises: jellyfish (Rhopilemaesculentum Kishinouye) of the jellyfish genus of Rhizostomalidae (Rhizostomalidae); Rhopilema hispidum and Lobonma Smithi of the genus Lobonema of the family Lobonematidae. 3. according to the preparation method of the described jellyfish collagen of claim 1, it is characterized in that: the alkaline solution that adopts in the pretreatment of described (2) step raw material is sodium hydroxide solution, sodium carbonate solution, sodium bicarbonate solution . 4. The preparation method of jellyfish collagen according to claim 1, characterized in that: the protease used in the dissociation of the middle gel layer in the step (4) is either acid protease or alkaline protease. 5. according to the preparation method of the described jellyfish collagen of claim 1, it is characterized in that: after the described (4) step dissociates the middle glue layer, the wet collagen of concentration can also be hydrolyzed through collagenase depth again, the The amount of collagenase is 0.1-2.0%, the dissociation temperature is 20-40°C, and the dissociation time is 10-48h, to obtain hydrolyzed polypeptide, that is, to obtain a structural collagen polypeptide containing about 30 amino acid residues, and the molecular length of the polypeptide is 30 -50 nanometers, molecular weight 3000-20000 Daltons. 6. according to the preparation method of the described jellyfish collagen of claim 1, it is characterized in that: adopt ultrafiltration membrane in the described (5) step ultrafiltration separation and concentration, or adopt polymer film Diaflo, or adopt polyarylether sulfone Membranes, or hollow cellulose membranes, or asymmetric ultrafiltration UF membranes, or asymmetric ultrafiltration FS membranes, or asymmetric ultrafiltration HF membranes, or asymmetric ultrafiltration T membranes.