A method for extracting fish scale collagen and its application

Abstract

The invention discloses a method for extracting fish scale collagen and its application, and belongs to the technical field of collagen extraction. Add 20 to 30 times the volume of water to the fish scale powder that has been washed, demineralized, defatted and washed to neutrality for homogenization, cook at 90 to 110°C for 20 to 30 minutes, cool to room temperature, and obtain fish scale homogenate. Add 0.1 to 0.3% ammonium citrate to the fish scale homogenate, and add mixed bacteria of Bacillus subtilis and Lactobacillus, and ferment at 35 to 38°C for 24 to 48 hours to obtain fermentation liquid; add papain, pepsin and trypsin to the fermentation liquid, enzymolyze at 50 to 60°C for 1 to 2 hours, and kill the enzyme in a boiling water bath to obtain an enzymolysis liquid, use a filter to perform preliminary filtration, and perform high-speed centrifugation separation on the filtrate to obtain a supernatant, and freeze-dry the supernatant to obtain fish scale collagen. More than 80% of the molecular weight of the fish scale collagen extracted by the method of the invention is concentrated in 500 to 1000Da, which can maximize the efficacy of collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen extraction, and specifically relates to a method for extracting fish scale collagen and application thereof. Background Art

[0002] my country is rich in fish resources. During the processing and consumption of fish-related products, a large amount of waste will be generated, accounting for about 50% of the quality of fresh fish, including fish bones, fish fins and fish scales, of which fish scales account for 6-10%. Processing fish scale byproducts can not only improve the utilization value of fish scales, reduce the pollution of fish scales to the environment, but also promote the development of the fish processing industry. Fish scales are mainly composed of organic matter and minerals, among which collagen and keratin account for more than 90% of the total organic content, and minerals are mainly calcium phosphate.

[0003] Collagen is an important structural protein, and its microstructure contains one or several triple helical regions, which are composed of α-peptide chains. Fish scale collagen has a unique molecular structure and good intermolecular cross-linking ability, good film-forming properties, biodegradability, and contains a variety of essential amino acids for the human body. It has health functions and high nutritional value, and can be added to food and food packaging.

[0004] At present, the methods for extracting collagen from fish scales are mainly divided into acid method, alkali method, enzyme method, hot water method and composite method. Physical-mechanical methods or the addition of specific chemical reagents are used to change the existence environment of collagen in fish scales, and finally separate collagen from other components to obtain collagen. However, different extraction methods have different extraction rates and main forms of extracts. The traditional method is acid-base extraction. The fish scales are washed, defatted and decolorized, and then soaked in hydrochloric acid (pH 4~5) for decalcification (2~3 weeks). After taking out, wash and soak for 15~20 days to make the raw material tissue loose. After washing, heat at 60~70℃ for 2~3h to boil the glue for extraction. This method has many steps and is time-consuming. The collagen is seriously lost during the extraction process.

[0005] At present, the molecular weight distribution of fish scale collagen obtained from fish scales is relatively wide, and there are problems of too large and too small molecular weight. Patent CN107723331 A discloses a process for extracting fish scale collagen from fish scales, wherein the fish scales are sequentially demineralized, defatted, steamed, expanded, and fermented, and then the protease-producing bacteria are cultured for protein decomposition, thereby obtaining low-molecular-weight collagen polypeptides while improving the extraction rate. However, the obtained fish scale collagen has a high content of molecular weights below 500Da (Daltons). When it is used in food, medicine, and other processing, it is particularly easy to penetrate the cell membrane in the body, has a fast metabolism rate, is very easy to be digested and excreted from the body, and cannot exert its efficacy. Collagen with a molecular weight that is too large is difficult to be absorbed by the body and cannot exert its efficacy. Therefore, it is extremely important to control the molecular weight of the fish scale collagen peptides obtained from fish scales. Summary of the invention

[0006] In view of the problem that the molecular weight distribution range of fish scale collagen obtained from fish scales in the prior art is large, and the efficacy of collagen with too high or too low molecular weight cannot be exerted, the present invention provides a method for extracting fish scale collagen and application thereof, wherein more than 80% of the molecular weight of the extracted fish scale collagen is concentrated in the range of 500-1000Da, thereby maximizing the efficacy of the collagen.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for extracting fish scale collagen comprises the following steps:

[0009] (1) Add 20 to 30 times the volume of water to the fish scale powder that has been washed, demineralized, defatted and washed to neutrality for homogenization, cook at 90 to 110°C for 20 to 30 minutes, and cool to room temperature to obtain fish scale homogenate;

[0010] (2) Fermentation: Add 0.1-0.3% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus, and ferment at 35-38°C for 24-48 hours to obtain a fermentation liquid;

[0011] (3) Enzymatic hydrolysis: add papain, pepsin and trypsin to the fermentation broth of step (2), perform enzymatic hydrolysis at 50-60°C for 1-2h, inactivate the enzymes in a boiling water bath, and obtain an enzymatic hydrolyzate;

[0012] (4) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter, and the filtrate is subjected to high-speed centrifugation to obtain a supernatant, which is freeze-dried to obtain fish scale collagen.

[0013] Preferably, in step (2), the amount of Bacillus subtilis used is 2-5% of the mass of the fish scale powder; and the amount of Lactobacillus used is 0.8-1.5% of the mass of the fish scale powder.

[0014] Preferably, in step (3), 1×10 4 ~2×10 4 U papain, 2×10 4 ~5×10 4 U pepsin and 1 × 10 3 ~5×10 3 UTriespase.

[0015] Preferably, the high-speed centrifugal separation speed in step (4) is 10000-18000 rpm.

[0016] Preferably, the homogenization condition in step (1) is homogenization at 2000-2500 rpm for 8-10 min.

[0017] Preferably, the method for washing, demineralizing and defatting the fish scales in step (1) is as follows: drying the cleaned fish scales, crushing them into fish scale powder with a particle size of 100-200 mesh, adding them to an acid solution, ultrasonically soaking them for 30-40 minutes, filtering them, washing the solids with water to a pH of 6-7, and then adding the washed fish scale powder to an alkaline solution, ultrasonically soaking them for 30-40 minutes, filtering them, and washing the solids with water to a pH of 6-8.

[0018] Preferably, the acid solution is a citric acid solution with a mass concentration of 1-4%; and the alkali solution is a sodium hydroxide solution with a mass concentration of 0.05-0.1%.

[0019] The present invention also discloses fish scale collagen obtained by the above extraction method; further, the mass proportion of the fish scale collagen with a molecular weight between 500 and 1000 Da is greater than 80%.

[0020] In the present invention, the fish scale collagen is used in the processing of food, medicine and health care products.

[0021] Beneficial Effects

[0022] The present invention adds ammonium citrate to the fish scale homogenate, and through fermentation and enzyme treatment, the molecular weight of the obtained fish scale collagen is concentrated in the range of 500-1000 Da. The collagen with this molecular weight does not need to be digested, can be directly absorbed by the blood, and is transmitted to the skin, and quickly reaches the dermis in 2-4 hours. The collagen is not easily digested and excreted from the body due to excessive metabolism, and cannot exert its efficacy. The collagen is suitable for absorption and utilization by organisms, and the effect of the collagen is maximized.

[0023] The invention adopts mixed bacteria of Bacillus subtilis and Lactobacillus for fermentation, cooperates with the effect of ammonium citrate, controls the molecular weight range of collagen; adds papain, pepsin and trypsin into the fermentation liquid, controls the type, addition amount and enzymolysis time of the enzymes, makes more than 80% of the molecular weight of the obtained fish scale collagen concentrate in 500-1000Da, and exerts the effect of collagen to the maximum extent. DETAILED DESCRIPTION

[0024] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0025] The fish scales used in the following examples and comparative examples have a protein content of 66.3% and a collagen content of 37.2%.

[0026] Example 1

[0027] (1) Pretreatment: The cleaned fish scales were dried and crushed into fish scale powder with a particle size of 100-200 mesh, added to a 3% mass concentration citric acid solution, and ultrasonicated (ultrasonic frequency 40kHz, ultrasonic sound intensity 0.6W / cm 2 ) for 35 min, filtered, and the solid was washed with water to pH 6.2. The washed fish scale powder was then added to a 0.08% mass concentration sodium hydroxide solution and ultrasonically treated (ultrasonic frequency of 35 kHz and ultrasonic sound intensity of 0.3 W / cm 2 ) soak for 35 min, filter, and wash the solid with water to pH 7.6;

[0028] (2) Homogenization and cooking: Add 25 times the volume of water to the cleaned, demineralized, defatted and neutralized fish scale powder for homogenization (10 min at 2000 rpm), cook at 100°C for 25 min, and cool to room temperature to obtain fish scale homogenate;

[0029] (3) Fermentation: Add 0.25% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus (the amount of Bacillus subtilis is 4% of the weight of the fish scale powder, and the amount of Lactobacillus is 1.2% of the weight of the fish scale), and ferment at 37°C for 36 hours to obtain a fermentation liquid;

[0030] (1) Enzymatic hydrolysis: Add papain, pepsin and trypsin (2×10 4 U papain, 2×10 4 U pepsin and 1 × 10 3U trypsin), hydrolyze at 60℃ for 1.5h, inactivate the enzyme in boiling water bath, and obtain the enzymatic solution;

[0031] (2) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter to remove large particles of impurities. The filtrate is subjected to high-speed centrifugation (15,000 rpm) to obtain a supernatant, which is freeze-dried to obtain fish scale collagen.

[0032] Example 2

[0033] (1) Pretreatment: The cleaned fish scales were dried and crushed into fish scale powder with a particle size of 100-200 mesh, added to a 4% mass concentration citric acid solution, and ultrasonicated (ultrasonic frequency 40kHz, ultrasonic sound intensity 0.6W / cm 2 ) for 30 min, filtered, and the solid was washed with water to pH 6.3. The washed fish scale powder was then added to a 0.06% sodium hydroxide solution and ultrasonicated (ultrasonic frequency of 35 kHz and ultrasonic intensity of 0.3 W / cm 2 ) soak for 30 min, filter, and wash the solid with water to pH 7.5;

[0034] (2) Homogenization and cooking: Add 20 times the volume of water to the fish scale powder that has been washed, demineralized, defatted and washed to neutrality for homogenization (8 min at 2500 rpm), cook at 100°C for 25 min, and cool to room temperature to obtain fish scale homogenate;

[0035] (3) Fermentation: Add 0.2% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus (the amount of Bacillus subtilis is 5% of the weight of the fish scale powder, and the amount of Lactobacillus is 0.8% of the weight of the fish scale powder), and ferment at 36°C for 42 hours to obtain a fermentation liquid;

[0036] (4) Enzymatic hydrolysis: Add papain, pepsin and trypsin (10 4 U papain, 4×10 4 U pepsin and 2 × 10 3 U trypsin), hydrolyze at 60℃ for 1.5h, inactivate the enzyme in boiling water bath, and obtain the enzymatic solution;

[0037] (5) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter to remove large particles of impurities. The filtrate is subjected to high-speed centrifugation (15,000 rpm) to obtain a supernatant, which is freeze-dried to obtain fish scale collagen.

[0038] Example 3

[0039] (1) Pretreatment: The cleaned fish scales were dried and crushed into fish scale powder with a particle size of 100-200 mesh, added to a 2.5% mass concentration citric acid solution, and ultrasonicated (ultrasonic frequency 40 kHz, ultrasonic sound intensity 0.6 W / cm 2 ) for 40 min, filtered, and the solid was washed with water to pH 6.4. The washed fish scale powder was then added to a 0.06% mass concentration sodium hydroxide solution and ultrasonically treated (ultrasonic frequency of 35 kHz and ultrasonic sound intensity of 0.3 W / cm 2 ) soak for 40 min, filter, and wash the solid with water until the pH value reaches 7.6;

[0040] (2) Homogenization and cooking: Add 30 times the volume of water to the cleaned, demineralized, defatted and neutralized fish scale powder for homogenization (8 min at 2500 rpm), cook at 100°C for 25 min, and cool to room temperature to obtain fish scale homogenate;

[0041] (3) Fermentation: Add 0.3% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus (the amount of Bacillus subtilis is 3% of the weight of the fish scale powder, and the amount of Lactobacillus is 1.5% of the weight of the fish scale powder), and ferment at 37°C for 30 hours to obtain a fermentation liquid;

[0042] (4) Enzymatic hydrolysis: Add papain, pepsin and trypsin (2×10 4 U papain, 2×10 4 Pepsin and 2 × 10 3 U trypsin), enzymolysis at 55℃ for 2h, inactivate the enzyme in boiling water bath, and obtain enzymolysis solution;

[0043] (5) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter to remove large particles of impurities. The filtrate is subjected to high-speed centrifugation (15,000 rpm) to obtain a supernatant, which is freeze-dried to obtain fish scale collagen.

[0044] Example 4

[0045] (1) Pretreatment: The cleaned fish scales were dried and crushed into fish scale powder with a particle size of 100-200 mesh, added to a 3% mass concentration citric acid solution, and ultrasonicated (ultrasonic frequency 40kHz, ultrasonic sound intensity 0.6W / cm 2 ) for 35 min, filtered, and the solid was washed with water to pH 6.2. The washed fish scale powder was then added to a 0.08% sodium hydroxide solution and ultrasonically treated (ultrasonic frequency of 35 kHz and ultrasonic intensity of 0.3 W / cm 2 ) soak for 35 min, filter, and wash the solid with water until the pH value reaches 7.4;

[0046] (2) Homogenization and cooking: Add 25 times the volume of water to the cleaned, demineralized, defatted and neutralized fish scale powder for homogenization (10 min at 2000 rpm), cook at 100°C for 25 min, and cool to room temperature to obtain fish scale homogenate;

[0047] (3) Fermentation: Add 0.25% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus (the amount of Bacillus subtilis is 3% of the weight of the fish scale powder, and the amount of Lactobacillus is 1.2% of the weight of the fish scale powder), and ferment at 37°C for 36 hours to obtain a fermentation liquid;

[0048] (4) Enzymatic hydrolysis: Add papain, pepsin and trypsin (2×10 4 U papain, 4×10 4 U pepsin and 1 × 10 3 U trypsin), hydrolyze at 60℃ for 1.5h, inactivate the enzyme in boiling water bath, and obtain the enzymatic solution;

[0049] (5) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter to remove large particles of impurities. The filtrate is subjected to high-speed centrifugation (15,000 rpm) to obtain a supernatant, which is freeze-dried to obtain fish scale collagen.

[0050] Comparative Example 1

[0051] Comparative Example 1 Except for step (3) of fermentation, the remaining steps are the same as those of Example 1, and fish scale collagen is obtained through pretreatment, homogenization, fermentation, enzymolysis and filtration;

[0052] Fermentation (without adding ammonium citrate): add mixed bacteria of Bacillus subtilis and Lactobacillus (the amount of Bacillus subtilis is 4% of the mass of fish scale powder, and the amount of Lactobacillus is 1.2% of the mass of fish scale) to the fish scale homogenate, and ferment at 37°C for 36 hours to obtain fermentation liquid.

[0053] Comparative Example 2

[0054] The operations of each step of Comparative Example 1 are the same as those of Example 1, except that the operation order of each step of Comparative Example 2 is that the fish scales are pretreated, homogenized, enzymatically hydrolyzed, fermented, and filtered to obtain fish scale collagen.

[0055] Comparative Example 3

[0056] Compared with Example 1, in Comparative Example 3, fish scale collagen is obtained by pretreatment, homogenization, fermentation and filtration without enzymatic hydrolysis.

[0057] test

[0058] (1) Yield calculation

[0059] The calculation method of fish scale collagen yield is:

[0060] Yield (%) = m1 / m2;

[0061] Wherein, m1 is the actual mass of fish scale collagen, g;

[0062] m2 is the theoretical mass of fish scale collagen, g.

[0063] (2) Molecular weight distribution test

[0064] The fish scale collagen was dissolved in 20 times the mass of water, and membrane separation was used to obtain fish scale collagen with molecular weights less than 500Da, 500-1000Da and greater than 1000Da, respectively, and dried. After drying, the mass proportion in each molecular weight range was calculated.

[0065] (3) The yield and molecular weight distribution of the fish scale collagen extracted in Examples 1 to 4 and Comparative Examples 1 to 3 were tested. The results are shown in Table 1 below:

[0066] Table 1 Fish scale collagen yield and molecular weight distribution

[0067]

[0068] As can be seen from Table 1, by adopting the technical solution of the present invention, the molecular weight of the fish scale collagen extracted is concentrated between 500 and 1000 Da (accounting for more than 80%), and the proportion of low molecular weight (<500 Da) and high molecular weight (>1000 Da) is relatively small. The molecular weight of the obtained collagen is controlled by adding ammonium citrate, fermentation and enzymolysis sequence, and the type and addition amount of fermentation bacteria and strains. The fish scale collagen obtained by the method of the present invention does not need to be digested and can be directly absorbed by the blood, but it is not easily digested and excreted from the body due to excessive metabolism, and is suitable for absorption and utilization by organisms, maximizing the efficacy of collagen.

Claims

1. A method for extracting fish scale collagen, characterized in that: The following steps are involved: (1) Add 20 to 30 times the volume of water to the fish scale powder that has been washed, demineralized, defatted and washed to neutrality for homogenization, cook at 90 to 110°C for 20 to 30 minutes, and cool to room temperature to obtain fish scale homogenate; (2) Fermentation: Add 0.1-0.3% ammonium citrate to the fish scale homogenate, and add a mixed bacteria of Bacillus subtilis and Lactobacillus acidophilus, and ferment at 35-38°C for 24-48 hours to obtain a fermentation liquid; (3) Enzymatic hydrolysis: add papain, pepsin and trypsin to the fermentation broth of step (2), perform enzymatic hydrolysis at 50-60°C for 1-2h, inactivate the enzymes in a boiling water bath, and obtain an enzymatic hydrolyzate; (4) Filtration: The enzymatic hydrolysate in step (3) is initially filtered using a filter, the filtrate is subjected to high-speed centrifugation to obtain a supernatant, and the supernatant is freeze-dried to obtain fish scale collagen; In step (2), the amount of Bacillus subtilis used is 3 to 5% of the mass of the fish scale powder; the amount of Lactobacillus used is 0.8 to 1.5% of the mass of the fish scale powder; In step (3), add 1×10 4 ~ 2×10 4 U papain, 2×10 4 ~ 4×10 4 U pepsin and 1 × 10 3 ~ 2×10 3 UTriespase.

2. The method for extracting fish scale collagen according to claim 1, characterized in that In step (4), the high-speed centrifugal separation speed is 10000 ~ 18000rpm.

3. The method for extracting fish scale collagen according to claim 1, characterized in that The homogenization condition in step (1) is 2000-2500 rpm for 8-10 min.

4. The method for extracting fish scale collagen according to claim 1, characterized in that The method for washing, demineralizing and defatting the fish scales in step (1) is as follows: drying the cleaned fish scales, crushing them into fish scale powder with a particle size of 100 to 200 meshes, adding them to an acid solution, ultrasonically soaking them for 30 to 40 minutes, filtering them, washing the solids with water to a pH of 6 to 7, and then adding the washed fish scale powder to an alkaline solution, ultrasonically soaking them for 30 to 40 minutes, filtering them, and washing the solids with water to a pH of 6 to 8.

5. The method for extracting fish scale collagen according to claim 4, characterized in that, The acid solution is a citric acid solution with a mass concentration of 1 to 4%; the alkali solution is a sodium hydroxide solution with a mass concentration of 0.05 to 0.1%.

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