A fish glue polypeptide, and a preparation method and application thereof

Abstract

The application belongs to the technical field of food processing, and particularly discloses fish glue polypeptide, a preparation method and application thereof. The preparation method comprises three procedures of acid immersion pretreatment, repeated homogenization and gelatinization treatment and ultrasonic wave assisted immobilized enzyme degradation treatment. The fish glue polypeptide has an average molecular weight of 3.2-8.4 KDa, a molecular weight dispersion coefficient of 0.2-0.5, a small molecular weight, a concentrated molecular weight distribution, a good polypeptide activity, and is beneficial to promoting the absorption of trace elements when applied to fish glue polypeptide-trace element supplement products. When the concentration in the aqueous solution is 2 g / L, the Zeta potential of the micelle is-24 mV to-30 mV, the average particle size of the micelle is 10-20 nm, the particle size dispersion coefficient of the micelle is 0.1-0.2, the average particle size of the micelle is small, the particle size distribution of the micelle is concentrated, the Zeta potential of the micelle is high, and the micelle has good stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing, and particularly relates to a fish glue polypeptide and a preparation method and application thereof. BACKGROUND

[0002] Fish glue, also known as flower glue, fish belly, fish bubble and fish bubble, is a dried product of swim bladder of various fish. As a precious traditional Chinese medicine, fish glue has a long history of medicinal use in China. Although fish glue is given different names in different medical classics, such as "Qimin Yaoshu", "Bencao Shiyi", "San Yin Fang", "Baiqi", "Bencao Gangmu" and the like (Chen Dan-kun, Li Yan-fen, "Fish Glue Appreciation", Shantou University Press, 2018, 1-2), these medical classics all indicate that fish glue has the effects of "tonifying yin and nourishing qi" and "tonifying blood and stopping metrorrhagia". In folk, fish glue is classified as one of "eight treasures of the ocean" and enjoys the reputation of "ocean ginseng".

[0003] There are also a few enterprises that process it into instant cans for sale in Guangdong. In sharp contrast, people have developed secondary processed goods such as bird's nest polysaccharide, bird's nest milkshake, shark fin protein and collagen cake for tonics such as bird's nest, shark fin and donkey-hide gelatin, greatly enriching the types and market of tonics. However, due to the lack of high-value processing technology for fish glue, there is a lack of fish glue product categories.

[0004] In the previous research, the applicant found that the main component of fish glue is protein (dry content of more than 90%), and among the amino acids that constitute fish glue protein, the content of glycine is about 30-35%, and the content of hydroxyproline is about 8-10% (Xiaofeng He et al. The Structural Characteristics of Collagen in Swim Bladders with 25-Year Sequence Aging: The Impact of Age, Applied science, 2021, 11, 4578). Hydroxyproline is a unique amino acid of collagen protein, in addition to which, the amino acid sequence of collagen protein usually has the repeating feature of [Gly-X-Y] n The repeating short peptide fragment feature makes the content of glycine (Gly) in collagen protein usually about 30-35%. Thus, it can be preliminarily judged that the protein in fish glue is mainly collagen protein. The applicant observed that the structure of these proteins has the morphological feature of periodic cross striations (as shown in Figure 1 The above results show that the protein in fish glue is essentially collagen protein.

[0005] The polypeptide products have been widely used for the collagen in animal tissues such as pigskin, cowhide, chicken skin, tendon, fish bone and the like. Among them, the collagen polypeptide-metal ion chelate is a good trace element supplement product, such as cowhide collagen peptide-calcium, chicken skin collagen-zinc and the like. In addition, the collagen peptides prepared from fish skin, cowhide, fish bone and the like are confirmed to have the effects of antioxidant, antibacterial and anti-skin photoaging. Some scholars even develop surface active agents, cosmetic additives, molecular probes and high molecular materials with biological functions by using the collagen peptides. It can be seen that, as a kind of natural biomass, the collagen polypeptide product has significant development value.

[0006] At present, there is no development and utilization of fish glue collagen polypeptide, and therefore, it is urgent to develop a method for preparing fish glue polypeptide by using fish glue as raw material, so as to improve the utilization value. SUMMARY

[0007] The present application provides a fish glue polypeptide and a preparation method and application thereof, so as to solve one or more technical problems in the prior art and at least provide a beneficial choice or create conditions.

[0008] In order to overcome the above technical problems, the first aspect of the present application provides a preparation method of fish glue polypeptide.

[0009] Specifically, the preparation method of fish glue polypeptide comprises three processes of acid immersion pretreatment, repeated homogenization gelatinization treatment and ultrasonic wave assisted-immobilized enzyme degradation treatment.

[0010] The present application mainly adopts three processes of acid immersion pretreatment, repeated homogenization gelatinization treatment and ultrasonic wave assisted-immobilized enzyme degradation treatment to prepare fish glue polypeptide according to the characteristics of fish glue collagen, and the prepared fish glue polypeptide has the advantages of low molecular weight, concentrated molecular weight distribution and good stability of fish glue polypeptide in aqueous solution. The specific preparation process is as follows:

[0011] Firstly, the solid fish glue is converted into liquid collagen by the method of acid immersion pretreatment.

[0012] Specifically, the structure of collagen has the following characteristics: three polypeptide chains constitute a triple helix structure of tropocollagen, five tropocollagens are connected end to end and 1 / 4 staggered to form microfibrils, the microfibrils are intertwined to form fibrils, the fibrils are combined to form fiber bundles through hydrogen bonds and van der Waals forces, and the structures at all levels are arranged in order and are tightly combined. The collagen-rich tissues of terrestrial animals (such as cowhide, pigskin, and cow tendon) are relatively stable in an acidic environment, but the applicant found in the research process that fish glue collagen is very susceptible to acid and loses the order of the structures at all levels. When the pH is lower than 3.0, part of the fish glue can be converted from a solid state to a liquid state without heating. The main reason is that the content of acid-sensitive amino acids such as histidine, lysine, and glutamic acid in fish glue collagen is relatively high, which can cause protonation of the side chain groups of acid-sensitive amino acids and changes in the surface charge of the peptide chain, ultimately destroying the ordered high-level structure of fish glue collagen, making the hydrophilic side chain fully exposed, and causing it.

[0013] It is worth noting that those skilled in the art know that the collagen in the tissues of terrestrial animals will not be directly degraded in an acidic environment, but will produce an "acid swelling" phenomenon. The applicant first observed the acid instability of fish glue collagen and its susceptibility to dissolution, and developed a fish glue polypeptide product using this characteristic. The fish glue is soaked in water and then subjected to acid immersion pretreatment, which can dissolve the fish glue and convert it into a liquid collagen.

[0014] Then, the prepared liquid collagen is subjected to repeated homogenization treatment to convert it into collagen micelles.

[0015] Specifically, the liquid collagen after acid immersion pretreatment is subjected to homogenization treatment. Under the action of the shear effect, impact effect, and cavitation effect generated by high-pressure homogenization, the liquid collagen will undergo partial hydrolysis. Research and analysis show that the average molecular weight of the liquid fish glue collagen obtained after acid immersion treatment is about 230 KDa, and the molecular weight can be reduced to about 120 KDa after multiple homogenization and emulsification treatments. The average particle size of the fish glue collagen before homogenization is about 4200 nm, and the average particle size is reduced to about 860 nm after homogenization treatment. No complete degradation of collagen protein into short peptides has been observed. This conclusion is not entirely consistent with the conclusion in the prior art that the collagen in the tissues of terrestrial animals will be degraded into short peptides during high-pressure homogenization (see George, Preparation Method of Small Molecule Animal Collagen Protein Based on High-Pressure Homogenization Technology, Master's Thesis, Harbin Institute of Technology, 2015). Therefore, the preparation method of small molecule animal collagen protein is not suitable for fish glue collagen.

[0016] Finally, the collagen micelles subjected to homogenization treatment are subjected to ultrasonic-assisted immobilized enzyme degradation treatment to prepare the fish glue polypeptide of the present application.

[0017] Specifically, the enzyme degradation process of the conventional protein is usually directly degraded by the enzyme treated by activation or immobilization technology. Some immobilized enzyme technology loads the enzyme on the surface of nanoparticles to increase the specific surface area of the enzyme and increase the effect of enzyme degradation treatment. However, the preparation conditions of the enzyme nanocarrier are very strict, and the yield is very small, which is not suitable for large-scale production. The present application can greatly improve the degradation efficiency of the protease by homogenizing the collagen to prepare nanocolloids. This technology uses the increased specific surface area of collagen instead of the specific surface area of enzyme, which avoids the problem that the protease immobilization technology is not suitable for large-scale production. Meanwhile, the use of ultrasonic assistance can increase the contact frequency of the protease and the collagen colloids, and further improve the degradation effect of the protease on the fish glue collagen colloids.

[0018] As a further improvement of the above-mentioned scheme, the preparation method of the fish glue polypeptide comprises the following steps:

[0019] (1) After the fish glue is soaked in water, it is pretreated by acid immersion to obtain liquid collagen;

[0020] (2) The liquid collagen prepared in step (1) is taken, and a homogenization aid is added to the liquid collagen for repeated homogenization treatment to obtain collagen colloids;

[0021] (3) The collagen colloids prepared in step (2) are mixed with the immobilized enzyme, and then subjected to ultrasonic treatment to obtain the fish glue polypeptide.

[0022] As a further improvement of the above-mentioned scheme, the acid solution is selected from at least one of sulfuric acid, hydrochloric acid, formic acid and acetic acid. These acids have good solubility for fish glue, and can convert solid fish glue into liquid collagen.

[0023] Preferably, the fish glue is selected from at least one of the following: Diapterus kneri, Archosargus probatocephalus, Chanodichthys erythropterus, Nibea diacanthus, Nibea albiflora and Anisakis.

[0024] Preferably, the mass ratio of the fish glue to the acid solution is 100:(0.2-10). The addition of an appropriate amount of acid solution can ensure the sufficient dissolution of the solid fish glue into liquid collagen.

[0025] Preferably, the concentration of the sulfuric acid solution is 88-92%, the concentration of the hydrochloric acid solution is 25-35%, the concentration of the formic acid solution is 90-95%, and the concentration of the acetic acid solution is 90-95%.

[0026] Preferably, the mass ratio of the fish glue to the water is 100:(200-5000).

[0027] As a further improvement of the above-mentioned solution, the fish glue is soaked in water for 1-24 hours; the temperature of the acid pickling pretreatment is 25-50℃, and the time is 1-6 hours. Specifically, the fish glue is soaked in water for a certain period of time and then subjected to heat treatment, which not only ensures the degree of dissolution of the fish glue but also helps to improve its dissolution speed.

[0028] As a further improvement of the above-mentioned solution, the homogenization aid is selected from at least one of propylene glycol alginate, succinylated monoglycerides, polyglycerol ricinoleate, Tween, and lactic acid fatty acid glyceride.

[0029] Preferably, the Tween is Tween 60.

[0030] Preferably, the mass ratio of the liquid collagen to the homogenization aid is 100:(0.01-5). The addition of an appropriate amount of homogenization aid not only helps to reduce the molecular weight of the micelles but also helps to maintain good stability of the micelles.

[0031] Preferably, the process conditions of the repeated homogenization treatment are as follows: the feed temperature is 20-80℃, the flow rate is 5-30 mL / min, and the pressure is 10-100 MPa.

[0032] Preferably, the repeated homogenization treatment is repeated for 3-8 times.

[0033] Preferably, the carrier used by the immobilized enzyme is cellulose, which is selected from epoxidized dialdehyde oxidized cellulose or epoxidized cellulose.

[0034] Preferably, the enzyme used by the immobilized enzyme is selected from at least one of papain, pepsin, bromelain, and trypsin.

[0035] Preferably, the mass ratio of the collagen micelles to the immobilized enzyme is 100:(0.05-1).

[0036] Preferably, the preparation method of the immobilized enzyme is to introduce conventional immobilized enzyme degradation treatment technology into the degradation treatment of collagen micelles.

[0037] Further preferably, the preparation method of the immobilized enzyme can refer to the research by Guo Qingqi et al. (Guo Qingqi, Zhang Na, Fang Guizhen, Research on Epoxidized Dialdehyde Oxidized Cellulose Immobilized β-Galactosidase, Food Science, 2011, 32: 204-208).

[0038] Specifically,

[0039] Put 4.0 g of alkali-activated cellulose into a 250 mL flask, add 48 mL of distilled water, and swell for 30 min. Then add 32 mL of 30 g / 100 mL NaOH solution and 24 mL of epichlorohydrin, and stir at 40℃ for 2.5 h. Wash the product with distilled water and ethanol until it is neutral to obtain epoxidized cellulose. Add 8.0 g of sodium periodate to the flask with constant stirring, and adjust the pH to 1-2 with sulfuric acid. Then quickly add 4.0 g of epoxidized cellulose, and react for 3.5 h. Add a small amount of ethylene glycol, and continue to react for 1 h. Filter and wash with distilled water to obtain epoxidized dialdehyde oxidized cellulose. Take 0.3 g of the epoxidized dialdehyde oxidized cellulose obtained by vacuum filtration, add 2 mL of 10 g / L protease aqueous solution, and stir at 4℃ for 4 h. Filter and wash with a large amount of distilled water to obtain immobilized enzyme.

[0040] Because the collagen colloid after homogenization treatment is substantially not charged (zeta potential is 0), and the cellulose carrier surface is charged with a large amount of negative charge under neutral pH conditions (the hydroxyl groups on the surface of the cellulose carrier for embedding are dissociated under neutral pH conditions). Therefore, the collagen colloid can contact the immobilized enzyme, and the cavitation effect of the ultrasonic wave is used to improve the contact efficiency of the two. The collagen belongs to amphoteric substances, which have both acidic amino acids and basic amino acids. After being degraded by the protease, the collagen molecules are broken, the secondary structure is destroyed, and the acidic amino acids (negatively charged under neutral conditions) are fully exposed, so that the surface of the collagen colloid is charged with negative charge. After the catalytic reaction is completed, the collagen colloid is affected by the negative charge and leaves the surface of the immobilized enzyme. The immobilized enzyme is in contact with the collagen colloid that is not degraded for reaction (as shown in the figure). Figure 4 Compared with the traditional immobilized enzyme technology, the present application fully utilizes the amphoteric properties of collagen and the surface charge change of the colloid before and after degradation, and improves the degradation efficiency.

[0041] As a further improvement of the above scheme, in step (3), the process conditions of ultrasonic treatment are as follows: ultrasonic frequency is 50-200 MHz, power is 10-300 W, temperature is 20-50℃, and treatment time is 10-120 min.

[0042] The second aspect of the present application provides a fish glue polypeptide.

[0043] Specifically, a fish glue polypeptide is prepared by the preparation method of the fish glue polypeptide, the molecular weight of the fish glue polypeptide is 3.2-8.4 KDa, and the molecular weight dispersion coefficient is 0.2-0.5. The fish glue polypeptide prepared by the preparation method of the fish glue polypeptide has the characteristics of small molecular weight and concentrated molecular weight distribution, has good polypeptide activity, and when it is applied to fish glue polypeptide products such as fish glue polypeptide-microelement supplement products, it is beneficial to promote the absorption of microelements.

[0044] Meanwhile, the Zeta potential of the micelle of the fish glue polypeptide is -24mV to -30mV, the average particle size of the micelle is 10-20nm, and the particle size dispersion coefficient of the micelle is 0.1-0.2 when the concentration of the fish glue polypeptide in the aqueous solution is 2g / L. The fish glue polypeptide prepared by the preparation method of the fish glue polypeptide has a small average particle size of the micelle, a concentrated particle size distribution, a high Zeta potential of the micelle, and good stability.

[0045] The third aspect of the present application provides a fish glue polypeptide.

[0046] Specifically, a fish glue polypeptide product comprises the fish glue polypeptide of the present application.

[0047] Preferably, the fish glue polypeptide product is a fish glue polypeptide-mineral element preparation, and the mineral element comprises at least one of calcium, iron and zinc.

[0048] The above technical solution of the present application has at least the following technical effects or advantages compared with the prior art:

[0049] The present application mainly adopts three processes of acid immersion pretreatment, repeated homogenization micellization treatment and ultrasonic wave assisted-immobilized enzyme degradation treatment according to the characteristics of fish glue collagen to prepare the fish glue polypeptide. The prepared fish glue polypeptide has a molecular weight of 3.2-8.4KDa and a molecular weight dispersion coefficient of 0.2-0.5, has the characteristics of small molecular weight and concentrated molecular weight distribution, has good polypeptide activity, and is beneficial to promote the absorption of mineral elements when applied to fish glue polypeptide products such as fish glue polypeptide-mineral element supplement products. Meanwhile, the Zeta potential of the micelle of the fish glue polypeptide is -24mV to -30mV, the average particle size of the micelle is 10-20nm, and the particle size dispersion coefficient of the micelle is 0.1-0.2 when the concentration of the fish glue polypeptide in the aqueous solution is 2g / L. The fish glue polypeptide prepared by the preparation method of the fish glue polypeptide has a small average particle size of the micelle, a concentrated particle size distribution, a high Zeta potential of the micelle, and good stability. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 for observing the morphology of the protein in the fish glue by atomic force microscopy;

[0051] Figure 2 for the molecular weight distribution and particle size distribution of the liquid collagen prepared in Example 1;

[0052] Figure 3 for the molecular weight distribution and particle size distribution of the collagen micelle prepared in Example 1;

[0053] Figure 4 for the surface charge of the collagen micelle before and after the fish glue polypeptide prepared in Example 1 is subjected to immobilized enzyme degradation treatment;

[0054] Figure 5 Molecular weight distribution, particle size distribution and Zeta potential of the products prepared in Example 1 and Comparative Examples 1-3 are shown in the following figures. DETAILED DESCRIPTION

[0055] The present application will be described in detail below with reference to the examples, so as to facilitate the understanding of the present application by those skilled in the art. It is necessary to point out here that the examples are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Non-essential improvements and adjustments of the present application made by those skilled in the art according to the above description should still fall within the scope of protection of the present application. Meanwhile, the raw materials mentioned below which are not described in detail are all commercially available products; the process steps or preparation methods which are not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

[0056] Example 1

[0057] A preparation method of fish glue polypeptide, comprising three processes of acid immersion pretreatment, homogenized gelatinization treatment and immobilized enzyme degradation treatment, comprising the following steps:

[0058] (1) 100 parts by mass of the striped large-spine fish glue is added to a reactor, 200 parts by mass of distilled water is added, and after soaking for 24 hours, acid immersion pretreatment is carried out, 0.2 parts by mass of sulfuric acid with a concentration of 90wt% is added, and heating treatment is carried out at 25℃ for 6 hours to obtain liquid collagen;

[0059] (2) 100 parts by mass of the supernatant of the liquid collagen prepared in step (1) is transferred to a high-pressure homogenizer, 0.01 parts by mass of homogenization aid propylene glycol alginate is added, and under the conditions of a feed temperature of 20℃, a flow rate of 5mL / min and a pressure of 10Mpa, repeated treatment is carried out for 8 times to obtain homogenized collagen gel;

[0060] (3) 100 parts by mass of the collagen gel prepared in step (2) is mixed with 0.05 parts by mass of papain treated by epoxy bis-aldehyde oxidized cellulose carrier immobilization, and ultrasonic treatment is carried out under the conditions of an ultrasonic frequency of 50MHz, a power of 10W and a temperature of 20℃ for 10 minutes to obtain the fish glue polypeptide product of the present example.

[0061] The preparation method of the papain treated by epoxy bis-aldehyde oxidized cellulose carrier immobilization is as follows:

[0062] Take 4.0 g of base-activated cellulose, put it into a 250 mL bottle, add 48 mL of distilled water, and swell in water for 30 min. Then add 32 mL of 30 g / 100 mL NaOH solution and 24 mL of epichlorohydrin, and stir at 40°C for 2.5 h. Wash the product with distilled water and ethanol until it is neutral to obtain epoxidized cellulose. Add distilled water to the flask, and continuously stir in 8.0 g of sodium periodate. Adjust the pH to 1-2 with sulfuric acid, quickly add 4.0 g of epoxidized cellulose, and react for 3.5 h. Add a small amount of ethylene glycol and continue to react for 1 h. Filter and wash with distilled water to obtain epoxidized dialdehyde oxidized cellulose. Take 0.3 g of the epoxidized dialdehyde oxidized cellulose obtained after vacuum filtration, add 2 mL of 10 g / L papain aqueous solution, and stir at 4°C for 4 h. Filter and wash with a large amount of distilled water to obtain the product.

[0063] The molecular weight distribution and particle size distribution of the liquid collagen obtained in step (1) of Example 1 are shown in Figure 2 , wherein: Figure 2 -A is the molecular weight distribution graph of the liquid collagen; Figure 2 -B is the particle size distribution graph of the liquid collagen. The molecular weight distribution and particle size distribution of the collagen colloid obtained in step (2) are shown in Figure 3 , wherein: Figure 3 -A is the molecular weight distribution graph of the collagen colloid; Figure 3 -B is the particle size distribution graph of the collagen colloid. As can be seen from Figure 2 and Figure 3 , the average molecular weight of the liquid collagen pretreated by acid immersion is about 230 KDa, and the average particle size is about 4200 nm. After repeated homogenization treatment, the molecular weight is reduced to 120 KDa, and the average particle size is reduced to 860 nm, indicating that repeated homogenization treatment can effectively reduce the molecular weight and average particle size of the liquid collagen.

[0064] The surface charge of the collagen colloid before and after the immobilized enzyme degradation treatment of the fish gel polypeptide obtained in Example 1 in step (3) is shown in Figure 4 , as can be seen from Figure 4 , because the surface of the collagen colloid has a negative charge, after the enzyme catalysis is completed, the collagen colloid is affected by the negative charge and leaves the surface of the immobilized enzyme, and the immobilized enzyme is in contact with the undegraded collagen colloid for reaction, thereby greatly improving the degradation efficiency.

[0065] Examples 2-12

[0066] Examples 2-12 differ from Example 1 in that the process conditions for acid immersion pretreatment, homogenization colloid treatment, or immobilized enzyme degradation treatment are different, as shown in Tables 1-3. In Tables 1-3, the concentration of sulfuric acid is 90 wt%, the concentration of hydrochloric acid is 30 wt%, the concentration of formic acid is 95 wt%, and the concentration of acetic acid is 95 wt%.

[0067] Table 1: Process conditions for acid soaking pretreatment of Examples 2-12

[0068]

[0069] Table 2: Process conditions for homogenization treatment of Examples 2-12

[0070]

[0071] Table 3: Process conditions for immobilized enzyme degradation treatment of Examples 2-12

[0072]

[0073] Comparative Example 1

[0074] A method for preparing a fish glue polypeptide, comprising the following steps:

[0075] (1) 100 parts by mass of the big diagonal fish glue was added to the reactor, 200 parts by mass of distilled water was added, and after soaking for 24 hours, acid soaking pretreatment was carried out, 0.2 parts by mass of sulfuric acid with a concentration of 90wt% was added, and heating treatment was carried out at 25℃ for 6 hours, to obtain liquid collagen;

[0076] (2) 100 parts by mass of the supernatant of the liquid collagen prepared in step (1) was transferred to a high-pressure homogenizer, 0.01 parts by mass of a homogenization aid propylene glycol alginate was added, and under the conditions of a feed temperature of 20℃, a flow rate of 5mL / min, and a pressure of 10Mpa, repeated treatment was carried out for 8 times, to obtain the fish glue polypeptide product of the present comparative example.

[0077] Comparative Example 2

[0078] A method for preparing a fish glue polypeptide, comprising the following steps:

[0079] (1) 100 parts by mass of the big diagonal fish glue was added to the reactor, 200 parts by mass of distilled water was added, and after soaking for 24 hours, acid soaking pretreatment was carried out, 0.2 parts by mass of sulfuric acid with a concentration of 90wt% was added, and heating treatment was carried out at 25℃ for 6 hours, to obtain liquid collagen;

[0080] (2) 100 parts by mass of the supernatant of the liquid collagen prepared in step (1) was mixed with 0.05 parts by mass of papain, and under the condition of a temperature of 20℃, enzymolysis was carried out for 60 minutes, to obtain the fish glue polypeptide product of the present comparative example.

[0081] Comparative Example 3

[0082] A method for preparing a fish glue polypeptide, comprising the following steps:

[0083] (1) Add 100 parts by weight of the large-spined sea bass glue to the reactor, add 200 parts by weight of distilled water, soak for 24 hours and then perform acid pretreatment, add 0.2 parts by weight of sulfuric acid with a concentration of 90wt%, heat at 25℃ for 6 hours to obtain liquid collagen.

[0084] (2) Take 100 parts by weight of the supernatant of the liquid collagen obtained in step (1), transfer it to a high-pressure homogenizer, add 0.01 parts by weight of the homogenizing agent propylene glycol alginate, and repeat the process 8 times under the conditions of feed temperature of 20℃, flow rate of 5mL / min and pressure of 10MPa to obtain homogenized collagen micelles.

[0085] (3) Mix 100 parts by weight of the collagen micelles obtained in step (2) with 0.05 parts by weight of papain and hydrolyze them at 20°C for 60 minutes to obtain the fish collagen polypeptide product of this comparative example.

[0086] Performance testing

[0087] The molecular weight, particle size, and zeta potential of the fish gelatin polypeptide products prepared in Examples 1-12 and Comparative Examples 1-3 were tested using gel filtration chromatography-evaporative light detection (HPSEC-ELSD) combined with dynamic light scattering (DLS), as shown in Table 4.

[0088] Table 4: Performance of fish gelatin polypeptide products prepared in Examples 1-12 and Comparative Examples 1-3

[0089]

[0090]

[0091] The molecular weight distribution (chromatogram) of the fish collagen polypeptide products prepared in Example 1 and Comparative Examples 1-3 was determined by HPSEC-ELSD, and the particle size distribution and zeta potential were obtained by DLS analysis. The results are as follows: Figure 5 As shown. Wherein: Figure 5 -A、 Figure 5 -B and Figure 5 -C represents the molecular weight distribution, particle size distribution, and zeta potential of the fish collagen polypeptide product prepared in Example 1, respectively. Figure 5 -D、 Figure 5 -E and Figure 5 -F are the molecular weight distribution, particle size distribution and zeta potential diagrams of the fish gelatin polypeptide product prepared in Comparative Example 1, respectively. Figure 5 -G、 Figure 5 -H and Figure 5 -I are the molecular weight distribution, particle size distribution and zeta potential diagrams of the fish collagen polypeptide product prepared in Comparative Example 2, respectively. Figure 5 -J、 Figure 5- K and Figure 5 - L are the molecular weight distribution, particle size distribution and Zeta potential diagram of the fish gel polypeptide product prepared in Comparative Example 3, respectively. Figure 5 - A, Figure 5 - D, Figure 5 - G and Figure 5 The horizontal axis Retention time in - J represents retention time; Figure 5 - B, Figure 5 - E, Figure 5 - H and Figure 5 The horizontal axis Size in - K represents particle size, and the vertical axis Volume represents volume; Figure 5 - C, Figure 5 - F, Figure 5 - I and Figure 5 The horizontal axis Apparent Zeta potential in - L represents apparent Zeta potential, and the vertical axis Total counts represents total counts.

[0092] As shown in Table 4 and Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 It can be seen that the fish gel polypeptide products prepared by simultaneously using the acid immersion pretreatment, repeated homogenate gelation treatment and immobilized enzyme degradation treatment of the present application (Examples 1-12) have an average molecular weight of 3.2-8.4 KDa, a molecular weight dispersion coefficient of 0.2-0.5, a Zeta potential of -24 mV to -30 mV, an average particle size of the gelatinous particles of 10-20 nm, and a particle size dispersion coefficient of the gelatinous particles of 0.1-0.2. The fish gel polypeptide products prepared by other preparation methods (Comparative Examples 1-3) have a larger average molecular weight of 65.2-120 KDa, a larger molecular weight dispersion coefficient of 0.8-0.9, a smaller Zeta potential of -0.2 mV to +0.1 mV, a larger average particle size of the gelatinous particles of 572-860 nm, and a larger particle size dispersion coefficient of the gelatinous particles of 1.0. It is shown that the fish gel polypeptide prepared by the present application has better polypeptide activity and stability.

[0093] For those skilled in the art to which the present application belongs, without departing from the concept of the present application, some simple deductions or substitutions can be made without creative labor. Therefore, the simple improvements made by those skilled in the art to the present application according to the disclosure of the present application should be within the protection scope of the present application. The above examples are preferred embodiments of the present application, and any similar processes and equivalent changes made should be within the protection scope of the present application.

Claims

1. A method for preparing fish gelatin polypeptide, characterized in that, The process includes three steps: acid soaking pretreatment, repeated homogenization and colloidal treatment, and ultrasound-assisted immobilized enzyme degradation treatment. Specifically, it includes the following steps: (1) After soaking fish maw in water, it is subjected to acid pretreatment to obtain liquid collagen; The fish maw is selected from the maw of the large-spined sea bass, the maw of the small-scaled croaker, and the long-snout maw. At least one of the following: colloid, croaker colloid glue, croaker colloid glue, and toad croaker glue; (2) Take the liquid collagen obtained in step (1), add a homogenizing agent to it, and perform repeated homogenization to obtain collagen micelles. The homogenizing agent is selected from at least one of propylene glycol alginate, glyceryl monosuccinate, polyglycerol ricinoleate, Tween, and lactic acid fatty acid glycerides. (3) The collagen micelles obtained in step (2) are mixed with immobilized enzymes and then subjected to ultrasonic treatment to obtain the fish collagen polypeptide. The carrier used for the immobilized enzyme is cellulose, which is selected from epoxidized cellulose or epoxidized fiber; the enzyme used for the immobilized enzyme is selected from at least one of papain, pepsin, figase, and trypsin.

2. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, The acid solution used in the acid immersion pretreatment is selected from at least one of sulfuric acid, hydrochloric acid, formic acid, and acetic acid; The mass ratio of the fish glue to the acid solution is 100:(0.2-10).

3. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, In step (1), the fish glue is soaked in water for 1-24 hours; The acid immersion pretreatment is performed at a temperature of 25-50℃ for 1-6 hours.

4. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, The mass ratio of the liquid collagen to the homogenizing agent is 100:(0.01-5).

5. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, In step (2), the process conditions for the repeated homogenization treatment are: feed temperature 20-80℃, flow rate 5-30mL / min, and pressure 10-100MPa; The homogenization process is repeated 3-8 times.

6. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, The mass ratio of the collagen micelles to the immobilized enzyme is 100:(0.05-1).

7. The method for preparing fish gelatin polypeptide according to claim 1, characterized in that, In step (3), the ultrasonic treatment process conditions are: ultrasonic frequency of 50-200MHz, power of 10-300W, temperature of 20-50℃, and treatment time of 10-120min.

8. A fish gelatin polypeptide, characterized in that, The fish collagen polypeptide is prepared by the method for preparing fish collagen polypeptide according to any one of claims 1 to 7; The fish collagen polypeptide has a molecular weight of 3.2-8.4 kDa and a molecular weight dispersion index of 0.2-0.

5. When the concentration of the fish collagen polypeptide in aqueous solution is 2 g / L, the zeta potential of its micelles is -24 mV to -30 mV, the average particle size of the micelles is 10-20 nm, and the particle size distribution coefficient of the micelles is 0.1-0.

2.

9. A fish gelatin polypeptide product, characterized in that, The fish glue polypeptide product comprises the fish glue polypeptide according to claim 8.

Images