Silver carp fishbone polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polysaccharide preparation, and particularly relates to chub fishbone polysaccharide as well as a preparation method and application thereof. The method comprises the following steps: drying and crushing pretreated silver carp processing byproducts to obtain fish bone meal; then mixing and stirring with petroleum ether, degreasing and drying to obtain degreased silver carp bone meal; the preparation method comprises the following steps: dissolving degreased silver carp bone meal in water, adjusting the pH value to 4-10, adding papain, carrying out water bath heating extraction, centrifugation and rotary evaporation concentration, adding absolute ethyl alcohol, standing, centrifuging, redissolving, dialyzing and freeze-drying to obtain the silver carp bone polysaccharide. The preparation process is simple, the silver carp fishbone polysaccharide is high in purity and extraction rate, and the prepared silver carp fishbone polysaccharide has good free radical scavenging capacity and can be suitable for extraction of fishbone polysaccharide in processing by-products of grass carp and other freshwater fishes, and the utilization rate of the processing by-products of freshwater fish products is increased.

Description

Technical Field

[0001] This application belongs to the field of polysaccharide preparation technology, specifically relating to a silver carp bone polysaccharide, its preparation method, and its application. Background Technology

[0002] my country boasts abundant fishery resources, with the scale of freshwater fish fishing and aquaculture continuously expanding and annual processing volumes increasing year by year. These byproducts are rich in collagen, lipids, minerals, and acidic mucopolysaccharides, among other nutrients and functional components. However, due to limitations in taste, safety, and digestibility, they are difficult to utilize directly as food, resulting in the ineffective conversion of their high-value bioactive substances into forms that can be absorbed and utilized by the human intestine. Specifically, the distribution of bioactive components varies significantly among different byproducts: collagen is mainly concentrated in fish scales, possessing excellent potential for skin repair and tissue regeneration, and has been widely used in the pharmaceutical and health care fields; fat is mainly concentrated in visceral tissues, rich in ω-3 and other unsaturated fatty acids, which have the function of assisting in regulating blood pressure and improving cardiovascular health. Furthermore, with in-depth research on freshwater fish processing byproducts, more and more bioactive macromolecules have been discovered, including polysaccharides, glycoproteins, and polysaccharide-protein complexes. These components have been proven to possess multiple physiological functions such as antioxidation, anticoagulation, immunomodulation, and antibacterial activity, showing broad development prospects. Therefore, systematically exploring the bioactive substances in freshwater fish processing by-products not only helps improve resource utilization efficiency but also provides new raw material sources for the development of functional foods, pharmaceuticals, and cosmetics.

[0003] Silver carp ( Hypophthalmichthys molitrix Silver carp (also known as silver carp) is one of my country's "four major freshwater fish," belonging to the Cyprinidae family and the genus *Silver carp*. It is widely distributed in large reservoirs, lakes, and river systems. This filter-feeding fish has low farming costs, requires no artificial feeding, and accounts for more than 15% of my country's total freshwater aquaculture production annually. Currently, the byproducts (especially the heads) generated after removing the meat from silver carp are mostly used as low-value feed or disposed of as waste, resulting in serious resource waste. The high-value-added active ingredients contained within these byproducts have not yet been fully developed and utilized.

[0004] Studies have shown that silver carp skulls contain a variety of functional components, among which polysaccharides have attracted widespread attention both domestically and internationally in recent years due to their significant antioxidant, antiproliferative, anticoagulant, and immunomodulatory biological activities. However, current research on silver carp bone polysaccharides is still in its early stages, particularly lacking in efficient extraction methods, fine structural analysis, structure-activity relationships, and mechanisms of action. Therefore, in-depth research on the optimization of extraction processes, physicochemical characterization, and bioactivity evaluation of silver carp bone polysaccharides will not only help improve the comprehensive utilization of silver carp processing byproducts but also provide theoretical basis and technical support for the development of novel bioactive substances and their applications in medicine, health care, and other fields. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a silver carp bone polysaccharide, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing polysaccharides from silver carp bones, comprising the following steps: S1. Add the by-products of silver carp processing to boiling water, remove the bits of meat attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, dry it in an oven after the ethanol evaporates, and pulverize it to obtain fish bone powder. S2. Mix the fish bone powder obtained in S1 with petroleum ether, stir to defatt the mixture, and obtain defatted fish bone powder after the petroleum ether evaporates. S3. Disperse the defatted fish bone powder obtained in S2 in water, adjust the pH to 4-10, add papain, heat and extract, centrifuge, concentrate the supernatant, slowly add anhydrous ethanol to the concentrate, let stand overnight, centrifuge, reconstitute, dialyze, and freeze dry to obtain the silver carp bone polysaccharide.

[0006] This invention uses an enzyme-assisted extraction method to extract polysaccharides from silver carp bones. The resulting fish bone polysaccharides have high purity and good free radical scavenging ability.

[0007] As a further preferred embodiment, the ratio of fish bone powder to petroleum ether is 1:5 g / mL to 1:10 g / mL.

[0008] As a further preferred embodiment, the ratio of silver carp skull powder to water is 1:10 g / mL to 1:60 g / mL.

[0009] As a further preferred embodiment, the amount of papain added is 1% to 10% of the silver carp skull powder.

[0010] As a further preferred embodiment, the heating extraction temperature is 30℃~50℃, and the extraction time is 15 min~180 min.

[0011] As a further preferred embodiment, the dialysis uses a dialysis bag with a molecular weight cutoff of 1000~5000 Da; The processed by-products of silver carp include the head, fins, spine, or tail.

[0012] Secondly, the present invention provides a silver carp bone polysaccharide, which is prepared by the above-described preparation method.

[0013] As a further preferred embodiment, the molecular weight of the silver carp bone polysaccharide is 3000 Da-8000 Da.

[0014] As a further preferred embodiment, the total sugar content of the silver carp bone polysaccharide is 20%~60%, the uronic acid content is 3%~30%, and the protein content is 1%~15%.

[0015] Thirdly, the present invention provides the application of the above-mentioned silver carp bone polysaccharide in the preparation of antioxidant and / or anti-aging products.

[0016] The beneficial effects of this invention are as follows: The preparation process provided by this invention is simple. First, a defatting treatment is performed to avoid the influence of lipids on subsequent extraction and purification processes, thus improving the purity of the prepared silver carp bone polysaccharide. Then, enzymatic hydrolysis is used to extract the silver carp bone polysaccharide, effectively breaking the binding bonds between proteins and polysaccharides, allowing for full extraction of the polysaccharide and improving resource utilization. The silver carp bone polysaccharide prepared by this invention has high purity and extraction rate, and also exhibits good free radical scavenging ability. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The figure shows the elution curve of silver carp bone polysaccharide by high performance liquid chromatography gel permeation chromatography. Figure 2 The image shows the infrared spectrum of polysaccharides from silver carp bones. Figure 3 The figure shown is a graph of the ABTS free radical scavenging rate of silver carp bone polysaccharide (5 mg / mL). Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] Example 1 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp head to boiling water, remove the meat scraps attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 60°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:10 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 50°C to obtain defatted fish head bone powder.

[0021] (2) Weigh 50 g of defatted silver carp skull powder, add 1500 mL of distilled water, adjust the pH to 5 with 1M HCl solution, add 2.5 g of papain (5% addition), heat in a 40℃ water bath for 30 min, centrifuge (4000 r / min, 10 min) to remove residue. Concentrate the supernatant to 100 mL by rotary evaporation, add 380 mL of anhydrous ethanol and let stand overnight. After 12 h, centrifuge to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h, and then freeze dry to obtain silver carp skull crude polysaccharide CBP-1 with a yield of 0.78%.

[0022] Example 2 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp spine to boiling water, remove the meat scraps attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 55°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:5 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 55°C to obtain defatted fish spine powder.

[0023] (2) Weigh 50 g of defatted silver carp spine powder, add 1000 mL of distilled water, adjust the pH to 6 with 1M HCl solution, add 3 g of papain (addition amount is 6%), heat in a water bath at 41℃ for 30 min, centrifuge (4000 r / min, 10 min) to remove residue, concentrate the supernatant to 110 mL by rotary evaporation, add 440 mL of anhydrous ethanol and let stand overnight, centrifuge after 12 h to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h and freeze dry to obtain silver carp spine crude polysaccharide CBP-2, with a yield of 0.18%.

[0024] Example 3 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp fins to boiling water, remove the meat scraps attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 50°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:8 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 55°C to obtain defatted fish fin bone powder.

[0025] (2) Weigh 30 g of defatted silver carp fin bone powder, add 960 mL of distilled water, adjust the pH to 7 with 1M HCl solution, add 3 g of papain (addition amount is 6%), heat in a water bath at 40℃ for 30 min, centrifuge (4000 r / min, 10 min) to remove residue, concentrate the supernatant to 60 mL by rotary evaporation, add 240 mL of anhydrous ethanol and let stand overnight, centrifuge after 12 h to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h and freeze dry to obtain silver carp fin bone crude polysaccharide CBP-3, with a yield of 0.16%.

[0026] Example 4 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp tail to boiling water, remove the scraps of meat attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 50°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:7 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 60°C to obtain defatted fish tail bone powder.

[0027] (2) Weigh 25 g of defatted silver carp tailbone powder, add 800 mL of distilled water, adjust the pH to 6 with 1M HCl solution, add 3 g of papain (6% addition), heat in a water bath at 42℃ for 30 min, centrifuge (3500 r / min, 10 min) to remove residue. Concentrate the supernatant to 52 mL by rotary evaporation, add 220 mL of anhydrous ethanol and let stand overnight. After 12 h, centrifuge to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h, and then freeze dry to obtain crude silver carp tailbone polysaccharide CBP-4 with a yield of 0.10%.

[0028] Comparative Example 1 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp head to boiling water, remove the meat scraps attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 60°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:10 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 50°C to obtain defatted fish head bone powder.

[0029] (2) Weigh 50 g of defatted silver carp skull powder, add 1500 mL of distilled water, adjust the pH to 10.5 with 1M NaOH solution, add 2.5 g of alkaline protease (addition amount is 5%), heat in a water bath at 40℃ for 30 min, centrifuge (4000 r / min, 10 min) to remove residue. Concentrate the supernatant to 100 mL by rotary evaporation, add 380 mL of anhydrous ethanol and let stand overnight. After 12 h, centrifuge to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h, and freeze dry to obtain crude polysaccharide from silver carp skull, with a yield of 0.45%, which is lower than the result of using papain in Example 1.

[0030] Comparative Example 2 A method for preparing polysaccharides from silver carp bones, which specifically includes the following steps: (1) Add fresh silver carp head to boiling water, remove the meat scraps attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, and dry it in an oven at 60°C after the ethanol evaporates, and then pulverize it to obtain fish bone powder; mix the fish bone powder with petroleum ether at a material-to-liquid ratio of 1:10 g / mL and stir to defatt it, and dry the petroleum ether in an oven at 50°C to obtain defatted fish head bone powder.

[0031] (2) Weigh 50 g of silver carp skull powder, add 1500 mL of distilled water, adjust the pH to 3 with 1M HCl solution, add 2.5 g of pepsin (5% addition), heat in a 40℃ water bath for 30 min, centrifuge (4000 r / min, 10 min) to remove residue. Concentrate the supernatant to 100 mL by rotary evaporation, add 380 mL of anhydrous ethanol and let stand overnight. After 12 h, centrifuge to collect the precipitate, redissolve in water, dialyze (molecular weight cutoff: 3500 Da) for 48 h, and then freeze dry to obtain crude polysaccharide from silver carp skull, with a yield of 0.21%, which is lower than the result of using papain in Example 1.

[0032] Example 5 Physicochemical properties and primary structure analysis of polysaccharides from silver carp bones (1) Determination of the chemical composition of polysaccharides from silver carp bones The results from the comparative and examples show that different enzymes have a significant impact on the yield of silver carp bone polysaccharides. The yields of extraction using papain, alkaline protease, and pepsin, in descending order, are papain, alkaline protease, and pepsin.

[0033] The chemical composition of silver carp bone polysaccharides prepared in Examples 1 to 4 was determined. Total sugar was determined using the phenol sulfate method, uronic acid using the carbazole sulfate method, and protein using the Coomassie brilliant blue method. Specific results are shown in Table 1. As can be seen from the table, the silver carp bone polysaccharides prepared by this method were all white or pale yellow flocculent substances. The total sugar, uronic acid, and protein contents varied somewhat among different parts of the silver carp. The total sugar and uronic acid contents of the four types of fish bone polysaccharides ranged from 30% to 70%. The crude polysaccharide from the head bone had the highest protein content, followed by the fins and spine, with the tail bone having the lowest.

[0034] Table 1. Appearance and chemical composition of silver carp bone polysaccharides (2) Determination of characteristic molecular weight distribution and size of polysaccharides from silver carp bones The molecular weight distribution and size of the silver carp bone polysaccharides prepared according to Examples 1 to 4 of this invention were determined by high performance liquid chromatography-gel permeation chromatography. The results are as follows: Figure 1 As shown. The main components of the polysaccharide extracted from silver carp bones were eluted after 16 min, and the molecular weight order was CBP-2, CBP-4, CBP-3, and CBP-1, with the molecular weight of polysaccharides from different parts ranging from approximately 3 kDa to 8 kDa.

[0035] (3) Analysis of characteristic functional groups of silver carp bone polysaccharides The infrared scanning spectra of silver carp bone polysaccharides prepared according to Examples 1 to 4 of the present invention are as follows: Figure 2 As shown. The four samples were at 3200–3600 cm⁻¹. -1 There is a broad and strong absorption peak, which is associated with carbohydrates. The stretching vibration absorption peak of OH is also present at 2900 cm⁻¹. -1 There is a relatively weak absorption peak near the 1637 cm⁻¹, which is the stretching vibration of the CH functional group of carbohydrates. These two sets of peaks together confirm that CBP is a carbohydrate. In addition, there is a peak at 1637 cm⁻¹. -1 The strong absorption peak near 1550 cm⁻¹ is due to the stretching vibration of the C=O group of the carboxyl group in -NHCOCH₃. -1 This is the variable-angle vibration of NH, 1415 cm. -1 The stretching vibration of the carboxyl group C=O is located near the point 1234 cm. -1 The stretching vibration near the point of S=O indicates that the silver carp polysaccharide contains sulfate ions. (1039 cm⁻¹) -1 and 927 cm -1The absorption peak indicates that the sugar ring is of the pyran type.

[0036] (4) Free radical scavenging effect of silver carp bone polysaccharide The ABTS free radical scavenging ability of the silver carp bone polysaccharide prepared according to Examples 1 to 4 of the present invention is as follows: Figure 3 As shown, under the condition of a concentration of 5 mg / mL, the ABTS free radical scavenging rates of fish bone polysaccharides obtained from different parts were as follows: CBP-2 (83.8±1.1%), CBP-3 (68.3±0.2%), CBP-1 (66.0±1.3%), and CBP-4 (62.1±1.2%), indicating that the fish bone polysaccharides obtained by this invention have good antioxidant activity.

[0037] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for preparing polysaccharides from silver carp bones, characterized in that, Includes the following steps: S1. Add the by-products of silver carp processing to boiling water, remove the bits of meat attached to the fish bones while cooking, add anhydrous ethanol to cover the fish bones, soak at room temperature for more than 12 hours, filter, place the filter residue in a fume hood, dry it in an oven after the ethanol evaporates, and pulverize it to obtain fish bone powder. S2. Mix the fish bone powder obtained in S1 with petroleum ether, stir to defatt the mixture, and obtain defatted fish bone powder after the petroleum ether evaporates. S3. Disperse the defatted fish bone powder obtained in S2 in water, adjust the pH to 4-10, add papain, heat and extract, centrifuge, concentrate the supernatant, slowly add anhydrous ethanol to the concentrate, let stand overnight, centrifuge, reconstitute, dialyze, and freeze dry to obtain the silver carp bone polysaccharide.

2. The preparation method according to claim 1, characterized in that, The ratio of fish bone powder to petroleum ether in S2 is 1:5 g / mL to 1:10 g / mL.

3. The preparation method according to claim 1, characterized in that, The ratio of silver carp bone powder to water in S3 is 1:10 g / mL to 1:60 g / mL.

4. The preparation method according to claim 3, characterized in that, The amount of papain added in S3 is 1% to 10% of the mass of silver carp bone powder.

5. The preparation method according to claim 1, characterized in that, The temperature for heating and extraction in S3 is 30℃~50℃, and the extraction time is 15 min~180 min.

6. The preparation method according to claim 1, characterized in that, In S3, dialysis is performed using dialysis bags with a molecular weight cutoff of 1000-5000 Da.

7. A silver carp bone polysaccharide, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

8. The silver carp bone polysaccharide according to claim 7, characterized in that, The molecular weight of the silver carp bone polysaccharide is 3000 Da-8000 Da.

9. The silver carp bone polysaccharide according to claim 8, characterized in that, The total sugar content of the silver carp bone polysaccharide is 20%~60%, the uronic acid content is 3%~30%, and the protein content is 1%~15%.

10. The use of silver carp bone polysaccharide according to any one of claims 7-9 in the preparation of antioxidant and / or anti-aging products.

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