Chondroitin sulfate C derived from fish bones, preparation method and application thereof

Abstract

The invention relates to a chondroitin sulfate C derived from fish bones, a preparation method and an application thereof. The specific preparation method of the chondroitin sulfate C derived from fish bones comprises the following steps: dissecting a fish body, removing the skin and flesh, obtaining fish bones, and performing defatting, alkali treatment, papain enzymolysis, ethanol precipitation and anion exchange resin elution purification to obtain high-purity chondroitin sulfate C. The high-purity chondroitin sulfate C exhibits good biocompatibility and the effect of promoting embryo hatching in the activity evaluation of a zebrafish embryo model. The chondroitin sulfate C derived from fish bones of the invention has the characteristics of rich raw material sources and high-value reuse of by-products, and provides an important source for the preparation of novel chondroitin sulfate C.

Description

Technical Field

[0001] The invention relates to chondroitin sulfate C derived from fish bones, a preparation method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] The repeating disaccharide units of chondroitin sulfate (CS) are mainly composed of D-GlcA and D-GalNAc. According to the different sulfation sites and degrees, it is divided into CSA, CSC, CSD and CSE, with a molecular weight between 5-100 kDa. CS is widely present in mammals and invertebrates. CS mainly comes from the cartilage of terrestrial animals, such as cattle, pigs and poultry, or from the cartilage of marine fish, such as sharks, rays and sturgeons. The sulfation site and degree of CS are mainly affected by factors such as the source, tissue structure and collection time of the organism. The dominance of GalNAc sulfation at position 4 (CS-4S) is observed in terrestrial animal CS, and the structural diversity of marine CS is observed from the limited data of marine species. Fish CS contains CS-4S and CS-6S, but the ratio of 4S / 6S varies greatly, ranging from 2 in tuna and monkfish, 1.0-1.4 in rays, and generally less than 0.5 in sharks. Based on these data, sharks appear to be the only source of CS-6S; however, its production raw materials are highly restricted because sharks are endangered species. Therefore, we have an urgent need to find more advantageous alternatives, and fish by-products with high CS-6S content should be the best source of CS-6S.

[0003] Flatfish are bony fish with an asymmetrical, flat body. They lie on their side on the sandy, gravel or mixed bottom seas. Their classification is based on which side of the body the eyes are on. Those with eyes on the left are called "flounder" and those on the right are called "flatfish". Deep-sea flatfish are fixed fishing resources, and the fishing areas are mostly in the northern Atlantic and northern Pacific waters. Since 2012, the annual catch has exceeded 1 million tons (FAO, 2020). In 1992, my country introduced the farming of flatfish for the first time. The flatfish farming industry has risen rapidly. By 2019, the annual output of flatfish aquaculture in my country has exceeded 120,000 tons (2020 China Fisheries Statistical Yearbook). In general, the natural resources and breeding resources of flatfish are very abundant.

[0004] Currently, the fish tissues disclosed for preparing chondroitin sulfate are mostly cartilage tissues. In addition, a Chinese patent application (publication number: CN111647098) discloses a chondroitin sulfate / dermatan sulfate derived from fish skin and its application. The chondroitin sulfate / dermatan sulfate derived from the skin of three kinds of fish (longli fish, greenfin pufferfish, flatfish) is mainly type A. Therefore, the preparation and application of chondroitin sulfate C derived from bony fish bones have not been reported. Chondroitin sulfate C has many active functions such as good treatment of osteoarthritis, enhancement of body immunity, anti-oxidation and anti-tumor, which makes it have broad potential in the development of functional foods and drugs.

[0005] Through the above analysis, the problems and defects of the existing technology are: shark as a traditional source of chondroitin sulfate C is facing a crisis of resource shortage, and the content of CS-6S in chondroitin sulfate from other sources is relatively low. At present, there is no new source of chondroitin sulfate C to replace shark.

[0006] The significance of solving the above problems and defects is that the present invention utilizes flounder bones as a new source, and obtains high-purity chondroitin sulfate C through defatting, alkali treatment, papain hydrolysis, ethanol precipitation and anion exchange resin elution purification. It has the characteristics of rich raw material sources and high-value reuse of by-products, thereby expanding the source of chondroitin sulfate C. Summary of the invention

[0007] In view of the shortcomings of the prior art in preparing chondroitin sulfate C, the present invention provides a method for preparing chondroitin sulfate C from fish bones, which can efficiently utilize fish bone waste generated during fish meat processing to obtain high-purity chondroitin sulfate C to supplement the market demand for safe sources of medicinal chondroitin sulfate.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0009] A chondroitin sulfate C derived from fish bones, with a molecular weight of 24kD-28KD, a disaccharide composition mainly composed of CS-6S, with a content of 49%-66%, and also containing CS-4S, CS-0S, and CS-2S6S, with contents of 23%-39%, 6%-10%, and 1%-3% respectively.

[0010] A chondroitin sulfate C derived from fish bones, wherein the fish bones come from one or more of Canadian gray flounder bones, Canadian yellowtail flounder bones, stone flounder bones, and stone flounder bones.

[0011] Preferably, the fish bone is chondroitin sulfate C derived from Canadian grey flounder fish bone, with a molecular weight of 24kD, and the disaccharide composition is mainly CS-6S, accounting for 66%, and there are also CS-4S, CS-0S, and CS-2S6S, with contents of 23%, 9%, and 2%, respectively.

[0012] Preferably, the fish bone is chondroitin sulfate derived from Canadian yellowtail flounder fish bone, with a molecular weight of 28kD, and the disaccharide composition is mainly CS-6S, accounting for 55%, and there are also CS-4S, CS-0S, and CS-2S6S, with contents of 35%, 9%, and 2%, respectively.

[0013] Preferably, the fish bone is chondroitin sulfate C derived from flounder fish bone, with a molecular weight of 23kD, and the disaccharide composition is mainly CS-6S, accounting for 58%, and there are also CS-4S, CS-0S, and CS-2S6S, with contents of 34%, 6%, and 2%, respectively.

[0014] Preferably, the fish bone is chondroitin sulfate C derived from turbot fish bone, with a molecular weight of 25kD, and the disaccharide composition is mainly CS-6S, accounting for 49%, and there are also CS-4S, CS-0S, and CS-2S6S, with contents of 39%, 10%, and 2%, respectively.

[0015] A method for preparing chondroitin sulfate C derived from fish bones comprises the following steps:

[0016] (1) defatting: dissecting a fresh fish, removing the skin and flesh to obtain fish bone tissue, drying and crushing the fish bone tissue to obtain fish bone powder, soaking the fish bone powder in an organic solvent to defatted the fish bone powder, taking it out and drying it to obtain defatted fish bone powder;

[0017] (2) Alkali treatment: add sodium hydroxide solution to defatted fish bone powder, stir at room temperature for 12 hours, and neutralize with hydrochloric acid after alkali treatment to obtain a neutralized solution;

[0018] (3) Enzymolysis: Add enzymolysis buffer to the neutralized solution obtained in (2), adjust the pH to 6.0-7.0, adjust the temperature to 65° C., add papain, stir for enzymolysis, and then boil to inactivate the enzyme to obtain an enzymolysis solution;

[0019] (4) ethanol precipitation: the enzymatic hydrolyzate obtained in (3) is centrifuged, the supernatant is concentrated to obtain a concentrated solution, and ethanol is added for precipitation. After centrifugation, the precipitate is collected, and the precipitate is re-dissolved in distilled water, dialyzed, and freeze-dried to obtain crude chondroitin sulfate polysaccharide;

[0020] (5) Purification: The crude polysaccharide obtained in (4) was re-dissolved in distilled water, and gradient eluted using distilled water and sodium chloride solutions of different concentrations as mobile phases. The product was separated and purified using Q-Sepharose Fast Flow strong anion exchange resin. The eluate was dialyzed, concentrated under reduced pressure, and freeze-dried to obtain purified chondroitin sulfate. The product was detected using LC-MS technology to determine the component with a high content of chondroitin sulfate C.

[0021] Preferably, in step (1), the organic solvent is acetone or chloroform / methanol (3:1, V / V), 20 to 25 times the volume of the organic solvent is added, and degreasing is performed by immersion for 12 to 24 hours or by stirring for 10 to 12 hours.

[0022] Preferably, in step (2), the concentration of the alkaline solution is 1% to 3% (m / V), the concentration of the hydrochloric acid is 3 to 5M, and the base is sodium hydroxide.

[0023] Preferably, in step (3), the buffer solution used for enzymolysis is a mixed solution containing 0.2M sodium acetate, 10mM EDTA-2Na and 10mM cysteine, and the amount of papain used is 1-10% of the mass of defatted fish bone powder.

[0024] Preferably, in step (4), the volume of ethanol added during ethanol precipitation is 3 to 5 times that of the concentrated solution, and the molecular weight cutoff of the dialysis bag used for dialysis is 1000 to 3000 Da.

[0025] Preferably, in step (5), Q-Sepharose Fast Flow strong anion exchange resin is used for separation and purification with a gradient elution of a sodium chloride aqueous solution with a concentration of 0.6 to 1.0 mol / L, and 3 to 8 column volumes are eluted successively. The eluate is dialyzed, desalted, concentrated and freeze-dried to obtain chondroitin sulfate.

[0026] The invention discloses an application of fish bone-derived chondroitin sulfate C in promoting the growth of an organism, wherein the organism is a zebrafish.

[0027] The advantages and beneficial effects of the present invention are:

[0028] 1. The present invention discloses for the first time chondroitin sulfate from fish bones of four kinds of flatfish (including Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot), and the chondroitin sulfate structure and disaccharide composition prepared from different types of fish bones are also different. In the present invention, the four kinds of fish bones are all inedible scraps, which are usually discarded as waste or made into low value-added products. The fish bones of Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot are used to prepare chondroitin sulfate C, which can increase its added value, turn waste into treasure, and expand the raw material source of chondroitin sulfate C.

[0029] 2. Chondroitin sulfate C is a special sulfated form of chondroitin sulfate. Previous reports have all used fish cartilage as the main source, and only shark cartilage is its commercial source. The present invention discloses for the first time a method for preparing chondroitin sulfate from the bones of four bony fish, namely, Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot. The structure of all of them is mainly CS-6S, with a molecular weight between 23-28 kDa, and can be used as a substitute for shark CSC.

[0030] 3. The chondroitin sulfate prepared by the present invention from the bones of Canadian gray flounder, Canadian yellowtail flounder, stone flounder and turbot is specific in structure and disaccharide composition, has an embryo survival rate of more than 90% in the activity evaluation using zebrafish as a model, and an embryo hatching rate of more than 40% higher than that of the control group, thereby ensuring the safety of the product and providing raw materials and theoretical data support for the research and development of new functional foods and drugs that promote growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The molecular weight detection spectrum of chondroitin sulfate C derived from fish bones of four kinds of flatfish (A Canadian gray flounder, B Canadian yellowtail flounder, C stone flounder, D turbot) prepared in the present invention: the abscissa in the figure is the retention time, the ordinate is the relative proportion, LS represents eighteen-angle laser scattering, and dRI represents the difference;

[0032] Figure 2 H NMR spectra of chondroitin sulfate C derived from fish bones of four kinds of flatfish (A Canadian gray flounder, B Canadian yellowtail flounder, C stone flounder, D turbot) prepared in the present invention: the abscissa in the figure is the chemical shift;

[0033] Figure 3 MRM chromatograms of disaccharide labeled by chondroitinaseABC and 2-AMAC derivatization of chondroitin sulfate C derived from fish bones of four kinds of flatfish (A Canadian gray flounder, B Canadian yellowtail flounder, C stone flounder, D turbot) prepared in the present invention;

[0034] Figure 4 A bar graph showing the effects of four types of flounder bone-derived chondroitin sulfate C prepared by the present invention and commercial CSA and CSC on the survival rate of zebrafish embryos;

[0035] Figure 5 A bar graph showing the effect of chondroitin sulfate C prepared from 4 types of flatfish bones and commercial CSA and CSC on the hatching of zebrafish embryos; DETAILED DESCRIPTION

[0036] The following is a comprehensive disclosure of the technical solutions for implementing the present invention in conjunction with the embodiments, but the contents of the embodiments do not limit the application scope of the present invention.

[0037] Source of raw materials:

[0038] Canadian yellowtail flounder (Limandaferruginea) and Canadian grey flounder (Cleisthenes herzensteini, Grey Plaice) were purchased from Qingdao Deep Sea Diary Qingdao Deep Sea Diary Refrigeration Co., Ltd.; turbot (Scophthalmusmaximus) and stone flounder (Platichthysbicoloratus) were purchased from Qingdao Tuandao Aquatic Products Market.

[0039] Example 1

[0040] The method for preparing chondroitin sulfate C from fish bones of four kinds of flatfish (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot) comprises the following steps:

[0041] (1) Defatting: fish bones are obtained by dissecting fresh fish bodies, and the fish bones are dried and crushed to obtain fish bone powder. 10 g of the fish bone powder is soaked in 20 to 25 volumes of chloroform / methanol (3:1, V / V) for 12 to 24 hours for defatting, and then taken out and dried to obtain defatted fish bone powder;

[0042] (2) Alkali treatment: add 1% to 3% (m / V) sodium hydroxide solution to the defatted fish bone powder, stir at room temperature for 12 hours, and neutralize with 3 to 5M hydrochloric acid after the alkaline treatment.

[0043] (3) Enzymatic hydrolysis: add enzymatic hydrolysis buffer (0.2 M sodium acetate, 10 mM EDTA-2Na and 10 mM cysteine) to the neutralized solution obtained in (2), adjust the pH to 6.0-7.0, adjust the temperature to 65° C., add papain at 1-10% of the dry weight of defatted fish bone powder, stir for enzymatic hydrolysis and then boil to inactivate the enzyme;

[0044] (4) Ethanol precipitation: centrifuge the enzymatic hydrolysate obtained in (3), concentrate the supernatant, add 3 to 5 times the amount of ethanol to precipitate the supernatant, collect the precipitate after centrifugation, re-dissolve the precipitate with distilled water, dialyze with a 1000 to 3000 Da dialysis bag, freeze-dry, and weigh to obtain crude chondroitin sulfate polysaccharide;

[0045] (5) Purification: The crude polysaccharide obtained in (4) was re-dissolved in distilled water, and gradient eluted with distilled water and sodium chloride solutions of different concentrations as mobile phases. The polysaccharide was separated and purified by Q-Sepharose Fast Flow strong anion exchange resin, and gradient eluted with 0.6-1.0 mol / L sodium chloride aqueous solution for 3-8 column volumes. The eluate was dialyzed with a dialysis bag having a molecular weight cutoff of 3000-10000 Da, concentrated under reduced pressure, and freeze-dried to obtain purified chondroitin sulfate from bones of four fish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot).

[0046] Example 2

[0047] Analysis of the molecular weight of chondroitin sulfate in bones of four flatfish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot)

[0048] Chondroitin sulfate from bones of four flatfish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot) was quenched with 0.1 mol / L NaNO 3 The solution was prepared into 5 mg / mL, filtered through a 0.22 μm water membrane, and then analyzed by HPLC.

[0049] The detection conditions are as follows: chromatographic columns: ShodexOhpak SB-803HQ (8.0μm×300mm) and ShodexOhpak PB-802.5HQ (8.0μm×300mm) chromatographic columns in series, mobile phase: 0.1mol / L NaNO 3 , solution, flow rate: 0.6 mL / min, column temperature: 35 °C, injection volume: 100 μL, detector: differential detector (RI) and multi-angle laser light scattering detector (MALLS) used in series, signal acquisition: 45 min, software processing: Astra 5.3.4.20 software.

[0050] Table 1 is a statistical table of the molecular weight determination results of purified chondroitin sulfate components from fish bones of four species of flatfish (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot)

[0051]

[0052] The experimental results are as follows Figure 1 As shown, the peaks of the four chondroitin sulfate HPGPC-MALLS detections were symmetrical and single, with a molecular weight of 23-28 kDa and a polydispersity index (PDI) below 1.2, further indicating that the products had high purity and high molecular weight uniformity.

[0053] Example 3

[0054] Nuclear magnetic resonance spectroscopy analysis of chondroitin sulfate in bones of four flatfish species (Canadian gray sole, Canadian yellowtail sole, stone sole, turbot)

[0055] 10 mg of chondroitin sulfate from the bones of four types of flatfish (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot) was weighed and mixed with 400 μL D 2 D O, and concentrated to complete dryness under reduced pressure. Repeat the concentration three times, and use 600 μL D for the last time. 2 O was dissolved, and the supernatant was transferred to a nuclear magnetic resonance tube after high-speed centrifugation for 10 min. Nuclear magnetic resonance spectroscopy was analyzed using an Agilent DD2600MHz superconducting nuclear magnetic resonance spectrometer.

[0056] Test results such as Figure 2As shown, 4 types of chondroitin sulfate 1 The H-NMR spectrum is consistent with that of standard chondroitin sulfate C and is concentrated in two spectral regions. One is between 1.9 and 2.0 ppm, showing the methyl signal of the acetyl group, and the two acetamidomethyl signals come from GalNAc 4S (1.99 ppm) and GalNAc 6S (1.98 ppm), respectively. The other is between 3 and 5 ppm, where the signals of hydrogen on all sugar rings are shown. The GalNAc6S H4 and GalNAc4SH4 signals are the signature peaks of CS-6S and CS-4S, with chemical shift values ​​of 4.14 ppm and 4.71 ppm, respectively. There are no signals of 0.5-1.5 ppm (aliphatic amino acids) and 7.0-8.5 ppm (aromatic amino acids), indicating that the purity of CS is very high.

[0057] Example 4

[0058] Fine structure analysis of chondroitin sulfate in bones of four flatfish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot)

[0059] 1 μg of chondroitin sulfate from different sources was dissolved in 100 μL buffer, 30 mU of Chondroitinase ABC was added, and the mixture was enzymatically hydrolyzed overnight at 37°C on a shaker. After the reaction was completed, the mixture was incubated at 100°C for 10 min, and the supernatant was centrifuged and freeze-dried for later use. Unsaturated chondroitin sulfate disaccharide standards (CS-0S, CS-2S, CS-4S, CS-6S, CS-NS, CS-2S4S, CS-2S6S, CS-4S6S, CS-TriS) were prepared into 1 μg / μL aqueous solution, and then 5 μL of each was taken into a 1.5 mL EP tube, and the liquid was freeze-dried for later use. The freeze-dried sample was derivatized with 2-AMAC (2-aminoacridone) and then analyzed by reversed-phase high performance chromatography with multiple reaction detection scanning mass spectrometry (RP-C18-MS / MS-MRM) mode to analyze the types of unsaturated disaccharides degraded by CS. Among them, Chondroitinase ABC can completely degrade chondroitin sulfate into unsaturated disaccharides. The detection conditions are as follows: the liquid chromatograph is a Thermo UltiMate 3000 liquid chromatograph, the chromatographic column is a Phenomenex Luna Omega 1.6μm Polar C18 column (50x 2.1mm, 1.6μm), the column temperature is 45°C, the mobile phase is A: 10mM ammonium bicarbonate (pH=6) and methanol, the flow rate is 0.12mL / min, and the elution gradient is 10-100% methanol. The mass spectrometer is a TSQ Quantiva mass spectrometer, negative ion detection mode, ESI ion source, and the scanning mode is multiple reaction monitoring (MRM) mode.

[0060] Test results such as Figure 3As shown in the figure, the disaccharide composition of chondroitin sulfate from the bones of four kinds of flatfish (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot) is the same, mainly CS-6S, all of which are chondroitin sulfate C, but the disaccharide composition of chondroitin sulfate C from different sources is different. In the disaccharide composition of chondroitin sulfate from the bones of Canadian gray flounder, CS-6S, CS-4S, CS-0S and CS-2S6S account for 66%, 35%, 9% and 2%, respectively. In the disaccharide composition of chondroitin sulfate from the bones of Canadian yellowtail flounder, CS-6S, CS-4S, CS-0S and CS-2S6S account for 55%, 35%, 9% and 2%, respectively. In the disaccharide composition of chondroitin sulfate from the bones of stone flounder, CS-6S, CS-4S, CS-0S and CS-2S6S account for 58%, 34%, 6% and 2%, respectively. The chondroitin sulfate disaccharide composition from turbot bones included CS-6S, CS-4S, CS-0S and CS-2S6S, accounting for 49%, 39%, 10% and 2%, respectively.

[0061] Example 5

[0062] Analysis of the effects of chondroitin sulfate C from bones of four flatfish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, turbot) on zebrafish embryonic development

[0063] Six chondroitin sulfate samples were selected, including bovine CSA, shark cartilage CSC, and chondroitin sulfate C from fish bones of four flatfish species (Canadian gray flounder, Canadian yellowtail flounder, stone flounder, and turbot). The samples were prepared into 20 μg / mL using embryo culture medium ERS. Wild-type (WT) zebrafish embryos at the two-cell stage were incubated, and the survival and growth status were observed and the hatching rate was statistically analyzed at the 48h developmental stage (48hpf). The survival rate was statistically analyzed at the 72h developmental stage (72hpf). All experiments were repeated three times in parallel.

[0064] The results are shown in Figure 4 and Figure 5 The statistical results of the survival rate of WT zebrafish embryos incubated with chondroitin sulfate from different sources at 72 hpf are shown in Figure 4In the experiment, the survival rate of all zebrafish incubated with chondroitin sulfate was close to that of the embryo culture medium control group (ERS), all of which were higher than 90%. The observation showed that the life characteristics of zebrafish were normal, indicating that chondroitin sulfate had no fatal toxicity to the growth and development of zebrafish and had good biocompatibility. The chondroitin sulfate from different sources was observed and statistically analyzed for the hatching (embryo rupture) of WT zebrafish embryos at 48hpf. The analysis showed that the hatching rate of all chondroitin sulfate groups (>40%) was higher than that of the ERS control group (<20%), indicating that chondroitin sulfate from different sources can accelerate the hatching of zebrafish embryos and have a certain promoting effect on the growth of zebrafish. In addition, the analysis found that the ability of chondroitin sulfate from flounder bones to promote the hatching of zebrafish embryos was slightly higher than that of commercial CSA and CSC. The possible reason is that the molecular weight of flounder chondroitin sulfate is between 23-28kDa, which is smaller than the molecular weight of commercial chondroitin sulfate (about 50kDa), which is more conducive to the absorption and utilization of zebrafish embryos. The above results show that chondroitin sulfate from different sources has no biological toxicity and good biocompatibility. Moreover, compared with the control group, chondroitin sulfate from flatfish bones has a significant effect on promoting the hatching of zebrafish and the growth of zebrafish, which expands the function and medical application range of chondroitin sulfate and provides raw materials and theoretical basis for the development of new growth-promoting carbohydrate functional foods and drugs.

[0065] The chondroitin sulfate prepared by the present invention from the bones of Canadian gray flounder, Canadian yellowtail flounder, stone flounder and turbot has an embryo survival rate of more than 90% in an activity evaluation using zebrafish as a model, and an embryo hatching rate that is more than 40% higher than that of the control group, thereby ensuring the safety of the product and providing raw materials and theoretical data support for the research and development of new growth-promoting functional foods and drugs.

[0066] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. The application of chondroitin sulfate C derived from fish bones in promoting biological growth is characterized by: The organism is zebrafish; the molecular weight of the chondroitin sulfate C is 23kD-28KD, the disaccharide composition is mainly CS-6S, with a content of 49%-66%, and there are also CS-4S, CS-0S, and CS-2S6S, with contents of 23%-39%, 6%-10%, and 1%-3%, respectively; the fish bones are derived from one or more of Canadian gray flounder bones, Canadian yellowtail flounder bones, and stone flounder bones.

2. The use of chondroitin sulfate C derived from fish bones in promoting biological growth according to claim 1, characterized in that: The fish bones are chondroitin sulfate C derived from Canadian gray flounder bones, with a molecular weight of 24 kD. The disaccharide composition is mainly CS-6S, accounting for 66%. In addition, CS-4S, CS-0S, and CS-2S6S are also present, with contents of 23%, 9%, and 2%, respectively.

3. The use of chondroitin sulfate C derived from fish bones in promoting biological growth according to claim 1, characterized in that: The fish bones are chondroitin sulfate derived from Canadian yellowtail flounder bones, with a molecular weight of 28 kD. The disaccharide composition is mainly CS-6S, accounting for 55%. In addition, CS-4S, CS-0S, and CS-2S6S are also present, with contents of 35%, 9%, and 2%, respectively.

4. The use of chondroitin sulfate C derived from fish bones in promoting biological growth according to claim 1, characterized in that: The fish bone is chondroitin sulfate C derived from flounder fish bone, with a molecular weight of 23 kD. The disaccharide composition is mainly CS-6S, accounting for 58%. In addition, CS-4S, CS-0S, and CS-2S6S are also present, with contents of 34%, 6%, and 2%, respectively.

5. The method for preparing chondroitin sulfate C from fish bones for use as claimed in claim 1, characterized in that: The steps include: (1) Defatting: fresh fish is dissected, the skin and meat are removed to obtain fish bone tissue, which is then dried and crushed to obtain fish bone powder, which is then soaked in an organic solvent for defatting, and then taken out and dried to obtain defatted fish bone powder; (2) Alkali treatment: add sodium hydroxide solution to defatted fish bone powder and stir at room temperature for 12 h. After alkali treatment, neutralize with hydrochloric acid to obtain a neutralized solution. (3) Enzymatic hydrolysis: add enzymatic hydrolysis buffer to the neutralized solution obtained in (2), adjust the pH to 6.0-7.0, adjust the temperature to 65°C, add papain, stir for enzymatic hydrolysis, and then boil to inactivate the enzyme to obtain an enzymatic hydrolyzate; (4) Ethanol precipitation: The enzymatic hydrolysate obtained in (3) is centrifuged, and the supernatant is concentrated to obtain a concentrated solution, and then ethanol is added for precipitation. After centrifugation, the precipitate is collected, and the precipitate is re-dissolved in distilled water, dialyzed, and freeze-dried to obtain crude chondroitin sulfate polysaccharide; (5) Purification: The crude polysaccharide obtained in (4) was re-dissolved in distilled water, and gradient eluted using distilled water and sodium chloride solutions of different concentrations as the mobile phase. The polysaccharide was separated and purified using Q-Sepharose Fast Flow strong anion exchange resin. The eluate was dialyzed, concentrated under reduced pressure, and freeze-dried to obtain purified chondroitin sulfate. The LC-MS technique was used to detect and identify the component with a high content of chondroitin sulfate C.

6. The method for preparing chondroitin sulfate C derived from fish bones for use according to claim 5, characterized in that: In step (1), the organic solvent is acetone or chloroform / methanol, the volume ratio of chloroform to methanol is 3:1, 20 to 25 times the volume of the organic solvent is added, and the degreasing is carried out by immersion for 12 to 24 hours or by stirring for 10 to 12 hours; in step (2), the concentration of the alkaline solution is 1% to 3% m / V, the concentration of the hydrochloric acid is 3 to 5 M, and the alkali is sodium hydroxide; in step (3), the buffer solution used for enzymatic hydrolysis is a mixed solution containing 0.2 M sodium acetate, 10 mM EDTA-2Na and 10 mM cysteine, and the amount of papain used is 1 to 10% of the mass of the defatted fish bone powder.

7. The method for preparing chondroitin sulfate C derived from fish bones for use according to claim 5, characterized in that: In step (4), the volume of ethanol added during ethanol precipitation is 3 to 5 times that of the concentrated solution, and the dialysis bag used for dialysis has a molecular weight cutoff of 1000 to 3000 Da; in step (5), Q-Sepharose Fast Flow strong anion exchange resin is used for separation and purification with a gradient elution of a sodium chloride aqueous solution with a concentration of 0.6 to 1.0 mol / L, and elution is performed for 3 to 8 column volumes. The eluate is dialyzed, desalted, concentrated and freeze-dried to obtain chondroitin sulfate.

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