A method for large-scale preparation of high-purity chondroitin sulfate and chondroitin sulfate calcium from heavy metal contaminated squid cartilage

Abstract

The present application relates to a kind of chondroitin sulfate calcium and high purity chondroitin sulfate from heavy metal pollution of squid cartilage The present application is with heavy metal pollution of different sea areas as raw material, first remove chondroitin and mucopolysaccharide, and complete deacetylation reaction, obtain the deacetylation degree ≥90% of squid cartilage chitosan;Then squid cartilage chitosan is degraded into chondroitin, after hydrolysis reaction, one side stirring heat dissipation, one side with saturated calcium hydroxide solution stirring chelation reaction, further low temperature centrifugation method removes metal precipitate;Supernatant is obtained by cation exchange chromatography / resin fractionation chondroitin sulfate calcium component and chondroitin component, then obtain the high purity chondroitin sulfate calcium and chondroitin sulfate with content ≥95%.The present application method is widely used, both not affected by the geographical source of squid cartilage raw material, also not affected by heavy metal pollution of squid cartilage raw material, all obtain good preparation effect.

Description

Technical Field

[0001] The invention relates to a large-scale preparation method of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate from heavy metal-contaminated squid cartilage, involves chemical extraction and metal chelation technology, and belongs to the field of medicines. Background Art

[0002] Squid is a common cephalopod from the phylum Mollusc and is a good raw material for aquatic product processing. During the squid processing, a large amount of waste by-product - squid cartilage - is often produced. Although some cartilage by-products can be converted into fishmeal feed, these cartilage scraps generated during the aquatic product processing process are often not fully utilized. Due to its perishable nature, the usual method of disposal is to bury it, which may have an adverse impact on the environment. Therefore, strengthening the efficient and comprehensive utilization of the waste by-product squid cartilage can not only turn waste into treasure and increase its scientific and technological added value, but also reduce the cost of waste treatment, which is of great significance to promoting the development of the squid industry and protecting the environment.

[0003] With the rapid growth of squid imports, my country has become the world's largest offshore squid fishing nation, and has further transformed into the world's largest squid importer, processor, and domestic marketer. Currently, squid caught primarily comes from the North Pacific, South Pacific, and North Indian Oceans. In recent years, with the development of industry, marine environmental pollution, particularly heavy metal pollution, has become increasingly serious. Squid organisms absorb liquid heavy metal ions from marine waters, which are easily accumulated and fixed in squid cartilage, further exacerbating the heavy metal contamination of squid cartilage.

[0004] Relevant research shows that squid cartilage accounts for about 2% of its body weight, and its active ingredients are proteins (collagen, core protein, etc.) and carbohydrates (chondroitin sulfate, chitin, etc.). Currently, literature and patents related to squid cartilage are essentially focused on the separation, purification, and utilization of the active ingredients of squid cartilage (chondroitin sulfate, chitin, chitosan, collagen, etc.) (e.g., CN202110439789.0, CN202011521963.8, CN202110439798.X, CN201811558088.3, ​​etc.). Few people have noticed that discarded squid cartilage can actually be used as a raw material source for glucosamine (glucosamine). More importantly, how to effectively utilize heavy metal-contaminated squid cartilage and develop an efficient, environmentally friendly, and low-cost production process to make it an effective raw material source for glucosamine is a scientific problem that urgently needs to be solved and has important research value. Summary of the Invention

[0005] In view of this, the present invention is based on the goal of achieving high-value utilization of heavy metal-contaminated squid cartilage from different sea areas, and provides a large-scale preparation method of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate with low production cost, simple production operation, feasible production process, and environmentally friendly production process. It not only solves the problem of environmental pollution and resource waste caused by squid processing waste - squid cartilage, but also develops a high-purity glucosamine sulfate that can be used in new anti-inflammatory and analgesic active drugs.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A large-scale preparation method of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate from heavy metal-contaminated squid cartilage comprises the following steps:

[0008] (1) High-speed crushing and high-temperature alkali treatment

[0009] The squid cartilage detected to be contaminated with heavy metals is washed with clean water, dried in a hot air drying oven at 70-90°C for 2-5 hours, cut into 3-5 cm small segments, placed in a traditional Chinese medicine grinder, and pulverized for 5-8 minutes; the pulverized squid cartilage powder is added to a reactor, and a sodium hydroxide solution 4-10 times the weight of the squid cartilage is added. The reaction temperature is 85-100°C, and the reaction is stirred for 2-6 hours, wherein the mass volume concentration of the sodium hydroxide solution is 10-30%.

[0010] (2) Cooling, centrifugation and cleaning and desalting

[0011] After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to 5-6.5 with sulfuric acid solution, and then centrifuged at high speed using a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of ≥90%; the solid was then washed with deionized water until there was no sodium ion (Na + ) and chloride ions (Cl - ) until it is detected;

[0012] (3) High-temperature sulfuric acid hydrolysis reaction, chelation reaction of glucosamine and calcium during the cooling stage, and low-temperature centrifugation to remove metal precipitates

[0013] The cleaned squid cartilage chitosan solid is placed in a reactor, and a sulfuric acid solution 2 to 5 times the weight of the squid cartilage is added. The reaction temperature is 85 to 100° C., and the mixture is stirred for 2 to 6 hours, wherein the mass volume concentration of the sulfuric acid solution is 10 to 30%. After the hydrolysis reaction is completed, the pH value is adjusted to 7 to 8.5 with a saturated calcium hydroxide solution while stirring to dissipate heat, until the temperature is lowered to 0 to 10° C., and then the calcium sulfate precipitate and heavy metal precipitate are removed by high-speed centrifugation in a continuous flow centrifuge under low temperature (0 to 10° C.).

[0014] (4) Cation exchange chromatography / resin fractionation of glucosamine chelated calcium fraction and glucosamine fraction

[0015] The centrifuged supernatant is adsorbed using a cation exchange chromatography / resin, and then desorbed by gradient elution using a 2.5-5.0% sodium chloride solution; a glucosamine chelated calcium component and a glucosamine component are sequentially fractionated using a differential or ultraviolet detector and a fraction collector;

[0016] (5) Nanofiltration membrane desalination concentration and spray drying

[0017] The glucosamine chelate calcium component and the glucosamine component are respectively desalted and concentrated using a nanofiltration membrane with a relative molecular weight of 200 Da to obtain a glucosamine chelate calcium sulfate solution and a glucosamine sulfate solution with a content of ≥95%; finally, the glucosamine chelate calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried using a spray drying technology to obtain glucosamine chelate calcium sulfate powder and glucosamine sulfate powder.

[0018] The squid cartilage used in the step (1) is respectively from the Pacific fringefish (Todarodes pacificus) from the North Pacific, the American giant squid (Dosidicus gigas) from the South Pacific, and the kite squid (Symplectoteuthisoualaniensis) from the North Indian Ocean.

[0019] The detection method for the deacetylation degree of chitosan in step (2) is the detection method for the deacetylation degree of the national food safety standard "Food Additive Deacetylated Chitosan (Chitosan)" (GB 29941-2013); the detection method for the sodium ion is the detection method of the national food safety standard "Determination of Potassium and Sodium in Foods" (GB 5009.91-2017); the detection method for the chloride ion is the detection method of the national food safety standard "Determination of Chloride in Foods" (GB 5009.44-2016).

[0020] The centrifuge used in step (2) and step (3) is a continuous flow centrifuge with a speed of 10,000 to 15,000 rpm.

[0021] The cation exchange chromatography / resin used in step (4) is a preparative chromatography or resin with sulfonic acid group or carboxyl group.

[0022] The nanofiltration membrane with a relative molecular weight of 200Da used for separation in step (5) has a flow rate of 10 to 18 cubic meters per hour, a membrane pressure of 1.0 to 2.5 MPa, and a temperature of 20 to 50°C.

[0023] The inlet air temperature of the spray drying used in step (5) is 120-180°C, and the outlet air temperature is 70-95°C.

[0024] The glucosamine chelate calcium sulfate having a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) reaches a chromatographically pure level; the glucosamine sulfate having a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) is a high-purity product of mass spectrometry pure level. The content of glucosamine chelate calcium sulfate is the sum of the contents of (glucosamine+calcium+sulfate); the content of high-purity glucosamine sulfate is the sum of the contents of (glucosamine+sulfate); the method for detecting glucosamine content adopts the detection method of the light industry industry standard of the People's Republic of China "Glucosamine" (QB / T 5853-2023); the method for detecting sulfate content adopts the national standard of the People's Republic of China "General Test Method for Salt Manufacturing Industry - Determination of Sulfate" (GB / T 13025.8-2012); and the method for detecting calcium content adopts the national food safety standard "Determination of Calcium in Food" (GB 5009.92-2016).

[0025] The present invention also provides a glucosamine chelated calcium sulfate from heavy metal-contaminated squid cartilage and a high-purity glucosamine sulfate prepared by a large-scale preparation method thereof.

[0026] The present invention also provides the use of the high-purity glucosamine sulfate in the preparation of novel medicines with anti-inflammatory and analgesic activities.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. The present invention uses squid cartilage, which is often contaminated by heavy metals in reality, as raw material. Considering that heavy metals (lead, arsenic, cadmium, mercury, chromium) will significantly reduce the enzyme activity of endo-chitosanase and exo-chitosanase (β-D-glucosaminidase) in the aminoglycoside enzymatic reaction, Figure 3 and Figure 4 ) and seriously interfere with the actual situation of chitosan enzymatic hydrolysis reaction ( Figure 7 ), firstly, high temperature alkali treatment is used to remove the protein and mucopolysaccharide of squid cartilage in one step, and the squid cartilage chitin is deacetylated to obtain squid cartilage chitosan with a deacetylation degree of ≥90%, and then high temperature sulfuric acid hydrolysis process is used to degrade the squid cartilage chitosan into squid cartilage glucosamine monomer ( Figure 11 and Figure 13 ).

[0029] 2. Due to the different types and contents of heavy metals in squid cartilage raw materials from different sea areas, the enzyme activities of endo-chitosanase and exo-chitosanase (β-D-glucosaminidase) are reduced to different degrees ( Figure 3 and Figure 4 ), and the conditions that interfere with the enzymatic hydrolysis of chitosan vary ( Figure 7); The present application is aimed at this case, and the chemical extraction method adopted may not be as "fashionable" as the chitosanase degradation method and the microbial fermentation chitosanase degradation method universally recommended in the previous literature reports and patent technologies, but can stably prepare chromatographically pure glucosamine calcium sulfate chelate and mass spectrometrically pure glucosamine sulfate high-purity product in batches, and form a "large-scale preparation method of heavy metal-polluted glucosamine calcium sulfate chelate and glucosamine sulfate high-purity product from squid cartilage" with good process stability. Therefore, the present application is a patent technology that can solve practical problems.

[0030] 3. The present application is based on the fact that heavy metal elements in squid cartilage are not only distributed on the surface of the cartilage, but also have considerable content in the interior of the cartilage; and chitin and its degradation product chitosan in squid cartilage are both extremely easy to adsorb heavy metal elements, and chitooligosaccharide, glucosamine, which are further degradation products of chitin, are also extremely easy to form relatively stable chelates with heavy metal elements, which makes it extremely difficult to remove heavy metal elements in squid cartilage. In particular: the stability constants of each chelate formed by glucosamine and heavy metal elements are relatively high, and the stability is relatively good, which makes the heavy metal removal method universally adopted in the previous literature reports and patent technologies, i.e. membrane separation technology or chelating resin adsorption method, difficult to effectively separate glucosamine and heavy metal elements. Therefore, on the one hand, the present application takes advantage of the fact that sulfates and hydroxides of heavy metals (such as arsenic, cadmium, mercury, lead and chromium) are insoluble in water, and on the other hand, based on the fact that the stability constant of each chelate formed by glucosamine and heavy metal elements is definitely lower than the precipitation coefficient of each precipitate formed by heavy metal elements and sulfate ions, hydroxide ions, and combined with the basic chemical principle that substances generally tend to be in a stable state with low energy, the chemical precipitation method can effectively remove heavy metals, not only can effectively shorten the process flow and reduce the time cost; but also can achieve the effect that the previous membrane separation technology and chelating resin adsorption heavy metal removal method cannot achieve.

[0031] 4. The present application is different from the pretreatment decalcification method universally adopted in the previous literature reports and patent technologies, which adopts high-speed pulverization, which can not only expose chitin, protein and mucopolysaccharide in squid cartilage to subsequent high-temperature alkali, but also can retain calcium elements in squid cartilage as endogenous calcium source for subsequent glucosamine and calcium chelation reaction; In the previous patent literature of glucosamine and calcium chelate / complex, the calcium source participating in the chelation reaction is basically exogenous calcium source (such as calcium chloride, etc.), however, for the chelation reaction of glucosamine and calcium in the present application, the calcium source comes from two parts: endogenous calcium source - calcium in squid cartilage itself; exogenous calcium source - calcium hydroxide for adjusting the pH of enzymatic reaction; Therefore, the preparation method of the present application can not only realize the self-chelation conversion of endogenous calcium source in squid waste, but also can effectively reduce the addition of excessive exogenous calcium source, avoid unnecessary waste of calcium source, obviously the process of the present application is more environmentally friendly.

[0032] 5. Since squid lives in the marine environment, its cartilage contains a large amount of salt (NaCl). At the same time, the high temperature alkali treatment step of the present invention will also introduce a large amount of sodium ions (Na + ), therefore, the present invention specifically points out in step "(2) cooling, centrifugation and washing and desalting" that the squid cartilage chitosan solid obtained by centrifugation needs to be washed with deionized water until there is no sodium ion (Na + ) and chloride ions (Cl - ) are detected, thus avoiding the large amount of sodium ions (Na + ) and chloride ions (Cl - ) The high salt solution formed interferes with the adsorption of glucosamine molecules and glucosamine-chelated calcium molecules on the cation exchange preparative chromatography / cation exchange resin in the subsequent "cation exchange preparative chromatography / cation exchange resin adsorption and desorption" step.

[0033] 6. The present invention utilizes the characteristics of high stability constant and good stability of the chelate formed by glucosamine molecules and metal elements, and specially arranges the chelate reaction of glucosamine molecules and calcium ions in the cooling stage after the high-temperature sulfuric acid hydrolysis reaction. Only through a simple stirring process (stirring to dissipate heat while adjusting the appropriate pH with saturated calcium hydroxide solution), glucosamine chelate calcium ( Figure 10 and Figure 11 ); At the same time, since calcium sulfate is slightly soluble in water at room temperature, and its solubility is even lower at low temperature (0-10°C), the present invention adopts a method of further cooling to 0-10°C and then high-speed centrifugation in the cooling stage, which can ensure that a large amount of sulfate ions added in steps (2) and (3) of the present invention are precipitated in the form of calcium sulfate and removed by the continuous flow centrifugation technology in step (3) of the present invention, thereby avoiding a large amount of sulfate ions (SO4 2- ) The high salt solution formed interferes with the adsorption of glucosamine molecules and glucosamine-chelated calcium molecules on the cation exchange preparative chromatography / cation exchange resin in the subsequent "cation exchange preparative chromatography / cation exchange resin adsorption and desorption" step.

[0034] 7. Compared with the chitosanase degradation method and microbial fermentation chitosanase degradation method generally recommended in previous literature reports and patent technologies, the present invention adopts a method combining chemical extraction and chemical precipitation. Its production cost is lower (no need to add expensive endo-chitosanase and exo-chitosanase), its production operation is simpler (no need to add special fermentation microorganisms and control complex fermentation conditions), and its production equipment is relatively simple (the extraction process only requires an ordinary reactor and does not require complex fermentation equipment). In addition, unlike previous literature reports and patent technologies, a set of production processes can only produce one product: glucosamine chelated calcium or glucosamine. The present invention can achieve efficient extraction of glucosamine from squid cartilage and rapid chelation of glucosamine with calcium with a single production process, and then efficiently and cost-effectively prepare two high-quality products at one time: chromatographically pure glucosamine chelated calcium sulfate and mass-spectrographically pure glucosamine sulfate. The large-scale production cycle of the present invention can be basically completed within 24 hours, the process method is simple and practical, the process flow is efficient and reliable, and the overall process can meet the requirements of industrial scale-up and large-scale standardized production.

[0035] 8. Although the present invention uses a large amount of sulfuric acid, sodium hydroxide and calcium hydroxide for acid-base high temperature reaction or neutralization solution pH, the large amount of sulfate ions (SO4 2- ) Most of it is in the form of calcium sulfate (gypsum), and a small part is fixed as a solid in the form of heavy metal sulfuric acid precipitation; a large amount of hydrogen ions (H + ) can not only convert the calcium salt in the squid cartilage into the endogenous calcium source required for the chelation reaction between glucosamine molecules and metal calcium ions, but also degrade the squid cartilage chitosan after high temperature alkali treatment into small molecule glucosamine, which can be oxidized by the hydroxide ions (OH) of sodium hydroxide and calcium hydroxide. - ) is neutralized into water (H20); a large amount of hydroxide ions (OH - ) In addition to neutralizing hydrogen ions (H + ) can also precipitate heavy metals (such as arsenic, cadmium, mercury, lead and chromium) into solids in the form of hydroxides. Therefore, the process of the present invention does not cause environmental pollution or waste of resources.

[0036] 9. Since the heavy metal elements in the squid cartilage are all enriched and fixed in the squid cartilage by the squid organism absorbing liquid heavy metal ions in the marine water body, if the heavy metal elements are still washed out into liquid heavy metal ions according to the general treatment method in the previous literature reports and patent technologies, the heavy metal waste liquid is probably still in the marine environment, which undoubtedly equals to circulating pollution; therefore, the chemical precipitation method adopted in the present application can fix all the heavy metal elements into stable heavy metal precipitates and uniformly disperse them in calcium sulfate (gypsum), and the gypsum containing the heavy metal elements, although cannot be directly eaten, can be used as industrial materials and building materials, thereby cutting off the way of liquid heavy metal ion circulating pollution; therefore, the present application has relatively great environmental protection significance.

[0037] 10. The method of the present application has wide application range and is not affected by the geographical source of the squid cartilage raw material (can be applied to the squid cartilage raw material from the North Pacific Ocean (taking the Pacific Oegopsida as an example), the South Pacific Ocean (taking the American Dosidicus gigas as an example) and the North Indian Ocean (taking the Sepioteuthis lessoniana as an example)), and is not affected by the heavy metal pollution of the squid cartilage raw material, and good preparation effect is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is the process flow chart of the method of the present application: a large-scale preparation method of high-purity chondroitin sulfate calcium and glucosamine sulfate from heavy metal contaminated squid cartilage.

[0039] Figure 2 is the process flow chart of the method of the comparative experiment 1 reference application patent "Amino glucose produced by enzyme method and preparation method thereof" (patent application number: 201410067600.X) for preparing glucosamine (amino sugar).

[0040] Figure 3 is the influence of heavy metal pollution of squid cartilage from different sea areas on the enzyme activity of endochitosanase. The detection method of the enzyme activity of endochitosanase adopts the industry standard of the People's Republic of China "Protease activity determination method" (SB / T10317-1999).

[0041] Figure 4 is the influence of heavy metal pollution of squid cartilage from different sea areas on the enzyme activity of exochitosanase. The detection method of the enzyme activity of exochitosanase adopts the industry standard of the People's Republic of China "Protease activity determination method" (SB / T10317-1999).

[0042] Figure 5The following are chromatograms of four standards: 1. Dextran (Mw 405700); 2. Dextran (Mw 45800); 3. Dextran (Mw 1200); and 4. Dextran (Mw 342). Chromatographic conditions: Column: TSKgel GMPWXL; Mobile phase: deionized water; Isocratic elution over 40 minutes; Differential refractive index detector; Flow rate: 1 mL / min; Sample volume: 10 μL; Sample concentration: 5 mg / mL.

[0043] Figure 6 is the standard curve of four standards.

[0044] Figure 7 This is a comparative experiment 1 using squid cartilage from different sea areas polluted by heavy metals and squid cartilage not polluted by heavy metals as raw materials, with reference to the method of the invention patent "An enzymatic production of glucosamine and its preparation method" (patent application number: 201410067600.X), to prepare chromatograms of glucosamine (1 - chromatogram of glucosamine prepared using squid cartilage from the North Pacific as raw material; 2 - chromatogram of glucosamine prepared using squid cartilage from the South Pacific as raw material; 3 - chromatogram of glucosamine prepared using squid cartilage from the North Indian Ocean as raw material; 4 - chromatogram of glucosamine prepared using squid cartilage not polluted by heavy metals from the North Pacific as raw material; 5 - chromatogram of glucosamine prepared using squid cartilage not polluted by heavy metals from the South Pacific as raw material; 6 - chromatogram of glucosamine prepared using squid cartilage not polluted by heavy metals from the North Indian Ocean as raw material). Chromatographic conditions: chromatographic column: TSKgel GMPWXL column; mobile phase: deionized water; 40-minute isocratic elution; flow rate: 1 mL / min; sample volume: 10 μL; sample concentration: 5 mg / mL; detection was performed in series with a differential detector and a UV detector, and the detection wavelengths of the UV detector were 280 nm, 220 nm, and 214 nm.

[0045] Figure 8 This is the process flow chart of comparative experiment 2, which uses membrane separation technology to remove heavy metals and prepare glucosamine.

[0046] Figure 9 This is the process flow chart of comparative experiment 3, which uses chelating resin adsorption method to remove heavy metals and prepare glucosamine.

[0047] Figure 10This is a chromatogram of the cation exchange chromatography / resin fractionation of a glucosamine chelate calcium component and a glucosamine component according to the present invention (1—unadsorbed impurity component; 2—desorbed glucosamine chelate calcium component; 3—desorbed glucosamine component). Chromatographic conditions: Chromatographic column: preparative sulfonic acid cation exchange chromatography column; Mobile phase A: deionized water; Mobile phase B: 4.0% sodium chloride solution; Elution pattern: ① First 20 minutes: Isocratic elution with mobile phase A; ② 20-80 minutes: Gradient elution with mobile phase A-mobile phase B (0-100%); ③ Last 10 minutes: Isocratic elution with mobile phase B; Detection was performed using an ultraviolet detector with a detection wavelength of 195 nm; Flow rate: 1 mL / min.

[0048] Figure 11 The chromatograms of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate prepared by the method of the present invention are shown below (1—Chromatogram of high-purity glucosamine sulfate prepared by the method of the present invention; 2—Chromatogram of glucosamine chelated calcium sulfate prepared by the method of the present invention). Chromatographic conditions: Column: TSKgel GMPWXL column; Mobile phase: deionized water; 40-minute isocratic elution; Flow rate: 1 mL / min; Sample volume: 10 μL; Sample concentration: 5 mg / mL; Detection was performed using a differential detector in series with a UV detector, with detection wavelengths of 280 nm, 220 nm, and 214 nm.

[0049] Figure 12 It is a structural diagram of the glucosamine chelated calcium prepared by the present invention.

[0050] Figure 13 This is a mass spectrum of the high-purity glucosamine sulfate prepared by the present invention. Mass spectrometry conditions: Instrument: Waters XevoG2QTof mass spectrometer; Weigh an appropriate amount of high-purity glucosamine sulfate sample, dissolve it in methanol / water at 25°C, and measure it using an ESI ionization source in positive ion mode. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0052] Example 1:

[0053] A large-scale preparation method of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate from heavy metal-contaminated squid cartilage comprises the following steps:

[0054] (1) High-speed crushing and high-temperature alkali treatment

[0055] The squid cartilage detected to be contaminated with heavy metals was washed with clean water, dried in a hot air drying oven at 80°C for 2 hours, cut into 5 cm small segments, and pulverized in a traditional Chinese medicine grinder for 5 minutes. The pulverized squid cartilage powder was placed in a reactor, and a sodium hydroxide solution 10 times the weight of the squid cartilage was added. The reaction temperature was 90°C, and the reaction was stirred for 5 hours. The mass volume concentration of the sodium hydroxide solution was 20%.

[0056] (2) Cooling, centrifugation and cleaning and desalting

[0057] After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to 6.5 with sulfuric acid solution. The mixture was then centrifuged at 14,000 rpm using a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of 95.72%. The solid was then washed with deionized water until free of sodium ions (Na + ) and chloride ions (Cl - ) until it is detected;

[0058] (3) High-temperature sulfuric acid hydrolysis reaction, chelation reaction of glucosamine and calcium during the cooling stage, and low-temperature centrifugation to remove metal precipitates

[0059] The cleaned squid cartilage chitosan solid is placed in a reactor, and a sulfuric acid solution 5 times the weight of the squid cartilage is added. The reaction temperature is 85° C., and the mixture is stirred for 5 hours. The mass volume concentration of the sulfuric acid solution is 20%. After the hydrolysis reaction is completed, the pH is adjusted to 8 with a saturated calcium hydroxide solution while stirring to dissipate heat until the temperature drops to 5° C., and then the mixture is centrifuged at 14,000 rpm in a continuous flow centrifuge at a low temperature (5° C.) to remove calcium sulfate precipitates and heavy metal precipitates.

[0060] (4) Cation exchange chromatography / resin fractionation of glucosamine chelated calcium fraction and glucosamine fraction

[0061] The centrifuged supernatant is adsorbed using a cation exchange resin with a sulfonic acid group, and then desorbed by gradient elution using a 4.0% sodium chloride solution; a glucosamine chelated calcium component and a glucosamine component are sequentially fractionated using a differential detector and a fraction collector;

[0062] (5) Nanofiltration membrane desalination concentration and spray drying

[0063] The inorganic salt sodium chloride in the chitosan calcium chelate component and the chitosan component is removed by a nanofiltration membrane with a relative molecular weight of 200 Da, a flow rate of 10 cubic meters per hour, a membrane pressure of 1.0 Mpa, and a temperature of 30 DEG C, and the chitosan calcium chelate sulfate solution with a content of 96.27% and the chitosan sulfate solution with a content of 98.94% are obtained by further concentrating respectively; finally, the chitosan calcium chelate sulfate solution and the chitosan sulfate solution are quickly dried by a spray drying technology to obtain chitosan calcium chelate sulfate powder and chitosan sulfate powder; the inlet air temperature of the spray drying is 160 DEG C, and the outlet air temperature is 85 DEG C.

[0064] Example 2:

[0065] A large-scale preparation method of high-purity chitosan calcium chelate sulfate and chitosan sulfate from heavy metal contaminated squid cartilage, comprising the following steps:

[0066] (1) high-speed pulverization and high-temperature alkali treatment

[0067] The heavy metal contaminated squid cartilage is washed with clean water, dried in a hot air drying oven at 70 DEG C for 3 hours, cut into small pieces of 3 cm, put into a traditional Chinese medicine pulverizer, and pulverized for 8 minutes; the pulverized squid cartilage powder is put into a reaction kettle, 6 times the weight of sodium hydroxide solution is added, the reaction temperature is 88 DEG C, and the stirring reaction is carried out for 4 hours; the mass concentration of the sodium hydroxide solution is 25%;

[0068] (2) cooling centrifugation and washing desalination

[0069] After the alkali treatment, the temperature is lowered to room temperature, the pH is adjusted to 6 with a sulfuric acid solution, and then the continuous flow centrifuge is used for high-speed centrifugation at 12500 rpm to obtain chitosan solid with a deacetylation degree of 96.78%; then the deionized water is used for washing until no sodium ion (Na + ) and chloride ion (Cl - ) are detected;

[0070] (3) high-temperature sulfuric acid hydrolysis reaction, chelation reaction of chitosan and calcium in the cooling stage, and low-temperature centrifugation to remove metal precipitates

[0071] The washed squid cartilage chitosan solid is put into a reaction kettle, 2 times the weight of sulfuric acid solution is added, the reaction temperature is 90 DEG C, and the stirring reaction is carried out for 6 hours; the mass concentration of the sulfuric acid solution is 30%; after the hydrolysis reaction, the stirring and heat dissipation are carried out, the pH is adjusted to 7.5 with a saturated calcium hydroxide solution, and then the temperature is lowered to 10 DEG C, and then the continuous flow centrifuge is used for high-speed centrifugation at 12500 rpm under low temperature (10 DEG C) to remove calcium sulfate precipitates and heavy metal precipitates;

[0072] (4) Cation exchange chromatography / resin fractionation of chitosan- calcium complex component and chitosan component

[0073] The supernatant is adsorbed using a cation exchange resin with carboxyl groups, and then desorbed using a gradient elution with a 2.5% sodium chloride solution; the chitosan- calcium complex component and the chitosan component are sequentially fractionated by means of a UV detector at a wavelength of 192 nm and with the aid of a fraction collector;

[0074] (5) Nanofiltration membrane desalination and concentration and spray drying

[0075] The chitosan- calcium complex component and the chitosan component are desalted using a nanofiltration membrane with a relative molecular mass of 200 Da, at a flow rate of 13.5 cubic meters per hour, a membrane pressure of 1.8 MPa, and a temperature of 30°C; and are further concentrated to obtain a chitosan- calcium complex sulfate solution with a content of 95.66% and a chitosan sulfate solution with a content of 97.79%; and finally, the chitosan- calcium complex sulfate solution and the chitosan sulfate solution are rapidly dried using a spray drying technique to obtain chitosan- calcium complex sulfate powder and chitosan sulfate powder; the inlet air temperature for the spray drying is 170°C, and the outlet air temperature is 88°C.

[0076] Example 3:

[0077] A method for large-scale production of high-purity chitosan- calcium complex sulfate and chitosan sulfate from heavy metal contaminated squid cartilage, comprising the following steps:

[0078] (1) High-speed pulverization and high-temperature alkali treatment

[0079] The heavy metal contaminated squid cartilage is washed with clean water, then placed in a hot air drying oven at 90°C for 2 hours, taken out and cut into small pieces of 4 cm, then placed in a traditional Chinese medicine pulverizer and pulverized for 5 minutes; the pulverized squid cartilage powder is placed in a reaction kettle, 10 times the weight of sodium hydroxide solution is added, the reaction temperature is 95°C, and the stirring reaction is carried out for 6 hours; the mass concentration of the sodium hydroxide solution is 15%;

[0080] (2) Cooling centrifugation and washing desalination

[0081] After the alkali treatment is completed, the temperature is lowered to room temperature, the pH is adjusted to 5.5 with a sulfuric acid solution, and then a continuous flow centrifuge is used for high-speed centrifugation at 15000 rpm to obtain squid chitosan solid with a degree of deacetylation of 94.36%; next, the squid chitosan solid is washed with deionized water until no sodium ions (Na + ) and chloride ions (Cl - ) are detected;

[0082] (3) High-temperature sulfuric acid hydrolysis reaction, chelation reaction of chitosan and calcium in the cooling stage, and low-temperature centrifugation to remove metal precipitates

[0083] The cleaned squid cartilage chitosan solid is placed in a reactor, and a sulfuric acid solution 4 times the weight of the squid cartilage is added. The reaction temperature is 95° C., and the mixture is stirred for 4 hours. The mass volume concentration of the sulfuric acid solution is 15%. After the hydrolysis reaction is completed, the pH is adjusted to 7 with a saturated calcium hydroxide solution while stirring to dissipate heat until the temperature is lowered to 8° C., and then the mixture is centrifuged at 15,000 rpm in a continuous flow centrifuge at a low temperature (8° C.) to remove calcium sulfate precipitates and heavy metal precipitates.

[0084] (4) Cation exchange chromatography / resin fractionation of glucosamine chelated calcium fraction and glucosamine fraction

[0085] The centrifuged supernatant was adsorbed using a preparative cation exchange chromatography with a sulfonic acid group, and then desorbed by gradient elution using a 5.0% sodium chloride solution; an ultraviolet detector was used at a wavelength of 195 nm and a fraction collector was used to sequentially fractionate to obtain a glucosamine chelated calcium component and a glucosamine component;

[0086] (5) Nanofiltration membrane desalination concentration and spray drying

[0087] The glucosamine chelate calcium component and the glucosamine component are respectively subjected to a nanofiltration membrane with a relative molecular weight of 200 Da to remove the inorganic salt sodium chloride at a flow rate of 16 cubic meters per hour, a membrane pressure of 2.20 MPa, and a temperature of 40°C; and are further concentrated to obtain a glucosamine chelate calcium sulfate solution with a content of 95.48% and a glucosamine sulfate solution with a content of 98.95%; finally, the glucosamine chelate calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried using a spray drying technology to obtain glucosamine chelate calcium sulfate powder and glucosamine sulfate powder; the inlet air temperature of the spray drying is 180°C, and the outlet air temperature is 93°C.

[0088] Comparative Example 1

[0089] Reference to the invention patent "An enzymatically produced glucosamine and its preparation method" (patent application number: 201410067600.X), and Figure 2 Process flow chart for preparing glucosamine (sugar ammonia)

[0090] Comparative Example 2

[0091] See the process diagram Figure 8 , using membrane separation technology to remove heavy metals and produce glucosamine

[0092] The preparation process is briefly as follows:

[0093] (1) Pretreatment decalcification and high temperature alkali treatment

[0094] The squid cartilage detected to be contaminated with heavy metals was washed with clean water, placed in a reactor, and a dilute hydrochloric acid solution 10 times the weight of the squid cartilage was added, and stirred at room temperature for 2 hours. The mass volume concentration of the dilute hydrochloric acid solution was 1%. After the pretreatment, the squid cartilage was washed with clean water until the pH was neutral, placed in a reactor, and a sodium hydroxide solution 10 times the weight of the squid cartilage was added. The reaction temperature was 90°C, and the reaction was stirred for 5 hours. The mass volume concentration of the sodium hydroxide solution was 20%.

[0095] (2) Cooling centrifugation

[0096] After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to neutral with a dilute hydrochloric acid solution. The mixture was then centrifuged at 14,000 rpm using a continuous flow centrifuge to obtain a squid cartilage chitosan solid with a deacetylation degree of 95.35%. The mass volume concentration of the dilute hydrochloric acid solution was 1%.

[0097] (3) High-temperature hydrochloric acid hydrolysis reaction

[0098] The cleaned squid cartilage chitosan solid was placed in a reactor, and a hydrochloric acid solution 5 times the weight of the squid cartilage was added. The reaction temperature was 85° C., and the mixture was stirred for 5 hours. The mass volume concentration of the hydrochloric acid solution was 20%. After the hydrolysis reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to 4.0 with a sodium hydroxide solution. The mass volume concentration of the sodium hydroxide solution was 2%. The mixture was then centrifuged at 14,000 rpm in a continuous flow centrifuge to obtain a supernatant.

[0099] (4) Membrane separation technology for heavy metal removal

[0100] A nanofiltration membrane with a relative molecular mass of 5 kDa was used to remove macromolecular impurities in the supernatant at a flow rate of 8 cubic meters per hour, a membrane pressure of 0.72 MPa, and a temperature of 35°C. The resulting supernatant was finally filtered through a nanofiltration membrane with a relative molecular mass of 200 Da to remove small molecular impurities and inorganic salts (including heavy metal inorganic salts) at a flow rate of 5 cubic meters per hour, a membrane pressure of 0.65 MPa, and a temperature of 35°C, and concentrated to obtain a squid glucosamine solution with a content of 94.25%.

[0101] (5) Spray drying

[0102] The glucosamine solution was quickly dried using spray drying technology to obtain glucosamine sample powder with heavy metals removed using membrane separation technology; the inlet air temperature of the spray drying was 160°C and the outlet air temperature was 85°C.

[0103] Comparative Example 3

[0104] See the process diagram Figure 9 , using chelating resin adsorption method to remove heavy metals and prepare glucosamine

[0105] The preparation process is briefly as follows:

[0106] (1) Pretreatment decalcification and high-temperature alkali treatment

[0107] The heavy metal-contaminated squid cartilage was washed with clean water and then put into a reaction kettle, 10 times the weight of the squid cartilage of dilute hydrochloric acid solution was added, and stirred at room temperature for 2 hours. The mass concentration of the dilute hydrochloric acid solution was 1%; after the pretreatment was completed, the squid cartilage was washed with clean water until the pH was neutral, and then put into a reaction kettle, 10 times the weight of the squid cartilage of sodium hydroxide solution was added, the reaction temperature was 90℃, and the stirring reaction was carried out for 5 hours. The mass concentration of the sodium hydroxide solution was 20%;

[0108] (2) Cooling and centrifugation

[0109] After the alkali treatment was completed, the temperature was lowered to room temperature, the pH was adjusted to neutral with dilute hydrochloric acid solution, and then the squid cartilage chitosan solid with a degree of deacetylation of 95.35% was obtained by high-speed centrifugation with a continuous flow centrifuge at 14000 rpm. The mass concentration of the dilute hydrochloric acid solution was 1%;

[0110] (3) High-temperature hydrochloric acid hydrolysis reaction

[0111] The washed squid cartilage chitosan solid was put into a reaction kettle, 5 times the weight of the squid cartilage of hydrochloric acid solution was added, the reaction temperature was 85℃, and the stirring reaction was carried out for 5 hours. The mass concentration of the hydrochloric acid solution was 20%; after the hydrolysis reaction was completed, the temperature was lowered to room temperature, the pH was adjusted to 4.0 with sodium hydroxide solution, the mass concentration of the sodium hydroxide solution was 2%, and then the supernatant was obtained by high-speed centrifugation with a continuous flow centrifuge at 14000 rpm;

[0112] (4) Removal of heavy metals by chelating resin adsorption method

[0113] The centrifugal supernatant was adsorbed and eluted using a chromatography column packed with D401 macroporous chelating resin, and the squid chitosamine eluate was collected by means of a fraction collector under the detection of an ultraviolet detector at a wavelength of 195 nm.

[0114] (5) Spray drying

[0115] The chitosamine sample powder obtained by the chelating resin adsorption method for removing heavy metals was obtained by rapidly drying the chitosamine eluate by spray drying. The inlet air temperature of the spray drying was 160℃, and the outlet air temperature was 85℃.

[0116] As Figure 1As shown, the present invention uses squid cartilage contaminated by heavy metals from different sea areas as raw material, first adopts high-speed crushing and high-temperature alkali treatment processes to remove cartilage protein and mucopolysaccharide, and completes the deacetylation reaction of squid cartilage chitin to obtain squid cartilage chitosan with a deacetylation degree of ≥90%; then uses high-temperature sulfuric acid hydrolysis to degrade the squid cartilage chitosan into squid cartilage glucosamine. After the hydrolysis reaction, while stirring to dissipate heat, the pH is adjusted to a suitable pH with a saturated calcium hydroxide solution to allow a stirring chelation reaction between calcium ions and glucosamine molecules, and then a low-temperature centrifugation method is further used to remove metal precipitates (calcium sulfate precipitates and heavy metal precipitates); the supernatant after centrifugation is fractionated by cation exchange chromatography / resin to obtain a glucosamine chelate calcium component and a glucosamine component, and then the two fractions are respectively desalted and concentrated by nanofiltration membrane and spray-dried to obtain a chromatographically pure glucosamine chelate calcium sulfate with a content of ≥95% and a mass spectrometry-pure glucosamine sulfate with a content of ≥95%.

[0117] like Figure 2 As shown, the present invention refers to the method of the invention patent "An enzymatically produced glucosamine and its preparation method" (patent application number: 201410067600.X), using heavy metal contaminated / uncontaminated squid cartilage as raw material, first decalcification by pretreatment, then using a high-temperature alkali treatment process to remove cartilage protein and mucopolysaccharide, and complete the deacetylation reaction of squid cartilage chitin; after the alkali treatment, the pH is adjusted to neutral, and continuous flow centrifugation is used to obtain a squid cartilage chitosan solid with a deacetylation degree of ≥90%; then it is dissolved into a colloid with glacial acetic acid, and then the chitosan is enzymatically hydrolyzed into glucosamine with endo-chitosanase and exo-chitosanase, and finally the glucosamine sample is obtained by high-temperature enzyme inactivation and spray drying technology.

[0118] As shown in Table 1, the heavy metal pollution of squid cartilage raw materials from different sea areas is different, which is manifested in the great differences in the types and contents of heavy metals in squid cartilage. Among them, the heavy metal pollution of squid cartilage from the North Indian Ocean is the most serious. Not only do the contents of arsenic, cadmium, mercury, lead and chromium all exceed the limit standards of heavy metals in the national food safety standard "Limits of Contaminants in Food", but the contents of arsenic, cadmium and mercury are 10.19 times, 8.72 times and 5.60 times the limit standards of heavy metals in the national food safety standard "Limits of Contaminants in Food" respectively. The heavy metal pollution of squid cartilage from the North Pacific is also relatively serious. The contents of arsenic, cadmium and lead exceed the limit standards of heavy metals in the national food safety standard "Limits of Contaminants in Food". In comparison, the heavy metal pollution of squid cartilage from the North Pacific is relatively good, with the contents of arsenic and cadmium exceeding the limit standards of heavy metals in the national food safety standard "Limits of Contaminants in Food".

[0119] like Figure 3 andFigure 4 As shown, whether it is squid cartilage from the North Pacific Ocean, the South Pacific Ocean, or the North Indian Ocean, when the squid cartilage is not contaminated by heavy metals, the enzyme activities of the two chitosanases, endochitosanase and exochitosanase, which must be used in the composite enzymatic hydrolysis method in Comparative Experiment 1, are basically the same; however, as long as the squid cartilage is contaminated by heavy metals, the enzyme activities of the endochitosanase and exochitosanase used in Comparative Experiment 1 will respectively decrease significantly, and as the heavy metal pollution of the squid cartilage (the types and contents of heavy metals in the squid cartilage) from the South Pacific Ocean to the North Pacific Ocean and then to the North Indian Ocean becomes more serious, the degree of decrease in the enzyme activities of endochitosanase and exochitosanase also becomes more serious.

[0120] As shown in Table 2, using the chemical extraction method of the present invention, the extraction yields of the obtained samples (glucosamine chelated calcium sulfate and high-purity glucosamine sulfate) were essentially the same (21.64% to 21.66%), regardless of whether the squid cartilage was sourced from the North Pacific, the South Pacific, or the North Indian Ocean, and regardless of whether the squid cartilage was contaminated with heavy metals. This result demonstrates that the chemical extraction method employed in the present invention is not affected by either the geographical origin of the squid cartilage raw material or the heavy metal contamination of the squid cartilage raw material, and thus has excellent applicability.

[0121] At the same time, from the data in Table 2, it can also be seen that: whether it is squid cartilage from the North Pacific Ocean, squid cartilage from the South Pacific Ocean, or squid cartilage from the North Indian Ocean, as long as the squid cartilage is contaminated with heavy metals, the extraction rate of the sample (glucosamine) obtained by the combined enzymatic hydrolysis method of Comparative Experiment 1 (endochitosanase and exochitosanase) will be significantly reduced, and as the heavy metal pollution becomes more serious (the types of heavy metals in the squid cartilage increase and the heavy metal content increases), the downward trend in the extraction rate of the glucosamine sample obtained by the combined enzymatic hydrolysis method of Comparative Experiment 1 becomes more and more significant. The order of the extraction rate of glucosamine samples obtained by the combined enzymatic hydrolysis method of Comparative Experiment 1 from heavy metal-contaminated squid cartilage raw materials from different sea areas is South Pacific Ocean > North Pacific Ocean > North Indian Ocean Ocean. These results can be attributed to the significant inhibitory effect of heavy metals in squid cartilage from different sea areas on the enzymatic activity of endochitosanase and exochitosanase, which are necessary for the enzymatic hydrolysis reaction (see Figure 3 and Figure 4 ), which resulted in a significant decrease in the extraction rate of glucosamine samples.

[0122] On the contrary, when the squid cartilage is not contaminated by heavy metals, the extraction rate data of the samples (glucosamine chelated calcium sulfate and glucosamine sulfate high-purity) obtained by the chemical extraction method of the present invention are slightly higher than the extraction rate data of the samples (glucosamine) obtained by the composite enzymatic hydrolysis method of comparative experiment 1, but there is no significant difference between the two groups of data; these results can prove that as long as there is no heavy metal interference to inhibit the enzymatic activity of the endo-chitosanase and exo-chitosanase necessary for the enzymatic hydrolysis reaction, the composite enzymatic hydrolysis method of comparative experiment 1 can also have a higher sample (glucosamine) extraction rate. Combined with the fact that the extraction rates of the samples (glucosamine chelated calcium sulfate and glucosamine sulfate high-purity) obtained by the chemical extraction method of the present invention are basically the same regardless of whether the squid cartilage is contaminated by heavy metals, these results show that the chemical extraction method used in the present invention can obtain a higher sample extraction rate obtained by the enzymatic hydrolysis reaction when the squid cartilage is not affected by heavy metal contamination without the influence of heavy metal contamination of the squid cartilage raw material. Therefore, the chemical extraction method used in the present invention is also highly efficient.

[0123] Finally, considering that the two chitosanases (endo-chitosanase and exo-chitosanase) required for the composite enzymatic hydrolysis method of comparative experiment 1 are relatively expensive (the price of 10 units is more than RMB 5,000 yuan), in comparison, the concentrated sulfuric acid raw material used in the chemical extraction method adopted by the present invention is extremely cheap (the price of 1,000 mL is only RMB 30 yuan). Therefore, although the chemical extraction method adopted by the present invention is not as "glamorous and fashionable" as the chitosanase degradation method or microbial fermentation chitosanase degradation method generally recommended in previous literature reports and patent technologies, it can provide two products at the same time: glucosamine chelated calcium sulfate and glucosamine sulfate, which further proves that the present invention is a low-cost, practical and efficient method with significant progress.

[0124] like Figure 5 As shown in the figure, four dextran standards with different molecular weights have separate peaks on the TSKgel GMPWXL column and do not overlap. Figure 6 As shown, the regression equation of the standard curve is: y = -1.0306x + 13.973 (R 2 =0.998), indicating that the molecular weight is in the range of 405700 to 342, and the accuracy of the measurement result is high.

[0125] like Figure 7As shown in the figure, at wavelengths of 280nm, 220nm and 214nm, the glucosamine samples prepared using the process of Comparative Experiment 1 did not show any peaks, regardless of whether the squid cartilage was sourced from the North Pacific Ocean, the South Pacific Ocean, or the North Indian Ocean, and regardless of whether the squid cartilage raw materials were contaminated with heavy metals. This indicates that the glucosamine samples prepared using the process of Comparative Experiment 1 did not contain proteins or peptides. These results further indicate that the chromatographic peaks of the glucosamine samples prepared using the process of Comparative Experiment 1 on the TSKgel GMPWXL column using squid cartilage from different waters that was contaminated with heavy metals or was not contaminated with heavy metals were not affected by the presence of proteins or peptides.

[0126] like Figure 7 As shown, under differential conditions, all glucosamine samples prepared using the process of Comparative Experiment 1, using squid cartilage from the North Pacific, South Pacific, and North Indian Ocean sources without heavy metal contamination, exhibited similar single peak shapes (4-6) on a TSKgel GMPWXL column. The peak shape of a sample on a chromatographic column is an important indicator of its purity. Therefore, this result indicates that, using squid cartilage without heavy metal contamination, the combined enzymatic hydrolysis method and subsequent processes of Comparative Experiment 1 can produce glucosamine of high purity. However, using squid cartilage contaminated with heavy metals as the raw material, the chromatographic peak shapes of the glucosamine samples prepared using the process of Comparative Experiment 1 exhibited a series of peak bundles (1-3), suggesting that these glucosamine samples likely contain a significant amount of impurities. As the heavy metal contamination of squid cartilage from the South Pacific to the North Pacific and then to the North Indian Ocean becomes more serious (the types and contents of heavy metals in squid cartilage), the peak package shapes of the glucosamine samples prepared by the process of comparative experiment 1 on the chromatographic column become increasingly discrete. In particular, in the chromatogram of the glucosamine sample prepared using the heavy metal-contaminated squid cartilage from the North Indian Ocean as raw material, the chromatographic peak representing the glucosamine sample has a large peak package and a small miscellaneous peak in front. This is because the squid cartilage from the North Indian Ocean is the most seriously contaminated by heavy metals (the most heavy metal elements and the highest heavy metal content are detected), which has the greatest impact on the enzyme activity of the endo-chitosanase and exo-chitosanase that must be used in the composite enzymatic hydrolysis method of comparative experiment 1, resulting in the worst composite enzymatic hydrolysis effect and the presentation of more impurities.

[0127] like Figure 8As shown, the present invention refers to previous literature reports and patent technologies, uses heavy metal-contaminated squid cartilage as raw material, washes it, and then decalcifies it by acid treatment at room temperature, then uses a high-temperature alkali treatment process to remove cartilage protein and mucopolysaccharide, and completes the deacetylation reaction of squid cartilage chitin; after the alkali treatment, continuous flow centrifugation is used to obtain squid cartilage chitosan; then, high-temperature hydrochloric acid hydrolysis reaction is used to degrade the squid cartilage chitosan into squid cartilage glucosamine, and then continuous flow centrifugation is used in conjunction with membrane separation technology and spray drying technology to obtain a glucosamine sample with heavy metals removed by membrane separation technology.

[0128] like Figure 9 As shown, the present invention refers to previous literature reports and patent technologies, uses heavy metal-contaminated squid cartilage as raw material, and after washing, first decalcifies by acid treatment at room temperature, then uses a high-temperature alkali treatment process to remove cartilage protein and mucopolysaccharide, and completes the deacetylation reaction of squid cartilage chitin; after the alkali treatment, continuous flow centrifugation is used to obtain squid cartilage chitosan; then, a high-temperature hydrochloric acid hydrolysis reaction is used to degrade the squid cartilage chitosan into squid cartilage glucosamine, and then a continuous flow centrifugation combined with a chelate resin adsorption method and a spray drying technology is used to obtain a glucosamine sample with heavy metals removed by the chelate resin adsorption method.

[0129] As shown in Table 3, no heavy metals were detected in the samples (glucosamine chelated calcium sulfate and high-purity glucosamine sulfate) obtained after treatment by the chemical precipitation method of the present invention. This result proves that the chemical precipitation method used in the present invention can effectively remove heavy metals carried by squid cartilage raw materials. At the same time, the content of most heavy metals in the glucosamine samples obtained after treatment with membrane separation technology in Comparative Experiment 2 showed a significant increase compared to the heavy metal content of the squid cartilage raw material; while the content of heavy metals in the glucosamine samples obtained after treatment with chelating resin adsorption method in Comparative Experiment 3 showed almost no significant change compared to the heavy metal content of the squid cartilage raw material. On the one hand, these results prove that the heavy metal removal methods commonly used in previous literature reports and patented technologies - membrane separation technology and chelating resin adsorption methods - are unable to effectively remove heavy metals carried by squid cartilage raw materials; on the other hand, these results also suggest that the glucosamine after degradation of squid cartilage has a strong chelating effect with heavy metal ions, and the glucosamine-heavy metal chelate formed is stable, so that neither membrane separation technology nor chelating resin adsorption methods can effectively separate glucosamine molecules from heavy metal ions. In addition, the glucosamine-metal chelate formed by the chelation reaction between glucosamine and metal ions is actually a chelate formed by multiple glucosamine molecules and one metal ion (such as Figure 12), the molecular weight of which is obviously greater than that of glucosamine, resulting in that during the membrane separation implementation, the un-chelated glucosamine molecules and other small molecules are more likely to be removed by penetrating the separation membrane (equivalent to the content of un-chelated glucosamine in the sample being reduced), while the glucosamine-heavy metal chelate is less likely to penetrate the separation membrane and thus is retained (equivalent to the content of glucosamine-heavy metal chelate in the sample being increased); thus, the phenomenon that the content of most heavy metals in the glucosamine sample obtained after the comparative experiment is treated by the membrane separation technology is significantly increased.

[0130] As can be seen from the experimental results of Tables 1 and 3, no heavy metals are detected in the samples (glucosamine chelated calcium sulfate and high-purity glucosamine sulfate) obtained after being treated by the chemical precipitation method of the present application, which meets the limit standard of heavy metals in the National Food Safety Standard "Limit of Contaminants in Foods"; and the contents of heavy metals in the glucosamine samples obtained after being treated by the membrane separation technology of Comparative Experiment 2 and the chelating resin adsorption method of Comparative Experiment 3 both exceed the limit standard of heavy metals in the National Food Safety Standard "Limit of Contaminants in Foods". These results prove that the samples (glucosamine chelated calcium sulfate and high-purity glucosamine sulfate) obtained after being treated by the chemical precipitation method of the present application can be applied in the food field; while the glucosamine samples obtained after being treated by the membrane separation technology of Comparative Experiment 2 and the chelating resin adsorption method of Comparative Experiment 3 cannot be applied in the food field. These results again prove the practicability and significant progressiveness of the present application.

[0131] As shown in Table 4, whether the squid cartilage is from the North Pacific Ocean, the South Pacific Ocean or the North Indian Ocean, the extraction rate of the samples (glucosamine chelated calcium sulfate and high-purity glucosamine sulfate) obtained by the chemical precipitation method of the present application is not only slightly higher than that of the samples (glucosamine) obtained by the membrane separation technology of Comparative Experiment 2, but also significantly higher than that of the samples (glucosamine) obtained by the chelating resin adsorption method of Comparative Experiment 3, which on the one hand indicates that the chemical precipitation method adopted by the present application for removing heavy metals has greater advantages in sample extraction rate compared with the membrane separation technology or the chelating resin adsorption method commonly used in the prior art; and on the other hand, the experimental results that the extraction rate of the samples (glucosamine) obtained by the chelating resin adsorption method of Comparative Experiment 3 is significantly low also indicate that the stability of the glucosamine-heavy metal chelate formed by the degradation of squid cartilage and heavy metal ions is good, and the prior chelating resin adsorption method cannot effectively separate the glucosamine molecules from the heavy metal ions, i.e., the substances adsorbed by the chelating resin are not only heavy metals, but also the glucosamine-heavy metal chelate; which leads to the phenomenon that the extraction rate of the samples (glucosamine) obtained by the chelating resin adsorption method of Comparative Experiment 3 is significantly low.

[0132] The following results of the present invention are taken as example 1:

[0133] like Figure 10 As shown, the impurity component, the glucosamine chelate calcium component and the glucosamine component can be well separated on the preparative sulfonic acid cation exchange chromatography column. Therefore, the present invention can sequentially fractionate the glucosamine chelate calcium component and the glucosamine component by means of a fraction collector;

[0134] like Figure 11 As shown in the figure, at wavelengths of 280 nm, 220 nm and 214 nm, the glucosamine chelated calcium sulfate and glucosamine sulfate prepared by the present invention did not show any peaks. This result indicates, on the one hand, that the glucosamine chelated calcium sulfate and glucosamine sulfate samples prepared by the present invention do not contain protein or peptide substances; on the other hand, it also indicates that the high-speed crushing and high-temperature alkali treatment process used in the present invention can successfully remove cartilage protein.

[0135] like Figure 5 and Figure 11 As shown in Figure 2, both glucosamine chelated calcium and glucosamine exhibited sharp single peaks on the TSKgel GMPWXL column, and their peak shapes were comparable to those of dextran standards ( Figure 5 ), this result shows that the glucosamine chelated calcium sulfate and glucosamine sulfate prepared by the present invention both reach chromatographically pure level;

[0136] like Figure 7 and Figure 11 As shown, the peak shapes of glucosamine chelated calcium sulfate and glucosamine sulfate prepared by the present invention on the TSKgel GMPWXL column are comparable to, or even superior to, the peak shapes of the glucosamine samples prepared by the process of Comparative Experiment 1 using squid cartilage from different sea areas that is not contaminated by heavy metals on the TSKgel GMPWXL column. This result proves that the technical process of the present invention can completely replace the composite enzymatic hydrolysis method previously reported in literature and patented technologies, which is expensive and has strict raw material requirements (squid cartilage must be free of heavy metal contamination), to prepare chromatographically pure products.

[0137] like Figure 11 As shown, the peak time of glucosamine chelated calcium is greater than the peak time of dextran standard with a molecular weight of 342, and is within the range of molecular weight 405700 to 342 ( Figure 5 and Figure 6 ), and the peak time of glucosamine is less than the peak time of dextran standard with molecular weight of 342, and is not within the range of molecular weight 405700~342, then the molecular weight of glucosamine chelated calcium can be determined according to Figure 6 The molecular weight was calculated to be 398.

[0138] like Figure 12As shown, according to the molecular weight results of glucosamine chelated calcium (398), it can be inferred that glucosamine chelated calcium is a chelate composed of one calcium ion and two glucosamine molecules.

[0139] As shown in Table 1, the glucosamine sulfate prepared by the method of the present application has a purity of 99.5% or above. Figure 13 As shown in Table 1, the glucosamine sulfate prepared by the method of the present application has a purity of 99.5% or above. 13 The theoretical accurate mass number of glucosamine plus sodium cation [C6H + NO5Na] in glucosamine sulfate is m / z 202.0686, and the accurate mass number of glucosamine plus sodium cation measured by high resolution mass spectrometry is m / z 202.0692, which is consistent with the data of glucosamine plus sodium cation. This result proves that the high-purity glucosamine sulfate prepared by the method of the present application reaches the mass spectrometry purity level.

[0140] As shown in Table 5, in terms of the number of writhing, the number of writhing of the high-, medium- and low-dose glucosamine groups was significantly less than that of the blank control group, and the number of writhing induced by acetic acid in mice gradually decreased with the increase of the dose of glucosamine. Meanwhile, compared with the blank control group, the high-, medium- and low-dose glucosamine groups had statistical differences (P<0.05), and the high- and medium-dose glucosamine groups had extremely significant statistical differences (P<0.01). In terms of the pain inhibition rate, the high-, medium- and low-dose glucosamine groups were 31.12%, 24.04% and 19.63%, respectively, compared with the blank control group. This result shows that the high-purity glucosamine sulfate prepared by the method of the present application has good analgesic effect on the writhing-induced pain experiment of mice induced by acetic acid, and the analgesic effect gradually increases with the increase of the concentration of the high-purity glucosamine sulfate.

[0141] As shown in Table 6, in terms of the swelling rate, compared with the blank control group, the swelling rate of the auricle of mice in the high-, medium- and low-dose glucosamine groups was reduced, and the difference was extremely significant (P<0.01). In terms of the swelling inhibition rate, the swelling inhibition rate of the high-purity glucosamine sulfate prepared by the method of the present application increased with the increase of the dose. This result proves that the high-purity glucosamine sulfate prepared by the method of the present application has good anti-inflammatory activity on the auricle swelling experiment of mice induced by soybean oil.

[0142] Table 1

[0143]

[0144]

[0145] Note: Table 1 shows the heavy metal contamination of squid cartilage raw materials from different sea areas (n=3). Among them, the detection method for the content of heavy metal arsenic in squid cartilage adopts the national food safety standard "Determination of total arsenic and inorganic arsenic in foods" (GB5009.11-2024); the detection method for the content of heavy metal cadmium in squid cartilage adopts the national food safety standard "Determination of cadmium in foods" (GB 5009.15-2023); the detection method for the content of heavy metal lead in squid cartilage adopts the national food safety standard "Determination of lead in foods" (GB 5009.12-2023); the detection method for the content of heavy metal mercury in squid cartilage adopts the national food safety standard "Determination of total mercury and organic mercury in foods" (GB 5009.17-2021); the detection method for the content of heavy metal chromium in squid cartilage adopts the national food safety standard "Determination of chromium in foods" (GB The limits for the content of heavy metals arsenic, cadmium, lead, mercury and chromium in squid cartilage are based on the national food safety standard "Limits of Contaminants in Food" (GB2762-2022).

[0146] Table 2

[0147]

[0148] a - The samples obtained by the chemical extraction method of the present invention include glucosamine chelated calcium sulfate and high-purity glucosamine sulfate;

[0149] b —The sample obtained by the composite enzymatic hydrolysis method of comparative experiment 1 was glucosamine.

[0150] Note: Table 2 compares the extraction rates of squid cartilage samples from different sea areas after being processed by various methods (n=3). The calculation formula for the extraction rate of the samples obtained by the chemical extraction method of the present invention is as follows:

[0151]

[0152] The calculation formula for the sample extraction rate obtained by the composite enzymatic hydrolysis method of comparative experiment 1 is as follows:

[0153]

[0154] Table 3

[0155]

[0156] *—Compared with the heavy metal content of squid cartilage raw materials, P<0.05, there is a significant difference;

[0157] **—Compared with the heavy metal content of squid cartilage raw materials, P<0.01, there is an extremely significant difference;

[0158] a - The samples obtained by the chemical precipitation method of the present invention include glucosamine chelated calcium sulfate and high-purity glucosamine sulfate;

[0159] b —The samples obtained using the membrane separation technology in comparative experiment 2 and the chelate resin adsorption method in comparative experiment 3 were both glucosamine. Note: Table 3 compares the heavy metal content of squid cartilage samples obtained from different sea areas after being treated by various methods (n=3). Among them, the detection method for the heavy metal arsenic content in the sample adopts the national food safety standard "Determination of Total Arsenic and Inorganic Arsenic in Food" (GB 5009.11-2024); the detection method for the heavy metal cadmium content in the sample adopts the national food safety standard "Determination of Cadmium in Food" (GB 5009.15-2023); the detection method for the heavy metal lead content in the sample adopts the national food safety standard "Determination of Lead in Food" (GB 5009.12-2023); the detection method for the heavy metal mercury content in the sample adopts the national food safety standard "Determination of Total Mercury and Organic Mercury in Food" (GB 5009.17-2021); the detection method for the heavy metal chromium content in the sample adopts the national food safety standard "Determination of Chromium in Food" (GB 5009.123-2023).

[0160] Table 4

[0161]

[0162] *—Compared with the extraction rate of the sample obtained after the chemical precipitation method of the present invention, P<0.05, there is a significant difference;

[0163] **—Compared with the extraction rate of the sample obtained after the chemical precipitation method of the present invention, P<0.01, there is an extremely significant difference; a - The samples obtained by the chemical precipitation method of the present invention include glucosamine chelated calcium sulfate and high-purity glucosamine sulfate;

[0164] b —The samples obtained using the membrane separation technology in comparative experiment 2 and the chelate resin adsorption method in comparative experiment 3 were both glucosamine. Note: Table 4 compares the extraction rates of squid cartilage samples from different sea areas after being processed by various methods (n=3). Among them, the calculation formula for the sample extraction rate obtained by the chemical precipitation method of the present invention is as follows:

[0165]

[0166] The calculation formulas for the sample extraction rates obtained using the membrane separation technology in comparative experiment 2 and the chelate resin adsorption method in comparative experiment 3 are as follows:

[0167]

[0168] Table 5

[0169]

[0170] *—Compared with the control group, P<0.05, there is a significant difference;

[0171] **—Compared with the control group, P<0.01, there is an extremely significant difference.

[0172] Note: Table 5 shows the effect of glucosamine prepared by the present invention on the writhing reaction of mice induced by acetic acid (n=10). The present invention verifies the analgesic effect of glucosamine of the present invention by observing and evaluating the writhing reaction of mice induced by acetic acid. The specific experimental steps are as follows:

[0173] ① Experimental animal grouping

[0174] Forty SPF Kunming mice, half male and half female, were randomly divided into four groups: high, medium, and low doses of glucosamine groups (500 mg / kg, 250 mg / kg, 125 mg / kg) and a blank control group (0 mg / kg), with 10 mice in each group.

[0175] ②Dosage method

[0176] Each group of mice was orally administered with a set dose of glucosamine once for seven consecutive days. On the eighth day, 30 minutes after orally administering the set dose of glucosamine, each mouse was intraperitoneally injected with 0.1 mL / 10 g of 0.6% glacial acetic acid solution.

[0177] ③Evaluation and statistical methods

[0178] The number of writhing times of the mice within 20 minutes was observed and recorded, and the pain inhibition rate was calculated. The calculation formula for the pain inhibition rate was as follows:

[0179]

[0180] Table 6

[0181]

[0182] **—Compared with the control group, P<0.01, there is an extremely significant difference.

[0183] Note: Table 6 shows the effect of glucosamine prepared in the present invention on soybean oil-induced ear swelling in mice (n=10). The present invention verifies the anti-inflammatory effect of glucosamine in the present invention by evaluating its effect on soybean oil-induced ear swelling in mice. The specific experimental steps are as follows:

[0184] ① Experimental animal grouping

[0185] Forty SPF Kunming mice, half male and half female, were randomly divided into four groups: high, medium, and low doses of glucosamine groups (500 mg / kg, 250 mg / kg, 125 mg / kg) and a blank control group (0 mg / kg), with 10 mice in each group.

[0186] ②Dosage method

[0187] Each group of mice was gavaged with a set dose of glucosamine once for seven consecutive days. On the eighth day, 30 minutes after gavage with a set dose of glucosamine, 20 μL of acetone solution was applied to the left ear and 20 μL of 5% soybean oil was applied to the right ear of each mouse. Four hours later, the mice were killed, and the ears were cut off and samples were taken from the ears one by one using a puncher.

[0188] ③Evaluation and statistical methods

[0189] After sampling, the weight of the left and right ears of the mice was recorded, and the swelling rate and swelling inhibition rate were calculated. The calculation formulas for the swelling rate and swelling inhibition rate are as follows:

[0190] .

Claims

1. A large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage, comprising the following steps: (1) High-speed crushing and high-temperature alkali treatment The squid cartilage detected to be contaminated with heavy metals is washed with clean water, dried in a hot air drying oven at 70-90°C for 2-5 hours, cut into 3-5 cm small segments, placed in a traditional Chinese medicine grinder, and pulverized for 5-8 minutes; the pulverized squid cartilage powder is added to a reactor, and a sodium hydroxide solution 4-10 times the weight of the squid cartilage is added. The reaction temperature is 85-100°C, and the reaction is stirred for 2-6 hours, wherein the mass volume concentration of the sodium hydroxide solution is 10-30%. (2) Cooling, centrifugation and cleaning and desalting After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to 5-6.5 with sulfuric acid solution. The solid squid cartilage chitosan with a deacetylation degree of ≥90% was obtained by high-speed centrifugation in a continuous flow centrifuge. The solid was then washed with deionized water until there was no sodium ion Na + and chloride ions Cl - Until it is detected; (3) High-temperature sulfuric acid hydrolysis reaction, chelation reaction of glucosamine and calcium during the cooling stage, and low-temperature centrifugation to remove metal precipitates The cleaned squid cartilage chitosan solid is placed in a reactor, and a sulfuric acid solution 2 to 5 times the weight of the squid cartilage is added. The reaction temperature is 85 to 100° C., and the reaction is stirred for 2 to 6 hours. The mass volume concentration of the sulfuric acid solution is 10 to 30%. After the hydrolysis reaction, while stirring to dissipate heat, the pH is adjusted to 7 to 8.5 with a saturated calcium hydroxide solution until the temperature is lowered to 0 to 10° C., and then the calcium sulfate precipitate and heavy metal precipitate are removed by high-speed centrifugation in a continuous flow centrifuge at a low temperature of 0 to 10° C. (4) Cation exchange chromatography fractionation of glucosamine chelated calcium fraction and glucosamine fraction The centrifuged supernatant is adsorbed using cation exchange chromatography, and then desorbed by gradient elution using a 2.5-5.0% sodium chloride solution; a glucosamine chelated calcium component and a glucosamine component are sequentially fractionated using a differential or ultraviolet detector and a fraction collector; (5) Nanofiltration membrane desalination concentration and spray drying The glucosamine chelate calcium component and the glucosamine component are respectively desalted and concentrated using a nanofiltration membrane with a relative molecular weight of 200 Da to obtain a glucosamine chelate calcium sulfate solution and a glucosamine sulfate solution with a content of ≥95%; finally, the glucosamine chelate calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried using a spray drying technology to obtain glucosamine chelate calcium sulfate powder and glucosamine sulfate powder.

2. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The squid cartilage used in step (1) is from at least one of the Pacific squid from the North Pacific, the American giant squid from the South Pacific, and the kite squid from the North Indian Ocean.

3. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The detection method for the deacetylation degree of chitosan in step (2) is the detection method for the deacetylation degree of the national food safety standard GB 29941-2013 "Food Additive Deacetylated Chitosan (Chitosan)"; the detection method for the sodium ion is the detection method of the national food safety standard GB 5009.91-2017 "Determination of Potassium and Sodium in Foods"; the detection method for the chloride ion is the detection method of the national food safety standard GB 5009.44-2016 "Determination of Chloride in Foods".

4. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The centrifuges used in step (2) and step (3) are continuous flow centrifuges with a speed of 10,000 to 15,000 rpm, and the centrifugal speeds are respectively 10,000 to 15,000 rpm.

5. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The cation exchange chromatography used in step (4) is a preparative chromatography with sulfonic acid group or carboxyl group.

6. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: In step (5), a nanofiltration membrane with a relative molecular weight of 200 Da is used at a flow rate of 10 to 18 cubic meters per hour, a membrane pressure of 1.0 to 2.5 MPa, and a temperature of 20 to 50°C.

7. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The inlet air temperature of the spray drying used in step (5) is 120-180°C, and the outlet air temperature is 70-95°C.

8. The method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal contaminated squid cartilage according to claim 1, characterized in that: The glucosamine chelated calcium sulfate with a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) reaches chromatographic purity level; the glucosamine sulfate with a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) is mass spectrometry purity level.

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