Large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity product from heavy metal polluted squid cartilage

Abstract

The invention relates to a large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products from heavy metal polluted squid cartilage. The method comprises the following steps: by taking squid cartilage polluted by heavy metals in different sea areas as a raw material, firstly removing chondroprotein and mucopolysaccharide, and completing deacetylation reaction to obtain squid cartilage chitosan with the deacetylation degree of more than or equal to 90%; then squid cartilage chitosan is degraded into squid cartilage glucosamine, after the hydrolysis reaction is finished, stirring heat dissipation is conducted while a saturated calcium hydroxide solution is used for stirring chelation reaction, and then metal precipitates are further removed through a low-temperature centrifugation method; and performing cation exchange chromatography / resin fractionation on the supernate to obtain a glucosamine chelated calcium component and a glucosamine component, and then obtaining glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products with the content of more than or equal to 95%. The method is wide in application range, is not influenced by regional sources of the squid cartilage raw materials and heavy metal pollution conditions of the squid cartilage raw materials, and has good preparation effects.

Description

Technical Field

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

[0002] Squid is a common cephalopod of the phylum mollusc and a good raw material for aquatic product processing. During the processing of squid, a large amount of waste by-products - squid cartilage - is often produced. Although some cartilage by-products can be converted into fish meal feed, the cartilage scraps produced 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 enhance 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 environmental protection.

[0003] With the rapid growth of squid imports, my country has become the world's largest squid offshore fishing country, and has further transformed into the world's largest squid importer, processor and domestic sales country. Currently, the squid caught mainly comes from the North Pacific Ocean, the South Pacific Ocean and the North Indian Ocean. In recent years, with the development of industry, marine environmental pollution represented by heavy metal pollution has become more and more serious; squid organisms absorb liquid heavy metal ions in marine waters, which are easily enriched and fixed in squid cartilage, making the heavy metal pollution of squid cartilage more serious.

[0004] Related studies have shown that squid cartilage accounts for about 2% of its body weight, and its effective ingredients are proteins (collagen, core protein, etc.) and carbohydrates (chondroitin sulfate, chitin, etc.). At present, the literature and patents related to squid cartilage are basically aimed at the separation, purification and utilization of the effective ingredients of squid cartilage (chondroitin sulfate, chitin, chitosan, collagen, etc.) (for example, CN202110439789.0, CN202011521963.8, CN202110439798.X, CN201811558088.3, ​​etc.), and few people pay attention to the fact that discarded squid cartilage can actually be used as a source of raw materials for glucosamine (samine). More importantly, how to effectively utilize heavy metal-contaminated squid cartilage and develop a set of efficient, environmentally friendly and low-cost production processes to make it an effective source of raw materials for glucosamine is a scientific problem that needs to be solved urgently and has important research value. Summary of the invention

[0005] In view of this, the present invention is based on achieving the goal of 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 of squid processing waste - squid cartilage, but also develops a high-purity glucosamine sulfate that can be used for new functional foods or medicines with anti-inflammatory and analgesic activity.

[0006] 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 glucosamine sulfate high-purity products 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, placed in a hot air drying oven at 70-90°C for 2-5 hours, taken out and cut into small pieces of 3-5 cm, placed in a Chinese medicine grinder, and crushed for 5-8 minutes; the crushed squid cartilage powder is put into a reaction kettle, and a sodium hydroxide solution of 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, and 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 temperature was lowered to room temperature, and the pH was adjusted to 5-6.5 with sulfuric acid solution, and then centrifuged at high speed with a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of ≥90%; then washed with deionized water until there was no sodium ion (Na + ) and chloride ions (Cl - ) is detected;

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

[0013] The cleaned squid cartilage chitosan solid is put into a reactor, and a sulfuric acid solution of 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, and the mass volume concentration of the sulfuric acid solution is 10 to 30%; after the hydrolysis reaction is completed, while stirring to dissipate heat, a saturated calcium hydroxide solution is used to adjust the pH to 7 to 8.5 until the temperature drops to 0 to 10° C., and then a continuous flow centrifuge is used at a low temperature (0 to 10° C.) to remove calcium sulfate precipitation and heavy metal precipitation;

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

[0015] Adsorbing the centrifuged clear liquid with a cation exchange chromatography / resin, and then desorbing it with a gradient elution solution containing 2.5-5.0% sodium chloride; fractionating in sequence to obtain a glucosamine chelated calcium component and a glucosamine component by 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 200Da 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) comes from Pacific squid (Todarodes pacificus) from the North Pacific Ocean, American giant squid (Dosidicus gigas) from the South Pacific Ocean, and 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 sodium ions is the detection method of the national food safety standard "Determination of Potassium and Sodium in Food" (GB 5009.91-2017); the detection method for chloride ions is the detection method of the national food safety standard "Determination of Chloride in Food" (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 the step (4) is a preparative chromatography or resin with a sulfonic acid group or a 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 spray drying used in step (5) has an inlet air temperature of 120-180°C and an outlet air temperature of 70-95°C.

[0024] The glucosamine chelated calcium sulfate with a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) reaches the chromatographic purity level; the glucosamine sulfate with a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) is a high-purity product with a mass spectrometry purity level. The content of glucosamine chelated 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 detection method of 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 detection method of sulfate content adopts the national standard of the People's Republic of China "General Test Method for Salt Industry Determination of Sulfate" (GB / T 13025.8-2012); the detection method of calcium content adopts the national food safety standard "Determination of Calcium in Food" (GB 5009.92-2016).

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

[0026] The present invention also provides the use of the high-purity glucosamine sulfate product in the preparation of novel functional foods or 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 polluted by heavy metals as raw materials. In view of the fact that each heavy metal (lead, arsenic, cadmium, mercury, chromium) will significantly reduce the enzyme activity of endo-chitosanase and exo-chitosanase (β-D-glucosaminidase) in the aminoglucose 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 monomers ( Fig.11 and Fig.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 chemical extraction method adopted by the present invention may not be as "glamorous and fashionable" as the chitosanase degradation method and the chitosanase degradation method produced by microbial fermentation, which are generally recommended in previous literature reports and patent technologies, but it can stably prepare chromatographically pure glucosamine chelated calcium sulfate and mass spectrometry-pure glucosamine sulfate in batches, and form a "large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate from heavy metal-contaminated squid cartilage with good process stability". Therefore, the present invention is a patented technology that can solve practical problems.

[0030] 3. The present invention takes into account that the heavy metal elements in squid cartilage are not only distributed on the surface of the cartilage, but also contain a considerable amount of heavy metals inside the cartilage; and the chitin of squid cartilage and its degradation product chitosan are very easy to adsorb heavy metal elements, and the further degradation products of chitin, chitosan oligosaccharides and glucosamine, are also very 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 constant of each chelate formed by glucosamine and heavy metal elements is high and the stability is good, which leads to the fact that the heavy metal removal methods commonly used in previous literature reports and patent technologies - membrane separation technology or chelate resin adsorption method, are difficult to effectively separate glucosamine from heavy metal elements. Therefore, the present invention utilizes the characteristics that sulfates and hydroxides of heavy metals (such as arsenic, cadmium, mercury, lead and chromium) are insoluble in water on the one hand, and on the other hand, based on the fact that the stability constants of various chelates formed by glucosamine and heavy metal elements must be lower than the precipitation coefficients of various precipitates formed by heavy metal elements and sulfate ions and hydroxide ions, and combined with the basic chemical principle that substances generally tend to a stable state with low energy, the chemical precipitation method is used to effectively remove heavy metals, which can not only effectively shorten the process flow and reduce time costs; but also achieve effects that cannot be achieved by previous membrane separation technology and chelating resin adsorption methods for removing heavy metals.

[0031] 4. The present invention is different from the pretreatment decalcification method commonly used in previous literature reports and patent technologies. It adopts a high-speed crushing method, which can not only expose the chitin, protein and mucopolysaccharide in the squid cartilage to the subsequent high-temperature alkali solution, but also retain the calcium element in the squid cartilage as an endogenous calcium source for the subsequent glucosamine and calcium chelation reaction; in the previous patent literature on glucosamine and calcium chelates / complexes, the calcium source participating in the chelation reaction is basically an exogenous calcium source (such as calcium chloride, etc.), however, for the chelation reaction of glucosamine and calcium in the present invention, its calcium source comes from two parts: endogenous calcium source - calcium in the squid cartilage itself; exogenous calcium source - calcium hydroxide for adjusting the pH of the enzymatic hydrolysis reaction; therefore, the preparation method of the present invention can not only realize the self-chelation conversion of the endogenous calcium source in the squid scraps, but also effectively reduce the supplementation of excessive exogenous calcium sources, and avoid unnecessary waste of calcium sources. Obviously, the process of the present invention is more economical and 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 - ) is detected, thus avoiding the large amount of sodium ions (Na + ) and chloride ions (Cl - ) The high salt solution formed by the reaction 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 a saturated calcium hydroxide solution), glucosamine chelate calcium ( Fig.10 and Fig.11 ); at the same time, since calcium sulfate is slightly soluble in water at room temperature, and the solubility of calcium sulfate is even lower under 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 by the reaction 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 with 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 the production equipment is relatively simple (the extraction process only requires an ordinary reactor, and no complex fermentation equipment is required). 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 in squid cartilage and rapid chelation of glucosamine and calcium with only one set of production processes, and then prepare two high-quality products at one time with high efficiency and low cost: chromatographically pure glucosamine chelated calcium sulfate and mass spectrometry pure glucosamine sulfate high-purity products; the large-scale production cycle of the present invention can basically be controlled to be completed within 24 hours, and the process method is simple and pragmatic; the process flow is efficient and reliable, and the overall process can meet the requirements of industrialization amplification 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, a large amount of sulfate ions (SO4 2- ) is mostly 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 the amino sugar molecules and the metal calcium ions, but also degrade the squid cartilage chitosan after high temperature alkali treatment into small molecule amino sugars, which can be oxidized by the hydroxide ions (OH - ) 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 and waste of resources.

[0036] 9. Since the heavy metal elements in squid cartilage are all absorbed by the squid organisms from the ocean water and enriched and fixed in the squid cartilage, if the general treatment method for heavy metal elements in previous literature reports and patent technologies is still followed - eluting them into liquid heavy metal ions, the destination of these heavy metal waste liquids is likely to be the marine environment, which is undoubtedly equivalent to circulating pollution; in view of this, the chemical precipitation method adopted in the present invention can fix all heavy metal elements into stable heavy metal precipitates and evenly disperse them in calcium sulfate (gypsum). Although these gypsums containing heavy metal elements cannot be eaten directly, they can be used as industrial materials and building materials, thereby cutting off the path of circulating pollution of liquid heavy metal ions; therefore, the present invention has a relatively significant environmental significance.

[0037] 10. The method of the present invention has a wide range of applications and is not affected by the geographical source of the squid cartilage raw materials (it can be applied to squid cartilage raw materials from the North Pacific Ocean (taking Pacific pleated squid as an example), squid cartilage raw materials from the South Pacific Ocean (taking American giant red squid as an example), and squid cartilage raw materials from the North Indian Ocean (taking kite squid as an example)), nor is it affected by the heavy metal pollution of the squid cartilage raw materials, and achieves good preparation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention is a process flow chart of a method for large-scale preparation of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products from heavy metal-contaminated squid cartilage.

[0039] Figure 2 This is a process flow chart for preparing glucosamine (sugar amino) by referring to the method of the invention patent "An enzymatic production of glucosamine and its preparation method" (patent application number: 201410067600.X) in comparative experiment 1.

[0040] Figure 3 The invention relates to 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 The effect of heavy metal pollution in squid cartilage from different sea areas on the enzyme activity of exochitosanase was investigated. The detection method of exochitosanase enzyme activity was based on the industry standard of the People's Republic of China "Protease Activity Determination Method" (SB / T10317-1999).

[0042] Figure 5The chromatograms of four standards are as follows: 1. Dextran (Mw 405700); 2. Dextran (Mw 45800); 3. Dextran (Mw 1200); 4. Dextran (Mw 342). Chromatographic conditions: Column: TSKgel GMPWXL column; Mobile phase: deionized water; 40-minute isocratic elution; Differential 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 chromatogram of glucosamine prepared by 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, referring to the method of the invention patent “An enzymatic production of glucosamine and preparation method thereof” (patent application number: 201410067600.X) (1 - chromatogram of glucosamine prepared using squid cartilage polluted by heavy metals from the North Pacific as raw material; 2 - chromatogram of glucosamine prepared using squid cartilage polluted by heavy metals from the South Pacific as raw material; 3 - chromatogram of glucosamine prepared using squid cartilage polluted by heavy metals 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 minutes isocratic elution; flow rate: 1 mL / min; sample volume: 10 μL; sample concentration: 5 mg / mL; differential detector and UV detector were used in series for detection, 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] Fig. 9 This is the process flow chart of comparative experiment 3, which uses the chelating resin adsorption method to remove heavy metals and prepare glucosamine.

[0047] Fig.10The present invention is a chromatogram of the cation exchange chromatography / resin fractionation of glucosamine chelated calcium component and glucosamine component (1 - unadsorbed impurity component; 2 - desorbed glucosamine chelated 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 mode: ① first 20 minutes: isocratic elution of mobile phase A; ② 20 to 80 minutes: gradient elution of mobile phase A-mobile phase B (0-100%); ③ last 10 minutes: isocratic elution of mobile phase B; detection by ultraviolet detector, the detection wavelength of the ultraviolet detector is 195nm; flow rate: 1mL / min.

[0048] Fig.11 The chromatograms of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate prepared by the method of the present invention (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: 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; differential detector and ultraviolet detector connected in series for detection, the detection wavelengths of the ultraviolet detector are 280 nm, 220 nm and 214 nm.

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

[0050] Fig.13 This is the mass spectrum of the high-purity glucosamine sulfate prepared by the present invention. Mass spectrometry conditions: Instrument: Waters Xevo G2QTof mass spectrometer; weigh an appropriate amount of high-purity glucosamine sulfate sample, dissolve it in methanol / water at 25°C, and use an ESI ionization source to measure in positive ion mode. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Embodiment 1:

[0053] A large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products 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, placed in a hot air drying oven at 80°C for 2 hours, cut into 5 cm small segments after being taken out, placed in a Chinese medicine grinder, and crushed for 5 minutes; the crushed squid cartilage powder was put into 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, and 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, and then centrifuged at 14000 rpm using a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of 95.72%; then, the mixture was washed with deionized water until there was no sodium ion (Na + ) and chloride ions (Cl - ) is detected;

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

[0059] The cleaned squid cartilage chitosan solid is put into a reactor, and a sulfuric acid solution of 5 times the weight of the squid cartilage is added, the reaction temperature is 85° C., and the reaction is stirred for 5 hours, and the mass volume concentration of the sulfuric acid solution is 20%; after the hydrolysis reaction is completed, while stirring to dissipate heat, a saturated calcium hydroxide solution is used to adjust the pH to 8 until the temperature drops to 5° C., and then a continuous flow centrifuge is used at a low temperature (5° C.) Under high-speed centrifugation at 14000 rpm to remove calcium sulfate precipitation and heavy metal precipitation;

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

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

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

[0063] The glucosamine chelated calcium component and the glucosamine component are respectively subjected to a nanofiltration membrane with a relative molecular weight of 200Da to remove the inorganic salt sodium chloride, with a flow rate of 10 cubic meters per hour, a membrane pressure of 1.0 MPa, and a temperature of 30°C, and are further concentrated to obtain a glucosamine chelated calcium sulfate solution with a content of 96.27% and a glucosamine sulfate solution with a content of 98.94%. Finally, the glucosamine chelated calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried by spray drying technology to obtain glucosamine chelated calcium sulfate powder and glucosamine sulfate powder. The inlet air temperature of the spray drying is 160°C, and the outlet air temperature is 85°C.

[0064] Embodiment 2:

[0065] A large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products from heavy metal contaminated squid cartilage comprises the following steps:

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

[0067] The squid cartilage detected to be contaminated with heavy metals was washed with clean water, placed in a hot air drying oven at 70°C for 3 hours, cut into 3 cm small pieces after being taken out, placed in a Chinese medicine grinder, and crushed for 8 minutes; the crushed squid cartilage powder was put into a reactor, and a sodium hydroxide solution of 6 times the weight of the squid cartilage was added, the reaction temperature was 88°C, and the reaction was stirred for 4 hours, and the mass volume concentration of the sodium hydroxide solution was 25%;

[0068] (2) Cooling, centrifugation and cleaning and desalting

[0069] After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to 6 with sulfuric acid solution. Then, the mixture was centrifuged at 12500 rpm using a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of 96.78%. The solid was then washed with deionized water until there was no sodium ion (Na + ) and chloride ions (Cl - ) is detected;

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

[0071] The cleaned squid cartilage chitosan solid is put into a reactor, and a sulfuric acid solution twice the weight of the squid cartilage is added, the reaction temperature is 90° C., and the reaction is stirred for 6 hours, and the mass volume concentration of the sulfuric acid solution is 30%; after the hydrolysis reaction is completed, while stirring to dissipate heat, a saturated calcium hydroxide solution is used to adjust the pH to 7.5 until the temperature drops to 10° C., and then a continuous flow centrifuge is used at a low temperature (10° C.) Under high-speed centrifugation at 12500 rpm to remove calcium sulfate precipitation and heavy metal precipitation;

[0072] (4) Cation exchange chromatography / resin fractionation of glucosamine chelated calcium component and glucosamine component

[0073] Adsorb the centrifuged clear liquid using a cation exchange resin with a carboxyl group, and then desorb it by gradient elution using a 2.5% sodium chloride solution; fractionate the solution in sequence to obtain a glucosamine chelated calcium component and a glucosamine component using an ultraviolet detector at a wavelength of 192 nm and a fraction collector;

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

[0075] The glucosamine chelated calcium component and the glucosamine component are respectively subjected to a nanofiltration membrane with a relative molecular weight of 200Da to remove the inorganic salt sodium chloride, with a flow rate of 13.5 cubic meters per hour, a membrane pressure of 1.8 MPa, and a temperature of 30°C; and further concentrated to obtain a glucosamine chelated calcium sulfate solution with a content of 95.66% and a glucosamine sulfate solution with a content of 97.79%; finally, the glucosamine chelated calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried by spray drying technology to obtain glucosamine chelated calcium sulfate powder and glucosamine sulfate powder; the inlet air temperature of the spray drying is 170°C, and the outlet air temperature is 88°C.

[0076] Embodiment 3:

[0077] A large-scale preparation method of glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products from heavy metal contaminated squid cartilage comprises the following steps:

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

[0079] The squid cartilage detected to be contaminated with heavy metals was washed with clean water, placed in a hot air drying oven at 90°C for 2 hours, cut into 4 cm small pieces after being taken out, placed in a Chinese medicine grinder, and crushed for 5 minutes; the crushed squid cartilage powder was put into a reactor, and a sodium hydroxide solution 10 times the weight of the squid cartilage was added, the reaction temperature was 95°C, and the reaction was stirred for 6 hours, and the mass volume concentration of the sodium hydroxide solution was 15%;

[0080] (2) Cooling, centrifugation and cleaning and desalting

[0081] After the alkali treatment, the mixture was cooled to room temperature, and the pH was adjusted to 5.5 with sulfuric acid solution, and then centrifuged at 15000 rpm using a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of 94.36%; then, the mixture was washed with deionized water until there was no sodium ion (Na + ) and chloride ions (Cl - ) is detected;

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

[0083] The cleaned squid cartilage chitosan solid is put into a reactor, and a sulfuric acid solution of 4 times the weight of the squid cartilage is added, the reaction temperature is 95° C., and the reaction is stirred for 4 hours, and the mass volume concentration of the sulfuric acid solution is 15%; after the hydrolysis reaction is completed, while stirring to dissipate heat, a saturated calcium hydroxide solution is used to adjust the pH to 7 until the temperature drops to 8° C., and then a continuous flow centrifuge is used under low temperature (8° C.) conditions to remove calcium sulfate precipitation and heavy metal precipitation at a high speed of 15000 rpm;

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

[0085] The centrifuged clear liquid is adsorbed by using a preparative cation exchange chromatography with a sulfonic acid group, and then desorbed by gradient elution with a 5.0% sodium chloride solution; the glucosamine chelated calcium component and the glucosamine component are sequentially fractionated by using an ultraviolet detector at a wavelength of 195 nm and with the aid of a fraction collector;

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

[0087] The glucosamine chelated calcium component and the glucosamine component are respectively subjected to a nanofiltration membrane with a relative molecular weight of 200Da to remove the inorganic salt sodium chloride, with a flow rate of 16 cubic meters per hour, a membrane pressure of 2.20 MPa, and a temperature of 40°C; and further concentrated to obtain a glucosamine chelated calcium sulfate solution with a content of 95.48% and a glucosamine sulfate solution with a content of 98.95%; finally, the glucosamine chelated calcium sulfate solution and the glucosamine sulfate solution are respectively quickly dried by spray drying technology to obtain glucosamine chelated 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] Referring 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 amino)

[0090] Comparative Example 2

[0091] See the process diagram Figure 8 , using membrane separation technology to remove heavy metals and prepare 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, put into a reactor, added with a dilute hydrochloric acid solution 10 times the weight of the squid cartilage, and stirred at room temperature for 2 hours, wherein 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, put into a reactor, added with a sodium hydroxide solution 10 times the weight of the squid cartilage, the reaction temperature was 90°C, stirred for 5 hours, and the mass volume concentration of the sodium hydroxide solution was 20%;

[0095] (2) Cooling and centrifugation

[0096] After the alkali treatment, the temperature was lowered to room temperature, and the pH was adjusted to neutral with a dilute hydrochloric acid solution, and then high-speed centrifugation was performed at 14000 rpm using a continuous flow centrifuge to obtain a squid cartilage chitosan solid with a deacetylation degree of 95.35%, and 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 put into a reactor, and a hydrochloric acid solution of 5 times the weight of the squid cartilage was added, the reaction temperature was 85° C., and the reaction was stirred for 5 hours, and the mass volume concentration of the hydrochloric acid solution was 20%; after the hydrolysis reaction was completed, the temperature was lowered to room temperature, and then the pH was adjusted to 4.0 with a sodium hydroxide solution, and the mass volume concentration of the sodium hydroxide solution was 2%, and then a continuous flow centrifuge was used to centrifuge at 14000 rpm to obtain a supernatant;

[0099] (4) Membrane separation technology to remove heavy metals

[0100] A nanofiltration membrane with a relative molecular mass of 5 kDa is used to remove large molecular impurities in the supernatant, with a flow rate of 8 cubic meters per hour, a membrane pressure of 0.72 MPa, and a temperature of 35°C; the resulting clear liquid is 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), with 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 by spray drying technology to obtain the glucosamine sample powder with heavy metals removed by 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 Fig. 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 squid cartilage detected to be contaminated with heavy metals was washed with clean water, put into a reactor, added with a dilute hydrochloric acid solution 10 times the weight of the squid cartilage, and stirred at room temperature for 2 hours, wherein 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, put into a reactor, added with a sodium hydroxide solution 10 times the weight of the squid cartilage, the reaction temperature was 90°C, stirred for 5 hours, and the mass volume concentration of the sodium hydroxide solution was 20%;

[0108] (2) Cooling and centrifugation

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

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

[0111] The cleaned squid cartilage chitosan solid was put into a reactor, and a hydrochloric acid solution of 5 times the weight of the squid cartilage was added, the reaction temperature was 85° C., and the reaction was stirred for 5 hours, and the mass volume concentration of the hydrochloric acid solution was 20%; after the hydrolysis reaction was completed, the temperature was lowered to room temperature, and then the pH was adjusted to 4.0 with a sodium hydroxide solution, and the mass volume concentration of the sodium hydroxide solution was 2%, and then a continuous flow centrifuge was used to centrifuge at 14000 rpm to obtain a supernatant;

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

[0113] The centrifuged clear liquid was adsorbed and eluted using a chromatography column filled with D401 macroporous chelating resin, and the squid aminoglucose eluate was collected and combined by an ultraviolet detector at a wavelength of 195 nm and with the aid of a fraction collector.

[0114] (5) Spray drying

[0115] The glucosamine eluate was quickly dried by spray drying technology to obtain glucosamine sample powder with heavy metals removed by chelating resin adsorption method; the inlet air temperature of the spray drying was 160°C and the outlet air temperature was 85°C.

[0116] like Figure 1As shown, the present invention uses squid cartilage polluted by heavy metals in different sea areas as raw materials, firstly adopts high-speed crushing and high-temperature alkali treatment process 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 reaction to degrade squid cartilage chitosan into squid cartilage glucosamine, after the hydrolysis reaction is completed, while stirring and dissipating heat, saturated calcium hydroxide solution is used to adjust to a suitable pH to cause 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 glucosamine chelated calcium component and glucosamine component, and then the two fractions are respectively desalted and concentrated by nanofiltration membrane and spray-dried to obtain chromatographically pure glucosamine chelated calcium sulfate with a content of ≥95% and mass spectrometry pure glucosamine sulfate high-purity product with a content of ≥95%.

[0117] like Figure 2 As shown, the present invention refers to the method of the invention patent "An enzymatic production of glucosamine and its preparation method" (patent application number: 201410067600.X), using squid cartilage contaminated by heavy metals / not contaminated by heavy metals as raw materials, first decalcification by pretreatment, and 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, adjust the pH to neutral, and use continuous flow centrifugation to obtain squid cartilage chitosan solid with a deacetylation degree of ≥90%; then use glacial acetic acid to dissolve it into a colloid, and then use endo-chitosanase and exo-chitosanase to hydrolyze chitosan into glucosamine, and finally obtain a glucosamine sample 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 the contents of five heavy metals, 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 also the contents of three heavy metals, arsenic, cadmium and mercury, are 10.19 times, 8.72 times and 5.60 times of 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, with the contents of three heavy metals, arsenic, cadmium and lead, exceeding the limit standards of heavy metals in the national food safety standard "Limits of Contaminants in Food"; in contrast, the heavy metal pollution of squid cartilage from the North Pacific is better, with the contents of two heavy metals, 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 and Figure 4 As shown, 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, when the squid cartilage is not contaminated by heavy metals, the enzyme activities of the two chitosanases that must be used in the composite enzymatic hydrolysis method in Comparative Experiment 1, endochitosanase and exochitosanase, 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 decrease significantly, and as the heavy metal pollution of squid cartilage from the South Pacific Ocean to the North Pacific Ocean and then to the North Indian Ocean (the types and contents of heavy metals in squid cartilage) becomes more serious, the degree of decrease in the enzyme activities of endochitosanase and exochitosanase becomes more serious.

[0120] As shown in Table 2, as long as the chemical extraction method of the present invention is used, no matter the squid cartilage is from the North Pacific Ocean, the squid cartilage is from the South Pacific Ocean, or the squid cartilage is from the North Indian Ocean, and no matter whether the squid cartilage is contaminated by heavy metals, the extraction rate of the obtained samples (glucosamine chelated calcium sulfate and glucosamine sulfate high-purity product) is basically the same (21.64% to 21.66%). This result shows that the chemical extraction method used in the present invention is not affected by the regional source of the squid cartilage raw material, nor by the heavy metal contamination of the squid cartilage raw material, and has good 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 composite 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 squid cartilage increase and the heavy metal content increases), the downward trend of the extraction rate of glucosamine samples obtained by the composite enzymatic hydrolysis method of Comparative Experiment 1 becomes more and more obvious. The order of the extraction rate of glucosamine samples obtained by the composite enzymatic hydrolysis method of Comparative Experiment 1 for squid cartilage raw materials contaminated by heavy metals from different sea areas is South Pacific>North Pacific>North Indian Ocean. These results can be attributed to the significant inhibitory effect of heavy metals in squid cartilage from different sea areas on the enzyme activity of endochitosanase and exochitosanase necessary for the enzymatic reaction (see Figure 3 and Figure 4 ), which resulted in a significant decrease in the extraction rate of amino sugar 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 is 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 enzyme 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 pollution without the influence of heavy metal pollution of the squid cartilage raw material, and therefore, the chemical extraction method used in the present invention is also highly efficient.

[0123] Finally, considering that the two chitosanases (endochitosanase and exochitosanase) that must be used in 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 price of concentrated sulfuric acid raw material used in the chemical extraction method adopted by the present invention is extremely low (the price of 1000 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 by 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 advancement.

[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, under the conditions of wavelengths of 280nm, 220nm and 214nm, regardless of whether the squid cartilage is from the North Pacific Ocean, the South Pacific Ocean, or the North Indian Ocean, regardless of whether the squid cartilage raw material is contaminated by heavy metals, the glucosamine samples prepared by the process of Comparative Experiment 1 did not show any peaks, which indicates that there are no proteins or peptides in the glucosamine samples prepared by the process of Comparative Experiment 1; and these results further indicate that the chromatographic peaks of the glucosamine samples prepared by the process of Comparative Experiment 1 using squid cartilage from different waters that is contaminated by heavy metals and that is not contaminated by heavy metals as raw materials on the TSKgel GMPWXL column are not affected by proteins or peptides.

[0126] like Figure 7 As shown in the figure, under differential conditions, 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, all the glucosamine samples prepared by the process of comparative experiment 1 using squid cartilage not contaminated by heavy metals as raw materials show similar single peak shapes (4-6) on the TSKgel GMPWXL column; as is known to all, the separation peak shape of the sample on the chromatographic column is an important indicator for determining its purity. Therefore, this result shows that under the condition that the squid cartilage raw material is not contaminated by heavy metals, comparative experiment 1 using the composite enzymatic hydrolysis method and its subsequent process can prepare glucosamine with higher purity. However, the chromatographic peak shape of each glucosamine sample prepared by the process of comparative experiment 1 using squid cartilage contaminated by heavy metals as raw materials is a series of peak package shapes (1-3), which suggests that these glucosamine samples should contain a lot of impurities. As the heavy metal pollution 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 packages of the glucosamine samples prepared by the process of comparative experiment 1 on the chromatographic column become more and more discrete, especially in the chromatogram of the glucosamine sample prepared with the squid cartilage contaminated by heavy metals from the North Indian Ocean as raw material. In addition to a large peak package, the chromatographic peak representing the glucosamine sample also has a small miscellaneous peak in front. This is because the squid cartilage from the North Indian Ocean is most seriously polluted 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 presence 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 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, uses continuous flow centrifugation to obtain squid cartilage chitosan; then uses a high-temperature hydrochloric acid hydrolysis reaction to degrade the squid cartilage chitosan into squid cartilage glucosamine, and then uses continuous flow centrifugation 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 Fig. 9 As shown, the present invention refers to previous literature reports and patent technologies, uses heavy metal-contaminated squid cartilage as raw material, washes it and 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, uses continuous flow centrifugation to obtain squid cartilage chitosan; then uses a high-temperature hydrochloric acid hydrolysis reaction to degrade the squid cartilage chitosan into squid cartilage glucosamine, and then uses a continuous flow centrifugation synergistic chelating resin adsorption method and spray drying technology to obtain a glucosamine sample with heavy metals removed by chelating resin adsorption method.

[0129] As shown in Table 3, no heavy metals were detected in the samples (glucosamine chelated calcium sulfate and glucosamine sulfate high-purity product) obtained 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 the heavy metals carried by the squid cartilage raw material. At the same time, the content of most heavy metals in the glucosamine samples obtained by the membrane separation technology in the comparative experiment 2 showed a significant increase compared with the heavy metal content of the squid cartilage raw material; while the content of heavy metals in the glucosamine samples obtained by the chelating resin adsorption method in the comparative experiment 3 was almost unchanged compared with 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 chelate 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 chelation effect with heavy metal ions, and the formed glucosamine-heavy metal chelate is stable, so that neither membrane separation technology nor chelate 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 essentially a chelate formed by multiple glucosamine molecules and one metal ion (such as Fig.12), whose molecular weight is significantly greater than that of glucosamine, resulting in that during the membrane separation process, unchelated glucosamine molecules and other small molecules are more likely to pass through the separation membrane and be removed (equivalent to a decrease in the content of unchelated glucosamine in the sample), while glucosamine-heavy metal chelates are less likely to pass through the separation membrane and are therefore retained (equivalent to an increase in the content of glucosamine-heavy metal chelates in the sample); this results in a significant increase in the content of most heavy metals in the glucosamine samples obtained after the membrane separation technology was used in the comparative experiment.

[0130] It can be seen from the experimental results of Table 1 and Table 3 that no heavy metals were detected in the samples (sugar chelated calcium sulfate and high-purity sugar sulphate) obtained after the chemical precipitation method of the present invention, which meets the limit standard requirements of heavy metals in the national food safety standard "Limit of Contaminants in Food"; and the content of heavy metals in the sugar sulphate samples obtained after the membrane separation technology of comparative experiment 2 and the sugar sulphate samples obtained after the chelating resin adsorption method of comparative experiment 3 exceeded the limit standard of heavy metals in the national food safety standard "Limit of Contaminants in Food". These results prove that the samples (sugar chelated calcium sulfate and high-purity sugar sulphate) obtained after the chemical precipitation method of the present invention can be applied to the food field; and the sugar sulphate samples obtained after the membrane separation technology of comparative experiment 2 and the sugar sulphate samples obtained after the chelating resin adsorption method of comparative experiment 3 can not be applied to the food field. These results once again prove the practicality and significant progress of the present invention.

[0131] As shown in Table 4, 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, the extraction rate of the sample (glucosamine chelated calcium sulfate and glucosamine sulfate high-purity product) obtained by the chemical precipitation method of the present invention is not only slightly higher than the extraction rate of the sample (glucosamine) obtained by the membrane separation technology of Comparative Experiment 2, but also significantly higher than the extraction rate of the sample (glucosamine) obtained by the chelate resin adsorption method of Comparative Experiment 3. These results show that the heavy metal removal method used in the present invention, the chemical precipitation method, is superior to the heavy metal removal method commonly used in previous literature reports and patent technologies, namely, the chelate resin adsorption method. -Membrane separation technology or chelate resin adsorption method has great advantages in sample extraction rate; the experimental result that the extraction rate of the sample (glucosamine) obtained by the chelate resin adsorption method of comparative experiment 3 is significantly lower also shows that the glucosamine-heavy metal chelate formed by the glucosamine after degradation of squid cartilage and heavy metal ions has good stability, and the previous chelate resin adsorption method cannot effectively separate the glucosamine molecules from the heavy metal ions, that is, the substances adsorbed by the chelate resin may not only be heavy metals, but the glucosamine-heavy metal chelate should be adsorbed together; this leads to the phenomenon that the extraction rate of the sample (glucosamine) obtained by the chelate resin adsorption method of comparative experiment 3 is significantly lower.

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

[0133] like Fig.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 Fig.11 As shown, under the wavelength conditions of 280nm, 220nm and 214nm, the glucosamine chelated calcium sulfate and glucosamine sulfate prepared by the present invention do not show any peak shape. This result shows on the one hand that there are no protein or peptide substances in the glucosamine chelated calcium sulfate and glucosamine sulfate samples prepared by the present invention; on the other hand, it also shows that the high-speed crushing and high-temperature alkali treatment process of the present invention can successfully remove cartilage protein.

[0135] like Figure 5 and Fig.11 As shown in Figure 2, both glucosamine chelated calcium and glucosamine showed 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 chromatographic purity level;

[0136] like Figure 7 and Fig.11 As shown, the peak shape of glucosamine chelated calcium sulfate and glucosamine sulfate prepared by the present invention on the TSKgel GMPWXL column is not inferior to, or even better than, the peak shape of various glucosamine samples prepared by the process of comparative experiment 1 using squid cartilage from different sea areas that is not contaminated by heavy metals as raw materials on the TSKgel GMPWXL column. This result proves that the technical process of the present invention can completely replace the composite enzymatic hydrolysis method that is generally recommended by previous literature reports and patent technologies but is expensive and has strict requirements on raw materials (squid cartilage must be free of heavy metal pollution) to prepare chromatographically pure products.

[0137] like Fig.11 As shown, the peak time of glucosamine chelated calcium is greater than the peak time of the 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 the dextran standard with a molecular weight of 342, and is not within the range of molecular weight 405700-342. The molecular weight of glucosamine chelated calcium can be determined according to Figure 6 The molecular weight was calculated to be 398 based on the standard curve.

[0138] like Fig.12As shown, based on the molecular weight 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] like Fig.13 As shown, the glucosamine in glucosamine sulfate plus sodium cation [C6H 13 NO5Na] + The theoretical accurate mass number 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 present invention reaches the mass spectrometry pure level.

[0140] As shown in Table 5, in terms of the number of writhing, the number of writhing in the high, medium and low dose groups of glucosamine was significantly less than that in the blank control group, and with the increase of the dose of glucosamine, the number of writhing of mice caused by acetic acid gradually decreased; at the same time, compared with the blank control group, the high, medium and low dose groups of glucosamine had statistical differences (P<0.05), among which the high and medium dose groups of glucosamine also had extremely significant statistical differences (P<0.01); in terms of pain inhibition rate, the high, medium and low dose groups of glucosamine were 31.12%, 24.04% and 19.63% respectively compared with the blank control group. This result shows that: for the acetic acid-induced mouse writhing pain experiment, the high-purity glucosamine sulfate prepared by the present invention has a good analgesic effect; and with the continuous increase of the concentration of the high-purity glucosamine sulfate, the analgesic effect is gradually enhanced.

[0141] As shown in Table 6, in terms of swelling rate, compared with the blank control group, the auricle swelling rates of mice in the high, medium and low dose groups of glucosamine were all reduced, and the difference was extremely significant (P<0.01); in terms of swelling inhibition rate, the swelling inhibition rate of the high-purity glucosamine sulfate prepared by the present invention increased with increasing dose. The results show that: for the soybean oil-induced mouse auricle swelling experiment, the high-purity glucosamine sulfate prepared by the present invention has good anti-inflammatory activity.

[0142] Table 1

[0143]

[0144]

[0145] Note: Table 1 shows the heavy metal pollution of squid cartilage raw materials from different sea areas (n=3). Among them, the detection method of heavy metal arsenic content in squid cartilage adopts the national food safety standard "Determination of total arsenic and inorganic arsenic in food" (GB5009.11-2024); the detection method of heavy metal cadmium content in squid cartilage adopts the national food safety standard "Determination of cadmium in food" (GB 5009.15-2023); the detection method of heavy metal lead content in squid cartilage adopts the national food safety standard "Determination of lead in food" (GB 5009.12-2023); the detection method of heavy metal mercury content in squid cartilage adopts the national food safety standard "Determination of total mercury and organic mercury in food" (GB 5009.17-2021); the detection method of heavy metal chromium content in squid cartilage adopts the national food safety standard "Determination of chromium in food" (GB 5009.123-2023); the limit standards 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 is amino sugar.

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

[0151]

[0152] The calculation formula of 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 by using the membrane separation technology in comparative experiment 2 and the chelate resin adsorption method in comparative experiment 3 are both glucosamine. Note: Table 3 is a comparison of the heavy metal content of the samples obtained by treating squid cartilage from different sea areas with 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 by using the membrane separation technology in comparative experiment 2 and the chelate resin adsorption method in comparative experiment 3 are both glucosamine. Note: Table 4 is a comparison of the extraction rates of the samples obtained by treating squid cartilage from different sea areas with 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 by 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, with 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 dose 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] ②Method of administration

[0176] Each group of mice was intraperitoneally administered with a set dose of glucosamine once for seven consecutive days. On the eighth day, 30 minutes after intraperitoneal administration of 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 times the mice twisted 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, with extremely significant difference.

[0183] Note: Table 6 shows the effect of glucosamine prepared by the present invention on soybean oil-induced ear swelling in mice (n=10). The present invention verifies the anti-inflammatory effect of glucosamine by evaluating the 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 dose 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] ②Method of administration

[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 of each mouse and 20 μL of 5% soybean oil was applied to the right ear. Four hours later, the mice were killed and their ears were cut off and sampled one by one using a hole 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 high-purity products 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, placed in a hot air drying oven at 70-90°C for 2-5 hours, taken out and cut into small pieces of 3-5 cm, placed in a Chinese medicine grinder, and crushed for 5-8 minutes; the crushed squid cartilage powder is put into a reaction kettle, and a sodium hydroxide solution of 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, and the mass volume concentration of the sodium hydroxide solution is 10-30%; (2) Cooling, centrifugation and cleaning and desalting After the alkali treatment, the temperature was lowered to room temperature, and the pH was adjusted to 5-6.5 with sulfuric acid solution, and then high-speed centrifugation was performed in a continuous flow centrifuge to obtain squid cartilage chitosan solid with a deacetylation degree of ≥90%; then, the solid was washed with deionized water until there was no sodium ion Na + and chloride ion Cl - Until it is detected; (3) High-temperature sulfuric acid hydrolysis reaction, chelation reaction of glucosamine and calcium in the cooling stage, and low-temperature centrifugation to remove metal precipitates. The cleaned squid cartilage chitosan solid is put into a reactor, and a sulfuric acid solution of 2 to 5 times the weight of the squid cartilage is added. The reaction temperature is 85 to 100° C. and stirred for 2 to 6 hours. The mass volume concentration of the sulfuric acid solution is 10 to 30%. After the hydrolysis reaction is completed, the pH is adjusted to 7 to 8.5 with a saturated calcium hydroxide solution while stirring to dissipate heat until the temperature drops 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 / resin fractionation of glucosamine chelated calcium component and glucosamine component Adsorbing the centrifuged clear liquid with a cation exchange chromatography / resin, and then desorbing it with a gradient elution solution containing 2.5-5.0% sodium chloride; fractionating in sequence to obtain a glucosamine chelated calcium component and a glucosamine component by 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 200Da 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 preparing glucosamine chelated calcium sulfate and glucosamine sulfate high-purity products 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 Pacific squid from the North Pacific Ocean, American giant red squid from the South Pacific Ocean, or kite squid from the North Indian Ocean.

3. The method for large-scale preparation of glucosamine chelated calcium sulfate and high-purity 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 "Food Additive Deacetylated Chitosan (Chitosan)" (GB 29941-2013); the detection method for sodium ions is the detection method of the national food safety standard "Determination of Potassium and Sodium in Food" (GB 5009.91-2017); the detection method for chloride ions is the detection method of the national food safety standard "Determination of Chloride in Food" (GB5009.44-2016).

4. The method for large-scale preparation of glucosamine chelated calcium sulfate and high-purity 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 of 10,000 to 15,000 rpm, and the centrifugal speeds are 10,000 to 15,000 rpm, respectively.

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

6. The method for large-scale preparation of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate from heavy metal-contaminated squid cartilage according to claim 1, characterized in that: 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.

7. The method for large-scale preparation of glucosamine chelated calcium sulfate and high-purity 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 high-purity 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 the chromatographic purity level; the glucosamine sulfate with a content of ≥95% obtained by nanofiltration membrane desalination and concentration in step (5) is a high-purity product with a mass spectrometry purity level.

9. High-purity glucosamine sulfate prepared according to the large-scale preparation method of glucosamine chelated calcium sulfate and high-purity glucosamine sulfate from heavy metal-contaminated squid cartilage according to any one of claims 1 to 8.

10. Use of the highly purified glucosamine sulfate according to claim 9 in the preparation of novel functional foods or medicines with anti-inflammatory and analgesic activities.

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