Method for extracting, by using low-salinity-water immersion, glycosaminoglycans from pig small intestines and improving quality of sundae casing

Abstract

The present invention relates to a method for extracting glycosaminoglycans (GAGs) without damaging a casing by immersing, in a predetermined concentration or less of brine, pig small intestines from which the mucosa has not been removed, and the method comprises the steps of: (S1) washing, with water, pig small intestines from which the mucosa has not been removed, and then immersing same in a predetermined concentration or less of brine so as to prevent degeneration of the tissue of the pig small intestines, thereby obtaining a brine mixture; (S2) causing the brine mixture to be an alkaline environment; (S3) adding an enzyme to the brine mixture so as to hydrolyze the brine mixture; (S4) lowering the pH of the hydrolyzed brine mixture and separating fat therefrom; (S5) adding an ion-exchange resin to the brine mixture, from which fat has been separated, so as to cause a GAG-based polymer included in the brine mixture to be adsorbed onto the ion-exchange resin; (S6) collecting the ion-exchange resin onto which the GAG-based polymer is adsorbed, washing same and preparing an eluate with brine; and (S7) mixing the eluate with an organic solvent so as to recover a generated precipitate, and then drying same to obtain a GAG-based polymer.

Description

Extraction of glycosaminoglycans from pig intestines using a low-salt immersion method and improvement of the quality of sundae casings

[0001] The present invention relates to a method for extracting glycosaminoglycans from a brine mixture obtained by soaking pig intestines in low-salt water.

[0002]

[0003] Heparin sodium is the sodium salt of aminodextran sulfate extracted from porcine intestinal mucosa. It exhibits anticoagulant activity as a viscous polysaccharide sulfate ester, making it one of the most widely used and most effective anticoagulants worldwide. Due to these properties, heparin sodium is primarily used for the treatment of cardiovascular and cerebrovascular diseases, hemodialysis, and other conditions. It is also used as a raw material for pharmaceuticals such as low-molecular-weight heparin sodium and heparin sodium injection.

[0004] This heparin sodium is usually extracted from the lungs of cattle or the small intestines of pigs. When using the small intestines of pigs, the casing (intestine) and the mucosa (offal) are physically separated, and the mucosa is used to extract heparin sodium, while the casing is used to make sausage casings, medical suture threads, etc.

[0005] Heparin sodium can be obtained by adding a protein hydrolytic enzyme to a physically separated mucosa to hydrolyze it, separating the fat, adsorbing the active ingredient to an ion exchange resin, and then desorbing and precipitating the adsorbed ingredient.

[0006] Meanwhile, in Korea, where sundae consumption is high, pork small intestines are used in sundae production, resulting in high demand not only in the pharmaceutical industry but also in the food industry. However, physically separating the casing and mucosa as described above leads to problems such as mixing visceral fat and outer skin, which increases nucleic acid content and reduces purity, ultimately lowering yield. Consequently, the supply of small intestines suitable for sundae production is reduced, driving up unit prices.

[0007]

[0008] The present invention is intended to solve the above problems, and to provide a method for easily extracting heparin while maintaining the shape of the intestines and not conflicting with the sundae industry.

[0009]

[0010] A method for extracting heparin from pig intestines and improving the quality of sundae casing using a low-salt immersion method according to an embodiment of the present invention is as follows.

[0011] (S1) A step of washing the pig intestines with water from which the mucosa has not been removed, and then immersing them in saline solution having a concentration below a certain level to prevent degeneration of the pig intestine tissue, thereby obtaining a saline mixture;

[0012] (S2) A step of creating an alkaline environment by mixing the brine mixture;

[0013] (S3) A step of adding an enzyme to the brine mixture to hydrolyze the brine mixture;

[0014] (S4) A step of lowering the pH of the hydrolyzed brine mixture and separating fat;

[0015] (S5) A step of adding an ion exchange resin to the salt water mixture from which the fat has been separated to adsorb the glycosaminoglycan (GAG) polymer contained in the salt water mixture onto the ion exchange resin;

[0016] (S6) A step of collecting and washing the ion exchange resin on which the above glycosaminoglycan polymer is adsorbed and preparing an eluate using saline water;

[0017] (S7) A step of obtaining a glycosaminoglycan polymer by mixing an organic solvent into the above-mentioned eluate, recovering the resulting precipitate, and then drying the same may be included.

[0018] In addition, the above step S1 may be a step of immersing the washed pig intestines in a saline solution having a concentration of 1 to 20%, more preferably 3 to 8%, to obtain a saline mixture containing glycosaminoglycans.

[0019] Additionally, the S2 step may be a step of adjusting the pH to 8.8 to 9.3 by adding NaOH to the brine mixture.

[0020] In addition, the S3 step may be a step of hydrolyzing the brine mixture by adding 0.1 to 5 parts by weight of a protein-decomposing enzyme to 100 parts by weight of the brine mixture.

[0021] Additionally, the S4 step may be a step of adding sodium bisulfite to the hydrolyzed brine mixture to lower the pH to 6.5 to 7.5 and separate fat.

[0022] In addition, the above step S5 may be a step of adding a strong anion exchange resin to the salt water mixture from which the fat has been separated to adjust the pH to 8.4 to 8.6, and adsorbing the glycosaminoglycan polymer contained in the salt water mixture to the ion exchange resin.

[0023]

[0024] The present invention provides a method for extracting heparin from process water generated in a washing process of pig intestines, thereby enabling heparin to be extracted without damaging the casing.

[0025] Accordingly, the quality of sundae skin can be improved, wastewater treatment costs can be reduced by recovering and using process water generated during the washing process of pig intestines, and it can also contribute to reducing the cost of heparin sodium.

[0026]

[0027] Figure 1 is a flow chart of the extraction method of the present invention.

[0028] Figure 2 is a drawing showing the color development of uronic acid and the content of glycosaminoglycans analyzed by reacting raw materials according to concentration using the Carbazole Assay analysis method in Example and Comparative Example 1.

[0029] Figure 3 is a photograph of sundae manufactured using the casings of Example 1 and Comparative Example 1.

[0030] Figure 4 is a diagram showing the HPLC analysis results of Example and Comparative Example 2.

[0031] Figure 5 is a diagram showing the results of anticoagulant activity analysis of Experimental Example and Comparative Example 2.

[0032] Figure 6 is a diagram showing the results of H-NMR analysis of an experimental example.

[0033] Figure 7 is a diagram showing the results of tensile strength and elasticity analysis of pig intestines of Experimental Example and Comparative Example 3.

[0034] Figure 8 is a photograph of a sundae manufactured using the casing of Example 1 and Comparative Example 3.

[0035]

[0036] (S1) A step of washing the pig intestines with water from which the mucosa has not been removed, and then immersing them in saline solution having a concentration below a certain level to prevent degeneration of the pig intestine tissue, thereby obtaining a saline mixture;

[0037] (S2) A step of creating an alkaline environment by mixing the brine mixture;

[0038] (S3) A step of adding an enzyme to the brine mixture to hydrolyze the brine mixture;

[0039] (S4) A step of lowering the pH of the hydrolyzed brine mixture and separating fat;

[0040] (S5) A step of adding an ion exchange resin to the salt water mixture from which the fat has been separated to adsorb the glycosaminoglycan (GAG) polymer contained in the salt water mixture onto the ion exchange resin;

[0041] (S6) A step of collecting and washing the ion exchange resin on which the above glycosaminoglycan polymer is adsorbed and preparing an eluate using saline water;

[0042] (S7) A method for extracting glycosaminoglycans from pig intestines and improving the quality of sundae casings using a low-salt immersion method, including a step of mixing an organic solvent into the above-mentioned eluate, recovering the resulting precipitate, and then drying it to obtain a glycosaminoglycan polymer.

[0043]

[0044] The following description of the present invention with reference to the drawings is not limited to specific embodiments, and various modifications and embodiments may be made. Furthermore, the following description should be understood to encompass all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0045] In the following description, terms such as first, second, etc. are used to describe various components, and are not limited in meaning in themselves, but are used only for the purpose of distinguishing one component from another.

[0046] The same reference numbers used throughout this specification represent the same components.

[0047] As used herein, singular expressions include plural expressions unless the context clearly dictates otherwise. In addition, terms such as "comprise," "include," or "have" used herein should be interpreted to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, and should be understood to not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0048] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0049] In addition, when describing with reference to the attached drawings, identical components will be assigned the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing the present invention, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

[0050] Hereinafter, with reference to FIGS. 1 to 8, a method for extracting glycosaminoglycans from pig intestines and improving the quality of sundae casings using a low-salt immersion method according to an embodiment of the present invention will be described in detail.

[0051]

[0052] Figure 1 is a flow chart of the extraction method of the present invention.

[0053] Referring to FIG. 1, the method for extracting glycosaminoglycans from pig intestines and improving the quality of sundae casing using the low-salt immersion method of the present invention is as follows.

[0054] (S1) A step of washing the pig intestines with water from which the mucosa has not been removed, and then immersing them in saline solution having a concentration below a certain level to prevent degeneration of the pig intestine tissue, thereby obtaining a saline mixture;

[0055] (S2) A step of creating an alkaline environment by mixing the brine mixture;

[0056] (S3) A step of adding an enzyme to the brine mixture to hydrolyze the brine mixture;

[0057] (S4) A step of lowering the pH of the hydrolyzed brine mixture and separating fat;

[0058] (S5) A step of adding an ion exchange resin to the salt water mixture from which the fat has been separated to adsorb the glycosaminoglycan (GAG) polymer contained in the salt water mixture onto the ion exchange resin;

[0059] (S6) A step of collecting and washing the ion exchange resin on which the above glycosaminoglycan polymer is adsorbed and preparing an eluate using saline water;

[0060] (S7) A step of obtaining a glycosaminoglycan polymer by mixing an organic solvent into the above-mentioned eluate, recovering the resulting precipitate, and then drying the same may be included.

[0061]

[0062] (S1) Step may be a step of obtaining a saline mixture by washing the pig intestines from which the mucosa has not been removed with purified water and then immersing them in saline solution of a certain concentration or lower so as to prevent denaturation of the pig intestine tissue.

[0063] Specifically, after first washing the foreign substances on the surface of the pig intestines with water, the pig intestines are immersed in low-salt brine having a temperature of 2 to 8°C and a concentration of 1 to 20%, more preferably 3 to 8%, to obtain a brine mixture.

[0064] It is known that glycosaminoglycans (hereinafter referred to as GAGs) are easy to extract when dissolved in a salty substance. Therefore, the present invention dissolves GAGs by immersing pig intestines in salt water.

[0065] At this time, if the concentration of the brine is less than 3%, heparin may stick to the GAGs finally obtained, which may lower the purity of the heparin. If it exceeds 8%, when filling the sundae casing and performing post-aging, the tissue may degenerate, which may lower the quality of the casing.

[0066] When pork intestines are immersed in low-salt brine as described above, many substances including GAGs can be dissolved in the brine, and the quality of the casing can be improved by not physically separating the casing and the mucosa.

[0067]

[0068] (S2) Step may be a step of creating an alkaline environment for the brine mixture.

[0069] Specifically, it may be a step of adjusting the pH to 8.8 to 9.3 by adding NaOH to the brine mixture obtained in step (S1).

[0070] At this time, the substance added to create an alkaline environment is not limited, but it is most preferable to use NaOH.

[0071] In addition, it is desirable to adjust the pH to 8.8 to 9.3 because an alkaline environment must be created in step (S2) so that hydrolysis can easily occur when enzymes are added in step (S3) to be performed later, thereby increasing the recovery rate of GAGs.

[0072]

[0073] (S3) Step may be a step of hydrolyzing the brine mixture by adding an enzyme to the brine mixture.

[0074] Specifically, the brine mixture obtained in step (S2) is heated to 50 to 60°C, and then 0.1 to 5 parts by weight of alkaline protease, a protein-decomposing enzyme, is added based on 100 parts by weight of the brine mixture, and biologically hydrolyzed for 2 to 16 hours, more preferably 2 to 8 hours, and most preferably 4 hours.

[0075] At this time, it is most preferable to use a protein-decomposing enzyme including alkaline protease as the enzyme added to the brine mixture, but it is not limited thereto, and any enzyme capable of hydrolyzing the brine mixture can be used.

[0076] Additionally, if the reaction time is less than 2 hours, sufficient enzymatic reaction may not occur, and if it exceeds 16 hours, unnecessary process costs may occur.

[0077] However, since enzyme activity is optimal when the substrate and enzyme react for a certain period of time to obtain the maximum product, it was confirmed that it is most desirable to react the protein-decomposing enzyme around 4 hours when it exhibits maximum activity.

[0078] As described above, when the hydrolysis of the brine mixture is completed, the protein-decomposing enzyme is inactivated and then filtered to obtain a filtrate.

[0079] Specifically, the protein-degrading enzyme and un-degraded mucosal protein in the hydrolyzed saline mixture can be rapidly heated to 75 to 95°C and then heat-treated for 5 to 15 minutes to denature and inactivate them.

[0080] As the above inactivation method is a known technology, further detailed description will be omitted.

[0081] Once the inactivation of the proteolytic enzyme is complete, the insoluble substances in the saline mixture, i.e. the impurities remaining in the mucosa, can be filtered through a 60 mesh sieve.

[0082]

[0083] (S4) Step may be a step of lowering the pH of the hydrolyzed brine mixture and separating fat.

[0084] Specifically, sodium bisulfite can be added to the hydrolyzed brine mixture to lower the pH to 6.5–7.5 and separate the fat.

[0085] Sodium bisulfite is a substance used as a preservative in the industry when added to small intestine during distribution. However, the present invention confirmed that the pH was lowered when sodium bisulfite was added to a saline mixture, and used sodium bisulfite as a pH regulator.

[0086] As will be explained later, the present invention has confirmed that the extraction efficiency of GAGs is best when the pH is approximately 8.5 when adsorbing GAGs onto an ion exchange resin. Therefore, considering that the pH increases when the ion exchange resin is added, sodium bisulfite was added to the hydrolyzed brine mixture to adjust the final pH to 8.5, thereby adjusting the pH to 6.5-7.5.

[0087] Additionally, when sodium bisulfite is added to the hydrolyzed brine mixture, the pH gradually becomes neutral or slightly acidic, allowing the lipid layer to separate from the brine mixture.

[0088] In the past, the casing and mucosa of the small intestine were physically separated, thereby reducing the occurrence of a fat layer in the brine mixture during GAG extraction. However, the present invention uses a brine mixture in which the small intestine is immersed without separating the casing and mucosa, resulting in the brine mixture containing a large amount of impurities, including fat.

[0089] To address this issue, the present invention adds sodium bisulfite to a hydrolyzed brine mixture, thereby lowering the pH and simultaneously separating fat, thereby purifying the brine mixture. Accordingly, compared to a brine mixture from which fat has not been separated, the adsorption efficiency of GAG polymers in the subsequent ion exchange resin adsorption step can be increased.

[0090]

[0091] (S5) Step may be a step of adding an ion exchange resin to the salt water mixture from which fat has been separated, thereby adsorbing GAG polymers contained in the salt water mixture onto the ion exchange resin.

[0092] Specifically, the temperature of the brine mixture from which fat has been separated is lowered to 62°C, and then FPA98 Cl-type strong anion exchange resin is added to adjust the pH to 8.4 to 8.6, and the mixture is stirred for 4 hours to adsorb GAGs onto the ion exchange resin.

[0093] At this time, it is most preferable to use FPA98 Cl-type strong anion exchange resin as the ion exchange resin, but it is not limited to this, and any ion exchange resin that can adsorb GAG type polymers can be used.

[0094] In addition, when a strong anion exchange resin is added, the pH increases. The present invention confirmed that the optimal GAG extraction efficiency is derived when the pH is adjusted to about 8.5. In order to adjust the increased pH to 8.5, sodium bisulfite was added to the brine mixture in step (S4) to lower the pH to 6.5 to 7.5 in advance.

[0095]

[0096] (S6) The step may be a step of collecting and washing the ion exchange resin on which GAGs are adsorbed and preparing an eluate using brine.

[0097] Specifically, the ion exchange resin can be collected by passing the brine mixture with the ion exchange resin through a 100 mesh sieve and washing it twice with 4% brine. Then, the first elution can be performed for 3 hours with 14% brine, which is 4 times the volume of the ion exchange resin, and the second elution can be performed for 2 hours with 16% brine, which is 2 times the volume of the ion exchange resin, to prepare an eluate.

[0098] At this time, it is most preferable that the brine be saline, and when the first elution is performed with 14% saline as described above, about 85% of GAGs are desorbed from the ion exchange resin, and by performing the second elution with 16% saline, about 10% of GAGs can be additionally desorbed.

[0099] At this time, in the past, a phenomenon occurred in which salt was easily precipitated by desorbing GAGs using high-salt saline solution, but the present invention uses a method of eluting twice with low-salt saline solution, so that salt is not easily precipitated and the cost can be reduced.

[0100]

[0101] (S7) Step may be a step of obtaining a GAG polymer by mixing an organic solvent into the eluent, recovering the precipitate generated, and then drying it.

[0102] (S7) Step may be performed by mixing an organic solvent with the eluate obtained in step (S6) to prepare an organic solvent mixture of 15 to 60%, more specifically 18 to 25%, and then precipitating the mixture at room temperature for 8 to 24 hours.

[0103] At this time, it is preferable to use ethanol as the organic solvent, but the type of organic solvent is not limited, and any organic solvent used for the purpose of recovering precipitates, such as methanol and acetic acid, can be used.

[0104] However, organic solvents including methanol are not recommended for worker safety, and it is most desirable to use edible alcohol ethanol.

[0105] Additionally, it was confirmed that the yield was best when the organic solvent mixture, especially edible alcohol ethanol, was prepared at a concentration of 18 to 25%, and there may be a risk of salt precipitation when it exceeds 25%.

[0106] Once the precipitation of the organic solvent mixture is complete, the precipitate can be collected and vacuum dried, ultimately obtaining a powder containing GAGs.

[0107] The GAGs obtained here may be composed of one or more of chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, and hyaluronan.

[0108]

[0109] Hereinafter, the present invention will be described in detail with reference to examples and experimental examples.

[0110]

[0111] [Example]

[0112] After washing the pig intestines without removing the mucosa with purified water, they were immersed in 5% saline at 5°C to obtain a saline mixture.

[0113] NaOH was added to the above brine mixture to adjust the pH to 9.0, and after heating to 55°C, 1.5 parts by weight of alkaline protease was added relative to 100 parts by weight of the brine mixture, and biological hydrolysis was performed for 4 hours.

[0114] The hydrolyzed brine mixture was heat treated at 90°C for 15 minutes to inactivate the proteolytic enzyme and then filtered through a 60-mesh sieve.

[0115] Sodium bisulfite was added to the filtered brine mixture to adjust the pH to 7.0 and separate the fat.

[0116] After lowering the temperature of the brine mixture from which the fat was separated to 62°C, FPA98 Cl-type strong anion exchange resin was added to adjust the pH to 8.5, and the mixture was stirred for 4 hours to adsorb GAGs onto the ion exchange resin.

[0117] The ion exchange resin was collected by passing the brine mixture containing the ion exchange resin through a 100 mesh sieve and washed twice with 4% brine. Then, the first elution was performed with 14% brine, which was 4 times the resin volume, for 3 hours, and then the second elution was performed with 16% brine, which was 2 times the resin volume, for 2 hours to prepare an eluate.

[0118] A 20% ethanol mixture was prepared by mixing edible ethanol into the extract, precipitating it at room temperature for 16 hours, and then collecting the precipitate and vacuum-drying it to obtain a powder containing GAGs.

[0119]

[0120] [Comparative Example 1]

[0121] After washing the pig intestines without removing the mucosa with purified water, they were immersed in 5% saline at 5°C and then treated with 30 kHz ultrasound to obtain a saline mixture.

[0122] A powder containing GAGs was obtained in the same manner as in the example, except for the process of obtaining the above brine mixture.

[0123]

[0124] [Comparative Example 2]

[0125] The casing and mucosa of the pig intestine without the mucosa removed were physically separated, and the mucosa was immersed in purified water to obtain a mixture.

[0126] A powder containing GAGs was obtained in the same manner as in the example, except for the process of obtaining the above mixture.

[0127]

[0128] [Comparative Example 3]

[0129] After washing the pig intestines without removing the mucosa with purified water, they were immersed in 20% saline at 5°C to obtain a saline mixture.

[0130] A powder containing GAGs was obtained in the same manner as in the example, except for the process of obtaining the above brine mixture.

[0131]

[0132] [Experimental Example 1] Analysis of glycosaminoglycan content

[0133] Experimental Example 1 is an experiment to confirm the effect of ultrasonic treatment during brine immersion of pig intestines on the content of GAGs and the quality of casings.

[0134] The contents of GAGs and their derivatives were analyzed using a carbazole reagent for the samples of Example and Comparative Example 1.

[0135] For quantitative analysis of GAGs, the carbazole analysis method was used, which reacts carbazole, a dye source of D-glucuronic acid among GAGs composed of amino sugars and uronic acid, to create a red compound, which was then observed using a spectrophotometer.

[0136] The amount of glycosaminoglycan was analyzed by preparing a standard solution at each concentration using a D-glucuronic acid standard and creating a calibration curve to analyze the amount of GAG contained in the powder.

[0137] The sample solution collected step by step was centrifuged at 13,000 rpm for 10 minutes to remove impurities and collect the supernatant.

[0138] The carbazole assay method is as follows.

[0139] First, 200 μl of H2SO4 reagent containing 25 mM sodium tetraborate was added to 50 μl of sample solution (1 mg / ml), and then heated at 100°C for 15 minutes. After cooling to room temperature for 10 minutes, 50 μl of 0.125% carbazole reagent was carefully added. After heating again at 100°C for 15 minutes and cooling to room temperature for 15 minutes, the sample was transferred to a 96-well plate, and the absorbance was measured at a wavelength of 550 nm using a microplate reader.

[0140] Figure 2 is a photograph of raw materials reacted using the Carbazole Assay analysis method and a drawing analyzing the content of glycosaminoglycans, and Figure 3 is a photograph of sundae manufactured using the casings of Example and Comparative Example 1.

[0141] Referring to Figure 2, it can be seen that the absorbance measurement results of the examples and comparative examples show that the value of comparative example 1 is measured slightly higher than that of the examples, but there is no significant difference.

[0142] However, referring to Fig. 3, which is a photograph taken immediately after the casing was filled and the sundae was steamed, it can be seen that the sundae was manufactured normally in the case of the embodiment (Fig. 3a), but in the case of Comparative Example 1 (Fig. 3b), it can be seen that the sundae casing is opaque and the filling burst out even though it is hot. This is because the protein structure of the sundae casing, which has been subjected to physical stimulation such as ultrasonic waves, is affected, reducing the bonding strength between the filling and the casing. In the case of Comparative Example 1, not only is it difficult to maintain the original taste because the sundae casing and the filling are separated, but also the commercial value is reduced in terms of appearance, making it difficult to apply to the food industry.

[0143]

[0144] [Experimental Example 2] HPLC Analysis

[0145] Experimental Example 2 is an experiment in which heparin and heparinoids included in Example and Comparative Example 2 were analyzed using HPLC analysis.

[0146] HPLC analysis was performed with reference to the USP (United States Pharmacopeia) and modified and supplemented to meet our own standards, as shown in [Table 1] below.

[0147] Detection system: SHIMADZU product, UV detector column and guard column: IonPac AS11-HC (2 mm × 250 mm), IonPac AS11-HC (2 mm × 5 mm) Column temperature: 40°C Mobile phases: Both A and B were prepared with 2.6 mM sodium phosphate (pH 3), and 1 M sodium perchlorate was added to solvent B. Separation flow rate: 0.22 ml / min

[0148] FIG. 4 is a diagram showing the HPLC analysis results of Example and Comparative Example 2. Referring to FIG. 4, Comparative Example 2 shows a generally unclear peak, indicating that fat is not removed when the casing and the mucosa are physically separated, and thus contains a lot of impurities, and that it must be used after going through an additional purification process.

[0149] However, in the case of the example, the DS and HP peaks are clearly separated, and only two peaks similar to the standard are confirmed, indicating that high-purity heparin with impurities removed can be obtained.

[0150] In addition, the Nucleic acid value, which is one of the indicators of impurities in two samples, was measured, and the Example was measured at 7.30, while Comparative Example 2 was measured at 31.55, confirming that the impurity content of Comparative Example 2 was much higher.

[0151] That is, it can be seen that the embodiment can contribute to process simplification and process cost reduction because no additional process for removing impurities is required.

[0152]

[0153] [Experimental Example 3] Anticoagulant activity analysis

[0154] Experimental Example 3 is an experiment that analyzed the anticoagulant activity of Example and Comparative Example 2.

[0155] Anticoagulant activity assays were performed using the BIOPHEN™ ANTI-IIa Assay Kit from HYPHEN BioMed.

[0156] Crude heparin was diluted in distilled water to a concentration of 1 mg / ml, vortexed until sufficiently dissolved, and filtered using a syringe filter. The heparin standard and the powders of Examples and Comparative Examples 2 were diluted in 0.9% saline and 1% BSA solution to concentrations of 0, 0.5, 1, 2, 4, and 6 IU / ml, respectively, and the prepared samples were analyzed as follows according to the manual provided by the manufacturer.

[0157] 20 μl of the standard and the samples of Example and Comparative Example 2 were dispensed into a 96-well microplate, and 20 μl of R1 (Human Antithrombin), 100 μl of R4 (Assay reaction buffer), and 40 μl of R2 (Chromogenic substrate specific for Thrombin) were sequentially added, and the mixture was incubated at 37°C for 2 minutes. After that, 40 μl of R3 (Human thrombin) was added, and the mixture was incubated at 37°C for 5 minutes to induce a color reaction. 80 μl of citric acid was added to the sample in which the color reaction occurred to terminate the reaction, and the OD value was measured at a wavelength of 405 nm using a microplate reader. The measured wavelength value was used to calculate the concentration of the analyzed sample compared to the standard using the CombiStats program.

[0158] Figure 5 is a diagram showing the results of anticoagulant activity analysis of Experimental Example and Comparative Example 2.

[0159] Referring to Fig. 5, it can be confirmed that the heparin contained in the powder of the example has an anticoagulant activity measured to be 20 IU / mg or higher compared to the comparative example.

[0160] That is, it can be confirmed that the embodiment can obtain heparin of higher quality compared to the conventional technology, Comparative Example 2.

[0161]

[0162] [Experimental Example 4] H-NMR Analysis

[0163] Experimental Example 4 was to confirm the heparin sodium of the experimental example, and a heparin standard product from Sigma was used as a control group.

[0164] Figure 6 is a diagram showing the results of H-NMR analysis of an experimental example.

[0165] Referring to Figure 6, the H-NMR analysis results of the control group show peaks at 2.04 ppm, 3.27 ppm (Doublet), 4.34 ppm, 5.22 ppm, and 5.42 ppm, which are the peak values ​​of heparin sodium in the United States Pharmacopoeia, the Collegiate Pharmacopoeia, and the European Pharmacopoeia. The H-NMR of the example also shows the same peak values, demonstrating ideal results.

[0166] Although some peaks in Fig. 6 are cut off here, it is not related to the peak of heparin sodium, and it is stated that a part of the overall analysis results was edited to clearly show the peak of heparin sodium.

[0167]

[0168] [Experimental Example 5] Measurement of tensile strength and elasticity

[0169] Experimental Example 5 is an experiment to measure the tensile strength and elastic modulus of pig intestines according to the salt concentration using Example and Comparative Example 3. As a control group, pig intestines immersed in water without salting were used.

[0170] Measurements were conducted at the Jeongeup Atomic Energy Research Institute, maximizing the freshness of the pig intestines and increasing reliability through 10 repetitions. Tensile strength and elasticity at each processing stage were measured using a Shimadzu (AGX-100KNV, Japan), at a test speed of 120 mm / min.

[0171] Fig. 7 is a drawing showing the results of tensile strength and elasticity analysis of pig intestines of Experimental Example and Comparative Example 3, and Fig. 8 is a photograph showing sundae manufactured using the casings of Example and Comparative Example 3.

[0172] Referring to Fig. 7(a), the tensile strengths of the control group, example, and comparative example 3 were measured to be 392.1 kPa, 549.2 kPa, and 622.8 kPa, respectively, confirming that the tensile strength increases depending on the concentration of the brine in which the pig intestines were immersed.

[0173] Referring to Fig. 7(b), the elastic moduli of the control group, example, and comparative example 3 were measured to be 1.66 N / mm², 2.41 N / mm², and 2.41 N / mm², confirming that the elasticity does not change significantly when a certain salt concentration is exceeded.

[0174] Accordingly, as the brine concentration increases, the tensile strength increases, and it can be expected that the quality of the sundae will improve. However, referring to the photograph of the sundae immediately after steaming in Fig. 8, unlike the sundae of the normally manufactured example (Fig. 8a), the sundae of the comparative example (Fig. 8b) has a separated casing and filling, and it can be confirmed that the casing is opaque even though it is hot.

[0175] This shows that even if the tensile strength increases, the elastic modulus does not increase, so when heat is applied, the tissue degenerates and becomes damaged, and immersing the casing in excessively high concentration brine does not properly bond the casing and the filling material, making it unsuitable for the food industry.

[0176] That is, in order to easily apply it to the food industry, it is desirable to immerse it in an appropriate concentration of 3 to 8% as in the example.

[0177]

[0178] As described above, by soaking pig intestines in low-salt water and extracting GAGs from the low-salt water, high-quality GAG powder and casings can be simultaneously obtained. This has resolved the conflict between the traditional sundae and GAG extraction industries, establishing a plan for their mutual growth.

[0179]

[0180] Although the embodiments of the present invention have been described above with reference to the attached drawings, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above are illustrative in all respects and are not limiting.

[0181]

[0182] [Explanation of symbols]

[0183] S1: Step of obtaining a brine mixture

[0184] S2: Step of creating an alkaline environment for the brine mixture

[0185] S3: Step of hydrolyzing the brine mixture

[0186] S4: Step to lower the pH of the brine mixture and separate the fat.

[0187] S5: Step of adsorbing GAGs onto ion exchange resin

[0188] S6: Step for preparing the eluent

[0189] S7: Steps to obtain GAGs

Claims

1. (S1) A step of washing pig intestines with water from which the mucosa has not been removed, and then immersing them in saline solution having a certain concentration or lower so as to prevent denaturation of the pig intestine tissue, thereby obtaining a saline mixture; (S2) a step of creating an alkaline environment in the brine mixture; (S3) a step of adding an enzyme to the brine mixture to hydrolyze the brine mixture; (S4) a step of lowering the pH of the hydrolyzed brine mixture and separating fat; (S5) A step of adding an ion exchange resin to the brine mixture from which the fat has been separated to adsorb glycosaminoglycan (GAG) polymers contained in the brine mixture onto the ion exchange resin; (S6) A step of collecting and washing the ion exchange resin to which the glycosaminoglycan polymer is adsorbed and preparing an effluent using saline water; (S7) A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings by utilizing a low-salt immersion method, including a step of mixing an organic solvent into the above-mentioned extract, recovering the resulting precipitate, and drying it to obtain a glycosaminoglycan polymer.

2. In paragraph 1, The above S1 step, A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings by utilizing a low-salt immersion method, characterized in that the washed pig intestines are immersed in brine having a concentration of 1 to 20% to obtain a brine mixture containing glycosaminoglycans.

3. In paragraph 2, The above S1 step, A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings by utilizing a low-salt immersion method characterized in that the washed pig intestines are immersed in brine having a concentration of 3 to 8% to obtain a brine mixture containing glycosaminoglycans.

4. In paragraph 1, The above S2 step is, A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings using a low-salt immersion method, characterized in that the step comprises adding NaOH to the brine mixture to adjust the pH to 8.8 to 9.

3.

5. In paragraph 1, The above S3 step is, A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings using a low-salt immersion method, characterized in that 0.1 to 5 parts by weight of a protein-decomposing enzyme is added to 100 parts by weight of the brine mixture to hydrolyze the brine mixture.

6. In paragraph 1, The above S4 step, A method for extracting glycosaminoglycans from pig intestines and improving the quality of sundae casings by utilizing a low-salt immersion method characterized by adding sodium bisulfite to the hydrolyzed brine mixture to lower the pH to 6.5 to 7.5 and separate fat.

7. In paragraph 1, The above S5 step, A method for extracting glycosaminoglycans from pig intestines and improving the quality of blood sausage casings using a low-salt immersion method, characterized in that a strong anion exchange resin is added to the salt water mixture from which the fat has been separated to adjust the pH to 8.4 to 8.6, and the glycosaminoglycan polymer contained in the salt water mixture is adsorbed to the ion exchange resin.

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