Method for extracting taurine from abalone viscera through magnetic induction electric field assisted enzymolysis

Abstract

The invention discloses a method for extracting taurine from abalone viscera through magnetic induction electric field assisted enzymolysis, which comprises the following steps: adding distilled water into abalone liver freeze-dried powder, and carrying out magnetic induction electric field treatment; adding an enzyme preparation, carrying out water bath treatment in a water bath kettle, carrying out enzyme deactivation, cooling, filtering and centrifuging, and collecting supernate to obtain an abalone viscera enzymolysis stock solution; concentrating and separating the stock solution by using a cation exchange resin column or absolute ethyl alcohol, then adding absolute ethyl alcohol, standing, and collecting a precipitate; adding hot water, dissolving the precipitate, adding activated carbon, and filtering to obtain filtrate; and crystallizing and recrystallizing to obtain high-purity taurine. According to the method, the extraction rate and the purity of the taurine are improved, the problems of waste of abalone viscera by-product resources, environmental pollution and the like are solved, the maximum extraction amount of the taurine extracted from the abalone viscera through the method reaches 12.36 mg / g, a natural taurine product with the purity of 96.78% is obtained, and the method has important significance for improving the yield and the quality of the taurine.

Description

Technical Field

[0001] The present invention relates to the technical field of taurine production, and in particular to a method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis. Background Art

[0002] Taurine (2-aminoethanesulfonic acid) is an amino acid. The pure product is colorless or white crystals, odorless and tasteless, with stable chemical properties, soluble in water and insoluble in organic solvents such as ethanol and ether. Taurine is a sulfur-containing amino acid, which generally exists in various tissues of animals in the form of free amino acids, and has physiological functions such as promoting metabolism, enhancing the body's immunity, participating in the regulation of neuroendocrine function, and protecting the retina. Supplementing taurine in the diet can effectively reduce the risk of diseases such as cardiovascular diseases, fatty liver, diabetes and diabetic complications, so it is widely used in pharmaceuticals and health foods. Since the activity of the rate-limiting enzyme (cysteine decarboxylase) in taurine biosynthesis is relatively low, the amount of taurine required by the human body must be obtained from the outside. There are mainly two production methods of taurine: natural extraction method and chemical synthesis method. Chemical synthesis using highly toxic raw materials such as ethylene oxide is the most commonly used synthesis method at present. However, the chemical synthesis method involves expensive equipment and causes environmental pollution. With the increasingly strict requirements for food and drug safety, it is more advocated to extract taurine from natural foods using green and low-toxic extraction technologies.

[0003] Although there are methods for extracting taurine from meat products and seafood in the prior art, there are still problems such as complex operation, low extraction rate and low purity. Therefore, it is necessary to provide an extraction method that can improve the extraction rate and purity of taurine and has a simple operation. Summary of the Invention

[0004] The present invention provides a method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis to solve the above problems.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] A method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis, comprising the following steps:

[0007] S1: Collect abalone liver, wash, homogenize, freeze-dry, pulverize and sieve it to obtain abalone liver freeze-dried powder;

[0008] S2: Add distilled water to the abalone liver freeze-dried powder and mix evenly, and perform magnetic induction electric field treatment;

[0009] S3: After magnetic induction electric field treatment, add enzyme preparation and perform water bath treatment in a water bath pot. After the water bath enzymatic hydrolysis is completed, inactivate the enzyme with boiling water, cool, filter, centrifuge, collect the supernatant to obtain abalone viscera enzymatic hydrolysis stock solution;

[0010] S4: Concentrate the crude enzyme solution of abalone viscera by a rotary evaporator to obtain a first concentrated solution; adsorb the first concentrated solution with a cation exchange resin column, elute with distilled water, collect the taurine eluate, add absolute ethanol to the taurine eluate, let it stand at room temperature and centrifuge, take the supernatant and concentrate it by a rotary evaporator to obtain a second concentrated solution; or,

[0011] Cool the crude enzyme solution of abalone viscera to room temperature, filter it through a gauze, further centrifuge to collect the first supernatant, add absolute ethanol to the first supernatant, let it stand and centrifuge, then take the second supernatant, and concentrate the second supernatant by a rotary evaporator to obtain a concentrated solution;

[0012] S5: Add absolute ethanol to the second concentrated solution or the concentrated solution and let it stand, collect the precipitate; add hot water to the precipitate, dissolve it and then treat it with activated carbon, filter to remove the activated carbon to obtain a filtrate;

[0013] S6: Immediately filter to remove impurities after adding absolute ethanol to the filtrate, let it stand, and the obtained solid is the crude taurine crystal;

[0014] Dissolve the crude taurine crystal with distilled water and then add absolute ethanol, mix and perform recrystallization to obtain high-purity taurine.

[0015] Further, the conditions for magnetic induction electric field treatment are: excitation voltage 800 - 1400V, electric field frequency 50 - 60kHz, sample injection flow rate 15 - 25L / h, and treatment time 0.5 - 1.5h.

[0016] Further, the enzyme preparation is papain;

[0017] The enzyme digestion conditions are: enzyme preparation addition amount 1000 - 2500U / g, water bath treatment at 40 - 60°C for 2 - 4h;

[0018] The centrifugation conditions are: centrifuge at 10000r / min for 10min.

[0019] Further, in S1, when homogenizing, the volume ratio of distilled water added to the mass of the homogenate is 1:5 (mL:g);

[0020] The crushing method is intermittent treatment with a blender for 3 - 5min;

[0021] The freeze-drying conditions are freeze-drying at -40°C for 48h; the mesh number of the sieve for sieving is not less than 40 meshes.

[0022] Further, in S4, the first stock solution is concentrated to less than 10% of the volume of the crude enzyme solution of abalone viscera, and the concentration conditions are: heating temperature 75 - 85°C, vacuum degree -0.1 to 0.09MPa;

[0023] The amount of the absolute ethanol added to the taurine eluate is 4 - 5 times the volume of the taurine eluate;

[0024] The supernatant is concentrated to a soluble solid content of 50%, and the concentration conditions of the supernatant are a heating temperature of 55 - 65°C and a vacuum degree of -0.1 to 0.09 MPa.

[0025] Further, in S4, 4 times the volume of absolute ethanol is added to the first supernatant, and after standing and centrifuging, the second supernatant is taken. The second supernatant is concentrated to about 50 mL by a rotary evaporator to obtain a concentrated solution with a soluble solid content of 51%.

[0026] Further, in S5, the addition amount of the absolute ethanol is 4 - 5 times the volume of the second concentrated solution or the concentrated solution, and it is left standing at 4°C for more than 12 h;

[0027] The activated carbon treatment step is as follows: after adding 5 - 6 times the volume of hot water to the precipitate, it is treated in a water bath at 70 - 90°C for 0.5 - 1 h. After dissolution, activated carbon is added, and the addition amount of the activated carbon is 3% - 5% based on the w / v of the solution formed by the activated carbon and the hot water added to the precipitate.

[0028] Further, in S6, during the crystallization process, the addition amount of the absolute ethanol is 3 - 5 times the volume of the filtrate. After suction filtration to remove impurities, it is left standing at 4°C for more than 24 h to obtain crude taurine crystals.

[0029] Further, in S6, the specific steps of recrystallization are as follows: the crude taurine crystals are dissolved in distilled water at 70 - 90°C for crystallization, and then absolute ethanol is added. After mixing, it is placed in a 4°C refrigerator for recrystallization. The number of recrystallization extraction times is 3 - 5 times. During each crystallization extraction process, the addition amount of the absolute ethanol is 4 - 6 times the volume of the solution formed by the crude taurine crystals, distilled water, and absolute ethanol.

[0030] The beneficial effects of the present invention are:

[0031] A method for extracting taurine from abalone viscera assisted by a magnetic induction electric field disclosed in the present invention. Through steps such as raw material pretreatment, magnetic induction electric field treatment, enzymatic hydrolysis, concentration and separation, filtration, purification and crystallization, a natural taurine product is obtained. Taurine is effectively extracted from abalone viscera, and the production process is green and efficient, and the experimental operation is simple. It not only improves the extraction rate and purity of taurine, but also solves problems such as waste of abalone viscera by-products and environmental pollution. The extraction amount of taurine from abalone viscera by the method of the present invention is up to 12.36 mg / g at most, and a natural taurine product with a purity of 96.78% is obtained, which is of great significance for improving the yield and quality of taurine. Description of the Drawings

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 It is a graph showing the influence of the excitation voltage of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0034] Figure 2 It is a graph showing the influence of the electric field frequency of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0035] Figure 3 It is a graph showing the influence of the sample injection flow rate of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0036] Figure 4 It is a graph showing the influence of the treatment time of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0037] Figure 5 It is a graph showing the influence of the enzyme addition amount of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0038] Figure 6 It is a graph showing the influence of the enzymatic hydrolysis temperature of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0039] Figure 7 It is a graph showing the influence of the enzymatic hydrolysis time of the present invention on the extraction amount of taurine in abalone viscera stock solution;

[0040] Figure 8 It is an HPLC graph of abalone viscera taurine samples extracted by two concentration and separation methods in Example 1 and Example 11 of the present invention;

[0041] Figure 9 It is an infrared spectrum graph of abalone viscera taurine samples extracted by two concentration and separation methods in Example 1 and Example 11 of the present invention, where A is the taurine standard, B is the taurine sample extracted by the ethanol extraction method used in Example 11, and C is the taurine sample extracted by the ion exchange resin method used in Example 1;

[0042] Figure 10 It is a positive and negative ion mass spectrum graph of abalone viscera taurine samples extracted by two concentration and separation methods in Example 1 and Example 11 of the present invention, where Figure 10 A is the taurine sample extracted by the ethanol extraction method used in Example 11, Figure 10 B is the taurine sample extracted by the ion exchange resin method used in Example 1;

[0043] Figure 11 This is the nuclear magnetic resonance spectrum of taurine samples extracted from abalone viscera by the two concentration and separation methods of Example 1 and Example 11 of the present invention. Among them Figure 11 A is the taurine standard Figure 11 B is the taurine sample extracted by the ethanol extraction method used in Example 11 Figure 11 C is the taurine sample extracted by the ion exchange resin method used in Example 1;

[0044] Figure 12 This is the result graph of the taurine extraction amounts by different methods in Example 1 of the present invention and Comparative Examples 1-6. Detailed implementation manners

[0045] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] The abalone viscera used in the present invention was purchased from Fuzhou Deep Sea Food Co., Ltd.;

[0047] Taurine standard: CAS: 107-35-7, purity ≥ 98%; purchased from Sinopharm Chemical Reagent Co., Ltd.

[0048] Other reagents are common reagents in the laboratory.

[0049] Examples:

[0050] Example 1:

[0051] A method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis (MIEF-EH) includes the following steps:

[0052] (1) Raw material pretreatment:

[0053] Collect the liver part of abalone viscera, wash the liver, add distilled water at a ratio of 1:5 (mL / g) to the mass of the homogenate, intermittently process with a blender for 3-5 min, freeze-dry in a freeze dryer at -40°C for 48 h after homogenization, and pulverize and sieve for later use. The mesh number of the sieve is not less than 40 meshes;

[0054] (2) Magnetic induction electric field treatment:

[0055] The abalone viscera freeze-dried powder and distilled water were added in a ratio of 1:50 (g / mL), and mixed evenly. Magnetic induction electric field treatment was carried out with an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, and a treatment time of 1 h;

[0056] (3) Enzymatic hydrolysis:

[0057] After magnetic induction electric field treatment, 2000 U / g of papain was added, and water bath treatment was carried out in a water bath at 60 °C for 4 h. After the enzymatic hydrolysis in the water bath was completed, the enzyme was inactivated by boiling water. After inactivation, it was cooled to room temperature, filtered through gauze, and centrifuged at 10000 r / min for 10 min to collect the supernatant, obtaining the abalone viscera enzymatic hydrolysis stock solution (AVE-MIEF-EH);

[0058] (4) Concentration and separation:

[0059] The abalone viscera enzymatic hydrolysis stock solution was concentrated to 10% using a rotary evaporator. The concentration conditions were a heating temperature of 80 °C and a vacuum degree of 0.0013 MPa to obtain the first concentrated solution. A 732-type Na + cation exchange resin column was used. The first concentrated solution was passed through the column and eluted with distilled water to collect the taurine eluate; 4 - 5 times the volume of absolute ethanol was added to the first eluate, allowed to stand at room temperature, centrifuged, and the supernatant was concentrated by a rotary evaporator to obtain a second concentrated solution with a soluble solid content of 50%. The concentration conditions were a heating temperature of 65 °C and a vacuum degree of 0.0022 MPa;

[0060] (5) Filtration:

[0061] 4 - 5 times the volume of absolute ethanol was added to the second concentrated solution and allowed to stand at 4 °C for more than 12 h. The precipitate was collected, 5 - 6 times the volume of hot water was added to the precipitate, dissolved, and treated with activated carbon. The addition amount of activated carbon was 5% based on the w / v of activated carbon and supernatant. The activated carbon was removed by suction filtration to obtain the filtrate;

[0062] (6) Purification and crystallization:

[0063] 3 times the volume of absolute ethanol was added to the above filtrate, and impurities were immediately removed by suction filtration. The filtrate was allowed to stand at 4 °C for more than 24 h for crystallization, and the obtained solid was the crude taurine crystal; the crude taurine crystal was dissolved in hot water and then 4 times the volume of absolute ethanol was added. After mixing, it was recrystallized in a 4 °C refrigerator for more than three times to obtain high-purity taurine.

[0064] It was determined that the content of taurine in the abalone viscera enzymatic hydrolysis stock solution was 12.36 mg / g, and the purity of taurine was 96.78% detected by infrared spectroscopy and high performance liquid chromatography.

[0065] Example 2:

[0066] A method for extracting taurine from abalone viscera assisted by magnetic induction electric field, comprising the following steps:

[0067] (1) Raw material pretreatment:

[0068] Collect the liver part of abalone viscera, wash the liver, add distilled water in a ratio of 1:5 (mL / g) to the mass of the homogenate, treat it intermittently with a blender for 3 - 5 min, freeze-dry it in a freeze dryer at -40 °C for 48 h after homogenization, pulverize and sieve it for later use, and the mesh number of the sieve is not less than 40 meshes;

[0069] (2) Magnetic induction electric field treatment:

[0070] Add abalone viscera freeze-dried powder and distilled water in a ratio of 1:50 (g / mL), and mix evenly. Conduct magnetic induction electric field treatment, with an excitation voltage of 1200 V, an electric field frequency of 60 kHz, a sample injection flow rate of 20 L / h, and a treatment time of 1.5 h;

[0071] (3) Enzymatic hydrolysis:

[0072] After magnetic induction electric field treatment, add 2000 U / g of papain, conduct water bath treatment in a water bath at 60 °C for 4 h, inactivate the enzyme with boiling water after water bath enzymatic hydrolysis is completed, cool to room temperature after enzyme inactivation, filter through gauze, centrifuge at 10000 r / min for 10 min to collect the supernatant, and obtain the abalone viscera enzymatic hydrolysis stock solution;

[0073] (4) Concentration and separation:

[0074] Use a rotary evaporator to concentrate the abalone viscera enzymatic hydrolysis stock solution to 10%, with the concentration conditions being a heating temperature of 80 °C and a vacuum degree of 0.0013 MPa to obtain the first concentrated solution. Use a 732-type Na + cation exchange resin column, pass the first concentrated solution through the column and elute it with distilled water to collect the taurine eluate; add 4 - 5 times the volume of absolute ethanol to the first eluate, let it stand at room temperature, centrifuge and take the supernatant, and concentrate it with a rotary evaporator to obtain a second concentrated solution with a soluble solid content of 50%, with the concentration conditions being a heating temperature of 65 °C and a vacuum degree of 0.0022 MPa;

[0075] (5) Filtration:

[0076] Add 4 - 5 times the volume of absolute ethanol to the second concentrated solution, place it at 4 °C and let it stand for more than 12 h, and collect the precipitate. Add 5 - 6 times the volume of hot water to the precipitate, dissolve it and then treat it with activated carbon. The addition amount of activated carbon is 5% based on the w / v of activated carbon and the supernatant. Filter to remove the activated carbon to obtain the filtrate;

[0077] (6) Purification and crystallization:

[0078] Add three volumes of absolute ethanol to the above filtrate and immediately filter to remove impurities. Place the filtrate at 4°C and let it stand for more than 24 hours to crystallize. The resulting solid is the crude taurine crystal. Dissolve the crude taurine crystal in hot water and then add four volumes of absolute ethanol. After mixing, place it in a refrigerator at 4°C for recrystallization three times or more to obtain high-purity taurine.

[0079] Under these conditions, the content of taurine in the enzymatically hydrolyzed stock solution of abalone viscera is 12.08 mg / g, and the purity of taurine detected by infrared spectroscopy and high-performance liquid chromatography is 95.48%.

[0080] Example 3:

[0081] A method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis, comprising the following steps:

[0082] (1) Raw material pretreatment:

[0083] Collect the liver part of abalone viscera, wash the liver, add distilled water at a ratio of 1:5 (mL / g) to the mass of the homogenate, and process it intermittently with a blender for 3 - 5 minutes. After homogenization, freeze-dry it in a freeze dryer at -40°C for 48 hours, pulverize it and pass it through a sieve for later use, and the mesh number of the sieve is not less than 40 meshes;

[0084] (2) Magnetic induction electric field treatment:

[0085] Add abalone viscera freeze-dried powder and distilled water at a ratio of 1:50 (g / mL), and mix evenly. Conduct magnetic induction electric field treatment, with an excitation voltage of 1400 V, an electric field frequency of 50 kHz, an injection flow rate of 25 L / h, and a treatment time of 1 hour;

[0086] (3) Enzymatic hydrolysis:

[0087] After magnetic induction electric field treatment, add 1500 U / g of papain and conduct water bath treatment in a water bath at 50°C for 3 hours. After the water bath enzymatic hydrolysis is completed, inactivate the enzyme with boiling water. After inactivating the enzyme, cool it to room temperature, filter it through a gauze, and centrifuge it at 10000 r / min for 10 minutes to collect the supernatant to obtain the enzymatically hydrolyzed stock solution of abalone viscera;

[0088] (4) Concentration and separation:

[0089] Use a rotary evaporator to concentrate the enzymatically hydrolyzed stock solution of abalone viscera to 10%, and the concentration conditions are a heating temperature of 80°C and a vacuum degree of 0.0013 MPa to obtain the first concentrated solution. Use a 732-type Na +The cation exchange resin column is used. The first concentrated solution is passed through the column and eluted with distilled water, and the taurine eluate is collected. Add 4 - 5 times the volume of absolute ethanol to the first eluate, let it stand at room temperature, centrifuge, and take the supernatant. Concentrate it with a rotary evaporator to obtain a second concentrated solution with a soluble solid content of 50%. The concentration conditions are a heating temperature of 65°C and a vacuum degree of 0.0022 MPa.

[0090] (5) Filtration:

[0091] Add 4 - 5 times the volume of absolute ethanol to the second concentrated solution, place it at 4°C and let it stand for more than 12 h, and collect the precipitate. Add 5 - 6 times the volume of hot water to the precipitate, dissolve it, add activated carbon for treatment. The addition amount of activated carbon is 5% based on the w / v of activated carbon and supernatant. Filter to remove the activated carbon to obtain the filtrate.

[0092] (6) Purification and crystallization:

[0093] Add 3 times the volume of absolute ethanol to the above - mentioned filtrate and immediately filter to remove impurities. Let the filtrate stand at 4°C for more than 24 h to crystallize. The obtained solid is the crude taurine crystal. Dissolve the crude taurine crystal in hot water and then add 4 times the volume of absolute ethanol. Mix and put it in a 4°C refrigerator for recrystallization three times or more to obtain high - purity taurine.

[0094] Under these conditions, the content of taurine in the abalone viscera enzymatic hydrolysis stock solution is 11.88 mg / g. The purity of taurine detected by infrared spectroscopy and high - performance liquid chromatography is 93.28%.

[0095] Example 4:

[0096] Experiment on the effect of different excitation voltages on the extraction amount of taurine from abalone viscera

[0097] According to the extraction methods of Examples 1 - 3, for 5 groups of 10 g abalone viscera sample solutions, under the conditions of an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, a treatment time of 1 h, an enzyme addition amount of 2000 U / g, an enzymatic hydrolysis temperature of 60°C, and an enzymatic hydrolysis time of 4 h, extract taurine from abalone viscera under the electric field conditions of excitation voltages of 600, 800, 1000, 1200 respectively, and compare with Example 1.

[0098] The effect of the excitation voltage on the extraction amount of taurine from abalone viscera is as Figure 1 shown. It can be seen from the figure that the extraction amount of taurine is relatively high in the range of 800 - 1400 V of the excitation voltage. When the excitation voltage is 600 V, the extraction amount of taurine is relatively low. Moreover, when the excitation voltage is 1400 V, the extraction amount of taurine from abalone viscera in the stock solution is the highest. Therefore, the optimal excitation voltage is 1400 V.

[0099] Example 5:

[0100] Experiment on the Effect of Different Electric Field Frequencies on the Extraction Yield of Taurine from Abalone Viscera

[0101] According to the extraction methods of Examples 1 - 3, for 5 groups of 10 g abalone viscera sample solutions, under the conditions of excitation voltage of 1400 V, injection flow rate of 25 L / h, treatment time of 1 h, enzyme addition amount of 2000 U / g, enzymatic hydrolysis temperature of 60 °C, and enzymatic hydrolysis time of 4 h, the electric field frequencies of 40, 60, 70, and 80 kHz were respectively used to extract taurine from abalone viscera, and a comparison was made with Example 1;

[0102] The effect of electric field frequency on the extraction yield of taurine from abalone viscera is as Figure 2 shown. It can be seen from the figure that the extraction yield of taurine is relatively high in the range of electric field frequencies from 50 to 70 kHz. When the electric field frequency is 40 kHz or 80 kHz, the extraction yield of taurine decreases significantly. Moreover, when the electric field frequency is 50 kHz, the extraction yield of taurine from the original abalone viscera solution is the highest. Therefore, the optimal electric field frequency is 50 kHz.

[0103] Example 6:

[0104] Experiment on the Effect of Different Injection Flow Rates on the Extraction Yield of Taurine from Abalone Viscera

[0105] According to the extraction methods of Examples 1 - 3, for 5 groups of 10 g abalone viscera sample solutions, under the conditions of excitation voltage of 1400 V, electric field frequency of 50 kHz, treatment time of 1 h, enzyme addition amount of 2000 U / g, enzymatic hydrolysis temperature of 60 °C, and enzymatic hydrolysis time of 4 h, the injection flow rates of 10, 15, 20, and 30 L / h were respectively used to extract taurine from abalone viscera, and a comparison was made with Example 1;

[0106] The effect of injection flow rate on the extraction yield of taurine from abalone viscera is as Figure 3 shown. It can be seen from the figure that the extraction yield of taurine is relatively high in the range of injection amounts from 15 to 25 L / h. When the injection amount is 10 L / h and 30 L / h, the extraction yield of taurine decreases significantly. Moreover, when the injection amount is 25 L / h, the extraction yield of taurine from the original abalone viscera solution is the highest. Therefore, the optimal injection amount is 25 L / h.

[0107] Example 7:

[0108] Experiment on the Effect of Different Treatment Times (Enzymatic Hydrolysis Times) on the Extraction Yield of Taurine from Abalone Viscera

[0109] According to the extraction methods of Examples 1-3, five groups of 10 g abalone viscera sample solutions were respectively used to extract taurine from abalone viscera under the conditions of an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, an enzyme addition amount of 2000 U / g, an enzymatic hydrolysis temperature of 60 °C, and an enzymatic hydrolysis time of 4 h, with the magnetic induction electric field treatment times being 0.5 h, 1.5 h, 2 h, and 2.5 h respectively, and a comparison was made with Example 1;

[0110] The results of the influence of the magnetic induction electric field treatment time on the extraction amount of taurine from abalone viscera are as Figure 4 shown. It can be seen from the figure that the extraction amount of taurine is relatively high within the range of 0.5 - 1.5 h of the magnetic induction electric field treatment time. However, when the treatment time is 2 h and 2.5 h, the extraction amount of taurine decreases. Moreover, when the treatment time is 1 h, the extraction amount of taurine from the original abalone viscera solution is the highest. Therefore, the optimal magnetic induction electric field treatment time is 1 h.

[0111] Example 8:

[0112] Experiment on the influence of different enzyme addition amounts on the extraction amount of taurine from abalone viscera

[0113] According to the extraction methods of Examples 1-3, five groups of 10 g abalone viscera sample solutions were respectively used to extract taurine from abalone viscera under the conditions of an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, a treatment time of 1 h, an enzymatic hydrolysis temperature of 60 °C, and an enzymatic hydrolysis time of 4 h, with the enzyme addition amounts being 1000 U / g, 1500 U / g, 2500 U / g, and 3000 U / g respectively, and a comparison was made with Example 1;

[0114] The results of the influence of the enzyme addition amount on the extraction amount of taurine from abalone viscera are as Figure 5 shown. It can be seen from the figure that the extraction amount of taurine is relatively high within the range of 1000 - 2500 U / g of the enzyme addition amount. However, when the treatment amount is 2500 U / g and 3000 U / g, the extraction amount of taurine decreases. Moreover, when the enzyme addition amount is 2000 U / g, the extraction amount of taurine from the original abalone viscera solution is the highest. Therefore, the optimal enzyme addition amount is 2000 U / g.

[0115] Example 9:

[0116] Experiment on the influence of different enzymatic hydrolysis temperatures on the extraction amount of taurine from abalone viscera

[0117] According to the methods of Examples 1 - 3, five groups of 10 g abalone viscera sample solutions were used to extract taurine from abalone viscera at enzyme hydrolysis temperatures of 30, 40, 50, and 70 °C respectively under the conditions of an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, a treatment time of 1 h, an enzyme addition amount of 2000 U / g, and an enzyme hydrolysis time of 4 h, and were compared with Example 1;

[0118] The results of the influence of enzyme hydrolysis temperature on the extraction amount of taurine from abalone viscera are as Figure 6 shown. It can be seen from the figure that the extraction amount of taurine is relatively high in the range of 40 - 60 °C for enzyme hydrolysis temperature. When the treatment temperature is 30 °C and 70 °C, the extraction amount of taurine decreases. Moreover, when the enzyme temperature is 60 °C, the extraction amount of taurine from the original abalone viscera solution is the highest. Therefore, the optimal enzyme hydrolysis temperature is 60 °C.

[0119] Example 10:

[0120] Experiment on the influence of different enzyme hydrolysis times on the extraction amount of taurine from abalone viscera

[0121] According to the extraction methods of Examples 1 - 3, five groups of 10 g abalone viscera sample solutions were used to extract taurine from abalone viscera at enzyme hydrolysis times of 1 h, 2 h, 3 h, and 5 h respectively under the conditions of an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, a treatment time of 1 h, an enzyme addition amount of 2000 U / g, and an enzyme hydrolysis temperature of 60 °C, and were compared with Example 1;

[0122] The results of the influence of enzyme hydrolysis time on the extraction amount of taurine from abalone viscera are as Figure 7 shown. It can be seen from the figure that the extraction amount of taurine is relatively high in the range of 2 - 4 h for enzyme hydrolysis time. When the enzyme hydrolysis time is 1 h and 5 h, the extraction amount of taurine decreases significantly. Moreover, when the enzyme time is 4 h, the extraction amount of taurine from the original abalone viscera solution is the highest. Therefore, the optimal enzyme hydrolysis time is 4 h.

[0123] Verification test on the extraction of taurine from abalone viscera by magnetic induction electric field assisted enzymatic hydrolysis: Based on the results of the single factor experiment, 30 verification experiments were carried out under the optimal extraction conditions in Example 1, and the results are shown in Table 1;

[0124] Table 1 Comparison of predicted values and experimental values under the optimal extraction conditions of magnetic induction electric field assisted enzymatic hydrolysis

[0125]

[0126]

[0127] As can be seen from Table 1, under the conditions of an excitation voltage of 1400 V, an electric field frequency of 50 kHz, a sample injection flow rate of 25 L / h, a treatment time of 1 h, an enzyme addition amount of 2000 U / g, an enzymatic hydrolysis temperature of 60 °C, and an enzymatic hydrolysis time of 4 h, the taurine content in the enzymatic hydrolysis stock solution of abalone viscera was 12.36 mg / g, which was basically consistent with the predicted value, and the maximum error between the predicted value and the experimental value was less than 2%, indicating that this method has good reproducibility, feasibility, and credibility.

[0128] Example 11:

[0129] S1: The same as Example 1;

[0130] S2: The same as Example 1;

[0131] S3: The same as Example 1;

[0132] S4: Ethanol extraction method was used for concentration and separation. The collected enzymatic hydrolysis stock solution of abalone viscera was cooled to room temperature, filtered through gauze, and the first supernatant was further collected by centrifugation; 4 times the volume of absolute ethanol was added to the first supernatant, and after standing and centrifugation, the upper second supernatant was taken; the second supernatant was concentrated to about 50 mL by a rotary evaporator to obtain a concentrated solution with a soluble solid content of 51%;

[0133] S5: The same as Example 1;

[0134] S6: The same as Example 1.

[0135] The purity of taurine detected by high performance liquid chromatography was 94.43%, and the concentration of the taurine standard was 98%. The results of high performance liquid chromatography are as Figure 8 shown (the figure shows the product purity results of the ion exchange resin used in Example 1 for concentration and separation).

[0136] The taurine extracted in Example 1 and Example 11 was identified and its physical and chemical indexes were determined:

[0137] (1) Identification of taurine extracted by two different concentration and separation methods in Example 1 and Example 11

[0138] Fourier Transform Infrared Spectrometry (FT-IR), Mass Spectroscopy (MS), and Nuclear Magnetic Resonance Hydrogen Spectrum ( 1 H NMR) were used to characterize the purified taurine in Example 1 and Example 11. The specific operations are as follows:

[0139] The purified taurine sample was mixed with potassium bromide powder, ground, and then pressed into a thin slice. FT-IR measurement was carried out using an FT-IR spectrometer in the wavenumber range of 4000–400 cm -1 . For the mass spectrometry analysis of taurine, a mass spectrometry system equipped with an electrospray ionization source (ESI) was used. The ion mass spectra in the positive and negative modes of the mass spectrometry were obtained under the following mass spectrometry conditions: positive ion mode (ESI+) voltage 5.5 kV; negative ion mode (ESI-) voltage 4.5 kV; nebulizer gas temperature 450 °C; curtain gas 40 psi; gas ion source 1 130 psi; gas ion source 2 30 psi; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile solution; flow rate: 0.2 mL / min, injection volume: 5 μL, mass spectrometry scanning range: 100 - 1500 m / z. The 1H NMR spectrum of taurine was recorded on a 400 MHz nuclear magnetic resonance spectrometer using D2O as the solvent.

[0140] As Figure 9 shown, Figure 9 in which A is the taurine standard, B is the taurine sample extracted by the ethanol extraction method used in Example 11, and C is the taurine sample extracted by the ion exchange resin method used in Example 1. According to the infrared spectrum of the taurine standard (black spectrum), structural groups such as NH2, CH2, C-S-O, and S=O exist in this substance. This result is consistent with that of pure taurine, indicating that the obtained product is preliminarily identified as taurine (NH2CH2CH2SO3H);

[0141] Furthermore, a mass spectrometer was used to identify the structure of the taurine sample. The ion mass spectra of the sample in the positive and negative modes are as Figure 10 shown. The mass spectrum of the taurine sample extracted by the ethanol extraction method used in Example 11 is as Figure 10 shown in A, and the mass spectrum of the taurine sample extracted by the ion exchange resin method used in Example 1 is as Figure 10 shown in B. The taurine obtained by the two concentration separation (purification) methods in Example 1 and Example 11 shows consistent characteristic peaks in the mass spectrum; for the taurine product purified by the ethanol extraction method in Example 11, the mass-to-charge ratios (m / z) of 125.81 ([M+H] + ) and 124.13 ([M-H] - ) were detected, and the calculated molecular weight was 124.97; for the taurine product purified by the ion exchange resin method in Example 1, the mass-to-charge ratios (m / z) of 125.93 ([M+H] + ) and 124.18 ([M-H] -), the calculated molecular weight is 125.06. The molecular weights of taurine purified by the two methods used in Example 1 and Example 11 are highly consistent with the theoretical molecular weight of taurine (125.15). The above results further indicate that the products purified by the two methods are both taurine;

[0142] The nuclear magnetic resonance results of taurine extracted by the two purification methods are as Figure 11 shown, Figure 11 A is the taurine standard, Figure 11 B is the taurine sample extracted by the ethanol extraction method used in Example 11, Figure 11 C is the taurine sample extracted by the ion exchange resin method used in Example 1. The chemical formula of taurine is NH2CH2CH2SO3H, and protonation peaks of SCH2 and NCH2 appear at 3.34 ppm and 3.16 ppm respectively. This result is consistent with the taurine standard.

[0143] (2) Determine the physical and chemical indexes of taurine extracted in Example 1 and Example 11

[0144] Determine the physical and chemical indexes of the high-purity taurine extracted in Example 1 and Example 11. The detection of conductivity, pH, sulfate (calculated as SO4 2- ), ignition residue (w / %), drying loss (w / %), and clarity refers to GB 14759—2010 "National Food Safety Standard Food Additive Taurine". The contents of heavy metals (As, Hg, Pb, Sn, Cd) are detected by inductively coupled plasma mass spectrometry (ICP-MS), and chlorides (calculated as Cl - ), ammonium salts (calculated as NH4 + ) are detected by ion chromatography (IC).

[0145] The results are shown in Table 2. According to the technical index requirements of "National Food Safety Standard Food Additive Taurine" (GB 14759—2010), both taurine products meet the national standards in key indexes such as heavy metal residues and can be safely used as food additives in the food industry.

[0146] Table 2 Physical and Chemical Indexes of Taurine Samples from Abalone Viscera

[0147]

[0148]

[0149] Comparative Example

[0150] Comparative Example 1:

[0151] A method for extracting taurine from abalone viscera by hydrothermal extraction (HE), comprising the steps:

[0152] S1: Collect the liver part of abalone viscera, wash the liver, add distilled water in a volume-to-homogenate mass ratio of 1:5 (mL / g), place it in a constant temperature water bath at 100 °C for hydrothermal treatment for 0.5 h to obtain the abalone viscera enzymatic hydrolysis stock solution (AVE-HE);

[0153] There is no S2;

[0154] There is no S3;

[0155] S4: The same as in Example 1;

[0156] S5: The same as in Example 1;

[0157] S6: The same as in Example 1.

[0158] Comparative Example 2:

[0159] Extracting taurine from abalone viscera by enzymatic hydrolysis (EH), comprising the steps:

[0160] S1: The same as in Example 1;

[0161] There is no S2;

[0162] S3: Add 2000 U / g of papain, place it in a constant temperature water bath at 60 °C for enzymatic hydrolysis for 4 h, inactivate the enzyme, cool to room temperature after inactivating the enzyme, filter through gauze, centrifuge at 10000 r / min for 10 min to collect the supernatant to obtain the abalone viscera enzymatic hydrolysis stock solution (AVE-EH);

[0163] S4: The same as in Example 1;

[0164] S5: The same as in Example 1;

[0165] S6: The same as in Example 1.

[0166] Comparative Example 3:

[0167] Extracting taurine from abalone viscera by magnetic induction electric field extraction (MIEFE), comprising the steps:

[0168] S1: The same as in Example 1;

[0169] S2: The same as in Example 1, and the obtained abalone viscera stock solution is denoted as AVE-MIEFE;

[0170] There is no S3;

[0171] S4: Same as Example 1;

[0172] S5: Same as Example 1;

[0173] S6: Same as Example 1.

[0174] Comparative Example 4:

[0175] Extracting taurine from abalone viscera by microwave extraction method (Microwave extraction, ME), including the steps:

[0176] S1: Same as Example 1;

[0177] S2: It is microwave treatment, and the equipment parameters are microwave power 60W, microwave time 25 min), and microwave jacket temperature 70 °C. The obtained abalone viscera stock solution is denoted as AVE-ME;

[0178] There is no S3;

[0179] S4: Same as Example 1;

[0180] S5: Same as Example 1;

[0181] S6: Same as Example 1.

[0182] Comparative Example 5:

[0183] Extracting taurine from abalone viscera by hydrothermal-enzyme hydrolysis coupling extraction method (Hydrothermal-Enzyme hydrolysis extraction, H-EH), including the steps:

[0184] S1: Collect the liver part of abalone viscera, wash the liver, add distilled water at a ratio of 1:5 (mL / g) to the mass of the homogenate, and place it in a constant temperature water bath at 100 °C for 0.5 h;

[0185] There is no S2;

[0186] S3: Add 2000 U / g papain, place it in a constant temperature water bath at 60 °C for 4 h, inactivate the enzyme, cool it to room temperature after inactivating the enzyme, filter it through gauze, and centrifuge it at 10000 r / min for 10 min to collect the supernatant to obtain the abalone viscera enzyme hydrolysis stock solution (AVE-H-EH);

[0187] S4: Same as Example 1;

[0188] S5: Same as Example 1;

[0189] S6: Same as Example 1.

[0190] Comparative Example 6:

[0191] Microwave-assisted enzymatic extraction of taurine from abalone viscera, including the steps:

[0192] S1: The same as in Example 1;

[0193] S2: For microwave treatment, equipment parameters: microwave power 60W, microwave time 25 min, microwave jacket temperature 70 °C;

[0194] S3: The same as in Example 1, and the obtained abalone viscera stock solution is denoted as AVE-M-EH;

[0195] S4: The same as in Example 1;

[0196] S5: The same as in Example 1;

[0197] S6: The same as in Example 1.

[0198] (1) Measure the taurine content in the enzymatic hydrolysis stock solution of abalone viscera in Comparative Examples 1-6 respectively, and compare it with the taurine content in Example 1. The results of three parallel experiments are as Figure 12 shown, Figure 12 where different lowercase letters a-f indicate significant differences (p < 0.05);

[0199] (2) LSD multiple comparison analysis of the effects of different extraction methods in Example 1 and Comparative Examples 1-6 on the extraction amount of taurine from abalone viscera, and the results are shown in Table 3 below:

[0200] Table 3 Results of LSD multiple comparison analysis of the effects of different extraction methods in Example 1 and Comparative Examples 1-6 on the extraction amount of taurine from abalone viscera

[0201]

[0202]

[0203] From the data in the above table and Figure 12 the results, it can be seen that compared with the extraction methods used in Comparative Examples 1-6, the extraction amount of taurine extracted by the magnetic induction electric field-enzymatic hydrolysis coupling extraction method adopted in the present invention is the highest. Compared with microwave-assisted enzymatic extraction, the taurine content is increased by about 12.7%. Compared with magnetic field extraction, the taurine content is increased by about 52.2%. Compared with microwave extraction, the taurine content is increased by about 74.6%. Compared with biological enzyme method, the taurine content is increased by about 91.0%. Compared with hydrothermal method, the taurine content is increased by about 119%. Compared with hydrothermal-enzymatic hydrolysis coupling extraction method, the taurine content is increased by about 49.1%.

[0204] In summary, taking the taurine extraction amount as an index, by optimizing seven factors including electric field voltage, electric field frequency, sample injection flow rate, magnetic induction electric field treatment time, enzyme addition amount, enzymatic hydrolysis temperature, and enzymatic hydrolysis time, the optimal extraction parameter range was determined, significantly increasing the taurine extraction amount from abalone viscera. The highest taurine extraction amount from abalone viscera by the method of the present invention is 12.36 mg / g, and a natural taurine product with a purity of 96.8% is obtained.

[0205] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis, characterized in that, It includes the following steps: S1: Collect abalone livers, wash, homogenize, freeze-dry, and pulverize and sieve them to obtain abalone liver freeze-dried powder; S2: Add distilled water to the abalone liver freeze-dried powder and mix evenly, then perform magnetic induction electric field treatment; S3: After magnetic induction electric field treatment, add enzyme preparation and perform water bath treatment in a water bath pot. After the water bath enzymatic hydrolysis is completed, inactivate the enzyme with boiling water, cool, filter, and centrifuge, and collect the supernatant to obtain abalone viscera enzymatic hydrolysis stock solution; S4: Concentrate the abalone viscera enzymatic hydrolysis stock solution with a rotary evaporator to obtain a first concentrated solution; adsorb the first concentrated solution with a cation exchange resin column, elute with distilled water, collect the taurine eluate, add absolute ethanol to the taurine eluate, stand and centrifuge at room temperature, and take the supernatant and concentrate it with a rotary evaporator to obtain a second concentrated solution; or, Cool the abalone viscera enzymatic hydrolysis stock solution to room temperature, filter it through a gauze, further centrifuge to collect the first supernatant, add absolute ethanol to the first supernatant, stand and centrifuge, take the second supernatant, and concentrate the second supernatant with a rotary evaporator to obtain a concentrated solution; S5: Add absolute ethanol to the second concentrated solution or the concentrated solution and stand, collect the precipitate; add hot water to the precipitate, dissolve it and then treat it with activated carbon, filter and remove the activated carbon by suction filtration to obtain a filtrate; S6: Immediately filter and remove impurities by suction filtration after adding absolute ethanol to the filtrate, stand, and the obtained solid is crude taurine crystal; Dissolve the crude taurine crystal with distilled water and then add absolute ethanol, mix and perform recrystallization to obtain high-purity taurine.

2. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymolysis according to claim 1, characterized in that: The conditions for magnetic induction electric field treatment are: excitation voltage 800 - 1400V, electric field frequency 50 - 60kHz, sample injection flow rate 15 - 25L / h, and treatment time 0.5 - 1.5h.

3. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, characterized in that The enzyme preparation is papain; The enzymatic hydrolysis conditions: the addition amount of the enzyme preparation is 1000 - 2500U / g, and perform water bath treatment at 40 - 60°C for 2 - 4h; The centrifugation conditions are: centrifuge at 10000r / min for 10min.

4. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymolysis according to claim 1, characterized in that In S1, when homogenizing, the volume ratio of the added distilled water to the mass of the homogenate is 1:5 (mL:g); The pulverization method is intermittent treatment with a blender for 3 - 5min; The freeze-drying conditions are freeze-drying at -40°C for 48h; the mesh number of the sieve for sieving is not less than 40 meshes.

5. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, wherein In S4, the first stock solution is concentrated to less than 10% of the volume of the abalone viscera enzymatic hydrolysis stock solution, and the concentration conditions are: heating temperature 75 - 85°C, vacuum degree -0.1 to 0.09MPa; The amount of absolute ethanol added to the taurine eluate is 4 - 5 times the volume of the taurine eluate; The supernatant is concentrated to a soluble solid content of 50%, and the concentration conditions of the supernatant are: heating temperature 55 - 65°C, vacuum degree -0.1 to 0.09MPa.

6. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, wherein In S4, add 4 times the volume of absolute ethanol to the first supernatant, stand and centrifuge, take the second supernatant, and concentrate the second supernatant with a rotary evaporator to about 50mL to obtain a concentrated solution with a soluble solid content of 51%.

7. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, wherein In S5, the addition amount of absolute ethanol is 4 - 5 times the volume of the second concentrated solution or the concentrated solution, and stand at 4°C for more than 12h; The activated carbon treatment steps are as follows: After adding hot water with a volume 5-6 times that of the precipitate, treat it in a water bath at 70-90 °C for 0.5-1 h. After dissolution, add activated carbon, and the addition amount of activated carbon is 3%-5% based on the w / v of the solution formed by the activated carbon and the hot water added to the precipitate.

8. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, wherein In S6, during the crystallization process, the addition amount of the absolute ethanol is 3-5 times the volume of the filtrate. After suction filtration to remove impurities, let it stand at 4 °C for more than 24 h to obtain the crude taurine crystal.

9. The method for extracting taurine from abalone viscera by magnetic induction electric field-assisted enzymatic hydrolysis according to claim 1, wherein In S6, the specific steps of recrystallization are as follows: Dissolve the crude taurine crystal with distilled water at 70-90 °C for crystallization, then add absolute ethanol. After mixing, place it in a refrigerator at 4 °C for recrystallization. The number of recrystallization extractions is 3-5 times. During each crystallization extraction process, the addition amount of absolute ethanol is 4-6 times the volume of the solution formed by the crude taurine crystal, distilled water, and absolute ethanol.

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