CN111675637B - Method for preparing taurine by enzymolysis of freshwater mussel meat under assistance of high-voltage pulse electric field - Google Patents
Method for preparing taurine by enzymolysis of freshwater mussel meat under assistance of high-voltage pulse electric field Download PDFInfo
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Abstract
The invention relates to a method for preparing taurine by carrying out enzymolysis on freshwater mussel meat under the assistance of a high-voltage pulse electric field, and aims to solve the problem that a method for extracting taurine from freshwater mussel meat is lacked at present; the method effectively extracts the high-purity natural taurine product from the freshwater mussel meat through the steps of raw material pretreatment, wall breaking treatment, high-voltage pulse electric field treatment, enzymolysis, filtration, purification, crystallization and the like, not only has green and high-efficiency production process, but also has simple test operation, improves the extraction effect of the taurine, solves the problems of the freshwater mussel by-product resource waste, environmental pollution and the like, optimizes four factors of electric field intensity, pulse number, enzyme addition amount and enzymolysis time by taking the extraction amount of the taurine as an index, and greatly improves the extraction amount of the taurine in the freshwater mussel meat.
Description
Technical Field
The invention relates to a method for preparing taurine, in particular to a method for preparing taurine by carrying out enzymolysis on mussel meat under the assistance of a high-voltage pulse electric field.
Background
Taurine, known as beta-aminoethanesulfonic acid, is a sulfur-containing amino acid, is ubiquitous in animal tissues in the form of free amino acids, and has physiological activity and multiple nutritional functions. At present, two methods for producing taurine are provided: chemical synthesis and natural extraction methods, which are more advocated to extract taurine by using green and safe natural extraction methods because of the toxic substances in the raw materials and production processes of chemical synthesis.
Although there is a method for extracting taurine from meat products and marine products in the prior art, there is no special extraction method for extracting taurine from freshwater mussels, and there is a technical blank, if the existing extraction technology is directly applied to the process of extracting taurine from freshwater mussel meat, the extraction rate is low and unstable, so many parameters and steps in the extraction process need to be further innovated, optimized and improved.
Disclosure of Invention
The permeability of cell membranes can be changed instantly by high-voltage Pulse Electric Field (PEF) treatment, so that treated cells are irreversibly damaged, and active substances in the cells flow out, therefore, the PEF can be used as a high-efficiency novel extraction technology and widely applied to the research of extracting active ingredients of animal and plant tissues. The invention provides a method for preparing taurine by carrying out enzymolysis on freshwater mussel meat under the assistance of a high-voltage pulse electric field, which comprises the following steps of:
(1) pretreatment of raw materials:
removing shell of Carnis Anodonta Seu Crislaria, separating viscera and meat, cleaning meat, draining, homogenizing, and packaging;
(2) wall breaking treatment:
adding distilled water into the pretreated mussel meat homogenate, processing with a wall breaking machine to obtain mussel meat sample liquid, and subpackaging for use; adding distilled water at a ratio of 1:1(mL/g) and homogenizing, and performing intermittent treatment for 2-3min by using a wall breaking machine;
(3) high-voltage pulse electric field treatment:
adding distilled water into the sample liquid for homogenization, wherein the addition amount of the distilled water and the mass of the sample liquid are added according to a ratio of 8:1, the addition amount of the distilled water is mL, and the mass unit of the sample liquid is g; adjusting the flow rate of a peristaltic pump to be 7mL/min, pumping the homogeneous liquid to a pulse electric field processing chamber for high-voltage pulse electric field processing, wherein the number of pulses is 6-10, and the electric field intensity is 20-25 kV/cm; the pulse number and the electric field intensity are respectively adjusted by adjusting two instrument parameters of frequency and input voltage;
(4) and (3) enzymatic hydrolysis:
after the treatment of the pulsed electric field, adopting a sodium hydroxide solution and a hydrochloric acid solution to adjust the pH value of the homogenized solution to 6.5, preheating the homogenized solution for 10min at 50 ℃, adding 4000U/g of an enzyme preparation into the homogenized solution, carrying out water bath treatment on the homogenized solution for 2.5-3.5h in a water bath kettle at 50 ℃, inactivating enzyme for 10min in boiling water after the water bath enzymolysis is finished, and cooling to room temperature after the enzyme inactivation; the enzyme preparation is papain;
(5) removing proteins:
adding 2.5-3% (v/v) of a precipitant I into the sample solution after enzymolysis: 15.0g/100mL potassium ferrocyanide solution, vortex mixing, then adding 2.5-3% (v/v) precipitant II: mixing 30.0g/100mL of zinc acetate solution in a vortex mode, centrifuging after mixing to obtain supernatant, and centrifuging for 10min under the centrifugal condition of 5000 r/min;
(6) and (3) filtering:
adding active carbon into the supernatant, wherein the addition amount of the active carbon is 2-3% w/v of the supernatant, standing, decoloring and filtering;
(7) separation and purification:
introducing the filtrate into cation exchange resin column, eluting with distilled water, and collecting taurine eluate;
(8) and (3) crystallization:
concentrating the collected eluent by using a rotary evaporator to below 10%, adding absolute ethyl alcohol, wherein the addition amount of the absolute ethyl alcohol is 4 times of the volume of the solution, mixing, putting the mixture into a refrigerator at 4 ℃, crystallizing, centrifuging, collecting crystals, dissolving the crystals by using a small amount of distilled water, adding the absolute ethyl alcohol, wherein the addition amount of the absolute ethyl alcohol is 4 times of the volume of the solution, mixing, and putting the mixture into the refrigerator at 4 ℃ for recrystallization; and repeating the crystallization and extraction for 3-4 times to obtain the high-purity taurine.
The invention has the beneficial effects that:
the method effectively extracts the high-purity natural taurine product from the freshwater mussel meat through the steps of raw material pretreatment, wall breaking treatment, high-voltage pulse electric field treatment, enzymolysis, filtration, purification, crystallization and the like, and the high-voltage pulse electric field is a new extraction technology and has the characteristics of non-thermal processing, uniform transfer, short-time treatment, low energy consumption and the like.
The method takes the extraction amount of taurine as an index, adopts a high-voltage pulse electric field to assist an enzymatic method to extract the taurine from the freshwater mussel meat, researches the influence of four factors of electric field intensity, pulse number, enzyme addition amount and enzymolysis time on the extraction amount of the taurine, and optimizes the optimal extraction process to obtain the extraction method; the highest extraction amount of the taurine extracted from the freshwater mussel meat by the method is 13.77mg/g, and a natural taurine product with the purity of 98.2 percent is obtained.
Drawings
FIG. 1 is a schematic diagram showing the effect of the electric field intensity of the present invention on the extraction amount of taurine from mussel meat;
FIG. 2 is a schematic diagram showing the effect of pulse number on the amount of taurine extracted from mussel meat according to the present invention;
FIG. 3 is a schematic diagram showing the effect of the amount of enzyme added on the amount of taurine extracted from mussel meat according to the present invention;
FIG. 4 is a schematic diagram showing the influence of the enzymolysis time on the extraction amount of taurine from freshwater mussel meat according to the present invention;
Detailed Description
The first embodiment is as follows:
a method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of a high-voltage pulse electric field comprises the following steps:
(1) pretreatment of raw materials:
removing shell of Carnis Anodonta Seu Crislaria, separating viscera and meat, cleaning meat, draining, homogenizing, and packaging; (2) wall breaking treatment:
adding distilled water into the pretreated freshwater mussel meat homogenate, adding the amount of the distilled water and the quality of the sample liquid according to a ratio of 1:1(mL/g), performing intermittent treatment for 2-3min by using a wall breaking machine to obtain a freshwater mussel meat sample liquid, and subpackaging for later use;
(3) high-voltage pulse electric field treatment:
taking 4g of clam meat sample liquid, adding 32mL of distilled water into the sample liquid for homogenization, adjusting the flow rate parameter of a peristaltic pump to be 7mL/min after homogenization, and then performing pulsed electric field treatment, wherein the number of pulses is 10, and the electric field intensity is 25 kV/cm;
(4) and (3) enzymatic hydrolysis:
adjusting the pH value of the homogenized solution after high-voltage pulse electric field treatment to 6.5 by using a sodium hydroxide solution and a hydrochloric acid solution, preheating the homogenized solution in a water bath kettle at 50 ℃ for 10min, adding 4000U/g of papain preparation into the homogenized solution, carrying out water bath treatment on the homogenized solution in the water bath kettle at 50 ℃ for 2.95h, carrying out enzyme deactivation in boiling water for 10min after water bath enzymolysis is finished, and cooling the solution to room temperature after enzyme deactivation;
(5) removing proteins:
adding 2.5% (v/v) precipitator I, namely 15.0g/100mL potassium ferrocyanide solution, into the sample solution after enzymolysis, carrying out vortex mixing, adding 2.5% (v/v) precipitator II, namely 30.0g/100mL zinc acetate solution, carrying out vortex mixing, and centrifuging for 10min under the condition of 5000r/min revolution number to obtain a supernatant after mixing;
(6) and (3) filtering:
adding active carbon into the supernatant, wherein the adding amount of the active carbon is 3% w/v of the supernatant, standing, decoloring and filtering;
(7) separation and purification:
introducing 732 type Na into the filtrate+Eluting with distilled water through cation exchange resin column, and collecting taurine eluate;
(8) and (3) crystallization:
concentrating the collected eluent to 10% by using a rotary evaporator, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, mixing, putting the mixture into a refrigerator at 4 ℃, crystallizing and separating out, centrifugally collecting crystals, dissolving the crystals by using distilled water which just enables the crystals to be completely dissolved, adding absolute ethyl alcohol with the volume 4 times that of the solution, putting the crystals into the refrigerator at 4 ℃ for recrystallization, and repeating the crystallization and extraction for three times to obtain the high-purity taurine crystals.
Under the condition, the extraction amount of the taurine in the freshwater mussel meat is 13.77mg/g, and the purity of the taurine is 98.2% by infrared spectrum and high performance liquid chromatography detection.
Example two:
a method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of a high-voltage pulse electric field comprises the following steps:
(1) pretreatment of raw materials:
removing shell of Carnis Anodonta Seu Crislaria, separating viscera and meat, cleaning meat, draining, homogenizing, and packaging; (2) wall breaking treatment:
adding distilled water into the pretreated freshwater mussel meat homogenate, adding the amount of the distilled water and the quality of the sample liquid according to a ratio of 1:1(mL/g), performing intermittent treatment for 2-3min by using a wall breaking machine to obtain a freshwater mussel meat sample liquid, and subpackaging for later use;
(3) high-voltage pulse electric field treatment:
taking 4g of clam meat sample liquid, adding 32mL of distilled water into the sample liquid for homogenization, adjusting the flow rate parameter of a peristaltic pump to be 7mL/min after homogenization, and then performing pulsed electric field treatment, wherein the number of pulses is 8, and the electric field intensity is 20 kV/cm;
(4) and (3) enzymatic hydrolysis:
adjusting the pH value of the homogenized solution after high-voltage pulse electric field treatment to 6.5 by using a sodium hydroxide solution and a hydrochloric acid solution, preheating the homogenized solution in a water bath kettle at 50 ℃ for 10min, adding 4000U/g of papain preparation into the homogenized solution, carrying out water bath treatment on the homogenized solution in the water bath kettle at 50 ℃ for 3h, carrying out enzyme deactivation in boiling water for 10min after water bath enzymolysis is finished, and cooling the solution to room temperature after enzyme deactivation;
(5) removing proteins:
adding 2.5% (v/v) precipitator I, namely 15.0g/100mL potassium ferrocyanide solution, into the sample solution after enzymolysis, carrying out vortex mixing, adding 2.5% (v/v) precipitator II, namely 30.0g/100mL zinc acetate solution, carrying out vortex mixing, and centrifuging for 10min under the condition of 5000r/min revolution number to obtain a supernatant after mixing;
(6) and (3) filtering:
adding active carbon into the supernatant, wherein the adding amount of the active carbon is 3% w/v of the supernatant, standing, decoloring and filtering;
(7) separation and purification:
introducing 732 type Na into the filtrate+Eluting with distilled water through cation exchange resin column, and collecting taurine eluate;
(8) and (3) crystallization:
concentrating the collected eluent to 10% by using a rotary evaporator, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, putting the concentrated solution into a refrigerator with the temperature of 4 ℃, crystallizing and separating out, centrifugally collecting crystals, dissolving the crystals by using distilled water which just enables the crystals to be completely dissolved, adding absolute ethyl alcohol with the volume 4 times that of the solution, putting the crystals into the refrigerator with the temperature of 4 ℃ for recrystallization, and repeating the crystallization and extraction for three times to obtain the high-purity taurine crystals.
Under the condition, the extraction amount of the taurine in the freshwater mussel meat is 13.46 mg/g.
Example three:
a method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of a high-voltage pulse electric field comprises the following steps:
(1) pretreatment of raw materials:
removing shell of Carnis Anodonta Seu Crislaria, separating viscera and meat, cleaning meat, draining, homogenizing, and packaging; (2) wall breaking treatment:
adding distilled water into the pretreated freshwater mussel meat homogenate, adding the amount of the distilled water and the quality of the sample liquid according to a ratio of 1:1(mL/g), performing intermittent treatment for 2-3min by using a wall breaking machine to obtain a freshwater mussel meat sample liquid, and subpackaging for later use;
(3) high-voltage pulse electric field treatment:
taking 4g of clam meat sample liquid, adding 32mL of distilled water into the sample liquid for homogenization, adjusting the flow rate parameter of a peristaltic pump to be 7mL/min after homogenization, and then performing pulsed electric field treatment, wherein the number of pulses is 10, and the electric field intensity is 25 kV/cm;
(4) and (3) enzymatic hydrolysis:
adjusting the pH value of the homogenized solution after high-voltage pulse electric field treatment to 6.5 by using a sodium hydroxide solution and a hydrochloric acid solution, preheating the homogenized solution in a water bath kettle at 50 ℃ for 10min, adding 4000U/g of an enzyme preparation, wherein the enzyme preparation is papain, performing water bath treatment on the homogenized solution in the water bath kettle at 50 ℃ for 2.5h, inactivating enzymes in boiling water for 10min after water bath enzymolysis is finished, and cooling the enzyme to room temperature after enzyme inactivation;
(5) removing proteins:
adding 2.5% (v/v) precipitator I, namely 15.0g/100mL potassium ferrocyanide solution, into the sample solution after enzymolysis, carrying out vortex mixing, adding 2.5% (v/v) precipitator II, namely 30.0g/100mL zinc acetate solution, carrying out vortex mixing, and centrifuging for 10min under the condition of 5000r/min revolution number to obtain a supernatant after mixing;
(6) and (3) filtering:
adding active carbon into the supernatant, wherein the addition amount of the active carbon is 2% w/v of the supernatant, standing, decoloring and filtering;
(7) separation and purification:
introducing 732 type Na into the filtrate+Eluting with distilled water through cation exchange resin column, and collecting taurine eluate;
(8) and (3) crystallization:
concentrating the collected eluent to 10% by using a rotary evaporator, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, putting the concentrated solution into a refrigerator at 4 ℃, crystallizing and separating out, centrifugally collecting crystals, dissolving the crystals by using distilled water which just enables the crystals to be completely dissolved, adding absolute ethyl alcohol with the volume 4 times that of the dissolved solution, putting the crystals into the refrigerator at 4 ℃ for recrystallization, and repeating the crystallization and extraction for three times to obtain the high-purity taurine crystals.
Under the condition, the extraction amount of the taurine in the freshwater mussel meat is 13.65 mg/g.
The optimization test process of the invention is as follows:
the single-factor test of extracting taurine from freshwater mussel meat by high-voltage pulse electric field assisted enzymolysis:
(1) influence of electric field intensity on extraction amount of taurine in freshwater mussel meat
According to the extraction method, 6 groups of 4g of freshwater mussel meat sample liquid are respectively subjected to research on the influence of the electric field strength of 5, 10, 15, 20, 25 and 30kV/cm on the extraction amount of taurine in the freshwater mussel meat under the conditions of 8 pulse numbers, 4000U/g enzyme addition amount and 3h enzymolysis time.
The influence of the electric field strength on the extraction amount of taurine from freshwater mussel meat in the one-factor test is shown in fig. 1: the extraction amount is higher when the electric field intensity is between 15 and 25kV/cm, so that the electric field intensity is selected to be between 15 and 25kV/cm according to the result to carry out subsequent optimization tests.
(2) Influence of pulse number on extraction amount of taurine in freshwater mussel meat
According to the extraction method, 6 groups of 4g of freshwater mussel meat sample liquid are respectively subjected to research on the influence of 2, 4, 6, 8, 10 and 12 pulse numbers on the extraction amount of taurine in freshwater mussel meat under the conditions of 20kV/cm of electric field intensity, 4000U/g of enzyme addition amount and 3h of enzymolysis time.
The influence of the pulse number on the extraction amount of taurine in freshwater mussel meat in the single-factor test is shown in fig. 2: the extraction amount is high when the number of pulses is 6-10, so that the number of pulses is selected to be 6-10 for response surface optimization test.
(3) Influence of enzyme addition on taurine extraction amount in freshwater mussel meat
According to the extraction method, 6 groups of 4g of freshwater mussel meat sample liquid are respectively studied on the influence of the addition of the enzyme on the extraction amount of taurine in the freshwater mussel meat under the conditions of the electric field intensity of 20kV/cm, the pulse number of 8 and the enzymolysis time of 3h, wherein the addition amount of the enzyme is respectively 1000, 2000, 3000, 4000, 5000 and 6000U/g.
The effect of the enzyme addition in the one-factor test on the amount of taurine extracted from mussel meat is shown in fig. 3: when the enzyme addition amount is between 1000-4000U/g, the taurine extraction amount shows a rising trend along with the increase of the enzyme addition amount, and the taurine extraction amount does not change greatly after the enzyme addition amount is increased to 4000U/g. Therefore, the enzyme addition amount is 4000U/g for subsequent optimization test.
(4) Influence of enzymolysis time on extraction amount of taurine in freshwater mussel meat
According to the extraction method, about 6 groups of about 4g of mussel meat sample liquid are respectively subjected to the conditions of 20kV/cm of electric field intensity, 8 pulse numbers and 4000U/g of enzyme addition, and the influence of enzymolysis time of 1.5, 2, 2.5, 3, 3.5 and 4 hours on the extraction amount of taurine in the mussel meat is studied.
The influence of the enzymolysis time on the extraction amount of taurine in freshwater mussel meat in the single-factor test is shown in fig. 4: the extraction amount is higher when the enzymolysis time is between 2.5 and 3.5 hours, so that the subsequent optimization test is carried out by selecting the enzymolysis time to be 2.5 to 3.5 hours according to the result.
The optimization test of the taurine response surface in the clam meat extracted by the high-voltage pulse electric field assisted enzymolysis:
on the basis of a single-factor test result, main factors and excellent levels are screened out, the process conditions for extracting taurine by carrying out enzymolysis on the mussel meat under the assistance of a high-voltage pulse electric field are optimized according to the principle of Central Composite Design (CCD) of a response surface design method, and the factor level table is shown in table 1.
TABLE 1 response surface design factor level coding scheme
According to the results of the single-factor test, three factors and levels of electric field intensity, pulse number and enzymolysis time which have obvious influence on the extraction amount of taurine in the freshwater mussel meat are selected, and 20 groups of tests (including 6 repeated tests) are carried out in total according to the central composite design principle of the response surface. Selecting electric field intensity (X) by taking taurine extraction amount (Y) as dependent variable1) Pulse number (X)2) And enzymolysis time (X)3) As a test independent variable, the technological conditions for extracting taurine by the enzymolysis of the freshwater mussel meat assisted by the high-voltage pulse electric field are optimized. In the research, Design Expert 8.0.6 software is used for analyzing test data, a quadratic polynomial regression model is established, and the optimal process parameters are obtained after predictive analysis. The optimized test protocol and results are shown in table 2 and the analysis of variance table is shown in table 3.
Table 2 optimization of the experimental protocols and results
TABLE 3 response surface design ANOVA
Note: indicates extreme significance (P < 0.01); significances (P < 0.05)
R299.87%; adjusting R299.75%; prediction of R297.68 percent; signal to noise ratio of 87.500
Performing multivariate analysis on the test data of the table 2 by using Design Expert 8.0.6 test software to obtain a regression equation between the extraction amount and the variable when the high-voltage pulse electric field assisted enzymatic method is used for extracting taurine, wherein the regression equation comprises the following steps:
Y＝13.40+0.12X1+0.35X2+0.086X3+0.83X1X2-0.052X1X3-0.082X2X3-0.31X1 2-0.51X2 2-0.27X3 2
wherein Y is the predicted taurine extraction amount (mg/g); x1，X2And X3The electric field intensity (kV/cm), the pulse number (number) and the enzymolysis time (h) are respectively.
As can be seen from the analysis of variance in Table 3, analysis of variance (ANOVA) in response to the surface test resulted in a high F value (857.12) and a very low p value (p)<0.0001), F-value and p-value indicate that the model is significant. ANOVA of taurine extraction yields also a considerable determination coefficient (R)299.87%), which shows that the model has good fitting degree and the linear correlation between the response value and the variable is significant. The regression equation is a high fit since the "lack of fit P value" is 0.9495 > 0.05.
Electric field intensity (X)1) Pulse number (X)2) And enzymolysis time (X)3) Has extremely obvious influence on the maximum taurine content (P is less than 0.01), and the pulse number (X) can be seen from the F value2) Has the most obvious effect on the taurine content and the electric field intensity (X)1) Second, the enzymolysis time (X)3) The effect is minimal. Electric field strength/pulse number (X)1X2) Electric field intensity/enzymolysis time (X)1X3) And number of pulses/time of enzymolysis (X)2X3) The interaction is very significant (P < 0.01). In addition, the quadratic term (X)1 2、X2 2、X3 2) Has very obvious influence on the content of taurine (P is less than 0.01).
Data analysis shows that the optimal test conditions for extracting taurine by a high-voltage pulse electric field assisted enzymolysis method are that the electric field strength is 25kV/cm, the pulse number is 10, the enzymolysis time is 2.95 hours, and the theoretical maximum value of the extraction amount of the taurine is 13.87 mg/g. In order to prove the feasibility of the CCD response surface design test, 3 groups of parallel tests are carried out under the optimal extraction process condition, the actual taurine extraction amount is measured to be 13.77mg/g, the actual taurine extraction amount is basically consistent with the theoretical value, the relative error is 0.72 percent, and the reliability of the optimization model is explained.
Comparative test of taurine extraction from mussel meat:
comparing five methods of extracting taurine from freshwater mussel meat by a direct wall breaking method, extracting taurine from freshwater mussel meat by an enzyme method, extracting taurine from freshwater mussel meat by an ultrahigh pressure auxiliary enzyme method, extracting taurine from freshwater mussel meat by a high-voltage pulse electric field and extracting taurine from freshwater mussel meat by a high-voltage pulse electric field auxiliary enzyme method:
1) test materials and reagents
Freshwater mussel meat: wild freshwater mussels purchased from Songhua river basin of Jilin province.
A taurine standard: CAS: 107-35-7, the purity is more than or equal to 99 percent; purchased from the institute of food and drug testing in China.
Other reagents are reagents commonly used in laboratories.
2) Extraction step
(1) Extracting taurine from the freshwater mussel meat by a direct wall breaking method: the method process is basically the same as the first embodiment of the invention, and the difference is that the step (5) treatment is directly carried out after the step (3) homogenization;
(2) extracting taurine from freshwater mussel meat by an enzyme method: the method process is basically the same as the first embodiment of the invention, and the difference is that the step (4) treatment is directly carried out after the step (3) homogenization, and the high-voltage pulse electric field treatment is not carried out;
(3) extracting taurine from freshwater mussel meat by using an ultrahigh pressure auxiliary enzyme method: the method process is basically the same as the first embodiment of the invention, and the difference is that after the homogenization in the step (3), the ultrahigh pressure treatment is carried out on the sample liquid by adjusting the equipment parameters such as the pressure (150 MPa), the pressure maintaining time (2-4min) and the like;
(4) extracting taurine from the freshwater mussel meat by using a high-voltage pulse electric field: the method is basically the same as the first embodiment of the invention, except that the step (5) is directly carried out after the step (3) is carried out with the pulsed electric field;
(5) extracting taurine from freshwater mussel meat by a high-voltage pulse electric field assisted enzyme method: the method is the first embodiment of the present invention.
The method takes the extraction amount of taurine, the extraction time and the extraction temperature as evaluation indexes, and contrastively analyzes the effects of five methods, namely the method for extracting the taurine from the freshwater mussel meat by a direct wall-breaking method, the method for extracting the taurine from the freshwater mussel meat by an enzyme method, the method for extracting the taurine from the freshwater mussel meat by an ultrahigh-pressure auxiliary enzyme method, the method for extracting the taurine from the freshwater mussel meat by a high-voltage pulse electric field and the method for extracting the taurine from the freshwater mussel meat by an auxiliary enzyme method of a high-voltage pulse electric field under the optimal conditions. The results are shown in Table 4.
TABLE 4 taurine extraction yields under three extraction methods
The extraction amount of the taurine obtained by the wall breaking method extraction, the enzyme method extraction, the ultrahigh pressure auxiliary extraction, the high voltage pulse electric field extraction and the high voltage pulse electric field auxiliary enzyme method extraction is represented as an average value of three parallel tests, and as can be seen from the table, the extraction amount of the taurine extracted from the mussel meat by the high voltage pulse electric field auxiliary enzyme method provided by the invention is the highest and reaches 13.77mg/g, the extraction amount is improved by 16.4% compared with the high voltage pulse electric field extraction, the extraction amount is improved by 18.4% compared with the ultrahigh pressure auxiliary enzyme method, the extraction amount is improved by 28.2% compared with the enzyme method extraction, and the extraction amount is improved by 56.3% compared with the taurine extracted from the mussel meat by the direct wall breaking method.
Claims (4)
1. A method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of a high-voltage pulse electric field is characterized by comprising the following steps: the method comprises the following steps:
(1) pretreatment of raw materials:
removing shell of Carnis Anodonta Seu Crislaria, separating viscera and meat, cleaning meat, draining, homogenizing, and packaging;
(2) wall breaking treatment:
adding distilled water into the pretreated freshwater mussel meat homogenate, wherein the addition amount of the distilled water and the mass of the homogenate are added according to a ratio of 1:1, the addition amount of the distilled water is mL, and the mass unit of the homogenate is g; treating with a wall breaking machine to obtain Carnis Anodonta Seu Crislaria sample liquid, treating with the wall breaking machine intermittently for 2-3min, and packaging;
(3) high-voltage pulse electric field treatment:
adding distilled water into the sample liquid for homogenization, wherein the addition amount of the distilled water and the mass of the sample liquid are added according to a ratio of 8:1, the addition amount of the distilled water is mL, and the mass unit of the sample liquid is g; adjusting the flow rate of a peristaltic pump to be 7mL/min, pumping the homogeneous liquid to a pulsed electric field processing chamber for high-voltage pulsed electric field processing, wherein the number of pulses is 10, and the electric field intensity is 25 kV/cm;
(4) and (3) enzymatic hydrolysis:
after the treatment of the pulsed electric field, adjusting the pH value of the homogeneous liquid to 6.5, adding 4000U/g of enzyme preparation into the homogeneous liquid, carrying out water bath treatment on the homogeneous liquid in a water bath kettle at 50 ℃ for 2.95h, carrying out enzyme deactivation by boiling water after the water bath enzymolysis is finished, and cooling to room temperature after the enzyme deactivation;
(5) removing proteins:
adding a precipitant I into the sample solution after enzymolysis: 15.0g/100mL potassium ferrocyanide solution, 2.5-3% v/v of precipitant I, mixing by vortex; and adding a precipitator II: 30.0g/100mL of zinc acetate solution, 2.5-3% v/v of precipitator II, mixing by vortex, and centrifuging to obtain supernatant;
(6) and (3) filtering:
adding active carbon into the supernatant, wherein the addition amount of the active carbon is 2-3% w/v of the supernatant, standing, decoloring and filtering;
(7) separation and purification:
introducing the filtrate into cation exchange resin column, eluting with distilled water, and collecting taurine eluate;
(8) and (3) crystallization:
concentrating the collected eluent to below 10% by using a rotary evaporator, adding absolute ethyl alcohol, mixing, putting into a refrigerator at 4 ℃, crystallizing, centrifuging, collecting crystals, dissolving the crystals with distilled water, adding absolute ethyl alcohol, mixing, putting into the refrigerator at 4 ℃, recrystallizing, and co-crystallizing and extracting for 3-4 times to obtain the high-purity taurine.
2. The method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of the high-voltage pulse electric field according to claim 1, which is characterized in that: in the step (4), adjusting the pH value of the treatment solution by adopting a sodium hydroxide solution and a hydrochloric acid solution; the enzyme preparation is papain.
3. The method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of the high-voltage pulse electric field according to claim 1, which is characterized in that: in the step (5), the centrifugation is carried out for 10min under the centrifugation condition of 5000 r/min.
4. The method for preparing taurine by enzymolysis of freshwater mussel meat under the assistance of the high-voltage pulse electric field according to claim 1, which is characterized in that: in the step (8), in each crystallization extraction process, the addition amount of the absolute ethyl alcohol is 4 times of the volume of the solution.
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