CN113088506B - Method for separating myrosinase from rapeseeds - Google Patents
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- CN113088506B CN113088506B CN202110356287.1A CN202110356287A CN113088506B CN 113088506 B CN113088506 B CN 113088506B CN 202110356287 A CN202110356287 A CN 202110356287A CN 113088506 B CN113088506 B CN 113088506B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/04—Pretreatment of vegetable raw material
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
- C11B1/106—Production of fats or fatty oils from raw materials by extracting using ultra-sounds
Abstract
The invention discloses a method for separating myrosinase from rapeseeds, which comprises the following steps: grinding rapeseed, sieving, adding normal hexane, performing ultrasonic treatment, removing upper fat, adding buffer solution, performing ultrasonic extraction, performing low-temperature treatment on the filtered solution, centrifuging at low temperature, taking precipitate, redissolving, and performing dialysis, and then using glucose gel to obtain concentrated enzyme solution.
Description
Technical Field
The invention relates to the field of food engineering, in particular to a method for separating myrosinase from rapeseeds.
Background
The rape seed is the seed of the cruciferous crop rape, the oil content is 35% -42%, the rape seed is one of main oil materials in China, and the annual output is inferior to that of soybean. Myrosinase (academic name: beta-thioglucosidase) is an enzyme that hydrolyzes thioglucosides, which is mainly present in cruciferous plants. In the rapeseed oil processing process, myrosinase in the seeds can partially hydrolyze thioglucoside distributed in cell vacuoles to generate thiocyanate, isothiocyanate and other substances, so that pungent and pungent flavor of the rapeseed oil is endowed. Meanwhile, the glucosinolate in the seeds can be reduced by degrading glucosinolate in the seeds by myrosinase, so that the quality of the rapeseed cakes is improved. The activity of myrosinase in rapeseed is one of the important factors affecting the degree of hydrolysis of thioglucosides in rapeseed.
During the extraction process of myrosinase, the activity of myrosinase is affected by improper extraction mode and condition. In addition, in the case of seeds of rapeseed which are high in oil content, the extraction purity of myrosinase is easily disturbed by impurities such as grease. Therefore, there are problems of low operability, large usage amount of raw materials such as reagents, and high cost in the current preparation of myrosinase for high oil seeds of rapeseed.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems with existing myrosinase products.
Accordingly, one of the objects of the present invention is to overcome the deficiencies of the prior art methods for obtaining myrosinase and to provide a method for separating myrosinase from rapeseed.
In order to solve the technical problems, according to one aspect of the present invention, the following technical solutions are provided: a method for separating myrosinase from rapeseed, comprising the steps of:
taking the rapeseeds subjected to vacuum freeze drying, grinding the rapeseeds with liquid nitrogen, and sieving the rapeseeds;
adding n-hexane, performing ultrasonic treatment, placing an ultrasonic treatment system in a constant temperature environment, standing after the treatment is finished, and discarding upper liquid;
adding the rest solid of the ultrasonic treatment discarded liquid into ethylenediamine tetraacetic acid, dithiothreitol and a buffer solution of tris hydrochloride of polyvinylpyrrolidone, adding into defatted and low-temperature vacuum dried desolventized rapeseed, carrying out ultrasonic extraction, centrifuging again, taking the supernatant, and filtering with gauze;
adding ammonium sulfate powder into the filtered solution to dissolve completely, and then carrying out ice water bath for 1h;
adding tris hydrochloride buffer solution into the precipitate after low-temperature centrifugation, re-dissolving, transferring to a dialysis bag for ice water bath dialysis until the dialysate does not contain sulfate ions;
the dialysis bag was buried in dried glucose gel G15 at 4℃to obtain a concentrated enzyme solution.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: the drying mode is vacuum freeze drying, and the vacuum freeze drying time is 48 hours.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: adding the filtered solution into ammonium sulfate powder, completely dissolving, and then carrying out ice water bath for 1h, wherein the mass fraction of the ammonium sulfate after dissolution is 10-30%.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: the rapeseeds are ground and then screened, and the grinding mode is liquid nitrogen grinding.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: the mixed solution added with n-hexane in the ultrasonic treatment is placed in a constant-temperature water bath at the temperature of 4 ℃.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: the ultrasonic treatment is set to 80-100W for 30min.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: the setting of the sonication was 90w,30min.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: grinding with liquid nitrogen, sieving with 60-100 mesh sieve.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: in the ultrasonic treatment, the mass ratio of the rapeseed to the normal hexane in the ultrasonic treatment mixed liquid is 1:3-5, and the ultrasonic treatment is repeated for 4-6 times.
As a preferred embodiment of the method for separating myrosinase from rapeseed of the present invention, wherein: grinding with liquid nitrogen, sieving with 100 mesh sieve, ultrasonic treating with mass ratio of semen Brassicae campestris to n-hexane of 1:4, and repeating ultrasonic treatment for 4 times.
The method for separating the myrosinase in the rapeseeds, which is provided by the invention, can effectively obtain the myrosinase product, and meanwhile, the obtained myrosinase product has higher concentration, less loss and simple and convenient operation.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a graph of allyl thioglucoside;
fig. 2 is a flow chart of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The sources of the raw material reagents selected in the embodiment part of the invention are as follows:
allyl thioglucoside was purchased from Shanghai Annotation; acetonitrile is chromatographic purity, J & K chemical company in the united states; other reagents were chromatographic pure, all purchased from national pharmaceutical agents.
The instrument and equipment adopted in the embodiment part of the invention are as follows:
waters1525 high performance liquid chromatograph, waters2996 ultraviolet detector.
Example 1
15g of rapeseeds which are respectively subjected to three drying modes, namely hot air drying at 60 ℃, vacuum freeze drying for 24 hours and vacuum freeze drying for 48 hours, are ground by liquid nitrogen and then are sieved by a 60-mesh sieve. 60g of n-hexane is added, then the ultrasonic treatment is carried out for 30min under the power of 90W, the temperature of the ultrasonic treatment process is controlled by adopting a 4 ℃ circulating water bath, the ultrasonic treatment is kept stand, the upper liquid is discarded, and the ultrasonic treatment is repeated for 4 times. 55mL of a buffer solution containing ethylenediamine tetraacetic acid, dithiothreitol and polyvinylpyrrolidone and having a concentration of 0.1mol/L, pH =7.5 of tris (hydroxymethyl) aminomethane hydrochloride is added into defatted and low-temperature vacuum-dried desolventized rapeseed for ultrasound, the ultrasound power is 90W, the ultrasound time is 30min, and the temperature of the ultrasound process is controlled by using a circulating water bath at 4 ℃. Centrifuging at 12000r/min at 4deg.C for 10min after ultrasonic treatment, and filtering the supernatant with 6 layers of gauze. Adding 12% ammonium sulfate powder, and completely dissolving in ice water for 1 hr. Centrifuging at 4deg.C and 12000r/min for 20min, adding 8mL of tris hydrochloride buffer solution into the precipitate, dissolving again, and transferring into a dialysis bag with molecular weight cut-off of 12kDa for ice water bath dialysis until the dialysate is free of sulfate ions. The dialysis bag was buried in dried sephadex G15 at 4℃for 25min to obtain a concentrated enzyme solution.
Measuring the enzyme activity of the concentrated enzyme solution: the concentrated enzyme solution was fixed to 20mL with neutral phosphate buffer. To 400. Mu.L of neutral phosphate buffer solution were added 10. Mu.L of enzyme solution, 25. Mu.L of 15mM allylthioglucoside solution and 100. Mu.L of 1mM ascorbic acid solution, and the mixture was reacted at 37℃for 30 minutes and then rapidly filtered through a 0.25 μm needle filter to determine the residual substrate concentration by high performance liquid chromatography, wherein the conditions of liquid chromatography: chromatographic column: HSS T3 reversed phase chromatographic column; mobile phase: ultrapure water: trifluoroacetic acid=100:0.1; column temperature: 30 ℃; a detector: an ultraviolet detector; detection wavelength: 227nm; flow rate: 1mL/min. Total protein content was determined by reference to coomassie brilliant blue method (Bradford method). The myrosinase activity calculation formula:
wherein, U: enzyme activity unit, enzyme required for hydrolyzing 1. Mu. Mol allylthioglucoside per minute; u/mg: specific activity unit, activity of myrosinase per mg protein; s: allyl thioglucoside hydrolyzed in enzymolysis reaction; t: enzymolysis time; m: total protein content in the reaction enzyme solution.
The measured enzyme activity data are shown in Table 1.
TABLE 1 enzyme Activity of myrosinase from different drying methods
| Hot air drying at 60 DEG C | Vacuum freeze drying for 24h | Vacuum freeze drying for 48h | |
| Specific enzyme activity (U/mg) | 22.5 | 69.2 | 78.8 |
As can be seen from Table 1, hot air drying significantly reduces the enzyme activity of the end product, black mustard, compared to vacuum freeze drying, and vacuum freeze drying for 48 hours has a certain increase but approaches 80% of the value compared to vacuum freeze drying for 24 hours, and the increase of enzyme activity above 80% is more difficult, and vacuum freeze drying for 48 hours already consumes a certain space and energy to meet the storage conditions, and is not suitable for prolonging the duration of vacuum freeze drying, so the preferred vacuum freeze drying market of the invention is 48 hours.
Example 2
15g of rapeseed are taken and respectively subjected to vacuum freeze drying for 48 hours, and are ground by liquid nitrogen and then are sieved by a 60-mesh sieve. 60g of n-hexane is added, then the ultrasonic treatment is carried out for 30min under the power of 90W, the temperature of the ultrasonic treatment process is controlled by adopting a 4 ℃ circulating water bath, the ultrasonic treatment is kept stand, the upper liquid is discarded, and the ultrasonic treatment is repeated for 4 times. 55mL of a buffer solution containing ethylenediamine tetraacetic acid, dithiothreitol and polyvinylpyrrolidone and having a concentration of 0.1mol/L, pH =7.5 of tris hydrochloride is added into defatted and low-temperature vacuum-dried desolventized rapeseed for ultrasonic treatment with an ultrasonic power of 90W, and the ultrasonic treatment is carried out
| Mass fraction of ammonium sulfate addition | 10％ | 15％ | 20％ | 25％ | 30％ |
| Specific enzyme activity (U/mg) | 62.6 | 75.5 | 73.9 | 50 | 42 |
And controlling the temperature of the ultrasonic process by adopting a circulating water bath at 4 ℃ for 30min. Centrifuging at 12000r/min at 4deg.C for 10min after ultrasonic treatment, and filtering the supernatant with 6 layers of gauze. Adding ammonium sulfate powder with mass fractions of 10%, 12%, 15%, 20%, 25% and 30% respectively, and completely dissolving in ice water for 1h. Centrifuging at 4deg.C and 12000r/min for 20min, adding 8mL of tris hydrochloride buffer solution into the precipitate, dissolving again, and transferring into a dialysis bag with molecular weight cut-off of 12kDa for ice water bath dialysis until the dialysate is free of sulfate ions. The dialysis bag was buried in dried sephadex G15 at 4℃for 25min to obtain a concentrated enzyme solution.
Measuring the enzyme activity of the concentrated enzyme solution: the concentrated enzyme solution was fixed to 20mL with neutral phosphate buffer. To 400. Mu.L of neutral phosphate buffer solution were added 10. Mu.L of enzyme solution, 25. Mu.L of 15mM allylthioglucoside solution and 100. Mu.L of 1mM ascorbic acid solution, and the mixture was reacted at 37℃for 30 minutes and then rapidly filtered through a 0.25 μm needle filter to determine the residual substrate concentration by high performance liquid chromatography, wherein the conditions of liquid chromatography: chromatographic column: HSS T3 reversed phase chromatographic column; mobile phase: ultrapure water: trifluoroacetic acid=100:0.1; column temperature: 30 ℃; a detector: an ultraviolet detector; detection wavelength: 227nm; flow rate: 1mL/min. Total protein content was determined by reference to coomassie brilliant blue method (Bradford method). The myrosinase activity calculation formula:
wherein, U: enzyme activity unit, enzyme required for hydrolyzing 1. Mu. Mol allylthioglucoside per minute; u/mg: specific activity unit, activity of myrosinase per mg protein; s: allyl thioglucoside hydrolyzed in enzymolysis reaction; t: enzymolysis time; m: total protein content in the reaction enzyme solution.
The measured enzyme activity data are shown in Table 2.
TABLE 2 myrosinase enzyme Activity obtained with different mass fractions of ammonium sulfate treatments
According to Table 2, it is found that, in the ammonium sulfate treatment step, when the mass fraction of ammonium sulfate added is increased, the specific enzyme activity of myrosinase produced tends to increase first and then decrease, and the maximum value of the specific enzyme activity occurs at 12% (in example 1), and the optimum content of ammonium sulfate added to sodium sulfate at the time of the ammonium sulfate addition treatment is 12% of the mass fraction.
Example 3
15g of rapeseed were each subjected to vacuum freeze-drying for 48 hours, and ground with liquid nitrogen and conventionally ground, respectively, and then sieved through a 60-mesh sieve. 60g of n-hexane is added, then the ultrasonic treatment is carried out for 30min under the power of 90W, the temperature of the ultrasonic treatment process is controlled by adopting a 4 ℃ circulating water bath, the ultrasonic treatment is kept stand, the upper liquid is discarded, and the ultrasonic treatment is repeated for 4 times. 55mL of a buffer solution containing ethylenediamine tetraacetic acid, dithiothreitol and polyvinylpyrrolidone and having a concentration of 0.1mol/L, pH =7.5 of tris (hydroxymethyl) aminomethane hydrochloride is added into defatted and low-temperature vacuum-dried desolventized rapeseed for ultrasound, the ultrasound power is 90W, the ultrasound time is 30min, and the temperature of the ultrasound process is controlled by using a circulating water bath at 4 ℃. Centrifuging at 12000r/min at 4deg.C for 10min after ultrasonic treatment, and filtering the supernatant with 6 layers of gauze. Adding 12% ammonium sulfate powder, and completely dissolving in ice water for 1 hr. Centrifuging at 4deg.C and 12000r/min for 20min, adding 8mL of tris hydrochloride buffer solution into the precipitate, dissolving again, and transferring into a dialysis bag with molecular weight cut-off of 12kDa for ice water bath dialysis until the dialysate is free of sulfate ions. The dialysis bag was buried in dried sephadex G15 at 4℃for 25min to obtain a concentrated enzyme solution.
Measuring the enzyme activity of the concentrated enzyme solution: the concentrated enzyme solution was fixed to 20mL with neutral phosphate buffer. To 400. Mu.L of neutral phosphate buffer solution were added 10. Mu.L of enzyme solution, 25. Mu.L of 15mM allylthioglucoside solution and 100. Mu.L of 1mM ascorbic acid solution, and the mixture was reacted at 37℃for 30 minutes and then rapidly filtered through a 0.25 μm needle filter to determine the residual substrate concentration by high performance liquid chromatography, wherein the conditions of liquid chromatography: chromatographic column: HSS T3 reversed phase chromatographic column; mobile phase: ultrapure water: trifluoroacetic acid=100:0.1; column temperature: 30 ℃; a detector: an ultraviolet detector; detection wavelength: 227nm; flow rate: 1mL/min. Total protein content was determined by reference to coomassie brilliant blue method (Bradford method). The myrosinase activity calculation formula:
wherein, U: enzyme activity unit, enzyme required for hydrolyzing 1. Mu. Mol allylthioglucoside per minute; u/mg: specific activity unit, activity of myrosinase per mg protein; s: allyl thioglucoside hydrolyzed in enzymolysis reaction; t: enzymolysis time; m: total protein content in the reaction enzyme solution.
The measured enzyme activity data are shown in Table 3.
TABLE 3 preparation of myrosinase Activity by different grinding modes
| Conventional grinding | Liquid nitrogen milling | |
| Specific enzyme activity (U/mg) | 52.1 | 80.9 |
As can be seen from Table 3, the different milling modes have an effect on the enzyme activity of the produced myrosinase, and the enzyme activity of the myrosinase after liquid nitrogen milling is significantly higher than that of the myrosinase produced by conventional milling.
Example 4
15g of rapeseed are taken and respectively subjected to vacuum freeze drying for 48 hours, and are ground by liquid nitrogen and then are sieved by a 60-mesh sieve. 60g of n-hexane is added, then the ultrasonic treatment is carried out for 30min under the power of 90W, the temperature of the ultrasonic treatment process is controlled by adopting a 4 ℃ circulating water bath, the ultrasonic treatment is kept stand, the upper liquid is discarded, and the ultrasonic treatment is repeated for 4 times. 55mL of a buffer solution containing 0.1mol/L, pH =7.5 of ethylenediamine tetraacetic acid, dithiothreitol and polyvinylpyrrolidone is added into defatted and low-temperature vacuum dried desolventized rapeseed to carry out ultrasonic treatment, the ultrasonic conditions are respectively set to be 80W of ultrasonic efficiency, 30min of ultrasonic efficiency or 90W of ultrasonic efficiency, 30min of ultrasonic efficiency or 100W of ultrasonic efficiency, 30min of ultrasonic time, and the temperature of the ultrasonic process is controlled by adopting a 4 ℃ circulating water bath. Centrifuging at 12000r/min at 4deg.C for 10min after ultrasonic treatment, and filtering the supernatant with 6 layers of gauze. Adding 12% ammonium sulfate powder, and completely dissolving in ice water for 1 hr. Centrifuging at 4deg.C and 12000r/min for 20min, adding 8mL of tris hydrochloride buffer solution into the precipitate, dissolving again, and transferring into a dialysis bag with molecular weight cut-off of 12kDa for ice water bath dialysis until the dialysate is free of sulfate ions. The dialysis bag was buried in dried sephadex G15 at 4℃for 25min to obtain a concentrated enzyme solution.
Measuring the enzyme activity of the concentrated enzyme solution: the concentrated enzyme solution was fixed to 20mL with neutral phosphate buffer. To 400. Mu.L of neutral phosphate buffer solution were added 10. Mu.L of enzyme solution, 25. Mu.L of 15mM allylthioglucoside solution and 100. Mu.L of 1mM ascorbic acid solution, and the mixture was reacted at 37℃for 30 minutes and then rapidly filtered through a 0.25 μm needle filter to determine the residual substrate concentration by high performance liquid chromatography, wherein the conditions of liquid chromatography: chromatographic column: HSS T3 reversed phase chromatographic column; mobile phase: ultrapure water: trifluoroacetic acid=100:0.1; column temperature: 30 ℃; a detector: an ultraviolet detector; detection wavelength: 227nm; flow rate: 1mL/min. Total protein content was determined by reference to coomassie brilliant blue method (Bradford method). The myrosinase activity calculation formula:
wherein, U: enzyme activity unit, enzyme required for hydrolyzing 1. Mu. Mol allylthioglucoside per minute; u/mg: specific activity unit, activity of myrosinase per mg protein; s: allyl thioglucoside hydrolyzed in enzymolysis reaction; t: enzymolysis time; m: total protein content in the reaction enzyme solution.
The measured enzyme activity data are shown in Table 5.
TABLE 5 specific enzyme activities of myrosinase obtained by different ultrasonic condition settings
| Ultrasonic conditions | 80W/30min | 90W/30min | 100W/30min |
| Specific enzyme activity (U/mg) | 61.2 | 74.4 | 76.5 |
As can be seen from Table 5, the specific enzyme activities of the myrosinases produced under different ultrasonic conditions are shown in Table 5, and there is a tendency that the specific enzyme activities of the myrosinases produced are increased when the ultrasonic efficiency is increased, and the increased value is smaller when the ultrasonic conditions are increased from 90W to 100W, and in this case, a large amount of energy is required to be input for the ultrasonic conditions of 100W and the equipment more specialized than 90W meets the use requirement of 100W, which is not suitable in terms of economical efficiency of production, and the ultrasonic treatment conditions of 90W and 30min are preferable for the ultrasonic treatment step.
Example 5
15g of rapeseeds which are respectively subjected to vacuum freeze drying for 48 hours are taken and respectively sieved by a 60-mesh sieve, a 80-mesh sieve and a 100-mesh sieve after being ground by liquid nitrogen. Adding 45, 60 and 75g of n-hexane, performing ultrasonic treatment at 90W power for 30min, controlling the ultrasonic process temperature by using a 4 ℃ circulating water bath, standing, removing the upper layer liquid, and repeating for 4, 5 and 6 times. The obtained liquid is used for measuring the residual oil rate, and the residual oil rate measuring method comprises the following steps: measurement of fat in GB 5009.6-2016 food, data of the measured residual oil rates are recorded in Table 6.
TABLE 6 residual oil Rate data for liquids obtained under different degreasing conditions
| Mesh number (mesh) | Feed-to-liquid ratio | Number of repetitions (times) | Residual oil Rate (%) |
| 60 | 3 | 4 | 2.4 |
| 60 | 4 | 5 | 2 |
| 60 | 5 | 6 | 1.9 |
| 80 | 3 | 5 | 1.2 |
| 80 | 4 | 6 | 1.1 |
| 80 | 5 | 4 | 1.3 |
| 100 | 3 | 6 | 0.9 |
| 100 | 4 | 4 | 1 |
| 100 | 5 | 5 | 0.8 |
According to table 6, it is possible to obtain different liquid residual oil rates under different degreasing conditions, and a 100 mesh sieve can achieve a good effect of separating grease and the residual oil rate can reach 1% or less, which already has a good level, and a 100 mesh sieve is preferable in view of the cost of continuously increasing degreasing ability; with the increase of the feed liquid ratio, the residual oil effect is correspondingly optimized, and in view of the fact that the residual oil rate of the improvement of the feed liquid ratio from 4 to 5 is lower and the rapid increase of the n-hexane addition amount when the feed liquid ratio is increased, the preferable feed liquid ratio is 1:4; the degreasing effect is improved with the increase of the repetition number, but the repetition number means that the raw material, the energy and the time of the ultrasonic treatment step are excessive, the residual oil rate is not greatly reduced due to the excessive repetition number, and the performance of the residual oil rate of 1% can be achieved under other proper conditions, and the significant improvement of the performance is not obtained due to the large input cost of the repetition, so that the repetition number of 4 is preferable in the invention.
The embodiment of the invention in the embodiment has certain error due to the arrangement of the factors such as raw materials, the environment temperature of the preparation and the like when the data parameters are set optimally, but the error is smaller when the embodiment is combined, and meanwhile, the specific enzyme activity in the invention can reach 80.9U/mg.
Compared with the traditional method, the myrosinase enzyme activity obtained by the method for separating myrosinase activity in rapeseeds has extremely high enzyme activity and less loss, and meanwhile, compared with the traditional method, the method for separating myrosinase has fewer steps, fewer used substrates and high speed.
Claims (1)
1. A method for separating myrosinase from rapeseeds, which is characterized in that: the method comprises the following steps:
taking the rapeseeds subjected to vacuum freeze drying, grinding the rapeseeds with liquid nitrogen, and sieving the rapeseeds;
adding n-hexane, performing ultrasonic treatment, placing an ultrasonic treatment system in a constant temperature environment, standing after the treatment is finished, and discarding upper liquid;
adding the buffer solution of the tris hydrochloride of ethylenediamine tetraacetic acid, dithiothreitol and polyvinylpyrrolidone into the remaining solid from which the liquid is removed by ultrasonic treatment, extracting by ultrasonic waves, centrifuging, taking the supernatant, and filtering with gauze;
adding ammonium sulfate powder into the filtered solution to dissolve completely, and then carrying out ice water bath 1h;
adding tris hydrochloride buffer solution into the precipitate after low-temperature centrifugation, re-dissolving, transferring to a dialysis bag for ice water bath dialysis until the dialysate does not contain sulfate ions;
embedding the dialysis bag into dried glucose gel G15 at 4deg.C to obtain concentrated enzyme solution;
the rapeseeds are ground and then screened, and the grinding mode is liquid nitrogen grinding;
in the ultrasonic treatment, the mass ratio of the rapeseeds to the n-hexane in the mixed liquid is 1:4, and the ultrasonic treatment is repeated for 4 times;
the drying mode is vacuum freeze drying, and the vacuum freeze drying time is 48 hours;
adding the filtered solution into ammonium sulfate powder, completely dissolving the solution into an ice water bath for 1h, wherein the mass fraction of the ammonium sulfate after dissolution is 10-30%, and the concentration of the ammonium sulfate powder is 12%;
the mixed solution added with n-hexane in the ultrasonic treatment is in a constant-temperature water bath at the temperature of 4 ℃;
the setting of the ultrasonic treatment is 90W and 30min;
the liquid nitrogen is ground and then screened, and the mesh number of the screen is 100 meshes.
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|---|---|---|---|---|
| CN103533842A (en) * | 2011-05-03 | 2014-01-22 | 哥本哈根大学 | A process for the manufacture of products from cruciferous crops |
| WO2017091891A1 (en) * | 2015-12-02 | 2017-06-08 | Agrisoma Biosciences Inc. | Method for removing glucosinolates from oilseed meals |
| CN108624575A (en) * | 2018-05-21 | 2018-10-09 | 北京化工大学 | The method that myrosin purifies co-immobilization |
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