Device for catalytically synthesizing fructosyl stevioside by using fructosidase mutant

Abstract

The utility model discloses a device for catalytically synthesizing fructosyl stevioside by fructosidase mutants, which comprises a reaction kettle, a crude enzyme liquid adding port and a reaction substrate adding port are arranged on the reaction kettle, and the reaction kettle is sequentially communicated with a cooling tank, a first plate-and-frame filter and a macroporous adsorption resin column. The macroporous adsorption resin column is respectively communicated with a low-alcohol desorption solution storage tank, a high-alcohol desorption solution storage tank, a water-top alcohol solution storage tank and a water storage tank; the low-alcohol desorption solution storage tank is sequentially communicated with the first concentration tank, the second plate and frame filter, the spray dryer, the buffer tank, the crystallizing tank, the centrifugal machine, the drying tank, the crushing tank and the fructosyl stevioside product storage tank. According to the device, biological catalysis is carried out through the fructosidase mutant, and subsequent purification is carried out on reaction liquid, so that high-yield and high-purity fructosyl stevioside is obtained.

Description

TECHNICAL FIELD

[0001] The utility model relates to the technical field of chemical industry, specifically relates to a device for catalytic synthesis of fructosyl steviol glycoside by fructosidase mutant. BACKGROUND

[0002] Steviol glycosides (SGs) extracted from Stevia rebaudiana Bertoni can be used as natural dietary supplements and sweeteners with high sweetness, and it is called the third sugar source next to sugarcane and sugar beet. The chemical structure of SGs is all centered on a glycoside aglycone named steviol, which is connected with different numbers and types of sugar groups at C19 and C13 positions. Research has found that the length of the sugar chain, pyranose substitution and C16 double bond are important structural features for distinguishing the taste characteristics of SGs. In order to improve the taste of SGs, modifying the carbohydrate part by enzymatic method is an efficient method. At present, the device for biocatalytic preparation of steviol glycoside in the prior art has a single function, and the product yield is low. SUMMARY

[0003] The technical problem to be solved by the utility model is: in view of the deficiencies in the prior art, a device for catalytic synthesis of fructosyl steviol glycoside by fructosidase mutant is provided, which carries out biological catalysis by fructosidase mutant and carries out subsequent purification on the reaction liquid, and high-yield and high-purity fructosyl steviol glycoside is obtained.

[0004] To solve the above technical problems, the technical scheme of the utility model is:

[0005] A device for catalytic synthesis of fructosyl steviol glycoside by fructosidase mutant, comprising a reaction kettle, a crude enzyme liquid adding port and a reaction substrate adding port are arranged on the reaction kettle, the reaction kettle is sequentially connected with a cooling tank, a first plate and frame filter, and a macroporous adsorption resin column, the macroporous adsorption resin column is respectively connected with a low alcohol eluent storage tank, a high alcohol eluent storage tank, a water top alcohol solution storage tank, and a water storage tank, the low alcohol eluent storage tank is sequentially connected with a first concentration tank, a second plate and frame filter, a spray dryer, a buffer tank, a crystallization tank, a centrifuge, a drying tank, a crushing tank, and a fructosyl steviol glycoside product storage tank.

[0006] Preferably, the high alcohol eluent storage tank is sequentially connected with a second concentration tank and a material recovery tank.

[0007] Preferably, the water top alcohol solution storage tank is connected with the buffer tank.

[0008] Preferably, the macroporous adsorption resin column is respectively connected with a high alcohol eluent storage tank and a low alcohol eluent storage tank.

[0009] Preferably, the first concentration tank and the second concentration tank are provided with a first stirring device, the first stirring device comprising a first stirring shaft and a plurality of first stirring rods arranged on the first stirring shaft, and the first stirring shaft is connected with a first stirring motor.

[0010] Preferably, the buffer tank is provided with a steam coil pipe, and the steam coil pipe is connected with a steam storage tank.

[0011] Preferably, the crystallization tank is provided with a second stirring device, the second stirring device comprising a second stirring shaft and a plurality of second stirring blades arranged on the second stirring shaft, the second stirring shaft is connected with a second stirring motor, and the plurality of second stirring blades are arranged obliquely.

[0012] Preferably, the second stirring shaft is further provided with a stirring frame, and the stirring frame is provided with a scraper matched with the inner wall of the crystallization tank.

[0013] Preferably, the crushing tank is provided with a crushing mechanism, the crushing mechanism comprising a third stirring shaft arranged in the crushing tank and a third stirring motor connected with the third stirring shaft, a plurality of third stirring rods are arranged on the third stirring shaft, and a plurality of main crushing teeth are arranged on the third stirring rods.

[0014] Preferably, a plurality of auxiliary crushing teeth are arranged on the inner wall of the crushing tank.

[0015] Due to the adoption of the above technical scheme, the present application has the following beneficial effects:

[0016] The utility model provides a kind of fructosylsucrose mutant catalytic synthesis of rebaudioside, including reaction kettle, and reaction kettle is provided with crude enzyme liquid adding port and reaction substrate adding port, and the reaction kettle is sequentially connected cooling tank, first plate and frame filter, macroporous adsorption resin column, and macroporous adsorption resin column is connected with low alcohol eluent storage tank, high alcohol eluent storage tank, water top alcohol solution storage tank, water storage tank respectively;Low alcohol eluent storage tank is sequentially connected with first concentration tank, second plate and frame filter, spray dryer, buffer tank, crystallization tank, centrifuge, drying tank, crushing tank, fructosylsucrose product storage tank.The above-mentioned device is added by adding crude enzyme liquid and reaction substrate to reaction kettle, and under certain conditions, fructosylsucrose solution is synthesized, and after cooling, filtrate is adsorbed by macroporous adsorption resin column, then fructosylsucrose solution on resin is resolved using low alcohol solution, low alcohol eluent solution is sequentially processed by first concentration tank, second plate and frame filter, spray dryer, buffer tank, crystallization tank, centrifuge, drying tank, crushing tank, and the obtained product is temporarily stored in fructosylsucrose product storage tank, improve the production efficiency and yield of product.

[0017] The high alcohol analysis liquid storage tank of the device is sequentially communicated with the first concentration tank and the material recovery tank, and the unreacted reaction substrate adsorbed on the macroporous adsorption resin column is analyzed out by the high alcohol analysis agent, concentrated in the second concentration tank, and then enters the material recovery tank to carry out the next round of biological catalytic reaction, so that the waste of resources is effectively avoided.

[0018] The water top alcohol solution storage tank of the device is communicated with the buffer tank, and the water top alcohol solution enters the buffer tank as a crystallization solvent to dissolve the crude product, so that the energy consumption is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 is a structural schematic diagram of the embodiment 1 of the present application;

[0021] Figure 2 is Figure 1 an enlarged structural schematic diagram of A in the embodiment 1 of the present application;

[0022] In the figure, 1 is a reaction kettle, 2 is a crude enzyme liquid adding port, 3 is a reaction substrate adding port, 4 is a cooling tank, 5 is a first plate and frame filter, 6 is a macroporous adsorption resin column, 7 is a low alcohol analysis liquid storage tank, 8 is a high alcohol analysis liquid storage tank, 9 is a water top alcohol solution storage tank, 10 is a water storage tank, 11 is a high alcohol analysis agent storage tank, 12 is a low alcohol analysis agent storage tank, 13 is a first concentration tank, 14 is a second plate and frame filter, 15 is a spray dryer, 16 is a buffer tank, 17 is a crystallization tank, 18 is a centrifuge, 19 is a drying tank, 20 is a crushing tank, 21 is a fructosyl steviol glycoside product storage tank, 22 is a second concentration tank, 23 is a material recovery tank, 24 is a first stirring shaft, 25 is a first stirring rod, 26 is a first stirring motor, 27 is a steam coil, 28 is a steam storage tank, 29 is a second stirring shaft, 30 is a second stirring blade, 31 is a second stirring motor, 32 is a stirring frame, 33 is a scraper, 34 is a third stirring shaft, 35 is a third stirring motor, 36 is a third stirring rod, 37 is a main crushing tooth, and 38 is an auxiliary crushing tooth. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application

[0024] Embodiment 1

[0025] As shown in Figure 1 and Figure 2 A device for synthesizing fructosyl steviol glycoside catalyzed by a fructosidase mutant, comprising a reaction kettle 1, a crude enzyme liquid adding port 2 and a reaction substrate adding port 3 are arranged on the reaction kettle 1, the reaction kettle 1 is sequentially connected with a cooling tank 4, a first plate and frame filter 5, and a macroporous adsorption resin column 6, the macroporous adsorption resin column 6 is respectively connected with a low-alcohol eluent storage tank 7, a high-alcohol eluent storage tank 8, a water-alcohol solution storage tank 9, and a water storage tank 10; the macroporous adsorption resin column 6 is respectively connected with a high-alcohol eluent agent storage tank 11 and a low-alcohol eluent agent storage tank 12.

[0026] The low-alcohol eluent storage tank 7 is sequentially connected with a first concentration tank 13, a second plate and frame filter 14, a spray dryer 15, a buffer tank 16, a crystallization tank 17, a centrifuge 18, a drying tank 19, a crushing tank 20, and a fructosyl steviol glycoside product storage tank 21.

[0027] The high-alcohol eluent storage tank 8 is sequentially connected with a second concentration tank 22 and a material recovery tank 23. The high-alcohol eluent solution is concentrated through the second concentration tank 22, and then recovered for the next round of biological catalytic reaction.

[0028] The water-alcohol solution storage tank 9 is connected with the buffer tank 16; the water-alcohol solution is recovered and used as a crystallization solvent to dissolve the crude product, thereby reducing energy consumption.

[0029] In this embodiment, a first stirring device is arranged in the first concentration tank 13 and the second concentration tank 22, the first stirring device comprises a first stirring shaft 24 and a plurality of first stirring rods 25 arranged on the first stirring shaft 24, and the first stirring shaft 24 is connected with a first stirring motor 26.

[0030] In this embodiment, a steam coil 27 is arranged in the buffer tank 16, the steam coil 27 is connected with a steam storage tank 28, steam is introduced into the steam coil 27 to heat the liquid in the buffer tank 16, so that the crude product is dissolved in the crystallization solvent, thereby facilitating subsequent cooling and crystallization purification treatment.

[0031] In the embodiment, the crystallization tank 17 is provided with a second stirring device, which comprises a second stirring shaft 29 and a plurality of second stirring blades 30 arranged on the second stirring shaft 29, and the second stirring shaft 29 is connected with a second stirring motor 31, and the plurality of second stirring blades 30 are arranged obliquely.

[0032] In the embodiment, the second stirring shaft 29 is further provided with a stirring frame 32, and the stirring frame 32 is provided with a scraper 33 abutting against the inner wall of the crystallization tank 17. The stirring frame 32 and the scraper 33 can scrape off the crystals adhered to the inner wall of the crystallization tank 17.

[0033] In the embodiment, the crushing tank 20 is provided with a crushing mechanism, which comprises a third stirring shaft 34 arranged in the crushing tank 20 and a third stirring motor 35 connected with the third stirring shaft 34, the third stirring shaft 34 is provided with a plurality of third stirring rods 36, and the third stirring rod 36 is provided with a plurality of main crushing teeth 37; and the inner wall of the crushing tank 20 is provided with a plurality of auxiliary crushing teeth 38. The main crushing teeth 37 and the auxiliary crushing teeth 38 cooperate with each other to improve the crushing efficiency, thereby improving the production efficiency of the fructosyl-steviol glycoside.

[0034] The process of biocatalytic synthesis of fructosyl-steviol glycoside by using the above device is as follows:

[0035] The reaction substrate and crude enzyme solution are added into the reaction kettle 1, and the biocatalytic reaction is carried out under certain conditions. After the reaction is completed, the reaction solution is inactivated after being heated and cooled in the cooling tank 4, and then filtered by the first plate and frame filter 5. The filtrate is adsorbed in the macroporous adsorption resin, and then resolved by using low-alcohol resolving agent and high-alcohol resolving agent respectively. The low-alcohol resolving liquid, the high-alcohol resolving liquid and the water-alcohol solution are collected. The solution in the low-alcohol resolving liquid storage tank 7 is concentrated by the first concentration tank 13, and the concentrated solution is filtered by the second plate and frame filter 14. The filtrate is dried by the spray dryer 15, and the dry powder is dissolved in the buffer tank 16 by adding a crystallization solvent. Then, the solution is cooled and crystallized in the crystallization tank 17. Finally, the crystallization solution is centrifuged by the centrifuge 18, dried by the drying tank 19, and crushed by the crushing tank 20. The qualified material is temporarily stored in the fructosyl-steviol glycoside product storage tank 21. The solution in the high-alcohol resolving liquid storage tank 8 is concentrated by the second concentration tank 22 and then enters the material recovery tank 23 for the next batch of biocatalytic reaction.

[0036] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A device for catalytic synthesis of fructosyl-steviol glycoside by fructosidase mutant, comprising a reaction kettle, characterized in that: The reaction kettle is provided with a crude enzyme liquid adding port and a reaction substrate adding port, and is sequentially connected with a cooling tank, a first plate and frame filter, and a macroporous adsorption resin column.

2. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The macroporous adsorption resin column is respectively connected with a low-alcohol eluent storage tank, a high-alcohol eluent storage tank, a water top alcohol solution storage tank, and a water storage tank.

3. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The low-alcohol eluent storage tank is sequentially connected with a first concentration tank, a second plate and frame filter, a spray dryer, a buffer tank, a crystallization tank, a centrifuge, a drying tank, a crushing tank, and a fructose-based steviol glycoside product storage tank.

4. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The high-alcohol eluent storage tank is sequentially connected with a second concentration tank and a material recovery tank.

5. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The water top alcohol solution storage tank is connected with the buffer tank.

6. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The macroporous adsorption resin column is respectively connected with a high-alcohol eluent storage tank and a low-alcohol eluent storage tank.

7. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The first concentration tank and the second concentration tank are respectively provided with a first stirring device, and the first stirring device comprises a first stirring shaft and a plurality of first stirring rods arranged on the first stirring shaft.

8. The device for catalytic synthesis of fructosyl steviol glycoside according to claim 7, characterized in that: The buffer tank is provided with a steam coil pipe, and the steam coil pipe is connected with a steam storage tank.

9. The apparatus for catalytic synthesis of fructosyl steviol glycoside according to claim 1, wherein: The crystallization tank is provided with a second stirring device, and the second stirring device comprises a second stirring shaft and a plurality of second stirring blades arranged on the second stirring shaft.

10. The device for catalytic synthesis of fructosyl-steviol glycoside according to claim 9, characterized in that: The second stirring shaft is further provided with a stirring frame, and the stirring frame is provided with a scraper matched with the inner wall of the crystallization tank. The crushing tank is provided with a crushing mechanism, and the crushing mechanism comprises a third stirring shaft arranged in the crushing tank and a third stirring motor connected with the third stirring shaft. The third stirring shaft is provided with a plurality of third stirring rods, and the third stirring rods are provided with a plurality of main crushing teeth. The inner wall of the crushing tank is provided with a plurality of auxiliary crushing teeth.

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