Device for producing steviol glycoside d by using genetically recombined bacteria

Abstract

The utility model belongs to the stevia sugar production technical field discloses a device for producing steviol glycoside D by using gene recombination bacteria, which comprises a seed tank, a fermentation tank, a first centrifuge, a homogenizer, a second centrifuge, an enzyme reactor, a ceramic membrane ultrafiltration device, a purification device, a third centrifuge and a drying device that are sequentially connected by pipelines, pumps and valves. The utility model can improve the yield and purity of steviol glycoside D and meet market demand.

Description

Technical Field

[0001] This utility model relates to the field of stevia production technology, specifically to an apparatus for producing steviol glycoside D using recombinant bacteria. Background Technology

[0002] Steviol glycosides are a new type of natural sweetener extracted from the leaves of the stevia plant (Stevia repens), a member of the Asteraceae family. They are characterized by high sweetness, low calories, good taste, and safety, and are non-toxic. They can be widely used in the food, beverage, pharmaceutical, and daily chemical industries. Steviol glycosides are a mixture of multiple glycosides, and their sweetness and taste depend on their glycoside components and content. Steviol glycoside D has a relatively low content, accounting for about 0.8% of the total sugar, but its sweetness is the highest, approximately 450 times that of white sugar. It also has the best taste, without any unpleasant aftertaste, and is hailed as the "gold among steviol glycosides" by industry professionals. Its price is also 3-5 times that of other glycosides, making it an ideal natural sweetener. However, producing steviol glycoside D using traditional methods of extraction from stevia leaves is complex and yields low output. Furthermore, steviol glycoside compositions obtained from plant-derived stevia extracts often contain components of the stevia plant that can impart an off-flavor. Using recombinant bacteria to produce steviol glycoside D can largely overcome the shortcomings of existing production technologies, greatly increase the yield and purity of steviol glycoside D, and meet market demand. Summary of the Invention

[0003] The technical problem to be solved by this utility model is to provide an apparatus for producing steviol glycoside D using recombinant bacteria, which can improve the yield and purity of steviol glycoside D and meet market demand.

[0004] To solve the above-mentioned technical problems, the technical solution of this utility model is as follows:

[0005] An apparatus for producing steviol glycoside D using recombinant bacteria includes a seed tank, a fermenter, a first centrifuge, a homogenizer, a second centrifuge, an enzyme reactor, a ceramic membrane ultrafiltration device, a purification device, a third centrifuge, and a drying device, which are connected in sequence by pipes, pumps, and valves.

[0006] Preferably, the process before the seed tank is equipped with a shaking incubator.

[0007] Preferably, both the seed tank and the fermentation tank are equipped with a temperature exchange device, a stirring device, and an online pH / DO monitoring device. The seed tank is a BIOF-50L model, and the fermentation tank is a Sartorius model. Cplus.

[0008] Preferably, the homogenizer is an E. coli high-pressure homogenizer, model SRH4000-100. Shanghai Shenlu Homogenizer Co., Ltd.

[0009] Preferably, the first centrifuge and the second centrifuge are both disc centrifuges, and the third centrifuge is a flat plate centrifuge.

[0010] Preferably, the enzyme reactor is an immobilized enzyme reactor, which is equipped with a constant temperature circulation system. The model is PBR-1000.

[0011] Preferably, the ceramic membrane ultrafiltration device is a TAMI 0.1μm model. It removes enzymes and bacterial proteins.

[0012] Preferably, the purification device is a crystallization tank, which is equipped with a temperature exchange device and a stirring device.

[0013] Preferably, the drying device is an oven.

[0014] Preferably, the shaker incubator is equipped with a temperature control device and a shaker. The model is ZQZY-70BS.

[0015] Due to the adoption of the above technical solution, the beneficial effects of this utility model are:

[0016] This invention involves culturing recombinant bacteria containing genetically modified glycosyltransferases in a shaker incubator, then transferring them to a seed tank for further cultivation. The bacteria are then obtained through fermentation and solid-liquid separation, followed by homogenization and separation using a homogenizer and a separator to obtain an enzyme solution. The enzyme solution and substrate are then introduced into an enzyme reactor for reaction, converting the substrate into steviol glycoside D. After ultrafiltration, purification, and drying using a ceramic membrane, pure steviol glycoside D is obtained.

[0017] In summary, this invention utilizes recombinant bacteria to produce steviol glycoside D, which can improve the yield and purity of steviol glycoside D and meet market demand. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the equipment process according to an embodiment of the present utility model;

[0019] In the diagram, 1 is a seed tank; 2 is a fermentation tank; 3 is a first centrifuge; 4 is a homogenizer; 5 is a second centrifuge; 6 is an enzyme reactor; 7 is a ceramic membrane ultrafiltration device; 8 is a purification device; 9 is a third centrifuge; 10 is a drying device; and 11 is a shaker incubator. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] like Figure 1As shown, an apparatus for producing steviol glycoside D using recombinant bacteria includes a seed tank 1, a fermentation tank 2, a first centrifuge 3, a homogenizer 4, a second centrifuge 5, an enzyme reactor 6, a ceramic membrane ultrafiltration device 7, a purification device 8, a third centrifuge 9, and a drying device 10, which are connected in sequence by pipes (not shown), pumps (not shown) and valves (not shown); a shaking incubator 11 is provided in the process preceding the seed tank 1.

[0022] In actual production, the recombinant Escherichia coli (containing genetically modified glycosyltransferase) production strain is first cultured on plates and then transferred to shake flasks (LB medium containing 50 μg / mL kanamycin). It is then cultured in a shaker incubator 10 at 37°C and 200 rpm for 12 hours. The bacterial solution is then sequentially transferred to seed tank 1 (TB medium supplemented with 0.5 mM IPTG) and cultured at 30°C, pH 7.0, and DO ≥ 30% for 18 hours (OD600 = 15). The culture is then transferred to fermenter 2 for further cultivation (specific cultivation process omitted). After cultivation, the bacteria / liquid is separated using a first centrifuge 3. The resulting bacterial cells are then homogenized using a homogenizer 4. The homogenized liquid is then separated from the enzyme solution using a second centrifuge 5. The solid bacterial residue is treated as solid waste. The filtrate, containing the enzyme solution, is transported to enzyme reactor 6, where the substrates steviol glycoside and UDP are added. Glucose and sucrose are reacted at 35°C and pH 7.5 with stirring for 12 hours. The reacted material is then transferred to a ceramic membrane ultrafiltration device 7 for further removal of enzymes and residual bacterial proteins, and then transferred to a purification device 8 (crystallization tank). The liquid is cooled and stirred for crystallization and recrystallization. The crystallized liquid is then separated into solid and liquid by a third centrifuge 9. The resulting solid is the wet product of steviol glycoside D. Finally, it is dried by a drying device 10 (oven) at 80-150°C to obtain the finished product of steviol glycoside D with a moisture content of less than 5%.

[0023] The production line of this Rebaudioside D is rationally designed, simple to operate, highly automated, low in energy consumption, and has a short process route, which is conducive to achieving high-efficiency production.

[0024] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An apparatus for producing steviol glycoside D using recombinant bacteria, characterized in that: It includes a seed tank, a fermentation tank, a first centrifuge, a homogenizer, a second centrifuge, an enzyme reactor, a ceramic membrane ultrafiltration device, a purification device, a third centrifuge, and a drying device, which are connected in sequence by pipes, pumps, and valves.

2. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The seed tank pre-process is equipped with a shaker culture box.

3. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: Both the seed tank and the fermentation tank are equipped with a temperature exchange device, a stirring device, and an online pH / DO monitoring device.

4. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The homogenizer is a high-pressure homogenizer for breaking down Escherichia coli.

5. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The first and second centrifuges are disc centrifuges, and the third centrifuge is a flat plate centrifuge.

6. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The enzyme reactor is an immobilized enzyme reactor, which is equipped with a constant temperature circulation system.

7. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The purification device is a crystallization tank, which is equipped with a temperature exchange device and a stirring device.

8. The apparatus for producing steviol glycoside D using recombinant bacteria as described in claim 1, characterized in that: The drying device is an oven.

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