阅读说明：本技术 一种超声辅助酶合成海藻糖的方法及装置 (Method and device for synthesizing trehalose by using ultrasonic-assisted enzyme ) 是由 梁承� 陈成 余义发 邹林君 吴宁 邹仕刚 闭革林 吴志雄 于 2020-11-10 设计创作，主要内容包括：本发明涉及酶制备领域,具体公开了一种超声辅助酶合成海藻糖的方法及装置,包括以下步骤往反应釜中加入水、淀粉、α-淀粉酶进行混合,然后调节pH值,接着加入无水氯化钙,启动反应釜的搅拌轴搅拌上述混合物、多个超声换能器、加热器和测温器,启动循环通路将反应釜底部的混合物先向外转移粉碎过滤,再送回反应釜之内；分阶段加热混合物以及保温；当液化DE值为15之后停止加热,混合物在搅拌和循环冷却至常温,调节混合物的pH值,然后加入普鲁兰酶、MTSase、MTHase和α/β-CGTase,重新加热并在45℃下保温、调节混合物的pH值,最后加入糖化酶进行混合,最终得到产物。通过超声辅助酶促合成海藻糖,对提高以大米淀粉为基本原料合成海藻糖转化率效果明显。(The invention relates to the field of enzyme preparation, and particularly discloses a method and a device for synthesizing trehalose by using ultrasonic-assisted enzyme, which comprises the following steps of adding water, starch and alpha-amylase into a reaction kettle for mixing, then adjusting the pH value, then adding anhydrous calcium chloride, starting a stirring shaft of the reaction kettle to stir the mixture, a plurality of ultrasonic transducers, a heater and a temperature detector, starting a circulation passage to transfer, crush and filter the mixture at the bottom of the reaction kettle outwards, and then sending the mixture back into the reaction kettle; heating the mixture in stages and preserving heat; stopping heating when the liquefied DE value is 15, stirring and circularly cooling the mixture to normal temperature, adjusting the pH value of the mixture, then adding pullulanase, MTSase, MTHase and alpha/beta-CGTase, reheating, keeping the temperature at 45 ℃, adjusting the pH value of the mixture, finally adding saccharifying enzyme for mixing, and finally obtaining the product. The trehalose is synthesized through ultrasonic-assisted enzymatic synthesis, and the effect of improving the conversion rate of the trehalose synthesized by taking rice starch as a basic raw material is obvious.)

1. A method for synthesizing trehalose by using ultrasonic-assisted enzyme is characterized by comprising the following steps:

adding water, 150g/L rice starch and alpha-amylase with the activity of 15U/g into a reaction kettle, adjusting the pH value to be 6.2 by using acid, and simultaneously adding anhydrous calcium chloride to ensure that the final concentration is 2 g/L;

starting a stirring shaft of the reaction kettle to stir the mixture, simultaneously starting an ultrasonic transducer immersed by the mixture in the reaction kettle, wherein the actual power of the ultrasonic transducer is 100-200w and the vibration frequency is 30kHz, starting a heater in the reaction kettle, starting a circulating passage to transfer, crush and filter the mixture at the bottom of the reaction kettle outwards, and then sending the mixture back to the reaction kettle;

heating the mixture to 75 ℃, keeping the temperature for a plurality of times when the temperature of the mixture is equal to 75 ℃, heating the mixture to 90 ℃, and keeping the temperature for a plurality of times;

stopping heating when the liquefied DE value is 15, stirring and circularly cooling the mixture to the normal temperature, and simultaneously adjusting the pH value of the mixture to be 6;

adding 0.75U/ml pullulanase, adding 2.4U/ml MTSase and MTHase, adding 1.4U/ml alpha/beta-CGTase, reheating, keeping the temperature at 45 ℃ for 25-30h, and adjusting the pH value of the mixture to 4.5;

adding saccharifying enzyme, simultaneously raising the temperature to 60 ℃, and keeping the temperature for 10-12 h;

the product is obtained.

2. The method for synthesizing trehalose by using the ultrasonic-assisted enzyme according to claim 1, wherein the method comprises the following steps: the MTSase is obtained by B.subtilis/pHY300PLK-PgsiB-Y fermentation, the MTHase is obtained by B.subtilis/pHY300PLK-Pxyl-Z fermentation, and the alpha/beta-CGTase is obtained by B.subtilis/pHY300 PLK-PhpaII-PamyQ' -CGT fermentation.

3. The method for synthesizing trehalose by using the ultrasonic-assisted enzyme according to claim 2, wherein the method comprises the following steps: in step S4, the vent of the reaction kettle is opened to dissipate heat, and the mixture is naturally cooled during stirring and circulation.

4. An apparatus for the ultrasonic-assisted enzymatic synthesis of trehalose according to any one of claims 1 to 3, comprising a reaction kettle, wherein the reaction kettle is provided with a stirring paddle, a heater and a temperature detector inside, the upper end of the reaction kettle is provided with a liquid inlet and a valve thereof, and the lower end of the reaction kettle is provided with a liquid outlet and a valve thereof, and is characterized in that: the liquid outlet at the lower end of the reaction kettle is provided with two branches which are a first branch and a second branch respectively, the first branch is used for discharging the mixture to the outside, the second branch is folded and returned to the liquid inlet of the reaction kettle, the second branch is provided with a pump body and a pulverizer, the side wall of the reaction kettle is provided with a plurality of ultrasonic transducers, and the transmitting ends of the ultrasonic transducers face the inner side of the reaction kettle.

5. The device for synthesizing trehalose by using an ultrasonic-assisted enzyme according to claim 4, wherein: the second branch is provided with a third branch and a valve thereof at a position avoiding the crusher for passing through the mixture without further crushing.

6. The device for synthesizing trehalose by using an ultrasonic-assisted enzyme according to claim 5, wherein: and a sampling port is arranged on the second branch.

7. The device for synthesizing trehalose by using an ultrasonic-assisted enzyme according to claim 4, wherein: the blade of stirring rake is helical blade when the stirring rake is rotatory, helical blade drives the mixture and rises, and the mixture descends from reation kettle near inboard position.

Technical Field

The invention belongs to the field of enzyme preparation, and particularly relates to a method and a device for synthesizing trehalose by using ultrasonic-assisted enzyme.

Background

Some insects and plants, which live in desert areas for a long time, are almost dehydrated and dried at high temperature in the noon, and are in a physiologically pseudo-dead state, but can revive after several hours as soon as rainfall replenishes water. Studies of these saphenous organisms by scholars at cambridge university in england have shown that this revival phenomenon is due to the presence of high concentrations of trehalose in their bodies. In addition, some frogs and other organisms can survive in severe cold conditions, and the important reason is the protective effect of trehalose on cells. Many organisms exhibit stress tolerance in stress environments (e.g., starvation, high temperature, freezing, desiccation, hypertonicity, radiation, toxic substances, etc.) that is directly related to the trehalose content in the body. The yeast can be reactivated after 90 percent of water is removed and rehydration is carried out, and the survival rate of the yeast depends on the content of trehalose in a body under the condition of long-term hunger. It has been reported that heat shock and ethanol shock treatment of yeast can cause significant increases in intracellular trehalose content.

Trehalose is a substance having high resistance to stress states resulting from environmental changes, and is a typical stress metabolite in organisms. Under various severe environments, trehalose shows good protection effect on biological macromolecules such as biological membranes, proteins and nucleic acids of species. Recent studies have shown that exogenous trehalose has good nonspecific protection, and thus trehalose is called "vital sugar".

With the improvement of the social living standard, the demand of trehalose is continuously increasing. Because the trehalose yield is low, the production process is complex, the product price is still high, the popularization and the application of the trehalose are limited, and how to improve the yield of the trehalose, how to improve the production method and how to reduce the production cost are important subjects for researching the trehalose.

Disclosure of Invention

The invention aims to provide a method and a device for synthesizing trehalose by using ultrasonic-assisted enzyme, which improve the efficiency of synthesizing trehalose.

In order to achieve the above object, the present invention provides a method for synthesizing trehalose by using an ultrasound-assisted enzyme, comprising the following steps:

s1, adding water, 150g/L rice starch and 15U/g alpha-amylase into a reaction kettle, adjusting the pH value to 6.2 by using acid, and simultaneously adding anhydrous calcium chloride to enable the final concentration to be 2 g/L;

s2, starting a stirring shaft of the reaction kettle to stir the mixture, simultaneously starting an ultrasonic transducer immersed by the mixture in the reaction kettle, wherein the actual power of the ultrasonic transducer is 100-200w, the vibration frequency is 30kHz, starting a heater in the reaction kettle, starting a circulating passage to transfer, crush and filter the mixture at the bottom of the reaction kettle outwards, and then sending the mixture back to the reaction kettle;

s3, heating the mixture to 75 ℃, preserving heat for a plurality of times when the temperature of the mixture is equal to 75 ℃, heating the mixture to 90 ℃, and preserving heat for a plurality of times;

s4, stopping heating after the liquefied DE value is 15, stirring and circularly cooling the mixture to normal temperature, and simultaneously adjusting the pH value of the mixture to 6;

s5, adding 0.75U/ml pullulanase, adding 2.4U/ml MTSase and MTHase, adding 1.4U/ml alpha/beta-CGTase, reheating, keeping the temperature at 45 ℃ for 25-30h, and adjusting the pH value of the mixture to 4.5;

s6, adding saccharifying enzyme, raising the temperature to 60 ℃ and preserving the temperature for 10-12 h;

and S7, obtaining a product.

As an improvement of the above scheme, the MTSase is obtained by B.subtilis/pHY300PLK-PgsiB-Y fermentation, the MTHase is obtained by B.subtilis/pHY300PLK-Pxyl-Z fermentation, and the alpha/beta-CGTase is obtained by B.subtilis/pHY300 PLK-PhpaII-PamyQ' -CGT fermentation.

As a modification of the above scheme, in step S4, the vent of the reaction kettle is opened to dissipate heat, and the mixture is naturally cooled in stirring and circulation.

In order to achieve the purpose, the invention provides a device for synthesizing trehalose by using ultrasonic-assisted enzyme, which comprises a reaction kettle, wherein the inner side of the reaction kettle is provided with a stirring paddle, a heater and a temperature detector, the upper end of the reaction kettle is provided with a liquid inlet and a valve thereof, and the lower end of the reaction kettle is provided with a liquid outlet and a valve thereof, and the device is characterized in that: the liquid outlet at the lower end of the reaction kettle is provided with two branches which are a first branch and a second branch respectively, the first branch is used for discharging the mixture to the outside, the second branch is folded and returned to the liquid inlet of the reaction kettle, the second branch is provided with a pump body and a pulverizer, the side wall of the reaction kettle is provided with a plurality of ultrasonic transducers, and the transmitting ends of the ultrasonic transducers face the inner side of the reaction kettle.

As a modification of the above, the second branch is provided with a third branch and its valve at a position avoiding the pulverizer for passing through the mixture that does not require further pulverization.

As an improvement of the scheme, a sampling port is arranged on the second branch path.

As an improvement of the scheme, the blades of the stirring paddle are helical blades, and when the stirring paddle rotates, the helical blades drive the mixture to ascend, and the mixture descends from the position, close to the inner side, of the reaction kettle.

The invention has the following beneficial effects:

1. the method for enzymatically synthesizing the trehalose by using the ultrasonic wave assisted enzyme effectively improves the trehalose conversion rate, saves resources, reduces the cost, has high trehalose yield and is beneficial to industrial production and application.

2. Starch liquefaction and enzyme catalysis synthesis reaction are carried out in the same reaction kettle, so that the trouble of material transfer is saved, the labor time is saved, and the efficiency is improved.

3. Under the conditions of superfine grinding and material circulating flow, the starch is liquefied, the liquefying efficiency is improved, the heating and the cooling are uniform, the slurry can circularly flow up and down, and no stirring dead angle exists.

Drawings

FIG. 1 is a schematic diagram of a reaction vessel and its interior according to an embodiment.

Description of reference numerals: 10. a reaction kettle; 11. a liquid inlet; 21. a liquid discharge port; 13. a stirring paddle; 14. a heater; 15. a temperature detector; 16. an ultrasonic transducer; 21. a pump body; 22. a pulverizer; 31. a first shunt; 32. a second branch circuit; 33. a third branch circuit; 34. a sampling port.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Referring to fig. 1, the invention discloses a method and a device for synthesizing trehalose by using ultrasonic-assisted enzyme.

The device is based on the existing reaction kettle 10, the inner side of the reaction kettle 10 is provided with a stirring paddle 13, a heater 14 and a temperature detector 15, the upper end of the reaction kettle 10 is provided with a liquid inlet 11 and a valve thereof, and the lower end of the reaction kettle 10 is provided with a liquid outlet 12 and a valve thereof. Further, the liquid discharge port 12 at the lower end of the reaction kettle 10 is provided with two branches, namely a first branch 31 and a second branch 32, the first branch 31 is used for discharging the mixture to the outside, the second branch 32 is turned back to the liquid inlet 11 of the reaction kettle 10, the second branch 32 is provided with a pump body 21 and a pulverizer 22, the side wall of the reaction kettle 10 is provided with a plurality of ultrasonic transducers 16, and the emitting end of each ultrasonic transducer 16 faces the inner side of the reaction kettle 10. The arrows in FIG. 1 indicate the flow direction of the mixture, and after the mixture is stirred in the reaction vessel 10, the mixture is driven by the pump 21 to return to the liquid inlet 11 along the left second branch path 32, and is then fed into the reaction vessel 10 again.

In this embodiment, a plurality of holes are drilled in the sidewall of the reaction kettle 10 for inserting a plurality of ultrasonic transducers 16, then the inner side of the reaction kettle 10 is sealed, and a sealing ring is disposed at the gap. The ultrasonic transducers 16 are uniformly distributed to provide uniform vibration, and the wires and signal wires of the ultrasonic transducers 16 are connected with a computer. The heater 14 is a heating rod, and is suspended or fixed in the reaction kettle 10.

Preferably, the second branch 32 is provided with a third branch 33 and its valve at a position avoiding the pulverizer 22 for passing through the mixture that does not require further pulverization. In this embodiment, the pulverizer 22 is a disk-type ultrafine high-speed shearing pulverizer. If the mixture is already fine enough, it may not pass through the pulverizer 22; in this case, the second branch passage 32 is provided with a third branch passage 33 and a valve thereof at a position avoiding the crusher 22, and it can be seen in fig. 1 that the starting point of the third branch passage 33 is before the crusher 22 and the end point of the third branch passage 33 is after the crusher 22.

Preferably, the second branch 32 is provided with a sampling port 34 so that the reaction condition of the mixture in the reaction vessel 10 can be clearly monitored.

Preferably, the blades of the stirring paddle 13 are helical blades, and when the stirring paddle 13 rotates, the helical blades lift the mixture, and the mixture descends from a position near the inner side of the reaction kettle 10. The arrows marked in fig. 1 indicate the flow state of the mixture, and the mixture located in the middle of the reaction tank 10 is pulled by the spiral blades while the mixture located at the edge of the reaction tank 10 naturally descends. Therefore, the uniformity of the materials and the enzyme in the reaction process can be ensured, and the temperature gradient between the materials and the inside of the enzyme is eliminated, so that the reaction speed and the reaction efficiency are improved.

Preferably, the temperature detector 15 is a thermocouple thermometer.

The method comprises the following steps: a

S1, adding water, 150g/L rice starch and alpha-amylase with the activity of 15U/g into a reaction kettle 10, adjusting the pH value to 6.2 by using acid, and simultaneously adding anhydrous calcium chloride to enable the final concentration to be 2 g/L.

S2, starting a stirring shaft of the reaction kettle 10 to stir the mixture, simultaneously starting an ultrasonic transducer 16 immersed by the mixture in the reaction kettle 10, wherein the actual power of the ultrasonic transducer 16 is 100-200w, the vibration frequency is 30kHz, starting a heater 14 in the reaction kettle 10, starting a circulation passage to transfer, crush and filter the mixture at the bottom of the reaction kettle 10 outwards, and then sending the mixture back to the reaction kettle 10.

S3, heating the mixture to 75 ℃, preserving heat for 15min when the temperature of the mixture is equal to 75 ℃, heating the mixture to 90 ℃, and preserving heat to wait for the change of the liquefied DE value. To ensure the stability of the heating process, the heating was carried out at a rate of 2 ℃/min at a temperature below 75 ℃ and at a rate of 3 ℃/min at a temperature above 75 ℃.

And S4, stopping heating after the liquefaction DE value is 15, stirring and circularly cooling the mixture to the normal temperature (the mixture can be cooled naturally in an open mode or can be cooled by inserting a refrigerating rod), and meanwhile, adjusting the pH value of the mixture to 6.0.

S5, adding 0.75U/ml pullulanase, adding 2.4U/ml MTSase and MTHase, adding 1.4U/ml alpha/beta-CGTase, reheating, keeping the temperature at 45 ℃ for 25-30h, and adjusting the pH value of the mixture to 4.5.

S6, adding saccharifying enzyme, raising the temperature to 60 ℃, and keeping the temperature for 10-12 h.

And S7, obtaining a product.

The above-mentioned B.subtilis/pHY300PLK-PgsiB-Y, B.subtilis/pHY300PLK-Pxyl-Z and B.subtilis/pHY300 PLK-PhoII-PamyQ' -CGT all come from the national emphasis laboratories of the south China university of food science and technology.

The fermentation method of MTSase, MTHase and alpha/beta-CGTase comprises the following steps:

selecting a single colony, culturing in liquid LB (containing neomycin at 20ug/ml) for 8-10h, transferring the seed solution into TB culture medium (containing neomycin at 20ug/ml) according to the inoculum size of 5%, culturing in a shaker at 37 deg.C for 2.5h, and continuously shake-culturing at 33 deg.C for about 48 h. Centrifuging the fermentation liquor at 4 deg.C and 8000r/min for 15min to collect thallus, and concentrating and breaking cell wall to obtain crude enzyme solution.

For the culture medium:

LB liquid medium: 5.0g of yeast powder, 10.0g of peptone and 10.0g of sodium chloride;

LB solid medium: adding 1.5-2.0 g of agarose into 100ml of LB liquid culture medium;

TB culture medium: peptone 12.0g, yeast powder 24.0g, glycerin 5.0g, KH₂PO₄Is 2.31g, K₂HPO₄•3H₂O was 16.43 g.

Based on the above steps, the comparative example obtained 71.4% conversion of the product without the ultrasonic transducer 16 and without any change elsewhere.

Based on the above steps, in example 1, the actual frequency of the ultrasonic transducer 16 is set to 100w, and no change is made elsewhere, so that the conversion rate of the product is 81.6%.

Based on the above steps, in example 2, the actual frequency of the ultrasonic transducer 16 is set to 150w, and no change is made elsewhere, so that the conversion rate of the product is 82.1%.

Based on the above steps, in example 2, the actual frequency of the ultrasonic transducer 16 is set to 150w, and no change is made elsewhere, so that the conversion rate of the product is 82.3%.

Therefore, the trehalose synthesized by the ultrasonic-assisted enzymatic synthesis has obvious effect of improving the conversion rate of the trehalose synthesized by using the rice starch as the basic raw material.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.