阅读说明：本技术 一种低温淀粉酶菌株、低温淀粉酶的生产方法 (Low-temperature amylase strain and production method of low-temperature amylase ) 是由 马露 陈康 曹杰 于 2020-06-12 设计创作，主要内容包括：本发明低温淀粉酶生产方法技术领域,具体的说是一种低温淀粉酶菌株、低温淀粉酶的生产方法,包括如下步骤：S1:于顶部入口处添加玉米粉、小麦粉、酵母膏、中蜂蜜、蛋白胨的混合物,然后再将盛放有中间气单孢菌的惰性气体罐上的加料管与发酵机的加料口连接,使得中间气单孢菌进入发酵机的内部,最后加入适量的去离子水,发酵时长为2-3d,温度为15-25℃；S2:发酵完后进行发酵液的取用时,将注射针管从发酵机的加料口插入,此时调节驱动橡胶圆环使得橡胶筒的底部上移,用注射针管吸出菌落；本发明大大减小了橡胶筒内溶液与空气的接触机会,从而可防止发酵液发生污染,且进行下一批的菌落的培养时,可直接从加料口注入原料,进而可实现持续化生产。(The invention relates to the technical field of low-temperature amylase production methods, in particular to a low-temperature amylase strain and a production method of low-temperature amylase, which comprise the following steps: s1, adding a mixture of corn flour, wheat flour, yeast extract, medium honey and peptone at a top inlet, connecting a feeding pipe on an inert gas tank containing the medium aeromonas with a feeding port of a fermentation machine to enable the medium aeromonas to enter the fermentation machine, and finally adding a proper amount of deionized water, wherein the fermentation time is 2-3d, and the temperature is 15-25 ℃; s2, when taking the fermentation liquor after fermentation, inserting an injection needle tube from a feed inlet of the fermentation machine, adjusting and driving the rubber ring to move the bottom of the rubber cylinder upwards, and sucking out bacterial colonies by using the injection needle tube; the invention greatly reduces the contact chance of the solution in the rubber cylinder and the air, thereby preventing the fermentation liquor from being polluted, and when the bacterial colony of the next batch is cultured, the raw material can be directly injected from the feed inlet, thereby realizing the continuous production.)

1. A low-temperature amylase strain and a production method of low-temperature amylase are characterized in that: the method comprises the following steps:

s1, opening a sealing plug at the top of the fermentation machine, adding a mixture of corn flour, wheat flour, yeast extract, medium honey and peptone at an inlet at the top, then adding inert gas, discharging oxygen in the fermentation machine, then connecting a feeding pipe on an inert gas tank containing the intermediate gas monospore with a feeding port of the fermentation machine to enable the intermediate gas monospore to enter the fermentation machine, and finally adding a proper amount of deionized water, wherein the deionized water is rich in Ca²⁺The fermentation time is 2-3d, and the temperature is 15-25 ℃;

s2, when taking the fermentation liquor after fermentation, firstly pulling out the sealing plug, inserting the injection needle tube after sterilization and disinfection from the feed inlet of the fermentation machine, adjusting and driving the rubber ring to make the bottom of the rubber cylinder move upwards, observing the liquid level condition inside the fermentation machine through the annular visual window, sucking out bacterial colony by the injection needle tube when the injection needle tube can contact with the fermentation liquor inside the fermentation machine, and plugging the sealing plug;

s3, centrifuging the sucked colony-inducing cells in a centrifuge, taking supernate and separating silk protein to obtain crude enzyme liquid;

s4, adding ammonium sulfate into the crude enzyme solution obtained in the step S3, stirring the mixed solution of the crude enzyme solution and the ammonium sulfate in ice bath, fully precipitating, and collecting precipitates;

s5, treating the precipitate with sulfuric acid buffer solution and gel column for chromatographic separation, performing activity determination, collecting active part, desalting, and concentrating to obtain low temperature amylase product;

the fermentation machine in S1 comprises a fermentation box (1), a rubber cylinder (2), a sealing cover (3), a driving rubber ring (10) and a driven rubber ring (25); the top of the fermentation box (1) is provided with a sealing cover (3); a feed inlet (12) is formed in the center of the sealing cover (3); a rubber diaphragm (11) is arranged inside the feed inlet (12); the edge of the rubber diaphragm (11) is fixedly connected with the inner wall of the feeding port (12); an opening is formed in the rubber diaphragm (11); a sealing plug (7) is inserted into the charging opening (12) and positioned above the rubber diaphragm (11); a first annular groove (24) is formed in the bottom edge of the sealing cover (3); the section of the first annular groove (24) is of an arc-shaped structure; a driven rubber circular ring (25) is arranged inside the first annular groove (24); an arc-shaped support rod (23) for supporting the driven rubber circular ring (25) is fixedly arranged on the inner wall of the first annular groove (24); the arc-shaped supporting rods (23) are provided with a plurality of groups, and the arc-shaped supporting rods (23) are uniformly arranged on the inner wall of the first annular groove (24); the inner walls of the first annular groove (24) and the arc-shaped supporting rod (23) are both fixedly provided with an elastic telescopic rod (26); the free end of the elastic telescopic rod (26) is tightly pressed on the edge of the driven rubber circular ring (25), and the free end of the elastic telescopic rod (26) is provided with a ball; a rubber cylinder (2) is arranged inside the fermentation box (1); the rubber cylinder (2) is a cylindrical cylinder with an arc-shaped bottom; the top end of the rubber cylinder (2) penetrates through a second annular groove (20) at the bottom of the sealing cover (3) and is fixedly connected to the edge of a driven rubber circular ring (25); the second annular groove (20) communicates with the first annular groove (24); a plurality of groups of square-shaped limiting plates (8) are arranged at the top of the sealing cover (3) and right above the first annular groove (24); a driving rubber ring (10) penetrates through the inside of the square limiting plate (8); the magnet groups (9) are fixedly arranged in the driving rubber circular ring (10) and the driven rubber circular ring (25); the magnets in the magnet group (9) are annularly arranged around the center points of the sections of the driving rubber ring (10) and the driven rubber ring (25) at equal intervals, and the polarities of the inner side ends of two adjacent groups of magnets on the magnet group (9) are opposite; the arrangement mode of the magnet group (9) on the driving rubber ring (10) is the same as that of the magnet group (9) on the driven rubber ring (25); the section diameter of the driving rubber circular ring (10) is larger than that of the driven rubber circular ring (25); an annular visible window is arranged on the sealing cover (3) and positioned at the outer edge of the feeding opening (12).

2. The low temperature amylase strain and the production method of the low temperature amylase according to claim 1, wherein the production method comprises the following steps: an annular through groove (13) is formed between the annular visible window and the second annular groove (20) on the sealing cover (3); an annular plate (6) is inserted into the annular through groove (13); an annular rubber pad is adhered to the inner wall of the annular through groove (13); the annular bulge arranged on the annular plate (6) is positioned in the annular slide rail on the inner wall of the annular rubber pad; the top end of the annular plate (6) extends out of the upper port of the annular through groove (13); the bottom end of the annular plate (6) is fixedly connected with the inner side wall of the fan-shaped scraper (14) through a compression spring; the compression spring presses the fan-shaped scraper (14) on the inner wall of the rubber cylinder (2); the fan-shaped scrapers (14) are provided with a plurality of groups, and the fan-shaped scrapers (14) are fixedly connected through rubber scrapers (33); the top end of the fan-shaped scraper (14) is movably hinged at the bottom of the sealing cover (3) through a hinged support.

3. The low temperature amylase strain and the production method of the low temperature amylase according to claim 1, wherein the production method comprises the following steps: a collecting tank (4) is arranged on the edge of the top of the fermentation box (1); and a scraper plate (22) is fixedly arranged on the movable rod part of the elastic telescopic rod (26) positioned below the driven rubber circular ring (25).

4. The low temperature amylase strain and the production method of the low temperature amylase according to claim 1, wherein the production method comprises the following steps: an inflator (16) is fixedly arranged on the side wall of the fermentation box (1); a piston plate (17) is movably arranged in the inflator (16); the outer side end face of the piston plate (17) is fixedly connected with one end of a push rod (18); the inner side end of the inflator (16) is communicated with an air cavity (21) in the rubber cylinder (2) through a spring pipe; micropores (5) are uniformly formed in the inner wall of the rubber cylinder (2); the micropores (5) are communicated with the air cavity (21); the inflator (16) is communicated with the bottom of the collecting tank (4) through a liquid pumping pipe (15); the interior of the liquid extracting pipe (15) is provided with a one-way valve.

5. The low temperature amylase strain and the production method of the low temperature amylase according to claim 1, wherein the production method comprises the following steps: a cylinder (30) is fixedly arranged in the bottom wall of the fermentation box (1); a sliding block (29) fixedly arranged at the top end of the air cylinder (30) is connected with a sliding rail at the bottom of the movable plate (27) in a sliding manner; the upper surface of the movable plate (27) is provided with a triangular conical bulge (28) which is clamped in a triangular conical clamping groove at the bottom of the rubber cylinder (2).

6. The low temperature amylase strain and the production method of the low temperature amylase according to claim 5, wherein the production method comprises the following steps: the bottom of the air cylinder (30) is communicated with the interior of the inflator (16) through an air outlet pipe (19); one end of the air outlet pipe (19) connected with the inflator (16) is close to the outer side end of the inflator (16).

Technical Field

The invention belongs to the technical field of low-temperature amylase production methods, and particularly relates to a low-temperature amylase strain and a production method of low-temperature amylase.

Background

Amylases are a generic term for a group of enzymes that catalyze the hydrolytic conversion of starch to glucose, maltose and other oligosaccharides. The low-temperature amylase is a relative concept, the optimal action temperature of the low-temperature amylase is 20-30 ℃ lower than that of the mesophilic amylase, and the low-temperature amylase has certain enzyme activity at 0 ℃. The low-temperature amylase can effectively play the role of catalysis at low temperature, so that the low-temperature amylase has wide application prospect in the field of bioengineering and solving of the modern energy crisis.

At present, the research on the influence of different production methods on enzyme activity is not exhaustive, and a patent with the patent number of CN1952115A provides a low-temperature amylase strain, a low-temperature amylase and a production method thereof, and mainly researches the influence of metal ions and temperature on the enzyme activity and the thermal stability of the enzyme under different temperature and pH conditions so as to obtain an environment suitable for enzyme growth. In the prior art, when the low-temperature amylase is produced, the raw materials are mostly directly added into a fermentation box, then a sealing cover is covered, the fermentation box is placed in an environment with a certain temperature for 2-3 days, after fermentation is finished, bacterial colonies are directly poured out of the fermentation box, and then subsequent low-temperature amylase preparation is carried out, the production method can greatly increase the contact area between the raw materials and air, so that excessive air in the fermentation box is caused, the probability of pollution of the raw materials is increased, and the probability of successful preparation of fermentation liquor is reduced; and the fermentation box in the prior art can continue to culture the next batch of bacterial colonies after sterilization and disinfection after being used, but can not continuously culture the bacterial colonies, thereby wasting production time.

Disclosure of Invention

The invention provides a low-temperature amylase strain and a production method of low-temperature amylase, which aims to make up for the defects of the prior art and solve the problems that the contact area of raw materials and air is large in the prior art, so that the raw materials are easily polluted and a fermentation box in the prior art cannot continuously culture bacterial colonies.

The technical scheme adopted by the invention for solving the technical problems is as follows: the invention relates to a low-temperature amylase strain and a production method of low-temperature amylase, which comprises the following steps:

s1, opening a sealing plug at the top of the fermentation machine, adding a mixture of corn flour, wheat flour, yeast extract, medium honey and peptone at an inlet at the top, then adding inert gas, discharging oxygen in the fermentation machine, then connecting a feeding pipe on an inert gas tank containing the intermediate gas monospore with a feeding port of the fermentation machine to enable the intermediate gas monospore to enter the fermentation machine, and finally adding a proper amount of deionized water, wherein the deionized water is rich in Ca²⁺The fermentation time is 2-3d, and the temperature is 15-25 ℃;

s2, when taking the fermentation liquor after fermentation, firstly pulling out the sealing plug, inserting the injection needle tube after sterilization and disinfection from the feed inlet of the fermentation machine, adjusting and driving the rubber ring to make the bottom of the rubber cylinder move upwards, observing the liquid level condition inside the fermentation machine through the annular visual window, sucking out bacterial colony by the injection needle tube when the injection needle tube can contact with the fermentation liquor inside the fermentation machine, and plugging the sealing plug;

s3, centrifuging the sucked colony-inducing cells in a centrifuge, taking supernate and separating silk protein to obtain crude enzyme liquid;

s4, adding ammonium sulfate into the crude enzyme solution obtained in the step S3, stirring the mixed solution of the crude enzyme solution and the ammonium sulfate in ice bath, fully precipitating, and collecting precipitates;

s5, treating the precipitate with sulfuric acid buffer solution and gel column for chromatographic separation, performing activity determination, collecting active part, desalting, and concentrating to obtain low temperature amylase product;

the fermentation machine in S1 comprises a fermentation box, a rubber cylinder, a sealing cover, a driving rubber ring and a driven rubber ring; the top of the fermentation box is provided with a sealing cover; a feed inlet is formed in the center of the sealing cover; a rubber diaphragm is arranged inside the feeding port; the edge of the rubber diaphragm is fixedly connected with the inner wall of the charging opening; the rubber diaphragm is provided with an opening; a sealing plug is inserted into the charging opening and positioned above the rubber diaphragm; a first annular groove is formed in the edge of the bottom of the sealing cover; the section of the first annular groove is of an arc-shaped structure; a driven rubber ring is arranged in the first annular groove; an arc-shaped supporting rod for supporting the driven rubber ring is fixedly arranged on the inner wall of the first annular groove; the arc-shaped supporting rods are provided with a plurality of groups, and the arc-shaped supporting rods are uniformly arranged on the inner wall of the first annular groove; elastic telescopic rods are fixedly arranged on the inner wall of the first annular groove and the inner wall of the arc-shaped supporting rod; the free end of the elastic telescopic rod is tightly pressed on the edge of the driven rubber circular ring, and the free end of the elastic telescopic rod is provided with a ball; a rubber cylinder is arranged inside the fermentation box; the rubber cylinder is a cylindrical cylinder with an arc-shaped bottom; the top end of the rubber cylinder penetrates through a second annular groove at the bottom of the sealing cover and is fixedly connected to the edge of the driven rubber circular ring; the second annular groove is communicated with the first annular groove; a plurality of groups of square-shaped limiting plates are arranged at the top of the sealing cover and right above the first annular groove; a driving rubber ring penetrates through the inside of the square limiting plate; magnet groups are fixedly arranged inside the driving rubber circular ring and inside the driven rubber circular ring; the magnets in the magnet group are arranged in an equidistant annular mode around the center points of the sections of the driving rubber ring and the driven rubber ring, and the polarities of the inner side ends of two adjacent groups of magnets on the magnet group are opposite; the arrangement mode of the magnet groups on the driving rubber ring is the same as that of the magnet groups on the driven rubber ring; the diameter of the section of the driving rubber ring is larger than that of the section of the driven rubber ring; an annular visible window is arranged on the sealing cover and positioned at the outer edge of the charging opening;

when the device works, the driving rubber ring is firstly turned towards the inner side, at the moment, the magnet group on the driving rubber ring attracts the magnet group on the driven rubber ring and enables the driven rubber ring to be turned towards the outer side, so that the edge of the rubber cylinder is wound on the driven rubber ring, when the driving rubber ring is continuously turned towards the inner side, the driven rubber ring can be continuously turned towards the outer side, so that the edge of the rubber cylinder is continuously wound, gas in the rubber cylinder is continuously discharged when the bottom of the rubber cylinder is continuously moved upwards, at the moment, the sealing plug at the top of the fermentation box is opened, a mixture of corn flour, wheat flour, yeast extract, medium honey and peptone is added at the top feeding port, the driving rubber ring is continuously turned towards the outer side while feeding, at the moment, the driven rubber ring is turned towards the inner side under the action of the driving rubber ring, the rubber cylinder wound on the driven rubber ring is released, the accommodation space of the rubber cylinder is increased, after feeding is completed, inert gas is added, oxygen in the fermentation machine is discharged, then a feeding pipe on an inert gas tank containing the Aeromonas intermedia is connected with a feeding port of the fermentation machine, so that the Aeromonas intermedia enters the fermentation machine, a proper amount of deionized water is added, finally, a sealing plug is plugged, fermentation can be performed, and the liquid level of the rubber cylinder can be observed from the annular visible window while feeding; when the fermentation is finished and the fermentation broth needs to be taken, the sterilized injection needle tube can be inserted from a feeding port at the top of the fermentation box for direct extraction, if the injection needle tube cannot be in contact with the liquid level in the rubber cylinder, the rubber ring can be wound inwards to drive the rubber ring, so that the rubber cylinder is wound by the driven rubber ring, the liquid level in the rubber cylinder moves upwards, the fermentation broth can be continuously extracted, the use is convenient, the preparation amount of the fermentation broth is controllable, meanwhile, the fermentation broth can be prepared by recycling, and meanwhile, the contact chance of the fermentation broth and air is reduced, and the fermentation broth is prevented from being infected.

Preferably, an annular through groove is formed in the sealing cover between the annular visible window and the second annular groove; an annular plate is inserted into the annular through groove; an annular rubber pad is adhered to the inner wall of the annular through groove; the annular bulge arranged on the annular plate is positioned in the annular slide rail on the inner wall of the annular rubber pad; the top end of the annular plate extends out of the upper port of the annular through groove; the bottom end of the annular plate is fixedly connected with the inner side wall of the fan-shaped scraper through a compression spring; the compression spring presses the fan-shaped scraper on the inner wall of the rubber cylinder; the fan-shaped scrapers are provided with a plurality of groups, and the groups of fan-shaped scrapers are fixedly connected through rubber scrapers; the top end of the fan-shaped scraper is movably hinged to the bottom of the sealing cover through a hinged support; when the rubber cylinder is wound on the driven rubber ring, the fan-shaped scraper and the rubber scraper can scrape the solution on the inner wall of the rubber cylinder and can rotate the annular plate back and forth, and the annular plate drives the fan-shaped scraper to swing back and forth through the compression spring, so that fermentation liquor on the inner wall of the rubber cylinder can be shaken off.

Preferably, a collecting tank is arranged on the edge of the top of the fermentation box; a scraping plate is fixedly arranged on the movable rod part of the elastic telescopic rod positioned below the driven rubber circular ring; during operation, when the rubber cylinder is wound on the driven rubber ring, the scraping plate positioned on the elastic telescopic rod can scrape the fermentation liquor on the inner wall of the rubber cylinder and fall into the collecting tank for collection.

Preferably, an inflator is fixedly arranged on the side wall of the fermentation box; a piston plate is movably arranged in the inflator; the end surface of the outer side of the piston plate is fixedly connected with one end of the push rod; the inner side end of the inflator is communicated with an air cavity in the rubber cylinder through a spring pipe; micropores are uniformly formed in the inner wall of the rubber cylinder; the micropores are communicated with the air cavity; the inflator is communicated with the bottom of the collecting tank through a liquid pumping pipe; a one-way valve is arranged inside the liquid pumping pipe; the during operation, when the push rod is to the outside when pulling, the inside of collecting fermentation liquor of collecting vat gets into the inside of inflater through the liquid suction pipe this moment, when the push rod promotes to the inboard of inflater, the inside gas of inflater and the fermentation liquor of drawing in from the collecting vat get into the inside air cavity of rubber tube through the spring pipe this moment, then the micropore blowout on the rubber tube, thereby not only can retrieve the inside of rubber tube again with the fermentation liquor in the collecting vat, and can be to the inside pump income gas of rubber tube, play the effect of stirring fermentation liquor, make the more even of mixture of fermentation liquor, thereby make the cultivation of bacterial colony more even.

Preferably, a cylinder is fixedly arranged in the bottom wall of the fermentation box; a sliding block fixedly arranged at the top end of the air cylinder is in sliding connection with a sliding rail at the bottom of the movable plate; the triangular pyramidal bulges arranged on the upper surface of the movable plate are clamped in the triangular pyramidal clamping grooves at the bottom of the rubber cylinder; the during operation, when the push rod promoted to the inboard of inflater, gas got into the inside of air cavity through the spring pipe this moment, air cavity one side inflation earlier to make the bottom of rubber tube take place the swing, because the protruding card of triangular pyramid shape that the upper surface of fly leaf was equipped with establishes in the triangular pyramid draw-in groove of rubber tube bottom, the swing of rubber tube drives the swing back and forth of fly leaf, thereby play the effect of vibrating the inside zymotic fluid of rubber tube, and then make the more even of mixture of zymotic fluid, avoid the bacterial colony to take place the large tracts of land cluster.

Preferably, the bottom of the air cylinder is communicated with the interior of the inflator through an air outlet pipe; one end of the air outlet pipe connected with the inflator is close to the outer side end of the inflator; during operation, when convoluteing the rubber cylinder on driven rubber ring, the inside gas that is located the air cavity this moment will extrude the inside that gets into the inflater, and then gets into the inside of cylinder through the outlet duct, and the cylinder shifts up and then upwards holds up the bottom of rubber cylinder through the fly leaf, can reduce the gravity that the driven rubber ring bore to the coiling of the rubber cylinder of being convenient for more.

The invention has the following beneficial effects:

1. according to the low-temperature amylase strain and the production method of the low-temperature amylase, the driving rubber ring and the driven rubber ring are arranged, the overturning of the driven rubber ring is controlled by the overturning of the driving rubber ring, so that the winding and the unfolding of the rubber cylinder are realized, the internal volume of the rubber cylinder is controllable, the culture amount is controllable, the distance from the bottom of the rubber cylinder to the port of the fermentation box is controllable, and bacterial colonies are conveniently extracted by the injection needle tube; meanwhile, when the materials are initially fed, the bottom of the rubber cylinder is very close to the port of the fermentation box, the internal volume of the rubber cylinder is very small, so that the air quantity is very small, the fed materials are fed from the feeding port, the taken materials are taken out from the feeding port through the injection needle tube, and the rubber diaphragm is arranged inside the feeding port, so that the contact chance of the solution in the rubber cylinder and the air is greatly reduced, the fermentation liquor can be prevented from being polluted, and when the bacterial colonies of the next batch are cultured, the raw materials can be directly injected from the feeding port, so that the continuous production can be realized, and the culture time can be saved.

2. According to the low-temperature amylase strain and the production method of the low-temperature amylase, the scraping plate is arranged, so that when the rubber cylinder is wound on the driven rubber ring, the fermentation liquor on the inner wall of the rubber cylinder can be scraped by the scraping plate on the elastic telescopic rod and falls into the collecting tank for collection.

3. According to the low-temperature amylase strain and the production method of the low-temperature amylase, the inflator is arranged, and further fermentation liquor and gas in the collecting tank can be pumped into the rubber cylinder by pushing the push rod, so that the fermentation liquor in the collecting tank can be recycled into the rubber cylinder again, the gas can be pumped into the rubber cylinder to play a role in stirring the fermentation liquor, the fermentation liquor is mixed more uniformly, and the bacterial colony is cultured more uniformly.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is a flow chart of the production process of the present invention;

FIG. 2 is a schematic view of the internal structure of the present invention;

FIG. 3 is an enlarged view of a portion of FIG. 2 at A;

FIG. 4 is an enlarged view of a portion of FIG. 2 at B;

FIG. 5 is a schematic view of the connection of the segment squeegee and the squeegee;

in the figure: fermentation box 1, rubber cylinder 2, sealed lid 3, collecting vat 4, micropore 5, annular plate 6, sealing plug 7, time shape limiting plate 8, magnet group 9, drive rubber ring 10, rubber diaphragm 11, charge door 12, annular through groove 13, fan-shaped scraper blade 14, liquid suction pipe 15, inflater 16, piston plate 17, push rod 18, outlet duct 19, second annular recess 20, air cavity 21, scrape flitch 22, arc bracing piece 23, first annular recess 24, driven rubber ring 25, elastic expansion pole 26, fly leaf 27, triangular pyramid arch 28, slider 29, cylinder 30, rubber scraper blade 33.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

As shown in fig. 1 to 5, the present invention provides a low temperature amylase strain and a method for producing low temperature amylase, the method comprising the following steps:

s1, opening a sealing plug at the top of the fermentation machine, adding a mixture of corn flour, wheat flour, yeast extract, medium honey and peptone at an inlet at the top, then adding inert gas, discharging oxygen in the fermentation machine, then connecting a feeding pipe on an inert gas tank containing the intermediate gas monospore with a feeding port of the fermentation machine to enable the intermediate gas monospore to enter the fermentation machine, and finally adding a proper amount of deionized water, wherein the deionized water is rich in Ca²⁺Fermenting for 2-3 days at 15-25 deg.C, wherein Ca²⁺Can activate the enzyme activity;

s2, when taking the fermentation liquor after fermentation, firstly pulling out the sealing plug, inserting the injection needle tube after sterilization and disinfection from the feed inlet of the fermentation machine, adjusting and driving the rubber ring to make the bottom of the rubber cylinder move upwards, observing the liquid level condition inside the fermentation machine through the annular visual window, sucking out bacterial colony by the injection needle tube when the injection needle tube can contact with the fermentation liquor inside the fermentation machine, and plugging the sealing plug;

s3, centrifuging the sucked colony-inducing cells in a centrifuge, taking supernate and separating silk protein to obtain crude enzyme liquid;

s4, adding ammonium sulfate into the crude enzyme solution obtained in the step S3, stirring the mixed solution of the crude enzyme solution and the ammonium sulfate in ice bath, fully precipitating, and collecting precipitates;

s5, treating the precipitate with sulfuric acid buffer solution and gel column for chromatographic separation, performing activity determination, collecting active part, desalting, and concentrating to obtain low temperature amylase product;

the fermentation machine in S1 comprises a fermentation box 1, a rubber cylinder 2, a sealing cover 3, a driving rubber ring 10 and a driven rubber ring 25; the top of the fermentation box 1 is provided with a sealing cover 3; a feed inlet 12 is formed in the center of the sealing cover 3; a rubber diaphragm 11 is arranged inside the feeding opening 12; the edge of the rubber diaphragm 11 is fixedly connected with the inner wall of the feeding port 12; an opening is formed in the rubber diaphragm 11; a sealing plug 7 is inserted into the charging opening 12 above the rubber diaphragm 11; a first annular groove 24 is formed in the bottom edge of the sealing cover 3; the section of the first annular groove 24 is of an arc-shaped structure; a driven rubber ring 25 is arranged in the first annular groove 24; an arc-shaped support rod 23 for supporting a driven rubber ring 25 is fixedly arranged on the inner wall of the first annular groove 24; the arc-shaped support rods 23 are provided with a plurality of groups, and the arc-shaped support rods 23 are uniformly arranged on the inner wall of the first annular groove 24; the inner walls of the first annular groove 24 and the arc-shaped support rod 23 are both fixedly provided with an elastic telescopic rod 26; the free end of the elastic telescopic rod 26 is tightly pressed on the edge of the driven rubber ring 25, and the free end of the elastic telescopic rod 26 is provided with a ball; a rubber cylinder 2 is arranged inside the fermentation box 1; the rubber cylinder 2 is a cylindrical cylinder with an arc-shaped bottom; the top end of the rubber cylinder 2 penetrates through the second annular groove 20 at the bottom of the sealing cover 3 and is fixedly connected with the edge of a driven rubber circular ring 25; the second annular groove 20 communicates with the first annular groove 24; a plurality of groups of square-shaped limiting plates 8 are arranged at the top of the sealing cover 3 and right above the first annular groove 24; a driving rubber ring 10 penetrates through the square limiting plate 8; the magnet groups 9 are fixedly arranged in the driving rubber circular ring 10 and the driven rubber circular ring 25; the magnets in the magnet group 9 are arranged in an equidistant annular shape around the center points of the sections of the driving rubber ring 10 and the driven rubber ring 25, and the polarities of the inner side ends of two adjacent groups of magnets on the magnet group 9 are opposite; the arrangement mode of the magnet group 9 on the driving rubber ring 10 is the same as that of the magnet group 9 on the driven rubber ring 25; the section diameter of the driving rubber ring 10 is larger than that of the driven rubber ring 25; an annular visible window is arranged on the outer edge of the sealing cover 3 positioned at the feeding hole 12;

when the fermentation box works, the driving rubber ring 10 is firstly turned towards the inner side, at the moment, the magnet group 9 on the driving rubber ring 10 attracts the magnet group 9 on the driven rubber ring 25 and enables the driven rubber ring 25 to be turned towards the outer side, the edge of the rubber cylinder 2 is wound on the driven rubber ring 25, when the driving rubber ring 10 is turned towards the inner side continuously, the driven rubber ring 25 can be turned towards the outer side continuously, the edge of the rubber cylinder 2 is wound continuously, the gas sealing plug positioned in the rubber cylinder 2 is discharged continuously while the bottom of the rubber cylinder 2 moves upwards continuously, at the moment, the sealing plug 7 at the top of the fermentation box 1 is opened, a mixture of corn flour, wheat flour, yeast cream, medium honey and peptone is added at the top feeding port 12, the driving rubber ring 10 is turned towards the outer side continuously while feeding, at the moment, the driven rubber ring 25 is turned towards the inner side under the action of the driving rubber ring 10, thereby releasing the rubber cylinder 2 wound on the driven rubber ring 25, improving the accommodation space of the rubber cylinder 2, adding inert gas after charging, discharging oxygen in the fermentation machine, then connecting a charging pipe on an inert gas tank containing the Aeromonas intermedia with a charging port 12 of the fermentation machine, enabling the Aeromonas intermedia to enter the fermentation machine, adding a proper amount of deionized water, finally plugging a sealing plug 7 to ferment, and observing the liquid level of the rubber cylinder 2 from the annular visual window while charging; when the fermentation is finished and the fermentation broth needs to be taken, the sterilized injection needle tube can be inserted from the feeding port 12 at the top of the fermentation box 1 for direct extraction, if the injection needle tube cannot be in contact with the liquid level in the rubber cylinder 2, the rubber ring 10 can be wound inwards at the moment, so that the driven rubber ring 25 winds the rubber cylinder 2, the liquid level in the rubber cylinder 2 moves upwards, the fermentation broth can be continuously extracted, the use is convenient, the preparation amount of the fermentation broth is controllable, meanwhile, the preparation method can be repeatedly used for preparing the fermentation broth, and meanwhile, the contact chance of the fermentation broth and the air is reduced, and the fermentation broth is prevented from being infected.

As an embodiment of the present invention, an annular through groove 13 is formed between the annular viewing window and the second annular groove 20 on the sealing cover 3; the annular through groove 13 is internally inserted with an annular plate 6; an annular rubber pad is adhered to the inner wall of the annular through groove 13; the annular bulge arranged on the annular plate 6 is positioned in the annular slide rail on the inner wall of the annular rubber pad; the top end of the annular plate 6 extends out of the upper port of the annular through groove 13; the bottom end of the annular plate 6 is fixedly connected with the inner side wall of the fan-shaped scraper 14 through a compression spring; the compression spring presses the fan-shaped scraper 14 on the inner wall of the rubber cylinder 2; the fan-shaped scrapers 14 are provided with a plurality of groups, and the plurality of groups of fan-shaped scrapers 14 are fixedly connected through rubber scrapers 33; the top end of the fan-shaped scraper 14 is movably hinged at the bottom of the sealing cover 3 through a hinged support; when the rubber cylinder 2 is wound on the driven rubber ring 25, the fan-shaped scraper 14 and the rubber scraper 33 can scrape off the solution on the inner wall of the rubber cylinder 2 and can rotate the annular plate 6 back and forth, and the annular plate 6 drives the fan-shaped scraper 14 to swing back and forth through the compression spring, so that the fermentation liquor on the inner wall of the rubber cylinder 2 can be shaken off.

As an embodiment of the invention, the top edge of the fermentation box 1 is provided with a collecting groove 4; a scraper plate 22 is fixedly arranged on the movable rod part of the elastic telescopic rod 26 positioned below the driven rubber ring 25; in operation, when the rubber cylinder 2 winds on the driven rubber ring 25, the material scraping plate 22 on the elastic telescopic rod 26 can scrape down the fermentation liquid on the inner wall of the rubber cylinder 2 and fall into the collecting tank 4 for collection.

As an embodiment of the invention, an air pump 16 is fixedly arranged on the side wall of the fermentation box 1; a piston plate 17 is movably arranged in the inflator 16; the end surface of the outer side of the piston plate 17 is fixedly connected with one end of a push rod 18; the inner side end of the inflator 16 is communicated with an air cavity 21 in the rubber barrel 2 through a spring pipe; micropores 5 are uniformly formed in the inner wall of the rubber cylinder 2; the micropores 5 are communicated with the air cavity 21; the inflator 16 is communicated with the bottom of the collecting tank 4 through a liquid pumping pipe 15; a one-way valve is arranged inside the liquid pumping pipe 15; the during operation, when the push rod 18 is to the outside when pulling, the inside fermentation liquid of collecting vat 4 this moment gets into the inside of inflater 16 through liquid suction pipe 15, when push rod 18 promotes to the inboard of inflater 16, the inside gas of inflater 16 and the inside fermentation liquid of extraction in from collecting vat 4 get into in the inside air cavity 21 of rubber tube 2 through the spring pipe this moment, then 5 blowout through micropore on the rubber tube 2, thereby not only can retrieve the inside of rubber tube 2 again with the fermentation liquid in the collecting vat 4, and can be to the inside pump income gas of rubber tube 2, play the effect of stirring fermentation liquid, make the more even of mixture of fermentation liquid, thereby make the cultivation of bacterial colony more even.

As an embodiment of the invention, a cylinder 30 is fixedly arranged in the bottom wall of the fermentation box 1; a sliding block 29 fixedly arranged at the top end of the air cylinder 30 is connected with a sliding rail at the bottom of the movable plate 27 in a sliding manner; the triangular pyramidal bulge 28 arranged on the upper surface of the movable plate 27 is clamped in the triangular pyramidal clamping groove at the bottom of the rubber cylinder 2; the during operation, when push rod 18 promoted to inflater 16's inboard, the inside of air cavity 21 is got into through the spring pipe to gas this moment, air cavity 21 one side is expanded earlier, thereby make the bottom of rubber tube 2 take place the swing, because the protruding 28 cards of triangular pyramid that the upper surface of fly leaf 27 was equipped with are established in the triangular pyramid draw-in groove of rubber tube 2 bottom, the swing of rubber tube 2 drives the swing back and forth of fly leaf 27, thereby play the effect of vibrating the inside zymotic fluid of rubber tube 2, and then make the mixture of zymotic fluid more even, avoid the bacterial colony to take place the large tracts of land cluster.

As an embodiment of the present invention, the bottom of the cylinder 30 is communicated with the inside of the pump 16 through an air outlet pipe 19; the end of the air outlet pipe 19 connected with the air pump 16 is close to the outer side end of the air pump 16; during operation, when convoluteing rubber cylinder 2 on driven rubber ring 25, the inside gas that is located air cavity 21 this moment will extrude the inside that gets into inflater 16, and then gets into the inside of cylinder 30 through outlet duct 19, and the cylinder 30 moves up and then upwards holds up the bottom of rubber cylinder 2 through fly leaf 27, can reduce the gravity that driven rubber ring 25 bore to the coiling of rubber cylinder 2 of being convenient for more.

The specific working process of the invention is as follows:

when the fermentation box works, the driving rubber ring 10 is firstly turned towards the inner side, at the moment, the magnet group 9 on the driving rubber ring 10 attracts the magnet group 9 on the driven rubber ring 25 and enables the driven rubber ring 25 to be turned towards the outer side, the edge of the rubber cylinder 2 is wound on the driven rubber ring 25, when the driving rubber ring 10 is turned towards the inner side continuously, the driven rubber ring 25 can be turned towards the outer side continuously, the edge of the rubber cylinder 2 is wound continuously, the gas sealing plug positioned in the rubber cylinder 2 is discharged continuously while the bottom of the rubber cylinder 2 moves upwards continuously, at the moment, the sealing plug 7 at the top of the fermentation box 1 is opened, a mixture of corn flour, wheat flour, yeast cream, medium honey and peptone is added at the top feeding port 12, the driving rubber ring 10 is turned towards the outer side continuously while feeding, at the moment, the driven rubber ring 25 is turned towards the inner side under the action of the driving rubber ring 10, thereby releasing the rubber cylinder 2 wound on the driven rubber ring 25, improving the accommodation space of the rubber cylinder 2, adding inert gas after charging, discharging oxygen in the fermentation machine, then connecting a charging pipe on an inert gas tank containing the Aeromonas intermedia with a charging port 12 of the fermentation machine, enabling the Aeromonas intermedia to enter the fermentation machine, adding a proper amount of deionized water, finally plugging a sealing plug 7 to ferment, and observing the liquid level of the rubber cylinder 2 from the annular visual window while charging; when the fermentation is finished and the fermentation broth needs to be taken, the sterilized injection needle tube can be inserted from the feeding port 12 at the top of the fermentation box 1 for direct extraction, if the injection needle tube cannot be in contact with the liquid level in the rubber cylinder 2, the rubber ring 10 can be wound inwards at the moment, so that the driven rubber ring 25 winds the rubber cylinder 2, the liquid level in the rubber cylinder 2 moves upwards, the fermentation broth can be continuously extracted, the use is convenient, the preparation amount of the fermentation broth is controllable, meanwhile, the preparation method can be repeatedly used for preparing the fermentation broth, and meanwhile, the contact chance of the fermentation broth and the air is reduced, and the fermentation broth is prevented from being infected.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.