Feed medium and feeding method for producing D-psicose-3 epimerase

Abstract

The present invention discloses a feed medium and a feeding method for producing D-psicose-3 epimerase, and belongs to the field of microbial fermentation technology. The present invention adopts a specific proportion of glucose, corn steep liquor, soybean cake powder and trace elements to make a feed medium with a certain carbon-nitrogen ratio and a quick-acting nitrogen source, a slow-acting nitrogen source and trace elements, and uses Bacillus subtilis as a fermentation strain. During the fermentation process, feed is added in a fed-batch manner, and pH-dissolved oxygen-feed coupling regulation is simultaneously performed during the fermentation process to increase the cell concentration and the activity of D-psicose-3 epimerase as much as possible. The method of the present invention is simple to operate, reduces production costs, shortens the fermentation cycle, and significantly improves the enzyme activity of the target enzyme.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and more particularly to a feed medium and a feed method for producing D-psicose-3 epimerase. Background Art

[0002] D-psicose is a rare sugar. With approximately 70% the sweetness of sucrose and less than 10% of its energy content, it offers a promising alternative to traditional sugar products for diabetics and obese individuals. Currently, the main technologies for producing D-psicose include extraction, chemical synthesis, and biotransformation. However, the natural abundance of D-psicose is extremely low, making extraction unsuitable for large-scale production. Chemical synthesis is not only costly and yields low yields, but also poses significant environmental risks. Biotransformation, which utilizes naturally occurring monosaccharides or certain byproducts of monosaccharide processing as raw materials, is not only less expensive but also offers far higher conversion efficiency than extraction, making it a key technology for large-scale production of D-psicose.

[0003] In bioconversion, the production of D-psicose via the epimerization of D-fructose at the C-3 position, catalyzed by D-psicose 3-epime-rase (DPE, EC5.3.1.3), offers numerous advantages, such as simplified purification and high product concentration. Existing research focuses on heterologous expression of D-psicose 3-epime-rase through genetic recombination or cloning, as well as cell immobilization. However, current production of D-psicose 3-epime-rase still suffers from low enzyme activity, short half-life, long production times, and high costs.

[0004] Therefore, providing a method for improving the enzymatic activity of D-psicose 3-epimerase is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a feed medium for producing D-psicose-3 epimerase and a feeding method thereof. By adding glucose, corn steep liquor, soybean cake powder and trace elements in different proportions, a feed medium with a certain carbon-nitrogen ratio and simultaneously containing a fast-acting nitrogen source, a slow-acting nitrogen source and trace elements is prepared. Through pH-dissolved oxygen-feeding feedback regulation, the purpose of increasing the strain concentration and the activity of the target protease is achieved.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A feed medium for producing D-psicose-3 epimerase comprises the following raw materials: glucose, corn steep liquor, soybean meal, and trace elements, wherein the trace elements are added in an amount of 0.15-0.5 g / L relative to glucose, the m:v ratio of soybean meal to corn steep liquor is (50-100):1, the carbon-nitrogen ratio of the final mixed feed is (5-10):1, and the pH is 4.06-4.51.

[0008] Glucose, with a purity of 30%-35%, also contains other substances such as maltose and fructose, which can provide a carbon source for fermentation. Corn steep liquor, with its main nutrients including protein, amino acids, reducing sugars, vitamins, and lactic acid, provides a fast-acting nitrogen source and growth factors during fermentation, promoting microbial growth and reproduction. The protein in soybean meal often exists in a large molecular form, which is slowly utilized by microorganisms. It often serves as a slow-acting nitrogen source, favoring the production of metabolites.

[0009] Furthermore, the glucose DS (soluble matter content) is 30-35 and the pH is 4.0-4.5; the corn steep liquor in the workshop has a Baume degree of 17-20, a pH of 4.0-5.0, and a protein content of 150-200 g / L; the total protein content of the soybean cake powder in the workshop is 50-55%; and the trace element is any one of manganese chloride, cobalt chloride and magnesium sulfate.

[0010] As an inventive concept identical to the above-mentioned technical solution, the present invention also seeks to protect a method for preparing a feed medium for producing D-psicose-3 epimerase, the process comprising: (1) first dissolving trace elements in glucose in a proportion, sterilizing at 115°C for 20-25 minutes, to obtain a mixed solution 1; (2) then dissolving soybean cake powder in corn steep liquor in a proportion, sterilizing at 115°C for 20-25 minutes, to obtain a mixed solution 2; (3) combining the above two mixed solutions in a feed bottle under sterile conditions to prepare a feed medium.

[0011] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of feed culture medium in feeding.

[0012] As an inventive concept identical to the above technical solution, the present invention further claims protection for a method for feeding a culture medium for producing D-psicose-3 epimerase, characterized in that a pH-dissolved oxygen-fed-batch coupled fermentation control mechanism is established, and the specific steps are as follows:

[0013] (1) Inoculate the recombinant Bacillus subtilis into LB medium at a 1% inoculum volume, culture at 37°C and 220 rpm for 6-10 h to obtain seed solution, and measure its OD 600nm =2.08-6.04;

[0014] (2) The seed liquid is inoculated into the fermentation medium for fermentation. The initial pH value of the fermentation is 6.02-6.5. In this stage, DO is controlled to make OD 600nm Increase at 1-3 / h, control fermentation parameters: temperature 37℃, ventilation ratio 0.5VVM, tank pressure 0.02 or 0.05Mpa, speed 150 or 200r / min;

[0015] (3) Fermentation to OD 600nm =5.04-14.97, the glucose in the initial batch is exhausted, the dissolved oxygen rises back to above 20.0%, and feeding begins; after feeding, the dissolved oxygen drops below 20%, and the cell enters a rapid growth stage, the pH is controlled to drop at 0.05-0.11 / h, and the feeding flow acceleration is 100-150g / L; the fermentation parameters controlled in this stage are: temperature 37±0.5℃, tank pressure 0.02-0.05Mpa, and DO is controlled at 10-20% by adjusting the rotation speed and air volume; when the dissolved oxygen rises back to above 20.0%, it indicates that the initial glucose is exhausted, which is a sign to start feeding; feeding can also be done when the dissolved oxygen is below 20.0%, but at this time the amount of initial glucose remaining is unknown, making it difficult to control the feeding rate;

[0016] The reason for controlling the pH drop rate is that if the drop rate is too fast, the bacterial OD600nm will grow slowly, and if the drop rate is too slow, the time will be extended. The reason for controlling the pH recovery rate is that if the rise rate is too fast, the enzyme production will be low, and if the rise rate is too slow, the time will be extended.

[0017] It can be explained in principle

[0018] (4) After 15-25 h of fermentation, the pH stopped decreasing and the D-psicose-3 epimerase production phase began. The pH was controlled to rise at a rate of 0.11-0.25 / h, and the feed flow acceleration was 100-170 g / L. The fermentation parameters in this phase were: temperature 37 ± 0.5°C, tank pressure 0.02-0.05 MPa, and DO 10-20% by adjusting the rotation speed and air volume.

[0019] (5) After 38-45h of fermentation, pH returned to 8.02-8.51, OD 600nm =151.4-197.4, stop feeding;

[0020] (6) After 45-50 h of fermentation, when the dissolved oxygen content rises to above 40% and the extracellular enzyme activity reaches 801.2-1002.3 U / mL by HPLC, the fermentation is terminated. When the feeding is stopped, the bacterial cells are broken and a large amount of enzyme is released. When the dissolved oxygen content rises to above 40%, the extracellular enzyme activity reaches the maximum value, which is the time point for the end of fermentation.

[0021] (7) The fermentation broth is centrifuged to obtain crude enzyme solution.

[0022] Studies have shown that low DO values ​​affect bacterial metabolism, and although too high DO values ​​can make the bacteria grow rapidly, they can easily lead to bacterial aging and reduced yields. Bacillus subtilis is an aerobic bacterium, and controlling the DO value in the fermentation process plays an important role in bacterial fermentation and enzyme production; pH can affect the permeability and metabolism of bacterial cell membranes, thereby affecting the fermentation process; if the feeding time is too late, the accumulation of adverse products during the fermentation process will affect the absorption of the feed by the bacteria to a certain extent. The present invention uses pH-dissolved oxygen-feeding feedback regulation to adjust the DO value in the rapid growth stage of the bacteria and the metabolic enzyme production stage respectively. 600nm The growth and metabolism of the bacteria can be monitored by adjusting the change rate of DO and feed flow, as well as fermentation conditions such as temperature, in order to increase the concentration of the bacteria and the activity of the target protease.

[0023] As a preferred technical solution of the above technical solution, the recombinant Bacillus subtilis in step (1) is obtained by constructing a recombinant plasmid with the DPE gene derived from (Clostridium scindens ATCC35704; the recombinant bacteria were constructed by Tianjin Industrial Biotechnology Institute), and then transferring the recombinant plasmid into Bacillus subtilis competent cells.

[0024] Bacillus subtilis is a Gram-positive bacterium and a traditional industrial production bacterium. The protein it produces can be released into the culture medium in large quantities, significantly reducing downstream protein purification processes and significantly lowering target protein production costs. Furthermore, Bacillus subtilis has a simple cell wall structure and is endotoxin-free, earning it a GRAS (Generally Recognized As Safe) designation from the US FDA. Therefore, using Bacillus subtilis for the production of D-psicose 3-epimerase offers significant advantages over Escherichia coli, the common host for DPEase enzymes.

[0025] As a preferred technical solution of the above technical solution, the preparation process of the fermentation medium in step (2) is: 100g of potassium dihydrogen phosphate, 80g of ammonium chloride, 50g of disodium hydrogen phosphate, 100g of workshop glucose, 2ml of polyoxypropylene glycerol ether and 9L of water are mixed and added to a 15L fermenter, sterilized at 115°C for 20-30min, and the sterilized fermenter is cooled to 37°C with circulating water to obtain a fermentation medium.

[0026] In summary, the technical effects achieved by the present invention are:

[0027] 1) The present invention achieves an extracellular enzyme activity of 800-1000 u / ml within 50 hours, overcoming the shortcomings of low enzyme activity (approximately 100 u / ml) and long production time commonly seen in the current production of D-psicose 3-epimerase using Bacillus subtilis as a host. Furthermore, the present invention uses a GRAS (Grade A Listerial System) host to produce D-psicose 3-epimerase, meeting national food safety requirements.

[0028] 2) The mixed feed prepared by the present invention not only provides sufficient carbon source, fast-acting nitrogen source, slow-acting nitrogen source, trace elements, and growth factors for the production of D-psicose 3-epimerase by recombinant Bacillus subtilis, but also the fermentation medium, which uses glucose, corn steep liquor, and soybean cake powder as raw materials for the mixed feed, is easy to obtain and inexpensive. Compared with other inventions that use yeast powder, peptone, etc. as fermentation medium, it has a greater cost advantage.

[0029] 3) The present invention adopts a feeding method to feed the bacteria, and combines the pH-dissolved oxygen-feeding coupling control during the fermentation process to invent a feeding method suitable for the high-density growth of Bacillus subtilis, which greatly increases the bacterial concentration, shortens the fermentation cycle and improves the activity of the target enzyme. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] In the embodiment, the glucose DS is 30-35%, and the pH is 4.0-4.5; the corn steep liquor has a Baume degree of 17-20, a pH of 4.0-5.0, and a protein content of 150-200 g / L; and the soybean meal has a total protein content of 50-55%.

[0032] Example 1

[0033] Seed liquid culture: Prepare seed culture medium (30 ml / 150 ml shake flask, 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, sterilized at 121 ° C for 30 min), inoculate recombinant Bacillus subtilis at a 1% inoculum size, culture at 37 ° C, 220 r / min for 6-10 h, obtain seed liquid, and measure its OD 600nm =2-6.

[0034] Example 2

[0035] Prepare fermentation medium: mix 100 g of potassium dihydrogen phosphate, 80 g of ammonium chloride, 50 g of disodium hydrogen phosphate, 100 g of glucose, 2 ml of polyoxypropylene glycerol ether and 9 L of water and add the mixture to a 15 L fermenter, sterilize at 115° C. for 20-30 min, and cool the sterilized fermenter to 37° C. with circulating water to obtain fermentation medium.

[0036] Fermentation with the recombinant Bacillus subtilis seed solution of Example 1 and the fermentation medium of Example 2 to produce D-psicose 3-epimerase, as in Examples 3-6;

[0037] Example 3

[0038] Prepare mixed feed: dissolve 0.6 g manganese chloride in 4 L glucose and sterilize at 115°C for 20 min; dissolve 50 g soybean cake powder in 1 L corn steep liquor and sterilize at 115°C for 20 min; aseptically combine the two in a 5 L feed bottle to prepare a feed with a carbon-nitrogen ratio of 6:1, pH = 4.06, and a ratio of fast-acting nitrogen source to slow-acting nitrogen source of 7:1.

[0039] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =4.82 seed solution, initial fermentation temperature 37 ° C, pH 6.02, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 5h, OD 600nm =14.97, DO rose to 25.5%, feeding was added, pH dropped at 0.07 / h, and the flow acceleration was controlled at 120g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 20h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.11 / h, and the feeding rate was controlled at 120g / h; after 42h of fermentation, OD 600nm =162.2, pH returned to 8.02, and feeding was stopped; fermentation lasted for 45 h, dissolved oxygen returned to 41%, and HPLC detection of extracellular enzyme activity reached 1002.3 U / ML, ending the fermentation; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0040] Example 4

[0041] Prepare mixed feed: dissolve 1.37g of cobalt chloride in 4.2L of glucose and sterilize at 115℃ for 25min; dissolve 50g of soybean cake powder in 0.8L of corn steep liquor and sterilize at 115℃ for 25min; under sterile conditions, combine the two in a 5L feeding bottle with a carbon-nitrogen ratio of 8.7:1, pH = 4.41, and fast-acting nitrogen source: slow-acting nitrogen source = 5.4:1.

[0042] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm=2.08 seed solution, initial fermentation temperature 37 ° C, pH 6.24, ventilation ratio 0.5VVM, tank pressure 0.05Mpa, speed 150r / min; fermentation 6h, OD 600nm =12.37, DO rose to 26.2%, feeding was added, pH dropped at 0.11 / h, and the flow acceleration was controlled at 100g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 15h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.18 / h, and the feeding rate was controlled at 100g / h; after 45h of fermentation, OD 600nm =151.4, pH returned to 8.39, and feeding was stopped; fermentation lasted for 48.5 h, dissolved oxygen returned to 40.4%, and HPLC detection of extracellular enzyme activity reached 901.8 U / ML, ending the fermentation; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0043] Example 5

[0044] Prepare mixed feed: dissolve 1.5 g magnesium sulfate in 4.2 L glucose and sterilize at 115°C for 20 min; dissolve 50 g soybean cake powder in 0.8 L corn steep liquor and sterilize at 115°C for 20 min; aseptically combine the two in a 5 L feeding bottle with a carbon-nitrogen ratio of 10:1, pH = 4.51, and a ratio of fast-acting nitrogen source to slow-acting nitrogen source of 4.4:1.

[0045] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =6.04 seed liquid, initial fermentation temperature 37 ° C, pH 6.5, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 6.5h, OD 600nm =12.97, DO rose to 22.3%, feeding was added, pH dropped at 0.05 / h, and the flow acceleration was controlled at 150g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 25h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.25 / h, and the feeding rate was controlled at 150g / h; after 38h of fermentation, OD 600nm =184.2, pH returned to 8.51, and feeding was stopped; after fermentation for 50 h, dissolved oxygen returned to 41.3%, and HPLC detection of extracellular enzyme activity reached 801.2 U / ML, and fermentation was terminated; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0046] Example 6

[0047] Prepare mixed feed: dissolve 1.0 g manganese chloride in 4.3 L glucose and sterilize at 115°C for 20 min; dissolve 50 g soybean cake powder in 0.7 L corn steep liquor and sterilize at 115°C for 20 min; aseptically combine the two in a 5 L feeding bottle with a carbon-nitrogen ratio of 8:1, pH = 4.27, and a ratio of fast-acting nitrogen source to slow-acting nitrogen source of 5:1.

[0048] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =5.17 seed liquid, fermentation initial temperature 37 ° C, pH 6.45, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 5.5h, OD 600nm =5.04, DO rose to 27.9%, feeding was added, pH dropped at 0.08 / h, and the flow acceleration was controlled at 130g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 22h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.22 / h, and the feeding rate was controlled at 170g / h; after 43h of fermentation, OD 600nm =197.4, pH returned to 8.49, and feeding was stopped; after fermentation for 47 h, dissolved oxygen returned to 40.6%, and HPLC detection of extracellular enzyme activity reached 952.3 U / ML, and fermentation was terminated; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0049] Comparative Example 1

[0050] The composition and proportion of the feed medium are: 17% ammonia solution, pH 13.0, and a volume of 5L.

[0051] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =4.82 seed liquid, initial fermentation temperature 37 ° C, pH 6.32, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 6.5h, OD 600nm =13.2, DO rose to 21.6%, feeding was added, pH dropped at 0.07 / h, and the flow acceleration was controlled at 120g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 27h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.14 / h, and the feeding rate was controlled at 120g / h; after 53h of fermentation, OD 600nm =126.5, pH returned to 8.35, and feeding was stopped; fermentation lasted for 58 h, dissolved oxygen returned to 41%, and HPLC detection of extracellular enzyme activity reached 462.4 U / ML, and fermentation was terminated; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0052] Comparative Example 2

[0053] The composition and proportion of the feed medium were as follows: 103 yeast peptone at a concentration of 100 g / L, a pH of 5.75, and a volume of 5 L.

[0054] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =2.08 seed solution, initial fermentation temperature 37 ° C, pH 6.44, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 6h, OD 600nm =12.37, DO rose to 24.2%, feeding was added, pH dropped at 0.11 / h, and the flow acceleration was controlled at 80g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 28h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.18 / h, and the feeding rate was controlled at 120g / h; after 52h of fermentation, OD 600nm =113.2, pH returned to 8.39, and feeding was stopped; after fermentation for 60 h, dissolved oxygen returned to 40.4%, and HPLC detection of extracellular enzyme activity reached 398.6 U / ML, and fermentation was terminated; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0055] Comparative Example 3

[0056] The composition and proportion of the feed medium are: glucose solution with a concentration of 300 g / L, a pH of 4.70, and a volume of 5 L.

[0057] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =6.04 seed liquid, initial fermentation temperature 37 ° C, pH 6.37, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 6.5h, OD 600nm =12.97, DO returned to 22.3%, fed feed was added, and pH decreased at a rate of 0.04 / h, with the flow rate controlled at 160 g / h. After feeding, DO dropped below 20%, and the rotational speed and ventilation ratio were adjusted to maintain DO between 10 and 20%. After 41 hours of fermentation, pH rebounded, and the enzyme production phase began. pH rebounded at a rate of 0.26 / h, with the feed rate controlled at 160 g / h. After 55 hours of fermentation, OD600nm = 152.5, and pH returned to 8.71, fed feed was stopped. After 66 hours of fermentation, dissolved oxygen returned to 41.3%, and extracellular enzyme activity reached 376.4 U / mL by HPLC, and fermentation was terminated. The fermentation broth was centrifuged to obtain a crude enzyme solution.

[0058] Comparative Example 4

[0059] The composition and proportion of the feed medium are: 100 g / L glycerol solution, pH 6.70, and a volume of 5 L.

[0060] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =5.17 seed liquid, fermentation initial temperature 37 ° C, pH 6.45, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 5.5h, OD 600nm =14.1, DO returned to 20.9%, fed feed was added, and pH decreased at a rate of 0.08 / h, with the flow rate controlled at 130 g / h. After feeding, DO dropped below 20%, and the rotational speed and ventilation ratio were adjusted to maintain DO between 10 and 20%. After 36 hours of fermentation, pH rebounded, and the enzyme production phase began. pH rebounded at a rate of 0.28 / h, with the feed rate controlled at 170 g / h. After 52 hours of fermentation, OD600nm = 139.8, and pH returned to 8.49, and fed feed was stopped. After 63 hours of fermentation, dissolved oxygen returned to 40.6%, and extracellular enzyme activity measured by HPLC reached 352.2 U / mL, terminating the fermentation. The fermentation broth was centrifuged to obtain a crude enzyme solution.

[0061] Comparative Example 5

[0062] Prepare mixed feed: dissolve 1.0 g manganese chloride in 4.3 L glucose and sterilize at 115°C for 20 min; dissolve 50 g soybean cake powder in 0.7 L corn steep liquor and sterilize at 115°C for 20 min; aseptically combine the two in a 5 L feeding bottle with a carbon-nitrogen ratio of 8:1, pH = 4.27, and a ratio of fast-acting nitrogen source to slow-acting nitrogen source of 5:1.

[0063] Establishing pH-dissolved oxygen-fed-batch coupled fermentation control mechanism: inoculation OD 600nm =5.17 seed liquid, fermentation initial temperature 37 ° C, pH 6.45, ventilation ratio 0.5VVM, tank pressure 0.02Mpa, speed 200r / min; fermentation 5.5h, OD 600nm =5.04, DO rose to 27.9%, feeding was added, pH dropped at 0.08 / h, and the flow acceleration was controlled at 130g / h; after feeding, DO dropped to below 20%, and the speed and ventilation ratio were adjusted to maintain DO at 10-20%; after 22h of fermentation, pH rose, and the enzyme production stage began, pH rose at 0.22 / h, and the feeding rate was controlled at 130g / h; after 43h of fermentation, OD 600nm =188.2, pH returned to 8.49, and feeding was stopped; after fermentation for 47 h, dissolved oxygen returned to 41.3%, and HPLC detection of extracellular enzyme activity reached 744.6 U / ML, and fermentation was terminated; the fermentation broth was centrifuged to obtain crude enzyme solution.

[0064] Table 1 OD of seed solution in Examples 3-6 and Comparative Examples 1-5 600nm Value, OD when stopping feeding 600nmValue, extracellular enzyme activity and fermentation time

[0065]

[0066]

[0067] As shown in Table 1, the feed medium and feed method for producing D-psicose-3 epimerase provided by the present invention can increase the OD of the seed solution. 600 nm value, the extracellular enzyme activity was increased to more than 800U / ML, and the fermentation time was controlled within 50h; From Comparative Example 5, it can be seen that if the pH change rate and feeding rate are not controlled in stages, the carbon and nitrogen sources will be insufficient in the late enzyme production stage, even if OD 600 When the nm value reaches 151.4 or above, the enzyme activity will be lower than that in the embodiment.

[0068] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0069] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for feeding a feed medium for producing D-psicose-3 epimerase, characterized in that: Establish a pH-dissolved oxygen-fed-batch coupled fermentation control mechanism. The specific steps are as follows: (1) Inoculate the recombinant Bacillus subtilis into LB medium at a 1% inoculum volume, culture at 37°C and 220 rpm for 6-10 h to obtain seed solution, and measure its OD 600nm =2.08-6.04; (2) The seed liquid is inoculated into the fermentation medium for fermentation. The initial pH value of the fermentation is 6.02-6.

5. In this stage, DO is controlled to make OD 600nm Increase at 1-3 / h, control fermentation parameters: temperature 37℃, ventilation ratio 0.5VVM, tank pressure 0.02 or 0.05Mpa, speed 150 or 200r / min; (3) Fermentation to OD 600nm =5.04-14.97, the glucose in the initial batch is exhausted, the dissolved oxygen rises to above 20.0%, and feeding begins; after feeding, the dissolved oxygen drops below 20%, and the cell enters a rapid growth phase. The pH is controlled to decrease at 0.05-0.11 / h, and the feed flow acceleration is 100-150g / L. The fermentation parameters controlled in this stage are: temperature 37±0.5℃, tank pressure 0.02-0.05MPa, and DO is controlled at 10-20% by adjusting the rotation speed and air volume; (4) After 15-25 h of fermentation, the pH stopped decreasing and the D-psicose-3 epimerase production phase began. The pH was controlled to rise at a rate of 0.11-0.25 / h, and the feed flow acceleration was 100-170 g / L. The fermentation parameters in this phase were: temperature 37 ± 0.5°C, tank pressure 0.02-0.05 MPa, and DO was controlled at 10-20% by adjusting the rotation speed and air volume. (5) After 38-45h of fermentation, pH returned to 8.02-8.51, OD 600nm =151.4-197.4, stop feeding; (6) After 45-50 h of fermentation, the dissolved oxygen content returned to above 40%, and the extracellular enzyme activity detected by HPLC reached 801.2-1002.3 U / mL, and the fermentation was terminated; (7) centrifuging the fermentation broth to obtain a crude enzyme solution; The feed medium for producing D-psicose-3 epimerase is composed of the following raw materials: glucose, corn steep liquor, soybean meal, and trace elements, wherein the trace elements are added in an amount of 0.15-0.5 g / L relative to glucose in the workshop, the m:v ratio of soybean meal to corn steep liquor is (50-100):1, the final mixed feed carbon-nitrogen ratio is (5-10):1, and the pH is 4.06-4.

51.

2. The method for feeding a feed medium for producing D-psicose-3 epimerase according to claim 1, wherein: The glucose DS is 30-35% and the pH is 4.0-4.5; the corn steep liquor has a Baume degree of 17-20, a pH of 4.0-5.0, and a protein content of 150-200 g / L; the soybean meal has a total protein content of 50-55%; and the trace element is any one of manganese chloride, cobalt chloride, and magnesium sulfate.

3. The method for feeding a feed medium for producing D-psicose-3 epimerase according to any one of claims 1 to 2, wherein: The specific preparation steps of the feed medium are as follows: (1) Dissolve the trace elements in glucose in a certain proportion, sterilize at 115°C for 20-25 minutes, and obtain mixed solution 1; (2) Dissolve the soybean cake powder in corn steep liquor in a certain proportion, sterilize at 115°C for 20-25 minutes, and obtain mixed solution 2; (3) Under sterile conditions, combine the above two mixed solutions and put them into the feeding bottle to prepare the feeding medium.

4. The method for feeding a feed medium for producing D-psicose-3 epimerase according to claim 1, wherein: The recombinant Bacillus subtilis in step (1) is obtained by transferring the DPE gene into Bacillus subtilis competent cells.

5. The method for feeding a feed medium for producing D-psicose-3 epimerase according to claim 1, wherein: The preparation process of the fermentation medium in step (2) is as follows: 100g of potassium dihydrogen phosphate, 80g of ammonium chloride, 50g of sodium dihydrogen phosphate, 100g of glucose, 2ml of polyoxypropylene glycerol ether and 9L of water are mixed and added to a 15L fermenter, sterilized at 115°C for 20-30min, and the sterilized fermenter is cooled to 37°C with circulating water to obtain the fermentation medium.

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