Compound enzyme fermentation process for improving yield of trehalose

Abstract

The invention belongs to the technical field of fermentation engineering, and particularly relates to a compound enzyme fermentation process for increasing the yield of trehalose. The process comprises the following steps: (1) respectively preparing a seed solution A, a seed solution B and a seed solution C; (2) simultaneously inoculating the seed solution A, the seed solution B and the seed solution C into a fermentation tank filled with a TB culture medium for culturing, and adding isopropyl-beta-D-thiogalactoside into the fermentation tank for induction when OD600 is measured to be 0.8-1.0, so as to obtain fermentation liquor; (3) adding colistin sulfate into the fermentation liquor, continuously culturing, centrifuging and discarding supernate after the culture is finished, centrifuging and discarding supernate after the treatment is finished, washing with a phosphate buffer solution, and resuspending to obtain a cell suspension; and (4) mixing the cell suspension with a maltose aqueous solution, carrying out a reaction, terminating the reaction after the reaction is finished, carrying out cooling, carrying out centrifugation, and taking a supernatant so as to obtain a trehalose-containing product. The trehalose obtained by the process is high in conversion rate and excellent in enzyme activity and repeated utilization rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation engineering, and particularly relates to a composite enzyme fermentation process for increasing the production of trehalose. Background Art

[0002] Trehalose is a functional sugar with stable properties and no reducing property. Due to its ability to protect bioactive substances, it has special functions and is widely used in many fields. However, there are some limitations in traditional trehalose production methods: the direct extraction method has a low yield, which not only has high production costs but also may cause certain pollution to the environment; although the enzyme synthesis method is feasible, the enzyme reagents are expensive, increasing the production burden. In recent years, the microbial fermentation method has gradually attracted attention and is regarded as a more potential way to produce trehalose.

[0003] Currently, most of the industrial production process routes of trehalose are to first ferment with engineering bacteria. After the cells are broken, the trehalose synthase system is separated and purified, and finally catalyzed with the substrate to produce trehalose. However, physical cell disruption methods are mostly used for cell disruption, and large-scale use will inactivate the enzymes, resulting in generally low enzyme use efficiency. Moreover, after cell disruption, intracellular complex components will be introduced, increasing the pressure of separation and purification, increasing the industrial cost of separation, and the raw material cost of fermentation is relatively high, further increasing the production cost.

[0004] Chinese Patent No. 201610413674.3 discloses a method for industrial production of trehalose by microbial fermentation, including the following steps: fermentation of trehalose hydrolase and trehalose-forming enzyme (X / Y), starch liquefaction, catalytic reaction, saccharification reaction, decolorization filtration, ion exchange, concentration, crystallization, centrifugation, drying, and membrane separation. This method breaks the bacterial cell wall of the fermentation broth through a high-pressure homogenizer twice, and the subsequent treatment steps are cumbersome and costly.

[0005] Duan Yingying et al. (Screening of trehalose synthase-producing strains and optimization of fermentation conditions) screened trehalose-producing strains from soil classification. After optimizing the fermentation conditions, the conversion rate of maltose to trehalose was 49.8%. Yang Meiyu et al. (Optimization of fermentation conditions for Escherichia coli to produce trehalose synthase) induced Escherichia coli to produce trehalose synthase, and the yield was 69.29%. However, there are still problems in the above methods, such as low conversion rate, low enzyme activity, and low reuse rate.

[0006] Therefore, there is an urgent need for a composite enzyme fermentation process for increasing the production of trehalose. Summary of the Invention

[0007] The purpose of the present invention is to provide a composite enzyme fermentation process for increasing the production of trehalose.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A composite enzyme fermentation process for increasing trehalose production, comprising the following steps:

[0010] (1) Inoculate Escherichia coli into LB medium and culture at 35 - 37 °C for 16 - 18 h to obtain seed liquid A; inoculate Bacillus subtilis into LB medium and culture at 35 - 37 °C for 10 - 12 h to obtain seed liquid B; inoculate yeast into LB medium and culture at 35 - 37 °C for 8 - 10 h to obtain seed liquid C;

[0011] (2) Inoculate seed liquid A, seed liquid B and seed liquid C into a fermenter containing TB medium for culture simultaneously. When the measured OD 600 is 0.8 - 1.0, add isopropyl-β-D-thiogalactoside to the fermenter for induction to obtain a fermentation broth;

[0012] (3) Add colistin sulfate to the fermentation broth and continue culturing. After culturing is completed, centrifuge to discard the supernatant, and after the treatment is completed, centrifuge to discard the supernatant, wash with phosphate buffer solution, and resuspend to obtain a cell suspension;

[0013] (4) Mix the cell suspension with a maltose aqueous solution and react at 25 - 27 °C and 220 - 250 r / min for 15 - 17 h. After the reaction is completed, terminate the reaction, cool, centrifuge, and take the supernatant to obtain a product containing trehalose.

[0014] In the present invention, by adding Bacillus subtilis to Escherichia coli, the conversion rate of trehalose can be improved. The compound synergistic effect of Bacillus subtilis and Escherichia coli can improve the activity of trehalose synthase in the fermentation broth. During the fermentation process, Bacillus subtilis can secrete various enzymes and produce intermediate metabolites beneficial to trehalose synthesis. These products can be directly utilized by Escherichia coli to promote the synthesis of trehalose. Bacillus subtilis can assist Escherichia coli in producing multiple trehalose synthases.

[0015] Further, in the step (3), add colistin sulfate to the fermentation broth to 1.3 - 1.4 g / L, continue culturing for 70 - 80 min, and the temperature is 32 - 34 °C.

[0016] The conditions of adding colistin sulfate for culturing in the present invention can improve the activity of trehalose synthase and improve the conversion rate of trehalose.

[0017] Further, the LB medium in the step (1) comprises the following components: peptone 10 - 12 g / L, yeast extract 1 - 3 g / L, NaCl 12 - 15 g / L; adjust the pH value to 7.3 - 7.6, and make up the balance with water.

[0018] Further, the TB medium in step (2) comprises the following components: peptone 10-12 g / L, yeast extract 20-24 g / L, glycerol 4-5 mL / L, potassium dihydrogen phosphate 2.3-2.4 g / L, and dipotassium hydrogen phosphate 0.2-0.4 g / L, and the balance is made up with water.

[0019] Further, the inoculation amounts (volume percentages) of seed liquid A, seed liquid B, and seed liquid C in the TB medium are 0.7%, 0.2%, and 0.1% respectively.

[0020] By compounding Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae for fermentation, the present invention can optimize the fermentation system, reduce side reactions, improve the reuse rate of the process. After 20 batches (12 h / batch) of reuse, the conversion rate can be maintained above 67%.

[0021] Further, the culture conditions in step (2) are: 35-37 °C, 220-260 rpm, and the ventilation volume is 7.2-7.4 L / min.

[0022] Further, the added concentration of isopropyl-β-D-thiogalactoside in step (2) is 0.8-1.0 mmol / L.

[0023] Further, the induction time in step (2) is 8-10 h.

[0024] Further, in step (3), for resuspension, the concentration of the fixed cell bacterial suspension is 10-12 wt%, and a cell suspension is obtained.

[0025] Further, the concentration of the maltose aqueous solution in step (4) is 30-32% (W / V).

[0026] Further, the volume ratio of the cell suspension to the maltose aqueous solution in step (4) is (3-4):10.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0028] 1. By adding Bacillus subtilis to Escherichia coli, the present invention can improve the conversion rate of trehalose. Bacillus subtilis and Escherichia coli have a synergistic effect and can improve the activity of trehalose synthase in the fermentation broth.

[0029] 2. By compounding Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae for fermentation, the present invention can optimize the fermentation system, reduce side reactions, and improve the reuse rate of the process.

[0030] 3. The conditions of adding polymyxin sulfate for culture in the present invention can improve the activity of trehalose synthase and the conversion rate of trehalose. Detailed implementation mode

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] All raw materials used in the following embodiments of the present invention are commercially available products:

[0033] Escherichia coli, preservation number CGMCC 4.1174, from the China General Microbiological Culture Collection Center.

[0034] Bacillus subtilis, preservation number CCTCC KB 20081355, purchased from the China Center for Type Culture Collection.

[0035] Yeast, preservation number CCTCC NY 20083094, purchased from the China Center for Type Culture Collection.

[0036] Isopropyl-β-D-thiogalactoside, Zhangjiajie Hengkang Biopharmaceutical Co., Ltd.

[0037] Colistin sulfate, Hunan Yunbang Biotechnology Co., Ltd.

[0038] Example 1

[0039] This example provides a composite enzyme fermentation process for improving the production of trehalose, including the following steps:

[0040] (1) Inoculate Escherichia coli in LB medium and culture at 36°C for 17 h to obtain seed liquid A; inoculate Bacillus subtilis in LB medium and culture at 36°C for 11 h to obtain seed liquid B; inoculate yeast in LB medium and culture at 36°C for 9 h to obtain seed liquid C; the LB medium includes the following components: peptone 11 g / L, yeast powder 2.4 g / L, NaCl 13 g / L; adjust the pH value to 7.5 and make up the balance with water.

[0041] (2) Inoculate seed liquid A, seed liquid B and seed liquid C into a fermenter filled with TB medium for culture at the same time. The inoculation amounts (volume percentages) of seed liquid A, seed liquid B and seed liquid C in the TB medium are 0.7%, 0.2% and 0.1% respectively; carry out fermentation culture; the culture conditions are: 36°C, 240 rpm, aeration rate 7.3 L / min. When the measured OD 600When it was 0.9, isopropyl-β-D-thiogalactoside was added to the fermenter to a concentration of 0.9 mmol / L for induction, and the induction time was 9 h to obtain a fermentation broth; the TB medium included the following components: peptone 11 g / L, yeast extract 22 g / L, glycerol 4.5 mL / L, potassium dihydrogen phosphate 2.3 g / L, and dipotassium hydrogen phosphate 0.3 g / L, and the balance was made up with water.

[0042] (3) Colistin sulfate was added to the fermentation broth to 1.35 g / L, and the culture was continued. After the culture was completed, the supernatant was discarded by centrifugation, and the continued culture time was 75 min, and the temperature was 33 °C; after the treatment was completed, it was centrifuged at 6500 r / min for 10 min, and then the supernatant was discarded, washed twice with phosphate buffer, and resuspended. The fixed cell suspension concentration was 10 wt% to obtain a cell suspension;

[0043] (4) A cell suspension with a volume ratio of 3:10 was mixed with a 30% (W / V) maltose aqueous solution, and reacted at 26 °C and 230 r / min for 16 h. After the reaction was completed, the reaction was terminated by heating in a boiling water bath for 10 min. After cooling, it was centrifuged at 5300 r / min for 10 min, and the supernatant was taken to obtain a product containing trehalose.

[0044] Example 2

[0045] This example provides a composite enzyme fermentation process for increasing the yield of trehalose, including the following steps:

[0046] (1) Escherichia coli was inoculated into LB medium and cultured at 37 °C for 18 h to obtain seed liquid A; Bacillus subtilis was inoculated into LB medium and cultured at 37 °C for 10 h to obtain seed liquid B; Saccharomyces cerevisiae was inoculated into LB medium and cultured at 37 °C for 10 h to obtain seed liquid C; the LB medium included the following components: peptone 12 g / L, yeast powder 1 g / L, NaCl 15 g / L; the pH value was adjusted to 7.3, and the balance was made up with water.

[0047] (2) Seed liquid A, seed liquid B, and seed liquid C were simultaneously inoculated into a fermenter filled with TB medium for culture. The inoculation amounts (volume percentages) of seed liquid A, seed liquid B, and seed liquid C in the TB medium were 0.7%, 0.2%, and 0.1% respectively; fermentation culture; the culture conditions were: 37 °C, 220 rpm, aeration rate 7.2 L / min. When the OD was measured 600When it is 1.0, isopropyl-β-D-thiogalactoside is added to the fermenter to a concentration of 0.8 mmol / L for induction, and the induction time is 10 h to obtain a fermentation broth; the TB medium comprises the following components: peptone 10 g / L, yeast extract 24 g / L, glycerol 4 mL / L, potassium dihydrogen phosphate 2.4 g / L, and dipotassium hydrogen phosphate 0.4 g / L, and the balance is made up with water.

[0048] (3) Colistin sulfate is added to the fermentation broth to a concentration of 1.4 g / L, and the cultivation is continued. After the cultivation is completed, the supernatant is discarded by centrifugation, and the continued cultivation time is 70 min, and the temperature is 34 °C; after the treatment is completed, it is centrifuged at 6500 r / min for 10 min, and then the supernatant is discarded, washed twice with phosphate buffer solution, and resuspended. The concentration of the fixed cell suspension is 10 wt% to obtain a cell suspension;

[0049] (4) A cell suspension with a volume ratio of 3:10 is mixed with a 30% (W / V) maltose aqueous solution, and the reaction is carried out at 27 °C and 220 r / min for 17 h. After the reaction is completed, the reaction is terminated by heating in a boiling water bath for 10 min. After cooling, it is centrifuged at 5500 r / min for 10 min, and the supernatant is taken to obtain a product containing trehalose.

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 1 is that Bacillus subtilis and yeast are not used. Escherichia coli is inoculated into LB medium for cultivation, and cultivated at 36 °C for 17 h to obtain seed liquid A; the seed liquid A is inoculated into a fermenter filled with TB medium for cultivation, and the inoculation amount (volume percentage) of the seed liquid A in the TB medium is 1%.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 1 is that yeast is not used.

[0054] Escherichia coli is inoculated into LB medium for cultivation, and cultivated at 36 °C for 17 h to obtain seed liquid A; Bacillus subtilis is inoculated into LB medium for cultivation, and cultivated at 36 °C for 11 h to obtain seed liquid B. The seed liquid A and the seed liquid B are simultaneously inoculated into a fermenter filled with TB medium for cultivation, and the inoculation amounts (volume percentages) of the seed liquid A and the seed liquid B in the TB medium are 0.8% and 0.2% respectively.

[0055] Comparative Example 3

[0056] The difference between this comparative example and Example 1 is that Bacillus subtilis is not used.

[0057] Escherichia coli was inoculated into LB medium and cultured at 36 °C for 17 h to obtain seed liquid A; Saccharomyces cerevisiae was inoculated into LB medium and cultured at 36 °C for 9 h to obtain seed liquid C. Seed liquid A and seed liquid C were simultaneously inoculated into a fermenter containing TB medium for culture. The inoculation amounts (volume percentages) of seed liquid A and seed liquid C in TB medium were 0.9% and 0.1% respectively.

[0058] Comparative Example 4

[0059] The difference between this comparative example and Example 1 is as follows: The process in Step 3 is different: Specifically, colistin sulfate was added to the fermentation broth to a concentration of 1.5 g / L, and the continued culture time was 50 min, and the temperature was 33 °C.

[0060] Comparative Example 5

[0061] The difference between this comparative example and Example 1 is as follows: The process in Step 3 is different: Specifically, colistin sulfate was added to the fermentation broth to a concentration of 1.35 g / L, and the continued culture time was 75 min, and the temperature was 33 °C.

[0062] Comparative Example 6

[0063] The difference between this comparative example and Example 1 is as follows: The process in Step 3 is different: Specifically, colistin sulfate was added to the fermentation broth to a concentration of 1.35 g / L, and the continued culture time was 75 min, and the temperature was 30 °C.

[0064] Performance Test

[0065] The following indexes of the fermentation processes of Examples 1-2 and Comparative Examples 1-6 were measured:

[0066] 1. Determination of the initial trehalose conversion rate

[0067] The determination was carried out with reference to the method for determining the trehalose conversion rate in "Optimization of Fermentation Conditions for Escherichia coli to Produce Trehalose Synthase" by Yang Meiyu et al.

[0068] 2. Enzyme activity: The enzyme activity of trehalase in the cell suspension was determined by the 3,5-dinitrosalicylic acid (DNS) colorimetric method.

[0069] 3. Reusability: The operation of the immobilized enzyme preparation was carried out by the adsorption method. The bacteria and enzymes were adsorbed on the surface of the adsorption carrier by physical adsorption. The adsorption carrier used was sodium alginate. The activated enzyme was fully mixed with the adsorption carrier and then made into gel beads. After one batch was completed, the fermentation was continued in the original fermenter and reused 20 batches (12 h / batch), and the trehalose conversion rate of the last batch was statistically analyzed.

[0070] Table 1 Performance Test Results

[0071] Trehalose conversion rate % Enzyme activity U / mL Reusability rate % Example 1 72.15 285 67.24 Example 2 71.63 281 67.01 Comparative Example 1 62.34 236 60.25 Comparative Example 2 64.57 251 61.81 Comparative Example 3 65.91 249 62.39 Comparative Example 4 66.42 263 63.62 Comparative Example 5 67.08 268 64.48 Comparative Example 6 65.23 254 62.54

[0072] As can be seen from Table 1, the trehalose conversion rate of the fermentation processes in Examples 1-2 is high, and the enzyme activity and reusability are high.

[0073] In Comparative Example 1, only Escherichia coli was used for fermentation. It can be seen that the trehalose conversion rate, enzyme activity, and reusability decreased significantly, indicating that Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae have a synergistic effect and can jointly optimize the fermentation system, resulting in a higher yield of trehalose synthase.

[0074] In Comparative Example 2, Saccharomyces cerevisiae was not used. From the results, it can be seen that the trehalose conversion rate, enzyme activity, and reusability decreased significantly, indicating that Saccharomyces cerevisiae plays a synergistic role in the system, which can reduce the occurrence of by-products and optimize the system.

[0075] In Comparative Example 3, Bacillus subtilis was not used. From the results, it can be seen that the trehalose conversion rate, enzyme activity, and reusability decreased significantly. It is analyzed that during the fermentation process, Bacillus subtilis can secrete various enzymes and produce some intermediate metabolites beneficial to trehalose synthesis. These products can be directly utilized by Escherichia coli to promote the synthesis of trehalose. Bacillus subtilis can assist Escherichia coli in producing more trehalose synthase.

[0076] In Comparative Examples 4-6, the conditions for culturing with polymyxin sulfate were different, indicating that the system after fermentation of the present invention is subjected to permeabilization treatment, and ideal results can be obtained only under specific conditions.

[0077] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composite enzyme fermentation process for increasing trehalose production, characterized in that: The following steps are involved: (1) inoculating Escherichia coli into LB medium and culturing at 35-37° C. for 16-18 hours to obtain seed solution A; inoculating Bacillus subtilis into LB medium and culturing at 35-37° C. for 10-12 hours to obtain seed solution B; inoculating yeast into LB medium and culturing at 35-37° C. for 8-10 hours to obtain seed solution C; (2) Seed solution A, seed solution B and seed solution C were inoculated into a fermentation tank containing TB medium for cultivation. When the OD 600 When the pH value is 0.8 to 1.0, isopropyl-β-D-thiogalactoside is added to the fermentation tank for induction to obtain a fermentation liquid; (3) adding colistin sulfate to the fermentation broth, continuing the culture, centrifuging and discarding the supernatant after the culture is completed, centrifuging and discarding the supernatant after the treatment is completed, washing with phosphate buffer, and resuspending to obtain a cell suspension; (4) The cell suspension is mixed with the maltose aqueous solution, and the mixture is reacted at 25-27° C. and 220-250 r / min for 15-17 h. After the reaction is completed, the reaction is terminated, the mixture is cooled, and centrifuged to obtain the supernatant to obtain a product containing trehalose.

2. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: In step (3), colistin sulfate is added to the fermentation broth to a concentration of 1.3-1.4 g / L, and the culture is continued for 70-80 min at a temperature of 32-34° C.

3. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: In step (1), the LB culture medium comprises the following components: 10-12 g / L of peptone, 1-3 g / L of yeast powder, and 12-15 g / L of NaCl; the pH value is adjusted to 7.3-7.6, and the balance is supplemented with water.

4. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: The TB culture medium in step (2) comprises the following components: 10-12 g / L of peptone, 20-24 g / L of yeast extract, 4-5 mL / L of glycerol, 2.3-2.4 g / L of potassium dihydrogen phosphate and 0.2-0.4 g / L of dipotassium hydrogen phosphate, and the balance is made up with water.

5. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: The culture conditions in step (2) are: 35-37° C., 220-260 rpm, and aeration volume of 7.2-7.4 L / min.

6. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: The concentration of isopropyl-β-D-thiogalactoside added in step (2) is 0.8-1.0 mmoL / L.

7. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: The induction time in step (2) is 8 to 10 hours.

8. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: In the step (3), the resuspending is performed, and the concentration of the fixed cell bacterial suspension is 10-12 wt % to obtain a cell suspension.

9. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: The concentration of the maltose aqueous solution in step (4) is 30-32% (W / V).

10. The composite enzyme fermentation process for increasing trehalose production according to claim 1, characterized in that: In the step (4), the volume ratio of the cell suspension to the maltose aqueous solution is (3-4):10.