Technological method for fermenting gentamicin C1a based on ultrasonic assistance and resin adsorption

Abstract

The invention relates to a process method for fermenting gentamicin C1a based on ultrasonic assistance and resin adsorption. The method comprises the following steps: firstly, determining an optimal ultrasonic condition through an ultrasonic single-factor experiment; after the optimal ultrasonic condition is determined, gentamicin is promoted to be released from intracellular to extracellular due to the ultrasonic cavitation effect, so that extracellular gentamicin C1a is accumulated. In addition, in order to explore the influence of extracellular gentamicin C1a accumulation on production titer, a product addition experiment is designed. Then, resin is added at different time points in the fermentation process to adsorb extracellular accumulated gentamicin C1a, so that the purposes of reducing the inhibition effect of a product and improving the yield are achieved. And finally, the operation process is reasonably designed, and the process improvement effect after combination is verified.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption. Background Art

[0002] Gentamicin is mainly a secondary metabolite produced by Micromonospora aculeatus and Micromonospora rubiginosa. Gentamicin is widely used in clinical practice because it has strong antibacterial and bactericidal abilities against most bacteria. The biosynthesis of gentamicin is based on 2-DOS, which is a multi-component mixture connected with rubiginosamine and galactosamine through glycosidic bonds. It is mainly classified into A, B, C, X and perilla mycin families. At present, in clinical practice, the C family components of gentamicin are mainly used, which include four main parts: C1, C1a, C2 and C2a. These four substances are all semi-synthetic antibiotics, and they all have the characteristics of broad spectrum, high efficiency and low toxicity. Among all the components, the side effects of C1 component are the mildest and it is not easy to cause drug resistance, but its efficacy is relatively weak; in contrast, although the components of C2 and C1a have significant efficacy, their toxicity is high and it is easy to cause drug resistance. In order to better ensure the quality and efficacy of the preparation, it is necessary to conduct compatibility studies on different component drugs to determine the reasonable proportion and effective parts of each component. Among them, the pharmacopoeias of various countries also have clear requirements and definitions for the proportions of different components of gentamicin.

[0003] Gentamicin is produced inside microbial cells and then released to the outside of the cells, and is partially adsorbed on the cell walls. In order to increase the extracellular secretion of gentamicin and thus improve production titer, researchers have proposed two strategies: one is to increase the permeability of bacterial cell walls to promote the release of gentamicin; the other is to use online mechanical vibration technology to improve the internal conditions of bacteria, enhance the exchange of intracellular nutrients, and thereby improve the release performance of gentamicin. Currently, there are very limited reports on the application of ultrasonic technology in the fermentation process of gentamicin, as well as its research on the metabolism and product synthesis of Micromonospora aculeatus. Some studies have shown that ultrasonic technology can improve the internal conditions of bacteria and enhance the exchange of nutrients in cells, thereby improving the release performance of gentamicin. [1] The report used ultrasonic online treatment technology to change the adsorption behavior of mycelium to gentamicin during the fermentation process, and also enhanced the transmission capacity of gentamicin from the inside to the outside of the cell. Subsequently, due to the non-specific binding of gentamicin to mycelium, although ultrasonic treatment helped the release of gentamicin, the effect was not complete.

[0004] In addition, previous studies [2] Focusing on the adsorption characteristics of gentamicin by cell wall peptidoglycan of Micromonas aculeatus var. ruby-red, it was found that the adsorption of peptidoglycan accounted for the vast majority of the total adsorption of the cell wall, and pointed out that metal ions such as Mg 2+ 、Na +This adsorption can be effectively removed. Another way is to improve the secretion efficiency of gentamicin by increasing the permeability of the cell membrane. The results show that the increase in cell membrane permeability may cause gentamicin in the membrane to bind to the cell wall and release it to the extracellular space after reaching a certain concentration. This technology not only helps the release of gentamicin, but also plays an important role in the separation process. Through these comprehensive research methods and strategies, it is expected to produce gentamicin more efficiently to meet the needs of the pharmaceutical field, while promoting the advancement and development of related technologies.

[0005] In addition, the application of resin adsorption technology in the fermentation process of gentamicin has also been reported. When the gentamicin solution comes into contact with the submicron strong acid resin, its adsorption behavior is mainly achieved through two mechanisms: electrostatic interaction, under acidic conditions, the amino group in the gentamicin molecule is converted into a proton and carries a positive charge, while the sulfonic acid group on the surface of the resin is negatively charged. These two groups with relative charges produce a high degree of electrostatic attraction, which in turn causes the gentamicin molecule to be adsorbed on the surface of the resin; hydrogen bond interaction, in addition to electrostatic interaction, the hydroxyl groups and other polar groups inside the gentamicin molecule may also establish hydrogen bonds with the polar groups on the surface of the resin. Although this interaction formed by hydrogen and oxygen is relatively weak, under certain specific conditions, they do cause significant changes in the adsorption process, thereby enhancing the adsorption capacity of gentamicin on the resin surface. The resins used in the fermentation process of gentamicin C1a include ion exchange resins and macroporous adsorption resins. Among them, ion exchange resins are the most widely used. Macroporous adsorption resins are widely used in the purification of C1a and the production of high-purity gentamicin C1a free base. In addition, the strong acid cation exchange resin and macroporous resin column chromatography separation technology are used to balance the proportion of each component and remove impurities to ensure that the standard requirements of the pharmacopoeias of various countries are met. It is worth noting that when the sodium type resin is added to the fermentation broth of gentamicin, it can promote the formation of gentamicin, and this effect is better than that of the ammonium type resin. In addition, in the separation and purification of gentamicin, the refined treatment of the original drug of gentamicin can be achieved by using the strong acid cation exchange resin and macroporous resin column chromatography technology.

[0006] At present, most of the research on gentamicin fermentation process is still in the optimization exploration of a single factor, and no systematic investigation has been conducted on the interaction between multiple operating variables. The cavitation bubbles generated by ultrasound in liquid can undergo physical, thermal, electromagnetic and chemical changes with the medium. [3]It is reported that it can promote the diffusion and formation of reactants and products. The positive effects of ultrasound on gentamicin fermentation are reflected in multiple aspects: First, ultrasound can change the adsorption capacity of mycelial cell wall to gentamicin through the cavitation effect it produces, prompting some gentamicin originally adsorbed by the cell wall to be released into the fermentation broth. This change helps to reduce the inhibitory effect of intracellular products, thereby promoting the synthesis process of gentamicin. Secondly, when ultrasound of appropriate intensity acts on the fermentation broth, it can effectively improve the permeability and selectivity of the cell membrane. This improvement helps the secretion of related enzymes and the enhancement of cell metabolism, which may shorten the fermentation cycle and improve the quality and output of biological products. However, in order to ensure the positive effect of ultrasound on the fermentation process, it is necessary to reasonably regulate the intensity and frequency of its action.

[0007] In addition, there are some research reports on the addition of resin during gentamicin fermentation. [4-6] However, the effects of resin addition time and extracellular products on the production performance of the strain were not investigated in depth.

[0008] References are as follows:

[0009] [1]Chu J.,Li B.,Zhang S.On-line ultrasound sitimulates the secretionand production of gentamicin by Micromonospora echinospora[J].ProcessBiochemistry,2000,35(6).

[0010] [2] Niu Wenzhe, Hu Hui, Chu Ju, et al. Preliminary study on the adsorption mechanism of gentamicin by cell wall peptidoglycan of Micromonospora aculeatus var. rubiaceae[J]. Chinese Journal of Antibiotics, 2003, 28(2): 65-69.

[0011] [3]Chen, Y., Tang,

[0012] [4] Jiang Deqi, Lan Xueye, Yang Dan. Study on the performance of macroporous weakly acidic ion exchange resin in adsorption and separation of gentamicin[J]. Food Industry Science and Technology, 2013, 34(23): 233-236.

[0013] [5] Li Xialan, Weng Lianjin, Chen Peiqin, et al. Study on the adsorption performance of gentamicin on carboxylic acid ion exchange fiber [J]. Chinese Journal of Pharmaceutical Industry, 2005, 36(5): 272-274.

[0014] [6] Zhan Jiahong, Yang Ziyan, Zhang Jianbin, et al. Application of resin in gentamicin C1a fermentation and related regulation[J]. Chinese Journal of Pharmaceutical Industry, 2021, 52(7): 908-909. Summary of the invention

[0015] The purpose of the present invention is to provide a process optimization method for the fermentation of gentamicin C1a based on ultrasound-assisted and resin adsorption. The present invention explores the optimal ultrasonic operating conditions in the fermentation process of gentamicin through a series of single-factor experiments, and explores the substrate inhibition level and the optimal resin addition time in the fermentation process of gentamicin C1a. In addition, the present invention constructs a fermentation-separation coupled gentamicin C1a production system by combining the optimal ultrasonic operating conditions and the resin addition time.

[0016] The purpose of the present invention can be achieved by the following technical solutions:

[0017] A process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption, the specific steps are as follows:

[0018] S1. Solid plate culture: inoculate the Micromonas aculeatus var. crimsonii on a solid plate medium for culture;

[0019] S2, seed shake flask culture: inoculate the colonies grown in the solid plate culture medium obtained in step S1 into the seed culture medium for culture to obtain a seed shake flask;

[0020] S3, fermentation shake flask culture: the seed liquid in the seed shake flask obtained in step S2 is inoculated into the fermentation medium, and the potency of gentamicin C1a is used as an evaluation index to perform optimized culture to obtain gentamicin C1a through fermentation.

[0021] Furthermore, in step S1, the components of the solid plate culture medium include 15-20 g / L bran, 12-18 g / L agar powder, 8-12 g / L soluble starch, 0.5-1.5 g / L calcium carbonate, 0.5-1.5 g / L potassium nitrate, 0.4-0.6 g / L sodium chloride, 0.4-0.6 g / L magnesium sulfate heptahydrate, 0.2-0.4 g / L potassium hydrogen phosphate trihydrate, 0.01-0.03 g / L asparagine, and the solvent is water.

[0022] Furthermore, in step S2, the components of the seed culture medium include 15-25 g / L corn flour, 12-18 g / L soluble starch, 0.5-1.5 g / L glucose, 12-18 g / L low-temperature soybean powder, 3-5 g / L calcium carbonate, 2-4 g / L feather powder, 0.4-0.6 g / L potassium nitrate, and the solvent is water.

[0023] Furthermore, in step S3, the components of the fermentation medium include 15-25 g / L corn flour, 25-35 g / L soluble starch, 4-6 g / L glucose, 24-28 g / L soybean cake powder, 6-8 g / L calcium carbonate, 8-12 g / L feather meal peptone, 0.4-0.6 g / L potassium nitrate, 0.5-1.5 g / L ammonium sulfate, 0.02-0.04 g / L cobalt chloride, and the solvent is water.

[0024] Furthermore, in step S3, during fermentation shake flask culture, the process optimization method is as follows:

[0025] S3-1, using the potency of gentamicin C1a as the evaluation index, single factor experiments were conducted on ultrasonic conditions, gentamicin C1a concentration, and timing of resin addition;

[0026] S3-2. Obtain the optimal fermentation process conditions based on the results of the single factor experiment, use the optimal fermentation process conditions for fermentation, and ultimately determine the optimal process conditions for gentamicin C1a fermentation.

[0027] Furthermore, in step S3-1, the potency of gentamicin C1a=2B+A, wherein B is the potency of gentamicin C1a in the resin adsorption, and A is the potency of gentamicin C1a in the fermentation supernatant.

[0028] Furthermore, in step S3-1, the ultrasound conditions include ultrasound timing, ultrasound duration and ultrasound power.

[0029] In the above, the ultrasonic timing is set to ferment the fermentation liquid for 12h, 24h, 36h, 48h, 60h, 74h, 84h, and 98h respectively;

[0030] The ultrasound duration is set to 2 min, 4 min, 6 min, and 8 min respectively;

[0031] The ultrasonic powers are set to 120W, 180W, 240W and 300W respectively.

[0032] Furthermore, in step S3-1, the concentration of gentamicin C1a is set to 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L, respectively.

[0033] Furthermore, in step S3-1, the timing of adding the resin is set to fermentation time 0h, 24h, 48h, 72h, and 96h, respectively.

[0034] Furthermore, in step S3-2, the optimal process conditions for the fermentation of gentamicin C1a are: ultrasound timing is 60h for fermentation broth fermentation time, ultrasound duration is 2min, ultrasound power is 180W, gentamicin C1a concentration is 100mg / L, and resin addition timing is 24h for fermentation time.

[0035] The principle of the present invention is as follows:

[0036] The present invention starts with the cultivation of an industrial Micromonospora aculeatus that is a single-component strain with high gentamicin C1a production. The optimal ultrasonic conditions are determined by ultrasonic single-factor experiments, and then the effects of different extracellular gentamicin concentrations on the titer of the production strain product are analyzed to determine the gentamicin concentration with the highest product inhibition. In addition, resin adsorption is used to reduce the extracellular product inhibition effect during the fermentation process, further improve the titer of gentamicin C1a, and obtain the optimal ultrasonic and resin delivery conditions that significantly improve the titer of gentamicin. The present invention solves the problem that some wastewater, waste residue and other wastes may be generated during the production process of gentamicin C1a. While improving the production titer, the amount of waste generated per unit product can be reduced, reducing the pollution pressure on the environment. In addition, by optimizing the production process, the reduction, resource utilization and harmless treatment of waste can also be achieved, further reducing the negative impact on the environment.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] 1. The present invention first determines the optimal ultrasonic conditions through an ultrasonic single-factor experiment. After determining the optimal ultrasonic conditions, the ultrasonic cavitation promotes the release of gentamicin from the intracellular to the extracellular, resulting in the accumulation of extracellular gentamicin C1a. In addition, in order to explore the effect of extracellular gentamicin C1a accumulation on production titer, the present invention designs a product addition experiment. Then, by adding resin at different time points during the fermentation process, the extracellular accumulated gentamicin C1a is adsorbed to achieve the purpose of reducing the product inhibition effect and increasing the yield. Finally, the above-mentioned operation process is reasonably designed and the process improvement effect after the combination is verified.

[0039] 2. The present invention systematically carried out ultrasonic single-factor experiments to determine the optimal ultrasonic conditions. On this basis, the effects of different gentamicin concentrations on the titer of the fermentation broth were further studied. By precisely controlling and adding different concentrations of gentamicin, the gentamicin concentration that can maximize the inhibition of product accumulation was determined. Next, in order to reduce the inhibitory effect of extracellular products during fermentation, the resin adsorption kinetics was deeply explored, and the resin adsorption method was successfully used to reduce the concentration of extracellular gentamicin C1a. This step not only optimizes the fermentation environment, but also significantly improves the titer of gentamicin C1a. After multiple experiments and data analysis, the present invention obtains the optimal ultrasonic conditions and resin delivery conditions: that is, when the fermentation time reaches 24h, 1mL of 001×8 resin with a diameter of 0.4mm is added to the fermentation broth. After the resin adsorbs extracellular gentamicin C1a, it effectively promotes the synthesis and release of more gentamicin. Subsequently, when the fermentation time reaches 60h, we ultrasonically treat the fermentation broth with an ultrasonic frequency of 40kHZ, an ultrasonic duration of 2min, and an ultrasonic power of 180W, and the secretion rate is increased to 29%. This treatment step further improved the production titer of gentamicin C1a, increasing the production titer of C1a by about 2.6 times during the shake flask fermentation stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the standard curve of gentamicin C1a in Example 1;

[0041] Figure 2 Schematic diagram of the ultrasonic treatment time experimental results in Example 1, wherein the fermentation time gradient in (a) is 12h, 24h, 36h, 48h, and the fermentation time gradient in (b) is 60h, 74h, 84h, 98h;

[0042] Figure 3 Schematic diagram of the ultrasound duration experimental results in Example 1 (* indicates a p value less than 0.05, ** indicates a p value less than 0.01), wherein (a) is the concentration of C1a at different ultrasound durations, and (b) is the secretion rate of C1a at different ultrasound durations;

[0043] Figure 4 Schematic diagram of the results of the optimal ultrasound power experiment in Example 1 (* indicates that the p value is less than 0.05, ** indicates that the p value is less than 0.01), wherein (a) is the concentration of C1a at different ultrasound powers, and (b) is the secretion rate of C1a at different ultrasound powers;

[0044] Figure 5 A schematic diagram of the experimental results of adding C1a in Example 1 (* indicates that the p value is less than 0.05);

[0045] Figure 6This is a schematic diagram of the experimental results of adding resin in Example 1 (* indicates a p value less than 0.05, ** indicates a p value less than 0.01);

[0046] Figure 7 This is a color comparison diagram of the fermentation broth in Example 1;

[0047] Figure 8 This is a schematic diagram of the results of the ultrasound-assisted combined resin adsorption experiment in Example 1 (* indicates a p value less than 0.05, ** indicates a p value less than 0.01, and *** indicates a p value less than 0.001). DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0049] In the following examples, the strains are as follows:

[0050] Micromospora echinosppra 49-92S-YB3 was provided by the National Biochemical Engineering Technology Research Center (Shanghai) Laboratory of East China University of Science and Technology. The strain was preserved in a glycerol tube and stored in a refrigerator at 80°C. It needs to be activated before use.

[0051] In the following examples, the culture medium is as follows:

[0052] (1) Solid plate culture medium (g / L)

[0053] Prepared with 18 grams of bran, 15 grams of agar powder, 10 grams of soluble starch, 1 grams of calcium carbonate, 1 grams of potassium nitrate, 0.5 grams of sodium chloride, 0.5 grams of magnesium sulfate heptahydrate, 0.3 grams of dipotassium hydrogen phosphate trihydrate, 0.02 grams of asparagine, and deionized water, and adjust the pH to 7.5-7.8.

[0054] (2) Seed shake flask medium (g / L)

[0055] Corn flour 20, soluble starch 15, glucose 1, low-temperature soybean powder 15, calcium carbonate 4, feather powder 3, potassium nitrate 0.5, deionized water, no need to adjust pH.

[0056] (3) Fermentation shake flask medium (g / L)

[0057] Corn flour 20, soluble starch 30, glucose 5, soybean cake powder 26, calcium carbonate 7, feather meal peptone 10, potassium nitrate 0.5, ammonium sulfate 1, cobalt chloride 0.03, deionized water, NaOH to adjust the pH to 8.0-8.1.

[0058] In the following examples, the culture method is as follows:

[0059] (1) Solid plate culture

[0060] The prepared solid plate culture medium was divided into 500mL shaking bottles, wrapped with four layers of gauze and kraft paper, sterilized at 121℃ for 30min, and dried in an oven. The culture medium was divided into plates in a clean bench, dried with UV light, and about 100uL of bacterial solution was pipetted into each plate with a pipette, evenly coated with a coating rod, and then placed in a 34℃ constant temperature incubator for 7 days.

[0061] (2) Seed shake flask culture

[0062] The prepared seed culture medium was divided into 500mL shake flasks, 55mL per bottle, and glucose was divided separately, and sterilized at 121℃ for 30min. A small piece of solid plate culture medium was dug out in the clean bench and placed in a seed shake flask, 5mL of glucose was added to each flask, and cultured in a shaker at 35.5℃ and 220rpm for 48h. The seed shake flask with loose outer layer and no foreign bacteria in the microscopic examination was selected for fermentation shake flask inoculation.

[0063] (3) Fermentation shake flask culture

[0064] The prepared fermentation medium was divided into 500 mL shake flasks, 50 mL per bottle, and sterilized at 121°C for 30 min. 5 mL of seed solution was drawn into the fermentation shake flask in the clean bench, 5 mL of glucose was added to each flask, and the flask was placed in a shaker at 35.5°C and 220 rpm for 120 h.

[0065] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0066] Example 1

[0067] This embodiment provides a process optimization method for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption, and the specific steps are as follows:

[0068] S1. Ultrasound single factor experiment

[0069] The experimental groups were subjected to ultrasound under different conditions, and the control group was not treated. Single-factor experiments were carried out in batches in an ultrasound instrument with a frequency of 40kHz. The single-factor conditions were ultrasound timing, ultrasound duration, and ultrasound power. In the ultrasound timing experiment, under the conditions of consistent ultrasound duration and ultrasound power, the first batch of ultrasound timing was 12h, 24h, 36h, and 48h of fermentation broth, and the second batch of ultrasound timing was 60h, 74h, 84h, and 98h of fermentation broth. There were 3 parallel controls in each group, and a blank control was set for each batch. In the ultrasound duration experiment, under the conditions of consistent ultrasound timing and ultrasound power, the ultrasound duration was set to 2min, 4min, 6min, and 8min, respectively, with 3 parallel controls in each group and a blank control. In the ultrasound power experiment, under the conditions of consistent ultrasound timing and ultrasound duration, the ultrasound power was set to 40%, 60%, 80%, and 100%, respectively. In each of the above single-factor experiments, each group was repeated 3 times. The titer of each group of experiments was determined 120h after the end of fermentation.

[0070] S2. C1a addition experiment

[0071] Different concentrations of C1a were added during the fermentation stage to simulate the effect of extracellular C1a accumulation on the yield during the fermentation of gentamicin C1a. Seven concentration gradients of 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L were set. At 72 hours of fermentation, the prepared C1a standard solutions of different concentrations were injected into the fermentation flasks through a sterile filter in a clean bench.

[0072] S3. Resin pretreatment

[0073] Use 2mol / L hydrochloric acid at a flow rate of 0.5BV / h to wash the resin through the chromatography column until the pH of the effluent is below 1.0, then wash with deionized water until the pH of the effluent is above 4.0, then rinse the resin several times with sterile water in a clean bench and irradiate with ultraviolet light for use.

[0074] S4. Resin addition experiment

[0075] The experimental group was fed with 1 mL of pre-treated 001×8 resin at different fermentation times, and the control group was not fed with resin. The timing of adding resin was set at 0h, 24h, 48h, 72h, and 96h, and a blank control group was set, with 3 parallel controls in each group.

[0076] S5, Ultrasound-assisted combined with resin adsorption

[0077] The experimental group was treated with the optimal ultrasonic parameters, and resin was added at the optimal resin addition time. Four groups of shake flasks were set up, including blank control, ultrasonic treatment, resin addition, and ultrasonic treatment combined with resin, with 3 parallel controls in each group, to verify the reliability of ultrasound-assisted combined with resin adsorption to increase the maximum yield of gentamicin C1a.

[0078] S6. Determination of gentamicin C1a

[0079] Preparation of gentamicin C1a standard curve: Prepare 1g / L gentamicin C1a standard solution, dilute to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000mg / L, and draw a standard curve, such as Figure 1 As shown. The potency of gentamicin C1a was determined by HPLC. The chromatographic column used for detection was a C18 column (4.6×150mm, 5μm), the detection wavelength was 330nm, the injection volume was 20μL, the flow rate was set to 1.0mL / min, and the column temperature was 30℃. The mobile phase was prepared in the ratio of methanol: aqueous phase: glacial acetic acid = 700:250:50. For sample processing, a certain amount of fermentation broth was first taken after the fermentation was completed, and the pH was adjusted to 1.9-2.0 with 6M H2SO4. After standing for 30min, centrifugation was performed, and 100μL of the supernatant was taken, and 900μL of ultrapure water, 400μL of derivatizing agent, and 1100μL of methanol were added in sequence, and the mixture was placed in a 60℃ water bath for 20min. After cooling, the mixture was injected into a liquid phase vial through a syringe, and the injection volume was 20μL.

[0080] S7. Resin treatment and determination of gentamicin C1a

[0081] The fermentation broth to which the resin was added was separated by a 100-mesh oscillating sieve, and the separated fermentation broth was titered according to the S6 method (fermentation supernatant A). The separated resin was washed with tap water to remove the fermentation broth residue, 10 mL of 1.5 mol / L NaOH was added, and the resin was shaken and soaked for about 15 hours. Subsequently, 0.5 mL of the soaking liquid was taken, 0.5 mL of 1 mol / L HCL was added, and the titer was determined according to the above method (resin adsorption B). At this time, the total titer of gentamicin C1a (mg / L) was:

[0082] Titer = 2B + A

[0083] The experimental results in this embodiment are as follows:

[0084] 1. Optimization of Ultrasonic Parameters

[0085] (1) Optimal ultrasonic treatment time

[0086] In the designed four fermentation time gradients of 12h, 24h, 36h, and 48h, Figure 2As shown in (a), the titer of gentamicin C1a was not significantly increased compared with the blank control CK, but the titer increased with the increase of fermentation time. The results showed that ultrasonic treatment can promote the secretion and production of gentamicin C1a. Then, this example designed four fermentation time gradients of 60h, 74h, 84h, and 98h. Figure 2 (b) It can be seen that the concentration of gentamicin C1a in the blank control is 186.5 mg / L. Among the four time gradients, ultrasonic treatment of the fermentation broth at 60 h can significantly increase the titer of the fermentation broth. The concentration of gentamicin C1a after ultrasonic treatment at this time is 233.5 mg / L, and the maximum increase in yield is about 25% compared with the control. Therefore, the fermentation time of 60 h is the best time for ultrasonic treatment.

[0087] (2) Optimal duration of ultrasound

[0088] In the designed four ultrasound duration gradients of 2min, 4min, 6min, and 10min, Figure 3 As shown in (a), the concentration of gentamicin C1a in the four experimental groups was significantly increased compared with the blank control. Among them, the gentamicin C1a concentration of the blank control was 214.3 mg / L, while the gentamicin C1a titer of the experimental group with an ultrasonic duration of 2 min was significantly increased compared with other groups. The concentration of gentamicin C1a treated with ultrasonic treatment under this duration was 308.6 mg / L, and the maximum yield was increased by about 44% compared with the control. At the same time, it was observed that after 2 min of treatment time, the yield of gentamicin C1a showed a gradual downward trend, indicating that the optimal ultrasonic treatment time is conducive to the production of gentamicin C1a and the transport of products from the intracellular to the extracellular. However, after the optimal treatment time, the ultrasonic treatment exceeded the limit, which may cause damage to the cell membrane and reduce the metabolic activity of the bacteria. Therefore, the optimal ultrasonic treatment duration is 2 min.

[0089] In addition, the C1a concentration measured in the extracellular supernatant of the fermentation broth was compared with that of the control, and the secretion rate was calculated. Figure 3 As shown in (b), it can be seen that the secretion rate is higher when the ultrasound duration is 2min and 4min, which are 24% and 26% respectively. Then, the secretion rate gradually decreases with the increase of ultrasound duration, indicating that under these two ultrasound duration conditions, the cavitation effect of ultrasound is most significant in promoting the release of intracellular gentamicin C1a. In summary, considering the purpose of improving the production titer and economy of C1a, the optimal ultrasound duration is 2min.

[0090] (3) Optimal ultrasound power

[0091] In the designed four ultrasonic power gradients of 40%, 60%, 80%, and 100%, the concentration of gentamicin C1a in the blank control was 234.0 mg / L. Figure 4As shown in (a), the titers of gentamicin C1a in the four experimental groups were increased compared with the control group, and the titer of gentamicin C1a in the experimental group with an ultrasonic power of 60% was significantly increased compared with other groups. The titer of gentamicin C1a treated with ultrasound at this power was 282.6 mg / L, and the maximum increase in yield was about 21% compared with the control.

[0092] In addition, the C1a concentration measured in the extracellular supernatant of the fermentation broth was compared with that of the control, and the secretion rate was calculated. Figure 4 As shown in (b), it can be seen that when the ultrasonic power is 60%, the secretion rate is the highest (25%). As the ultrasonic power increases, the secretion rate gradually decreases. The results show that when the ultrasonic power is 60%, the cavitation effect of ultrasound is most significant in promoting the release of intracellular gentamicin C1a. The optimal ultrasonic treatment power is 60%, and the maximum power of the ultrasonic instrument used is 300W, so the optimal ultrasonic power is 180W.

[0093] 2. Resin adsorption research

[0094] (1) C1a addition experiment

[0095] After determining the optimal ultrasonic conditions, the ultrasonic cavitation promoted the release of gentamicin from the cell to the extracellular space, resulting in the accumulation of extracellular gentamicin C1a. Excessive extracellular products may inhibit bacterial metabolism. In order to explore the effect of extracellular gentamicin C1a levels on bacterial fermentation, a gentamicin C1a addition experiment was then conducted. Figure 5 The value obtained by subtracting the added C1a concentration from the measured final C1a concentration of the fermentation broth. The results show that in the designed six concentration gradients of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L, the final titer of the fermentation broth showed an upward trend before the C1a addition concentration was 150 mg / L, and a significant decrease occurred at 150 mg / L. As the concentration of added C1a increased, the final titer decreased and was lower than that of the control group, indicating that the formation of the product would be inhibited when the concentration of extracellular C1a reached 150 mg / L, and the concentration of extracellular C1a was most conducive to the formation of the product when it was 100 mg / L. This result proves that when the extracellular C1a concentration reaches a certain level, feedback inhibition will indeed occur, inhibiting the further synthesis and release of gentamicin.

[0096] (2) Resin addition experiment

[0097] In order to further alleviate the accumulation of extracellular products caused by ultrasound and improve the titer, the effect of adding resin at different time points (0h, 24h, 48h, 72h, 96h) during the fermentation process on the production of gentamicin C1a was then investigated. Figure 6It can be seen that the concentration of gentamicin C1a in the experimental group with added resin is higher than that in the blank control group, indicating that adding resin to the fermentation broth can effectively adsorb extracellular gentamicin C1a and significantly reduce the inhibition of bacterial metabolism caused by product inhibition, and ultimately increase the production titer of gentamicin C1a. In addition, it can be seen that when the resin is added at a fermentation time of 24h, the C1a production titer is the highest and significantly higher than the blank control and other time points. The final C1a titer of the control group is 197.9mg / L, and the final C1a titer of the resin added at this time point is 276.2mg / L, which is about 73% higher than the C1a titer of the control group.

[0098] The experiment also found that the fermentation shake flasks with added resin showed a lighter color ( Figure 7 Right), while the fermentation broth in the control shake flask without the addition of resin showed higher viscosity and darker color ( Figure 7 Left). This may be because the resin can absorb most of the extracellular gentamicin and pigments and other byproduct impurities, causing the fermentation liquid to be relatively thin. Extracellular gentamicin and pigments and other byproduct impurities may cause product inhibition. By adding resin, the bacterial metabolism inhibition caused by product inhibition during the fermentation of gentamicin C1a can be effectively relieved, thereby more effectively improving the production titer of gentamicin C1a.

[0099] 3. Ultrasound-assisted resin adsorption

[0100] After exploring the optimal ultrasonic treatment parameters and the optimal resin addition time, it is necessary to apply the two together to the fermentation process of gentamicin C1a to explore the effect of the combination of the two on the enhancement of the gentamicin fermentation process. By designing three experimental groups, namely the ultrasonic group, the resin group, and the ultrasonic combined with resin group, and setting up a control group, a fermentation shake flask experiment was conducted.

[0101] Depend on Figure 8 It can be seen that the fermentation conditions of ultrasound-assisted combined with resin adsorption were significantly improved compared with single ultrasound treatment and single resin treatment, and its C1a production titer was 686.2 mg / L, which was about 1.2 times higher than that of single ultrasound treatment. In addition, compared with single resin treatment, the C1a production titer was about 58.7% higher. The C1a production titer of the control group was 187.8 mg / L, and the C1a production titer of the ultrasound-assisted combined with resin optimization group was about 2.6 times higher than that of the control group.

[0102] In addition, the secretion rate of the ultrasound group alone was calculated to be 29%, which was higher than the secretion rate obtained in the ultrasound single factor experiment, indicating that the effect of combining the optimal ultrasound parameters in the gentamicin C1a fermentation process was positive and significant, and was conducive to the further synthesis and release of gentamicin C1a.

[0103] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption, characterized in that: The specific steps are as follows: S1. Solid plate culture: inoculate the Micromonas aculeatus var. crimsonii on a solid plate medium for culture; S2, seed shake flask culture: inoculate the colonies grown in the solid plate culture medium obtained in step S1 into the seed culture medium for culture to obtain a seed shake flask; S3, fermentation shake flask culture: the seed liquid in the seed shake flask obtained in step S2 is inoculated into the fermentation medium, and the potency of gentamicin C1a is used as an evaluation index to perform optimized culture to obtain gentamicin C1a through fermentation.

2. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 1, characterized in that: In step S1, the components of the solid plate culture medium include 15-20 g / L of bran, 12-18 g / L of agar powder, 8-12 g / L of soluble starch, 0.5-1.5 g / L of calcium carbonate, 0.5-1.5 g / L of potassium nitrate, 0.4-0.6 g / L of sodium chloride, 0.4-0.6 g / L of magnesium sulfate heptahydrate, 0.2-0.4 g / L of potassium hydrogen phosphate trihydrate, 0.01-0.03 g / L of asparagine, and the solvent is water.

3. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 1, characterized in that: In step S2, the components of the seed culture medium include 15-25 g / L corn flour, 12-18 g / L soluble starch, 0.5-1.5 g / L glucose, 12-18 g / L low-temperature soybean powder, 3-5 g / L calcium carbonate, 2-4 g / L feather powder, 0.4-0.6 g / L potassium nitrate, and the solvent is water.

4. The process for fermenting gentamicin C1a based on ultrasound-assisted and resin adsorption according to claim 1, characterized in that: In step S3, the components of the fermentation medium include 15-25 g / L corn flour, 25-35 g / L soluble starch, 4-6 g / L glucose, 24-28 g / L soybean cake powder, 6-8 g / L calcium carbonate, 8-12 g / L feather powder peptone, 0.4-0.6 g / L potassium nitrate, 0.5-1.5 g / L ammonium sulfate, 0.02-0.04 g / L cobalt chloride, and the solvent is water.

5. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 1, characterized in that: In step S3, during fermentation shake flask culture, the process optimization method is as follows: S3-1, using the potency of gentamicin C1a as the evaluation index, single factor experiments were conducted on ultrasonic conditions, gentamicin C1a concentration, and timing of resin addition; S3-2. Obtain the optimal fermentation process conditions based on the results of the single factor experiment, use the optimal fermentation process conditions for fermentation, and ultimately determine the optimal process conditions for gentamicin C1a fermentation.

6. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 5, characterized in that: In step S3-1, the potency of gentamicin C1a = 2B + A, wherein B is the potency of gentamicin C1a in the resin adsorption, and A is the potency of gentamicin C1a in the fermentation supernatant; In step S3-1, the ultrasound conditions include ultrasound timing, ultrasound duration, and ultrasound power.

7. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 6, characterized in that: The ultrasonic timing was set to ferment the fermentation liquid for 12h, 24h, 36h, 48h, 60h, 74h, 84h, and 98h respectively; The ultrasound duration is set to 2 min, 4 min, 6 min, and 8 min respectively; The ultrasonic powers are set to 120W, 180W, 240W and 300W respectively.

8. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 6, characterized in that: In step S3-1, the concentration of gentamicin C1a is set to 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L, respectively.

9. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 6, characterized in that: In step S3-1, the timing of adding the resin is set to fermentation time 0h, 24h, 48h, 72h, and 96h, respectively.

10. The process for fermenting gentamicin C1a based on ultrasound assistance and resin adsorption according to claim 6, characterized in that: In step S3-2, the optimal process conditions for the gentamicin C1a fermentation are: ultrasonic timing is fermentation time of fermentation broth is 60h, ultrasonic duration is 2min, ultrasonic power is 180W, gentamicin C1a concentration is 100mg / L, and resin is added at fermentation time of 24h.

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