Defoaming agent for biological fermentation process, preparation method and application

Abstract

The invention provides a defoaming agent for a biological fermentation process as well as a preparation method and application thereof.The defoaming agent is prepared from the following components in parts by weight: 18-20 parts of silicon paste, 18-20 parts of polyether modified silicone oil, 2-5 parts of polysaccharide components, 2-5 parts of an emulsifier and 50-60 parts of deionized water, the mass ratio of the ginkgo biloba polysaccharide to the trehalose is (2: 1)-(3: 1). According to the defoaming agent prepared by the invention, the efficient defoaming performance of an organic silicon defoaming agent is maintained, the promotion effect on cell growth is also realized, and the yield of a fermentation product is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an antifoaming agent for use in bio-fermentation processes, its preparation method, and its application. Background Technology

[0002] During bio-fermentation, the fermentation broth is prone to producing a large amount of foam due to microbial metabolism, aeration, and stirring. Excessive foam can lead to overflow of the fermentation broth, a decrease in dissolved oxygen, and a reduction in mass transfer efficiency, which seriously affects fermentation efficiency and product yield. Therefore, it is necessary to add defoaming agents to inhibit foam production.

[0003] Currently, silicone-based defoamers are commonly used in industry due to their high defoaming efficiency and good stability. However, while traditional silicone defoamers exert their defoaming effect, they tend to adhere to the surface of microbial cells, disrupting cell membrane integrity and inhibiting cell growth and reproduction, thus reducing the yield of fermentation products. To address this issue, existing technologies often improve compatibility by optimizing the molecular weight of silicone or by compounding surfactants, but these methods still cannot achieve a synergistic effect of "defoaming" and "cell growth promotion." Therefore, there is an urgent need to develop a novel defoamer that combines highly efficient defoaming performance with cell proliferation promotion. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an antifoaming agent, preparation method and application for use in bio-fermentation processes, in order to solve the technical problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: An antifoaming agent for use in bio-fermentation processes, characterized in that it comprises the following components by weight: The ingredients are 18-20 parts silicone paste, 18-20 parts polyether modified silicone oil, 2-5 parts polysaccharide component, 2-5 parts emulsifier, and 50-60 parts deionized water, wherein the polysaccharide component is ginkgo polysaccharide and trehalose, and the mass ratio of ginkgo polysaccharide to trehalose is 2:1 to 3:1.

[0006] According to one aspect of the above technical solution, the main components of the silicone paste are polydimethyl silicone oil and silicon dioxide, the ratio of polydimethyl silicone oil to silicon dioxide is 8:1 to 10:1, and the polyether modified silicone oil is a polyoxyethylene-polyoxypropylene block modified silicone oil with an EO / PO molar ratio of 1.2:1-1.5:1 and a hydroxyl value ≤3mgKOH / g.

[0007] This invention also provides a method for preparing an antifoaming agent for a bio-fermentation process, comprising the following steps: S10 provides 18-20 parts by weight of silicone paste and 18-20 parts by weight of polyether-modified silicone oil, and adds them to a reaction vessel for stirring and mixing to obtain a basic defoamer; S20, add 2-5 parts by weight of polysaccharide component to the basic defoamer and continue stirring to obtain the reaction system; S30, add 2-5 parts by weight of emulsifier and 50-60 parts by weight of deionized water to the reaction system and stir to emulsify, so as to obtain the final defoamer; The polysaccharide component is ginkgo polysaccharide and trehalose, and the mass ratio of ginkgo polysaccharide to trehalose is 2:1 to 3:1.

[0008] According to one aspect of the above technical solution, in step S10, the temperature during stirring and mixing is 25~35℃, the stirring and mixing rate is 400~450r / min, and the stirring and mixing time is 20~30min.

[0009] According to one aspect of the above technical solution, in step S20, the stirring time is 30~40 minutes.

[0010] According to one aspect of the above technical solution, in step S30, the stirring and emulsification time is 25~30 minutes.

[0011] According to one aspect of the above technical solution, the pretreatment process of the polysaccharide component is as follows: Ginkgo polysaccharides were dissolved in water to prepare a ginkgo polysaccharide solution with a mass concentration of 6-7%. The ginkgo polysaccharide solution was treated with ultrasound at a power of 350-450W for 25-35 minutes, and then trehalose was added and stirred to dissolve, thus obtaining the reactant. The reactants were freeze-dried at -45 to -30°C and then pulverized to 120 to 180 mesh to obtain the polysaccharide component.

[0012] According to one aspect of the above technical solution, the emulsifier is compounded from Span 80 and Tween 80 in a mass ratio of 3:1, and the stirring and emulsification process is carried out in stages. The staged stirring is as follows: the stirring rate is 300 r / min for the first 10 minutes and 500 r / min for the next 15 to 20 minutes.

[0013] The present invention also provides an application of an antifoaming agent for a bio-fermentation process, wherein the antifoaming agent described above is applied to the bio-fermentation process.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses silicone paste as the base defoaming component and innovatively introduces pretreated polysaccharide components (ginkgo polysaccharide and trehalose) in a specific compound ratio. Ginkgo polysaccharide exhibits excellent biocompatibility and can form a protective film on the cell surface, reducing the irritation of cells by silicone. Trehalose stabilizes intracellular protein structure and enhances cell resistance; the synergistic effect of these two components significantly promotes cell growth and reproduction. Simultaneously, the introduction of the polysaccharide components improves the dispersibility of silicone, prolongs the defoaming effect, and achieves a triple synergistic function of "defoaming-foam suppression-growth promotion." Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation method of the defoamer for the bio-fermentation process in the first embodiment of the present invention; The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0017] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0019] Please see Figure 1 The image shows a method for preparing an antifoaming agent for a bio-fermentation process according to a first embodiment of the present invention, comprising the following steps: S10 provides a preset amount of silicone paste and polyether-modified silicone oil, and adds them to a reaction vessel for stirring and mixing to obtain a basic defoamer; S20, add a predetermined amount of polysaccharide component to the basic defoamer and continue stirring to obtain a reaction system; S30, add a preset amount of emulsifier and deionized water to the reaction system and stir to emulsify, so as to obtain the final defoamer; The polysaccharide component is ginkgo polysaccharide and trehalose, and the mass ratio of ginkgo polysaccharide to trehalose is 2:1 to 3:1.

[0020] In this embodiment, the mass ratio of ginkgo polysaccharide to trehalose is 2.5:1.

[0021] Understandably, this invention uses silicone paste as the base defoaming component and innovatively introduces pretreated polysaccharide components (ginkgo polysaccharide and trehalose) in a specific compound ratio. Ginkgo polysaccharide exhibits excellent biocompatibility and can form a protective film on the cell surface, reducing the irritation of cells by silicone. Trehalose stabilizes intracellular protein structures and enhances cell resistance; the synergistic effect of these two components significantly promotes cell growth and reproduction. Simultaneously, the introduction of the polysaccharide components improves the dispersibility of silicone, prolongs the defoaming effect, and achieves a triple synergistic function of "defoaming-foam suppression-growth promotion."

[0022] Further, in step S10, the silicone paste and the polyether-modified silicone oil are both 18-20 parts by weight. The mixing temperature is 25-35°C, the mixing speed is 400-450 r / min, and the mixing time is 20-30 min. The main components of the silicone paste are polydimethyl silicone oil and silica, with a ratio of 8:1 to 10:1. The polyether-modified silicone oil is a polyoxyethylene-polyoxypropylene block-modified silicone oil with an EO / PO molar ratio of 1.2:1-1.5:1 and a hydroxyl value ≤3 mgKOH / g.

[0023] In this embodiment, the weight parts of the silicone paste are 18 parts, the weight parts of the polyoxyethylene-polyoxypropylene block modified silicone oil are 18 parts, the EO / PO molar ratio is 1.3:1, the hydroxyl value is 2.5 mg KOH / g, the stirring temperature is 25°C, the stirring speed is 400 r / min, the stirring time is 25 min, and the ratio of polydimethyl silicone oil to the silica is 8:1.

[0024] Furthermore, in step S20, the polysaccharide component is 2 to 5 parts by weight, and the stirring time is 30 to 40 minutes.

[0025] In this embodiment, the polysaccharide component is 2 parts by weight, the stirring speed is adjusted to 450 r / min, and the stirring time is 40 min.

[0026] Furthermore, the pretreatment process for the polysaccharide component is as follows: Ginkgo polysaccharides were dissolved in water to prepare a ginkgo polysaccharide solution with a mass concentration of 6-7%. The ginkgo polysaccharide solution was treated with ultrasound at a power of 350-450W for 25-35 minutes, and then trehalose was added and stirred to dissolve, thus obtaining the reactant. The reactants were freeze-dried at -45 to -30°C and then pulverized to 120 to 180 mesh to obtain the polysaccharide component.

[0027] In this embodiment, the mass concentration of the ginkgo polysaccharide solution was 6.5%, the ultrasonic power was 400W, the processing time was 25min, the stirring rate after adding trehalose was 200r / min, the freeze-drying temperature was -35℃, and the powder was pulverized to 150 mesh.

[0028] Furthermore, in step S30, the emulsifier comprises 2-5 parts by weight, the deionized water comprises 50-60 parts by weight, and the emulsification time is 25-30 minutes. The emulsifier is a compound of Span 80 and Tween 80 in a mass ratio of 3:1, and the emulsification process employs staged stirring, wherein the stirring rate is 300 r / min for the first 10 minutes and 500 r / min for the next 15-20 minutes.

[0029] In this embodiment, the emulsifier is 2 parts by weight, the deionized water is 60 parts by weight, the stirring and emulsification time is 30 minutes, and the staged stirring is as follows: the stirring rate is 300 r / min for the first 10 minutes and 500 r / min for the next 20 minutes.

[0030] Testing revealed that the defoamer prepared using the method described in the first embodiment for the bio-fermentation process had a defoaming time of 3.8 seconds and a foam suppression time of 55 hours. When this defoamer was applied to penicillin fermentation (fermentation conditions: temperature 26℃, pH 6.8), it was added in stages at 0.2% of the total fermentation broth mass (50% initially, 30% in the middle stage, and 20% in the later stage). Compared to the blank control group, the average yeast biomass (based on OD600) in the fermentation system increased by 18%, and the final penicillin yield increased by 12%.

[0031] Animal cell culture application: In a CHO cell suspension culture system (using commercial serum-free medium), the defoamer was added at the beginning of the culture at a volume of 0.1% of the medium. Compared with the control group using a conventional polyether defoamer, by day 7 of culture, the viable cell density (VCD) of the defoamer group in this example was increased by approximately 25%, cell viability was maintained above 96%, and the expression level of the target recombinant protein was significantly increased.

[0032] The method for preparing the defoamer for the bio-fermentation process in the second embodiment of the present invention differs from that in the first embodiment in that: In this embodiment, 20 parts by weight of silicone paste and 20 parts by weight of polyoxyethylene-polyoxypropylene block-modified silicone oil are used. The EO / PO molar ratio is 1.2:1, the hydroxyl value is 3 mg KOH / g, and the mixing temperature is 25°C, the mixing speed is 450 r / min, and the mixing time is 25 min. The ratio of polydimethyl silicone oil to silica is 10:1. The mass ratio of ginkgo polysaccharide to trehalose is 3:1. The polysaccharide component was 5 parts by weight, and the stirring speed was 450 r / min for 40 min.

[0033] The mass concentration of the ginkgo polysaccharide solution was 6.5%, the ultrasonic power was 400W, the treatment time was 25min, the stirring rate after adding trehalose was 180r / min, the freeze-drying temperature was -40℃, and the powder was pulverized to 150 mesh.

[0034] The emulsifier was 5 parts by weight, the deionized water was 55 parts by weight, the emulsification time was 25 min, and the staged stirring was as follows: the stirring rate was 300 r / min for the first 10 min and 500 r / min for the next 15 min.

[0035] Testing revealed that the defoamer prepared using the method described in the second embodiment for the bio-fermentation process had a defoaming time of 4.8 seconds and a foam suppression time of 48 hours. This defoamer was applied to glutamic acid fermentation (fermentation conditions: temperature 35℃, pH 7.2) and added in stages at 0.3% of the total fermentation broth mass. Results showed that, compared to the blank control group, the average biomass of *E. coli* increased by 15%, and the final glutamic acid yield increased by 8%.

[0036] Animal cell culture application: In fed-batch culture of CHO cells, the initial addition amount was 0.08% of the culture medium volume, with 0.02% added on days 3 and 5 of culture, respectively. Compared with the control, this defoamer effectively controlled foaming throughout the culture process, increasing the viable cell density by approximately 20% by the end of the culture period, without inhibiting cell growth or product expression.

[0037] Comparative experiment: Using the defoamer prepared in the first embodiment (experimental group), a commercially available traditional organosilicon defoamer (control group 1), and the basic defoamer of this application without polysaccharide components (control group 2) as research objects, equal amounts (0.2%, w / w) of the above three defoamers were added to the same yeast fermentation system (used for penicillin production, fermentation conditions the same as in the first embodiment). Their defoaming and foam-inhibiting performance, effects on cell growth, and final product yield were tested. The results are shown in the table below.

[0038] The comparative data shows that by introducing polysaccharide components, this invention not only maintains the high-efficiency defoaming performance of silicone defoamers, but also promotes cell growth and significantly increases the yield of fermentation products. In contrast, traditional silicone defoamers and polysaccharide-free base solutions both inhibit cell growth and product synthesis, which fully demonstrates the innovation and superiority of this application.

[0039] The experimental group showed a significant promoting effect on yeast cell proliferation (+18.0%), directly resulting in a 12.0% increase in penicillin yield. In contrast, both control groups 1 and 2 exhibited varying degrees of inhibition on cell growth, leading to decreased yield. These results clearly demonstrate that the introduction of active polysaccharide components is a key innovation in overcoming the cytotoxicity of traditional organosilicon defoamers and achieving growth-promoting functions.

[0040] This application not only solves the technical pain point of traditional silicone defoamers inhibiting cell activity in biological processes, but also transforms defoamers from simple "process aids" into "performance enhancers" that can improve overall production efficiency through functional modification, demonstrating significant technological progress and application value.

[0041] Beneficial effects 1. Functional Synergy: By introducing specific polysaccharide components, this application enables the defoamer to simultaneously possess the functions of highly efficient defoaming (defoaming time 3.8-4.8s), long-lasting foam suppression (foam suppression time 48-55h), and cell growth promotion (proliferation promotion rate 15-18%), thus solving the problem of the single function of traditional organosilicon defoamers. 2. Wide applicability: This defoamer has good compatibility with a variety of microorganisms (bacteria, yeast, etc.) and can be applied to different types of biological fermentation processes such as antibiotics, enzyme preparations, and amino acids. Furthermore, the application effect can be further optimized by adding it in stages. 3. Simple preparation process: The entire preparation process requires no special equipment, the reaction conditions are mild (temperature 20-40℃, atmospheric pressure), the optimized parameter range is easier to control, and it is easy to scale up for industrial production; 4. Significant economic benefits: While improving defoaming efficiency, it can increase the yield of fermentation products by 8-12%, reduce the production cost of industrial fermentation, and has extremely high promotion value.

[0042] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0043] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

[0044] Therefore, the scope of protection of this invention patent shall be determined by the appended claims.

Claims

1. An antifoam agent for a biological fermentation process, characterized in that, The following components are included by weight parts: 18-20 parts of silicone paste, 18-20 parts of polyether modified silicone oil, 2-5 parts of polysaccharide component, 2-5 parts of emulsifier, 50-60 parts of deionized water, wherein the polysaccharide component is ginkgo biloba polysaccharide and trehalose, and the mass ratio of the ginkgo biloba polysaccharide to the trehalose is 2:1-3:

1.

2. The antifoam agent for a biological fermentation process according to claim 1, characterized by, The main components of the silicone paste are polydimethylsiloxane and silicon dioxide, and the ratio of the polydimethylsiloxane to the silicon dioxide is 8:1-10:1, and the polyether modified silicone oil is polyoxyethylene-polyoxypropylene block modified silicone oil with an EO / PO molar ratio of 1.2:1-1.5:1 and a hydroxyl value of ≤3 mgKOH / g.

3. A process for the preparation of an antifoam agent for biological fermentation processes, characterized in that, The following steps are included: S10, providing 18-20 parts of silicone paste and 18-20 parts of polyether modified silicone oil by weight parts, and adding them into a reaction kettle for stirring and mixing to obtain a basic defoaming agent; S20, adding 2-5 parts of polysaccharide component by weight parts into the basic defoaming agent and continuing to stir to obtain a reaction system; S30, adding 2-5 parts of emulsifier and 50-60 parts of deionized water by weight parts into the reaction system and stirring and emulsifying to obtain a final defoaming agent; wherein the polysaccharide component is ginkgo biloba polysaccharide and trehalose, and the mass ratio of the ginkgo biloba polysaccharide to the trehalose is 2:1-3:

1.

4. The process for the preparation of an antifoam agent for biological fermentation processes according to claim 3, characterized in that, In the step S10, the temperature during stirring and mixing is 25-35℃, the stirring and mixing rate is 400-450 r / min, and the stirring and mixing time is 20-30 min.

5. The method of preparing an antifoam agent for a biological fermentation process according to claim 3, characterized in that, In the step S20, the stirring time is 30-40 min.

6. The process for the preparation of an antifoam agent for biological fermentation processes according to claim 3, characterized in that, In the step S30, the stirring and emulsifying time is 25-30 min.

7. The process for the preparation of an antifoam agent for biological fermentation processes according to claim 3, characterized in that, The pretreatment process of the polysaccharide component is as follows: Dissolve ginkgo biloba polysaccharide in water to prepare a ginkgo biloba polysaccharide solution with a mass concentration of 6-7%; Use an ultrasonic wave with a power of 350-450 W to treat the ginkgo biloba polysaccharide solution for 25-35 min, and then add trehalose to stir and dissolve to obtain a reactant; Freeze dry the reactant at a temperature of -45--30℃, and then crush it to 120-180 mesh to obtain the polysaccharide component.

8. The process for the preparation of an antifoam agent for biological fermentation processes according to claim 3, characterized in that, The emulsifier is compounded by spandab 80 and tween 80 with a mass ratio of 3:1, and the stirring and emulsifying process adopts phased stirring, i.e., the stirring rate is 300 r / min in the first 10 min and 500 r / min in the next 15-20 min.

9. Use of an antifoam agent for a biological fermentation process, characterized in that, The defoaming agent for biological fermentation process in any one of claims 1-2 is applied in a biological fermentation process.

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