Process for promoting saccharopolyspora erythraea to produce erythromycin by adding surfactant

Abstract

The invention discloses a process for promoting saccharopolyspora erythraea to produce erythromycin by adding surfactants, 11 nonionic surfactants are screened, a nonionic surfactant S107 capable of promoting synthesis of erythromycin is obtained through single factor experiment comparison, the optimal adding concentration mass fraction is 0.2%, and the optimal adding time is 144 h. According to the process, the yield of erythromycin in a shake flask can be improved by 31.90% at most. The addition process of the surfactant is combined in a 5L fermentation tank, and through fed-batch fermentation, the result shows that the final erythromycin yield of an experimental group added with the surfactant reaches 4704.71 mg / L and is improved by 17.18% compared with that of a control group.

Description

Technical Field

[0001] The invention provides a process for adding a surfactant to a fermentation process, specifically, the process for adding a surfactant to a fermentation process for producing erythromycin by Saccharopolyspora erythraea based on different adding types, concentrations and times. Background Art

[0002] Nonionic surfactants refer to a type of surfactant that does not produce ions in aqueous solution, and the groups do not dissociate. Its hydrophobic groups are similar to those of ionic surfactants, while its hydrophilic groups are mainly composed of multiple oxygen-containing groups, commonly hydroxyl groups and oxygen-containing long-chain olefins and ether chains. Because the hydrophilicity of ether bonds, aldehyde groups, hydroxyl groups, and amide groups is slightly weaker, nonionic surfactants must contain multiple such groups to show hydrophilicity, while cationic and anionic surfactants only need one hydrophilic group. Nonionic surfactants have better performance than ionic surfactants, such as good solubility in organic solvents and water, not easily affected by strong electrolytes such as inorganic salts, acids and alkalis, and high stability in solution. Nonionic surfactants can be compounded and compatible with different types of surfactants, so they are suitable for scenarios with different performance requirements. Due to its low foaming and excellent hard water resistance, it is suitable for some special detergents. At the same time, it has many common properties such as emulsification, dispersion, solubilization, wetting, etc., so it has many uses in industry and life. The most commonly used non-ionic surfactants are divided into three series: Span, Tween, and Brij, as shown in the figure below.

[0003] Surfactants are widely used additives that can reduce the surface tension of solutions, improve gas exchange efficiency, and optimize the utilization of raw materials. They are widely used in the fields of petroleum, chemical industry, and materials. Surfactants can improve the fluidity of microbial fermentation liquid, change the permeability of cell membranes, and reduce the transmission resistance of gases and substrate raw materials. The fermentation industry is used in the production of organic acids and amino acids, colloids, antibiotics, pigments, active enzymes and other products.

[0004] The main strain for industrial production of erythromycin is Saccharopolyspora erythraea, which uses a variety of nutrients to increase carbon and nitrogen sources and the factors necessary for growth and metabolism in shake flasks. In batch fermentation, glucose, soybean oil and n-propanol are added to provide the necessary precursors and nutrients for the growth of S.erythraea and the synthesis of erythromycin. Summary of the invention

[0005] Preliminary screening in preliminary experiments: Among the 11 non-ionic surfactants S100, S101, S102, S103, S105, S107, S109, S110, S111, S113, and S115, 8 non-ionic surfactants, S100, S101, S102, S103, S105, S107, S109, and S108, were selected through preliminary screening in preliminary experiments.

[0006] Single factor screening of surfactant types: 8 surfactants were selected from the preliminary experiment, and various non-ionic surfactants with a mass fraction of 3‰ were added around 60h. The potential optimal surfactant type was determined by comparing with the blank shake flask without surfactants and sampling at the end of fermentation. Three parallel shake flasks were taken for each single factor variable. Three parallel shake flasks were taken for each group of surfactants, and three groups of blank shake flasks were added.

[0007] Single factor screening of surfactant concentration: By setting a more detailed and wider range of added concentrations, and in order to avoid the error caused by the gradient itself when setting the gradient, a 1‰ difference between groups is set, which can provide more data for subsequent research. In order to investigate the effect of the optimized S107 surfactant on S.erythraea's erythromycin production, after literature research, the surfactant mass fraction was set in the range of 1‰, 2‰, 3‰, 4‰, 5‰, 6‰, and 7‰, and the effect of different concentrations of surfactant on erythromycin synthesis was determined. Three shake flasks were taken for parallel experiments at each concentration point, and three groups of blank shake flasks were added for control.

[0008] Single factor screening of surfactant addition time: The long fermentation cycle of S.erythraea can be roughly divided into primary metabolism, platform phase, and secondary metabolism synthesis phase. In order to investigate the effect of the optimized S107 surfactant on the production of erythromycin by S.erythraea, the concentration of addition was 3‰, and the corresponding surfactant was added at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h, and 168h. These 15 time points correspond to the sampling points in laboratory research and industrial production, and the effect of adding surfactants in different periods of S.erythraea can be seen. Three shake flasks were taken for parallel experiments at each time point, and three groups of blank shake flasks were added.

[0009] Shake flask verification: By screening 11 non-ionic surfactants, the non-ionic surfactant S107 that can promote erythromycin synthesis was obtained from single factor experimental comparison. The optimal addition concentration was 2‰, and the optimal addition time was 144h. This process can increase the yield of erythromycin by 31.90% in a shake flask.

[0010] 5L fermentation tank verification: Secondly, the above-mentioned surfactant addition process was combined in a 5L fermentation tank through batch fed fermentation. The results showed that the final output of the experimental group with added surfactant reached 4704.71 mg / L, an increase of 17.18% over the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Measurement of erythromycin potency Figure 2 :Effects of different nonionic surfactants on erythromycin production by S.erythraea Figure 3 :Effects of different concentrations of surfactant S107 on erythromycin production in S.erythraea Figure 4 :Effects of different addition times of surfactant S107 on erythromycin production in S.erythraea Figure 5 :Verification of surfactant addition process in shake flask fermentation Figure 6 :Verification of surfactant addition process on 5L fermentation DETAILED DESCRIPTION

[0012] 1. First, the bacteria in the glycerol tube are revived and enriched in the plate, and the spore plate with a size of 2cm×2cm and agar block with spores is inoculated into the seed culture shake bottle to obtain high-quality red Saccharopolyspora seeds.

[0013] Second: Then, the seeds were transferred to a 500mL shaking flask at a 10% inoculation rate, shaken at 220rpm, and cultured in a culture room at 34°C for 7 days (about 168h). S100, S101, S102, S103, S105, S107, S109, and S108 were added to the fermentation process. Three parallel fermentation shaking flasks were taken, and a blank control was taken. After the fermentation was completed, the absorbance of the fermentation liquid was measured at a wavelength of 485nm with an enzyme marker, and the erythromycin potency was calculated according to Er=(ax+b)*N, where Er is the chemical potency of erythromycin (mg / L), ax+b is the standard curve, where x is the OD value, and N is the dilution multiple of the sample. The optimal surfactant S107 was obtained.

[0014] Three: The same culture method as step 2, set the concentration gradient to 1‰, 2‰, 3‰, 4‰, 5‰, 6‰, 7‰, and the time gradient to 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h, 168h. They must meet the different addition conditions of the single factor. Similarly, the absorbance of the fermentation liquid was measured after the fermentation was completed. The optimal surfactant S107 was obtained, and the optimal concentration and time were 2‰ and 144h.

[0015] Fourth: According to the new addition process, the erythromycin content in the fermentation broth was measured after fermentation in a 500mL shake flask at 220rpm and 34℃ for 168h. The new process increased the erythromycin content to 1286.86mg / L, an increase of 31.90%

[0016] Five: According to the new addition process, the culture was carried out in a 5L fermentation tank, with the ventilation range set at about 1vvm, tank pressure 0.05MPa, temperature 34℃, dissolved oxygen around 40%, cultured for 168h, and glucose, soybean oil, and n-propanol were added during the process. Samples were usually taken every 12h, and the residual alcohol concentration was controlled at 0.8g / L-1.0g / L, and the glucose concentration was controlled at 0.1-0.2g / L. Glucose and soybean oil were increased and decreased synchronously, and the pH was maintained at 7.0. After the fermentation was completed, the final yield of the fermentation tank with the addition of surfactant by the new process reached 4704.71mg / L, which was 17.18% higher than that of the control group.

Claims

1. (1) Preliminary screening: 8 nonionic surfactants, namely S100, S101, S102, S103, S105, S107, S109, S110, S111, S113, and S115, were initially screened out through preliminary experiments. (2) Single factor screening of surfactant types: In order to investigate the effects of different types of surfactants on the erythromycin production of S.erythraea, 8 surfactants were selected to observe their effects on erythromycin synthesis. Various non-ionic surfactants with a mass fraction of 3‰ were selected to be added around 60h. The potential optimal surfactant type was determined by comparing it with a blank shake flask without surfactant added and sampling at the end of fermentation. Three shake flasks were used for parallel experiments for each group of surfactants, and three groups of blank shake flasks were added. The optimal surfactant was S107. (3) Single factor screening of surfactant concentration: Different concentrations of S107 added will affect the synthesis of erythromycin. Based on the literature, a more detailed and wider range of added concentrations was set. At the same time, in order to avoid the error caused by the gradient itself, a 1‰ difference between groups was set when setting the gradient. S107 was added around 60h, and the surfactant mass fraction was set in the range of 1‰, 2‰, 3‰, 4‰, 5‰, 6‰, and 7‰ to measure the effect of different concentrations of surfactant on erythromycin synthesis. Three parallel experiments and a control group concentration single factor group were also taken. The optimal addition concentration of S107 is 2‰. (4) Single factor screening of the time for adding surfactants: The long fermentation cycle of S.erythraea can be roughly divided into primary metabolism, plateau phase, and secondary metabolism synthesis phase. By adding S107 at a concentration of 2‰, the corresponding surfactants were added at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h, and 168h. These 15 time points correspond to the sampling points in laboratory research and industrial production, and the effects of adding surfactants at different periods of S.erythraea can be seen. Three shake flasks were taken for parallel experiments at each time point, and three groups of blank shake flasks were added. The optimal addition time of S107 is 144h. By screening 11 non-ionic surfactants, the non-ionic surfactant S107 that can promote erythromycin synthesis was obtained from single factor experimental comparison. The optimal addition concentration was 2‰ and the optimal addition time was 144h. This process can increase the yield of erythromycin by 31.90% in a shake flask. Secondly, the above-mentioned surfactant addition process was combined with batch fermentation in a 5L fermenter. The results showed that the final yield of the experimental group with added surfactant reached 4704.71 mg / L, an increase of 17.18% over the control group.

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